# SVI ClinGen Familial Hypercholesterolemia Expert Panel Specifications to the ACMG/AMP Variant Classification Guidelines Version 1.2 (GN013) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MYH7 Version 2.0.0 (GN002) ClinGen PTEN Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PTEN Version 3.1.0 (GN003) ClinGen Hearing Loss Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CDH23, COCH, GJB2,KCNQ4, MYO6, MYO7A, SLC26A4, TECTA and USH2A Version 2 (GN005) ClinGen Phenylketonuria Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PAH Version 2.0.0 (GN006) ClinGen CDH1 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 3.1 (GN007) ClinGen Myeloid Malignancy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RUNX1 Version 3.1.0 (GN008) ClinGen TP53 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for TP53 Version 2.4.0 (GN009) ClinGen Lysosomal Storage Disorders Variant Curation Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 2 (GN010) ClinGen Platelet Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 2.1 (GN011) ClinGen Malignant Hyperthermia Susceptibility Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RYR1 Version 2 (GN012) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (GN001) ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_ntDNA (GN014) ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_ntDNA (GN014) ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_ntDNA (GN014) ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_ntDNA (GN014) ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_mtDNA (GN015) ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PALB2 Version 1.2.0 (GN077) ClinGen VHL Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for VHL Version 1.1.0 (GN078) ClinGen Platelet Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GP1BA Version 1.1.0 (GN079) ClinGen Coagulation Factor Deficiency Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for F9 Version 2.0.0 (GN080) ClinGen von Willebrand Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for VWF Version 1.0.0 (GN081) ClinGen Platelet Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GP1BB Version 1.1.0 (GN082) ClinGen Platelet Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GP9 Version 1.1.0 (GN083) ClinGen Thrombosis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SERPINC1 Version 1.1.0 (GN084) ClinGen Monogenic Diabetes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for HNF4A Version 4.0.0 (GN085) ClinGen Monogenic Diabetes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GCK Version 3.1.0 (GN086) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MRAS Version 1.4.0 (GN087) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RMRP Version 1.2.0 (GN088) ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for APC Version 2.1.0 (GN089) ClinGen von Willebrand Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for VWF Version 1.0.0 (GN090) ClinGen Lysosomal Diseases Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for IDUA Version 1.2.0 (GN091) ClinGen ENIGMA BRCA1 and BRCA2 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRCA1 Version 1.2.0 (GN092) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for LZTR1 Version 1.3.0 (GN094) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MYBPC3 Version 1.0.0 (GN095) ClinGen ENIGMA BRCA1 and BRCA2 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRCA2 Version 1.2.0 (GN097) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for TNNI3 Version 1.0.0 (GN098) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for TNNT2 Version 1.0.0 (GN099) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for TPM1 Version 1.0.0 (GN100) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ACTC1 Version 1.0.0 (GN101) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MYL2 Version 1.0.0 (GN102) ClinGen Cardiomyopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MYL3 Version 1.0.0 (GN103) ClinGen Glaucoma Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CYP1B1 Version 1.0.0 (GN104) ClinGen X-linked Inherited Retinal Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RPGR Version 1.0.0 (GN106) ClinGen Potassium Channel Arrhythmia Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for KCNQ1 Version 1.0.0 (GN112) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for FOXN1 Version 2.2.0 (GN113) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ADA Version 2.1.0 (GN114) ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0.0 (GN115) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for DCLRE1C Version 2.1.0 (GN116) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for IL7R Version 2.1.0 (GN119) ClinGen Leber Congenital Amaurosis/early onset Retinal Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RPE65 Version 1.0.0 (GN120) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for JAK3 Version 2.1.0 (GN121) ClinGen Antibody Deficiencies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CTLA4 Version 1.0.0 (GN122) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RAG1 Version 2.1.0 (GN123) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RAG2 Version 2.1.0 (GN124) ClinGen Pulmonary Hypertension Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BMPR2 Version 2.0.0 (GN125) ClinGen X-linked Inherited Retinal Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RS1 Version 1.0.0 (GN126) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RRAS2 Version 1.3.0 (GN127) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PPP1CB Version 1.3.0 (GN128) ClinGen Severe Combined Immunodeficiency Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for IL2RG Version 2.1.0 (GN129) ClinGen Hereditary Hemorrhagic Telangiectasia Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ACVRL1 Version 1.1.0 (GN135) ClinGen Hereditary Hemorrhagic Telangiectasia Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ENG Version 1.1.0 (GN136) ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH2 Version 2.0.0 (GN137) ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH6 Version 2.0.0 (GN138) ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PMS2 Version 2.0.0 (GN139) ClinGen Antibody Deficiencies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PIK3CD Version 1.0.0 (GN141) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for NEB Version 1.0.0 (GN146) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ACTA1 Version 2.0.0 (GN147) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for DNM2 Version 1.0.0 (GN148) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MTM1 Version 1.0.0 (GN149) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RYR1 Version 2.0.0 (GN150) ClinGen Urea Cycle Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for OTC Version 1.0.0 (GN156) ClinGen ABCA4 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ABCA4 Version 1.0.0 (GN164) ClinGen Leber Congenital Amaurosis/early onset Retinal Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GUCY2D Version 1.0.0 (GN167) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ACTA1 Version 1.0.0 (GN169) ClinGen Congenital Myopathies Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RYR1 Version 2.0.0 (GN179) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for DYSF Version 2.0.0 (GN180) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SGCB Version 2.0.0 (GN184) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SGCG Version 2.0.0 (GN185) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SGCD Version 2.0.0 (GN186) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CAPN3 Version 2.0.0 (GN187) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ANO5 Version 2.0.0 (GN188) ClinGen Limb Girdle Muscular Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SGCA Version 2.0.0 (GN189) ClinGen Monogenic Diabetes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for HNF1A Version 3.1.0 (GN017) ClinGen Brain Malformations Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1.1.0 (GN018) ClinGen Glaucoma Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MYOC Version 2.1.0 (GN019) ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ATM Version 1.5.0 (GN020) ClinGen ACADVL Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for ACADVL Version 2.1.0 (GN021) ClinGen FBN1 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1 (GN022) ClinGen Hearing Loss Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for OTOF and MYO15A Version 1 (GN023) ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for DICER1 Version 1.4.0 (GN024) ClinGen Cerebral Creatine Deficiency Syndromes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GATM Version 2.0.0 (GN025) ClinGen Cerebral Creatine Deficiency Syndromes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for GAMT Version 2.0.0 (GN026) ClinGen Cerebral Creatine Deficiency Syndromes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SLC6A8 Version 1.2.0 (GN027) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for TCF4 Version 5.0.0 (GN032) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SLC9A6 Version 5.0.0 (GN033) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for CDKL5 Version 5.0.0 (GN034) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for FOXG1 Version 5.0.0 (GN035) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MECP2 Version 5.0.0 (GN036) ClinGen Rett and Angelman-like Disorders Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for UBE3A Version 6.0.0 (GN037) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SHOC2 Version 2.3.0 (GN038) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for NRAS Version 2.3.0 (GN039) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RAF1 Version 2.3.0 (GN040) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SOS1 Version 2.3.0 (GN041) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SOS2 Version 2.3.0 (GN042) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PTPN11 Version 2.3.0 (GN043) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for KRAS Version 2.3.0 (GN044) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MAP2K1 Version 2.3.0 (GN045) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for HRAS Version 2.3.0 (GN046) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RIT1 Version 2.3.0 (GN047) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MAP2K2 Version 2.3.0 (GN048) ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRAF Version 2.3.0 (GN049) ClinGen Epilepsy Sodium Channel Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SCN1A Version 2.0.0 (GN067) ClinGen Epilepsy Sodium Channel Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SCN2A Version 2.0.0 (GN068) ClinGen Epilepsy Sodium Channel Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SCN3A Version 2.1.0 (GN069) ClinGen Epilepsy Sodium Channel Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SCN8A Version 2.0.0 (GN070) ClinGen Coagulation Factor Deficiency Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for F8 Version 2.0.0 (GN071) ClinGen Epilepsy Sodium Channel Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for SCN1B Version 2.0.0 (GN076) ClinGen Leber Congenital Amaurosis/early onset Retinal Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for AIPL1 Version 1.0.0 (GN208) # RuleSet 135640393 135640453 135640513 135640633 135640693 135640753 135640813 135640873 135640933 135640993 135641053 135641113 135641173 135641174 135641175 135641176 135641398 1526222369 1527855768 1527857191 1527859065 1528062428 1528071540 1528072656 1528074035 1528338164 1528339124 1528438503 1529230108 1529292804 1529372875 1529725542 1530970800 1532496151 1535304693 1535438847 1535821579 1535826201 1535827410 1535829283 1535830819 1535831955 1536422175 1552193489 1562365913 1562840339 1564418413 1564688423 1565017056 1566364251 1566365968 1567011294 1567102254 1567643924 1567863469 1568416535 1570648190 1571593874 1571595305 1571601262 1576018580 1576926556 1577628949 1578294898 1578297071 1610842302 1613756510 1615403533 1616103433 1616197519 1616389166 1622722972 1670131001 1733815125 1743586732 1795775094 1807907472 1811993358 1812015340 1812027476 1812056150 1812076198 1812104365 467903870 467907154 635003681 639508987 639509377 643243099 643243100 643243101 643243102 643243103 643243104 643243109 643243110 643243111 643243112 643243113 643243114 643243115 643243116 643243117 643243118 643243119 643243120 643243121 643243122 643243123 643243124 643243125 643243126 643243144 643243145 643243146 643243147 643243148 643243153 94412675 # GeneType nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear Nuclear nuclear nuclear nuclear nuclear mitochondrial nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear nuclear # Gene LDLR (undefined) MYH7 (undefined) PTEN (undefined) CDH23 (undefined), COCH (undefined), GJB2 (undefined), KCNQ4 (undefined), MYO6 (undefined), MYO7A (undefined), SLC26A4 (undefined), TECTA (undefined), USH2A (undefined) PAH (undefined) CDH1 (undefined) RUNX1 (undefined) TP53 (undefined) GAA (undefined) ITGA2B (undefined), ITGB3 (undefined) RYR1 (undefined) NA SLC19A3 (undefined) PDHA1 (undefined) POLG (undefined) ETHE1 (undefined) NA PALB2 (undefined) VHL (undefined) GP1BA (undefined) F9 (undefined) VWF (undefined) GP1BB (undefined) GP9 (undefined) SERPINC1 (undefined) HNF4A (undefined) GCK (undefined) MRAS (undefined) RMRP (undefined) APC (undefined) VWF (undefined) IDUA (undefined) BRCA1 (undefined) LZTR1 (undefined) MYBPC3 (undefined) BRCA2 (undefined) TNNI3 (undefined) TNNT2 (undefined) TPM1 (undefined) ACTC1 (undefined) MYL2 (undefined) MYL3 (undefined) CYP1B1 (undefined) RPGR (undefined) KCNQ1 (undefined) FOXN1 (undefined) ADA (undefined) MLH1 (undefined) DCLRE1C (undefined) IL7R (undefined) RPE65 (undefined) JAK3 (undefined) CTLA4 (undefined) RAG1 (undefined) RAG2 (undefined) BMPR2 (undefined) RS1 (undefined) RRAS2 (undefined) PPP1CB (undefined) IL2RG (undefined) ACVRL1 (undefined) ENG (undefined) MSH2 (undefined) MSH6 (undefined) PMS2 (undefined) PIK3CD (undefined) NEB (undefined) ACTA1 (undefined) DNM2 (undefined) MTM1 (undefined) RYR1 (undefined) OTC (undefined) ABCA4 (undefined) GUCY2D (undefined) ACTA1 (undefined) RYR1 (undefined) DYSF (undefined) SGCB (undefined) SGCG (undefined) SGCD (undefined) CAPN3 (undefined) ANO5 (undefined) SGCA (undefined) HNF1A (undefined) AKT3 (undefined), MTOR (undefined), PIK3CA (undefined), PIK3R2 (undefined) MYOC (undefined) ATM (undefined) ACADVL (undefined) FBN1 (undefined) MYO15A (undefined), OTOF (undefined) DICER1 (undefined) GATM (undefined) GAMT (undefined) SLC6A8 (undefined) TCF4 (undefined) SLC9A6 (undefined) CDKL5 (undefined) FOXG1 (undefined) MECP2 (undefined) UBE3A (undefined) SHOC2 (undefined) NRAS (undefined) RAF1 (undefined) SOS1 (undefined) SOS2 (undefined) PTPN11 (undefined) KRAS (undefined) MAP2K1 (undefined) HRAS (undefined) RIT1 (undefined) MAP2K2 (undefined) BRAF (undefined) SCN1A (undefined) SCN2A (undefined) SCN3A (undefined) SCN8A (undefined) F8 (undefined) SCN1B (undefined) AIPL1 (undefined) # Disease hypercholesterolemia, familial, 1 (MONDO:0007750) dilated cardiomyopathy (MONDO:0005021), hypertrophic cardiomyopathy (MONDO:0005045) NA Usher syndrome (MONDO:0019501), nonsyndromic genetic hearing loss (MONDO:0019497), nonsyndromic genetic hearing loss (MONDO:0019497), nonsyndromic genetic hearing loss (MONDO:0019497), nonsyndromic genetic hearing loss (MONDO:0019497), nonsyndromic genetic hearing loss (MONDO:0019497), Usher syndrome (MONDO:0019501), nonsyndromic genetic hearing loss (MONDO:0019497), Pendred syndrome (MONDO:0010134), nonsyndromic genetic hearing loss (MONDO:0019497), Usher syndrome (MONDO:0019501) phenylketonuria (MONDO:0009861) hereditary diffuse gastric adenocarcinoma (MONDO:0007648) hereditary thrombocytopenia and hematologic cancer predisposition syndrome (MONDO:0011071) Li-Fraumeni syndrome (MONDO:0018875) glycogen storage disease II (MONDO:0009290) Glanzmann thrombasthenia (MONDO:0100326), Glanzmann thrombasthenia (MONDO:0100326) malignant hyperthermia of anesthesia (MONDO:0018493) NA biotin-responsive basal ganglia disease (MONDO:0011841) pyruvate dehydrogenase deficiency (MONDO:0019169) mitochondrial disease (MONDO:0044970) ethylmalonic encephalopathy (MONDO:0011229) NA PALB2-related cancer predisposition (MONDO:0700272), Fanconi anemia complementation group N (MONDO:0012565) von Hippel-Lindau disease (MONDO:0008667) Bernard-Soulier syndrome (MONDO:0009276) hemophilia B (MONDO:0010604) von Willebrand disease type 2A (MONDO:0015628), von Willebrand disease type 2B (MONDO:0015629), von Willebrand disease type 2M (MONDO:0015630), von Willebrand disease 2 (MONDO:0013304), von Willebrand disease (hereditary or acquired) (MONDO:0024574) Bernard-Soulier syndrome (MONDO:0009276) Bernard-Soulier syndrome (MONDO:0009276) hereditary antithrombin deficiency (MONDO:0013144) monogenic diabetes (MONDO:0015967) monogenic diabetes (MONDO:0015967) RASopathy (MONDO:0021060) cartilage-hair hypoplasia (MONDO:0009595) familial adenomatous polyposis 1 (MONDO:0021056) von Willebrand disease type 2N (MONDO:0015631) mucopolysaccharidosis type 1 (MONDO:0001586) BRCA1-related cancer predisposition (MONDO:0700268) RASopathy (MONDO:0021060) hypertrophic cardiomyopathy (MONDO:0005045) BRCA2-related cancer predisposition (MONDO:0700269) hypertrophic cardiomyopathy (MONDO:0005045) hypertrophic cardiomyopathy (MONDO:0005045), dilated cardiomyopathy (MONDO:0005021) hypertrophic cardiomyopathy (MONDO:0005045) hypertrophic cardiomyopathy (MONDO:0005045) hypertrophic cardiomyopathy (MONDO:0005045) hypertrophic cardiomyopathy (MONDO:0005045) CYP1B1-related glaucoma with or without anterior segment dysgenesis (MONDO:0800472) RPGR-related retinopathy (MONDO:0100437) long QT syndrome 1 (MONDO:0100316) T-cell immunodeficiency, congenital alopecia, and nail dystrophy (MONDO:0011132) severe combined immunodeficiency, autosomal recessive, T cell-negative, B cell-negative, NK cell-negative, due to adenosine deaminase deficiency (MONDO:0007064) Lynch syndrome 2 (MONDO:0012249), mismatch repair cancer syndrome 1 (MONDO:0010159) severe combined immunodeficiency due to DCLRE1C deficiency (MONDO:0011225) immunodeficiency 104 (MONDO:0012163) RPE65-related recessive retinopathy (MONDO:0100368) T-B+ severe combined immunodeficiency due to JAK3 deficiency (MONDO:0010938) autoimmune lymphoproliferative syndrome due to CTLA4 haploinsufficiency (MONDO:0014493) recombinase activating gene 1 deficiency (MONDO:0000572) recombinase activating gene 2 deficiency (MONDO:0000573) pulmonary arterial hypertension (MONDO:0015924) X-linked retinoschisis (MONDO:0010725) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) T-B+ severe combined immunodeficiency due to gamma chain deficiency (MONDO:0010315) telangiectasia, hereditary hemorrhagic, type 2 (MONDO:0010880) telangiectasia, hereditary hemorrhagic, type 1 (MONDO:0008535) Lynch syndrome 1 (MONDO:0007356), mismatch repair cancer syndrome 1 (MONDO:0010159) Lynch syndrome (MONDO:0005835), mismatch repair cancer syndrome 1 (MONDO:0010159) Lynch syndrome 4 (MONDO:0013699), mismatch repair cancer syndrome 1 (MONDO:0010159) immunodeficiency 14 (MONDO:0014222) nemaline myopathy (MONDO:0018958) alpha-actinopathy (MONDO:0100084) centronuclear myopathy (MONDO:0018947) centronuclear myopathy (MONDO:0018947) RYR1-related myopathy (MONDO:0100150) ornithine carbamoyltransferase deficiency (MONDO:0010703) ABCA4-related retinopathy (MONDO:0800406) GUCY2D-related recessive retinopathy (MONDO:0100453) alpha-actinopathy (MONDO:0100084) RYR1-related myopathy (MONDO:0100150) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) autosomal recessive limb-girdle muscular dystrophy (MONDO:0015152) monogenic diabetes (MONDO:0015967) obsolete cerebral malformation (MONDO:0016054), obsolete cerebral malformation (MONDO:0016054), obsolete cerebral malformation (MONDO:0016054), obsolete cerebral malformation (MONDO:0016054) open-angle glaucoma (MONDO:0005338) ATM-related cancer predisposition (MONDO:0700270), ataxia telangiectasia (MONDO:0008840), ataxia - telangiectasia variant (MONDO:0018266) very long chain acyl-CoA dehydrogenase deficiency (MONDO:0008723) Marfan syndrome (MONDO:0007947) nonsyndromic genetic hearing loss (MONDO:0019497), nonsyndromic genetic hearing loss (MONDO:0019497) DICER1-related tumor predisposition (MONDO:0100216) AGAT deficiency (MONDO:0012996) guanidinoacetate methyltransferase deficiency (MONDO:0012999) creatine transporter deficiency (MONDO:0010305) Pitt-Hopkins syndrome (MONDO:0012589) Christianson syndrome (MONDO:0010278) CDKL5 disorder (MONDO:0100039) FOXG1 disorder (MONDO:0100040) Rett syndrome (MONDO:0010726) Angelman syndrome (MONDO:0007113) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) RASopathy (MONDO:0021060) Dravet syndrome (MONDO:0100135), generalized epilepsy with febrile seizures plus (MONDO:0018214), genetic developmental and epileptic encephalopathy (MONDO:0100062) complex neurodevelopmental disorder (MONDO:0100038) genetic developmental and epileptic encephalopathy (MONDO:0100062) complex neurodevelopmental disorder (MONDO:0100038) hemophilia A (MONDO:0010602) generalized epilepsy with febrile seizures plus (MONDO:0018214), genetic developmental and epileptic encephalopathy (MONDO:0100062) AIPL1-related retinopathy (MONDO:0100438) PM2_Moderate Variant has a PopMax MAF ≤0.0002 (0.02%) in gnomAD. Consider exceptions for known founder variants. NA NA NA NA NA NA NA Low frequency in population databases. * Minor allele frequency <0.1% (0.001) in all continental populations with >2000 alleles in gnomAD. Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium Not Applicable: PM2 is not used alone. Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.Caveat: Population data for indels may be poorly called by next generation sequencing. <0.00005 (<0.0050%) 0.0000092 (<0.00092%) <0.0005 (<0.05% ) <0.00002 (<0.0020%) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PM1_Moderate Missense variant located in exon 4, or a missense change in one of 60 highly conserved cysteine residues (listed in Supp. Table 4). Caveat: variant must also meet PM2. Applicable to missense variants in _MYH7_ in the specific regions listed below (Walsh _et al._ 2019[¹⁰](#pmid_30696458)). 1. Transcripts ENST00000355349 & NM\_000257.42. Codons 167-931\*Data from HCM case cohorts was used to derive these cluster regions. Therefore, this rule should NOT be applied when additional evidence for the variant supports that the variant causes a phenotype other than HCM (e.g., variant seen in multiple DCM cases).Enrichment was not observed for DCM in any genes.Rule should NOT be combined with PM5 because presence of pathogenic variants in the same codon/region were used to determine clustering and would be double-counting evidence._\* This region is updated from v1.0 (Kelly et al. 2018_[_¹¹_](#pmid_29300372)_)._ Located in a mutational hot spot and/or critical and well-established functional domain. Defined to include residues in catalytic motifs: 90-94, 123-130, 166-168 (NP_ 000305.3) Mutational hot spot or well-studied functional domain without benign variation (KCNQ4 pore-forming region).* KCNQ4 (NM_004700.4) gene - missense variants located within amino acids 271-292 can be awarded PM1. This region is the pore-forming intramembrane region where many variants that cause autosomal dominant hearing loss are located (Naito et al. 2013, PMID: 23717403; https://www.uniprot.org/uniprot/P56696). There are only two missense variants in this region in gnomAD, each with only single allele (http://gnomad.broadinstitute.org/; rs763326539: 1/33578 Latino chromosomes; rs55737429: 1/111720 European chromosomes). * Active site residues in PAH include: Tyr138, Arg158, Val245, Tyr268, Thr278, Pro279, Glu289, Ala300, Asp315, Phe331, Ala345, Gly346, Ser349, Tyr377* Substrate binding residues in PAH are: 46-48, 63-69* Cofactor binding residues in PAH are: His285, His290, Glu330, 246-266, 280-283, 322-326, 377-379* Do not apply if PP3\_Strong applies Not Applicable: Not applicable for CDH1. NA Missense variants within the following codons using transcript NM\_00546.4: 175, 245, 248, 249, 273, 282. This code weight can also be used for germline missense variants seen in cancerhotspots.org with ≥ 10 somatic occurrences for the same amino acid change. Located in a mutational hot spot and/or critical and well-established functional domain without benign variation. * Missense substitution or in frame deletion of residues important in the active site architecture and substrate binding of GAA:- D282, W376, D404, L405, I441, W481, W516, D518, M519, R600, W613, D616, W618, F649, L650, H674. Rule does not apply due to genes being highly polymorphic. Located in a mutational hot spot and/or critical and well established functional domain.* Residues 1-552 (N-terminal region) and 2,101-2,458 (central region) * PM1 should not be applied at a moderate weight with PS1/PM5, see PM1_Supporting. Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. Not Applicable Located in one of the following functional domains: * thiamine pyrophosphate (TPP) binding site (aa positions 118Y, 119R, 165G, 167V, 195G, 196D, 197G, 198A, 225N, 227Y, 292H). * α β heterodimer interface (aa positions 160F, 162G, 164N, 169A, 172P, 173L, 176G, 177I, 179L, 180A,183Y, 202G,203Q, 209N, 210M, 213L). * α2 β2 heterotetramer interface (aa positions 88R, 140G, 165G, 166I, 197G, 199A, 200N, 201Q, 202G, 205F, 209N, 213L, 228G, 229M, 230G, 231T, 245R, 296D, 300S). * Phosphorylation loop region (aa positions 287Y, 288R, 289Y, 290H, 291G, 292H, 293S, 295S, 296D, 297P, 298G, 299V, 300S, 301Y, 302R, 303T, 304R, 305E, 314S, 315D, 316P). Not Applicable Not Applicable Not Applicable Not Applicable: Do not use: Missense pathogenic variation in PALB2 is not yet confirmed as a mechanism of disease. Putative missense variants that are known germline hotspots AND/OR in key functional domains AND/OR somatic variants that have ≥10 instances for the same AA in cancerhotspots.org. See the table of Germline and Somatic Hotspots. Disulfide bonds in GPIb are well-established as critical to function, both for interaction with GPIX (PMID: 12036872) and receptor binding to von Willebrand factor (PMID: 18647229). PM1 can be applied when the following cysteine residues (at which there are no known benign variants) are altered: 20, 33, 225, 227, 264, 280, 526, 527. This code should be applied when the variant is within exons 3, 4 or 5. Not Applicable: Rule does not apply due to benign variation being present throughout the gene. Disulfide bonds in GPIb are well-established as critical to function, both for interaction with GPIX (PMID: 12036872) and receptor binding to von Willebrand factor (PMID: 18647229). PM1 can be applied when the following cysteine residues (at which there are no known benign variants) are altered: 93, 95, 118, 141, and 147. Not Applicable: Rule does not apply due to gene being polymorphic. This code is applicable for variants disrupting cystine residues involved in disulfide bridges (Cys40, Cys53, Cys127, Cys160, Cys279, Cys462)¹, variants that would impact heparin binding site residues (Ile39, Arg56, Pro73, Arg79), variants involving reactive site residues (Ala414 and Ala416)², and variants involving the N-glycosylation site (Asn224)³. Applicable to amino acids that directly bind DNA and are necessary for Zinc-finger or homodimer formation[²](#pmid_18829458) * Directly bind DNA: Asp43, His49, Tyr 50, Gly51, Asp56, Gly57, Lys59, Arg63, Arg64, Arg67, His70, Tyr72, Arg87, Asn88, Arg91, Arg94, Gln109, Arg112 * Homodimer: Arg75, Gln89, Glu111, Asp113 * Zinc finger: Cys38, Cys41, Cys55, Cys58, Cys74, Cys80, Cys90, Cys93Applicable to variants within the promoter region that are direct HNF1A/HNF1B binding sites [¹⁵](#pmid_23332764), [¹⁶](#pmid_38909044):* c.-170 to c.-173 and c.-178 to c.-181 (NM\_175914.4) Applicable for glucose- and ATP-binding sites (see attached chart for details).Glucose-binding sites: Ser151 - Pro153; Thr168 - Lys169; Asn204 - Thr206; Ile225 - Asn231; Asn254 - Gly258; Gln287; Glu290ATP-binding sites: Asp78 - Arg85; Ser151; Lys169; Asp205; Ile225 - Gly229; Gly295 - Lys296; Glu331 - Arg333; Ser336; Gly410 - His416 Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 20-27\], SW1 \[AA 35-50\], SW2 \[AA 67-74\], no SAK). Not applicable to specific amino acid residues (see PM5). NA Not Applicable Not Applicable: Rule does not apply due to benign variation being present throughout the gene. Studies on functional domains (Bie et al, 2013, PMID: 24036510; Maita et al, 2013, PMID: 23959878; Saito et al, 2014, PMID: 24480078; Figueiredo et al, 2014, PMID: 25459762) have shown that the following residues are important to the function of IDUA:Active site nucleophiles: Glu182 and Glu299Active site pocket and substrate binding: Arg89, His91, Asn181, His262, Lys264, Asp301, Gly305, Trp306, Asp349, Arg363, Asn372.PM1 will be applied to any missense substitutions or inframe deletions of the above residues.There are no benign or likely benign missense or infame deletions of these residues in ClinVar, or common missense or inframe deletions of these residues in gnomAD v4.1.0 (ClinVar and gnomAD v4.1.0 data accessed on October 30, 2024). Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Not Applicable: Not applicable at this time. Applicable to missense variants in _MYBPC3_ in the specific regions listed below (Walsh _et al._ 2019[¹⁴](#pmid_30696458)). 1. Transcripts ENST00000545968 and NM\_000256.32. Codons 485-502 and 1248-1266Data from HCM case cohorts was used to derive these cluster regions. Therefore, this rule should NOT be applied when additional evidence for the variant supports that the variant causes a phenotype other than HCM (e.g., variant seen in multiple DCM cases).Enrichment was not observed for DCM in any genes.Rule should NOT be combined with PM5 because presence of pathogenic variants in the same codon/region were used to determine clustering and would be double-counting evidence. Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Applicable to missense variants in _TNNI3_ in the specific regions listed below (Walsh _et al._ 2019[¹⁰](#pmid_30696458)). 1. Transcripts ENST00000344887 and NM\_000363.52. Codons 141-209Data from HCM case cohorts was used to derive these cluster regions. Therefore, this rule should NOT be applied when additional evidence for the variant supports that the variant causes a phenotype other than HCM (e.g., variant seen in multiple DCM cases).Enrichment was not observed for DCM in any genes.Rule should NOT be combined with PM5 because presence of pathogenic variants in the same codon/region were used to determine clustering and would be double-counting evidence. NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For TPM1, there is evidence for gene-level enrichment of rare missense variants (see PP2 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For ACTC1, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL2, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL3, there is insufficient evidence for regional enrichment of rare missense variants. Missense variants and in-frame indels in the hinge region (aa51-61) or the L-helix including the haem-binding domain (aa460-493). Not Applicable: See PM2_Supporting. Located in a mutational hot spot and/or critical and well-established functional domain (_e.g._ active site of an enzyme) without benign variation * The variant must be rare (meeting PM2\_Supporting) in order to be considered for PM1.* Met by a rare variant within the pore helix (amino acids 300 to 320). This region is known to be a critical region of KCNQ1 and has been confirmed to show an absence of likely benign or benign variants listed in gnomAD. Applicable for variants in the DNA binding forkhead domain (amino acids 270-367), which is a well-established functional domain (Newman et al., 2020; PMID: 31914405) of _FOXN1_ with low tolerance for benign variation. Not Applicable Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: Does not apply. No known missense variation hot spots in the DCLRE1C gene have been described. See PVS1 for the note about a known hotspot for DCLRE1C deletion variants. Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608). Not Applicable Located in a mutational hot spot and/or critical and well-established functional domain.* Met by variants encoding missense substitutions at His180, His182, His241, His313, Glu417, or His527, which are required residues located within the active site[¹⁶](#PMID_34492281).* Met by variants encoding missense substitutions between Ala107 and Gly125, which are known to mediate localization to the ER membrane[¹⁵](#PMID_36265895). Defined to include missense alterations of two JH2 domain residues: R651W and C759R (PMID: 11668610). * Not mutually exclusive with PM5.* Met by variants in the MYPPPY domain (residues 134-139), which is required for interaction with CD80 and CD86. Strength is dependent upon the location of the variant within specific functional domains (PMID: 26996199):* PM1\_Moderate: missense variant located in the **NBD domain** (amino acids 394-460) and **DDBD domain** (amino acids 461-517). Strength is dependent upon the location of the variant within specific functional domains (PMID: 26996199):* PM1\_Moderate: missense variant located in the **PHD domain** (amino acids 414-487); Variant changes an amino acid in the extracellular domain (aa 33-131) or kinase domain (aa 203-504) but without functional evidence indicating critical or non-critical. Applies to amino acids p.Glu72, p.Trp122, p.Trp163, p.Arg200, p.Glu215 which form salt bridges that are required for higher order structure of octamers and paired octamers. Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 21-28\], SW1 \[AA 36-51\], SW2 \[AA 68-75\], no SAK). Not applicable to specific amino acid residues (see PM5). Not Applicable: Not applicable for PPP1CB. NA Apply if variant is located in a critical residue:* _ACVRL1_: * glycine-rich loop: G209-V216 * phosphate anchor: K229 * C-helix E pairing the phosphate anchor: E242 * catalytic loop: R329-N335 * metal-binding loop: D348-L351 * _BMP10_ interaction cluster * (His40, Val54, Val56, Arg57, Glu58, Glu59, His66, Asn71, Leu72, His73, Glu75, Leu76, Arg78, Gly79, Arg80, Thr82, Glu83, Phe84, Val85, His87) Apply if variant is located in a critical residue:* ENG: * BMP9 binding sites: S278, F282 (PMIDs 28564608, 25312062) * Cysteine residues either previously reported to be likely pathogenic or pathogenic: * C207, C363, C382, C412, C549 * Or cysteine residues known to be important for ENG function: * C350 (C350-C382 disulfide in ZP-N domain of ENG is required for secretion of its ZP module) * C394 (makes a disulfide bond with C412 which is reported to be a mutated residue) * C516 (involved in forming intermolecular disulfides that hold ENG homodimer together) * C582 (involved in forming intermolecular disulfides that hold ENG homodimer together) Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: The PM1 code has been considered but does not apply to PIK3CD variant curation for this disease entity. Not Applicable: There are no defined hotspots or critical functional domains in NEB at this time. Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. Not Applicable: There are no defined hotspots or critical functional domains in DNM2 at this time. Not Applicable: There are no defined hotspots or critical functional domains in MTM1 at this time. The pore/transmembrane region of RYR1 is critical for protein function and missense variants that fall within this region (amino acids 4800-4950) The following residues are considered critical within OTC: Carbamoylphosphate binding site – amino acids: Ser-90, Thr-91, Arg-92, Thr-93, His-117, Arg-141, His-168, Gln-171, Leu-304, and Arg-330 Ornithine binding site – amino acids: Leu-163, Asn-198, Asn-199, Asp-263, Ser-267, Met 268 Catalytic site – amino acids: His-302, Cys-303, Leu-304 Conserved amino acids important for OTC structure and function: Arg-277 - Affect Km for ornithine \[PMID: 9175746, 9065786\] Pro-305 - cis-proline that stabilizes the HCLP motif \[PMID: 9852088\] Gly-269 - part of the mobile SMG loop \[PMID: 8544185\] Not Applicable: This code is not applicable as there is benign variation throughout the ABCA4 gene. Met by active site residues that bind the GTP substrate or the Mg²⁺ ion; Phe883, Asp885, Thr890, Leu905, Glu925, Ile927, Asp929, Met932, Arg976, Arg995, Cys997, Leu998, Phe999, Gly1000, Val1003, Asn1004, Arg1008, and Glu1010 [¹³](#pmid_9391039). Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. The pore/transmembrane region of RYR1 is critical for protein function and missense variants that fall within this region (amino acids 4800-4950) Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. This criterion can be used for variants in residues that directly bind DNA: Gln130, Arg131, Glu132, His143, Leu144, Ser145, Gln146, His147, Leu148, Asn149, Lys155, Thr156, Gln157, Lys158, Arg203, Phe204, Lys205, Trp206, Arg263, Val264, Tyr265, Asn270, Arg271, Arg272, Lys273 NA Not Applicable: MYOC has no mutational hot spot and benign variants are present though the well-characterised olfactomedin domain in exon 3. Not Applicable: Do not use: Benign and pathogenic variants are known to occur within the same domains and germline mutational hotspots are not well defined at this time * See table PM1, curators may seek approval from the expert panel for identifying additional hotspots or critical regions as discovered in literature searches for inclusion. * Moderate * p.1-40,mitochondrial signal peptide, PMIDs: 18227065\*, 20060901 * p.214-223, nucleotide/substrate binding, PMIDs: 18227065\*, 20060901 * p.249-251, nucleotide/substrate binding, PMIDs: 18227065\*, 20060901 * p.R326, CpG dinucleotide, PMID: 9973285 * p.381-382, FAD binding and salt-bridge interaction, PMID: 20060901 * p.R429, CpG dinucleotide, PMID: 9973285 * p.E441, Adjacent to FAD binding, on dimer formation loop, PMIDs: 20060901 * p.R459, dimerization, PMID: 14517516 * p.481-516, membrane binding, PMIDs: 18227065\*, 20060901 * p.562, nucleotide/substrate binding, PMIDs: 18227065\*, 20060901 * \*protein is described in mature protein nomenclature without signal peptide; add 40 amino acids to reach HGVS nomenclature * Cys in EGF-like domain, Cys in TB domain, Cys in hybrid domain, (D/N)-X-(D/N)-(E/Q)-Xm-(D/N)-Xn-(Y/F) substitution in cbEGF-like domain, invariant calcium-binding or hydroxylation residue in cbEGF-like domain, critical Gly between Cys2-Cys3 in cbEGF-like domain, Gly between Cys3-Cys4 if there is an upstream cbEGF domain, Cys-creating variants.* Add caveat: N to S substitution in the second N of de consensus sequence and G to A might be tolerated, PM1 should not be used in these cases. Mutational hot spot or well-studied functional domain without benign variation (None defined for OTOF and MYO15A). Putative missense variants at residues affecting metal ion-binding: codons p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, p.E1813 Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Not applicable Located in a mutational hot spot and/or critical and well-established functional domain.* Basic Helix-Loop-Helix domain (bHLH): aa 564-617[³](#PMID_17436254)^,[²](#PMID_22045651) Not Applicable: Not applicable for SLC9A6. Located in a mutational hot spot and/or critical and well-established functional domain.* ATP binding region: aa 19-43; TEY phosphorylation site: aa 169-171[¹](#PMID_28544139),[⁴](#PMID_17993579),[²](#PMID_23064044),[³](#PMID_29264392) Located in a mutational hot spot and/or critical and well-established functional domain.* Forkhead: aa 181-275[³](#PMID_18571142)^,[⁴](#PMID_28661489) Located in a mutational hot spot and/or critical and well-established functional domain.* Methyl-DNA binding (MBD): aa 90-162* Transcirptional repression domain (TRD): aa 302-306 Located in a mutational hot spot and/or critical and well-established functional domain.* 3’ cysteine binding site: aa 820[³](#PMID_9887341) Not Applicable: Not applicable. Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 10-17\], SW1 \[AA 25-40\], SW2 \[AA 57-64\], SAK \[AA 145-156\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (CR2 domain \[AA 251-266/exon7\], exon 14, exon 17). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (PH domain \[AA 420-500\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (PH domain \[AA 418-498\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (Directly interacting residues between N-SH2 and PTPN domains \[AA 4, AA 7-9, AA 58-63, AA 69-77, AA 247, AA 251, AA 255, AA 256, AA 258, AA 261, AA 265, AA 278-281, AA 284\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 10-17\], SW1 \[AA 25-40\], SW2 \[AA 57-64\], SAK \[AA 145-156\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (AA 43-61, AA 124-134). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 10-17\], SW1 \[AA 25-40\], SW2 \[AA 57-64\], SAK \[AA 145-156\]). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (P-loop \[AA 28-35\], SW1 \[AA 43-58\], SW2 \[AA 75-82\], no SAK). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (AA 47-65, AA 128-138). Not applicable to specific amino acid residues (see PM5). Applicable only to critical and well-established functional domains available in the supplementary table (exon 6, exon 11, P-loop \[AA 459-474\], CR3 activation segment \[AA 594-627\]). Not applicable to specific amino acid residues (see PM5). Variant is located within a Pathogenic Enriched Region. See specific amino acid residues noted in the attached “PM1 Table". Variant is located within a Pathogenic Enriched Region. See specific amino acid residues noted in the attached “PM1 Table". Variant is located within a Pathogenic Enriched Region. See specific amino acid residues noted in the attached “PM1 Table". Variant is located within a Pathogenic Enriched Region. See specific amino acid residues noted in the attached “PM1 Table". This code can be applied at the moderate level for variants involving the following residues: Residues affecting secretion: Arg1667, Arg1332FXa-binding residues: Gly2267-Gly2304 (with the exception of Ser2283) Not Applicable: Currently, insufficient numbers of pathogenic variants have been reported in SCN1B to calculate “mutational hotspots”. SCN1B does not belong to a gene family to utilize PERs. Not Applicable: Functional domains have not been definitively identified. PVS1_Very Strong See PVS1 flow diagram (Figure 1). Not Applicable: Not applicable for MYH7. Use PTEN PVS1 decision tree. Null variant in a gene with established LOF as a disease mechanism; see PVS1_Strong, PVS1_Moderate, PVS1_Supporting for reduced evidence applications. Applicable as described in Tayoun et al. 2018.* Any nonsense or frameshift variant occurring upstream of c.1285* Any canonical splice site predicted to disrupt reading frame and undergo nonsense mediated decayPVS1 (RNA): splicing assay data - assays demonstrating a variant leads to aberrant splicing profile that can be categorized against a PVS1 decision tree* Use the PVS1 decision tree to determine code strength* Applicable as described in Walker et al. (PMID: 36865205) Per modified CDH1 PVS1 decision tree. Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. Please utilize the PVS1 decision tree for application of PVS1 code. The decision tree details the specific strengths each type of null variant may be applied at. Please see below for some additional helpful summary details for application of PVS1 code:* Initiation codon: * PVS1 may be applied to initiation codon variants* Nonsense or frameshift variants: * PVS1 applies to variants predicted to result in nonsense-mediated decay (NMD) for nonsense variants upstream of p.Lys351 and for frameshift induced premature termination codon (PTC) upstream of p.Lys351 * PVS1\_Strong applies to variants not predicted to undergo NMD (nonsense variants downstream of codon 350 or frameshift induced PTC in exon 11 or in the 3’ most 50 nucleotides of exon 10) in variants located in the p.Lys351 to p.Ala 355 range * PVS1\_Moderate applies to variants not predicted to undergo NMD (nonsense variants downstream of codon 350 or frameshift induced PTC in exon 11 or in the 3’ most 50 nucleotides of exon 10) in variants located in the p.Gly356 to p.Asp393 range * PVS1\_Moderate may also be applied to frameshift induced PTC downstream of the natural stop codon* Canonical splice variants (+/- 1,2 intronic positions): * PVS1 applies to predicted splicing alterations that are PTC resulting in NMD (or in-frame but targeting critical domains or residues) * PVS1 applies to predicted splicing alterations that target the start codon (Exon 2 donor) * PVS1\_Moderate applied to splicing alterations that are predicted to shorten (\<10% of the protein removed) or expand a _TP53_ C-terminal end of unknown function (E10 donor or E11 acceptor)* Deletions * Full gene deletions: PVS1 * Single- to multi-exon deletions that target the initiation codon, preserving the potential rescue ATG (p.Met40) in exon 4: PVS1 * Single- to multi-exon deletions that target the initiation codon and the potential rescue ATG (p.Met40) in exon 4: PVS1 * Single- to multi-exon deletion that disrupts the reading frame and is predicted to undergo NMD (nonsense or frameshift induced PTC upstream of p.Lys351): PVS1 * Single- to multi-exon deletion that disrupts the reading frame and is **NOT** predicted to undergo NMD (nonsense or frameshift inducted PTC downstream of p.Leu350): PVS1 * Single- to multi-exon deletion including the last exon where the truncated/altered region is critical to protein function (any multi-exon combination targeting exon 11): PVS1 * If the role of the region in protein function is unknown, if the variant removed \< 10% of the protein (deletion of exon 11): PVS1\_Moderate * Single- to multi-exon deletion that preserves the reading frame where the truncated/altered region is critical to protein function: PVS1* Duplications (≥1 exon in size and must be completely contained within the _TP53_ gene) * Proven in tandem. Reading frame is disrupted and NMD predicted to occur (nonsense upstream of p.Lys351 or frameshift-induced PTC upstream of p.Lys351): PVS1 * Presumed in tandem. Reading frame presumed disrupted and NMD predicted to occur (nonsense upstream of p.Lys351 or frameshift-induced PTC upstream of p.Lys351): PVS1\_StrongFor variants inducing aberrant transcripts identified via mRNA assay, apply as PVS1\_Variable Weight (RNA) following recommendations from Walker et al., 2023 (PMID: 37352859), downgrading one strength level if the assay data indicates leakiness._Caveats_: PS3 should not be applied at any strength if PVS1 is applied at full strength. PP3 should not be used in combination with PVS1.For the purposes of unified curation, the TP53 domains/important motifs by amino acid range are defined as:**TAD1**: aa 17-25**TAD2**: aa 48-56**Proline residues**: aa 64-92**DNA binding domain**: aa 100-292**Hinge domain**: aa 293-324**Oligomerization domain**: aa 325-356**C-terminal domain (Basic domain)**: aa 368-387A disease-specific PVS1 decision tree incorporating the above bullets as well as a supplemental file for _TP53_ PVS1 Splicing Worksheet is also included as an additional curation tool and has more granular details. Null variant in a gene where loss of function is a known mechanism of disease or in frame loss of an exon that contains residues involved in the active site of GAA. * Any nonsense, frameshift, or splice variant creating a premature stop codon before codon 916. * In frame deletions of an entire exon containing critical active site/substrate binding residues (exons 8 and 10), or for which another variant removing the exon is known to be pathogenic (exons 2 and 18). Use decision tree as per SVI WG with specified “regions critical to protein function”. Not Applicable: PVS1 is not applicable. MHS is due to gain of function variants in RYR1. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. Applied per PVS1 flowsheet of Abou Toyoun et al. Applied per PVS1 flowsheet of Abou Toyoun et al. Applied per PVS1 flowsheet of Abou Toyoun et al. Applied per PVS1 flowsheet of Abou Toyoun et al. Large heteroplasmic mtDNA deletions, where at least one gene is completely deleted Use PALB2 PVS1 Decision Tree **LINK TO PVS1 DECISION TREE DOCUMENT:** [**https://drive.google.com/file/d/1mGfChgxbGVbzYn6Ggmb9rYvoGGah25ll/view?usp=sharing**](https://drive.google.com/file/d/1mGfChgxbGVbzYn6Ggmb9rYvoGGah25ll/view?usp=sharing)**Do not apply PVS1 for truncations that occur prior to Codon 54 (including frameshift events that start and end prior to Codon 54 but the truncation extends beyond Codon 54.)****Note1: Exon presence in biologically relevant transcripts:** In some transcripts exon 2 of _VHL_ is skipped and expressed at low levels. The function of this transcript is not fully known. Exon 2 comprises almost the entirety of the nuclear export function region of the Beta domain and is critical for known VHL function. Exon 1 contains the only initiator codons in _VHL_. Exon 3 contains the elongin binding function. _**All exons should be considered as "present in biologically relevant transcripts" in the PVS1 decision tree.**_**Note 2: The 10% PVS1 downgrade to Moderate cannot apply to VHL** because of the small size.**Nonsense Mediated Decay** [⁵](#pmid_22825683) [⁴](#pmid_20145706) **NMD experimental evidence in 1st exon after codon 54 and to 5' region of 2nd exon (codon 138)**.\*\***Critical Domains:**1st Beta (β) domain (63-154), especially Nuclear Export (114-155)Alpha (ɑ) domain (155-192), especially Elongin C binding (157 - 172)Second Beta domain (193-204) _Truncating variants after Met54 and predicted to undergo NMD (from AA55-AA136/c.408) or in the beta or alpha domains can receive PVS1, and those outside the second Beta domain (205-213) can receive PVS1\_Moderate downgrade to account for minimal loss of VHL protein. Notably a frame shift deletion at 205 is pathogenic in ClinVar (ID 18971) as are stop loss extension variants in the last codons (see PM4)._**SPLICE: If any canonical exon is skipped, the variant receives PVS1.** If a cryptic splice disrupts the reading frame, and is in a critical domain (AA63-AA204) or is predicted to undergo NMD (AA55-AA136) it receives PVS1. If it is outside a critical domain and predicted to undergo NMD (AA55-62), it receives PVS1\_Strong (the second site outside of critical domains AA205-213 is not predicted to undergo NMD). If a cryptic splice does not alter the reading frame, and is in a critical domain (AA 63-204), it can receive PVS1\_Strong, and if it is outside the critical domain (AA 205-213) or in an NMD prediction (AA 55-62), it receives PVS1\_Moderate. Note: There is a cryptic exon (E1) in intron 1 [⁷](#pmid_31996412) [⁶](#pmid_29891534), and silent variants in exon 2 that are reported to cause skipping of exon 1. If there is functional evidence of exon skipping (RNA splice assay) then PVS1 can apply. Do not double count evidence. Ex. PVS1 should be used in place of PS3 functional evidence confirming splice alteration, but PS3 evidence code could still apply to other relevant assays confirming effect on HIF1/2a presence etc. **EXON DELETION:** SVI PVS1 decision tree modified for whole exon deletions. There are only 3 exons in _VHL_ and each has an important functional domain. Any exon deletion of _VHL_ receives PVS1. **EXON DUPLICATION:** Follow PVS1 decision tree. Note: few pathogenic exon duplications are reported in ClinVar. (ID:417571, ID:584137). These have no literature cited. **INITIATION CODON:** VHL Met 1 (in VHL p30) truncation or missense would not affect VHL p19, as VHL has a second start at codon 54 (VHL p19), it cannot be scored in the PVS1 decision tree. After that, no other viable alternative starts are known. Start loss at codon 54 would presumably result in an impact, as VHL p30 and p19 would be truncated prior to any known functional domains (PVS1). Ong 2007 has 1 family (2 subjects) with Met54X and reports cerebellar hemangioblastomas. There is no functional study in the paper for this variant. Olschwang et al 1998 VHL Type 2A, one subject with 161insT (FS result). There is no functional study in the paper. Missense at Met 54 (VHL p19 initiation codon) would presumably not result in as strong an impact as the full length VHL p30 would still be produced (PVS1 decision tree = N/A). ClinVar has M54T (ID:819688) and M54L (ID:843990). M54T is uncertain, with no other evidence provided. M54L references M54I which segregates in homozygous state with erythrocytosis in individuals of Moroccan descent [⁹](#pmid_26224408) [⁸](#pmid_27578599), and those heterozygous for M54I did not present VHL phenotype [⁹](#pmid_26224408). Use _GP1BA_ modified decision tree as per SVI WG. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Not Applicable: VWD type 2 is defined by qualitative defects in the VWF protein and not caused by null variants. Use _GP1BB_ modified decision tree as per SVI WG. Use _GP9_ modified decision tree as per SVI WG. Use decision tree as per SVI WG with specified “regions critical to protein function”. Use _HNF4A_ PVS1 decision tree.* Variants generating PTCs in exon 10 and last 55 nucleotides of exon 9 (c.1162-1216) are not expected to cause NMD[¹](#pmid_24274751) * The most 3’ nonsense or frameshift variant is c.1256C>G, p.S419X in the last exon. This variant has been classified as Pathogenic by the MDEP. There are six other nonsense and frameshift variants in exon 10, none of which have case information and are all currently classified as VUS. The collective evidence supports applying PVS1 for variants at codon 419 (c.1257) and 5’ and PVS1\_Supporting for variants at c.1258 (G)/p.Gly420 and 3’.* “Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” * Exons 1, 2 (LRG 4), 3 (LRG 5), 4 (LRG 6), 6 (LRG 8): deletion or skipping causes frameshift: PVS1 * Exons 5 (LRG 7), 7 (LRG 9), 8 (LRG 10), 9 (LRG 11) - deletion or skipping causes in-frame deletion, 52/52/79/51-79 AA deleted, that is >10 % of the protein in each case - PVS1\_Strong * Exon 10 (LRG 12) - 46 AA, contains the transactivation domain, includes stop loss - PVS1\_Strong Use _GCK_ PVS1 decision tree created based on PVS1 decision tree from ClinGen SVI group[¹](#pmid_30096381)* Variants generating PTCs 3’ of c.1198 (p.Asp400) of NM\_000162.3, which includes the last 55 nucleotides of exon 9 and exon 10, are not expected to cause NMD[²](#pmid_24274751). The α13 helix (p.444-456), located at the C-end of the protein, has a critical role in GCK conformational change upon glucose binding. Individuals with PTCs in exon 10 have a MODY phenotype. Therefore, a “very strong” level of evidence will be applied for PTCs in exon 10.* “Exon skipping or “use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” * single exon deletions * deletion of exon 1 is in-frame but over 20 families with GCK-MODY phenotype and exon 1 deletion (some also have promoter deletions) --> PVS1 * deletions of single **exons 2,3,6 and 7** cause frameshift --> **PVS1** * deletions or skipping of **exons 8 and 9** are in-frame and the proportion is >10 % (52 AA and 78 AA, respectively) --> **PVS1** * deletions or skipping of **exons 4 and 5** are in-frame and the proportion is \<10 % (40 AA and 32 AA, respectively). Exon 4 (p.122-161) and exon 5 (p.162-193) contain each a part of the active site that binds glucose /p.151-180[³](#pmid_23957911) according to Beck et al., Biochemistry 2013/ --> **PVS1** * deletion of exon 10 (47 AA) – There are a number of patients with a GCK-MODY phenotype with reported with missense, frameshift, PTC, splice acceptor, and stop loss variants in exon 10 --> **PVS1*** Apply PVS1\_Supporting to initiation codon variants given MDEP has only reviewed one variant and classified as VUS (c.3G>A, PVS1\_Supporting + PM2\_Supporting; one case submitted, dx.53 and no other info provided to lab). The next methionine is at codon 8 and there are no variants classified as pathogenic 5' of p.Met8. * Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable: Not applicable. Not Applicable: Does not apply. Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (**Figure 1**) \[Reference 1\]. Not Applicable: VWD type 2N is caused by qualitative protein defects and not null variants. * All nonsense and frameshift variants will meet PVS1 (Very Strong), unless the variant is predicted to be undetected by nonsense-mediated decay (NMD) i.e. if the resulting premature termination codon is in the last exon (exon 14) or in the last 50 nucleotides of the penultimate exon (exon 13; after c.1778, codon 592). In this case, PVS1\_Moderate will be applied because \<10% of the primary amino acid sequence is predicted to be lost (in this case, ~9.3%).* For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1.* Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary).* If a single or multi-exon deletion results in an out-of-frame consequence, use PVS1 (Very Strong) if NMD is predicted to occur.* Initiation codon variants will meet PVS1. * The next in-frame methionine is at position 133. If used as a start signal, the signal sequence (amino acids 1-27) would be lost ([https://www.uniprot.org/uniprotkb/P35475/entry](https://www.uniprot.org/uniprotkb/P35475/entry)) * There are 6 variants, upstream of Met133, that are classified as pathogenic in ClinVar (data accessed July 20, 2022) (Table 1).* If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply.* For duplications, consult the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Null variant in a gene where loss of function is a known mechanism of disease. Currently only applicable to _MYBPC3_ where LOF is an established disease mechanism.Refer to SVI guidance for the interpretation of this criterion (Abou Tayoun _et al._ 2018[¹](#pmid_30192042)).[SpliceAI](https://spliceailookup.broadinstitute.org)[²](#pmid_30661751) is recommended for evaluation of predicted splice impacts.Factors to consider when assessing the consequences of putative LOF variants in the _MYBPC3_ gene:1. Codon p.1254 is located 50 nucleotides upstream of the most 3' exon-exon junction (exon 33:34) in _MYBPC3._ As such, nonsense variants introducing a premature termination codon after this point may escape nonsense mediated decay (NMD) and consequently not result in protein haploinsufficiency (Nagy & Maquat 1998[³](#pmid_9644970)).2. When assessing variants predicted to affect splicing of micro-exons (exons 10, 11 and 14), be aware that _in silico_ splice site predictions may be less reliable in this setting and the consequences of variants affecting splice sites at these exons less predictable (Frank-Hansen _et al._ 2008[⁴](#pmid_18337725)).3. When assessing variants affecting splice sites of in-frame exons (exons 2-4, 8-11, 14, 20, 22, 24-27), be aware that although most of these exons encode domains that have been shown to play critical roles in protein function, and/or harbor functionally important residues (Carrier _et al_. 2015[⁵](#pmid_26358504)), in general, the consequences of in-frame deletions are less predictable.For canonical splice site variants where other canonical splice variants have been reported, application of the PS1 rule may be considered if the other variant affecting the same slice site is 1) predicted to have a similar or more deleterious effect and 2) has been classified as pathogenic according to these modified guidelines without use of PS1 for other splice variants.For genes where haploinsufficiency is NOT an established mechanism, see PM4 for truncating variants that do NOT undergo NMD. Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Not Applicable: Not currently applicable to TNNI3. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TNNT2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TPM1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to ACTC1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL3. See PM4 for truncating variants that do NOT undergo NMD. Applies to:* Nonsense/frameshifts variants predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493)* GT-AG 1,2 splice sites variants leading to exon skipping or use of a cryptic splice site disrupting reading frame predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493)* Full gene deletions and deletions removing exon 2 and/or exon 3* Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame, predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493), AND proven in tandem Use SVI recommendations (PMID 30192042, and PMID 37352859) with modifications as shown in the RPGR PVS1 Decision Tree with splice guidance figures attached.[**NM\_001034853.2**](https://www.ncbi.nlm.nih.gov/nuccore/NM_001034853.2/) **\-** Transcript isoform C reads through the splice site at the end of exon 15 (ORF15) creating a longer protein sequence with a repetitive region in the 3’ end. This is the transcript expressed in retina.[**NM\_000328.3**](http://www.ncbi.nlm.nih.gov/nuccore/NM_000328.3) **\-** Transcript isoform A includes 19 exons and produces a shorter mRNA and protein sequence than isoform C. Variants in exons 16 to 19 are not associated with RPGR-related retinopathy.**PVS1\_Met** designation for variants predicted to undergo NMD, nonsense or frameshift variants that disrupt the critical function of glutamylation in ORF15, or variants causing skipped or deleted exons that affect regions critical to protein function. For truncating variants, apply PVS1 at default (VeryStrong) level if the variant affects codons 1-581, since nonsense-mediated decay is predicted.[https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1](https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1) Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* PVS1 can be applied to variants not predicted to undergo nonsense-mediated decay but removing/altering the critical forkhead domain (amino acids 270-367; Newman et al., 2020; PMID: 31914405) based on recommendations from Walker et. al., 2023 (PMID: 37352859). Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)). Nonsense/frameshift variant introducing Premature Termination Codon (PTC)^a at or before codon 753 in _MLH1._ORLarge genomic alterations^a of single or multi-exon size.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). If exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b then use PVS1\_Strong. If exon skipping or use of a cryptic splice site disrupts reading frame and is NOT predicted to undergo NMD then use PVS1\_Moderate.ORVariants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript) leading to premature stop codon or in-frame deletion disrupting a functional domain^b or protein conformation. Splicing aberration must be confirmed in a minigene assay or an additional RNA assay from an independent laboratory if it is a non-canonical splice site variant.ORVariants in the initiation codon of _MLH1_. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)). Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)). Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group [¹⁶](#pmid_30192042) and as updated by the ClinGen SVI Splicing Subgroup [¹⁷](#pmid_37352859)* Refer to RPE65-specific PVS1 Decision Tree, file attached. * PVS1: Predicted splice defects at +/- 1,2 in exons 1-14 * PVS1: Single to multi-exon deletions, with or without predicted NMD. All exons are considered critical to protein function. * PVS1: Nonsense or frameshift mutations from p.Ser2 through p.Gly528 * PVS1: Duplications of exons proven in tandem * PVS1(RNA): RNA splicing data with evidence of alternative transcript production at complete levels, relative to normal allele. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)). * For truncating variants located in the isoform-specific exon 3, apply PVS1 if nonsense mediated decay is predicted (truncation at codon 172 or lower) and PVS1\_strong if NMD is not predicted (truncation at codon 173 or higher). While this recommendation already matches the SVI guidance, it is specified here because similar clinical phenotypes are associated with null variants in exon 3 and exon 2 (PMID: 25329329, PMID: 34111452), despite the fact that exon 3 is omitted from an alternative transcript (NM\_001037631.3) that encodes the soluble isoform (PMID: 40168991).* If a missense, synonymous, or intronic variant outside of the canonical splice sites has a SpliceAI score greater than or equal to 0.2 and has been confirmed to cause complete or near-complete disruption of splicing within the mRNA in a study of patient RNA or minigene assay, avoid PP3 and PS3\_Supporting and evaluate PVS1 instead (PMID: 37352859).* In order to avoid over-weighing loss-of-function evidence, PP3 and PS3 are mutually exclusive with PVS1 at the default (very strong) level. These additional codes cannot be applied for a variant that has already met PVS1. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with two specifications:* Given that the _RAG1_ protein is encoded by a single exon (based on MANE Select transcript NM\_000448.3) and nonsense-mediated decay is not predicted for nonsense or frameshift variants, PVS1 cannot be applied at the default strength to RAG1 variants (indicated by the red boxes in the Flowchart attached), **except** in the case of full gene deletion **or** removing/altering critical domain for the protein (NBD domain, DDBD domain, and core domain, indicated by the purple in the Flowchart).* PVS1 can be applied to variants not predicted to undergo nonsense-mediated decay when removing/altering the critical NBD domain (aa 394-460), DDBD domain (aa 461-517), and core domain (aa 387-1011) based on recommendations from Walker et al., preprint. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with two specifications:* Given that the _RAG2_ protein is encoded by a single exon (based on MANE Select transcript NM\_000536.4) and nonsense-mediated decay is not predicted for nonsense or frameshift variants, PVS1 cannot be applied at the default strength to RAG2 variants (indicated by the red boxes in the Flowchart), **except** in the case of full gene deletion **or** removing/altering critical domain: the PHD domain and core domain (indicated by the purple in the Flowchart). * PVS1 can be applied to variants not predicted to undergo nonsense-mediated decay when removing/altering the critical PHD domain (spanning amino acids 414-487) and core domain (amino acids 1-383) based on recommendations from Walker et al., preprint. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats:* Use caution interpreting LOF variants at the extreme 3’ end of a gene.* Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact.\*\*Use the PVS1 decision tree guide. Use attached RS1-specific PVS1 Decision Tree file, which has been modified from Abou Tayoun, et al., 2018 (PMID 30192042) and incorporates splice site guidance from Walker et al, 2023 (PMID 37352859). This strength applies to nonsense, frameshift, splice site, and deletion variants in NM\_000330.4 c.1A to c.671C (p.Met1 to p.Cys223) and to duplications in NM\_000330.4 c.1 to c. 472 (p.Met1 to p.Asp158).The structure of RS1 is important for function. The RS1 monomers have cystine disulfide bonds within a RS1 monomer and bonds with the two neighboring monomers to form an octomer substructure and bonds to the second RS1 octomer that completes the paired octomer structure. Within this structure there are a number of sites that have been shown to function in other types of protein structure stabilization. Not Applicable: Not applicable. Not Applicable: Not applicable. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* PVS1 at default strength (Very Strong) can be applied to variants not predicted to undergo nonsense-mediated decay but truncating the transmembrane domain (which begins at amino acid 255) or any distal region (i.e. cytoplasmic domain) due to the lack of functionality of the protein expressed with this defect. Use ACVRL1 PVS1 Decision Tree (see attachments) Use ENG PVS1 Decision Tree (see attachments) Nonsense/frameshift variant introducing Premature Termination Codon (PTC)^a ≤ codon 891 in _MSH2._ Refer to Appendix for details.ORLarge genomic alterations^a of single or multi-exon size.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). If exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b then use PVS1\_Strong. If exon skipping or use of a cryptic splice site disrupts reading frame and is NOT predicted to undergo NMD then use PVS1\_Moderate.ORVariants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript) leading to premature stop codon or in-frame deletion disrupting a functional domain^b or protein conformation. Splicing aberration must be confirmed in a minigene assay or an additional RNA assay from an independent laboratory if it is a non-canonical splice site variant. Nonsense/frameshift variant introducing Premature Termination Codon (PTC)^a ≤ codon 1341 in _MSH6_. Refer to Appendix for details.ORLarge genomic alterations^a of single or multi-exon size.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). If exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b then use PVS1\_Strong. If exon skipping or use of a cryptic splice site disrupts reading frame and is NOT predicted to undergo NMD then use PVS1\_Moderate.ORVariants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript) leading to premature stop codon or in-frame deletion disrupting a functional domain^b or protein conformation. Splicing aberration must be confirmed in a minigene assay or an additional RNA assay from an independent laboratory if it is a non-canonical splice site variant. Nonsense/frameshift variant introducing Premature Termination Codon (PTC)^a ≤ codon 798 in _PMS2_. Refer to Appendix for details.ORLarge genomic alterations^a of single or multi-exon size.ORVariants at IVS±1 or IVS±2\*\*^a,c \*\* where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). If exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b then use PVS1\_Strong. If exon skipping or use of a cryptic splice site disrupts reading frame and is NOT predicted to undergo NMD then use PVS1\_Moderate.ORVariants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript) leading to premature stop codon or in-frame deletion disrupting a functional domain^b or protein conformation. Splicing aberration must be confirmed in a minigene assay or an additional RNA assay from an independent laboratory if it is a non-canonical splice site variant. Not Applicable: Does not apply given gain of function disease mechanism. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats:* Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7).* Use caution interpreting LOF variants at the extreme 3’ end of a gene.* Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact.* Use caution in the presence of multiple transcripts.Specified critical regions in NEB that should also receive PVS1 are listed below and in the PVS1 flowchart:In-frame deletions due to the repetitive nature of NEB, particularly in exon 55, are deleterious and pathogenic (Anderson 2004 PMID:15221447, Lehtokari 2009 PMID:19232495). Exons 161-183 (Pelin 1999 PMID:10051637). Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant alpha-actinopathy caused by variants in ACTA1. Not Applicable: Loss of function is not a mechanism of disease for DNM2-related AD Centronuclear myopathy See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant RYR1-related myopathy. See “OTC VCEP PVS1 Decision tree” file Please use the SVI WG decision tree modified for the _ABCA4_ gene. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (Abou Tayoun et al., 2018[¹⁶](#pmid_30192042)) and as updated by the ClinGen SVI Splicing Subgroup (Walker et al., 2023[¹⁷](#pmid_37352859)).* Refer to GUCY2D-specific PVS1 Decision Tree, file attached. * PVS1: Predicted splice defects at +/- 1,2 in exons 2-20 * PVS1: Single to multi-exon deletions, with or without predicted NMD. All exons are considered critical to protein function. * PVS1: Applied to variants in the initiation codon. The second in-frame methionine is located at residue 218 and use of this as a start codon would eliminate the leader sequence which targets the protein to the endoplasmic reticulum. There is no known study indicating that this methionine in GUCY2D can be used as start codon. Variants affecting Met1 can lead to a complete absence of the protein product. Additionally, there are multiple variants located upstream of p.Met218 that have been reported as a pathogenic in HGMD and ClinVar, evidence that this entire region of the protein is functionally important. * PVS1: Nonsense or frameshift mutations from p.Thr2 through p.Lys1068 * PVS1: Duplications of exons proven in tandem * PVS1(RNA): RNA splicing data with evidence of alternative transcript production at complete levels, relative to normal allele. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. Please see attached _DYSF_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCB_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCG_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCD_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _CAPN3_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _ANO5_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCA_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Use HNF1A PVS1 decision tree.* Apply PVS1 to nonsense or frameshift variants occurring 5' of c.1768. * Variants generating PTCs 3’ of c.1714 of NM\_000545.8, which includes the last 55 nucleotides of exon 9 and all of exon 10, are not expected to cause NMD [²](#pmid_24274751). The transactivation domain (TAD) of the protein overlaps with this region. The last 55 nucleotides of exon 9 (c.1714-1768) is enriched for disease-causing variants and loss-of function variants in this region have been found in patients/families with a MODY phenotype. Therefore, a “very strong” level of evidence will be used for loss-of-function variants 5’ of c.1768 regardless of where the premature termination codon occurs.* “Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” * Apply PVS1 for exon skipping or single to multi exon deletion involving exons 1-9 * Deletions of exon 1 would lead at least to loss of the initiation codon (see below for recommendations for initiation codon variants). Deletions of single exons 2, 3, 4, 5, 6, 8 or 9 all cause frameshift, and thus PVS1 would be used. In HNF1A, only exon 7 (LRG\_522t1) is surrounded by introns of the same phase. Skipping or deletion of exon 7 would remove 64 amino acids in the TAD, which is >10% of the protein and 18% of the TAD. Given the significance of the TAD, we support still using PVS1 instead of PVS1\_Strong in this situation.* Apply PVS1 to initiation codon variants. Four initiation codon variants have been identified in patients with a MODY phenotype. The closest potential in-frame start codon is p.Met118. Starting the protein at p.Met118 would remove 18% of the protein, including the entire dimerization domain. There are many P/LP variants upstream of p.Met118.* Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable: LOF and/or haploinsufficiency have not been clearly identified as disease mechanisms underlying brain malformations related to these genes, so in general this rule is not applicable. The disease mechanism for these genes is gain of function (GOF). Not Applicable: MYOC variants cause JOAG/POAG through a gain of function (GoF) disease mechanism and not loss of function (LoF). Truncating variants in exon 3 are expected to be pathogenic because they escape nonsense-mediated decay. Use ATM PVS1 Decision Tree Loss of function is a known mechanism for VLCAD Deficiency. The specifications below are based on published guidance for assigning strength of evidence for PVS1 (Abou Tayoun et al., (2018) PMID: 30192042). There are multiple transcripts for ACADVL. The major isoform, NM\_000018.4, encodes a 655 amino acid precursor protein that contains a 40 amino acid N-terminal target sequence that is removed during uptake (Aoyama et al., (1995) PMID: 7668252). In a joint project between NCBI and EMBL-EBI (MANE), NM\_000018.4 was designated as the most relevant transcript. Nonsense or Frameshift:* Use caution when interpreting LOF variants at the 3’ end of the gene.* NMD is not predicted if the variant is in the last exon (exon 20) or in the last 50 nucleotides of the penultimate exon (exon 19). * Canonical Splice Site (+1, +2, -1, -2): All donor/acceptor sites follow the GT/AG rule, except for the donor splice site of intron 8, which begins with GC. PVS1 should not be applied for variants in the splice donor site of intron 8 since the impact of GC donor splice sites is not well understood. For +1 or +2 GT donor splice site variants, the exon immediately 5’ of the variant is predicted to be skipped. For -1 or -2 AG acceptor splice site variants, the exon immediately 3’ of the variants is predicted to be skipped.* Initiation codon: The next in-frame methionine is at position 6 (on transcript NM\_000018). However, the first 40 amino acids comprise the leader sequence in the precursor peptide and are important for proper localization of the protein (Aoyama et al., (1995) PMID: 7668252). Therefore, initiator codon variants will meet PVS1\_Strong.* Well-established \_in vitro\_ or \_in vivo\_ functional studies supportive of a damaging effect \_as measured by effect on mRNA transcript profile (mRNA assay only).\_ Apply as PVS1 (RNA) at appropriate strength. * See ACADVL PVS1 decision tree; cannot be combined with PM1 * Nonsense/frameshift variants predicted to undergo NMD (not affecting last exon or 55 last nt of penultimate exon). * 1,2 splice site variants leading to exon skipping or use of a cryptic splice site disrupting the reading frame and predicted to undergo NMD. * Full gene deletion. * Single to multi-exon deletion disrupting the reading frame and predicted to undergo NMD. * Duplication (>=1 exon in size and completely contained within gene) proven in tandem and disrupting the reading frame and predicted to undergo NMD. Null variant in a gene with established LOF as a disease mechanism; see PVS1_Strong, PVS1_Moderate, PVS1_Supporting for reduced evidence applications.PVS1 should also be considered for both of the genes OTOF and MYO15A with variants falling in two exons being exceptions to this rule: OTOF: NM_194248.2 Exon 46 (c.5841 to c.5994; PMID: 19250381) and MYO15A: NM_016239.3 Exon 8 (c.4033 to c. 4038; PMID: 10552926) and Exon 26 (c.5911 to c.5964; PMID: 30096381 and high frequency LOF variant https://gnomad.broadinstitute.org/variant/17-18046894-G-A?dataset=gnomad_r2_1) Follow SVI guidance, using DICER1-specific information. Per the PVS1 workflow guidance provided in Tayoun et al. 2018[¹](#pmid_30192042), the following will apply:* Nonsense or frameshift variants:* PVS1 applies to variants predicted to result in nonsense-mediated decay (NMD); the predicted NMD cutoff for DICER1 occurs at p.Pro1850.* PVS1\_Moderate applies to variants resulting in protein truncation 3’ of this cutoff* Canonical splice variants (+/- 1,2 intronic positions): PVS1 applies with the following exceptions:* Exon 10 SDS/SAS: PVS1\_Strong (in-frame but exon includes >10% protein)* Exons 5, 15, 18, 22 SDS/SAS: PVS1\_Moderate (in-frame and each \<10% of protein)* Exon 27 SAS: PVS1\_Moderate (final exon)* Exon 1: no criteria (non-coding)* Variants that disrupt the translation start site (p.M1?): no criteria applied given p.M1 is not highly conserved, there are three in-frame possible alternate start codons (p.Met11, p.Met17, p.Met24), and multiple lab cases of p.Met1? without DICER1 phenotype. SDS = splice donor site; SAS = splice acceptor site. Refer to PS3 weight guidelines when a variant meets criterion for application of both PVS1 and PS3. A disease-specific PVS1 decision tree incorporating the above bullets is also included at the end of this document as an additional curation tool. **Nonsense-mediated decay predicted.**CCDS VCEP notes: Loss of function (LOF) of GATM is a known mechanism of disease for arginine:glycine amidinotransferase deficiency (AGAT-D). There are examples of various LOF variants, including nonsense and frameshift, in GATM in individuals with AGAT-D ([https://databases.lovd.nl/shared/variants/GATM/unique](https://databases.lovd.nl/shared/variants/GATM/unique)). The specifications below are based on the PVS1 decision tree (see Appendix 1) (based on Figure 1, Abou Tayoun et al, 2018, PMID 30192042).GATM specifications: **Nonsense and frameshift variants** \* All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 9) or the last 50 bases of the penultimate exon (exon 8, 3’ of c.1109). In that case, please refer to the PVS1 flowchart for guidance on PVS1 weight (Appendix 1).**Splice site variants (+1, +2, -1, -2)** \* All canonical splice site pairs in GATM are GT-AG. \* For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants. However, to apply PVS1 at the very strong level, splice site variants must have no detectable nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing, as predicted by SpliceAI. Otherwise, the PVS1 strength should be reduced accordingly. \* For the predicted in frame/out of frame consequences of exon skipping and considerations for strength of PVS1, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). \* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used. \* Non-canonical splice variants, such as +3 or -3, may also meet PVS1 - refer to Walker et al, PMID: 37352859.**Deletions (single or multi exon)** \* If a single or multi-exon deletion results in an out of frame consequence, use PVS1 at the very strong level unless not predicted to undergo NMD. If not predicted to undergo NMD, please refer to Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). \* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. For weight of PVS1, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss) . \* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.**Duplications** \* To assess the impact of duplications, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). **Nonsense-mediated decay predicted****Nonsense and frameshift variants*** All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1\_Strong or PVS1\_Moderate will be applied, depending on whether >10% or \<10% of the protein is lost.**Splice site variants (+1, +2, -1, -2)*** All canonical splice site pairs in GAMT are GT-AG.* For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants.* For the predicted in frame/out of frame consequences of exon skipping and assigned strength of PVS1, see Appendix 1.* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.* Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. Otherwise, the PVS1 strength should be reduced accordingly.* Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.**Deletions (single or multi exon)*** If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD. * If not predicted to undergo NMD, use PVS1\_Strong if >10% of the protein is predicted to be removed, and use PVS1\_Moderate if \<10% of the protein is predicted to be removed.* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1\_Strong if more than 10% of the protein is removed and PVS1\_Moderate if \<10% of the protein is removed.* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4. Appendix 1 can be used to predict the consequences of single exon deletions.**Duplications*** Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications. * Loss of function is a known mechanism of disease for Creatine Transporter Deficiency.* Specifications are based on the decision tree as outlined in Tayoun etal, 2018 (Hum Mutat. 2018 Nov;39(11):1517-1524; PMID: 30192042) SLC6A8: PVS1, at appropriate strength, is applicable as described in Abou Tayoun et al, 2018 (PMID: 30192042) Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 can be applied for: * Null variants up to p.E643, which corresponds to the distal most de novo truncating variant in an affected patient reported to date.[⁴](#PMID_29695756) * Any frameshift variant that results in a read-through of the stop codon * Canonical splice site variants predicted to result in an out-of-frame product * Canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exon 15) * In-frame deletions including the PM1 functional domains (p.E564\_V617 (bHLH)) * Deletions and duplications ≥1 exon in size (that are completely contained within the _TCF4_ gene) where the reading frame is disrupted and NMD is predicted to occur * Exon skipping or single exon deletion of exon 19 predicted to disrupt the reading frame but is not predicted to undergo NMD * A full gene deletion Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 is applicable for: * Null variants up to p.A563 * Canonical splice site variants predicted to result in an out-of-frame product * Canonical splice site variants predicted to preserve the reading frame (exon 10) * Multiple in-frame exon deletions that include exon 10. * Single exon 3 or 10 in-frame deletion that preserves the reading frame (Note: This gene has no PM1 functional domains) * Deletions and duplications ≥1 exon in size (that are completely contained within the _SLC9A6_ gene) where the reading frame is disrupted and NMD is predicted to occur * A full gene deletion Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 is applicable for: * Null variants up to p.R948 **when using the major brain isoform which has an alternative C-terminus (NM\_001323289.2)** * Frameshift variants that result in a read-through of the stop codon * Canonical splice site variants predicted to result in an out-of-frame product * Canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exons 7, 10, 13) and for the non-coding CDKL5 exon (exon 1) **(NM\_001323289.2).** * In-frame deletions including the PM1 functional domains (p.V19\_K43 ATP binding domain or p.T169\_Y171 TEY phosphorylation domain) * Deletions and duplications ≥1 exon in size (that are completely contained within the CDKL5 gene) where the reading frame is disrupted and NMD is predicted to occur * A full gene deletion Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 is applicable for * Null variants up to p.S468 [¹](#PMID_30525188) * A full gene deletion Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 is applicable for: * Null variants up to p.E472 * Any frameshift variant that results in a read-through of the stop codon * Canonical splice site variants predicted to result in an out-of-frame product * Canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exon 3) * A full gene deletion* PVS1 is **not** applicable for initiation codons. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (PMID:30192042).* PVS1 is applicable for: * Any truncating variant up to p.K841[³](#PMID_9887341) * Any frameshift variant that results in a read-through of the stop codon * Initiation codon variants * Canonical splice site variants predicted to result in an out-of-frame product * Intragenic deletions/duplications predicted to result in an out-of-frame product. * Full gene deletion Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: LOF and/or haploinsufficiency has not been clearly identified as disease mechanisms for these genes relative to the RASopathy spectrum phenotype, therefore in general this rule is not applicable. Note that PTPN11 is currently the only gene with a confirmed association to another non-RASopathy disorder due to LOF alleles. Variants in PTPN11 with predicted LOF should not be evaluated by these RASopathy specific criteria, but should defer to non-adjusted criteria. Given that some historical LOF variants (e.g. canonical splice sites) could potentially result in a gain of function, users should assess using these criteria and non-adjusted criteria to identify the highest likelihood of pathogenicity for all associated diseases. We recommend that the ClinGen Dosage Sensitivity Map Status (http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml) be reviewed for any new apparently LOF disease associations prior to classification assessment. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (Abou Tayoun et al., 2018[¹](#pmid_30192042)) and as updated by the ClinGen SVI Splicing Subgroup (Walker et al., 2023[²](#pmid_37352859)).* Refer to AIPL1-specific PVS1 Decision Tree, file attached. * PVS1: Predicted splice defects at +/- 1,2 in exons 1-6 * PVS1: Single to multi-exon deletions, with or without predicted NMD. All exons are considered critical to protein function[⁶](#pmid_33067476). * PVS1: Applied to variants in the initiation codon. The second in-frame methionine is located at residue 40 in exon 2. There is no known study indicating that this methionine in AIPL1can be used as start codon. There are multiple variants located upstream of p.Met40 that have been reported as a pathogenic in HGMD and ClinVar, evidence that this entire region of the protein is functionally important. * PVS1: Nonsense variants from p.Thr2 through p.Ser328 * PVS1: Frameshift variants from p.Thr2 though p.Glu337 (The presence of extensive long ORFs may lead to significant additional protein length after frameshift[⁷](#pmid_10615133).) * PVS1: Duplications of exons proven in tandem * PVS1(RNA): RNA splicing data with evidence of alternative transcript production at complete levels, relative to normal allele [⁵](#pmid_26650897). PVS1_Strong See PVS1 flow diagram (Figure 1). Not Applicable: Not applicable for MYH7. Use PTEN PVS1 decision tree. See PVS1 flow chart for PVS1_Strong variants in gene where LOF is a known mechanism of disease.* PVS1 should also be considered for the following genes with variants assessed in the Hearing Loss Variant Pilot: GJB2, CDH23, USH2A, SLC26A4, MYO6, MYO7A, TECTA, KCNQ4.* For other genes, LOF must be an established disease mechanism, and the gene/disease association must be Strong or Definitive clinical validity level as outlined in Strande et al. 2017 (PMID: 28552198).* If above criteria is met, follow PVS1 flowchart as recommended by the SVI. Use PVS1\_strong with:* Any nonsense or frameshift variant occurring downstream of c.1285* Any canonical splice site predicted to preserve reading frame (skipping of exons 1, 9, 10) or affect the last exon (exon 13)* Initiator codon variant Per modified CDH1 PVS1 decision tree.Other CDH1 caveats: * Use PVS1_Strong as the default strength of evidence for canonical splice site variants and follow the site-specific recommendations in the splicing table. * CDH1 Exonic deletions or tandem duplications of in-frame exons (exon 4,5,8,9,12,13,15). Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. See PVS1 flowchart for code application Null variant in a gene where loss of function is a known mechanism of disease. * In frame loss of an exon which is part of the catalytic barrel domain and contains pathogenic/likely pathogenic nontruncating variants (exons 6 and 9). * Initiator codon variant. NA Not Applicable: PVS1 is not applicable. MHS is due to gain of function variants in RYR1. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. NA NA NA NA Assessment of small deletions, nonsense, and frameshift variants in protein-coding genes should follow established guidelines (Abou Tayoun et al., 2018) Use PALB2 PVS1 Decision Tree. NA Use _GP1BA_ modified decision tree as per SVI WG. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Not Applicable: VWD type 2 is defined by qualitative defects in the VWF protein and not caused by null variants. Use _GP1BB_ modified decision tree as per SVI WG. Use _GP9_ modified decision tree as per SVI WG. Use decision tree as per SVI WG with specified “regions critical to protein function”. Use _HNF4A_ PVS1 decision tree.* “Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” * Exons 5 (LRG 7), 7 (LRG 9), 8 (LRG 10), 9 (LRG 11) - deletion or skipping causes in-frame deletion, 52/52/79/51-79 AA deleted, that is >10 % of the protein in each case - PVS1\_Strong * Exon 10 (LRG 12) - 46 AA, contains the transactivation domain, includes stop loss - PVS1\_Strong* Apply PVS1\_Strong to initiation codon variants. MDEP has classified two start codon variants as likely pathogenic (c.3G>A: PM2\_Supporting + PP4\_Moderate + PP1\_Strong + PVS1\_Moderate (c.1delA); c.1delA: PM2\_Supporting + PP1 + PP4\_Moderate + PVS1\_Moderate) and there are multiple P/LP variants before the next methionine, p.Met71. Use _GCK_ PVS1 decision tree.Per the SVI standard PVS1 decision tree, apply PVS1\_Strong to duplications ≥ 1 exon in size, contained completely within gene, presumed in tandem, reading frame presumed disrupted, and NMD predicted to occur. Not Applicable: Not applicable. Not Applicable: Does not apply. Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (**Figure 1**) \[Reference 1\]. Not Applicable: VWD type 2N is caused by qualitative protein defects and not null variants. * For frameshift variants at the 3’ end of IDUA that are not predicted to undergo NMD i.e. PTC downstream of c.1778, consider the length of abnormal amino acid sequence that is added due to the frameshift. If >10% of the length of the normal sequence is altered, PVS1 can be applied at strong.* For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1.* Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary).* If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply.* For duplications, consult the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Null variant in a gene where loss of function is a known mechanism of disease. NA Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Not Applicable: Not currently applicable to TNNI3. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TNNT2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TPM1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to ACTC1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL3. See PM4 for truncating variants that do NOT undergo NMD. Applies to:* Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame, predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493), AND presumed in tandem PVS1\_Strong designation for variants that remove more than 10% of the protein and are not expected to lead to NMD. NA Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with two specifications:* For variants not predicted to undergo nonsense-mediated decay, but removing >10% of protein, (i.e. variants in the last exon, exon 9, or variants in the last 50 nucleotides of the penultimate exon after c.1577, codon 526, in exon 8), at least one pathogenic variant must be present downstream in order to apply PVS1\_Strong* PVS1\_Strong can be applied to variants not predicted to undergo nonsense-mediated decay but removing/altering the transactivation domain (amino acids 511-563; Schlake et al., 2000 PMID: 10767081). Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 12, or variants in the last 50 nucleotides of the penultimate exon after c.1028, codon 343, in exon 11), at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong. Presumed by default in tandem duplication of ≥1 exon resulting in a frameshift before the last splice junction. This rule does not apply for variants that involve the UTR (i.e. exon 1 or last exon) and whole gene duplications.ORG>non-G at last base of exon if first 6 bases of the intron are not GTRRGT. If confirmed to cause a splice defect, then PVS1 should be used instead.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 14, or variants in the last 50 nucleotides of the penultimate exon after c.1106, codon 369, in exon 13), at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong._Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608)._ Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with two specifications:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 8, or variants in the last 50 nucleotides of the penultimate exon after c.826, codon 276, in exon 7), at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong.* PVS1\_Strong can be applied to variants not predicted to undergo nonsense-mediated decay but causing truncation of the transmembrane domain (which begins at amino acid 240) or any distal region (i.e. cytoplasmatic domain). Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group [¹⁶](#pmid_30192042) and as updated by the ClinGen SVI Splicing Subgroup [¹⁷](#pmid_37352859)* Refer to RPE65-specific PVS1 Decision Tree, file attached. * PVS1\_Strong:Applied to variants in the initiation codon. The second in-frame methionine is located at residue 93, and there is no known study indicating that this methionine in _RPE65_ can be used as start codon. Variants affecting Met1 can lead to a complete absence of the protein product. Even though we cannot exclude the possibility of a 2nd Met being used as start codon, the translation efficiency maybe significantly reduced due to lack of other important elements (Kozak sequence, etc.). Also, there are multiple variants located upstream of Met93 having been reported as a pathogenic variant in HGMD and ClinVar, evidence that this region of the protein is functionally important. * PVS1\_Strong: Nonsense or frameshift mutations from p.Leu529 through p.Ser533 * PVS1\_Strong: Duplications of exons presumed in tandem * PVS1(RNA)\_Strong: RNA splicing data with evidence of alternative transcript production at near complete levels, relative to normal allele. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 24, or variants in the last 50 nucleotides of the penultimate exon after c.3157, codon 1053, in exon 23), at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong. * For truncating variants located in the isoform-specific exon 3, apply PVS1 if nonsense mediated decay is predicted (truncation at codon 172 or lower) and PVS1\_strong if NMD is not predicted (truncation at codon 173 or higher). While this recommendation already matches the SVI guidance, it is specified here because similar clinical phenotypes are associated with null variants in exon 3 and exon 2 (PMID: 25329329, PMID: 34111452), despite the fact that exon 3 is omitted from an alternative transcript (NM\_001037631.3) that encodes the soluble isoform (PMID: 40168991). Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein, at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein, at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong. Use the PVS1 decision tree guide. Use attached RS1-specific PVS1 Decision Tree file, which has been modified from Abou Tayoun, et al., 2018 (PMID 30192042) and incorporates splice site guidance from Walker et al, 2023 (PMID 37352859). This strength applies to frameshift variants, GT--AG splice variants, deletions, duplications or nonsense variants in NM\_000330.4 c.672 to c.677 (p.Asp224 to p.\*225). Not Applicable: Not applicable. Not Applicable: Not applicable. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 8, or variants in the last 50 nucleotides of the penultimate exon after c.874, codon 292, in exon 7), at least one pathogenic variant **must be** present downstream in order to apply PVS1\_Strong Use ACVRL1 PVS1 Decision Tree (see attachments) Use ENG PVS1 Decision Tree (see attachments) Presumed by default in tandem duplication of ≥1 exon resulting in a frameshift before the last splice junction. This rule does not apply for variants that involve the UTR (i.e. exon 1 or last exon) and whole gene duplications.ORG>non-G at last base of exon if first 6 bases of the intron are not GTRRGT. If confirmed to cause a splice defect, then PVS1 should be used instead.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). Variants in the initiation codon of _MSH6_.OR Presumed by default in tandem duplication of ≥1 exon resulting in a frameshift before the last splice junction. This rule does not apply for variants that involve the UTR (i.e. exon 1 or last exon) and whole gene duplications.ORG>non-G at last base of exon if first 6 bases of the intron are not GTRRGT. If confirmed to cause a splice defect, then PVS1 should be used instead.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). Variants in the initiation codon of _PMS2_OR Presumed by default in tandem duplication of ≥1 exon resulting in a frameshift before the last splice junction. This rule does not apply for variants that involve the UTR (i.e. exon 1 or last exon) and whole gene duplications.ORG>non-G at last base of exon if first 6 bases of the intron are not GTRRGT. If confirmed to cause a splice defect, then PVS1 should be used instead.ORVariants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). Not Applicable: Does not apply given gain of function disease mechanism. In-frame deletions due to the repetitive nature of NEB, particularly in exon 55, are deleterious and pathogenic (Anderson 2004 PMID:15221447, Lehtokari 2009 PMID:19232495). The majority of NEB exons are in frame (exons 3-180, 182); thus, skipping of in-frame exons should be scored at PVS1\_Strong. Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant alpha-actinopathy caused by variants in ACTA1. Not Applicable: Loss of function is not a mechanism of disease for DNM2-related AD Centronuclear myopathy See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant RYR1-related myopathy. * PVS1\_Strong is applicable for nonsense frameshift, and splice variants affecting nucleotide c.1033 and downstream * Several variants downstream of c.1032 leading to frameshift, stop-loss, and protein extension are reported in affected individuals in the literature demonstrating the critical role of the C-terminal region of OTC for protein function \[PMID:9143919, 39256843, 23278509, 34014557, 34014569\]* See “OTC VCEP PVS1 Decision tree” file Please use the SVI WG decision tree modified for the _ABCA4_ gene. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (Abou Tayoun et al., 2018[¹⁶](#pmid_30192042)) and as updated by the ClinGen SVI Splicing Subgroup (Walker et al., 2023[¹⁷](#pmid_37352859)).* Refer to GUCY2D-specific PVS1 Decision Tree, file attached. * PVS1\_Strong: Nonsense or frameshift mutations from p.Pro1069 through p.Ser1103 * PVS1\_Strong: Duplications of exons presumed in tandem * PVS1(RNA)\_Strong: RNA splicing data with evidence of alternative transcript production at near complete levels, relative to normal allele. See PVS1 flowchart In-frame deletions or in frame exon-skipping variants in the pore/transmembrane region of RYR1 should be scored at PVS1\_strong. (Amino acids 4800-4950, Exons 100-103) Please see attached _DYSF_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCB_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCG_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCD_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _CAPN3_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _ANO5_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCA_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Use HNF1A PVS1 decision tree.* Apply PVS1\_Strong for nonsense variants at c.1803 (p.601) and 5’ and frameshift variants at c.1854 (p.618) and 5’. * The distinction of nonsense and frameshift variants was made following a careful review of the phenotypes of individuals with loss-of-function variants in exon 10, which lead to our prediction that the addition of new amino acids from a frameshift will disrupt the TAD and cause a MODY phenotype more so than the deletion of a small part of the end of the TAD. Moderate phenotypic evidence was applied to the c.1802del (p.601Ter) variant, but the individual with the next nonsense variant (p.Gln625Ter) was unaffected. Frameshift variants at p.Ile618 and 5’ have been identified in patients with a phenotype consistent with MODY.* “Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” * Apply PVS1\_Strong for deletions of exon 10 and splicing variants that would predict the skipping of exon 10. * A deletion of exon 10 would remove part of the TAD but less than 10% of the protein. Since the TAD is critical to protein function, and variants that disrupt all of exon 10 have been found in patients with a MODY phenotype, we will use This specification is in accordance with Tayoun’s recommendation to use PVS1\_Strong in cases in which the truncated region is critical to protein function.[¹⁷](#pmid_30192042)Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable: LOF and/or haploinsufficiency have not been clearly identified as disease mechanisms underlying brain malformations related to these genes, so in general this rule is not applicable. The disease mechanism for these genes is gain of function (GOF). Not Applicable: MYOC variants cause JOAG/POAG through a gain of function (GoF) disease mechanism and not loss of function (LoF). Truncating variants in exon 3 are expected to be pathogenic because they escape nonsense-mediated decay. Use ATM PVS1 Decision Tree. Loss of function is a known mechanism for VLCAD Deficiency. The specifications below are based on published guidance for assigning strength of evidence for PVS1 (Abou Tayoun et al., (2018) PMID: 30192042). There are multiple transcripts for ACADVL. The major isoform, NM\_000018.4, encodes a 655 amino acid precursor protein that contains a 40 amino acid N-terminal target sequence that is removed during uptake (Aoyama et al., (1995) PMID: 7668252). In a joint project between NCBI and EMBL-EBI (MANE), NM\_000018.4 was designated as the most relevant transcript. Nonsense or Frameshift:* Use caution when interpreting LOF variants at the 3’ end of the gene.* NMD is not predicted if the variant is in the last exon (exon 20) or in the last 50 nucleotides of the penultimate exon (exon 19). * Canonical Splice Site (+1, +2, -1, -2): All donor/acceptor sites follow the GT/AG rule, except for the donor splice site of intron 8, which begins with GC. PVS1 should not be applied for variants in the splice donor site of intron 8 since the impact of GC donor splice sites is not well understood. For +1 or +2 GT donor splice site variants, the exon immediately 5’ of the variant is predicted to be skipped. For -1 or -2 AG acceptor splice site variants, the exon immediately 3’ of the variants is predicted to be skipped.* Initiation codon: The next in-frame methionine is at position 6 (on transcript NM\_000018). However, the first 40 amino acids comprise the leader sequence in the precursor peptide and are important for proper localization of the protein (Aoyama et al., (1995) PMID: 7668252). Therefore, initiator codon variants will meet PVS1\_Strong.* Well-established \_in vitro\_ or \_in vivo\_ functional studies supportive of a damaging effect \_as measured by effect on mRNA transcript profile (mRNA assay only).\_ Apply as PVS1 (RNA) at appropriate strength. * See ACADVL PVS1 decision tree; cannot be combined with PM1 * Nonsense/frameshift variants predicted to escape NMD (affecting last exon, last 55nt of the penultimate exon). * 1,2 splice site variants leading to exon skipping or use of a cryptic splice site disrupting the reading frame and predicted to escape NMD. * 1,2 splice site variants leading to exon skipping or use of a cryptic splice site but preserving the reading frame. * Single to multi-exon deletion disrupting the reading frame and predicted to escape NMD. * Single to multi-exon deletion preserving the reading frame. * Duplication (>=1 exon in size and completely contained within gene) presumed in tandem and presumably disrupting the reading frame and predicted to escape NMD. See PVS1 flow chart for PVS1_Strong variants in gene where LOF is a known mechanism of disease. NA **In frame loss of >10% of the protein.** CCDS VCEP notes: Loss of function (LOF) of GATM is a known mechanism of disease for arginine:glycine amidinotransferase deficiency (AGAT-D). There are examples of various LOF variants, including nonsense and frameshift, in GATM in individuals with AGAT-D ([https://databases.lovd.nl/shared/variants/GATM/unique](https://databases.lovd.nl/shared/variants/GATM/unique)). The specifications below are based on the PVS1 decision tree (see Appendix 1) (Figure 1, Abou Tayoun et al, 2018, PMID 30192042).GATM specifications: **Nonsense and frameshift variants** \* All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 9) or the last 50 bases of the penultimate exon (exon 8, 3’ of c.1109). In that case, please refer to the PVS1 flowchart for guidance on PVS1 weight (Appendix 1).**Splice site variants (+1, +2, -1, -2)** \* All canonical splice site pairs in GATM are GT-AG. \* For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants. However, it is recommended to use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. \* For considerations for strength at which PVS1 may be applied see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss) . \* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used. \* Non-canonical splice variants, such as +3 or -3, may also meet PVS1 - refer to Walker et al, PMID: 37352859.**Deletions (single or multi exon)** \* If a single or multi-exon deletion results in an out of frame consequence, use PVS1 at the very strong level unless not predicted to undergo NMD. If not predicted to undergo NMD, please refer to Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). \* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. For weight of PVS1, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss) . \* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.**Duplications** \* To assess the impact of duplications, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). **Single exon or larger deletion resulting in loss of >10% of the protein.** **Nonsense and frameshift variants*** All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1\_Strong will be applied if >10% of the protein is lost.**Splice site variants (+1, +2, -1, -2)*** All canonical splice site pairs in GAMT are GT-AG.* For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants.* Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. * For considerations for strength at which PVS1 may be applied see Appendix 1.* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.* Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.**Deletions (single or multi exon)*** If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD.* If not predicted to undergo NMD, use PVS1\_Strong if >10% of the protein is predicted to be removed, and use PVS1\_Moderate if \<10% of the protein is predicted to be removed.* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1\_Strong if more than 10% of the protein is removed and PVS1\_Moderate if \<10% of the protein is removed.* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4. Appendix 1 can be used to predict the consequences of single exon deletions.**Duplications*** Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications. NA Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Strong is applicable for single to multi exon deletions that preserve the reading frame and the variant removes \<10% of the protein. Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Strong is applicable for: * Any truncating variant from p.C564 to p.T601 * Canonical splice site variants that flank exon 3 (in-frame exon).[³](#PMID_27256868)^,[²](#PMID_19377476)^,[⁴](#PMID_18342287) Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Strong is applicable for: * Cannonical splice site variants that flank exon 18 **(the final exon of NM\_001323289.2)** * Single to multi exon deletions that disrupt the reading frame such that exon 18 **(the final exon of NM\_001323289.2)** is truncated/altered * Duplications ≥1 exon in size (that are completely contained within the CDKL5 gene) where the reading frame is presumed to be disrupted and NMD is predicted to occur Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Strong is applicable for any truncating variant from p.G469 to p.Q480 [²](#PMID_29655203). * PVS1\_Strong is applicable for: * Canonical splice site variants or deletions (single exon to full gene deletion) resulting in exon skipping or use of a cryptic splice site that disrupts reading frame and is **NOT** predicted to undergo NMD, but the truncated/altered region is critical to protein function (exon 4). Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Strong is applicable for: * Any truncating variant from p.A842 to p.G850 * Canonical splice site variants that flank exons 7, 8 (in-frame exons) Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: LOF and/or haploinsufficiency has not been clearly identified as disease mechanisms for these genes relative to the RASopathy spectrum phenotype, therefore in general this rule is not applicable. Note that PTPN11 is currently the only gene with a confirmed association to another non-RASopathy disorder due to LOF alleles. Variants in PTPN11 with predicted LOF should not be evaluated by these RASopathy specific criteria, but should defer to non-adjusted criteria. Given that some historical LOF variants (e.g. canonical splice sites) could potentially result in a gain of function, users should assess using these criteria and non-adjusted criteria to identify the highest likelihood of pathogenicity for all associated diseases. We recommend that the ClinGen Dosage Sensitivity Map Status (http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml) be reviewed for any new apparently LOF disease associations prior to classification assessment. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (Abou Tayoun et al., 2018[¹](#pmid_30192042)) and as updated by the ClinGen SVI Splicing Subgroup (Walker et al., 2023[²](#pmid_37352859)).* Refer to AIPL1-specific PVS1 Decision Tree, file attached. * PVS1\_Strong: Nonsense variants from p.Glu329 through p.Ser346 * PVS1\_Strong: Frameshift variants from p.Pro338 through p.His384 * PVS1\_Strong: Duplications of exons presumed in tandem * PVS1(RNA)\_Strong: RNA splicing data with evidence of alternative transcript production at near complete levels, relative to normal allele [⁵](#pmid_26650897). PVS1_Moderate See PVS1 flow diagram (Figure 1). Not Applicable: Not applicable for MYH7. Use PTEN PVS1 decision tree. See PVS1 flowchart for PVS1_Moderate variants in gene where LOF is a known mechanism of disease. NA Per modified CDH1 PVS1 decision tree.Other CDH1 caveats: * G to non-G variants disrupting the last nucleotide of an exon. * Canonical splice sites predicted or demonstrated experimentally to result in in-frame partial skipping/insertion (e.g., Exon 3 donor site). Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. See PVS1 flowchart for code application Null variant in a gene where loss of function is a known mechanism of disease. * Premature termination codon in the 3’ end of GAA (3’ to codon 916), not predicted to be detected by nonsense-mediated decay. * Predicted exon-skipping due to canonical splice variant or exon deletion resulting in an in frame deletion of <10% of the gene product (exons 17, 19, and 20). NA Not Applicable: PVS1 is not applicable. MHS is due to gain of function variants in RYR1. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. NA NA NA NA Assessment of small deletions, nonsense, and frameshift variants in protein-coding genes should follow established guidelines (Abou Tayoun et al., 2018) Use PALB2 PVS1 Decision Tree. NA Use _GP1BA_ modified decision tree as per SVI WG. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Not Applicable: VWD type 2 is defined by qualitative defects in the VWF protein and not caused by null variants. Use _GP1BB_ modified decision tree as per SVI WG. Use _GP9_ modified decision tree as per SVI WG. Use decision tree as per SVI WG with specified “regions critical to protein function”. NA NA Not Applicable: Not applicable. Not Applicable: Does not apply. Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (**Figure 1**) \[Reference 1\]. Not Applicable: VWD type 2N is caused by qualitative protein defects and not null variants. * Any nonsense or frameshift variant that is predicted to be undetected by nonsense-mediated decay (NMD) i.e. if the resulting premature termination codon is in the last exon (exon 14) or in the last 50 nucleotides of the penultimate exon (exon 13; after c.1778, codon 592). In this case, PVS1\_Moderate will be applied because \<10% of the primary amino acid sequence is predicted to be lost ( ~9.3%).* For frameshift variants at the 3’ end of IDUA that are not predicted to undergo NMD i.e. PTC downstream of c.1778, consider the length of abnormal amino acid sequence that is added due to the frameshift. If \<10% of the length of the normal sequence is altered, PVS1 can be applied at moderate.* For all variants involving either the +1 or +2 position of GT donor splice sites, the exon immediately 5’ of the variant is predicted to be skipped, and for all variants involving either the -1 or -2 position of AG acceptor splice sites, the exon immediately 3’ of the variant is predicted to be skipped, unless indicated otherwise by RT-PCR or in silico prediction. For the predicted in frame/out of frame consequences for skipping any exon in IDUA, and recommended weight for PVS1, see Appendix 1.* Follow the recommendations of Walker et al (PMID: 37352859) for all variants occurring in splice motifs (the donor site motif = last 3 nucleotides of an exon and first 6 nucleotides of an intron; acceptor site motif = first nucleotide of an exon and 20 nucleotides upstream from the exon boundary).* If a deletion results in an in-frame consequence, the deletion must encompass one or more exons for PVS1 to apply. Consult Appendix 1 and use professional judgment regarding the strength of evidence to apply.* For duplications, see the PVS1 decision tree and Appendix 1 to assess the impact of single and multi-exon duplications and apply PVS1 at the appropriate strength. Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Null variant in a gene where loss of function is a known mechanism of disease. NA Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Not Applicable: Not currently applicable to TNNI3. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TNNT2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TPM1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to ACTC1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL3. See PM4 for truncating variants that do NOT undergo NMD. Applies to:* Nonsense/frameshifts variants NOT removing the haem-binding domain (aa494-Ter)* GT-AG 1,2 splice sites variants leading to exon skipping or use of a cryptic splice site disrupting reading frame and NOT predicted to undergo NMD (aa1-330) or removing the haem-binding domain (aa331-493)* Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame after the haem-binding domain (aa4934-Ter) AND proven in tandem* Initiation codon variants when there is no known alternative start codon in other transcripts and ≥1 pathogenic variant upstream of closest potential in-frame start codon PVS1\_Moderate designation for initiation codon variants with upstream pathogenic variants of closest potential in-frame start codon or for variants in NM\_00328.3 which remove less than 10% of the protein and are not expected to lead to NMD. Apply PVS1\_Moderate for truncating variants between codons 582 and 620, for which nonsense-mediated decay is not predicted. This region includes the subunits assembly domain (SAD) between residues 589-620, which mediates tetramerization, so that truncating variants that remove this region are reportedly unable to assemble and are disease-causing (PMID: 10654932).[https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1](https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1) Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay, but removing >10% of protein, (i.e. variants in the last exon, exon 9, or variants in the last 50 nucleotides of the penultimate exon after c.1577, codon 526, in exon 8), when at least one pathogenic variant is not present downstream downgrade to PVS1\_Moderate Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 12, or variants in the last 50 nucleotides of the penultimate exon after c.1028, codon 343, in exon 11), when at least one pathogenic variant is **not** present downstream downgrade to PVS1\_Moderate. Nonsense/frameshift variant introducing premature termination codon in codons 754, 755 or 756 in _MLH1._ Refer to Appendix for details. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 14, or variants in the last 50 nucleotides of the penultimate exon after c.1106, codon 369, in exon 13), when at least one pathogenic variant is **not** present downstream downgrade to PVS1\_Moderate._Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608)._ Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 8, or variants in the last 50 nucleotides of the penultimate exon after c.826, codon 276, in exon 7), when at least one pathogenic variant is **not** present downstream downgrade to PVS1\_Moderate. NA Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 24, or variants in the last 50 nucleotides of the penultimate exon after c.3157, codon 1053, in exon 23), when at least one pathogenic variant is **not** present downstream downgrade to PVS1\_Moderate. * Apply PVS1\_Moderate for nonsense or frameshift variants that introduce a premature stop codon between codons 202 and 223 of CTLA4, as these are predicted not to undergo nonsense-mediated decay and to result in C-terminal truncation of less than 10% of the protein product. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein, when at least one pathogenic variant is **not** present downstream, downgrade to PVS1\_Moderate. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein, when at least one pathogenic variant is **not** present downstream, downgrade to PVS1\_Moderate. Use the PVS1 decision tree guide. Use attached RS1-specific PVS1 Decision Tree file, which has been modified from Abou Tayoun, et al., 2018 (PMID 30192042) and incorporates splice site guidance from Walker et al, 2023 (PMID 37352859). Not Applicable: Not applicable. Not Applicable: Not applicable. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) with one specification:* For variants not predicted to undergo nonsense-mediated decay but removing >10% of protein (i.e. variants in the last exon, exon 8, or variants in the last 50 nucleotides of the penultimate exon after c.874, codon 292, in exon 7), when at least one pathogenic variant is **not** present downstream downgrade to PVS1\_Moderate. Use ACVRL1 PVS1 Decision Tree (see attachments) Use ENG PVS1 Decision Tree (see attachments) Nonsense/frameshift variant introducing premature termination codon between codons 892 & 934 in _MSH2_. Refer to Appendix for details. Nonsense/frameshift variant introducing premature termination codon between codons 1342 & 1360 in MSH6. Refer to Appendix for details. Nonsense/frameshift variant introducing premature termination codon between codons 799 & 862 in _PMS2_. Refer to Appendix for details. Not Applicable: Does not apply given gain of function disease mechanism. See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant alpha-actinopathy caused by variants in ACTA1. Not Applicable: Loss of function is not a mechanism of disease for DNM2-related AD Centronuclear myopathy See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant RYR1-related myopathy. See “OTC VCEP PVS1 Decision tree” file Please use the SVI WG decision tree modified for the _ABCA4_ gene. NA See PVS1 flowchart See PVS1 flowchart Please see attached _DYSF_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCB_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCG_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCD_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _CAPN3_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _ANO5_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCA_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. NA Not Applicable: LOF and/or haploinsufficiency have not been clearly identified as disease mechanisms underlying brain malformations related to these genes, so in general this rule is not applicable. The disease mechanism for these genes is gain of function (GOF). Not Applicable: MYOC variants cause JOAG/POAG through a gain of function (GoF) disease mechanism and not loss of function (LoF). Truncating variants in exon 3 are expected to be pathogenic because they escape nonsense-mediated decay. Use ATM PVS1 Decision Tree. Loss of function is a known mechanism for VLCAD Deficiency. The specifications below are based on published guidance for assigning strength of evidence for PVS1 (Abou Tayoun et al., (2018) PMID: 30192042). There are multiple transcripts for ACADVL. The major isoform, NM\_000018.4, encodes a 655 amino acid precursor protein that contains a 40 amino acid N-terminal target sequence that is removed during uptake (Aoyama et al., (1995) PMID: 7668252). In a joint project between NCBI and EMBL-EBI (MANE), NM\_000018.4 was designated as the most relevant transcript. Nonsense or Frameshift:* Use caution when interpreting LOF variants at the 3’ end of the gene.* NMD is not predicted if the variant is in the last exon (exon 20) or in the last 50 nucleotides of the penultimate exon (exon 19). * Canonical Splice Site (+1, +2, -1, -2): All donor/acceptor sites follow the GT/AG rule, except for the donor splice site of intron 8, which begins with GC. PVS1 should not be applied for variants in the splice donor site of intron 8 since the impact of GC donor splice sites is not well understood. For +1 or +2 GT donor splice site variants, the exon immediately 5’ of the variant is predicted to be skipped. For -1 or -2 AG acceptor splice site variants, the exon immediately 3’ of the variants is predicted to be skipped.* Initiation codon: The next in-frame methionine is at position 6 (on transcript NM\_000018). However, the first 40 amino acids comprise the leader sequence in the precursor peptide and are important for proper localization of the protein (Aoyama et al., (1995) PMID: 7668252). Therefore, initiator codon variants will meet PVS1\_Strong.* Well-established \_in vitro\_ or \_in vivo\_ functional studies supportive of a damaging effect \_as measured by effect on mRNA transcript profile (mRNA assay only).\_ Apply as PVS1 (RNA) at appropriate strength. * See ACADVL PVS1 decision tree; cannot be combined with PM1 * Initiation codon variant with 1 or more pathogenic variant(s) upstream of closest potential in-frame start codon. See PVS1 flowchart for PVS1_Moderate variants in gene where LOF is a known mechanism of disease. NA **Single exon or larger deletion resulting in loss of \<10% of the protein, and initiator codon variants.**CCDS VCEP notes: Loss of function (LOF) of GATM is a known mechanism of disease for arginine:glycine amidinotransferase deficiency (AGAT-D). There are examples of various LOF variants, including nonsense and frameshift, in GATM in individuals with AGAT-D ([https://databases.lovd.nl/shared/variants/GATM/unique](https://databases.lovd.nl/shared/variants/GATM/unique)). The specifications below are based on the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042). GATM specifications:**Nonsense and frameshift variants** \* All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 9) or the last 50 bases of the penultimate exon (exon 8, 3’ of c.1109). In that case, please refer to the PVS1 flowchart for guidance on PVS1 weight (Appendix 1).**Splice site variants (+1, +2, -1, -2)** \* All canonical splice site pairs in GATM are GT-AG. \* For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants. However, it is recommended to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. \* For considerations for strength at which PVS1 may be applied see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). \* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used. \* Non-canonical splice variants, such as +3 or -3, may also meet PVS1 (refer to Walker et al, PMID: 37352859).**Initiator codon variants** \* All initiator codon variants will meet PVS1\_Moderate. The next in-frame methionine is at amino acid position 130 (based on NP\_001473). **Deletions (single or multi exon)** \* If a single or multi-exon deletion results in an out of frame consequence, use PVS1 at the very strong level unless not predicted to undergo NMD. If not predicted to undergo NMD, please refer to Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). \* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. For weight of PVS1, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss) . \* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.**Duplications** \* To assess the impact of duplications, see Appendix 1 (PVS1 flowchart) and Appendix 2 (predicted impact of exon loss). **Single exon or larger deletion resulting in loss of \<10% of the protein.** **Nonsense and frameshift variants*** All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1\_Moderate will be applied if \<10% of the protein is lost.**Splice site variants (+1, +2, -1, -2)*** All canonical splice site pairs in GAMT are GT-AG.* Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. * For any canonical splice site variant (+1, +2, -1, -2), if RT-PCR data predicts in-frame loss of \<10% of the protein, apply PVS1\_Moderate.* If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.* Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.**Initiator codon variants*** All initiator codon variants will meet PVS1\_Moderate. The next in-frame methionine is at amino acid position 42 (based on NP\_000147.1).**Deletions (single or multi exon)*** If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD. * If not predicted to undergo NMD, use PVS1\_Strong if >10% of the protein is predicted to be removed, and use PVS1\_Moderate if \<10% of the protein is predicted to be removed.* If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1\_Strong if more than 10% of the protein is removed and PVS1\_Moderate if \<10% of the protein is removed.* If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4. Appendix 1 can be used to predict the consequences of single exon deletions.**Duplications*** Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications. NA Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Moderate is applicable for any truncating variant distal of p.E643 and for single exon deletions that involve just non-coding exon 20. Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Moderate is applicable for * Any truncating variant between p.Y602 to p.A669 * Any frameshift variant that results in a read-through of the stop codon Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Moderate is applicable for: * Any truncating variant distal of p.R948 (**when using the major brain isoform, NM\_001323289.2**) * Canonical splice site variants that flank exon 17 (in-frame exon) (**NM\_001323289.2**) Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Moderate is applicable for any truncating variant distal of p.Q480. * PVS1\_Moderate is applicable for any truncating variant distal of p.E472. Null variant in a gene where loss of function is a known mechanism of disease.* PVS1_Moderate is applicable for any truncating variant distal of p.G850. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: LOF and/or haploinsufficiency has not been clearly identified as disease mechanisms for these genes relative to the RASopathy spectrum phenotype, therefore in general this rule is not applicable. Note that PTPN11 is currently the only gene with a confirmed association to another non-RASopathy disorder due to LOF alleles. Variants in PTPN11 with predicted LOF should not be evaluated by these RASopathy specific criteria, but should defer to non-adjusted criteria. Given that some historical LOF variants (e.g. canonical splice sites) could potentially result in a gain of function, users should assess using these criteria and non-adjusted criteria to identify the highest likelihood of pathogenicity for all associated diseases. We recommend that the ClinGen Dosage Sensitivity Map Status (http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml) be reviewed for any new apparently LOF disease associations prior to classification assessment. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Null variant in a gene where loss of function is a known mechanism of disease.* Use as defined by ClinGen SVI working group (Abou Tayoun et al., 2018[¹](#pmid_30192042)) and as updated by the ClinGen SVI Splicing Subgroup (Walker et al., 2023[²](#pmid_37352859)).* Refer to AIPL1-specific PVS1 Decision Tree, file attached. * PVS1\_Moderate: Nonsense variants from p.Ser347 through p.His384 PP1_Strong Variant segregates with phenotype in ≥6 informative meioses in ≥1 family. Must include ≥2 affected relatives (LDL-C >75th centile) with the variant. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7 segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members, with ≥7 meioses observed across at least two families. Segregation in three affected relatives for recessive and five affected relatives for dominant. * 3 affected segregations + 0 unaffected segregations OR* 2 affected segregations + 3 unaffected segregations ≥Seven informative meioses across ≥2 families. **PP1\_Strong**: ≥ 7 meioses observed within one or across multiple families. Cosegregation must be observed in ≥ 7 meioses across > 1 family Not Applicable: Sib-ships large enough to meet this criterion are extremely rare. In addition, because GAA is the only gene involved in Pompe disease, all patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Segregations in proband plus >2 affected relatives. * Affected relatives must have both variants identified in proband. Co-segregation with disease in ≥7 reported meioses Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.Note: May be used as stronger evidence with increasing segregation data. NA NA NA NA NA * AD Condition: LOD ≥1.26 or Bayes Factor (LR) ≥18:1* AR Condition: Segregation in ≥3 affected relatives \>7 meioses across >=2 families See instructions below for scoring system. Total segregation score 3+ points. This code is applicable when there ≥4 meioses across ≥2 families. NA See instructions below for scoring system. Total segregation score 3+ points. See instructions below for scoring system. Total segregation score 3+ points. Appropriate to use there are 7 or more meioses across more than one family. Use thresholds suggested by Jarvik and Browning[⁵](#pmid_27236918)* Single Family : ≤ 1/32 (5 meioses)* \> 1 Family : ≤ 1/16 (4 meioses) Use thresholds suggested by Jarvik and Browning[⁸](#pmid_27236918)* Single Family : ≤ 1/32 (5 meioses)* \>1 Family : ≤ 1/16 (4 meioses) ≥7 informative meioses. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Variant segregates in ≥ 7 meioses in ≥ 2 families. Appropriate to use when a proband has three affected family members. NA Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1\_Strong – LR ≥18.7:1PP1\_Very Strong – LR ≥350:1 ≥7 informative meioses. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹⁶](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1\_Strong – LR ≥18.7:1PP1\_Very Strong – LR ≥350:1 Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥7** **segregations** (LOD score of 2.1) for **STRONG**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * STRONG evidence requires ≥5 segregations (LOD score of 1.5)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1.3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). ≥ 3 affected segregations or 2 affected segregations AND ≥ 3 unaffected segregations or 1 affected segregation AND ≥ 8 unaffected segregations. Use of this code requires ≥4 meioses in >1 familyOnly phenotype positive relatives with the same variant identified in the proband should be counted as segregations. Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease * Each affected family member must have Schwartz score (PMID: 36017572, Table 10) >3 or QTc greater than or equal to 480ms or syncope in order to be included / counted.* The proband + 7 affected family members with the variant meets PP1\_Strong.* The numbers above have been informed by PMID: 27236918.* If any family member is affected but does not harbor the variant, use BS4 (non-segregation) instead. Can be applied when there is a 32:1 likelihood ratio (LOD score ≥1.5, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7 in ≥2 families. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in multiple affected family members and evidence that this variant and another RPE65 variant are _in trans_.* Requires segregation in proband plus ≥3 similarly affected relatives Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. * Due to incomplete penetrance (45-70%), PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the PP1\_Strong level for co-segregation of the variant with the affected phenotype across at least 4 meioses in one family or combined across multiple unrelated families.* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in ≥7 affected family members. Use of this code requires ≥3 meioses in 1 or 2 families.Only phenotype positive male relatives with the same variant identified in the proband should be counted as segregations. ≥7 informative meioses. ≥7 informative meioses. Use ClinGen SVI recommendations for co-segregation criterion (PMID: 30311386), with strength dependent on number of affected segregations (see list below). 5+ meioses (1/32 likelihood) 5+ meioses (1/32 likelihood) Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7 in ≥2 families. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7 in ≥2 families. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7 in ≥2 families. * PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the PP1\_Strong level for co-segregation of the variant with the affected phenotype across at least 4 meioses in one family or combined across multiple unrelated families (PMID: 38103548).* The combined strength of the PP1 and PP4 codes is limited so that a variant that meets PP1\_Strong can meet PP4 but not PP4\_Moderate (PMID: 38103548).* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. See segregation chart See segregation chart At least five segregations At least five segregations See segregation chart ≥5 informative segregations Segregations in proband plus >2 affected relatives.* Affected relatives must have both variants identified in proband.. Co-segregation with disease in multiple affected family members and evidence that this variant and another GUCY2D variant are _in trans_.* Requires segregation in one proband plus ≥3 similarly affected relatives See segregation chart See segregation chart Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Strong for 3 affected segregations (in addition to proband) across ≥2 families. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use thresholds suggested by Jarvik and Browning[⁷](#pmid_27236918)* Single Family : ≤ 1/32 (5 meioses)* \>1 Family : ≤ 1/16 (4 meioses) Not Applicable: Not applicable since disease-causing variants are germline mosaic, de novo or mosaic. ≥7 meioses in >1 family AR Condition: Segregation in ≥3 affected relatives * See PP1 table. Probands are NOT counted toward segregation. Likewise, carrier parents DO NOT count as unaffected segregations. Affected segregations = # affected individuals in the family with the variants - 1. Affected segregations are defined as affected family members (typically siblings) who harbor the variant in question and a second variant on the remaining allele. Unaffected segregations are defined as unaffected family members, typically siblings, who are at risk to inherit the two variants identified in the proband. These individuals should be either wild-type for both variants identified in the proband, or a heterozygous carrier for a single variant. There may be scenarios where individuals other than siblings could be counted as segregations, such as in families where one parent is affected with the autosomal recessive disorder, in large families with multiple branches, or in consanguineous families. * Strong (32:1 Likelihood) * A LOD score ≥ 1.50 ≥5 affected individuals. Segregation in three affected relatives for recessive. ≥7 meioses across ≥2 families Not Applicable: CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GATM is the only gene involved in AGAT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Not Applicable: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GAMT is the only gene involved in GAMT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. * 3 affected segregations + 0 unaffected segregations OR* 2 affected segregations + 3 unaffected segregations Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis Co-segregation with disease in multiple affected family members.* ≥5 informative meiosis. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. ≥7 informative meioses. Co-segregation with disease in multiple affected family members.\>=7 independent meioses \>=7 independent meioses \>=7 independent meioses \>=7 independent meioses The code is application when there are ≥4 meioses across ≥2 families. Co-segregation with disease in multiple affected family membersAD: ≥7 independent meiosesAR: ≥3 affected segregations Co-segregation with disease in multiple affected family members and evidence that this variant and another AIPL1 variant are _in trans_.* Requires segregation in one proband plus ≥3 similarly affected relatives PP1_Moderate Variant segregates with phenotype in 4-5 informative meioses in ≥1 family. Must include ≥2 affected relatives (LDL-C >75th centile) with the variant. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members, with 5 or 6 meioses observed. Segregation in two affected relatives for recessive and 4 affected relatives for dominant. * 2 affected segregations + 0 unaffected segregations Five-six informative meioses across ≥1 family. **PP1\_Moderate**: 5 or 6 meioses observed within one or across multiple families. Cosegregation must be observed in 5-6 meioses in/across 1 or more families Not Applicable: Sib-ships large enough to meet this criterion are extremely rare. In addition, because GAA is the only gene involved in Pompe disease, all patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Segregation in proband plus 2 affected relatives. * Affected relatives must have both variants identified in proband. Co-segregation with disease in 5-6 reported meioses Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.Note: May be used as stronger evidence with increasing segregation data. NA NA NA NA Co-segregation with disease in 5+ maternal family members and level of heteroplasmy segregating with disease manifestations * AD Condition: LOD ≥.60 or Bayes Factor (LR) ≥4:1* AR Condition: Segregation in 2 affected relatives 5 – 6 meioses across ≥1 family. See instructions below for scoring system. Total segregation score 2-2.75 points. This code is applicable when there are at least 3 meioses across one or more families. Appropriate to use when there are multiple families each reported to have two or more meioses. See instructions below for scoring system. Total segregation score 2-2.75 points. See instructions below for scoring system. Total segregation score 2-2.75 points. Appropriate to use there are 4-6 meioses across one or more families. Use thresholds suggested by Jarvik and Browning[⁵](#pmid_27236918)* Single Family : ≤ 1/16 (4 meioses)* \> 1 Family : ≤ 1/8 (3 meioses) Use thresholds suggested by Jarvik and Browning[⁸](#pmid_27236918)* Single Family : ≤ 1/16 (4 meioses)* \>1 Family : ≤ 1/8 (3 meioses) ≥5 informative meioses. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Variant segregates in 5-6 meioses in ≥ 1 family. Appropriate to use when a proband has two affected family members. See Appendix 5 for points system and guidance (based on PMID: 38103548). Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1\_Moderate – LR ≥4.3:1 ≥5 informative meioses. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹⁶](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1\_Moderate – LR ≥4.3:1 Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥5** **segregations** (LOD score of 1.5) for **MODERATE**.Although rare for inherited cardiomyopathies, when the phenotype/presentation of a variant within and across families is highly specific (e.g., early-onset severe RCM in all affected individuals), the following thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) can be considered: * MODERATE evidence requires ≥4 segregations (LOD score of 1.2)Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). 2 affected segregations or 1 affected segregation AND ≥ 5 unaffected segregations or ≥ 10 unaffected segregations. ≥ 3 meiosesIn ≥ 1 familiesOnly phenotype positive relatives with the same variant identified in the proband should be counted as segregations. Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease * Each affected family member must have Schwartz score (PMID: 36017572, Table 10) >3 or QTc greater than or equal to 480ms or syncope in order to be included / counted.* The proband + 5 affected family members with the variant meets PP1\_Moderate.* The numbers above have been informed by PMID: 27236918.* If any family member is affected but does not harbor the variant, use BS4 (non-segregation) instead. Can be applied when there is a 16:1 likelihood ratio (LOD score 1.2-\<1.5, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >4.3 & ≤18.7. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in multiple affected family members and evidence that this variant and another RPE65 variant are _in trans_.* Requires segregation in proband plus 2 similarly affected relatives Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. * Due to incomplete penetrance (45-70%), PP1\_Moderate will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the PP1\_Moderate level for co-segregation of the variant with the affected phenotype across at least two meioses in one family or combined across multiple unrelated families.* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in ≥5 affected family members. 2 meioses in a family (e.g. 2 brothers and mother genotyped; 3 brothers without mother's genotype; or uncle and nephew)Only phenotype positive male relatives with the same variant identified in the proband should be counted as segregations. ≥5 informative meioses. ≥5 informative meioses. Use ClinGen SVI recommendations for co-segregation criterion (PMID: 30311386), with strength dependent on number of affected segregations (see list below). 4 meioses (1/16 likelihood) 4 meioses (1/16 likelihood) Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >4.3 & ≤18.7. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >4.3 & ≤18.7. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >4.3 & ≤18.7. * PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the PP1\_Moderate level for co-segregation of the variant with the affected phenotype across at least two meioses in one family or combined across multiple unrelated families (PMID: 38103548).* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. See segregation chart See segregation chart Four segregations 3-4 segregations See segregation chart 3-4 informative segregations Segregation in proband plus 2 affected relatives.* Affected relatives must have both variants identified in proband. Co-segregation with disease in multiple affected family members and evidence that this variant and another GUCY2D variant are _in trans_.* Requires segregation in one proband plus 2 similarly affected relatives See segregation chart See segregation chart Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1\_Moderate for 2 affected segregations (in addition to proband; may be from a single family). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use thresholds suggested by Jarvik and Browning[⁷](#pmid_27236918)* Single Family : ≤ 1/16 (4 meioses)* \>1 Family : ≤ 1/8 (3 meioses) Not Applicable: Not applicable since disease-causing variants are germline mosaic, de novo or mosaic. ≥ 5 meioses regardless of the number of families AR Condition: Segregation in 2 affected relatives * See PP1 table. Probands are NOT counted toward segregation. Likewise, carrier parents DO NOT count as unaffected segregations. Affected segregations = # affected individuals in the family with the variants - 1. Affected segregations are defined as affected family members (typically siblings) who harbor the variant in question and a second variant on the remaining allele. Unaffected segregations are defined as unaffected family members, typically siblings, who are at risk to inherit the two variants identified in the proband. These individuals should be either wild-type for both variants identified in the proband, or a heterozygous carrier for a single variant. There may be scenarios where individuals other than siblings could be counted as segregations, such as in families where one parent is affected with the autosomal recessive disorder, in large families with multiple branches, or in consanguineous families. * Moderate (16:1 Likelihood) * A LOD score \< 1.50 ≥ 1.20 4 affected individuals. Segregation in two affected relatives for recessive. 5 – 6 meioses across ≥1 family Not Applicable: CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GATM is the only gene involved in AGAT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Not Applicable: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GAMT is the only gene involved in GAMT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. * 2 affected segregations + 0 unaffected segregations. Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis Co-segregation with disease in multiple affected family members.* 3-4 informative meiosis. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. ≥5 informative meioses. Co-segregation with disease in multiple affected family members.5-6 independent meioses 5-6 independent meioses 5-6 independent meioses 5-6 independent meioses The code is application when there are at least 3 meioses across one or more families. Co-segregation with disease in multiple affected family membersAD: 5-6 independent meiosesAR: 2 affected segregations Co-segregation with disease in multiple affected family members and evidence that this variant and another AIPL1 variant are _in trans_.* Requires segregation in one proband plus 2 similarly affected relatives PP1_Supporting Variant segregates with phenotype in 2-3 informative meioses in ≥1 family. Must include ≥1 affected relative (LDL-C >75th centile) with the variant. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members, with 3 or 4 meioses observed. Segregation in one affected relative for recessive and two affected relatives for dominant. * 1 affected family member + 3 unaffected segregations Three-four informative meioses across ≥1 family. **PP1**: 3 or 4 meioses observed within one or across multiple families. Cosegregation must be observed in 3-4 meioses in/across 1 or more families Not Applicable: Sib-ships large enough to meet this criterion are extremely rare. In addition, because GAA is the only gene involved in Pompe disease, all patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Segregation in proband plus 1 affected relative. * Affected relatives must have both variants identified in proband. Co-segregation with disease in 3-4 reported meioses Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.Note: May be used as stronger evidence with increasing segregation data. For segregation, an affected is defined as an individual who 1) has brainstem or basal ganglia lesions compatible with SLC19A3-related Biotin-responsive basal ganglia disease OR 2) a person with neurodevelopmental regression or MRI lesions compatible with SLC19A3-related Biotin-responsive basal ganglia disease who had significant clinical improvement in either symptoms or MRI lesions from treatment with biotin and thiamine. Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease Further define “affected” as an individual in whom there is objective evidence of manifestations consistent with POLG-related disorders spectrum: Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), and/or progressive external ophthalmoplegia (PEO) Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease Co-segregation with disease in 2-4 maternal family members and level of heteroplasmy segregating with disease manifestations * AD Condition: LOD ≥0.3 or Bayes Factor (LR) ≥2:1* AR Condition: Segregation in 1 affected relative 3 – 4 meioses across ≥1 family. See instructions below for scoring system. Total segregation score 1-1.75 points. This code is applicable when there 2 meioses in one family **OR** 1 meiosis between 2 affected siblings. Appropriate to use when there are 2 or more meioses within a single family. See instructions below for scoring system. Total segregation score 1-1.75 points. See instructions below for scoring system. Total segregation score 1-1.75 points. Appropriate to use when there are 2-3 meioses across one or more families. Use thresholds suggested by Jarvik and Browning[⁵](#pmid_27236918)* Single Family : ≤ 1/8 (3 meioses)* \> 1 Family : ≤ ¼ (2 meioses) Use thresholds suggested by Jarvik and Browning[⁸](#pmid_27236918)* Single Family : ≤ 1/8 (3 meioses)* \>1 Family : ≤ ¼ (2 meioses) ≥3 informative meioses. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Variant segregates in 3-4 meioses in ≥ 1 family. Appropriate to use when a proband has one affected family member. See Appendix 5 for points system and guidance (based on PMID: 38103548). Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1 - LR ≥2.08:1 ≥3 informative meioses. Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹⁶](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:PP1 - LR ≥2.08:1 Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹²](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). Due to the genotypic and phenotypic heterogeneity of inherited cardiomyopathies, segregation thresholds have been conservatively set at **≥3** **segregations** (LOD score of 0.9) for **SUPPORTING**. The thresholds as proposed by Jarvik and Browning (2016)[¹¹](#pmid_27236918) are the same at ≥3 segregations (LOD score of 0.9) for supporting.Only genotype positive/phenotype positive individuals are counted as segregations, which can include affected obligate carriers. Genotype positive/phenotype negative individuals are generally less informative for cardiomyopathy genes due to variable age at onset and reduced penetrance.Phenotypes should be clinically confirmed, whenever possible, and should not include individuals with a suspected diagnosis. Important considerations include:1. Segregation of a variant within a single family or haplotype has the potential to represent linkage disequilibrium with another undetected variant. If linkage disequilibrium is a concern, consider downgrading strength of segregation. 2. Use of segregation criteria should be carefully evaluated if variant frequency meets criteria for BS1 (see below).3. Caution is needed when counting segregations in presence of other possible disease-causing variants, as both variants may be contributing to the phenotype. 4. Caution is needed when distantly related (≥3^rd degree) affected individuals are connected by unknown or unaffected relatives (raises possibility of multiple causes of disease). 1 affected segregation or ≥ 5 unaffected segregations. ≥ 2 meioses in the same familyOR proband with affected motherOnly phenotype positive relatives with the same variant identified in the proband should be countedas segregations. Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease * Each affected family member must have Schwartz score (PMID: 36017572, Table 10) >3 or QTc greater than or equal to 480ms or syncope in order to be included / counted.* The proband + 3 affected family members with the variant meets PP1.* The numbers above have been informed by PMID: 27236918.* If any family member is affected but does not harbor the variant, use BS4 (non-segregation) instead. Can be applied when there is a 4:1 likelihood ratio (LOD score 0.6-\<1.2, summed across all families with segregation evidence) per recommendations from PMID: 30311386 Table 4a. See instructions for calculating the estimated LOD score. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in multiple affected family members and evidence that this variant and another RPE65 variant are _in trans_.* Requires segregation in proband plus 1 similarly affected relative Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. * Due to incomplete penetrance (45-70%), PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the supporting level for co-segregation of the variant with the affected phenotype across at least one meiosis (i.e. the variant is present in the proband + an affected relative).* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Use recommendations for co-segregation criterion from PMID: 30311386, with strength dependent on number of affected segregations. Co-segregation with disease in ≥3 affected family members. 1 meiosis in a familyOnly phenotype positive male relatives with the same variant identified in the proband should be counted as segregations. ≥3 informative meioses. ≥3 informative meioses. Use ClinGen SVI recommendations for co-segregation criterion (PMID: 30311386), with strength dependent on number of affected segregations (see list below). 3 meioses (1/8 likelihood) 3 meioses (1/8 likelihood) Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3. Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3. * PP1 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting co-segregations.* Met at the supporting level for co-segregation of the variant with the affected phenotype across at least one meiosis (i.e. the variant is present in the proband + an affected relative, PMID: 38103548).* PP1 should not be applied when a variant also has population data meeting BA1 or BS1 since a common variant may appear to segregate with the disease by chance. Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.See segregation chart Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.See segregation chart At least 2 segregations 2 segregations Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.See segregation chart 2 informative segregations Segregation in proband plus 1 affected relative.* Affected relatives must have both variants identified in proband. Co-segregation with disease in multiple affected family members and evidence that this variant and another GUCY2D variant are _in trans_.* Requires segregation in one proband plus 1 similarly affected relative Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.See segregation chart Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.See segregation chart Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Segregations should be counted across families, with the total number of segregations determining the strength level. Apply PP1 (Supporting) for 1 affected segregation (in addition to proband). When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use thresholds suggested by Jarvik and Browning[⁷](#pmid_27236918)* Single Family : ≤ 1/8 (3 meioses)* \>1 Family : ≤ ¼ (2 meioses) Not Applicable: Not applicable since disease-causing variants are germline mosaic, de novo or mosaic. ≥ 3 meioses regardless of the number of families AR Condition: Segregation in 1 affected relative * See PP1 table. Probands are NOT counted toward segregation. Likewise, carrier parents DO NOT count as unaffected segregations. Affected segregations = # affected individuals in the family with the variants - 1. Affected segregations are defined as affected family members (typically siblings) who harbor the variant in question and a second variant on the remaining allele. Unaffected segregations are defined as unaffected family members, typically siblings, who are at risk to inherit the two variants identified in the proband. These individuals should be either wild-type for both variants identified in the proband, or a heterozygous carrier for a single variant. There may be scenarios where individuals other than siblings could be counted as segregations, such as in families where one parent is affected with the autosomal recessive disorder, in large families with multiple branches, or in consanguineous families. * Supporting (4:1 Likelihood) * An LOD score \< 1.20 ≥ 0.60 2-3 affected individuals. Segregation in one affected relative for recessive. 3 – 4 meioses across ≥1 family Not Applicable: CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GATM is the only gene involved in AGAT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Not Applicable: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GAMT is the only gene involved in GAMT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. * 1 affected family member + 3 unaffected segregations. Co-segregation with disease in multiple affected family members.* 2 informative meiosis Co-segregation with disease in multiple affected family members.* 2 informative meiosis Co-segregation with disease in multiple affected family members.* 2 informative meiosis Co-segregation with disease in multiple affected family members.* 2 informative meiosis Co-segregation with disease in multiple affected family members.* 2 informative meiosis Co-segregation with disease in multiple affected family members.* 2 informative meiosis. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. ≥3 informative meioses. Co-segregation with disease in multiple affected family members.3-4 independent meioses 3-4 independent meioses 3-4 independent meioses 3-4 independent meioses The code is application when there are 2 meioses in one family **OR** 1 meiosis between 2 affected siblings. Co-segregation with disease in multiple affected family membersAD: 3-4 independent meiosesAR: 1 affected segregation Co-segregation with disease in multiple affected family members and evidence that this variant and another AIPL1 variant are _in trans_.* Requires segregation in one proband plus 1 similarly affected relative BP3_Very Strong Not Applicable Not Applicable: Not applicable to the current genes. Not Applicable: This rule is not applicable to PTEN. NA Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: RUNX1 does not contain a repetitive region without known function. BP3 is therefore deemed not applicable. Not Applicable: Not applicable Not Applicable: There are no known repetitive regions without known function in GAA. NA Not Applicable: BP3 is not applicable. RYR1 does not have repetitive regions without known function. NA NA NA NA NA Not Applicable: There are a few locations in the mtDNA genome where indels within a repetitive region are observed outside of two common locations: one is in the hypervariable region 1 (around position 16,189) and the other in hypervariable region 2 (around position 310). These indels are well-known benign findings. Not Applicable: Do not use: small in-frame losses are neither confirmed nor refuted as a mechanism of pathogenicity for PALB2. In addition, PALB2 is not considered to have repetitive regions without known function NA NA Not Applicable: Not applicable for F9 gene. Not Applicable: There are no known repetitive regions in the VWF gene without a known function. NA NA Not Applicable: There are no known repetitive regions in the SERPINC1 gene without a known function. Not Applicable Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable: There are no known repetitive regions in the VWF gene without a known function. Not Applicable: There are no known repetitive regions without known function in IDUA. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Not applicable to the current genes. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: CYP1B1 does not have a repetitive region without a known function. NA NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. Not Applicable: Not applicable, as repetitive regions of unknown function are not known within CTLA4.The most relevant known variant, NM_005214.5(CTLA4):c.110_118del, may be better evaluated as a potential splice variant (splice acceptor loss and splice acceptor gain). Not Applicable: Does not apply. Not Applicable: Does not apply. NA Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable Not Applicable: There are no regions in NEB where BP3 would apply. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in DNM2 where BP3 would apply. Not Applicable: There are no regions in MTM1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. OTC does not contain repetitive regions without known function. Not Applicable: There are no known repetitive regions without known function. Not Applicable: No repetitive regions with unknown function. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in DYSF. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCB. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCG. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCD. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in CAPN3. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in ANO5. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCA. Not Applicable Not Applicable: This is not applicable for the genes specified since the exon regions do not have repetitive regions without a known function. Not Applicable: MYOC does not have a repetitive region without a known function. Not Applicable: Do not use. Not Applicable: There are no known repetitive regions without known function in ACADVL. Not Applicable NA Not Applicable: Not applicable at this time. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function. Not Applicable: There are no known repetitive regions without known function in GAMT. Not Applicable NA NA NA NA NA NA Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. NA NA NA NA Not Applicable: Not applicable for F8 gene. NA Not Applicable: No repetitive regions with unknown function. BP3_Strong Not Applicable Not Applicable: Not applicable to the current genes. Not Applicable: This rule is not applicable to PTEN. NA Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: RUNX1 does not contain a repetitive region without known function. BP3 is therefore deemed not applicable. Not Applicable: Not applicable Not Applicable: There are no known repetitive regions without known function in GAA. NA Not Applicable: BP3 is not applicable. RYR1 does not have repetitive regions without known function. In frame-deletions/insertions in a repetitive region without a known function. NA NA NA NA Not Applicable: There are a few locations in the mtDNA genome where indels within a repetitive region are observed outside of two common locations: one is in the hypervariable region 1 (around position 16,189) and the other in hypervariable region 2 (around position 310). These indels are well-known benign findings. Not Applicable: Do not use: small in-frame losses are neither confirmed nor refuted as a mechanism of pathogenicity for PALB2. In addition, PALB2 is not considered to have repetitive regions without known function NA NA Not Applicable: Not applicable for F9 gene. Not Applicable: There are no known repetitive regions in the VWF gene without a known function. NA NA Not Applicable: There are no known repetitive regions in the SERPINC1 gene without a known function. Not Applicable Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable: There are no known repetitive regions in the VWF gene without a known function. Not Applicable: There are no known repetitive regions without known function in IDUA. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Not applicable to the current genes. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: CYP1B1 does not have a repetitive region without a known function. NA NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. Not Applicable: Not applicable, as repetitive regions of unknown function are not known within CTLA4.The most relevant known variant, NM_005214.5(CTLA4):c.110_118del, may be better evaluated as a potential splice variant (splice acceptor loss and splice acceptor gain). Not Applicable: Does not apply. Not Applicable: Does not apply. NA Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable Not Applicable: There are no regions in NEB where BP3 would apply. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in DNM2 where BP3 would apply. Not Applicable: There are no regions in MTM1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. OTC does not contain repetitive regions without known function. Not Applicable: There are no known repetitive regions without known function. Not Applicable: No repetitive regions with unknown function. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in DYSF. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCB. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCG. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCD. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in CAPN3. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in ANO5. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCA. Not Applicable Not Applicable: This is not applicable for the genes specified since the exon regions do not have repetitive regions without a known function. Not Applicable: MYOC does not have a repetitive region without a known function. Not Applicable: Do not use. Not Applicable: There are no known repetitive regions without known function in ACADVL. Not Applicable NA Not Applicable: Not applicable at this time. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function. Not Applicable: There are no known repetitive regions without known function in GAMT. Not Applicable NA NA NA NA NA NA Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. NA NA NA NA Not Applicable: Not applicable for F8 gene. NA Not Applicable: No repetitive regions with unknown function. BP3_Moderate Not Applicable Not Applicable: Not applicable to the current genes. Not Applicable: This rule is not applicable to PTEN. NA Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: RUNX1 does not contain a repetitive region without known function. BP3 is therefore deemed not applicable. Not Applicable: Not applicable Not Applicable: There are no known repetitive regions without known function in GAA. NA Not Applicable: BP3 is not applicable. RYR1 does not have repetitive regions without known function. In frame-deletions/insertions in a repetitive region without a known function. NA NA NA NA Not Applicable: There are a few locations in the mtDNA genome where indels within a repetitive region are observed outside of two common locations: one is in the hypervariable region 1 (around position 16,189) and the other in hypervariable region 2 (around position 310). These indels are well-known benign findings. Not Applicable: Do not use: small in-frame losses are neither confirmed nor refuted as a mechanism of pathogenicity for PALB2. In addition, PALB2 is not considered to have repetitive regions without known function NA NA Not Applicable: Not applicable for F9 gene. Not Applicable: There are no known repetitive regions in the VWF gene without a known function. NA NA Not Applicable: There are no known repetitive regions in the SERPINC1 gene without a known function. Not Applicable Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable: There are no known repetitive regions in the VWF gene without a known function. Not Applicable: There are no known repetitive regions without known function in IDUA. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Not applicable to the current genes. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: CYP1B1 does not have a repetitive region without a known function. NA NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. Not Applicable: Not applicable, as repetitive regions of unknown function are not known within CTLA4.The most relevant known variant, NM_005214.5(CTLA4):c.110_118del, may be better evaluated as a potential splice variant (splice acceptor loss and splice acceptor gain). Not Applicable: Does not apply. Not Applicable: Does not apply. NA Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable Not Applicable: There are no regions in NEB where BP3 would apply. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in DNM2 where BP3 would apply. Not Applicable: There are no regions in MTM1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. OTC does not contain repetitive regions without known function. Not Applicable: There are no known repetitive regions without known function. Not Applicable: No repetitive regions with unknown function. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in DYSF. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCB. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCG. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCD. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in CAPN3. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in ANO5. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCA. Not Applicable Not Applicable: This is not applicable for the genes specified since the exon regions do not have repetitive regions without a known function. Not Applicable: MYOC does not have a repetitive region without a known function. Not Applicable: Do not use. Not Applicable: There are no known repetitive regions without known function in ACADVL. Not Applicable NA Not Applicable: Not applicable at this time. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function. Not Applicable: There are no known repetitive regions without known function in GAMT. Not Applicable NA NA NA NA NA NA Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. NA NA NA NA Not Applicable: Not applicable for F8 gene. NA Not Applicable: No repetitive regions with unknown function. BP3_Supporting Not Applicable Not Applicable: Not applicable to the current genes. Not Applicable: This rule is not applicable to PTEN. In-frame indels in repeat region without known function.No changes. Follow recommendations as outlined in Richard 2015 and/or ClinGen's Sequence Variant Interpretation working group. Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: RUNX1 does not contain a repetitive region without known function. BP3 is therefore deemed not applicable. Not Applicable: Not applicable Not Applicable: There are no known repetitive regions without known function in GAA. Use with no specification Not Applicable: BP3 is not applicable. RYR1 does not have repetitive regions without known function. In frame-deletions/insertions in a repetitive region without a known function. In-frame deletions/insertions in a repetitive region without a known function In-frame deletions/insertions in a repetitive region without a known function In-frame deletions/insertions in a repetitive region without a known function In-frame deletions/insertions in a repetitive region without a known function Not Applicable: There are a few locations in the mtDNA genome where indels within a repetitive region are observed outside of two common locations: one is in the hypervariable region 1 (around position 16,189) and the other in hypervariable region 2 (around position 310). These indels are well-known benign findings. Not Applicable: Do not use: small in-frame losses are neither confirmed nor refuted as a mechanism of pathogenicity for PALB2. In addition, PALB2 is not considered to have repetitive regions without known function BP3 can be applied to the 8x GXEEX AA repeat motif in the 5’ end of VHL p30 (AA14-AA48). Otherwise, the rest of the coding regions in VHL do not contain repeats (and none contain LINE/SINE, low complexity or other repeat types as identified by RepeatMasker) and BP3 is not applicable. Use with no specification Not Applicable: Not applicable for F9 gene. Not Applicable: There are no known repetitive regions in the VWF gene without a known function. Use with no specification Use with no specification Not Applicable: There are no known repetitive regions in the SERPINC1 gene without a known function. Not Applicable Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable: There are no known repetitive regions in the VWF gene without a known function. Not Applicable: There are no known repetitive regions without known function in IDUA. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Not applicable to the current genes. Not Applicable: Captured by bioinformatic tool prediction, and domain analysis. See Appendix J for details Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: Not applicable to the current genes. Not Applicable: CYP1B1 does not have a repetitive region without a known function. Use for ORF15 repetitive regions only (amino acids 585-1078). These events can be multiple and large (e.g. 9 plus 15 total 20-30 bp is known, a single 21bp event is common). Please note that ORF15 encompasses residues 585 through 1152, with UniProt identifying particularly disordered regions between 609-776, 790-906, and 989-1020. In-frame deletions/insertions in a repetitive region without a known function* Not applicable to _KCNQ1_ Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. Not Applicable: Not applicable, as repetitive regions of unknown function are not known within CTLA4.The most relevant known variant, NM_005214.5(CTLA4):c.110_118del, may be better evaluated as a potential splice variant (splice acceptor loss and splice acceptor gain). Not Applicable: Does not apply. Not Applicable: Does not apply. In frame-deletions/insertions in a repetitive region without a known function. Not Applicable Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: Does not apply. Not Applicable Not Applicable Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function is not used. Not Applicable Not Applicable: There are no regions in NEB where BP3 would apply. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in DNM2 where BP3 would apply. Not Applicable: There are no regions in MTM1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. OTC does not contain repetitive regions without known function. Not Applicable: There are no known repetitive regions without known function. Not Applicable: No repetitive regions with unknown function. Not Applicable: There are no regions in ACTA1 where BP3 would apply. Not Applicable: There are no regions in RYR1 where BP3 would apply. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in DYSF. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCB. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCG. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCD. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in CAPN3. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in ANO5. Not Applicable: Not applicable. Repetitive regions without a known function are not well described in SGCA. Not Applicable Not Applicable: This is not applicable for the genes specified since the exon regions do not have repetitive regions without a known function. Not Applicable: MYOC does not have a repetitive region without a known function. Not Applicable: Do not use. Not Applicable: There are no known repetitive regions without known function in ACADVL. Not Applicable In-frame indels in repeat region without known function.* No changes. Follow recommendations as outlined in Richard 2015 and/or ClinGen's Sequence Variant Interpretation working group. Not Applicable: Not applicable at this time. Not Applicable: In-frame deletions/insertions in a repetitive region without a known function. Not Applicable: There are no known repetitive regions without known function in GAMT. Not Applicable In-frame deletions/insertions in a repetitive region without a known function.* BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. In-frame deletions/insertions in a repetitive region without a known function* BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. In-frame deletions/insertions in a repetitive region without a known function. * BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. In-frame deletions/insertions in a repetitive region without a known function* Inframe expansions or deletions in _FOXG1_ repetitive regions: poly His (p.His47-p.His57), poly Gln (p.Gln70-p.Gln73) and poly Pro (p.Pro58-p.Pro61; p.Pro65-p.Pro69; p.Pro74-p.Pro80)* BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. In-frame deletions/insertions in a repetitive region without a known function. * BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. In-frame deletions/insertions in a repetitive region without a known function.* BP3 is applicable if there are in-frame deletions/duplications in a repetitive region where other in-frame deletions/duplications have been observed with an overall frequency commensurate with the BA1 threshold for this gene. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. Not Applicable: No known benign repetitive areas in RASopathy genes. In frame-deletions/insertions in a repetitive region without a known function. In frame-deletions/insertions in a repetitive region without a known function. In frame-deletions/insertions in a repetitive region without a known function. In frame-deletions/insertions in a repetitive region without a known function. Not Applicable: Not applicable for F8 gene. In frame-deletions/insertions in a repetitive region without a known function. Not Applicable: No repetitive regions with unknown function. PM3_Moderate This criterion can be used for a candidate LDLR variant observed in an individual with a homozygous FH phenotype when there is only one other pathogenic or likely pathogenic variant in LDLR (in trans), APOB or PCSK9. Caveat: variant must also meet PM2. Not Applicable: It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: This rule is not applicable to PTEN. 1 point awarded from tables 7a and 7b.Example: Detected in trans with a pathogenic variant (recessive). Applicable as described in SVI recommendations for in trans criterion Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes:FPD/AML is inherited in an autosomal dominant manner, thus PM3 is not applicable. Not Applicable: This rule does not apply to TP53/Li-Fraumeni syndrome. Detected in trans with a pathogenic variant. Points-based system. See main specifications document. Use proposed SVI point recommendations. Both variants must be classified using ITGA2B/ITGB3 Rule Specifications. Not Applicable: PM3 is not applicable. MHS is inherited as an autosomal dominant trait with reduced penetrance. For recessive disorders, detected in trans with a pathogenic variantNote: This requires testing of parents (or offspring) to determine phase. Use per SVI guidance Not Applicable Use per SVI guidance.Note: T251I and P587L are almost always in cis Use per SVI guidance Not Applicable: mtDNA variants are maternally inherited and not inherited in an autosomal recessive manner. PM3 = **2** pointsSee Fanconi Anemia PM3 tables for approach to assign points per proband. Not Applicable: Autosomal dominant. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications. Not Applicable: Not applicable for the F9 gene. Not Applicable: These are dominant conditions, so this rule code does not apply. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications, with the exception that a 22q11.2 deletion in trans ([https://www.ncbi.nlm.nih.gov/books/NBK1523/](https://www.ncbi.nlm.nih.gov/books/NBK1523/)) may be automatically scored 1pt with confirmation that deletion includes the _GP1BB_ gene. Use proposed SVI point recommendations (see below). Both variants must be classified using these rule specifications. Not Applicable: Variants in this gene are being curated as a dominant condition, so this rule code does not apply. Not Applicable Use SVI-recommended point-based system. Not Applicable: Not applicable. Use ClinGen SVI recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for RMRP. Not Applicable Use SVI recommended point system for this code for probands with a VWD type 2N diagnosis. Total of 1 point required. Detected in trans with a pathogenic variant. Points system based on [guidance](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene.Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3 = 2 points ≥1 points. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene. Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3 = 2 points Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. ≥ 1.0 points Not Applicable For recessive disorders, detected in _trans_ with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase* Can be used for variants seen in patients with Jervell and Lange-Nielsen syndrome, if the phenotype includes both long QT interval and congenital deafness.* This code is not considered mutually exclusive with PS4. For example, variants associated with both recessive and dominant cases can meet both PM3 and PS4.* Use the ClinGen SVI recommendations to determine evidence weight for this rule code.* Both variants must be classified using these rule specifications. Sum of case scores 2-3.75 points (see instructions below) Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _ADA._ 1 - 1.5 points Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _DCLRE1C._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _IL7R._ Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 1 to 1.75 points from Table 1 Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _JAK3._ Not Applicable: Not applicable, as this code is specific to recessive disorders. Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG1._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG2._ Not Applicable: PAH is autosomal dominant. Not Applicable Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Does not apply. Not Applicable: HHT is autosomal dominant disorder. Not Applicable: HHT is autosomal dominant disorder. 1 - 1.5 points 1 - 1.5 points 1 - 1.5 points Not Applicable: This criterion does not apply since the model of inheritance is autosomal dominant. For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.1.0 points per the PM3 chart Not Applicable: Biallelic cases should not be counted using the ACTA1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Biallelic case counts should not be used for DNM2. Not Applicable: Biallelic case counts should not be used for MTM1. Not Applicable: Biallelic cases should not be counted using the RYR1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Not applicable, as OTC is an X-linked gene and biallelic females are sufficiently rare Use proposed SVI point recommendations. Both variants must be classified using _ABCA4_ Rule Specifications. Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 1 to 1.75 points from Table 1 For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.1.0 points per the PM3 chart For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.1.0 points per the PM3 chart Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3 (Moderate) should be applied for ≥ 1 pt but \< 2 pts. Not Applicable Not Applicable: Not applicable since disease-causing variants are heterozygous. Not Applicable: MYOC variants have an autosomal dominant mode of inheritance. PM3 = **2** pointsSee ATM PM3/BP2 table for approach to assign points per proband. * Details of the cDNA change must be used to describe any variants used as evidence for PM3. If the variant is described only as an amino acid change, this is not sufficient. Probands must also meet PP4 criteria to be counted.* If more than one case has the same genotype and the variants are not confirmed in trans, then only one case should be used for assigning points to avoid overcounting evidence if the variants are actually in cis and hence inherited together in multiple individuals or potentially counting the same case twice. If the variants are confirmed to be in trans, more than one individual with the same genotype can be counted as long as the reports do not represent the same case.* These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign.* For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases.* See PM3 table * PM3 score ≥ 1.0 \< 2.0 Not Applicable 1 point awarded from tables 7a and 7bExample: Detected in trans with a pathogenic variant (recessive). Not Applicable: Autosomal dominant. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. Not Applicable: SLC6A8 is an X-linked gene, therefore PM3 is not applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: SCN1A is associated with autosomal dominant inheritance. Not Applicable: SCN2A is associated with autosomal dominant inheritance. Not Applicable: SCN3A is associated with autosomal dominant inheritance. Not Applicable: SCN8A is associated with autosomal dominant inheritance. Not Applicable: Not applicable for the F8 gene. For recessive disorders, points-based system based on confirmation of phase and classification of other variant. Total of **1.0 point** will arrive at **Moderate**. * Classification of other variant is Pathogenic/Likely Pathogenic * Confirmed in trans: 1 point * Phase unknown: 0.5 (P) or 0.25 (LP) points* Homozygous occurrence (max point 1.0) * Confirmed in trans: 0.5 points* Classification of other variant is Uncertain Significance * Confirmed in trans: 0.25 points * Phase unknown: 0 points Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 1 to 1.75 points from Table 1 BS1_Strong Variant has a PopMax FAF ≥0.002 (0.2%) in gnomAD. Allele frequency is **≥0.0001 for** _**MYH7**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁶](#pmid_29300386); Tavtigian _et al._ 2020[¹⁵](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%) MAF of ≥0.003 (0.3%) for autosomal recessive; MAF of ≥0.0002 (0.02%) for autosomal dominant. Likely benign, provided there is no conflicting evidence. Allele frequency ≥0.002 (0.2%) MAF cutoff of 0.1%. **MM-VCEP notes:**Similarly, for the **BS1** calculation, we utilized the Whiffin/Ware calculator (http://cardiodb.org/allelefrequencyapp/) with a prevalence of 1 in 40, a conservative unascertained penetrance estimate of 85%, an allelic heterogeneity of 100% and a maximum genetic heterogeneity of 1% (one magnitude lower than for **BA1**). A 95% confidence interval was used to develop the threshold. We developed a range for application of **BS1** for variants with a minor allele frequency between 0.00015 (0.015%) and 0.0015 (0.15%). 1. The MM-VCEP also adopted the SVI recommendation that the variant be present in any general continental population dataset with a minimum number of 2,000 alleles and variant present in ≥ 5 alleles.2. The variant can be classified as likely benign based on **BS1** alone if there is no contradictory evidence supporting pathogenicity._**RUNX1**_ **Specification:****BS1:** Minor allele frequency between 0.00015 (0.015%) and 0.0015 (0.15%) in any general continental population dataset with ≥ 2,000 alleles tested and variant present in ≥ 5 alleles. Filtering allele frequency (FAF) of ≥ 0.0003 but \< 0.001 in gnomAD continental subpopulations of a single genetic ancestry group (excluding genetic ancestry groups influenced by founder effects, such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”). Genetic ancestry group must have ≥2,000 alleles tested and a minimum of 2 alleles present. Caution should be exerted if the majority of alleles have a variant allele fraction ( “allele balance” in gnomAD) below 0.35. To set the strong benign FAF cutoff, we used a Whiffin-Ware calculation using prevalence of 1 in 5,000 (Lalloo, et al., 2006 PMID: 16644204). Genetic and allelic heterogeneity were set at 100% and penetrance at 30%. In general, the most recent version of gnomAD should be used when available; however, other population databases or earlier versions of gnomAD may be utilized if they are able to provide information the curator deems necessary for optimal variant classification (e.g, they would provide superior information for a particular variant type; have a larger sample size; or better representation for certain subpopulations, etc.) Allele frequency greater than expected for disease. * Highest minor allele frequency >0.005 (>0.5%) in any continental population in gnomAD with >2000 alleles. Frequency cutoff of 0.158% (>0.00158 at 99.99% CI w/subpopulation w/min of 5 alleles) Popmax allele frequency >0.0008 (0.08%) Allele frequency is greater than expected for disorder. >0.0005 (>0.050%) >0.000092 (>0.0092%) >0.005 (>0.5% - AR) >0.0002 (>0.020%) Allele frequency 0.005 - 0.0099 (0.5% - 0.99%) Grpmax Filtering AF **\>.01%** in gnomAD v4 dataset Use BS1 cut off of >=0.0000156 (0.00156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4). gnomAD MAF greater than or equal to 0.0005 but less than 0.001. MAF cutoff of greater than or equal to 0.00000556 (or 0.000556%). Appropriate to use for variants with a Popmax MAF of >0.01 in gnomAD. gnomAD MAF greater than or equal to 0.0005 but less than 0.001. gnomAD MAF of greater than or equal to 0.0007 but less than 0.001. Appropriate to use for variants with a Popmax MAF of >0.0002 in gnomAD. gnomAD Grpmax FAF ≥ 1:30,000 (≥0.0033% or 0.000033) gnomAD Grpmax FAF ≥ 1:25,000 (0.004% or 0.00004) GnomAD filtering allele frequency ≥0.025%. Allele frequency is greater than expected for disorder. * gnomAD popmax filtering allele frequency >0.00089 * Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator ([https://www.cardiodb.org/allelefrequencyapp/](https://www.cardiodb.org/allelefrequencyapp/)) and the following parameters: * Prevalence: 1:50,000 * Allelic heterogeneity: 1 * Genetic heterogeneity: 0.04 (based on the contribution of _RMRP_ variants to total SCID in the PIDTC 6901 cohort reported in Dvorak et al., 2019 (PMID: 30193840, Table 1): 3.6%, rounded to 4%) * Penetrance: 100%Use caution when applying BS1 based on allele frequencies derived from gnomAD exome sequencing given the reduced coverage of certain regions of _RMRP_. Ensure at least 20X read depth for allele frequencies derived from exome sequencing. GnomAD Popmax Filtering Allele Frequency (AF) **≥ 0.001%** (0.00001). Appropriate to use for variants with a Popmax MAF of >0.01 in gnomAD. Any variant with Grpmax >0.0025 in the most recent version of gnomAD (95% confidence interval, lower bound) (version # will be stated in the written summary).BS1 minor allele frequency cut-off calculated using [http://cardiodb.org/allelefrequencyapp](http://cardiodb.org/allelefrequencyapp) with prevalence = 1 in 40,000 (PMID: 33208168), genetic heterogeneity = 1.0 (IDUA is the only gene known to cause MPS1), allelic heterogeneity = ~0.5 (frequency of the most common known pathogenic variants in patients with MPS1 (PMID: 28595941, 29393969), and penetrance = 1.0. Filter allele frequency (FAF) is above 0.01% (FAF > 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. GnomAD filtering allele frequency ≥0.025%. Allele frequency is **≥0.0002 for** _**MYBPC3**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁸](#pmid_29300386); Tavtigian _et al._ 2020[¹⁹](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Filter allele frequency (FAF) is above 0.01% (FAF > 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. Allele frequency is **≥0.0001 for** _**TNNI3**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁵](#pmid_29300386); Tavtigian _et al._ 2020[¹⁶](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is **≥0.0001 for** _**TNNT2**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁵](#pmid_29300386); Tavtigian _et al._ 2020[¹⁶](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is **≥0.0001 for** _**TPM1**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁵](#pmid_29300386); Tavtigian _et al._ 2020[¹⁶](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is **≥0.0001 for** _**ACTC1**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁴](#pmid_29300386); Tavtigian _et al._ 2020[¹⁵](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is **≥0.0001 for** _**MYL2**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁴](#pmid_29300386); Tavtigian _et al._ 2020[¹⁵](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is **≥0.0001 for** _**MYL3**_ based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).Criterion BS1 may only be used as standalone evidence to classify a variant as Likely Benign in the absence of conflicting data. See SVI guidance (Tavtigian _et al._ 2018[¹⁴](#pmid_29300386); Tavtigian _et al._ 2020[¹⁵](#pmid_32720330)). See BA1 for additional specifications that also apply to BS1. Allele frequency is ≥ 0.01 (1%) in population databases. Allele frequency in males is greater than expected for the disorder. Use for alleles with frequency ≥8.3x10^-5 (based on the most frequent pathogenic allele). Allele frequency is greater than expected for disorder * Maximum allele frequency in gnomAD (in one of the 5 continental populations; African/African-American, East Asian, European non-Finnish, Latino/Admixed-American, or South Asian) greater than or equal to 0.0004 (0.04%). gnomAD Grpmax filtering allele frequency >0.00141 OR a bottle-necked population with a MAF >0.00141 may be used for this criterion. Caveat: If the variant is known to be a founder variant in the bottle-necked population do not consider the frequency in that population for BS1. gnomAD popmax filtering allele frequency >0.00161¹¹ Consider also bottleneck populations. GnomAD v4 Grpmax filtering allele frequency ≥ 0.0001 and \< 0.001 (0.01-0.1%) and variant is excluded as founder pathogenic variant. gnomAD popmax filtering allele frequency >0.00078¹¹ Consider also bottleneck populations. gnomAD popmax filtering allele frequency >0.00126¹¹Consider also bottleneck populations. Allele frequency greater than expected for disorder. Use gnomAD PopMax FAF if available.* Allele frequency of between 8x10^-3 and 8x10^-4 gnomAD popmax filtering allele frequency >0.00100¹¹Consider also bottleneck populations. * Met for GrpMax Filtering Allele Frequency greater than or equal to 1.11 x 10^-6 (0.00000111) in gnomAD, with at least 3 alleles total across all populations in gnomAD (gnomAD v4.1.0).* If GrpMax filtering allele frequency is not listed for the variant, this code is evaluated in relation to the maximum allele frequency among the five major continental populations (African / African-American, East Asian, European non-Finnish, Latino / Admixed-American, or South Asian), but requires **at least 3 alleles total** across all populations in gnomAD (gnomAD v4.1.0) to be met.* Threshold is based on the experts’ estimate of CTLA-4 insufficiency prevalence of 1/200,000 – 1/1,000,000 people and 45-70% penetrance. An intermediate prevalence estimate (1 in 500,000) and the lower end of the penetrance estimate (45%) were used for this calculation. Allelic heterogeneity of 1 and genetic heterogeneity of 0.5 were also assumed for the calculation. gnomAD popmax filtering allele frequency >0.00195¹¹ Consider also bottleneck populations. gnomAD popmax filtering allele frequency >0.00195¹¹ Consider also bottleneck populations. Allele frequency ≥0.1% among gnomAD controls, using the subpopulation with the highest frequency and at least 1,000 allele counts. Allele frequency ≥ 2x10^-5 in males in population databases. \- The highest allele frequency in population databases should be used GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. gnomAD popmax filtering allele frequency >0.00249¹¹ Consider bottleneck populations \>0.2% to \<1% in general population databases (e.g. gnomAD) based on Popmax FAF, **or** if variant meets BS1\_Supporting and has ≥2 homozygotes. \>0.2% to \<1% in general population databases (e.g. gnomAD) based on Popmax FAF, **or** if variant meets BS1\_Supporting and has ≥2 homozygotes. GnomAD v4 Grpmax filtering allele frequency ≥ 0.0001 and \< 0.001 (0.01-0.1%) and variant is excluded as founder pathogenic variant. GnomAD v4 Grpmax filtering allele frequency ≥ 0.00022 and \< 0.0022 (0.022-0.22%) and variant is excluded as founder pathogenic variant. GnomAD v4 Grpmax filtering allele frequency ≥ 0.00028 and \< 0.0028 (0.028-0.28%) and variant is excluded as founder pathogenic variant. * Applicable to variants with a GrpMax filtering allele frequency greater than or equal to 0.000316 in gnomAD v4.1.0.* If GrpMax filtering allele frequency is not listed for the variant, this code is applicable to the maximum allele frequency among the five major continental populations (African / African-American, East Asian, European non-Finnish, Latino / Admixed-American, or South Asian).* The BS1 threshold was derived by decreasing the BA1 cutoff (greater than or equal to 0.00316) by one order of magnitude. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is ≥0.000237 in continental populations. All populations used should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.00000781 for AD variants**. All continental populations used in gnomAD should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.00000015**. All continental populations used in gnomAD should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.0000016**. All continental gnomAD populations used should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes of continental populations in gnomAD is **≥0.00000486 for AD variants**. All populations used should have at least 2000 alleles and >1 observation. **BA1/BS1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 or BS1 threshold) are as follows: NM\_000540.3:c.14524G>A (p.Val4842Met)NM\_000540.3:c.325C>T (p.Arg109Trp) An allele frequency >0.002000 (0.2%), Grpmax Filtering Allele Frequency in the most current version of gnomAD available at the time of curation. Note: Both BS1 and BS2 cannot be applied if the same dataset is used to assess population data Grpmax allele frequency cutoff of greater than 0.0163 in gnomAD. Allele frequency greater than expected for disorder. Use gnomAD Grpmax FAF if available.* Allele frequency of 0.0016 - 0.016 The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.00025 for AR** **variants**. All continental populations used in gnomAD should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes of continental populations in gnomAD is **≥0.000697 for AR variants**. All populations used should have at least 2000 alleles and >1 observation. **BA1/BS1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 or BS1 threshold) are as follows: NM\_000540.3:c.6721C>T (p.Arg2241Ter)NM\_000540.3:c.10348-6C>G Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.001. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.0009. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.0009. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.0009. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.001. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.001. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.0009. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. gnomAD Grpmax FAF ≥ 1:30,000 (≥0.0033% or 0.000033) Allele frequency (>0.0185%). An allele frequency (>0.0185%) was approved. (Supplemental Table 3). Allele frequency ≥ 0.001 in population databases. Grpmax Filtering AF **\>.05%** in gnomAD v4 dataset * Variants with a highest population minor allele frequency (MAF) ≥0.0035 (0.35%) in any continental population with >2000 alleles in gnomAD will meet BS1. * Calculated using the Prevalence of 1:30,000, Allelic Contribution of 0.5, Genetic Contribution of 1, and Penetrance of 0.75 to allow for mild VLCAD that may develop in adulthood. Allele frequency greater than expected for disease (>0.005%).* Use the ethnic population with the highest allele frequency.* Caveat: Do not use Finnish, Ashkenazi Jewish, or “Other” populations in gnomAD.* Minimum amount of studied alleles should be 2000. NA Frequency >0.0003 (0.03%) in gnomAD subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present. **GrpMax >0.0001 (0.01%) in gnomAD*** Any variant with a GrpMax (lower bound 95%ile) >0.0001 in gnomAD. Use the current version recommended by SVI; version number will be stated in classification summary.* Threshold based on max allelic contribution = 25% and max genetic contribution = 100% based on estimated prevalence of 1 in 3,450,000, (PMID 27233232), and penetrance of 100%) (PMID 30311383) (see Appendix 3). * Allele frequency >0.001 (0.1%) in gnomAD v4.0 in any continental population with >2000 alleles (based on the estimated prevalence 1 in 114,000 (PMID: 24071436) in gnomAD v4.0 (max allelic contribution = 40%; max genetic contribution = 100%).* Use the highest population minor allele frequency (MAF) in any given continental population with >2,000 alleles (European non-Finnish, African, East Asian, South Asian, Latino) (PMID 30311383). Allele frequency > 0.0002 (0.02%) OR ≥ 5 hemizygotes in gnomAD Allele frequency greater than expected for disease.* Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. Allele frequency greater than expected for disease (0.025%).* Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.0000083 (0.00083%) and \<0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. GnomAD filtering allele frequency ≥0.025%. Allele frequency is above 0.0004% in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above 0.0002% in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above 0.0002% in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above 0.0002% in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. MAF cutoff of greater than or equal to 0.00333% (or 0.0000333). Allele frequency is above **0.01%** in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency greater than expected for disorder. Use gnomAD Grpmax FAF if available.* Allele frequency of 0.00057 - 0.00569 PP4_Supporting Any LDLR variant identified in an FH patient [diagnosis based on validated clinical criteria, e.g. Dutch Lipid Clinic Network (≥6), Simon Broome possible/definite), MEDPED], after alternative causes of high cholesterol are excluded.Caveat: variant must also meet PM2. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: PTEN EP Commentary: Phenotype specificity has been incorporated into the rule specifications for PS4 Use 2. Patient's phenotype highly specific for gene or fully sequenced gene set (see specifications in Table 7). A plasma phenylalanine concentration persistently above 120umol/L (2mg/dL) without analysis of urine pterins, DHPR activity, or sequencing to exclude defects of BH4 cofactor metabolism. Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The FPD/AML phenotype is rather unspecific and can be caused by a number of other inherited predisposition syndromes, somatic mutations or environmental factors that are insufficient to meet the original ACMG/AMP rule PP4. Observation of the variant with VAF 5-35% (i.e., once or multiple times with VAF >25-35% and/or once with VAF 5-25%) Phenotype specific for disease with single genetic etiology. * Points-based system. See main specifications document Patients phenotype or family history is highly specific for a disease with a single genetic etiology Not Applicable: PP4 is not applicable, variants in CACNA1S also result in MHS. Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. Patient has/had MRI features of Leigh syndrome with clinical response to biotin/thiamine One of the following criteria are met: (1) Pyruvate radioactive enzyme assay showing decreased (as defined as <3rd percentile of controls) for PDC, activated and decreased ratios (PDC/E3 and/or PDC/CS) in fibroblasts, muscle, and/or lymphocytes; (2) other assays showing decrease in PDC activity (ie: western blot, immunocapture, and activity; commercial kits for research); (3) abnormally high pyruvate and/or pyruvate/lactate ratio 1. Mitochondrial DNA depletion showing 20-50% of controls in children (< 18 years of age), AND/OR 2. COX negative fibers in muscle in children and/or adultsNote: Will only apply if other mtDNA maintenance disorders have been excluded (exome sequencing or comprehensive panel-based testing) Individual has abnoramlly high urinary ethylmalonic acid AND one of the following: (1) 3 of the following features present: -Acrocyanosis -Petechiae -Chronic diarrhea -Developmental delay (2) abnormal laboratory studies in 2 of the following biochemical studies: -Abnormally high blood C4-Acylcarnitine esters -Abnormally high blood C5-acylcarnitine -Abnormally high plasma thiosulphate -Abnormally low cytochrome oxidase activity in skeletal muscle, without evidence of other complexes decreased Decreased ETC enzyme activity (<20%) performed in a CLIA-approved (or equivalently-certified) laboratory in muscle, liver, and/or fibroblasts (for fibroblasts, must be seen in multiple unrelated probands and/or assayed in different individuals). Not Applicable: Do not use for AD disorder as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.For AR disorder, use PM3 for specific phenotype considerations Not Applicable: Combine with PS4 to avoid double counting probands. * Proband with platelet aggregation study absent for ristocetin and present for all other agonists, OR * Flow cytometry or Western blot less than 10% expression of GPIba. NA The patient must have a clinical phenotype of excessive mucocutaneous bleeding and required laboratory values to use the PP4 rule code at the supporting strength. See Table 2B for required and consistent laboratory values. * Proband with platelet aggregation study absent for ristocetin and present for all other agonists, OR * Flow cytometry or Western blot less than 10% expression of GPIba. * Proband with platelet aggregation study absent for ristocetin and present for all other agonists, OR * Flow cytometry or Western blot less than 10% expression of GPIba. Proband must have an antithrombin activity level of \< 0.8 IU/mL (or below the lower limit of a laboratory’s assays reference range). confirmed on repeated independent samples. An abnormal crossed immunoelectrophoresis assay demonstrating decreased antithrombin function may be used in lieu of low activity levels, which is typically caused by type II variants. MODY Probability Calculator (MPC)⁶ result ≥50% chance of testing positive AND negative _HNF1A_ testing HbA1C 5.6 – 7.6% (38-60 mmol/mol) (if given multiple results, use maximum value) AND fasting plasma glucose always (FPG) 5.5-8 mmol/L (100-144 mg/dL) Not Applicable: Not applicable, see PS4. A patient score of 1-\<2 points (see instructions below). Not Applicable NA 2 of the following criteria are met:* Deficient IDUA activity, within the affected range (usually non-detectable or stated to be in the affected range by lab) in fibroblasts, leukocytes, or plasma. * Do not count if one or more pseudodeficiency variants are reported to be present, or if the result was obtained on newborn screen without confirmatory enzyme testing.* Enzyme replacement therapy results in a significant reduction in urine GAGs (either total, or dermatan or heparan sulfate).* Elevated urinary and/or blood GAGs expressed as either total GAGs, specific GAG (heparan sulfate, dermatan sulfate, or endogenous biomarker) stated to be consistent with MPS I.* An individual with clinical features specific to MPS I; At minimum at least 2 of the following: dysostosis multiplex, hepatosplenomegaly, arthropathy, corneal involvement, valvular thickening; AND/OR case reported within the context of a larger clinically-diagnosed MPS I cohort, when published by groups with demonstrated experience (as determined at the discretion of the VCEP) in lysosomal disorders and the clinical care of affected individuals. When detailed clinical information is limited, confidence in the phenotypic classification is strengthened when the report originates from groups with established familiarity in the diagnosis and longitudinal management of MPS I.* Bone marrow transplant results in a significant reduction in urine GAGs (either total, or dermatan or heparan sulfate). Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4 - LR ≥2.08:1 See Specifications Table7 and Appendix B for details. Not Applicable: PP4 is not applicable due to genetic heterogeneity. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4 - LR ≥2.08:1 See Specifications Table7 and Appendix B for details. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: The phenotype associated with CYP1B1 variants is not highly specific and there is genetic heterogeneity. 4-7 phenotype points required; one specific criterion must be met. Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.* QT prolongation above 480ms AND* Swimming-associated events OR* Treadmill stress test result (PMID: 21699858) OR* T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844) Patient score of 1-\<2 points (see instructions below) A patient score of 1-\<2 points (see instructions below). 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4. For _MLH1_ variants, _MLH1_ promoter methylation is to be excluded in the tumor. A patient score of 1-\<2 points (see instructions below). A patient score of 1-\<2 points (see instructions below). * 4-7.5 phenotype points are required to use this code (see points list below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. A patient score of 1-\<2 points (see instructions below). * In order to be counted for PP4 at the default (supporting) level of strength, a proband must; * score at least 10 phenotype points in the PS4 counting rubric * AND * have been genotyped using a method able to detect _LRBA_ variants without biallelic variants found.* This proband cannot be used for the PS4 code.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. A patient score of 1-\<2 points (see instructions below). A patient score of 1-\<2 points (see instructions below). Not Applicable: PAH does not have a single genetic etiology. A male proband diagnosed with retinoschisis by the age of 13 with visual acuity impairment and showing schisis. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. A patient score of 1-\<2.5 points (see instructions below). NA NA 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4. 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4. 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4. * In order to be counted for this criterion, a proband must score greater than or equal to 10 phenotype points in the PS4 counting rubric **(Table 3)** AND have genotyping to rule out a variant in the _PIK3R1_ locus.* Please note that a proband used for PP4 cannot be included in PS4.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. PP4 is met with the presence of any of these features**Presence on Muscle Biopsy of:*** Nemaline rods* Shorter thin filaments**Functional assays performed upon patient muscle biopsy indicate:*** Significantly altered Calcium sensitivity* Significantly altered muscle mechanics (altered force-sarcomere length or reduced contractile strength and force generation) If a biopsy demonstrates a presence of nemaline rods, this is suggestive of ACTA1-related congenital myopathy and can be given PP4 at a supporting level. Not Applicable: PP4 is factored into the strength of PS4. See case counting specifications above. **Affected Males (must be negative for BIN1, RYR1, and DNM2 variants)*** Muscle biopsy with rounded muscle fibers with a single centrally located nucleus surrounded by a halo devoid of contractile elements, but containing mitochondria**Carrier Females (a panel test for neuromuscular disease to rule out other causes)*** Observation of myopathy (may be asymmetric) AND at least 1 other feature * Unilateral skeletal asymmetry * Narrow, elongated face * High arched palate Not Applicable: PP4 is factored into the weight of PS4 case counting and should not be applied separately. Proband phenotype points total >0.5 to \<1.5 points This code is applicable when a single proband can be awarded 3-7.5 phenotype points. * 4-7.5 phenotype points are required to use this code (see points list below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. If a biopsy demonstrates a presence of nemaline rods, this is suggestive of ACTA1-related congenital myopathy and can be given PP4 at a supporting level. **To meet PP4, a congenital myopathy testing panel should be performed without identification of other causative variants and AT LEAST TWO of these features should be present*** **Presence on Muscle Biopsy of:** mini cores or central cores (histology or electron microscopy)* Exercise, heat, or anesthetic induced rhabdomyolysis* Ophthalmoplegia* Characteristic muscle imaging (see Figure 8, Saade et al 2019 PMID: 31060725) Use the PP4 table (see supplementary file “PP4 table DYSF”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Apply PP4 only once, for a patient meeting the highest possible strength level. Use the PP4 table (see supplementary file “PP4 table SGCB”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table SGCG”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table SGCD”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table CAPN3”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Apply PP4 (Supporting) for a proband meeting both of the following criteria:* progressive limb-girdle pattern of muscle weakness observed over ≥6 mo OR clinical suspicion of LGMD¹* 2 presumed diagnostic variants in _ANO5_, 1 of which is the variant under curation²¹ May be accompanied by supporting EMG, MRI, muscle histology, elevated CK but not required.² Screening of all exons and exon/intron boundaries of _ANO5_ required. Screening of additional neuromuscular disease genes (e.g., through a panel) is recommended but not required. If variants have not yet been curated by the LGMD VCEP, confirm they cannot be classified as LB or B (e.g., through application of BA1, BS1, and/or BP4/BP7). If phase is unknown, do not apply if the identified variants were ever confirmed _in cis_ or if gnomAD co-occurrence data ([https://gnomad.broadinstitute.org/variant-cooccurrence](https://gnomad.broadinstitute.org/variant-cooccurrence)) predict the variants may be part of the same haplotype in at least one genetic ancestry group. Use the PP4 table (see supplementary file “PP4 table SGCA”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). MODY Probability Calculator (MPC) result ≥50% chance of testing positive https://www.diabetesgenes.org/mody-probability-calculator/) AND negative HNF4A testing Not Applicable: Not applicable since this criterion is accounted for under PS4. Not Applicable: The phenotype associated with MYOC variants is not highly specific and there is genetic heterogeneity. Not Applicable: Autosomal Dominant: do not use as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.Autosomal Recessive: do not use as a separate line of evidence. Such evidence is built into the Ataxia Telangiectasia PM3|BP2 table * Abnormal tests that are consistent with VLCAD deficiency include deficient VLCAD enzyme activity in patient cells (leukocytes, fibroblasts, liver, heart, or skeletal muscle, or amniocytes), abnormal C14:1 acylcarnitine values from newborn screening (NBS), and abnormal acylcarnitine values from follow-up plasma analysis. * 1 point (See PP4 Table) * Use if patient fulfils revised Ghent criteria.* Can be used if any of the family members have a highly specific phenotype. Patient's phenotype highly specific for gene or fully sequenced gene set (see specifications in Table 7).* The HL-EP applied this rule to HL syndromes if all causative genes have been sequenced and the detection rate at least doubles when the added clinical feature is present.* See table below for applicable gene-disease phenotypes.* Advise against using PP4 for patients with nonsyndromic or apparently nonsyndromic hearing loss, given genetic heterogeneity. Somatic tumor testing identifies somatic hotspot second hit and no additional somatic LOF variants. Tumor testing[⁶](#pmid_30311369) of a neoplasm with known DICER1 association in a proband who carries the germline variant under evaluation reveals the following:* A previously reported somatic second hit of DICER1 in an RNase IIIb-disrupting “hotspot” codon (p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813) AND* Retention of the germline DICER1 variant under evaluation. PP4 is NOT applicable if:* The germline variant is a missense variant in one of the seven RNase IIIb “hotspot” codons (see PM1), OR* Somatic sequencing reveals additional DICER1 non-hotspot variants (could be consistent with sporadic tumorigenesis). 1-2 points based on: • Low urine guanidinoacetate with or without low or low normal creatine (1 point) • Low plasma guanidinoacetate with or without low or low normal creatine (2 points) Variant must meet PM2\_Supporting for PP4 to apply at any strength. 1-2 points based on: * Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).* Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).* Variant must meet PM2\_Supporting for PP4 to apply at any strength. 1-2 or more points based on: * Elevated urine creatine/creatinine ratio on one occasion (1 point)* Elevated urine creatine/creatinine ratio on more than one occasion (2 points)Additional specifications:* An individual used to assign PP4, at any weight, cannot be also included for PS4 count. If multiple unrelated probands with the variant have been identified, it is recommended that the case with the highest PP4 points is assigned the appropriate weight for PP4, and the other cases are used for PS4.* Variant must meet PM2\_Supporting for PP4 to apply at any strength. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Phenotype specific for disease with single genetic etiology.* See gene specific clinical phenotype guidelines. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 NA Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 * 4-7.5 phenotype points are required to use this code (see points list below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. PP2_Very Strong Not Applicable Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYH7, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The recommended cutoff for PP2 by the SVI is a missense constraint z score of ≥ 3.09 which was not met by RUNX1 (2.48 on ExAC and 2.08 on gnomAD). In addition, there are 9 benign/likely benign missense RUNX1 variants in ClinVar. Not Applicable: Not applicable Not Applicable: Does not apply; there are benign and pathogenic missense variants in GAA. Not Applicable: This rule does not apply because benign missense variants are not rare. This rule does not apply to GT due to the fact that these genes are thought to be highly polymorphic (PMID: 25827233). Not Applicable: PP2 is not applicable. RYR1 does not appear to be constrained for missense variation with a z-score of 1.92 in gnomAD. NA Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: mtDNA exhibits lack of recombination and a relatively high mutation rate (due to lack of histones or other protective structures) that allows for mtDNA variants to accumulate over time. Not Applicable: Do not use. Missense is not yet confirmed or refuted as a mechanism of disease for PALB2 Not Applicable: Do not use. While there are known pathogenic missense in VHL, there is also evidence of benign or common missense in VHL. gnomAD shows VHL is not intolerant to missense (Z score = -0.39). Missense variants in VHL will need to achieve pathogenic interpretation via other evidence codes. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable for F9. Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable due to presence of benign variation throughout the SERPINC1 gene. Not Applicable: While missense variants in HNF4A are a common mechanism of monogenic diabetes, and the constraint score for HNF4A (gene) is 1.81, the MDEP does not support using this criterion at this time. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Does not apply. Not Applicable Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: Does not apply; there are benign and pathogenic missense variants in IDUA. Not Applicable: High frequency of benign missense variants. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYBPC3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: High frequency of benign missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNI3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNT2, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For ACTC1, although there is evidence for missense constraint in gnomAD (v2.1.1, Z=4.52), the levels of rare missense variant observed in this gene in the HCM cohort described in Walsh et al. 2019 PMID:30696458 did not meet the threshold selected for application of this rule (lower 95% CI of OR ≥10). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL2, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL3, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Although pathogenic missense variants are common in CYP1B1, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = -0.75) supporting tolerance to variation. Not Applicable NA Not Applicable: Does not apply, FOXN1 does not have a low rate of benign missense variation, with a missense constraint score of Z=0.66. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for ADA (Z = 0.12) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in ADA. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for DCLRE1C (Z = -0.68) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in DCLRE1C. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL7R (Z = -1.29) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL7R. Not Applicable: Not applicable for RPE65. Not Applicable: The gnomAD v2.1.1 missense Z score for JAK3 (Z = 2.81) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in JAK3. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease Not applicable, as analysis of the evolutionary constraint of CTLA4 (low missense Z-score) shows that some missense variation is tolerated (benign). Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG1 (Z = 0.58) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG1. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG2 (Z = 0.2) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG2. NA Not Applicable: Loss of function variants are underrepresented. The Z score in GnomAD is 0.97. The pLoF eligible alleles for this code is 0/303. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL2RG (Z = 1.49) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL2RG. Not Applicable: Does not apply to ACVRL1 (Z-score 2.45). Not Applicable: Does not apply to ENG (Z-score= 0.93). Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: The gnomAD v2.1.1 missense Z score for PIK3CD (Z = 4.27) suggests this gene is constrained for missense variation. However, given the gain of function disease mechanism, it is not considered reasonable to expect that a missense variant at any residue will lead to a gain of function. Not Applicable: NEB is not a gene that is constrained for missense variation. Hence PP2 is not applicable. NA NA Not Applicable: MTM1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable, gnomAD (05/2021) expected missense 131.9, observed missense 89, for Z=1.33 (o/e =0.67). Not Applicable: This rule does not apply because this gene is not constrained for missense variation. Not Applicable: Not applicable for GUCY2D. Not Applicable: PP2 may only be used for missense variants with an autosomal dominant mode of inheritance. See autosomal dominant specifications if this is applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable. DYSF is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCB is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCG is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCD is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. CAPN3 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. ANO5 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCA is not constrained for missense variation (Z-score <3). Not Applicable: Missense variants account for 55% of all published pathogenic variants in this gene (Colclough et al 2013), however the constraint score for HNF1A (gene) is 1.07, which is not significant; therefore, we do not support using this criterion at this time. The low constraint score is most likely due to high tolerance for missense variants in the transactivation domain (see PM1 section). There are significantly more pathogenic missense variants in the DNA binding and dimerization domains, which are much less tolerant to missense variation. We may update this in the future if we can generate domain-specific scores. NA Not Applicable: Although pathogenic missense variants are common in MYOC, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = 0.52) supporting tolerance to variation. Not Applicable: Do not use: ATM does not have a defined low rate of missense benign variation. Not Applicable: This rule does not apply as there are benign and pathogenic missense variants in ACADVL. NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable: While DICER1 does meet recommended cutoff for missense constraint z score of ≥3.09 established by the SVI (4.23 on gnomAD) the VCEP recommends this rule not be used for DICER1 due to the presence of various missense variants throughout the gene that are clinically interpreted as benign (9) or likely benign (30) in ClinVar. Not Applicable: CCDS VCEP notes for PP2:Does not apply; there are benign and pathogenic missense variants in GATM. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.CCDS VCEP notes for PP2: Does not apply; there are benign and pathogenic missense variants in GAMT Not Applicable: Not applicable, gnomAD (01/2019) expected missense 243.5, observed missense 117, for Z=2.94 (o/e =0.48). No constraint against missense variation. Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for F8. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for AIPL1. PP2_Strong Not Applicable Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYH7, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The recommended cutoff for PP2 by the SVI is a missense constraint z score of ≥ 3.09 which was not met by RUNX1 (2.48 on ExAC and 2.08 on gnomAD). In addition, there are 9 benign/likely benign missense RUNX1 variants in ClinVar. Not Applicable: Not applicable Not Applicable: Does not apply; there are benign and pathogenic missense variants in GAA. Not Applicable: This rule does not apply because benign missense variants are not rare. This rule does not apply to GT due to the fact that these genes are thought to be highly polymorphic (PMID: 25827233). Not Applicable: PP2 is not applicable. RYR1 does not appear to be constrained for missense variation with a z-score of 1.92 in gnomAD. NA Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: mtDNA exhibits lack of recombination and a relatively high mutation rate (due to lack of histones or other protective structures) that allows for mtDNA variants to accumulate over time. Not Applicable: Do not use. Missense is not yet confirmed or refuted as a mechanism of disease for PALB2 Not Applicable: Do not use. While there are known pathogenic missense in VHL, there is also evidence of benign or common missense in VHL. gnomAD shows VHL is not intolerant to missense (Z score = -0.39). Missense variants in VHL will need to achieve pathogenic interpretation via other evidence codes. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable for F9. Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable due to presence of benign variation throughout the SERPINC1 gene. Not Applicable: While missense variants in HNF4A are a common mechanism of monogenic diabetes, and the constraint score for HNF4A (gene) is 1.81, the MDEP does not support using this criterion at this time. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Does not apply. Not Applicable Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: Does not apply; there are benign and pathogenic missense variants in IDUA. Not Applicable: High frequency of benign missense variants. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYBPC3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: High frequency of benign missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNI3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNT2, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For ACTC1, although there is evidence for missense constraint in gnomAD (v2.1.1, Z=4.52), the levels of rare missense variant observed in this gene in the HCM cohort described in Walsh et al. 2019 PMID:30696458 did not meet the threshold selected for application of this rule (lower 95% CI of OR ≥10). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL2, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL3, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Although pathogenic missense variants are common in CYP1B1, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = -0.75) supporting tolerance to variation. Not Applicable NA Not Applicable: Does not apply, FOXN1 does not have a low rate of benign missense variation, with a missense constraint score of Z=0.66. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for ADA (Z = 0.12) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in ADA. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for DCLRE1C (Z = -0.68) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in DCLRE1C. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL7R (Z = -1.29) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL7R. Not Applicable: Not applicable for RPE65. Not Applicable: The gnomAD v2.1.1 missense Z score for JAK3 (Z = 2.81) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in JAK3. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease Not applicable, as analysis of the evolutionary constraint of CTLA4 (low missense Z-score) shows that some missense variation is tolerated (benign). Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG1 (Z = 0.58) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG1. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG2 (Z = 0.2) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG2. NA Not Applicable: Loss of function variants are underrepresented. The Z score in GnomAD is 0.97. The pLoF eligible alleles for this code is 0/303. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL2RG (Z = 1.49) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL2RG. Not Applicable: Does not apply to ACVRL1 (Z-score 2.45). Not Applicable: Does not apply to ENG (Z-score= 0.93). Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: The gnomAD v2.1.1 missense Z score for PIK3CD (Z = 4.27) suggests this gene is constrained for missense variation. However, given the gain of function disease mechanism, it is not considered reasonable to expect that a missense variant at any residue will lead to a gain of function. Not Applicable: NEB is not a gene that is constrained for missense variation. Hence PP2 is not applicable. NA NA Not Applicable: MTM1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable, gnomAD (05/2021) expected missense 131.9, observed missense 89, for Z=1.33 (o/e =0.67). Not Applicable: This rule does not apply because this gene is not constrained for missense variation. Not Applicable: Not applicable for GUCY2D. Not Applicable: PP2 may only be used for missense variants with an autosomal dominant mode of inheritance. See autosomal dominant specifications if this is applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable. DYSF is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCB is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCG is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCD is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. CAPN3 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. ANO5 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCA is not constrained for missense variation (Z-score <3). Not Applicable: Missense variants account for 55% of all published pathogenic variants in this gene (Colclough et al 2013), however the constraint score for HNF1A (gene) is 1.07, which is not significant; therefore, we do not support using this criterion at this time. The low constraint score is most likely due to high tolerance for missense variants in the transactivation domain (see PM1 section). There are significantly more pathogenic missense variants in the DNA binding and dimerization domains, which are much less tolerant to missense variation. We may update this in the future if we can generate domain-specific scores. NA Not Applicable: Although pathogenic missense variants are common in MYOC, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = 0.52) supporting tolerance to variation. Not Applicable: Do not use: ATM does not have a defined low rate of missense benign variation. Not Applicable: This rule does not apply as there are benign and pathogenic missense variants in ACADVL. NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable: While DICER1 does meet recommended cutoff for missense constraint z score of ≥3.09 established by the SVI (4.23 on gnomAD) the VCEP recommends this rule not be used for DICER1 due to the presence of various missense variants throughout the gene that are clinically interpreted as benign (9) or likely benign (30) in ClinVar. Not Applicable: CCDS VCEP notes for PP2:Does not apply; there are benign and pathogenic missense variants in GATM. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.CCDS VCEP notes for PP2: Does not apply; there are benign and pathogenic missense variants in GAMT Not Applicable: Not applicable, gnomAD (01/2019) expected missense 243.5, observed missense 117, for Z=2.94 (o/e =0.48). No constraint against missense variation. Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for F8. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for AIPL1. PP2_Moderate Not Applicable Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYH7, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The recommended cutoff for PP2 by the SVI is a missense constraint z score of ≥ 3.09 which was not met by RUNX1 (2.48 on ExAC and 2.08 on gnomAD). In addition, there are 9 benign/likely benign missense RUNX1 variants in ClinVar. Not Applicable: Not applicable Not Applicable: Does not apply; there are benign and pathogenic missense variants in GAA. Not Applicable: This rule does not apply because benign missense variants are not rare. This rule does not apply to GT due to the fact that these genes are thought to be highly polymorphic (PMID: 25827233). Not Applicable: PP2 is not applicable. RYR1 does not appear to be constrained for missense variation with a z-score of 1.92 in gnomAD. NA Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: mtDNA exhibits lack of recombination and a relatively high mutation rate (due to lack of histones or other protective structures) that allows for mtDNA variants to accumulate over time. Not Applicable: Do not use. Missense is not yet confirmed or refuted as a mechanism of disease for PALB2 Not Applicable: Do not use. While there are known pathogenic missense in VHL, there is also evidence of benign or common missense in VHL. gnomAD shows VHL is not intolerant to missense (Z score = -0.39). Missense variants in VHL will need to achieve pathogenic interpretation via other evidence codes. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable for F9. Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable due to presence of benign variation throughout the SERPINC1 gene. Not Applicable: While missense variants in HNF4A are a common mechanism of monogenic diabetes, and the constraint score for HNF4A (gene) is 1.81, the MDEP does not support using this criterion at this time. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Does not apply. Not Applicable Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: Does not apply; there are benign and pathogenic missense variants in IDUA. Not Applicable: High frequency of benign missense variants. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYBPC3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: High frequency of benign missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNI3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNT2, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For ACTC1, although there is evidence for missense constraint in gnomAD (v2.1.1, Z=4.52), the levels of rare missense variant observed in this gene in the HCM cohort described in Walsh et al. 2019 PMID:30696458 did not meet the threshold selected for application of this rule (lower 95% CI of OR ≥10). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL2, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL3, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Although pathogenic missense variants are common in CYP1B1, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = -0.75) supporting tolerance to variation. Not Applicable NA Not Applicable: Does not apply, FOXN1 does not have a low rate of benign missense variation, with a missense constraint score of Z=0.66. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for ADA (Z = 0.12) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in ADA. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for DCLRE1C (Z = -0.68) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in DCLRE1C. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL7R (Z = -1.29) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL7R. Not Applicable: Not applicable for RPE65. Not Applicable: The gnomAD v2.1.1 missense Z score for JAK3 (Z = 2.81) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in JAK3. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease Not applicable, as analysis of the evolutionary constraint of CTLA4 (low missense Z-score) shows that some missense variation is tolerated (benign). Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG1 (Z = 0.58) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG1. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG2 (Z = 0.2) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG2. NA Not Applicable: Loss of function variants are underrepresented. The Z score in GnomAD is 0.97. The pLoF eligible alleles for this code is 0/303. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL2RG (Z = 1.49) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL2RG. Not Applicable: Does not apply to ACVRL1 (Z-score 2.45). Not Applicable: Does not apply to ENG (Z-score= 0.93). Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: The gnomAD v2.1.1 missense Z score for PIK3CD (Z = 4.27) suggests this gene is constrained for missense variation. However, given the gain of function disease mechanism, it is not considered reasonable to expect that a missense variant at any residue will lead to a gain of function. Not Applicable: NEB is not a gene that is constrained for missense variation. Hence PP2 is not applicable. NA NA Not Applicable: MTM1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable, gnomAD (05/2021) expected missense 131.9, observed missense 89, for Z=1.33 (o/e =0.67). Not Applicable: This rule does not apply because this gene is not constrained for missense variation. Not Applicable: Not applicable for GUCY2D. Not Applicable: PP2 may only be used for missense variants with an autosomal dominant mode of inheritance. See autosomal dominant specifications if this is applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable. DYSF is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCB is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCG is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCD is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. CAPN3 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. ANO5 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCA is not constrained for missense variation (Z-score <3). Not Applicable: Missense variants account for 55% of all published pathogenic variants in this gene (Colclough et al 2013), however the constraint score for HNF1A (gene) is 1.07, which is not significant; therefore, we do not support using this criterion at this time. The low constraint score is most likely due to high tolerance for missense variants in the transactivation domain (see PM1 section). There are significantly more pathogenic missense variants in the DNA binding and dimerization domains, which are much less tolerant to missense variation. We may update this in the future if we can generate domain-specific scores. NA Not Applicable: Although pathogenic missense variants are common in MYOC, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = 0.52) supporting tolerance to variation. Not Applicable: Do not use: ATM does not have a defined low rate of missense benign variation. Not Applicable: This rule does not apply as there are benign and pathogenic missense variants in ACADVL. NA Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable: While DICER1 does meet recommended cutoff for missense constraint z score of ≥3.09 established by the SVI (4.23 on gnomAD) the VCEP recommends this rule not be used for DICER1 due to the presence of various missense variants throughout the gene that are clinically interpreted as benign (9) or likely benign (30) in ClinVar. Not Applicable: CCDS VCEP notes for PP2:Does not apply; there are benign and pathogenic missense variants in GATM. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.CCDS VCEP notes for PP2: Does not apply; there are benign and pathogenic missense variants in GAMT Not Applicable: Not applicable, gnomAD (01/2019) expected missense 243.5, observed missense 117, for Z=2.94 (o/e =0.48). No constraint against missense variation. Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. NA Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for F8. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for AIPL1. PP2_Supporting Not Applicable Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYH7, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The recommended cutoff for PP2 by the SVI is a missense constraint z score of ≥ 3.09 which was not met by RUNX1 (2.48 on ExAC and 2.08 on gnomAD). In addition, there are 9 benign/likely benign missense RUNX1 variants in ClinVar. Not Applicable: Not applicable Not Applicable: Does not apply; there are benign and pathogenic missense variants in GAA. Not Applicable: This rule does not apply because benign missense variants are not rare. This rule does not apply to GT due to the fact that these genes are thought to be highly polymorphic (PMID: 25827233). Not Applicable: PP2 is not applicable. RYR1 does not appear to be constrained for missense variation with a z-score of 1.92 in gnomAD. Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: mtDNA exhibits lack of recombination and a relatively high mutation rate (due to lack of histones or other protective structures) that allows for mtDNA variants to accumulate over time. Not Applicable: Do not use. Missense is not yet confirmed or refuted as a mechanism of disease for PALB2 Not Applicable: Do not use. While there are known pathogenic missense in VHL, there is also evidence of benign or common missense in VHL. gnomAD shows VHL is not intolerant to missense (Z score = -0.39). Missense variants in VHL will need to achieve pathogenic interpretation via other evidence codes. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable for F9. Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: This rule does not apply because BSS is a rare disease and this gene is not constrained for missense variation (gnomAD). Not Applicable: Not applicable due to presence of benign variation throughout the SERPINC1 gene. Not Applicable: While missense variants in HNF4A are a common mechanism of monogenic diabetes, and the constraint score for HNF4A (gene) is 1.81, the MDEP does not support using this criterion at this time. Apply to all missense variants in GCK. gnomAD missense constraint score for _GCK_ is 3.07 (observed/expected= 0.5), which is significant. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Does not apply. Not Applicable Not Applicable: Not applicable due to presence of benign variation throughout the VWF gene. Not Applicable: Does not apply; there are benign and pathogenic missense variants in IDUA. Not Applicable: High frequency of benign missense variants. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYBPC3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: High frequency of benign missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNI3, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For TNNT2, there is evidence for regional enrichment of rare missense variants (see PM1 specifications). Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh _et al._ 2019[¹²](#pmid_30696458)) rather than the missense constraint score in gnomAD. On the basis of data from Walsh _et al._ 2019[¹²](#pmid_30696458), **PP2 is currently** **only applicable to** _**TPM1**_ **for HCM** (transcripts ENST00000403994 and NM\_001018005.2)_._Data from HCM case cohorts was used to derive these cluster regions. Therefore, this rule should NOT be applied when additional evidence for the variant supports that the variant causes a phenotype other than HCM (e.g., variant seen in multiple DCM cases).Enrichment was not observed for DCM in any genes. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For ACTC1, although there is evidence for missense constraint in gnomAD (v2.1.1, Z=4.52), the levels of rare missense variant observed in this gene in the HCM cohort described in Walsh et al. 2019 PMID:30696458 did not meet the threshold selected for application of this rule (lower 95% CI of OR ≥10). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL2, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458) rather than the missense constraint score in gnomAD. For MYL3, there is insufficient evidence of gene-level enrichment of rare missense variants. Not Applicable: Although pathogenic missense variants are common in CYP1B1, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = -0.75) supporting tolerance to variation. Not Applicable Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease * Not applicable due to presence of benign variation throughout the _KCNQ1_ gene (since the missense constraint Z-score in gnomAD is 1.83, lower than 3). Not Applicable: Does not apply, FOXN1 does not have a low rate of benign missense variation, with a missense constraint score of Z=0.66. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for ADA (Z = 0.12) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in ADA. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for DCLRE1C (Z = -0.68) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in DCLRE1C. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL7R (Z = -1.29) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL7R. Not Applicable: Not applicable for RPE65. Not Applicable: The gnomAD v2.1.1 missense Z score for JAK3 (Z = 2.81) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in JAK3. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease Not applicable, as analysis of the evolutionary constraint of CTLA4 (low missense Z-score) shows that some missense variation is tolerated (benign). Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG1 (Z = 0.58) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG1. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for RAG2 (Z = 0.2) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in RAG2. PM2\_supporting and PP3 must be met. Not Applicable: Loss of function variants are underrepresented. The Z score in GnomAD is 0.97. The pLoF eligible alleles for this code is 0/303. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Missense z score is >3.09 in gnomAD. Not Applicable: Does not apply. The gnomAD v2.1.1 missense Z score for IL2RG (Z = 1.49) suggests this gene is not constrained for missense variation. Both benign and pathogenic missense variants are present in IL2RG. Not Applicable: Does not apply to ACVRL1 (Z-score 2.45). Not Applicable: Does not apply to ENG (Z-score= 0.93). Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: Missense variant in a gene with low rate of benign missense changes does not apply. Not Applicable: The gnomAD v2.1.1 missense Z score for PIK3CD (Z = 4.27) suggests this gene is constrained for missense variation. However, given the gain of function disease mechanism, it is not considered reasonable to expect that a missense variant at any residue will lead to a gain of function. Not Applicable: NEB is not a gene that is constrained for missense variation. Hence PP2 is not applicable. ACTA1 is a gene that is constrained for missense variation (gnomAD v4.1 z=6.09). PP2 may be used for missense variants with an autosomal dominant mode of inheritance. DNM2 is a gene that is constrained for missense variation (gnomAD v4.1 z=4.87). PP2 may be used for missense variants. Not Applicable: MTM1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable, gnomAD (05/2021) expected missense 131.9, observed missense 89, for Z=1.33 (o/e =0.67). Not Applicable: This rule does not apply because this gene is not constrained for missense variation. Not Applicable: Not applicable for GUCY2D. Not Applicable: PP2 may only be used for missense variants with an autosomal dominant mode of inheritance. See autosomal dominant specifications if this is applicable. Not Applicable: RYR1 is not a gene that is constrained for missense variation. Hence PP2 is not applicable. Not Applicable: Not applicable. DYSF is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCB is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCG is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCD is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. CAPN3 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. ANO5 is not constrained for missense variation (Z-score <3). Not Applicable: Not applicable. SGCA is not constrained for missense variation (Z-score <3). Not Applicable: Missense variants account for 55% of all published pathogenic variants in this gene (Colclough et al 2013), however the constraint score for HNF1A (gene) is 1.07, which is not significant; therefore, we do not support using this criterion at this time. The low constraint score is most likely due to high tolerance for missense variants in the transactivation domain (see PM1 section). There are significantly more pathogenic missense variants in the DNA binding and dimerization domains, which are much less tolerant to missense variation. We may update this in the future if we can generate domain-specific scores. Missense constraint computed in ExAC/gnomAD was utilized. Award PP2 if the z-score > 3.09. (applicable to MTOR, PIK3CA and AKT3 but not PIK3R2). Not Applicable: Although pathogenic missense variants are common in MYOC, the gene also has a significant amount of benign missense variants as shown by the missense constraint z score in gnomAD (z = 0.52) supporting tolerance to variation. Not Applicable: Do not use: ATM does not have a defined low rate of missense benign variation. Not Applicable: This rule does not apply as there are benign and pathogenic missense variants in ACADVL. Add caveat: if this argument is used pro-pathogenicity, there must be other arguments supporting pathogenicity, and no arguments supporting a benign assertion. Not Applicable: Advise against using this rule because there are few such genes that this would apply to, particularly genes associated to autosomal recessive hearing loss. Not Applicable: While DICER1 does meet recommended cutoff for missense constraint z score of ≥3.09 established by the SVI (4.23 on gnomAD) the VCEP recommends this rule not be used for DICER1 due to the presence of various missense variants throughout the gene that are clinically interpreted as benign (9) or likely benign (30) in ClinVar. Not Applicable: CCDS VCEP notes for PP2:Does not apply; there are benign and pathogenic missense variants in GATM. Not Applicable: Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.CCDS VCEP notes for PP2: Does not apply; there are benign and pathogenic missense variants in GAMT Not Applicable: Not applicable, gnomAD (01/2019) expected missense 243.5, observed missense 117, for Z=2.94 (o/e =0.48). No constraint against missense variation. Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Missense z score is >3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Missense z score is >3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Not Applicable: Not applicable because missense z score is <3.09 in gnomAD. Missense z score is >3.09 in gnomAD. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for F8. Not Applicable: Benign missense variants are common. Not Applicable: Not applicable for AIPL1. BA1_Stand Alone Variant has a PopMax FAF ≥0.005 (0.5%) in gnomAD. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. gnomAD Filtering allele frequency >0.00056 (0.056%) MAF of ≥0.005 (0.5%) for autosomal recessive; MAF of ≥0.001 (0.1%) for autosomal dominant. An allele frequency ≥0.015 (1.5%), which is calculated with genetic heterogeneity of 90% to account for defects of BH4 metabolism, and penetrance of 80% to account for individuals who come to attention after becoming clinically symptomatic. MAF cutoff of 0.2%. _**RUNX1**_ **Specification:****BA1:** Minor allele frequency ≥ 0.0015 (0.15%) in any general continental population dataset with ≥ 2,000 alleles tested and variant present in ≥ 5 alleles. Filtering allele frequency (FAF) of ≥ 0.001 or 0.1% in gnomAD continental subpopulations of a single genetic ancestry group (excluding genetic ancestry groups influenced by founder effects, such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”). Genetic ancestry group must have ≥2,000 alleles tested and a minimum of 2 alleles present. Caution should be exerted if the majority of alleles have a variant allele fraction ("allele balance" in gnomAD) below 0.35. To set the stand-alone benign FAF cutoff, we used the FAF cutoff established for BS1 (0.0003) and increased this cutoff by one order of magnitude to come to a value of 0.001. In general, the most recent version of gnomAD should be used when available; however, other population databases or earlier versions of gnomAD may be utilized if they are able to provide information the curator deems necessary for optimal variant classification (e.g, they would provide superior information for a particular variant type; have a larger sample size; or better representation for certain subpopulations, etc.) Common in population databases. * Highest minor allele frequency >0.01 (>1%) in any continental population in gnomAD with >2000 alleles. Frequency cutoff of 0.24% (>0.0024 at 99.99% CI w/subpopulation w/min of 5 alleles). Popmax allele frequency >0.0038 (0.38%) Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. >0.001 (>0.1%) >0.00092 (>0.092%) >0.01 (>1%) >0.001 (>0.1%) Top-level haplogroup defining variants in individuals that are members of that same top-level haplogroup OR Allele frequency > 0.01 (1%) Grpmax Filtering AF **\>.1%** in gnomAD v4 dataset Use a BA1 cut off of >=0.000156 (0.0156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4 release). gnomAD MAF of greater than or equal to 0.001 (or 0.1%). MAF cutoff of greater than or equal to 0.0000556 (or 0.00556%). Appropriate to use for variants with a Popmax MAF of >0.1 in gnomAD. gnomAD MAF greater than or equal to 0.001 (or 0.1%) in gnomAD. gnomAD MAF of greater than or equal to 0.001 (or 0.1%). Appropriate to use for variants with a popmax MAF of greater than or equal to 0.002 in gnomAD. gnomAD Grpmax FAF ≥ 1:10,000 (≥ 0.01% or 0.0001). gnomAD Grpmax FAF ≥ 1:10,000 (≥ 0.01% or 0.0001) GnomAD filtering allele frequency ≥0.05%. Common in population databases.* gnomAD popmax filtering allele frequency >0.00400 * Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator ([https://www.cardiodb.org/allelefrequencyapp/](https://www.cardiodb.org/allelefrequencyapp/)) and the following parameters: * Prevalence: 1:5,000 * Allelic heterogeneity: 1 * Genetic heterogeneity: 0.04 (based on the contribution of _RMRP_ variants to total SCID in the PIDTC 6901 cohort reported in Dvorak et al., 2019 (PMID: 30193840, Table 1): 3.6%, rounded to 4%) * Penetrance: 50% * Use caution when applying BA1 based on allele frequencies derived from gnomAD exome sequencing given the reduced coverage of certain regions of _RMRP_. Ensure at least 20X read depth for allele frequencies derived from exome sequencing. GnomAD Popmax Filtering Allele Frequency (AF) **≥ 0.1%** (0.001). Appropriate to use for variants with a Popmax MAF of >0.1 in gnomAD. Any variant with Grpmax >0.005 in the most recent version of gnomAD (95% confidence interval, lower bound) (version # will be stated in the written summary)BA1 minor allele frequency cut-off calculated using [http://cardiodb.org/allelefrequencyapp](http://cardiodb.org/allelefrequencyapp) with prevalence = 1 in 40,000 (PMID: 33208168), genetic heterogeneity = 1.0 (IDUA is the only gene known to cause MPS1), allelic heterogeneity = 1.0, and penetrance = 1.0. Filter allele frequency (FAF) is above 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. GnomAD filtering allele frequency ≥0.05%. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Filter allele frequency (FAF) is above 0.1% (FAF > 0.001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. * Caution should be applied when assessing variants in the _MYL2_ and _MYL3_ genes, as homozygous or compound heterozygous variants have been reported to cause a recessive HCM and heterozygous individuals show no sign of disease.gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency is **≥0.001** based on the **filtering allele frequency (FAF)** in **gnomAD** in the subpopulation with the highest frequency (popmax).The values used to calculate the BA1 threshold were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/300 or lower).The threshold is applicable when assessing variants in the context of autosomal dominant cardiomyopathy. * Caution should be applied when assessing variants in the _MYL2_ and _MYL3_ genes, as homozygous or compound heterozygous variants have been reported to cause a recessive HCM and heterozygous individuals show no sign of disease.gnomAD is the preferred database for this calculation. If a subpopulation specific FAF other than the popmax is needed, this value can be calculated using the AlleleFrequencyApp on the [CardioDB website](https://cardiodb.org/allelefrequencyapp/).1. Using the Inverse AF tab, enter in the population size and the number of alleles identified and it will calculate the FAF. 2. Set confidence to 0.95 (95%).3. If the FAF is ≥0.001, this rule can be applied.The FAF by platform (e.g., exome vs. genome; v.2.1.1 vs. v.3.1.1) should be considered, the larger population is most likely to have the most accurate representation of “true” population allele frequency.Caution is needed when considering any population cohorts that are smaller than the smallest subpopulations within gnomAD v.2.1.1 (e.g., ~5000 individuals or ~10,000 alleles). Despite this conservative nature of this threshold and approach, in smaller cohorts, the observed allele frequency may less accurately reflect the true allele frequency. Traditionally, once a variant is classified as Benign, it is rarely re-evaluated and so the highest confidence is needed to establish that classification on an allele frequency alone. Allele frequency ≥ 0.05 in population databases. Allele frequency in males is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium in the subpopulation with the highest frequency. Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.* Maximum allele frequency in gnomAD (in one of the 5 continental populations; African/African-American, East Asian, European non-Finnish, Latino/Admixed-American, or South Asian) greater than or equal to 0.004 (0.4%). gnomAD Grpmax filtering allele frequency >0.00447 gnomAD popmax filtering allele frequency >0.00721. GnomAD v4 Grpmax filtering allele frequency ≥ 0.001 (0.1%) and variant is excluded as founder pathogenic variant. gnomAD popmax filtering allele frequency >0.00346. gnomAD popmax filtering allele frequency >0.00566. Use gnomAD PopMax FAF if available, cutoff of ≥8 x 10^-3* Use large population databases (i.e. gnomAD). gnomAD popmax filtering allele frequency >0.00447 * Met for GrpMax Filtering Allele Frequency greater than or equal to 1.11 x 10^-5 (0.0000111) in gnomAD, with at least 5 alleles total across all populations in gnomAD (gnomAD v4.1.0).* Threshold is based on the experts’ estimate of CTLA-4 insufficiency prevalence of 1/200,000 – 1/1,000,000 people and 45-70% penetrance. A higher prevalence estimate (1 in 100,000) and the lower end of the penetrance estimate (45%) were used for this calculation. Allelic heterogeneity of 1 and genetic heterogeneity of 1 were also assumed for the calculation. gnomAD popmax filtering allele frequency >0.00872. gnomAD popmax filtering allele frequency >0.00872 Allele frequency is above 1% in gnomAD, including any sub-population with at least 1,000 allele counts. Allele frequency ≥ 2x10^-4 in males in population databases (Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium) in the subpopulation with the highest frequency. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. gnomAD popmax filtering allele frequency >0.01110. Allele frequency is ≥1% in general population databases (e.g. gnomAD) based on Popmax FAF. Allele frequency is ≥1% in general population databases (e.g. gnomAD) based on Popmax FAF. GnomAD v4 Grpmax filtering allele frequency ≥ 0.001 (0.1%) and variant is excluded as founder pathogenic variant. GnomAD v4 Grpmax filtering allele frequency ≥ 0.0022 (0.22%) and variant is excluded as founder pathogenic variant. GnomAD v4 Grpmax filtering allele frequency ≥ 0.0028 (0.28%) and variant is excluded as founder pathogenic variant. * Applicable to variants with a GrpMax filtering allele frequency greater than or equal to 0.00316 in gnomAD v4.1.0.* If GrpMax filtering allele frequency is not listed for the variant, this code is applicable to the maximum allele frequency among the five major continental populations (African / African-American, East Asian, European non-Finnish, Latino / Admixed-American, or South Asian).* Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator ([https://www.cardiodb.org/allelefrequencyapp/](https://www.cardiodb.org/allelefrequencyapp/)) and the following estimated parameters (with the prevalence estimated for PIK3CD-related immune disease and the inheritance tailored to the autosomal recessive rather than autosomal dominant mode of inheritance, associated with complete penetrance): * Inheritance: biallelic * Prevalence: 1 in 100,000 (calculated at 1 in 7,500,000 based on the USIDNET cohort, PMID: 34352450, but adjusted based on expectation that cases are actually more common, resulting in a much more frequent / more aggressive prevalence estimate of 1 in 100,000 in order to generate a more conservative estimate) * Allelic heterogeneity: 1 * Genetic heterogeneity: 1 * Penetrance: 1 The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is ≥0.00559. All populations used should have at least 2000 alleles and >1 observation. The Ashkenazi Jewish, European Finnish, and Other populations in gnomAD will not be used for BA1 application.**BA1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 threshold) are as follows: Exon 55 deletion common in the AJ population (NM\_001271208.2:c.7431+1919\_7536+374del)NM\_001271208.2:c.19097G>T (p.Ser6366Ile)NM\_001271208.2:c.22249A>C (p.Thr7417Pro) The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.0000781 for AD variants**. All continental populations in gnomAD used should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.0000015**. All continental populations used in gnomAD should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.000016**. All continental gnomAD populations used should have at least 2000 alleles and >1 observation. The minor allele frequency using the filtering allele frequency of either exomes or genomes of continental populations in gnomAD is **≥0.0000486** **for AD variants**. All populations used should have at least 2000 alleles and >1 observation. **BA1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 or BS1 threshold) are as follows: NM\_000540.3:c.14524G>A (p.Val4842Met)NM\_000540.3:c.325C>T (p.Arg109Trp) Allele frequency above 0.010000 (1.0%), Grpmax Filtering Allele Frequency OR ≥10 (female) homozygotes or (male) hemizygotes in the most current version of gnomAD available at the time of curation. Gprmax allele frequency cutoff of greater than 0.163 in gnomAD. Use gnomAD Grpmax FAF if available, cutoff of >0.016* Use large population databases (i.e. gnomAD). The minor allele frequency using the filtering allele frequency of either exomes or genomes in gnomAD is **≥0.0025 for AR variants**. All continental populations in gnomAD used should have at least 2000 alleles and >1 observation. **BA1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 threshold) are as follows: NM\_001100.4(ACTA1):c.541del (p.Asp181fs)NM\_001100.4(ACTA1):c.121C>T(p.Arg41\*) The minor allele frequency using the filtering allele frequency of either exomes or genomes of continental populations in gnomAD is **≥0.00697** **for AR variants**. All populations used should have at least 2000 alleles and >1 observation. **BA1 exclusion variants** (well-known pathogenic variants that are above the specified BA1 or BS1 threshold) are as follows: NM\_000540.3:c.6721C>T (p.Arg2241Ter)NM\_000540.3:c.10348-6C>G Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.003. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.002. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.002. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.002. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.003. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.003. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. Apply if the variant Grpmax FAF (the lower bound of the 95% confidence interval of the maximum credible genetic ancestry group allele frequency) is >0.002. This value can be taken directly from gnomAD, but do not use data for which the variant does not pass quality control filters. See supplementary file “benign frequency exceptions” for a list of variants defined as exceptions to the benign frequency rules. Ongoing updates to this list will be available at the LGMD VCEP webpage: [https://clinicalgenome.org/affiliation/50061/.](https://clinicalgenome.org/affiliation/50061/.) Variants whose frequency may not be reliable (e.g., variants that may reflect a sequencing artifact) should be critically evaluated and brought to the attention of the LGMD VCEP. gnomAD Grpmax FAF ≥ 1:10,000 (≥ 0.01% or 0.0001) Allele frequency (>0.0926%). An allele frequency (>0.0926%) was approved. Note: this was adjusted from ACMG Guidelines due to maintaining the 5x threshold for benign (consistent with previously established guidelines) Allele frequency ≥ 0.01 in population databases. Grpmax Filtering AF **\>.5%** in gnomAD v4 dataset * Variants with a highest population minor allele frequency (MAF) ≥0.007 (0.7%) in any continental population with >2000 alleles in gnomAD will meet BA1. * Calculated using the Prevalence of 1:30,000, Allelic Contribution of 1, Genetic Contribution of 1, and Penetrance of 0.75 to allow for mild VLCADD that may develop in adulthood. Allele frequency above 0.1% in ExAc and gnomAD* Use the ethnic population with the highest allele frequency.* Caveat: Do not use Finnish, Ashkenazi Jewish, or “Other” populations in gnomAD.* Minimum amount of studied alleles should be 2000. MAF of ≥0.005 (0.5%) for autosomal recessive. Frequency >0.003 (0.3%) in gnomAD subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present. **GrpMax >0.0005 (0.05%) in gnomAD*** Any variant with a GrpMax (lower bound 95%ile) >0.0005 in gnomAD. Use the current version recommended by SVI; version number will be stated in classification summary. * Threshold based on (max allelic contribution = 100% and max genetic contribution = 100% based on estimated prevalence of 1 in 3,450,000 (PMID 27233232), and penetrance of 100%) (PMID 30311383) (see Appendix 3) * Allele frequency >0.003 (0.3%) in gnomAD v4.0 in any continental population with >2000 alleles (based on the estimated prevalence 1 in 114,000, PMID 24071436) in gnomAD v4.0 (max allelic contribution = 100%; max genetic contribution = 100%).* Use the highest population minor allele frequency (MAF) in any given continental population with >2,000 alleles (European non-Finnish, African, East Asian, South Asian, Latino) (PMID 30311383). Allele frequency >0.0020 (0.2%) OR ≥10 hemizygotes in gnomAD Allele frequency above 0.05%.* Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. Allele frequency above 0.05%.* Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. * Use large population databases (i.e. gnomAD).* Use if variant is present at ≥0.000083 (0.0083%) in any sub-population.* Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. GnomAD filtering allele frequency ≥0.05%. Allele frequency is above 0.02% in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above **0.01%** is gnomAD or other large population databases, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above **0.01%** is gnomAD or other large population databases, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Allele frequency is above **0.01%** is gnomAD or other large population databases, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. MAF cutoff of greater or equal to 0.0333% (or 0.000333). Allele frequency is above **0.3%** in GnomAD or other large population database, must be greater than or equal to 5 alleles if a minimum of 10,000 alleles was assessed. Use gnomAD Grpmax FAF if available, cutoff of ≥0.0057* Use large population databases (i.e. gnomAD). PS3_Strong Variant meets Level 1 pathogenic functional study criteria. See Table 3.(1) Study of the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding, and LDL internalization) performed in heterologous cells (with noendogenous LDLR) transfected with mutant plasmid. Assay result of <70% ofwild-type activity in either expression/biosynthesis, binding OR internalization. **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product.* RNA, mini-gene, or other assay shows impact on splicing Knock-in mouse model demonstrates the phenotype. NA RNA assay demonstrating abnormal out-of-frame transcripts. **PS3:** Transactivation assays demonstrating altered transactivation (\<20% of wt, and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) AND data from a secondary assay demonstrating altered function. Not applicable if variant meets **PVS1**. If variant meets **PVS1\_strong**, upgrade to **PVS1**. Non-functional on Kato et al. data **AND** loss of function (LOF) by the majority of other eligible assays Well-established in vitro or in vivo functional studies supportive of a damaging effect. * RT-PCR evidence of mis-splicing for non-canonical intronic variants with no evidence of normal splice products. * In a transgenic animal model, must demonstrate minimal to no function. OR* In a model organism or heterologous cell line, EITHER (A) when expression is normal or reduced, disruption of protein function must be demonstrated OR (B) Absent surface protein expression (<5%). Well-established functional studies supportive of a damaging effect on protein function * Knock-in mouse showing MH reaction in response to RYR1 agonist AND increased sensitivity to RYR1 agonists in ex vivo tisue/cells Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. NA Not Applicable Not Applicable NA NA Not Applicable: ● Protein: Do not use: Lack of known positive controls ● RNA: Do not use: See code PVS1_Variable(RNA) NA In a transgenic animal model, must demonstrate minimal to no function. NA Either (1) In a transgenic animal model, must demonstrate minimal to no function.OR(2) The following types of assays using recombinant vWF are approved for each subtype:Subtype 2A = A multimerization assay in which the variant is expressed in a recombinant system (either independently or coexpressed with WT) resulting in abnormal multimers, with a reported loss of HMWM, AND, to confirm this is consistent with the variant's mechanism of disease, there must be a patient harboring the variant with a clinical assay also showing loss of HMWMs. This evidence must be published in a peer reviewed journal and a picture of the gel must be visible for evaluation.Subtype 2B = A GP1b or platelet binding assay indicating gain of function by increased binding at low doses of ristocetinSubtype 2M = Either (1) A GP1b or platelet binding assay OR (2) Collagen binding assay, indicating loss of function by decreased binding See attached spreadsheet for examples of approved assay instances to use for this rule code. There are no universal thresholds for these assays; however, the relevant results should be described as clinically significant if assays were performed in a clinical laboratory or statistically significant if pertaining to research findings. In a transgenic animal model, must demonstrate minimal to no function. In a transgenic animal model, must demonstrate minimal to no function. NA Applicable to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing. Applicable to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing. NA PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the Cartilage-hair hypoplasia (CHH) phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level. **RNA assays** show1. a premature stop codon **OR**2. inframe skipping of exon 13 or 14**AND** \< 10% of full-length transcript. Either (1) in a transgenic animal model, must demonstrate minimal to no function. (2) a Factor VIII binding assay using recombinant vWF resulting in decreased binding compared to WT.See attached spreadsheet for examples of approved assay instances to use for this rule code. There are no universal thresholds for these assays; however, the relevant results should be described as clinically significant if assays were performed in a clinical laboratory or statistically significant if pertaining to research findings. NA Well-established in vitro or in vivo functional studies supportive of a damaging effect. Apply PS3 for assays measuring effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. NA **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[¹](#pmid_30192042)). Well-established in vitro or in vivo functional studies supportive of a damaging effect. Apply PS3 for assays measuring effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). **In vitro splicing assays (e.g., RNA studies)**_In vitro_ splicing assays may be considered as **STRONG** evidence, providing the following criteria are met.* Prior knowledge of predominant transcripts in cardiac tissueAnalysis undertaken using RNA extracted from cardiac tissue from the individual with the variantAnalysis undertaken using RNA extracted from whole blood providing the relevant transcripts (isoforms) are expressed in blood and are at sufficient levels to assess splice disruption.Assay shows a clear, reproducible and convincing effect on splicing (i.e. a distinct splice product, present at a level comparable to the splice product from the wild-type allele), which is not observed in controls* Confirmation of abnormal splice product by Sanger sequencing**NOTE:** Mini-gene assay in non-patient derived cell lines are NOT considered to provide STRONG evidence.**NOTE:** Whether to activate this rule needs to be reconciled with the variant spectrum and disease mechanism for the gene at hand (i.e., consider whether the effect is likely to lead to LOF or an in-frame alteration and whether this type of effect is expected to be disease causing) (Abou Tayoun _et al._ 2018[³](#pmid_30192042)). Assays with OddsPath >18.7 as per the SVI recommendationsAnimal models that replicate the glaucoma phenotype NA Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1](https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet PS3 if the variant is co-expressed with KCNE1 and the current magnitude is outside the normal range defined by the paper and is statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for PS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for PS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) PS3 may be applied at the strong level for evidence from an animal model expressing the variant of interest and recapitulating FOXN1 deficiency (i.e. a mouse model with T cell lymphopenia). PS3 may potentially be applied at the default strength level of Strong for evidence from an animal model expressing the variant of interest and recapitulating the ADA-SCID phenotype. Calibrated functional assays with functional odds for pathogenicity > 18.7 PS3 may potentially be applied at the default strength level of Strong for evidence from an animal model expressing the variant of interest and recapitulating the DCLRE1C-SCID phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level. PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the IL7R-SCID phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level. NA PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the JAK3-SCID phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level. * PS3 will be applied at the default level of strength for an abnormal result in an approved _in vitro_ assay with a calculated OddsPath >18.7 (as recommended in PMID: 31892348). Example assays have not yet been found in published reports at the time of these specifications, but are anticipated in the future as additional studies are published. PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the RAG1-SCID phenotype. PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the RAG2-SCID phenotype. Use the BMPR2 functional assay document for guidance on allowable assays. The document is based on the recommendations by Brnich et al 2019 (PMID: 31892348). Known variant validation controls (i.e. established pathogenic and benign variants) are required. One exception is if the same functional assay has been performed for the same variant by two independent groups and demonstrated to have the same functional effect by both groups. Also applicable for non-canonical splice site variants when RNA splice site assay data is available demonstrating abnormal splicing; positive and negative controls are required, preferably from patients and matched unaffected individuals. Note that splicing assay results may be tissue-sensitive. Variants tested in high quality knock-out/knock-in variant mouse lines can meet PS3 at the strong level. NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for PPP1CB at this time. PS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and recapitulating the IL2RG-SCID phenotype. * mRNA splicing assays can be used as strong functional evidence. * **Note:** level of evidence used may differ depending on whether the abnormal transcript is in-frame or out-of-frame, and whether there is complete or incomplete splicing impact. * Do not use PS3 for splice variants that meet PVS1. * mRNA splicing assays can be used as strong functional evidence. * **Note:** level of evidence used may differ depending on whether the abnormal transcript is in-frame or out-of-frame, and whether there is complete or incomplete splicing impact. * Do not use PS3 for splice variants that meet PVS1. Calibrated functional assays with functional odds for pathogenicity > 18.7 Calibrated functional assays with functional odds forpathogenicity > 18.7 Calibrated functional assays with functional odds for Pathogenicity > 18.7 * PS3 will be applied at the default level of strength for evidence from an animal model expressing the variant of interest and recapitulating the APDS phenotype. This strength level is considered particularly appropriate if multiple mouse models of the same variant recapitulate APDS features.* PS3 will be applied at the default level of strength for an abnormal result in an approved _in vitro_ assay with a calculated OddsPath >18.7 (as recommended in PMID: 31892348). One example has been found in the literature: * Enrichment of the variant in high phospho-S6 and phospho-AKT T cells relative to low phospho-S6 and phospho-AKT T cells, within a next generation sequencing-based screen of donor T cells subjected to of CRISPR-mediated adenine base-editing (PMID: 40543502) Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. PS3 at a strong level is not applicable for OTC enzyme activity assays. Please see PS3\_Moderate and PS3\_supporting below. Loss of function of ABCA4 protein in transgenic mice measured by autofluorescence and/or A2E production. Well-established in vitro or in vivo functional studies supportive of a damaging effect. See attached table for acceptable functional studies. Eligible results include _Gucy2e−/−Gucy2f−/−_ knockout mice subretinally injected with adeno-associated virus-packaged constructs encoding a GUCY2D variant (Boye et al., 2016,[¹⁰](#pmid_27881908)) or mice with knock-in of a _GUCY2D_ variant (not yet available in the published literature). Results should include flat or nearly flat scotopic and photopic electroretinogram responses in comparison to wild-type injected control, absent or near-absent guanylate cyclase activity in comparison to wild-type injected control, and/or absent or near absent GUCY2D expression at the protein level despite approximately wild-type expression at the RNA level. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Strong may only be considered for variant-specific mouse models. Currently, no other assays are applicable at this strength. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor _CAPN3_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _CAPN3_. Assays that may be considered in the future include titin ectopic expression, titin degradation assay, baculovirus-based titin cleavage assay, and assays of autolytic activity.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor _ANO5_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _ANO5_. For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3 at Strong for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)* behavioral signs of muscle weakness* progression over timeFor any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Applicable to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing. Follow recommendations set forth by the SVI in conjunction with specifications added by the BMVCEP for quality metrics and minimum validation controls required. (Supplemental Document 1) Animal models are considered in a different manner.Award PS4_Strong if the animal model generated with the variant of interest expressed in neural progenitors shows a complementary brain phenotype.Award PS3 if the functional assay meets the acceptability criteria delimited in (PMID: 31892348) with specifications added by the BMVCEP. Quality metrics and minimum validation controls required can be found in Supplementary Document 1.Animal models are considered in a different manner. Award PS4_Strong if the animal model generated with the variant of interest expressed in neural progenitors show a complementary brain phenotype.Caveat: Studies of cell lines derived from the affected patient as the only source of functional characterization are by themselves insufficient to provide strong evidence of pathogenicity. This is because cells derived from patient affected tissue are likely to exhibit the desired phenotype since the patient tissue exhibits the phenotype. It is therefore impossible to determine whether the variant of interest was solely responsible for that phenotype. Instead, functional readout of patient-derived cells are now included in PS4. Assays with OddsPath >18.7 as per the SVI recommendations.[¹](#PMID_31892348) Do not use as strong. * Functional evidence from non-patient derived material with only a single variant best reflects the variant-level data. Apply patient-derived evidence in PP4.* Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart).* Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). Follow the ‘Funtional Assay SVI Documentation’ Knock-in mouse model demonstrates the phenotype.* Recommend that functional evidence, except for a variant specific mouse model, is not used as strong evidence, due to the absence of well-established functional studies for hearing loss genes.* There are no specific assays for OTOF or MYO15A. However, PS3_Supporting can be applied for other functional analyses if * The assay has been validated by a known pathogenic and benign variant AND * There is plausible reason that the function the assay is testing relates to the phenotype AND * The assay conditions are likely to mimic the physiological environment. RNA assay shows splicing impact that is out-of-frame, in-frame ≥193 residues, or in-frame with RNase IIIb disruption. (PS3\_Moderate if PVS1\_Strong is applied). NA NA **RT-PCR evidence of mis-splicing for non-canonical intronic variants.**For non-canonical splicing variants, RT-PCR evidence demonstrating transcripts of alternative length or specific intron or exon inclusion/exclusion. These studies can be performed in patient derived cells, by placing the mutant genomic DNA into plasmid vectors, or by over-expressing mutant transcript. Assays should demonstrate defective splicing with RT-PCR analysis or RNA sequencing to confirm alternative transcripts. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-offrame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-of-frame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-offrame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-of-frame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-offrame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an out-of-frame transcript.* Do not use for canonical splice site variants and when PVS1 is used. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for SHOC2 at this time. NA NA NA NA NA NA NA NA NA NA NA * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 72.7% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 135% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.2 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 4.1 mV (absolute value).* Mouse knock-in model displays spontaneous seizures. * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 72.7% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 135% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.2 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 4.1 mV (absolute value).* Mouse knock-in model displays spontaneous seizures. * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 72.7% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 135% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.2 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 4.1 mV (absolute value).* Mouse knock-in model displays spontaneous seizures. * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 72.7% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 135% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.2 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 4.1 mV (absolute value).* Mouse knock-in model displays spontaneous seizures. NA Mouse knock-in model displays spontaneous seizures. NA PS3_Moderate Variant meets Level 2 pathogenic functional study criteria. See Table 3.(1) Study of a) only part of the LDLR cycle following Level 1 methodology, or b) whole or part of the LDLR cycle in true homozygous patient cells. A variant with assay results of <70% of wild type activity in either LDLR expression/biosynthesis, LDL binding OR internalization.(2) RNA studies, using RNA extracted from heterozygous or true homozygous patient cells, where aberrant transcript is confirmed by sequencing and is quantified as >25% of total transcript from heterozygous cells or 50% of total transcript from homozygous cells.(3) Variants with two or more Level 3 functional studies (must be different assays); or any Level 3 functional study #1-4 performed by two or more independent labs with concordant results. **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product.* Phosphatase activity ≤ -1.11 per Mighell et al. 2018, PMID: 29706350. Validated functional studies show a deleterious effect (predefined list): GJB2: electrical coupling assays, dye transfer assays → PS3_Moderate* Dye Transfer Assays: Expect results that compare the fluorescence of a variant-transfected cell toboth a negative control (or H2O injected control) and a wildtype-transfected cell. PS3_Moderatewould be applied if the variant results in no dye transfer or significantly different dye transfer whencompared to the wildtype.* Electrical Coupling Assays: Expect results comparing the current of the variant-transfected cells to both a negative control (i.e. H2O injected control) and a wildtype-transfected cell. PS3_Moderate would be applied if the variant results in significantly different current compared to the wildtype, and the current is comparable to background levels/negative control. Functional studies with sufficient analyses to calculate OddsPath reaching strong have not been identified. Therefore, the strength of this criteria is modified to PS3\_moderate or PS3\_supporting for future or existing studies.In vitro enzyme activity \<50% compared to wild type controls.* Expression systems placing the mutant (and wild-type) cDNAs into plasmid vectors and introducing these into human or other mammalian host cells, which is the closest available approximation to the in vivo situation (e.g., COS cells) (Trunzo et al. Gene. 2016. 594:138-143. PMID: 27620137).* With ≥11 benign/pathogenic variant controls used in assay* NOTE: no papers that meet PS3\_Moderate criteria have been identified by the PAH VCEP at time of this specification update. However, there may be future studies that meet the above criteria where a moderate level of evidence can be applied. RNA assay demonstrating abnormal in-frame transcript. **PS3\_Moderate:** Transactivation assays demonstrating altered transactivation (\<20% of wt and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) OR ≥ 2 secondary assays demonstrating altered function. Partially functional on Kato et al. data **AND** loss of function (LOF) by the majority of other available assays Well-established in vitro or in vivo functional studies supportive of a damaging effect. * <5% wild type GAA activity when the variant is expressed in a heterologous cell type and evidence of abnormal GAA synthesis and/or processing. * RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products. In a model organism or heterologous cell line, significantly reduced surface protein expression (5 * 25%). Well-established functional studies supportive of a damaging effect on protein function * Increased sensitivity to RYR1 agonist in HEK293 in vitro assay, Ca2+ release significantly increased compared to WT, controls to include known pathogenic and benign variants, n≥3. * Three or more independent ex vivo studies all showing release of Ca2+ in response to RYR1 agonist * Knock-in mouse showing MH reaction in response to RYR1 agonist OR increased sensitivity to RYR1 agonists in ex vivo tissue/cells (but not both, which would be PS3_strong) Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. NA Not Applicable Not Applicable NA NA Not Applicable: ● Protein: Do not use: Lack of known positive controls ● RNA: Do not use: See code PVS1_Variable(RNA) NA NA NA NA NA NA NA NA See list of approved functional studies and guidelines for interpretation of data. Two or more different approved assays. NA **RNA assays** show 1. a premature stop codon AND reports of exon deletion/skipping/loss, insertion of intronic nucleotides **OR**2. inframe skipping of exon 13 or 14 AND reports of exon deletion/skipping/loss, insertion of intronic nucleotides **OR**3. other inframe skipping AND absent or \< 10% full-length transcript. NA When PS3\_Supporting is met for enzyme activity AND there is expression data (Western blot, pulse chase) showing a clear difference in synthesis and/or processing of alpha-iduronidase, PS3 will be applied at moderate (e.g. Matte et al, 2003, PMID: 12559846; see Appendix 3). NA Two or more different approved assays. **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) NA **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) **In vivo models (e.g., variant knock-in animal models)**Mammalian variant-specific knock-in animal models that produce a phenotype consistent with the clinical phenotype in humans (e.g., structural and/or functional cardiac abnormalities, premature death, arrhythmia) may be considered as **MODERATE** evidence**NOTE:** The following assays/models do NOT meet criteria1. Assays that are known to be associated with non-specific cardiac phenotypes (e.g., morpholino-induced pericardial edema in zebrafish)2. In vivo evidence that is not variant specific, such as whole gene alterations (i.e., cDNA or whole gene transgenic mice and whole or partial gene knock-out mice) Assays with OddsPath >4.3 as per the SVI recommendations NA Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1](https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet PS3 if the variant is co-expressed with KCNE1 and the current magnitude is outside the normal range defined by the paper and is statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for PS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for PS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) PS3 may be applied at the moderate level based on a luciferase assay showing reduced (\<50%) activity, as part of a validated assay with pathogenic and benign controls (PMID: 37419334). The strength of evidence from cellular models/_in vitro_ studies is dependent upon the level of expressed ADA enzyme activity based on levels defined in Arredondo-Vega et al., 1998 (PMID: 9758612): * PS3\_Moderate: ≤0.05% of wild-type activity (group I)_At least one previously observed proband with the expressed ADA variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._ Calibrated functional assays with functional odds for pathogenicity >4.3 and \<= 18.7OR MMR function defect following functional assay flowchart\*OR Variants with monoallelic expression: complete loss of expression (\<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. The strength of evidence from cellular models/_in vitro_ studies is dependent upon abnormal results in _in vitro_ DNA repair activity and V(D)J recombination assays: * PS3\_Moderate: Abnormal result in **both** an _in vitro_ DNA repair activity assay AND an _in vitro_ V(D)J recombination assay (defined as \<25% of wild-type activity for both assays)._At least one previously observed proband with the DCLRE1C variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._* Approved assay instances: * DNA repair activity assay * Felgentreff et al., 2015 (PMID: 25917813) * V(D)J recombination assay * Pannicke et al., 2004 (PMID: 15071507) * Ege et al., 2005 (PMID: 15731174) * Felgentreff et al., 2015 (PMID: 25917813) * Volk et al., 2015 (PMID: 26476407) NA NA NA * PS3 will be applied at the moderate level of strength for an abnormal result in an approved _in vitro_ assay with a minimum of 11 total pathogenic and benign variant controls (classified using these same specifications, as recommended in PMID: 31892348). Example assays with sufficient control variants have not yet been found in published reports at the time of these specifications, but are anticipated in the future as additional studies are published. The strength of evidence from cellular models/_in vitro_ studies is dependent upon the abnormal result in a V(D)J recombination assay:* PS3\_Moderate: \<25% of wild-type activity in Lee et al., 2014 (PMID: 24290284);_At least one previously observed proband with the expressed RAG1 variant meeting PP4 is required to apply PS3 at any strength._ Strength of evidence from cellular models/_in vitro_ studies is dependent upon abnormal result in a V(D)J recombination assay:* PS3\_Moderate: \<25% of wild-type activity in Tirosh et al., 2019 (PMID: 29772310)_At least one previously observed proband with the expressed RAG2 variant meeting PP4 is required to apply PS3 at any strength._ NA NA Two or more different approved assays. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for PPP1CB at this time. NA See PS3\_Supporting and Instructions below. See PS3\_Supporting and Instructions below. Calibrated functional assays with functional odds for pathogenicity >4.3 and \<= 18.7OR MMR function defect following functional assay flowchart\*OR Variants with monoallelic expression: complete loss of expression (\<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. Calibrated functional assays with functional odds for pathogenicity >4.3 and \<= 18.7OR MMR function defect following functional assay flowchart\*OR Variants with monoallelic expression: complete loss of expression (\<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. Calibrated functional assays with functional odds for pathogenicity >4.3 and \<= 18.7OR MMR function defect following functional assay flowchart\*OR Variants with monoallelic expression: complete loss of expression (\<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. * PS3 will be applied at the moderate level of strength for an abnormal result in an approved _in vitro_ assay with a minimum of 11 total pathogenic and benign variant controls (classified using these same specifications, as recommended in PMID: 31892348). Example assays with sufficient control variants have not yet been found in published reports at the time of these specifications, but are anticipated in the future as additional studies are published. The two assays from PS3\_Supporting may be stacked to reach a Moderate Strength The two assays from PS3\_Supporting may be stacked to reach a Moderate Strength The two assays from PS3\_Supporting may be stacked to reach a Moderate Strength Where indicated, some of the assays from PS3\_Supporting may be stacked to reach a Moderate Strength Some of the assays from PS3\_Supporting may be stacked to reach a Moderate Strength. One specific assay is currently suggested to be applied at Moderate:Calcium Release\*\*: An abnormal readout consists of increased calcium release compared to controls or WT RYR1.**\*\*Calcium Release assays and Single Channel Activity assays should not be stacked because they may not be assessing independent biological function.** Yeast growth assays -- applicable when growth of the ARG3 variant strain is ≤50% when compared to wildtype strains NA NA NA NA Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)PS3\_Moderate may also be applied for dysferlin membrane localization assays that have been clinically validated with ≥11 control variants that meet criteria specified in Brnich et al. 2020 (PMID: 31892348) for the number of pathogenic and benign control variants. * Dysferlin membrane localization functional score \<0.25 AND concordant immunocytochemistry assay result in Tominaga et al. 2022 (concordant readout of “non-functional” for both assays) (PMID: 35028538). See supplementary file “PS3 assays\_DYSF”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)PS3\_Moderate may be applicable for sarcoglycan complex membrane localization assays that have been clinically validated with ≥11 control variants that meet criteria specified in Brnich et al. 2020 (PMID: 31892348) for the number of pathogenic and benign control variants. * Sarcoglycan complex cell surface localization functional score \<-0.5 in Li et al. 2023 (PMID: 37317968). See supplementary file “PS3 assays\_SGCB”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)PS3\_Moderate may be applied for sarcoglycan complex membrane localization assays that have been clinically validated with ≥11 control variants that meet criteria specified in Brnich et al. 2020 (PMID: 31892348) for the number of pathogenic and benign control variants. See supplementary file “PS3 assays\_SGCG”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)PS3\_Moderate may be applied for sarcoglycan complex membrane localization assays that have been clinically validated with ≥11 control variants that meet criteria specified in Brnich et al. 2020 (PMID: 31892348) for the number of pathogenic and benign control variants. See supplementary file “PS3 assays\_SGCD”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)For _CAPN3_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _CAPN3_. Assays that may be considered in the future include titin ectopic expression, titin degradation assay, baculovirus-based titin cleavage assay, and assays of autolytic activity.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)For _ANO5_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _ANO5_. For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Variant-specific animal models are rare but may be assessed on a case-by-case basis and PS3 applied at a strength level that reflects how well the model recapitulates features observed in human patients. Apply PS3\_Moderate for a variant-specific animal model that meets all of the following conditions, regardless of species: * signs of myopathy or dystrophy are present in skeletal muscle * an effect on gene or protein function is demonstrated (e.g., decreased protein expression, impaired membrane localization, or other functional abnormality)PS3\_Moderate may be applied for sarcoglycan complex membrane localization assays that have been clinically validated with ≥11 control variants that meet criteria specified in Brnich et al. 2020 (PMID: 31892348) for the number of pathogenic and benign control variants. See supplementary file “PS3 assays\_SGCA”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. Currently applicable for variants with luciferase assay data (evidence of decreased transactivation (≤ 40% of wild type) by the Gloyn/Oxford group[¹⁰](#pmid_32910913) ([Althari et al. 2020).](https://pubmed.ncbi.nlm.nih.gov/32910913/) This upgrade from supporting is based on a validation conducted according to the guidelines by Brnich et al. 2019 [¹](#pmid_31892348). Follow recommendations set forth by the SVI in conjunction with specifications added by the BMVCEP for quality metrics and minimum validation controls required (PMID: 31892348). Animal models are considered in a different manner. Award PS4_Moderate if the animal model generated with the variant of interest expressed in non-neural tissues show an increased cancer burden. Assays with OddsPath >4.3 as per the SVI recommendations.[¹](#PMID_31892348) Use when a variant fails to rescue both an ATM specifc feature (e.g. phosphorylation of ATM-specific targets) AND radiosensitivity. * Functional evidence from non-patient derived material with only a single variant best reflects the variant-level data. Apply patient-derived evidence in PP4.* Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart).* Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). Follow the ‘Funtional Assay SVI Documentation’ Validated functional studies show a deleterious effect (none are defined for OTOF and MYO15A). RNA assay shows in-frame splicing impact with change in protein length \<193 residues AND RNase IIIb domain not disrupted. NA NA NA NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for SHOC2 at this time. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. Two or more different approved assays. * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 80.6% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 125% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.1 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 3.0 mV (absolute value).* Mouse knock-in model displays induced seizures* Zebrafish knock-in model displays spontaneous seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 80.6% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 125% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.1 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 3.0 mV (absolute value).* Zebrafish knock-in model displays spontaneous seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 80.6% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 125% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.1 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 3.0 mV (absolute value).* Mouse knock-in model displays induced seizures* Zebrafish knock-in model displays spontaneous seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies * In patch clamping experiments: Peak current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is less than or equal to 80.6% of wildtype.* In patch clamping experiments: Persistent current as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is greater than or equal to 125% of wildtype.* In patch clamping experiments: Voltage dependence of activation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 2.1 mV (absolute value).* In patch clamping experiments: Voltage dependence of inactivation as defined by FENICS ontology ([https://bioportal.bioontology.org/ontologies/FENICS](https://bioportal.bioontology.org/ontologies/FENICS)) is shifted by at least 3.0 mV (absolute value).* Mouse knock-in model displays induced seizures* Zebrafish knock-in model displays spontaneous seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies NA * Heterologous expression with voltage clamping shows statistically significant difference over wildtype in at least one parameter (https://bioportal.bioontology.org/ontologies/FENICS)* Mouse knock-in model displays induced seizures* Zebrafish knock-in model displays spontaneous seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies NA PS3_Supporting Variant meets Level 3 pathogenic functional study criteria. See Table 3. (1) Study of LDLR cycle (whole or part) in heterozygous patient cells, with assay results of <85% of wild-type activity in either LDLR expression/biosynthesis, LDL binding OR internalization. (2) Luciferase studies with transcription levels of <50% compared to wild-type (applicable to 5’UTR/promoter variants). (3) Minigene splicing assays with <10% wild-type transcript present where anaberrant transcript from the candidate variant is confirmed by sequencing. (4) High-throughput assays, which include alternative microscopy assays (e.g.,Thormaehlen et al., 2015), Multiplex Assays of Variant Effect (MAVE) (e.g.,Weile & Roth, 2018) and deep mutational scanning assays, can be considered here, only if assay has been validated with a minimum of four pathogenic and four benign variant controls in LDLR. Note: % activity thresholds will be defined by the FH VCEP as more data becomes available. (5) RNA studies, using RNA extracted from heterozygous or homozygous patient cells, with aberrant transcript confirmed by sequencing (but without transcript quantification). _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. Phosphatase activity \<50% of wild-type or abnormal _in vitro_ cellular assay or transgenic model with phenotype different from wild type that does not meet PS3\_moderate. SLC26A4: Radio isotope and fluorescence assays → PS3_Supporting* Radio Isotope Assays: PS3_Supporting would be applied when cells transfected with mutant SLC26A4 show a statistically significant decreased efflux of iodide compared to wildtype pendrin.* Fluorescence Assays: PS3_Supporting would be applied when a cell transfected with the mutant SLC26A4 shows a statistically significant difference in fluorescence (ΔFmax %) compared to the wildtype protein, and when the fluorescence is not significantly different from that of an empty vector control.COCH: Localization, secretion, and dimerization studies performed using immunofluorescence andWestern blotting techniques →PS3_Supporting* Localization: PS3_Supporting would be applied if the mutant cochlin protein does not aggregate into extracellular deposits or in the perinuclear region, comparable to the localization of wildtype cochlin.* Secretion: PS3_Supporting would be applied if cochlin protein containing the variant does not show secretion from transfected cells, but aggregates in cell regions such as the ER, Golgi and nucleus or is degraded.* Dimerization: In a non-reducing environment, wildtype cochlin migrate quickly and appear smaller than in the reduced state because the structure is maintained by disulfide bonds. PS3_Supporting would be applied if the cochlin protein containing the variant forms more, or less, stable disulfide bonds when compared to the wildtype in non-reducing conditions.* If not listed above, OK to use PS3_Supporting for other genes/functional analyses if * The assay has been validated by a known pathogenic and benign variant AND * There is plausible reason that the function the assay is testing relates to the phenotype AND * The assay conditions are likely to mimic the physiological environment. In vitro enzyme activity ≤50% compared to wild type controls* with ≤10 benign/pathogenic variant controls used in assay NA **PS3\_Supporting:** Transactivation assays demonstrating enhanced transactivation (>115% of wt). Non-functional on Kato et al. data **AND** abnormal on Kawaguchi et al. data regardless of other assaysPS3\_Supporting may also be applied to small deletions that demonstrate LOF on the majority of available assays Well-established in vitro or in vivo functional studies supportive of a damaging effect. * <30% wild type GAA activity when the variant is expressed in a heterologous cell type. * RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products. NA Well-established functional studies supportive of a damaging effect on protein function * Two independent ex vivo studies all showing release of Ca2+ in response to RYR1 agonist Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. Transporter assay showing loss of function Not Applicable Not Applicable Reduced ETHE1 persulfide dioxygenase Functional validation is present in cybrid studies or single fiber analysis Not Applicable: ● Protein: Do not use: Lack of known positive controls ● RNA: Do not use: See code PVS1_Variable(RNA) Acceptable assays that display functional effect in VHL are the following: • HIF 1/2a degradation assays -- HIF1/2a is not degraded and/or • VBC complex stability is affected and/or• Pathogenicity supported by abnormal ECM formation and impaired fibronectin binding [¹](#pmid_9651579) , [²](#pmid_11331613) , [³](#pmid_11358843) , [¹⁷](#pmid_14706840)Multiple studies and publications confirm the role of VHL in HIF1/2a regulation and VBC complex stability for VHL Type 1, and 2A/B, as well as fibronectin binding/deposition and assays evaluating extra-cellular matrix composition. In-vitro assays should display total loss of HIF1/2a degradation (i.e. HIF1/2a presence) for VHL Type 1, and 2A/B. VHL Type 2C should display presence of HIF1/2a (with VBC complex stability variably affected and fibronectin deposition/extra cellular matrix composition affected). These assays are typically performed in Renal Cell Carcinoma (RCC) cells lacking VHL, introducing normal pVHL as a control in addition to a variant-VHL, then comparing HIF1/2a levels to WT pVHL. Brnich et al proposed 10 controls to achieve PS3\_Supporting and 11 for PS3\_Moderate [¹⁰](#pmid_31892348). We propose to _follow the workflow outlined in Brnich et al._ Type 2C VHL variants are typically missense variants in the alpha domain of VHL, and do not usually affect HIF1/2a. If HIF1/2a maintains presence and VHL Type 2C is suspected, assays evaluating fibronectin deposition or extracellular matrix assembly should be used. **SPLICING:** **PS3:** RNA transcripts carrying splicing mutation display splicing defects in patient cells. **PS3\_Moderate**: RNA transcripts carrying splicing mutation display splicing defects in in-vivo or in-vitro assays. **Functional Assay Documentation** : [https://drive.google.com/file/d/1w8P8zs1fHUolaAYmBL1jw-vcjsX3N_yh/view?usp=sharing](https://drive.google.com/file/d/1w8P8zs1fHUolaAYmBL1jw-vcjsX3N_yh/view?usp=sharing) Functional assays measuring quantity of GP1ba expression on cell surface measured by flow cytometry analysis of GPIb and GPIX when there is absent or near absent expression, >75% reduction (see spreadsheet for more detail). Abnormal factor IX activity level (\<40 IU/dL or 40%) in a cell line and/or mouse model.\--OR--Abnormal factor IX activity level (\<40 IU/dL or 40%) studied in an animal model setting other than mouse (i.e. – bovine factor IX activity levels compared to factor X levels).\--OR--Absent or significantly reduced factor IX antigen level compared to wildtype using conformation-specific reporter assay in cell lines. Subtype 2A = Either (1) a multimerization assay in which the variant is expressed in a recombinant system (either independently or coexpressed with WT) resulting in abnormal multimers, with a reported loss of HMWM. This evidence must be published in a peer reviewed journal and a picture of the gel must be visible for evaluation. (2) a ADAMTS susceptibility assay indicating increased susceptibility relative to WT. Functional assays measuring quantity of GP1ba and/or GPIX expression on cell surface measured by flow cytometry (see spreadsheet for more detail). Functional assays measuring quantity of GP9 expression on cell surface measured by flow cytometry analysis of GPIb and GPIX when there is absent or near absent expression, >75% reduction (see spreadsheet for more detail). See attached spreadsheet for list of approved assays to use for this rule code. Below is a general description of assays that can be used: - _In vitro_ functional studies in COS-1, HEK293T, HEK-EBNA cells demonstrating abnormal activity levels:* Antithrombin activity levels measured by FXa inhibition activity assay* Antithrombin activity levels measured by thrombin inhibition activity assayOR\- _In vitro_ functional studies in COS-1, HEK293T, HEK-EBNA cells demonstrating abnormal antigen levels:* Antithrombin antigen levels measured by ELISA * Immunofluorescence assay – intracellular retention and secretion defectsOR\- _In vivo_ studies demonstrating rescue of antithrombin levels would be considered, but no studies are known to be available at this time. List of approved functional studies and guidelines for interpretation:* EMSA for DNA binding * “Decreased function” is defined as activity less than 60% of wildtype * Note: the effect of the variant on DNA binding will be highly dependent on whether the variant is located within the DNA binding domain.* Luciferase assays for transactivation * “Decreased function” is defined as activity less than 60% of wildtype * Note: this threshold is not 100% specific for transactivation (TA) activity and is complicated by the fact that TA activity will vary depend on many factors, for instance cell line that is used (HeLa, INS, MIN6 etc).* Western blotting and indirect immunofluorescence for protein expression (specifically levels and nuclear and cytoplasmic localization, respectively). * Determining appropriate thresholds for protein expression is more difficult due to variability in results due to the complexity of the technique.  Sample preparations, gel loading, transfer efficiency, specificity of the antibody, choice of internal control and inaccurate detection and quantification are some of the factors that can contribute to varying and inconsistent results. If a reduction in protein expression is seen by immunoblotting, then further testing by quantitative PCR (qPCR) is recommended in order to measure the mRNA level and assess whether a reduction in amount of protein is due to a reduced mRNA level.* To use PS3\_Supporting, functional study must have been performed on a transfected variant. If a study was performed on a cell line generated from a patient sample (and therefore contains the variant plus wild-type allele) does not count as PS3\_Supporting. See list of approved functional studies and guidelines for interpretation of data (below). One approved assay. PS3\_Supporting can be applied based on an abnormal result in **at least** one approved _in vitro_ assay.Approved assay instances:* Endonucleolytic cleavage activity assay (mRNA cleavage activity smaller than 0.9) * Thiel et al., 2005 (PMID: 16252239) * Thiel et al., 2007 (PMID: 17701897)) **OR*** Luciferase reporter assay * Hermanns et al., 2005 (PMID: 16254002)) (luciferase activity higher than 7) **RNA assays** show 1. inframe skipping of exons other than exon 13 or 14 AND reports of exon deletion/skipping/loss, insertion of intronic nucleotides **OR**2. over-expression of an alternative transcript (exons 10, 11 or 15)**Protein assays** showIncreased β-catenin regulated transcription activity and/or decreased binding to β-catenin by surface plasmon resonance (only for variants within the β-catenin binding domain, which refers to codons 959-2129 of _APC_) \[Reference 2\]. NA _In vitro_ expression studies: After assessment of the parameters listed below, results from such studies can be used at PS3\_Supporting (see Appendix 3 for thresholds for specific studies; \<2% activity (Beesley et al, 2001, PMID: 11735025; Yogalingam et al, 2004, PMID: 15300847; Matte et al, 2003); \<1% activity / enzyme abundance (Yu et al, 2020. PMID: 33198351).* Were clones sequenced to verify that the variant is present and that no artifacts have been introduced during the site-directed mutagenesis process? * Were appropriate controls included? Examples * Negative controls: Empty vector, antisense (at least one appropriate negative control is required); * Positive control: Wild type IDUA, normal cells (at least one appropriate positive control is required)* Was the experiment replicated?* If cells have intrinsic IDUA activity e.g., COS cells, the level of activity should be stated so that this can be taken into account. NA One approved assay. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. NA _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. _**In vitro**_ **assays (e.g., biochemical assays of myofilament function, motility assays, human iPSC-CM)**While some _in vitro_ assays may provide evidence that a variant in a cardiomyopathy gene has an effect on protein and/or myofilament function, at present, there are no validated “gold-standard” assays that are considered to reliably predict the clinical phenotype.As such, in the cardiomyopathy genes listed in these guidelines, data from individual _in vitro_ studies are unlikely to meet the criteria required to assign this rule at more than SUPPORTING level. Assays with OddsPath >2.1 as per the SVI recommendationsAs of 06/11/25, the following studies meet the criteria to apply PS3\_supporting: Chavarria-Soley et al. 2008[³¹](#PMID_18470941), Pasutto et al. 2010[³³](#PMID_19643970) and Mammen et al. 2003[³²](#PMID_12807732). Assays with OddsPath >2.1 as per the SVI recommendations¹Applies to the following functional studies:* Protein-protein interaction (PMIDs: 30622176, 20631154, 10958648, 23213406, 9990021, 36445968)* Protein localization to connecting cilia (PMIDs: 30622176, 25630948).* Glutamylation assays (only applicable to truncating variants in exon 15, PMIDs: 27162334, 36445968)\-OR-Animal models that replicate the phenotype. Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1](https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet PS3 if the variant is co-expressed with KCNE1 and the current magnitude is outside the normal range defined by the paper and is statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for PS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for PS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) PS3 may be applied at the supporting level based on a luciferase assay, without sufficient validation controls, showing reduced (\<50%) activity, such as those reported in PMIDs: 31566583, 33464451, 34860543. The strength of evidence from cellular models/_in vitro_ studies is dependent upon the level of expressed ADA enzyme activity based on levels defined in Arredondo-Vega et al., 1998 (PMID: 9758612):* PS3\_Supporting: 0.06-0.6% of wild-type activity (groups II and III)_At least one previously observed proband with the expressed ADA variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._ Calibrated functional odds for pathogenicity >2.08 and \<= 4.3 The strength of evidence from cellular models/_in vitro_ studies is dependent upon abnormal results in _in vitro_ DNA repair activity and V(D)J recombination assays: * PS3\_Supporting: Abnormal result in an _in vitro_ V(D)J recombination assay (same threshold, \<25% of wild-type activity)._At least one previously observed proband with the DCLRE1C variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._* Approved assay instances: * DNA repair activity assay * Felgentreff et al., 2015 (PMID: 25917813) * V(D)J recombination assay * Pannicke et al., 2004 (PMID: 15071507) * Ege et al., 2005 (PMID: 15731174) * Felgentreff et al., 2015 (PMID: 25917813) * Volk et al., 2015 (PMID: 26476407) PS3\_Supporting can be applied based on an abnormal result in **at least** **one** approved _in vitro_ assay (IL-7-induced Jak3 phosphorylation assay, IL-7 binding assay, IL-7-induced STAT5 DNA binding/transcriptional induction).* Approved assay instances: * IL-7-induced Jak3 phosphorylation assay * Roifman et al., 2000 (PMID: 11023514) * IL-7 binding assay * Puel et al., 1998 (PMID: 9843216) * IL-7-induced STAT5 DNA binding/transcriptional induction * Puel et al., 1998 (PMID: 9843216)_At least one previously observed proband with the expressed IL7R variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._ Well-established in vitro or in vivo functional studies supportive of a damaging effect. Not applicable for splicing effects (replaced by PVS1\_Strength (RNA))See attached table for acceptable functional studies.For studies reporting isomerhydrolase activity, cutoff is ≤10% of wild-type control. PS3\_Supporting can be applied based on an abnormal result in an _in vitro_ kinase activity assay.* Approved assay instance: Roberts et al., 2004 (PMID: 14615376)._At least one previously observed proband with the JAK3 variant meeting PP4 is required to apply PS3 at any strength on the basis of a cellular model/in vitro study._ * An abnormal functional result by a _CTLA4_ variant in non-patient cells in an approved assay should be applied at PS3\_Supporting strength. Further details on how to interpret the assays can be found in the associated manuscript.* Functional assays can be found in the attached Excel file entitled CTLA4\_Functional Assay\_Research\_PS3\_BS3.* Assays approved by the VCEP for PS3\_Supporting are non-patient cell-based: * (1) CD80 or CD86 transendocytosis / soluble CD80 or CD86 ligand uptake in non-patient cells (i.e. PMID: 25329329, PMID: 25632005, PMID: 25367873, PMID: 27102614, PMID: 29375547, PMID: 15814706, PMID: 20870175) * (2) Cell surface expression of CTLA4 expressed in non-patient cell lines (i.e. PMID: 25367873, PMID: 14578884, PMID: 29375547, PMID: 7559643, PMID: 25213377) * (3) Protein localization / translocation by CTLA4 expressed in non-patient cell lines (i.e. PMID: 7559643, PMID: 20870175, PMID: 27102614, PMID: 25367873, PMID: 29375547, PMID: 15814706) * (4) _In vitro_ T cell suppression by CTLA4 expressed in non-patient cell lines (i.e. PMID: 29375547, PMID: 26478010, PMID: 25213377)* Please note that patient cell-based assays of CTLA4 function were also considered for inclusion in PS3\_Supporting, but were instead viewed as part of the proband phenotype and were incorporated into the PP4\_Moderate code instead. The strength of evidence from cellular models/_in vitro_ studies is dependent upon the abnormal result in a V(D)J recombination assay:* PS3\_Supporting: * 25-60% of wild-type activity in Lee et al., 2014 (PMID: 24290284) **OR** * Reduced activity compared to wild type in Corneo et al., 2001 (PMID: 11313270);_At least one previously observed proband with the expressed RAG1 variant meeting PP4 is required to apply PS3 at any strength._ Strength of evidence from cellular models/_in vitro_ studies is dependent upon abnormal result in a V(D)J recombination assay:* PS3\_Supporting: * 25-60% of wild-type activity in Tirosh et al., 2019 (PMID: 29772310) **OR** * Reduced activity compared to wild type in Couëdel et al., 2010 (PMID: 20234091)_At least one previously observed proband with the expressed RAG2 variant meeting PP4 is required to apply PS3 at any strength._ Use the BMPR2 functional assay document for guidance on allowable assays as described above. If no known variant validation controls (i.e. established pathogenic and benign variants) were used, then score at the supporting strength. Use at the supporting strength for most assays (see Functional Assays Table). One approved assay. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for PPP1CB at this time. Strength modification based on an abnormal result in **at least** one approved _in vitro_ assay.* Approved assay instances: * Phosphorylation of JAK3/Co-Immunoprecipitation with JAK3 * Sharfe et al., 1997 (PMID: 9399950) * Kumaki et al., 1999 (PMID: 9933465) * Arcas-García et al., 2020 (PMID: 31799703) * Cytokine binding * Sharfe et al., 1997 (PMID: 9399950) * Kumaki et al., 1995 (PMID: 7632950) * Surface expression of the gamma chain * Kumaki et al., 1995 (PMID: 7632950) * Interaction profiling-BioID * Tuovinen et al., 2020 (PMID: 32072341)_At least one previously observed proband with the IL2RG variant meeting PP4 is required_ _to apply PS3 at any strength on the basis of a cellular model/in vitro study._ * Protein expression assays: Metabolic label & IP, WB & FACS HUVECs/BOECs, FACs activated monocytes, cDNA transfect, WB & ML HEK293T/COS/NIH3T3, cDNA transfect & luciferase HepG2 * **Note:** Decreased protein expression can be used as supporting pathogenic evidence if experiment was not done in a single assay, and the corresponding densitometry of western blot reflects the conclusion drawn.* Intracellular signaling assays: BRE/CAGA-luciferase, Gal4 Smad1/Smad3 for TGF-beta/BMP9 signaling* Binding assays: BMP9 binding, transcription factor Sp1, BMP9 protein-protein interaction (BLI)* Subcellular protein localization* Morphology: Morphology & actin cytoskeleton, tubulogenesis* Somatic variant 2^nd hit: In vivo evidence can be obtained as supporting functional evidence by identification of somatic variants in telangiectases´ biopsies using NGS, suggesting a second-hit mechanism leading to biallelic LOF (PMID: 31630786). * Protein expression assays: Metabolic label & IP, WB & FACS HUVECs/BOECs, FACs activated monocytes, cDNA transfect, WB & ML HEK293T/COS/NIH3T3, cDNA transfect & luciferase HepG2 * **Note:** Decreased protein expression can be used as supporting pathogenic evidence if experiment was not done in a single assay, and the corresponding densitometry of western blot reflects the conclusion drawn.* Intracellular signaling assays: BRE/CAGA-luciferase, Gal4 Smad1/Smad3 for TGF-beta/BMP9 signaling* Binding assays: BMP9 binding, transcription factor Sp1, BMP9 protein-protein interaction (BLI)* Subcellular protein localization* Morphology: Morphology & actin cytoskeleton, tubulogenesis* Somatic variant 2^nd hit: In vivo evidence can be obtained as supporting functional evidence by identification of somatic variants in telangiectases´ biopsies using NGS, suggesting a second-hit mechanism leading to biallelic LOF (PMID: 31630786). Calibrated functional odds for pathogenicity >2.08 and \<= 4.3 Calibrated functional odds for pathogenicity >2.08 and \<= 4.3 Calibrated functional odds for pathogenicity >2.08 and \<= 4.3 * PS3\_Supporting can be applied based on an abnormal result in an approved _in vitro_ assay. These are described in detail in the attached Excel file entitled “PIK3CD\_Functional\_Assays\_PS3\_BS3” * Approved assay classes and specific assay instances: * AKT kinase activity assay * PMID: 24136356, PMID: 24165795, PMID: 28414062 * Lipid kinase activity * PMID: 24136356; PMID: 28167755; PMID: 28414062 * Lipid vesicle affinity * PMID: 24136356 * Conformational dynamics: * PMID: 28167755; PMID: 28414062* Please note that protein binding assays testing PIK3CD binding to PIK3R1 were also considered for inclusion in PS3\_Supporting, but were excluded based on lack of evidence of altered binding by disease-associated variants compared to wild-type controls (PMID: 24165795; PMID: 28414062).* Patient cell-based functional assays directly showing abnormally high activity of the disease-relevant PI3K delta pathway were considered for inclusion in the PS3\_Supporting code but are viewed as part of the proband phenotype and have been recommended for consideration under the PP4\_Moderate code instead. Two specific assays are currently suggested to be applied at Supporting:1. Thin filament structure: An abnormal readout consists of a significant difference in intensity reflections of X-ray diffraction patterns generated by muscle fibers from patient biopsies compared to WT.2. _In vitro_ motility: An abnormal readout consists of a significant difference in speed of single fibers derived from patient muscle compared to WT.If not listed above, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. Three specific assays are currently suggested to be applied at Supporting:1. Actin Localization: An abnormal readout consists of integration of actin into cytoplasmic or intranuclear aggregates or rods.2. Actin Polymerization: An abnormal readout consists of a significant reduction in levels of actin in insoluble fraction or absent or short polymerized actin filaments compared to WT.3. Actin Motility Assay: An abnormal readout consists of percent motility, velocity, and force generation statistically different from WT.If not listed above, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. Two specific assays are currently suggested to be applied at Supporting:1. Oligomerization: An abnormal readout consists of integration of increased dynamin stability compared to WT dynamin.2. GTPase activity: An abnormal readout consists of increased GTPase activity or increased stability compared to wild type dynamin.If not listed above, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. Five specific assays are currently suggested to be applied at Supporting:1. Phosphatase Activity: An abnormal readout consists of reduced phosphatase activity (measured via levels of PtdIns and PtdIns5p or increased levels of precursors)2. Myotubularin Localization**\*\***: An abnormal readout consists of altered localization (presence in spots/aggregates/extensions, loss of cytoplasmic localization)3. Myotubularin Translocation**\*\***: An abnormal readout consists of loss of MTM1 recruitment to late endosomal compartment following EGF stimulation4. Intracellular Trafficking (Trafficking of endosomal cargo is thought to require phosphoinositide conversion and may be disrupted by defective MTM1 activity): An abnormal readout consists of reduced localization/trafficking of receptor proteins5. Protein and Lipid Association: An abnormal readout consists of decreased association with known binding partner (BIN1, Desmin, hVPS34-PI 3-Kinase, MTMR12, Phosphoinositide, or EEA Membrane association)**\*\*These two assays should not be stacked because they may not be assessing independent biological function**If not listed above, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. Two specific assays are currently suggested to be applied at Supporting:1. Single Channel Activity\*\*: An abnormal readout consists of increased opening probability compared to controls or WT RYR1.2. Calstabin1/FKBP12 Binding: An abnormal readout consists of significantly reduced RYR1-FKBP12 binding compared to controls or WT RYR1.If not listed above, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment.**\*\*Calcium Release assays and Single Channel Activity assays should not be stacked because they may not be assessing independent biological function.** Yeast growth assays -- applicable when growth of the ARG3 variant strain is >50 and ≤65% when compared to wildtype strainsEnzyme activity in non-patient derived cell lines -- applicable if enzyme activity of the variant under review is \<20% compared to wildtype. Measurement of ABCA4 expression or ATPase activity in HEK293 or HeLa cells. See list of approved functional assays in PS3/BS3 Guidance spreadsheet below. Well-established in vitro or in vivo functional studies supportive of a damaging effect. Not applicable for splicing effects (replaced by PVS1\_Strength (RNA))See attached table for acceptable functional studies.For studies reporting guanylate cyclase activity, cutoff is ≤10% of wild-type control. There are no specific functional assays listed for the ACTA1 AR specifications; all should be used for AD specifications. However, it is acceptable to use PS3\_Supporting for functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. There are no specified functional assay for AR RYR1-related myopathy. However, it is acceptable to use PS3\_Supporting for other functional analyses if* The assay has been validated by a known pathogenic and benign variant AND* There is plausible reason that the function the assay is testing relates to the phenotype AND* The assay conditions are likely to mimic the physiological environment. PS3\_Supporting may be applied if the variant is expressed in heterologous cell lines/model organisms and shows absent membrane localization of the dysferlin protein but fewer than 11 control variants were used, in accordance with Brnich et al. 2020 (PMID: 31892348). See supplementary file “PS3 assays\_DYSF”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level.Assays that may be considered in the future include membrane resealing activity assays and calcium signaling assays. PS3\_Supporting may be applied if the variant is expressed in heterologous cell lines/model organisms and shows absent membrane localization of the sarcoglycan protein complex and fewer than 11 control variants were used, in accordance with Brnich et al. 2020 (PMID: 31892348). See supplementary file “PS3 assays\_SGCB”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. PS3\_Supporting may be applied if the variant is expressed in heterologous cell lines/model organisms and shows absent membrane localization of the sarcoglycan protein complex and fewer than 11 control variants were used, in accordance with Brnich et al. 2020 (PMID: 31892348). See supplementary file “PS3 assays\_SGCG”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. PS3\_Supporting may be applied if the variant is expressed in heterologous cell lines/model organisms and shows absent membrane localization of the sarcoglycan protein complex and fewer than 11 control variants were used, in accordance with Brnich et al. 2020 (PMID: 31892348). See supplementary file “PS3 assays\_SGCD”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. For _CAPN3_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _CAPN3_. Assays that may be considered in the future include titin ectopic expression, titin degradation assay, baculovirus-based titin cleavage assay, and assays of autolytic activity.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. For _ANO5_, functional studies in heterologous systems are hard to conduct and rare in the literature. Therefore, PS3 is not applicable at this time for _in vitro_ assays for variants in _ANO5_. For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. PS3\_Supporting may be applied if the variant is expressed in heterologous cell lines/model organisms and shows absent membrane localization of the sarcoglycan protein complex and fewer than 11 control variants were used, in accordance with Brnich et al. 2020 (PMID: 31892348). See supplementary file “PS3 assays\_SGCA”.For any variant type, experimental evidence for altered splicing should be scored under PVS1 in accordance with the decision tree for RNA splicing assay results outlined in Walker et al. 2023 (PMID: 37352859).Apply PS3 only once, for the piece of evidence that meets the highest possible strength level. PS3 should be applied at the supporting level for the following approved functional studies and cutoffs except as noted in the PS3\_Moderate specification: 1. Luciferase assays for transactivation: less than 40% activity of wildtype (WT). Assays should include controls for WT, T2DM-risk, and known MODY variants. 2. EMSA for DNA binding: less than 40% activity of WT. We recommend that at least two of the following variants be used as positive controls for reduced DNA binding activity: c.335C>T (p.Pro112Leu), c.608G>A (p.Arg203His), c.787C>T (p.Arg263Cys) and c.686G>A (p.Arg229Gln) [⁴](#pmid_11162430), [⁵](#pmid_12574234), [⁶](#pmid_24915262). 3. Western blotting and indirect immunofluorescence for protein expression and localization - Determining appropriate thresholds for protein expression is more difficult due to variability in results between experimental protocols. Altered protein expression can be indirectly captured through the read-out frame from transactivation assay, and reduced protein expression can provide an explanation for reduced transactivation. When exploring protein mis-localization, we recommend that the c.589\_615del (p.Lys197\_Lys205del) variant is included as a positive control for impaired nuclear localization (cytosolic retention). Follow recommendations set forth by the SVI in conjunction with specifications added by the BMVCEP for quality metrics and minimum validation controls required (PMID: 31892348). Assays with OddsPath >2.1 as per the SVI recommendations.[¹](#PMID_31892348) Use when a variant fails to rescue an ATM specifc feature, only (e.g. phosphorylation of ATM-specific targets). Do not use for radiosensitivity-only as that is not a feature specific to ATM deficiency * Functional evidence from non-patient derived material with only a single variant best reflects the variant-level data. Apply patient-derived evidence in PP4.* Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart).* Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). If an enzyme activity assay has >20% activity it cannot be weighted above PS3\_supporting regardless of flowchart results. Use the ‘Funtional Assay SVI Documentation’. Functional studies with limited validation show a deleterious effect. In vitro cleavage assay shows failure or severely reduced capacity to produce either 5p or 3p microRNAs from a premiRNA (positive and negative controls also performed). AGAT activity data from an in vitro assay in which GATM variants were overexpressed in HeLa cells has been published (DesRoches et al, 2016; PMID 27233232). Any variant with AGAT activity at or below 15% of normal in this paper meets PS3\_Supporting (see Appendix 3 for further details on AGAT functional assays). PS3\_Supporting can be assigned if a variant is expressed in GAMT-deficient fibroblasts, HeLa cells and has \<15% of the control value, for values published in Mercimek-Mahmutoglu et al, 2014, PMID 24415674; Mercimek-Mahmutoglu et al, 2016, PMID 26319512; or DesRoches et al, 2016, PMID 26003046. * Creatine transport activity \<10% wild type with less than or equal to 125uM creatine used in SLC6A8 deficient fibroblasts* RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products. For non-canonical splicing variants, RT-PCR evidence demonstrating transcripts of alternative length or specific intron or exon inclusion/exclusion. These studies can be performed in patient derived cells, by placing the mutant genomic DNA into plasmid vectors, or by over-expressing mutant transcript. Assays should demonstrate defective splicing with RT-PCR analysis or RNA sequencing to confirm alternative transcripts. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section).* See included table for acceptable functional studies. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section). Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section).* See included table for acceptable functional studies. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section).* See included table for acceptable functional studies. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section).* See included table for approved functional studies. Well-established in vitro or in vivo functional studies supportive of a damaging effect.* RNA studies that demonstrate abnormal splicing and an inframe product (unless it affects an in-frame exon specified in the PVS1 section).* See included table for acceptable functional studies. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for SHOC2 at this time. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. One approved assay. Zebrafish knock-in model displays induced seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies Zebrafish knock-in model displays induced seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies Zebrafish knock-in model displays induced seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies Zebrafish knock-in model displays induced seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies Abnormal factor VIII activity (\<40 IU/dL or 40%) via one-stage or two-stage chromogenic assay in a cell line and/or mouse model.\--OR--Absent or significantly reduced factor VIII antigen levels compared to wildtype in a cell line by quantitative assay. Zebrafish knock-in model displays induced seizures, evidenced by hyperexcitability through electrophysiology or calcium imaging-based studies Well-established in vitro or in vivo functional studies supportive of a damaging effect. Not applicable for splicing effects (replaced by PVS1\_Strength (RNA))See attached table for acceptable functional studies. PS2_Strong Variant is de novo in a patient with the disease and no family history. Follow SVI guidance for de novo occurrences: https://clinicalgenome.org/working-groups/sequence-variant-interpretation/ Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11)).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. De novo (both maternity and paternity confirmed) observation in a patient with the disease and no family history. 2 points per tables 5a and 5b:Examples: 1 proven de novo occurrence; OR 2 assumed de novo occurrences. Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Only applicable when proband has a known pathogenic variant in trans with the de novo variant. One patient meets the HDGC individual phenotype criteria w/ parental confirmation. NA 4-7 points Not Applicable: De novo variants are rarely reported in GAA (PMIDs 7981676, 27142047). The occurrence of de novo variants in GAA is not a mechanism of disease for Pompe disease, and the observation that a variant in GAA has arisen de novo does not support its causality. Any de novo variants will be assessed based on the variant type, functional evidence, and in trans data as described in these guidelines. Use proposed SVI point recommendations.* Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 4-7 points De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history De novo (maternity confirmed or identical full mtDNA sequence) in a patient with the disease and no family history; with weighting per ClinGen SVI guidance Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase● Autosomal Dominant Disease: Do not use-Informative de novo occurrences have not yet been observed for autosomal dominant disease. As breast cancer is relatively common and occurs frequently as an apparently sporadic event, de novo is unlikely to ever be informative unless specific features of PALB2-related cancer predisposition are identified.● Autosomal Recessive Disease: Do not use - de novo occurrences are too rare to be informative at this time. In addition, in a biallelic state, de novo occurrences have an exceedingly low probability of being able to be confirmed as in trans because parental testing (and identification of one variant in each parent) is typically required without the use of long-range technologies. Phenotype highly specific for the gene (Danish Criteria) (≥2 but less than 4 _de novo_ points). Use proposed SVI point recommendations. See further instruction below. Total of 2 points. Use the SVI recommendations for de novo cases; 2 points. Use de novo guidance below to determine point value. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 2 points. Use proposed SVI point recommendations. See further instruction below. Total of 2 points. Use proposed SVI point recommendations. See further instruction below. Total of 2 points. Use proposed SVI point recommendations for “Phenotype highly specific for gene.” See de novo rule code guidance attached. Required 2 points. Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Use SVI-recommended point-based system with specifications for “Phenotype Consistency” per instructions. 2 Points. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 2-3.5 _de novo_ scores. For curation of _de novo_ score see **Tables 1** and **2**. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 2 points. Variant occurs de novo in an affected individual, and the biological relationship of the parent without the variant is confirmed e.g. if the father is not heterozygous for an IDUA variant that has been detected in the patient, paternity must be confirmed. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 2 Points. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[⁷](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to **Phenotype consistency: “Phenotype consistent with gene but not highly specific”**. Clinical judgment is required for shifting to a higher or lower category. For use as a STRONG or VERY STRONG criterion, ideally parents have been thoroughly clinically evaluated without evidence of cardiomyopathy (ideally using a combination of ECG and echocardiogram or cardiac MRI for maximum sensitivity).A family history consistent with _de novo_ inheritance should not have any clinical signs or symptoms suggestive of cardiomyopathy in a 1^st or 2^nd degree relative, for example: 1. Sudden death under 60 years of age2. Heart transplant3. Implantable cardiac defibrillator (ICD) under 60 years of age4. Features of cardiomyopathy (e.g., systolic dysfunction, hypertrophy, left ventricular enlargement in an individual without risk factors).5. Other related/overlapping cardiomyopathiesExamples of non-suspicious family history may include non-specific clinical features (e.g., palpitations, syncope, borderline/inconclusive echocardiogram findings, heart attack if age appropriate and suspected to result from coronary artery disease), but every attempt should be made to clarify features. Generally, this criterion is only applicable in the ABSENCE of any other possible disease-causing variants. If other pathogenic or likely pathogenic variants are present, consider decreasing points assigned or overall weight. ≥ 2.0 points Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 3 “Phenotype consistent with gene but not hightly specific and high genetic heterogeneity.” Confirmed _de novo_ with confirmed maternity worth 0.5 points per proband. Assumed de novo with assumed maternity worth 0.25 points per proband. Total 2.0 points to use at the strong strength. _De novo_ (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.* For PS2, both maternity and paternity must be confirmed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years). Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, _etc._ can contribute to non-maternity. Cases and parents genotyped by trio whole exome sequencing are considered to have confirmed maternity and paternity.* The _de novo_ variant in question must be coding or flanking.* The PS2 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 2-3.5 _de novo_ points Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). Use option 3 “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” (maximum 0.5 points/proband)* Total of 2.00 - 3.75 points required for Strong level Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:* (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.* (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.* (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 1 “Phenotype highly specific for gene.” Confirmed _de novo_ with confirmed maternity worth 2.0 points per proband. Assumed _de novo_ with assumed maternity worth 1.0 points per proband. Total 2.0 points to use at the strong strength. 2 Points. 2 Points. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. 2 - 3.5 _de novo_ points 2 - 3.5 _de novo_ points 2 - 3.5 _de novo_ points * Use PS2\_Moderate when there are 2 - 3 _de novo_ occurrences according to the attached Recommendation for determining the appropriate ACMG/AMP evidence strength level for de novo occurrences (Table 2).* The “phenotypic consistency” used on the attached Table 1 (Points awarded per _de novo_ occurrence) will be chosen for each proband by the number of phenotype points scored by the proband on the attached Phenotype scoring criteria per affected individual (Table 3):1. If the proband scores at least 4 and \<6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.2. If the proband scores at least 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.3. If the proband scores at least 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”.* PS2 can only be applied if the variant does not meet BA or BS1. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use SVI recommended scoring system, attached “PS2.PM6\_Scoring”. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific” for probands with Stargardt disease. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” for probands with other ABCA4-related retinopathies (e.g. - retinitis pigmentosa, cone-rod dystrophy, etc.). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 2.00 - 3.75 points required for Strong level De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. NA NA NA NA NA NA NA Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. Award the PS2_Strong point if Criteria 1 AND Criteria 2 are fulfilled. Criteria 1. The variant is present at a detectable allele fraction but is absent from parental samples with confirmed maternity and paternity. Criteria 2. The variant is present at a detectable allele fraction in an affected tissue sample but is absent from or detected at a lower allelic fraction in another tissue (e.g. if present in 5% of brain tissue but absent from the blood or skin this point can be awarded).For the sake of implementation, these criteria are intended to apply to high-confidence somatic mutations identified by the reporting CLIA laboratory. The expert panel recognizes that in practice there may be significant heterogeneity in the technical methods and thresholds used to identify such variants as 'high confidence', and flags the need to establish consensus statistical frameworks (e.g. Phred-scaled genotype qualities) or experimental approaches (e.g., confirmation of somatic variants by sequencing on orthogonal platforms) by which quality thresholds can be consistently applied. * ≥2 confirmed de novo in JOAG Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase Not Applicable Two-three points 2 points per tables 5a and 5b:Examples: 1 proven de novo occurrence; OR 2 assumed de novo occurrences.* OTOF and MYO15A are associated with autosomal recessive conditions. Therefore, de novo variants are expected to be an unlikely occurrence. It is recommended that de novo evidence is only awarded when phase with another variant (VUS, Likely Pathogenic, or Pathogenic) can be confirmed in trans. This is to avoid inappropriately awarding de novo evidence, which would lead to potentially incorrect classification.* Follow recommendations as specified by the Sequence Variant Interpretation working group within ClinGen, as outlined below * Determine number of points per proband using table 1 below. Sum the total number of points for all probands, and determine the strength of the evidence by using table 2. * Please note, the phenotype for de novo occurrences for MYO15A and OTOF are not considered “highly specific”. ≥2 but less than 4 de novo points Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity. Note: Confirmation of paternity in females only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.X-linked disorder. Only maternity needs to be confirmed. De novo (maternity and paternity confirmed) in a patient with the disease and no family history. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* 1 occurrence of PS2 De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* 1 occurrence of PS2. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* 1 occurrence of PS2. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* 1 occurrence of PS2. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* 1 occurrence of PS2. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use the SVI recommendations for de novo cases; 2 points. Use de novo guidance below to determine point value. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 2.00 - 3.75 points required for Strong level BS3_Strong Variant meets Level 1 benign functional study criteria. See Table 3.(1) Study of the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding, and LDL internalization) performed in heterologous cells (with no endogenous LDLR) transfected with mutant plasmid. Assay result of >90% of wild-type activity in expression/biosynthesis, binding AND internalization.Note: studies of only part of the LDLR cycle are not eligible for BS3 or BS3_Supporting. See PS3 specifications. Well-established _in vitro_ or _in vivo_ functional studies shows no damaging effect on protein function. To be applied to intronic or synonymous variants, RNA, mini-gene or other splicing assay demonstrating no splicing impact. NA NA Functional RNA studies demonstrating no impact on transcript composition. **BS3:** Transactivation assays demonstrating normal transactivation (80-115% of wt) AND data from a secondary assay demonstrating normal function. Functional on Kato et al. data **AND** no loss of function (LOF) by the majority of available eligible assays NA * Must demonstrate normal aggregometry in a transgenic mouse model. OR* In a heterologous cell line, must demonstrate BOTH normal expression and normal protein function NA Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. Not Applicable Not Applicable Not Applicable Not Applicable NA Not Applicable: Do not use: ● Protein functional studies (BS3) See PS3 for details ● RNA functional studies (Use BP7_Variable(RNA)) NA Must demonstrate normal aggregometry in a transgenic mouse model This code can be used for _F9_ gene variants studied in a cell line or mouse model setting that confer a normal factor IX activity AND normal factor IX antigen levels **OR** normal Western Blot. Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. Must demonstrate normal aggregometry in a transgenic mouse model Must demonstrate normal aggregometry in a transgenic mouse model Not Applicable: There are no available assays or model organisms that can recapitulate disease, and in vitro studies cannot dependably rule out pathogenicity. Applicable to non-canonical splice site variants that have RNA and in silico evidence of normal splicing. Applicable to non-canonical splice site variants that have RNA and in silico evidence of normal splicing (see BP4). Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. **RNA assay** of a synonymous or intronic variant in constitutional patient sample demonstrates no mRNA aberration**AND**biallelic expression is shown and/or nonsense-mediated decay inhibition was used. Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. NA Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. Assay measures effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of no damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as BP7 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. See PS3 specifications. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. Assay measures effect via protein only OR mRNA and protein combined. See Specifications Table 9 for code recommendations from calibrated published assays. Also see Figure1C and Appendix E for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of no damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as BP7 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. Not Applicable: Given that normal protein abundance and stability does not rule out impact on enzymatic activity, and that normal enzymatic activity for one substrate is not indicative of other substrates, the Glaucoma VCEP decided to not apply BS3. Not Applicable Well-established _in vitro_ or _in vivo_ functional studies show no damaging effect on protein function or splicing.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1](https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet BS3 if the variant is co-expressed with KCNE1 and if the variant current magnitude is within the normal range defined by the paper and is NOT statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for BS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for BS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05ORSynonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. * BS3 will be applied at the default level of strength for a normal result in an approved _in vitro_ assay with a calculated OddsPath \< 0.053 (as recommended in PMID: 31892348). Example assays have not yet been found in published reports at the time of these specifications, but are anticipated in the future as additional studies are published. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Use the BMPR2 functional assay document for acceptable assays and guidance. Note that Gly182Asp and Met186Val have been demonstrated _non-critical/not necessary_ for kinase activity based on a luciferase assay (apply BS3). We note that Glu503Asp has demonstrated lack of of effect on canonical signaling in one assay, this has not been replicated in an independent assay; in the absence of further investigation, this variants cannot be conclusively designated non-critical (do not apply BS3). While tested variants in the extracellular domain (p.Gln42Arg, Gly47Asn, Gln82His, Thr102Ala, Ser107Pro) have limited or no effect on canonical signaling, studies have indicated that they may play a role in disruption of non-canonical/SMAD-independent pathways (PMIDs: 14583445 and 16002577). In the absence of further investigation, these variants cannot be conclusively designated non-critical (do not apply BS3).Note that p.Cys34, Cys60, Cys66, Cys84, Cys94, Cys99, Cys116, Cys117, Cys118, Cys123, Gly210, Gly212, Lys230, Glu/Asn245, Asp333, Asn338, Asp351, Gly353 Glu386, Asp405, Gly410, Asp485, Gln486, Asp487, Ala488, Arg489, Ala490, Arg491, Arg491, and Leu492 have been demonstrated _critical_ (apply PM1\_strong). Not Applicable: The many secretion assays have not tested enough benign variants to meet the threshold in PMID 31892348 Supplemental Table 1 or Supplemental Table 2. NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. NA NA Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05ORSynonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05ORSynonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05ORSynonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. * BS3 may potentially be applied at the default strength level of strong for evidence from an animal model expressing the variant of interest and failing to recapitulate the APDS phenotype. Animal models will be reviewed on a case-by-case basis by the VCEP to determine the appropriate strength level. NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an MTM1 variant because of the numerous possible functions of myotubularin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. NA NA NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Applicable to non-canonical splice site variants that have RNA and in silico evidence of normal splicing. Follow recommendations set forth by the SVI in conjunction with specifications added by the Brain Malformation Group for quality metrics and minimum validation controls required. NA NA Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). Use the ‘Funtional Assay SVI Documentation’ (https://clinicalgenome.org/working-groups/sequence-variant-interpretation/).For step 2 assessment.* Functional studies deemed appropriate: * cDNA analyses showing altered FBN1 sequence. * Functional studies showing altered FBN1 protein or RNA expression, proteolysis, folding, assembly, trafficking, secretion, Ca2+ binding, matrix deposition (cfr Dave Hollister assay), microfibril fragmentation/catabolism in an in vitro engineered system.* Functional studies NOT deemed appropriate: non-specific altered TGF-beta signaling or histological hallmarks of medial degeneration, which are associated with many other types of variants in genes that are associated with MFS or HTAAD in general.For step 3 assessment: Studies should be performed in the presence of NMD inhibitor. NA For intronic or synonymous variants, no splicing impact observed via RNA assay. (Should be observed more than once.) NA NA NA Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.* RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data.* Not applicable for other functional studies. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic ID/ASD, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. This code can be used for _F8_ gene variants studied in a cell line or mouse model setting that confer a normal factor VIII activity level AND normal factor VIII antigen level OR normal Western Blot. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Not applicable for splicing effects (replaced by BP7_Strong (RNA)). BS3_Supporting Variant meets Level 3 benign functional study criteria. See Table 3.(1) Study of whole LDLR cycle in a) true homozygous patient cells, with assay result of >90% of wild-type activity in biosynthesis, binding AND internalization; or in b) heterozygous patient cells with assay result of >95% of wild-type activity in biosynthesis, binding AND internalization.(2) Luciferase studies with transcription levels of >90% when compared to wild-type (applicable to 5’UTR/promoter variants).(3) RNA studies, using RNA extracted from heterozygous or homozygous patientcells, with a) aberrant transcripts quantification, where aberrant transcript is<10% of total transcript OR b) without transcript quantification where noaberrant transcript is confirmed by sequencing.(4) Minigene splicing assay where only wild-type transcript is present and confirmed by sequencing.(5) High-throughput assays as defined above; only applicable when assay canindicate the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding AND internalization) is unaffected. See PS3 specifications. _In vitro_ or _in vivo_ functional study or studies showing no damaging effect on protein function.* Phosphatase activity >0 per Mighell et al. 2018, PMID: 29706350. Functional study shows no deleterious effect (predefined list).* Recommend that functional evidence is not used as strong evidence, due to the absence of well-established functional studies for hearing loss genes.* Guidance on functional evidence at supporting level is as follows (see functional spreadsheets attached): * GJB2: electrical coupling assays, dye transfer assays → BS3_Supporting * Dye Transfer Assays: Expect results that compare the fluorescence of a variant-transfected cell to both a negative control (or H2O injected control) and a wildtype-transfected cell. BS2_Supporting can be applied if the variant results in dye transfer comparable to the wildtype. * Electrical Coupling Assays: Expect results comparing the current of the variant-transfected cells to both a negative control (or H2O injected control) and a wildtype-transfected cell. BS2_Supporting would be applied if the variant results in a current comparable to the wildtype. * SLC26A4: Radio isotope and fluorescence assays → BS3_Supporting * Radio Isotope Assays: BS3_Supporting would be applied if the variant results in iodide efflux levels comparable to the wildtype. * Fluorescence assay: BS3_Supporting would be applied if the variant results in fluorescence comparable to the wildtype * COCH: Localization, secretion, and dimerization studies performed using immunofluorescence and Western blotting techniques → BS3_Supporting * Localization: BS3_Supporting would be applied if the variant results in extracellular deposits comparable to the wildtype. * Secretion: BS3_Supporting would be applied if the variant results in secretion comparable to the wildtype. * Dimerization: In a non-reducing environment, wildtype cochlin migrate quickly and appear smaller than in the reduced state because the structure is maintained by disulfide bonds. BS3_Supporting would be applied if the variant results in molecular weight and size comparable to the wildtype.* If not listed above, OK to use BS3_Supporting for other genes/functional analyses if * The assay has been validated by a known pathogenic and benign variant AND * There is plausible reason that the function the assay is testing relates to the phenotype AND * The assay conditions are likely to mimic the physiological environment. In vitro enzyme activity >85% compared to wild type* Expression systems: placing the mutant (and wildtype) cDNA into plasmid vectors and introducing these into host cells. Transiently transfected human or other mammalian host cells are the closest available approximation to the in vivo situation (e.g., COS cells) (Trunzo, et al. Gene. 2016. 594:138-143). NA **BS3\_Supporting:** Transactivation assays demonstrating normal transactivation (80-115% of wt). Partially functional on Kato et al. data **AND** no evidence of loss of function (LOF) by **all** available assaysBS3\_Supporting may also be applied to small deletions with available Kotler et al. data that are loss of function (LOF) by the majority of available assays Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. * >50% activity when the variant is expressed in a heterologous cell type, or >30% activity if there is also evidence of normal synthesis and processing. NA Well-established functional studies show no damaging effect on protein function * No significant increased sensitivity to RYR1 agonist in an approved in vitro assay, Ca2+ release measured, n≥3 * One or two independent ex vivo studies, NO significant release of Ca2+ in response to agonist * Knock-in mouse showing no MH reaction in response to RYR1 agonist AND no increased sensitivity to RYR1 agonists in ex vivo tissue/cells Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. Not Applicable Not Applicable Not Applicable Not Applicable No evidence of functional effect in cybrid studies or single fiber analysis is present (no statistically significant difference from control; mean values of <2 SD from control mean, or 50% enzyme activity compared to controls). Not Applicable: Do not use: ● Protein functional studies (BS3) See PS3 for details ● RNA functional studies (Use BP7_Variable(RNA)) • HIF 1/2a assay replicates WT function and/or• VBC complex stability is not affected and/or • ECM formation/fibronectin binding is unaffected This rule can be used and weighted as appropriate for functional tests of variants prior to codon 54 (which show the VHL19 product is not impacted). Evidence of benign effect for VHL Type 1 and 2A/B can be seen when HIF1/2a displays degradation (i.e. replicates WT function), and/or the VHL Elongin C, Elongin B, Cullin2 RBX1 (VCB-CR) E3 ubiquitin ligase complex stability is not affected and/or ECM formation/fibronectin binding is unaffected. Note: VHL Type 2C variants typically do not affect HIF1/2a; absence of HIF1/2a alone when testing a suspected VHL Type 2C variant should not be used for BS3. Functional studies of fibronectin and ECM formation are needed for VHL Type 2C. Follow modified SVI guidance for functional assays, general controls and benign controls. For splicing variants (and intronic/synonymous), RNA assays must demonstrate no impact on splicing. In a heterologous cell line, must demonstrate BOTH normal expression and normal protein function as compared to wildtype. NA Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. In a heterologous cell line, must demonstrate BOTH normal expression and normal protein function as compared to wildtype. In a heterologous cell line, must demonstrate BOTH normal expression and normal protein function as compared to wildtype. Not Applicable: There are no available assays or model organisms that can recapitulate disease, and in vitro studies cannot dependably rule out pathogenicity. List of approved functional studies and guidelines for interpretation of data.* EMSA for DNA binding * “No functional impact” is defined as ≥75% activity of wildtype * Note: the effect of the variant on DNA binding will be highly dependent on whether the variant is located within the DNA binding domain.* Luciferase assays for transactivation * “No functional impact” is defined as ≥75% activity of wildtype * Note: this threshold is not 100% specific for transactivation (TA) activity and is complicated by the fact that TA activity will vary depend on many factors, for instance cell line that is used (HeLa, INS, MIN6 etc). Assays should include controls for WT, T2DM and known MODY variants.* Western blotting and indirect immunofluorescence for protein expression (specifically levels and nuclear and cytoplasmic localization, respectively). * Determining appropriate thresholds for protein expression is more difficult due to variability in results due to the complexity of the technique.  Sample preparations, gel loading, transfer efficiency, specificity of the antibody, choice of internal control and inaccurate detection and quantification are some of the factors that can contribute to varying and inconsistent results. If a difference in protein expression compared to WT is seen by immunoblotting, then further testing by quantitative PCR (qPCR) is recommended in order to measure the mRNA level and assess whether the difference in amount of protein is due to a reduced mRNA level. Use GCK PS3 decision tree, which incorporates the relative activity index (RAI), relative stability index (RSI) and assays that measure the impact of variants on binding with GKRP and GKA.Evidence of no impact on function:* Normal RAI (>0.5) + normal RSI (>0.5) + normal inhibition/activation with GKRP/GKA = BS3\_Supporting * Normal RAI (>0.5) + normal RSI (>0.5) but no studies investigating GKRP/GKA = Cannot use PS3 or BS3Gloyn, et al. 2005 [⁵](#pmid_15677479); Beer, et al. 2012 [⁶](#pmid_22611063); Raimondo, et al. 2014 [⁷](#pmid_25015100); Gloyn, et al. (2004)¹². Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. **RNA assay** of a synonymous or intronic variant in constitutional patient sample demonstrates no mRNA aberration, without demonstration of biallelic expression or use of nonsense-mediated decay inhibition**OR****Protein assay** show retention of β-catenin regulated transcription activity comparable to wild-type (only for variants within the β-catenin binding domain, which refers to codons 959-2129 of _APC_, see PMID: 33348689) Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. The same assays outlined for PS3 will be used for BS3. Please see PS3 guidance for additional information on these assays. BS3\_Supporting can be applied for expression of IDUA sequence variants in cultured cells and subsequent measurement of enzyme activity provided that there is no other evidence to suggest that the variant could be disease-causing e.g. mislocalization (see Appendix 3 for thresholds for specific studies: >10% activity (Beesley et al, 2001, PMID: 11735025; Yogalingam et al, 2004, PMID: 15300847; Matte et al, 2003); >~10% activity / enzyme abundance (Yu et al, 2020. PMID: 33198351). NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. See PS3 specifications. NA See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. Not Applicable: Given that normal protein abundance and stability does not rule out impact on enzymatic activity, and that normal enzymatic activity for one substrate is not indicative of other substrates, the Glaucoma VCEP decided to not apply BS3. Not Applicable Well-established _in vitro_ or _in vivo_ functional studies show no damaging effect on protein function or splicing.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1](https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet BS3 if the variant is co-expressed with KCNE1 and if the variant current magnitude is within the normal range defined by the paper and is NOT statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for BS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for BS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. The strength of evidence from cellular models/_in vitro_ studies is dependent upon the level of expressed ADA enzyme activity based on levels defined in Arredondo-Vega et al., 1998 (PMID: 9758612): * BS3\_Supporting: Expressed ADA enzyme activity ≥4.8% of wild-type activity (based on group IV). Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48ORVariant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart\*. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. Not applicable for splicing effects (replaced by BP7\_Strong (RNA)).See attached table for list of acceptable functional studies.For studies reporting isomerohydrolase activity, activity must be ≥50% of wild-type control. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. * A normal result for a _CTLA4_ variant using one of the approved assays below cannot be interpreted to mean normal CTLA4 function by itself. BS3\_Supporting can only be applied when a second different assay also shows a normal result.* Functional assays can be found in the attached Excel file entitled CTLA4\_Functional Assay\_Research\_PS3\_BS3.* Although suspected benign variants are not present in all of the PMIDs below, assays approved by the VCEP for BS3\_Supporting are: * (1) CD80 or CD86 transendocytosis / soluble CD80 or CD86 ligand uptake in non-patient cells (i.e. PMID: 25329329, PMID: 25632005, PMID: 25367873, PMID: 27102614, PMID: 29375547, PMID: 15814706, PMID: 20870175) * (2) Cell surface expression of CTLA4 expressed in non-patient cell lines (i.e. PMID: 25367873, PMID: 14578884, PMID: 29375547, PMID: 7559643, PMID: 25213377) * (3) Protein localization / translocation by CTLA4 expressed in non-patient cell lines (i.e. PMID: 7559643, PMID: 20870175, PMID: 27102614, PMID: 25367873, PMID: 29375547, PMID: 15814706) * (4) _In vitro_ T cell suppression by CTLA4 expressed in non-patient cell lines (i.e. PMID: 29375547, PMID: 26478010, PMID: 25213377)* Please note that patient cell-based assays of CTLA4 function were also considered for inclusion in BS3\_Supporting, but were instead viewed as part of the proband phenotype. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. For variants that have demonstrated limited or no effect on canonical signaling but not tested for effect on non-canonical/SMAD-independent pathways (PMID: 14583445 and 16002577). Not Applicable: The many secretion assays have not tested enough benign variants to meet the threshold in PMID 31892348 Supplemental Table 1 or Supplemental Table 2. Assays approved by the VCEP sufficiently evaluate the functional impact of a given variant for the application of PS3\_Supporting. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. * mRNA splicing assays * Intracellular signaling assays: BRE/CAGA-luciferase, Gal4 Smad1/Smad3 for TGF-beta/BMP9 signaling* Binding assays: BMP9 binding, transcription factor Sp1, BMP9 protein-protein interaction (BLI)* Subcellular protein localization* Morphology: Morphology & actin cytoskeleton, tubulogenesis * mRNA splicing assays * Intracellular signaling assays: BRE/CAGA-luciferase, Gal4 Smad1/Smad3 for TGF-beta/BMP9 signaling* Binding assays: BMP9 binding, transcription factor Sp1, BMP9 protein-protein interaction (BLI)* Subcellular protein localization* Morphology: Morphology & actin cytoskeleton, tubulogenesis Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48ORVariant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart. Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48ORVariant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart. Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48ORVariant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart. * BS3\_Supporting can be applied based on a normal result in at least two different approved _in vitro_ assays (from two separate approved assay classes). These are described in detail in the attached Excel file entitled “PIK3CD\_Functional\_Assays\_PS3\_BS3”.* Approved assay classes and specific assay instances: * AKT kinase activity assay (PMID: 24136356, PMID: 24165795, PMID: 28414062) * Lipid kinase activity (PMID: 24136356, PMID: 28167755, PMID: 28414062) * Enrichment of the variant in high phospho-S6 and phospho-AKT T cells relative to low phospho-S6 and phospho-AKT T cells, within a next generation sequencing-based screen of donor T cells subjected to of CRISPR-mediated adenine base-editing (PMID: 40543502) * Lipid vesicle affinity (PMID: 24136356) * Conformational dynamics (PMID: 28167755; PMID: 28414062)* Please note that protein binding assays testing PIK3CD binding to PIK3R1 were also considered for inclusion in BS3\_Supporting, but were excluded based on lack of evidence of altered binding by disease-associated variants compared to wild-type controls (PMID: 24165795; PMID: 28414062).* Patient cell-based functional assays directly showing normal activity of the disease-relevant PI3K delta pathway were considered for inclusion in the BS3\_Supporting code but are viewed as part of the proband phenotype instead. Two specific assays are currently suggested to be applied at Supporting:1. Thin filament structure: A normal readout consists of no significant difference in intensity reflections of X-ray diffraction patterns generated by muscle fibers from patient biopsies compared to WT.2. _In vitro_ motility: A normal readout consists of no a significant difference in speed of single fibers derived from patient muscle compared to WT. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. BS3\_Supporting is met if **both** of these assays have WT readouts:Oligomerization Assay: DNM2 assembly/disassembly dynamics similar to wild type DNM2GTPase Activity Assay: GTPase activity and stability similar to wild type DNM2 Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an MTM1 variant because of the numerous possible functions of myotubularin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. Yeast growth assays in which the ARG3 open reading frame of a yeast strain is replaced with human OTC coding sequence, and site directed mutagenesis utilized to introduce variant of interest (PMID: 37146589) BS3\_Supporting is applicable when growth of the variant strain is >65% and \<80% growth when compared to strains containing the wildtype OTC gene. See list of approved functional assays in PS3/BS3 Guidance spreadsheet below. Not applicable for splicing effects (replaced by BP7\_Strong (RNA)).For studies reporting guanylate cyclase activity, cutoff is >50% of wild-type control. Studies included; Peshenko et al., 2020[¹](#pmid_33109612), Feng et al., 2020, Jacobson et al., 2021[²](#pmid_33997691), Jacobson et al., 2020[³](#pmid_36274938), Tucker et al., 2004[⁴](#pmid_15123990), Jacobson et al., 2013[⁵](#pmid_23035049), and Peshenko et al., 2015[⁷](#pmid_26100624)See attached table of Approved PS3 Functional Assays for a list of acceptable functional studies including those that:* Report failure of GUCY2D to localize to the cell surface : Zulliger et al., 2015[⁶](#pmid_25477517).* Report failure of GUCY2D to bind RD3: Zulliger et al., 2015[⁶](#pmid_25477517). * Report failure of GCAP1, GCAP2, or RD3 to co-localize with GUCY2D at the cell surface: Peshenko et al., 2020[¹](#pmid_33109612), Jacobson et al., 2020[³](#pmid_36274938), and Peshenko et al., 2015[⁷](#pmid_26100624). Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. BS3 should be applied at the supporting level for the following approved functional studies and cutoffs: 1. Luciferase assays for transactivation: ≥ 75% activity of wildtype 2. EMSA for DNA binding: ≥ 75% activity of wildtype. 3. Western blotting and indirect immunoflorescence for protein expression and localization - Determining appropriate thresholds for protein expression is more difficult due to variability in results between different experimental protocols. Altered protein expression can be indirectly captured through the read-out from a transactivation assay and reduced protein expression can provide an explanation for reduced transactivation. Follow recommendations set forth by the SVI in conjunction with specifications added by the Brain Malformation Group for quality metrics and minimum validation controls required. Applies to variants showing solubility or secretion in functional assays for studies with OddsPath \<0.48 as per the SVI recommendations. Use when a variant rescues EITHER an ATM specifc feature OR rescues radiosensitivity. Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). NA Functional study shows no deleterious effect (none are defined for OTOF and MYO15A).* Recommend that functional evidence is not used as strong evidence, due to the absence of well-established functional studies for hearing loss genes.* No specific assays are listed for OTOF or MYO15A. However, BS3_Supporting can be used for functional analyses if * The assay has been validated by a known pathogenic and benign variant AND * There is plausible reason that the function the assay is testing relates to the phenotype AND * The assay conditions are likely to mimic the physiological environment. An in vitro cleavage assay must demonstrate the variant produces both 5p and 3p microRNAs from a pre-miRNA (positive and negative controls also performed). An example of an appropriate assay to which criteria could be applied is Wu et al. 2018[⁷](#pmid_28862265). In vitro assays in which a variant is expressed in AGAT-deficient cultured cells (e.g. AGAT-deficient fibroblasts) or in-fusion High-Fidelity cloning of GATM transcript and site directed mutagenesis to generate missense variant overexpressed in HeLa cells and measurement of AGAT activity in cells for wild-type and missense variant. Any variant with enzyme activity at or above 30% of normal in DesRoches et al, 2016, PMID 27233232, meets BS3\_Supporting. In vitro assays in which a variant is expressed in GAMT-deficient cultured cells (e.g. GAMT-deficient fibroblasts) or in-fusion High-Fidelity cloning of GAMT transcript and site directed mutagenesis to generate missense variant overexpressed in HeLa cells and measurement of GAMT activity in cells for wild-type and missense variant. Any variant with enzyme activity at or above 30% of normal in the following publications meets BS3\_Supporting (Mercimek-Mahmutoglu et al, 2014; PMID 24415674; Mercimek-Mahmutoglu et al, 2016, PMID 26319512; DesRoches et al, 2016, PMID 26003046). * Creatine transport assay demonstrating ≥50% normal transport activity using physiological creatine concentrations (≤125μM creatine).* RT-PCR evidence demonstrating no observable effect of splicing.* Expression assay demonstrating wild type transcript levels NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic ID/ASD, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. This code can be used for _F8_ gene variants studied in a cell line or mouse model setting that confer the following results: * Normal factor VIII activity level, OR* Abnormal factor VIII activity level with abnormal 2N binding assay suggesting a diagnosis of VWD Normandy (VWD 2N) instead of hemophilia A. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Not applicable for splicing effects (replaced by BP7_Strong (RNA)). PP5_Very Strong Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PP5_Strong Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PP5_Supporting Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PP5_Moderate Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. BP7_Supporting Variant is synonymous.Caveat: variant must also meet BP4 (i.e. no predicted impact on splicing). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). A synonymous (silent) or intronic variant at or beyond +7/-21 for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice. Silent variant with no predicted impact to splicing.No changes. Follow recommendations as outlined in Richard 2015 and/or ClinGen's Sequence Variant Interpretation working group. Per SVI recommendations (PMID: 36865205), use BP7 only if BP4 is met; for variants with experimental evidence supporting that they do not alter splicing, use BP7\_strong (RNA)* intronic variants must be outside +7/-21 nt* exonic variants must be outside first and last 3 bases of exon Synonymous and intronic variants at or beyond +7 to -21 locations. **BP7:** Applicable for* Synonymous variants – excluding those in the last 3 nucleotides preceding a canonical donor splice site or the first nucleotide following a canonical acceptor splice site – with SpliceAI ∆ scores ≤ 0.20.* Intronic variants with SpliceAI ∆ scores ≤ 0.20.**BP7\_Variable (RNA):** * Applicable for variants with RNA data, with weighting based on the quality of the available RNA data. A synonymous (silent) outside of the core splice motif (last three nucleotides and first nucleotide of the exon) or intronic variant at or beyond +7 to -21 positions for which SpliceAI predicts no impact to the splice consensus nor the creation of a new splice site (BP4 is met, SpliceAI ≤ 0.1). No requirement to assess for nucleotide conservation for rule application as per evidence and recommendations in Walker et al., 2023 (PMID: 37352859). A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Use with no specification. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved A synonymous (silent) variant. * BP7: Use for synonymous and deep intronic variants defined as further than (but not including) +7 and further than (but not including) -21 at donor and acceptor sites, respectively.* BP7(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. To evaluate splice prediction, use the BP4 code. If BP4 is met for lack of splice effect, BP7 can be applied to silent or intronic variants where the PhyloP score is ≤0.2. Use SpliceAI to rule out possible splicing defect (score = 0.2 or less) and reference PhyloP (score = 1.5 or less) to assess conservation. Splicing prediction score of less than or equal to 0.1 is required. Conservation should be assess using PhyloP (cutoff less than 0.1) and PhastCons (cutoff less than 0.5). Use SpliceAI for splicing predictor with a cutoff score of \<0.1. Use SpliceAI to rule out possible splicing defect (score = 0.2 or less) and reference PhyloP (score = 1.5 or less) to assess conservation. Use SpliceAI to rule out possible splicing defect (score = 0.2 or less) and reference PhyloP (score = 1.5 or less) to assess conservation. Use Splice AI to rule out a predicted splicing effect (less than or equal to 0.1). Evolutionary conservation is defined as a PhyloP > 0.1 **OR** the reference nucleotide is present in 3 mammals or 1 primate. Apply BP7 when the predicted change from SpliceAI is below 0.2 AND phyloP100 way \< 2.0. Apply BP7 when the predicted change from SpliceAI is below 0.2 AND phyloP100 way \< 2.0. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. Not Applicable: Does not apply. A synonymous (silent) or intronic variant at or beyond +7/–21 for which multiple splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site. Use SpliceAI for splicing predictor with a cutoff score of 0. BP7 can be applied if the variant is synonymous, unless the variant is in the first nucleotide or last three nucleotides of an exon, AND BP4 is met i.e. SpliceAI predicts no impact on splicing (score ≤ 0.10) Silent variant inside a (potentially) clinically important functional domain, IF BP4 met.Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.See Specifications Figure1A and Appendix J for additional details.As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA1 RING aa 2-101; BRCA1 coiled-coil aa 1391-1424; BRCA1 BRCT repeats aa 1650-1857. See Specifications Figure1A and Appendix J for details. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[⁶](#pmid_25741868). Silent variant inside a (potentially) clinically important functional domain, IF BP4 met.Intronic variants located outside conserved donor or acceptor motif positions (at or beyond positions +7/-21) IF BP4 met.See Specifications Figure1A and Appendix J for additional details.As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure1A and Appendix J for details. Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Also applicable to **intronic variants outside the splice consensus sequence (-4 and +7 outward)** for which splicing prediction algorithms predict no impact to the splice consensus sequence NOR the creation of a new splice site AND the nucleotide is not highly conserved.Rule can be combined with BP4 to make a variant likely benign per Richards _et al._ 2015[¹](#pmid_25741868). Applies to intronic/noncoding variants outside the donor/ acceptor splice region (intronic variants at or beyond positions +7/-21) and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon if BP4 is met. Applies to:Synonymous variants or noncoding variants with no impact on splicing (SpliceAI ≤ 0.2)ANDPhyloP \< 0 for conservation A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Use SpliceAI to look for suspected splicing defects (cutoff Δ score \<0.1).* Use PhyloP to check for conservation (cutoff \<2.0).* Please note that SpliceAI Δ score \<0.1 AND PhyloP score \<2.0 must both be met.* This code can be met by synonymous variants except for variants in the last 3 nucleotides of an exon, or in the 1st nucleotide of an exon.* This code can be met by intronic variants except for variants in positions +1 to +6 or -1 to -20, which are considered part of the splice donor and splice acceptor sites (PMID: 37352859). A synonymous variant, or deep intronic variant affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions, for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site (SpliceAI ∆ score ≤0.1) AND the nucleotide is not highly conserved. * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is **not required** in order to apply BP7. A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4. * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is **not required** in order to apply BP7. * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is not required in order to apply BP7. Use for variants located outside of the donor/acceptor +/- 1,2 dinucleotide positions. If SpliceAI score ≤0.1, apply BP4 followed by assessment of BP7. See RPE65-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Positions **excluded** from BP7: * Synonymous substitutions at the first base of an exon * Synonymous substitutions in the last 3 bases of an exon * +1 through +7 of donor sequence * \-1 through -21 of acceptor sequence * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is not required in order to apply BP7. * Apply only if BP4 is met.* Applicable to both synonymous variants and intronic variants not predicted to impact splicing by SpliceAI (SpliceAI Δ score \<0.1, PMID: 37352859).* Only applicable for intronic/non-coding variants located outside the donor/acceptor splice region (conservatively designated as intronic variants at or beyond positions +7/−21) or for synonymous exonic variants located outside of the first nucleotide or the last 3 nucleotides of the exon (PMID: 37352859). * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is **not required** in order to apply BP7. * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* The variant should be predicted not to impact splicing by at least two out of three _in silico_ tools (freely available tools include GeneSplicer, MaxEntScan, NNSplice, SpliceAI, Splicing Sequences Finder (SSF), and varSEAK).* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is **not required** in order to apply BP7. A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Applicable after assignment of BP4 for no adverse splicing predictions, and inclusive of exonic and intronic variants. Not applicable for synonymous variants located at the first base or the last three bases of an exon. Applies to:Synonymous variants or noncoding variants with no impact on splicing (SpliceAI ≤ 0.1)ANDPhyloP \< 0.1 for conservation A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. * Applicable to both synonymous variants and deep intronic variants affecting nucleotides at or beyond the +7 (donor) and -21 (acceptor) positions.* Given the potential for poor conservation of genes related to T cell and B cell development among vertebrates, nucleotide conservation is **not required** in order to apply BP7. A synonymous or intronic variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site. Can be used together with BP4 evidence. A synonymous or intronic variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site. Can be used together with BP4 evidence. A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4. A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4. A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4. * Met for a synonymous variant if SpliceAI does not predict a splicing defect (cut-off \<0.1).* Synonymous variants in the first nucleotide of an exon or the final 3 nucleotides of an exon are not eligible for this code, as they are considered part of the splice region.* Met for an intronic variant if SpliceAI does not predict a splicing defect (cut-off \<0.1).* Intronic variants between +1 and +6 or between -1 and -20 are not eligible for this code, as they are considered part of the splice region (PMID: 37352859). A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Splicing / intronic variant -- applicable for intronic variants with spliceAI \<0.10 if the variant under review is outside splice region +7/-21 nucleotides.Synonymous variants -- applicable when BP4\_Supporting is met and only if the synonymous variant is outside the first base of an exon and last 3 bases of an exon. NA Use not only for synonymous variants but also for intronic variants located outside of the donor/acceptor +/- 1,2 dinucleotide positions. If SpliceAI score ≤0.1, apply BP4 followed by assessment of BP7. See GUCY2D-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Positions **excluded** from BP7: * Synonymous substitutions at the first base of an exon * Synonymous substitutions in the last 3 bases of an exon * +1 through +7 of donor sequence * \-1 through -21 of acceptor sequence A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which SpliceAI predicts no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). For splice predictions, use SpliceAI with a score ≤0.05. BP7 may be co-applied with BP4 for synonymous, UTR, and intronic variants located outside the splice donor/acceptor regions designated in Walker et al. 2023 (+6/-3 for donor; +1/-20 for acceptor). Apply BP7 when the predicted change from SpliceAI is below 0.2 AND phyloP100 way \< 2.0. For synonymous, intronic positions (except canonical splice sites) and non-coding variants in the UTRs, if the nucleotide is non-conserved award this point (PhyloP score <0.1). Apply to intronic/noncoding and synonymous (silent) exonic variants if BP4 is met * BP7: Use for synonymous and deep intronic variants defined as further than (but not including) +7 and further than (but not including) -21 at donor and acceptor sites, respectively.* BP7(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. * A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved* Can be used for intronic variants that fall outside the minimal splice region (≥+7 or ≤-21)* Can be used with BP4 code. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.BP7 applicable as described. Silent variant with no predicted impact to splicing.* No changes. Follow recommendations as outlined in Richard 2015 and/or ClinGen's Sequence Variant Interpretation working group. Silent variantOR Intronic variant at or beyond +7 to -21 positionsOR Other intronic or non-coding variant if the variant is the reference nucleotide in ≥1 primate and/or ≥4 mammalian species.Caveat: Variant must meet BP4 to apply BP7 A synonymous (silent) variant OR an intronic variant at or beyond positions +7 and -21, for which SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/), predicts no impact on splicing (score \<0.1) (see Walker et al, 2023, PMID: 37352859). A synonymous (silent) variant OR an intronic variant at or beyond positions +7 and -21, for which SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/), predicts no impact on splicing (score \<0.1). A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.* Defined 'not highly conserved' regions in BP7 as those with PhastCons score \<1 and/or PhyloP score \<0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species.* For splice site variants use SpliceAI with a score ≤ 0.1. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4. A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Splice AI should be used to suggest no splicing impact. Splicing prediction score of less than or equal to 0.1 is required. Conservation should be assess using PhyloP (cutoff less than 0.1) and PhastCons (cutoff less than 0.5). A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. Use not only for synonymous variants but also for intronic variants located outside of the donor/acceptor +/- 1,2 dinucleotide positions. If SpliceAI score ≤0.1, apply BP4 followed by assessment of BP7. See AIPL1-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Positions **excluded** from BP7: * Synonymous substitutions at the first base of an exon * Synonymous substitutions in the last 3 bases of an exon * +1 through +7 of donor sequence * \-1 through -21 of acceptor sequence PP3_Supporting REVEL score ≥0.75 (missense variants), or predicted impact to splicing using MaxEntScan (see Fig. 2 for suggested thresholds). As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹⁴](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. Multiple lines of computational evidence support a deleterious effect on the gene or gene product.* Splicing variants: Concordance of SpliceAl and VarSeak* Missense variants: REVEL score > 0.7 REVEL score ≥0.7, or predicted impact to splicing using MaxEntScan.* Use REVEL and MAXENTSCAN. * For missense variants, award PP3 if REVEL score is ≥0.7. * If splicing is predicted to be impacted, either creation of a cryptic splice site, or disruption of a native splice site, award PP3.* For splice variants (except for canonical -/+1 or 2), use MAXENTSCAN. * For -/+ 1 or 2 splice variants, do not use PP3 if you are using PVS1. Applicable as described in Pejaver et al. (PMID: 36413997):* REVEL score of 0.644 - 0.733 for missense variants* In frame deletion or insertion predicted deleterious by 2 out of 3 tools (PROVEAN, MutationTaster, MutPred-InDel)* Predicted impact on splicing by SpliceAI (score >0.5) At least three in silico splicing predictors in agreement (SpliceAI, MaxEntScan, SSF, GeneSplicer, HSF, TraP, varSEAK). **For missense variants:****PP3:** REVEL score ≥ 0.88 or SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites.**For synonymous and intronic (intron 4-8) variants:****PP3:** SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites.Caveats:\*Do not use for variants with a predicted splicing effect that is proven by RNA analysis. See **PVS1\_Variable (RNA)**.\*Do not use for for canonical splice site variants. **Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_aGVGD class C25-C55 and BayesDel score ≥ 0.16**Single amino acid inframe deletions** _(See single aa BayesDel spreadsheet)_BayesDel score ≥ 0.16**Exonic (including silent variants and apparent “missense” variants or “single amino acid inframe deletions” for which there is a predicted splice effect) or Intronic Splice Variants (excluding ± 1,2 positions):**SpliceAI ≥ 0.2 Multiple lines of computational evidence support a deleterious effect on the gene or gene product. * REVEL score >0.7 for missense variants. * In frame deletion or insertion predicted deleterious by 2 out of 3 tools (PROVEAN, MutationTaster, MutPred-InDel). * Predicted impact on splicing by SpliceAI (score >0.5). REVEL score of ≥ 0.7 OR>2 independent in silico missense predictors predict a damaging impact NA Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant. No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively Agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively * Will also utilize POLG pathogenicity prediction server if/when live again (PMID: 28480171); both tools (REVEL and server) will have to be in agreement to score No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 f or PP3 and BP4, respectively Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, etc) * Missense: Do not use.* Splicing: Predicted impact via splicing (SpliceAI **≥0.2**) for silent, missense/in-frame and for intronic variants outside of donor and acceptor 1,2 sites. For missense variants, use REVEL score >=0.664. For splice variants, concordance of Splice AI (>0.5) and VarSeak (class 4 or class 5). REVEL score of ≥0.644 (to \<0.773), based on recommendations of Pejaver et al., 2022 (PMID: 36413997).OR suggested splicing effect using SpliceAI greater than or equal to 0.5. Code can be applied for variants where the REVEL score is greater than or equal to 0.6 or a SpliceAI score of greater than or equal to 0.2. Appropriate to use for missense variants that have a REVEL score of greater or equal to 0.644 OR a SpliceAI score suggestive of a splicing defect (greater or equal to 0.5). REVEL score of ≥0.644 (to \<0.773), based on recommendations of Pejaver et al., 2022 (PMID: 36413997).OR suggested splicing effect using SpliceAI greater than or equal to 0.5. REVEL score of ≥0.644 (to \<0.773), based on recommendations of Pejaver et al., 2022 (PMID: 36413997),OR suggested splicing affect using SpliceAI greater than or equal to 0.5. Appropriate to use for missense variants that have a REVEL score of greater or equal to 0.6. For potential splicing variants, SpliceAI must predict a damaging impact with a score greater or equal to 0.5. Use REVEL score of ≥0.70 as supportive evidence of pathogenicity. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply PP3 when the predicted change is at least 0.2 [⁴](#pmid_32123317),[³](#pmid_30661751). Use REVEL score of ≥0.70 as supportive evidence of pathogenicity. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply PP3 when the predicted change is at least 0.2[⁹](#pmid_32123317),[¹⁰](#pmid_30661751) For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. Multiple lines of computational evidence support a deleterious effect on the gene orgene product (conservation, evolutionary, splicing impact, etc).* Use RNAsnp to access SNP effects on local RNA secondary structure ([https://rth.dk/resources/rnasnp/](https://rth.dk/resources/rnasnp/))* Use only for Single Nucleotide Polymorphism inside the gene (do not use for promoter sequence) * Parameters utilized: * Input sequence: FASTA (Ensembl NR\_\_003051.3) * Insert SNP details * Mode: 1 * Folding window: 100 nt * Measure: “Distance” option * Minimum length of the sequence interval: 50 * Cut-off the base pair probabilities: 0.01The p-value threshold significance should be considered 0.1 according to Sabarinathan et al., 2013. PMID: 23315997. If the value is less than 0.1, apply PP3 as a supporting level of evidence. **Missense variants:** Do not use computational prediction models for conservation, evolution, etc. _In silico_ splicing predictors should be used for presumed missense variants to reveal possible splicing effects.**Non-canonical splicing variants:** Multiple _in silico_ splicing predictors support a deleterious effect. Appropriate to use for missense variants that have a REVEL score of greater or equal to 0.644 OR a SpliceAI score suggestive of a splicing defect (greater or equal to 0.5). * Any missense changes with a REVEL score between 0.644 - 0.773 will meet PP3 (Pejaver et al., PMID: 3641399)* For in frame insertions and deletions, use Mutation Taster ([http://www.mutationtaster.org/](http://www.mutationtaster.org/) ; count if “disease-causing”), and MutPred-Indel ([https://mutpred2.mutdb.org/mutpredindel/](https://mutpred2.mutdb.org/mutpredindel/) ; score >0.5 for “pathogenic”). Apply PP3 if both predictors indicate that the variant is deleterious.* For non-canonical splice site variants (e.g., +3, -3), use SpliceAI ([https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) ). A score of >0.2 is taken to indicate disruption of the splice site allowing PP3 to be applied (based on Walker et al, PMID: 37352859). Evidence for use of a cryptic splice site and the impact on the gene product should also be assessed. PP3 for splicing variants will be applied only in the absence of functional (RNA analysis) data. Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.28). As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA1 RING aa 2-101; BRCA1 coiled-coil aa 1391-1424; BRCA1 BRCT repeats aa 1650-1857.Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.See Specifications Figure1A and Appendix J for details. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹⁷](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[²](#pmid_30661751) is recommended for evaluation of predicted splice impacts. Apply PP3 for missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain and predicted impact via protein change (BayesDel no-AF score ≥0.30). As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186.Apply PP3 for predicted splicing (SpliceAI ≥0.2) for silent, missense/in-frame (irrespective of location in clinically important functional domain) and for intronic variants outside of donor and acceptor 1,2 sites.See Specifications Figure1A and Appendix J for details. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≥0.70 for PP3**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. For missense variants: SpliceAI ≥ 0.2 OR REVEL score of 0.644-0.772For all other variants located outside of donor/acceptor ±1,2 dinucleotide positions, when splicing assay is not available: SpliceAI ≥ 0.2 Applies to missense variants with a REVEL score between 0.644 and 0.772 that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2).For variants with SpliceAI scores greater than or equal to 0.2, in the two first or last two bases in an exon or the 6 intronic bases flanking an exon, this code is met at the supporting level. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc)* REVEL and SpliceAI are the preferred predictors* Apply this code if REVEL score is greater than or equal to 0.75.* Use SpliceAI to look for a possible splicing defect. Apply this code if delta score for donor gain, donor loss, acceptor gain, or acceptor loss is greater than or equal to 0.2 (PMID: 37352859). * Supporting evidence can be applied for a REVEL score of ≥0.644 (to \<0.932), based on recommendations of Pejaver et al., 2022 (PMID: 36413997).* Also applicable to missense, synonymous, or intronic variants predicted to impact splicing by SpliceAI ∆ score ≥0.2 * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** Missense variant with HCI prior probability for pathogenicity >0.68 & ≤0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS)ORPredicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** * For a missense variant use REVEL, requires a score of 0.644 - 0.773.* For a predicted splicing variant use SpliceAI with max distance set to 500 bp. Highest delta score from SpliceAI must be ≥ 0.2 to use this code. See RPE65-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart. * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** * Met at the default (PP3) level by a missense variant with REVEL score greater than or equal to 0.75 and CADD PHRED score greater than or equal to 20.* The above requirement for agreement between two _in silico_ tools was reached following a pilot study of suspected pathogenic and suspected benign variants (assembled from ClinVar submissions and published assertions of pathogenicity). Some suspected pathogenic _CTLA4_ variants fall in REVEL’s benign range while some suspected benign _CTLA4_ variants fall in CADD’s pathogenic range. All false positive and false negative predictions in the test set could be avoided by requiring that both tools agree in order for PP3 or BP4 to be Met.* Met by missense, synonymous, or intronic variants outside the +/-1,2 dinucleotide positions that are predicted as damaging using SpliceAI (cutoff Δ score greater than or equal to 0.2). * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** * Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** Two out of three REVEL/AlphaMissense/CADD predictor scores must meet the thresholds for supporting as specified in Pejaver 2022 PMID: 36413997 (CADD) and Bergquist et al 2025 PMID: 40084623 (AlphaMissense, REVEL). CADD ≥25.3, AlphaMissense ≥0.792, REVEL ≥0.644. The criterion can also be used for non-canonical splice variants if SpliceAI ≥0.2. Applies to missense variants with a REVEL score between 0.644 and 0.772 that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2). For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. Only applicable to synonymous or intronic variants predicted to impact splicing by SpliceAI with a delta score greater than or equal to 0.2.* **Do not apply to missense variants.** * For missense variants: REVEL score ≥0.644 or SpliceAI score ≥0.2.* For synonymous and intronic variants: SpliceAI score ≥0.2. * For missense variants: REVEL score ≥0.644 or SpliceAI score ≥0.2.* For synonymous and intronic variants: SpliceAI score ≥0.2. Missense variant with HCI prior probability for pathogenicity >0.68 & ≤0.81 as per https://hci-priors.hci.utah.edu/PRIORSORPredicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. Missense variant with HCI prior probability for pathogenicity >0.68 & ≤0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) ORPredicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. Missense variant with “MAPP/PP2 Prior P” score >0.68 & ≤0.81 from [http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique](http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique) ORPredicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. * Met at the default (PP3) level by a missense variant with REVEL score greater than or equal to 0.644 and CADD PHRED score greater than or equal to 25.3.* Higher strength levels of PP3\_Moderate or PP3\_Strong have not been recommended due to pilot data indicating poor performance and significant discordance between REVEL and CADD predictions for established benign and gain-of-function _PIK3CD_ variants.* SpliceAI scores should be checked for missense variants, but there currently is no evidence of variants causing autosomal dominant disease through a splicing mechanism, so PP3 cannot be met by a variant located in the boundary exonic/intronic region with a predicted splicing impact. A variant with a predicted impact on splicing (SpliceAI Δ score greater than or equal to 0.2) should not be evaluated using these specifications, but rather using alternative specifications for autosomal recessive disease with a loss-of-function mechanism, unless functional evidence indicates a gain-of-function effect. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Missense variants - REVEL score ≥0.644 and \<0.773 Splice region and intronic variants - spliceAI delta score ≥0.20 This code is applicable for missense variants with a REVEL score between 0.644-0.772 or for synonymous or indel variants with a CADD score between 25.3-28.0 (https://www.medrxiv.org/content/10.1101/2023.04.24.23288782v1). This code is also applicable for variant with a SpliceAI score of greater than or equal to 0.2. * For a missense variant use REVEL, requires a score of 0.644 - 0.773.* For a variant in an untranslated region use CADD, requires a score of ≥ 20.0* For a predicted splicing variant use SpliceAI with max distance set to 500 bp. Highest delta score from SpliceAI must be ≥ 0.2 to use this code. See GUCY2D-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.PP3 is met if the REVEL score ≥ 0.7 or if the variant is predicted to impact splicing using SpliceAI score ≥0.5 For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≥0.7. For variants that may affect splicing, use SpliceAI with a score ≥0.5.For any variant with RNA or other experimental data indicating an impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Use REVEL score of ≥0.70 as supportive evidence of pathogenicity. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply PP3 when the predicted change is above 0.2[⁸](#pmid_32123317),[⁹](#pmid_30661751). Not Applicable: This criterion is not applicable since these variants are GOF, and traditional mutation pathogenicity prediction algorithms focus on LOF mechanisms. Use of this criterion can be revisited if there emerges additional published experience with predictive algorithms specifically designed to detect gain of function mutations. REVEL score of 0.644-0.772 * Missense: REVEL **\>.7333*** Splicing: Predicted impact via splicing (SpliceAI **≥0.2**) for silent, missense/in-frame and for intronic variants outside of donor and acceptor 1,2 sites. * Missense changes with a REVEL scores >0.75 will meet PP3* For in-frame deletions and insertions, use Mutation Taster.* For non-canonical splice site variants, use Splice AI. Based on data from Jaganathan et al., (2019) PMID: 30661751, Houdayer et al., (2012) PMID: 22505045 and Tang et al., (2016) PMID: 27313609 and Walker et al., (2023) PMID:37352859, PP3 can be applied if there is, a SpliceAI “high score” (Δ Score ≥ 0.5 “confidently predicted splice variants”) (exclude any results with Δ Score ≤ 0.2 from consideration of pathogenicity, \<0.2 are not “predicted to alter splicing”).* For SpliceAI’s cryptic splice-site rules, the creation of a new splice-site with Δ Score ≥ 0.5 may be enough to produce a large proportion of aberrant transcripts.* If a new splice site is predicted to be generated, this rule can be applied if the newly generated splice site is significantly stronger than the wild type site (Δ Score ≥ 0.5 using SpliceAI).* Do not apply this rule for canonical splice site changes meeting PVS1. * Recommended prediction program for missense variants: REVEL. Use 0.75 as a discriminatory cut-off value.* Recommended prediction programs for splice variants: GeneSplicer, MaxEntscan, and NNSPLICE. The outcome of all 3 prediction programs need to be in concordance. REVEL score ≥0.7, or predicted impact to splicing using MaxEntScan.* Use REVEL and MAXENTSCAN * For missense variants, award PP3 if REVEL score is ≥0.7 * If splicing is predicted to be impacted, either creation of a cryptic splice site, or disruption of a native splice site, award PP3* For splice variants (except for canonical -/+1 or 2), use MAXENTSCAN. * For -/+ 1 or 2 splice variants, do not use PP3 if you are using PVS1 For missense variants, REVEL score ≥ 0.750 OR agreement in splicing predictors predict splicing effects. For splicing variants, concordance of MaxEntScan and SpliceAI. * Missense variant with a REVEL score 0.644-0.773 (based on guidance from Pejaver et al, 2022, PMID: 36413997).* In frame deletion or insertion predicted deleterious by PROVEAN and MutationTaster. Results must be consistent to count.* For non-canonical splice site variants (e.g., +3, -3), predict the impact on splicing by using SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) , and apply PP3 for a score equal to or >0.2 (as indicated in PMID: 37352859, Table 1 and Figure 4). Assess the possibility of activation of cryptic splice sites. * Missense variant with a REVEL score 0.644-0.773 (based on guidance from Pejaver et al, 2022, PMID: 36413997).* In frame deletion or insertion predicted deleterious by PROVEAN and MutationTaster. Results must be consistent to count.* For non-canonical splice site variants (e.g., +3, -3), predict the impact on splicing by using SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) , and apply PP3 for a score equal to or >0.2 (as indicated in PMID: 37352859, Table 1 and Figure 4). Assess the possibility of activation of cryptic splice sites. * REVEL score >0.75 for missense variants* In frame deletion or insertion predicted deleterious by PROVEAN, MutPred indel, and MutationTaster.* Predicted impact on splicing by SpliceAI and varSEAK. Multiple lines of computational evidence support a deleterious effect on the gene or gene product.* For missense variants use REVEL with a score ≥ 0.644.* For splice site variants use SpliceAI with a score ≥ 0.2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product.* For missense variants use REVEL with a score ≥ 0.664.* For splice site variants use SpliceAI with a score ≥ 0.2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).* For missense variants use REVEL with a score ≥ 0.644.* For splice site variants use SpliceAI with a score ≥ 0.2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product.* For missense variants use REVEL with a score ≥ 0.644.* For splice site variants use SpliceAI with a score ≥ 0.2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).* For missense variants use REVEL with a score ≥ 0.644.* For splice site variants use SpliceAI with a score ≥ 0.2. Multiple lines of computational evidence support a deleterious effect on the gene or gene product.* For missense variants use REVEL with a score ≥ 0.644.* For splice site variants use SpliceAI with a score ≥ 0.2. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool, with the following stipulations: with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Code can be applied for variants where the REVEL score is greater than or equal to 0.6 or a SpliceAI score of greater than or equal to 0.2. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong * For a missense variant use REVEL, requires a score of 0.644 - 0.773.* For a variant in an untranslated region use CADD, requires a score of ≥ 20.0* For a predicted splicing variant use SpliceAI with max distance set to 500 bp. Highest delta score from SpliceAI must be ≥ 0.2 to use this code. See AIPL1-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart. BP2_Supporting If a FH patient with a heterozygous phenotype has a pathogenic or likely pathogenic variant in LDLR (in trans), APOB or PCSK9, BP2 is applicable to any additional LDLR variants. Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁷](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Observed in trans with a pathogenic or likely pathogenic PTEN variant OR at least three observations in cis and/or phase unknown with different pathogenic/likely pathogenic PTEN variants. Observed in trans with a dominant variant/observed in cis with a pathogenic variant (use with caution).Use with caution. For genes that are associated with both dominant and recessive hearing loss, consider whether an earlier onset/more severe phenotype could be present if variant is identified in trans with a dominant variant. Not Applicable Variant is observed in cis (or phase is unknown) w/ a pathogenic variantOR observed in the homozygous state in gnomAD. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Not Applicable: Not applicable Observed in cis with a pathogenic variant. Use as written for recessive variants (i.e. - variant must be observed in cis with a pathogenic variant) Observed in cis with a pathogenic variant in any inheritance pattern Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern Not Applicable Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern Other mtDNA variant is observed in individual’s mtDNA that has previously been confirmed to be pathogenic Not Applicable: Do not use: See Fanconi Anemia BS2 table \-variant is observed _in cis_ (or phase is unknown) w/ a pathogenic _VHL_ variant Use as written for recessive variants (i.e. - variant must be observed in cis with a pathogenic variant). Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). Use as written for recessive variants (i.e. - variant must be observed in cis with a pathogenic variant) Use as written for recessive variants (i.e. - variant must be observed in cis with a pathogenic variant) This rule can be applied when a _SERPINC1_ variant is _in cis_ with another pathogenic _SERPINC1_ variant. The pathogenic variant must be evaluated using ClinGen _SERPINC1_ specified rules. This rule cannot be applied to a variant _in trans_ with a pathogenic variant, as this scenario could reasonably occur and increase the risk of venous thrombosis. Also applicable when in cis or trans with a likely pathogenic variant. Also applicable when in cis or trans with a likely pathogenic variant. ≥ (-1) Point. Not Applicable: Does not apply. Observed _in trans_ with a (Likely) Pathogenic _APC_ variant **OR** ≥ 3 times in an unknown phase with different (Likely) Pathogenic _APC_ variants. Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). Not Applicable Not Applicable: Applied only in the context of BS2. ≥ (-1) Point. Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[²⁰](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Not Applicable: Applied only in the context of BS2. Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁷](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁷](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁷](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁶](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁶](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Other variants must be pathogenic as defined by these specifications.Testing of parents or other informative relatives is often required to determine _cis_/_trans_ status.If a variant is seen in _trans_ (or as double heterozygous) with another pathogenic variant in ≥2 cases and the phenotype is not more severe than when either of the two variants are seen in isolation, this rule may be applied (i.e., high confidence this variant is NOT contributing to disease).* \<1% of cases of HCM have >1 pathogenic or likely pathogenic variant (0.6%; Alfares _et al._ 2015[¹⁶](#pmid_25611685)).This rule cannot be applied when the variant has only been observed in _cis_ with a pathogenic variant as its significance in isolation is unknown in this scenario. Caution is needed if using this criterion as a primary piece of evidence for classifying a variant as likely benign/benign (i.e., only 2 SUPPORTING criteria are sufficient for a likely benign classification). Not Applicable: Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. Not Applicable: X-linked gene. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.* Not applicable to _KCNQ1_ due to biallelic cases (Jervell and Lange-Nielsen syndrome) Applicable only when observed in cis with a pathogenic variant in any inheritance pattern, with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _FOXN1._ Not Applicable Not Applicable: BS2 is used instead. Not Applicable Not Applicable Observed in cis with a Pathogenic variant.* Use code if the variant of interest is _in cis_ with a Pathogenic or Likely Pathogenic variant. The other variant must meet a Likely Pathogenic or Pathogenic classification using these rule specifications. Not Applicable Not Applicable: Do not use this criterion.BP2 would be more applicable to a very large, polymorphic gene, while it’s rarely going to come up for a short gene like CTLA4.Also, while biallelic cases have not yet been found, they exist for other IEI genes. Not Applicable Not Applicable No change - use as originally described Not Applicable: For this X-linked gene, only variants in cis with a pathogenic variant in RS1 in an affected male could be used, but these variants could have combined effect. The VCEP does not use this code. ≥ (-1) Point. ≥ (-1) Point. Not Applicable: Does not apply. Observed in trans with a pathogenic or likely pathogenic variant based on HHT VCEP rules. Observed in trans with a pathogenic or likely pathogenic variant based on HHT VCEP rules. Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BP2 is considered not applicable, as the field at present does not understand all of the potential allelic mechanisms associated with PIK3CD variants, so that the possibility of diverse combinatorial variant effects cannot be excluded. This has been described for other inborn errors of immunity genes. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Applicable in females for which OTC deficiency is diagnosed and the variant under review is identified in trans or in cis with a known pathogenic variant, or if the variant under review is identified in cis in a male with OTC deficiency. Not Applicable: Rule code does not apply because some modifier gene variants are in cis with known pathogenic variants. Observed in cis with a Pathogenic variant.* Use code if the variant of interest is _in cis_ with a Pathogenic or Likely Pathogenic variant. The other variant must meet a Likely Pathogenic or Pathogenic classification using these rule specifications. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Use when variant is found _in cis_ with a variant classified as pathogenic or likely pathogenic using the LGMD VCEP specifications. Also applicable when in cis or trans with a likely pathogenic variant. Observed in cis or trans with a known pathogenic variant in the same gene. Not Applicable: Biallelic variants (either compound heterozygotes or homozygotes) have been reported (with variable phenotype) and are not incompatible with life. Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. BP2 = **\-1** pointSee ATM PM3/BP2 table for approach to assign points per proband. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern. * Observed in trans in multiple cases (+2) with co-occuring pathogenic variants and phenotype is not more severe than when seen in isolation.* Observed in cis with a pathogenic variant, if the pathogenic variant has been seen in isolation in a patient with the disease phenotype. Observed in trans with a dominant variant/observed in cis with a pathogenic variant (use with caution).* Use with caution. For genes that are associated with both dominant and recessive hearing loss, consider whether an earlier onset/more severe phenotype could be present if variant is identified in trans with a dominant variant. ≥1 observation in trans with P/LP DICER1 variant or ≥3observations in cis or phase unknown with 2+ differentP/LP DICER1 variants. Observed in cis with a pathogenic variant (to take AR inheritance for AGAT deficiency into account). Observed in cis with a pathogenic variant (to take AR inheritance into account). Not Applicable Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.* Applicable for _TCF4_ for _in trans_ state Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.* BP2 is not applicable for SLC9A6 for _in trans_ state. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.* BP2 is not applicable for _in trans_ state. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.* Applicable for _in trans_ state Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.* BP2 is not applicable for UBE3A _in trans_ state. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. Observed in cis with a Pathogenic variant.* Use code if the variant of interest is _in cis_ with a Pathogenic or Likely Pathogenic variant. The other variant must meet a Likely Pathogenic or Pathogenic classification using these rule specifications. BP5_Very Strong Not Applicable Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. NA NA NA NA Not Applicable: BP5 is not applicable. In rare circumstances, a patient can carry two pathogenic variants in genes predisposing to hematologic malignancies. Not Applicable: Not applicable Not Applicable: * An individual could be a carrier of a pathogenic variant in GAA and have another disorder.* There is no known alternate molecular basis for deficiency of GAA activity, other than variants in GAA. NA Not Applicable: BP5 is not applicable as individuals have been described with MHS and two pathogenic variants in RYR1. NA NA NA NA NA NA Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, PALB2 has moderate penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There is no known alternate cause of isolated factor IX deficiency. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There are other genes that can be associated with decreased antithrombin activity levels, such as genes associated with the congenital disorders of glycosylation. NA NA NA Not Applicable: Does not apply. NA NA Not Applicable: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA. NA NA Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. NA Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable NA NA Not Applicable NA Not Applicable Not Applicable Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. Not Applicable NA Not Applicable Not Applicable NA Not Applicable NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Do not use this rule as an individual can be a carrier of a pathogenic ABCA4 variant while having another molecular etiology for their retinopathy. Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. NA NA Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Secondary deficiencies of related proteins are often seen in LGMD. Not Applicable: Not applicable. Not Applicable: Not applicable. NA NA Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, ATM has low penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. Not Applicable: An individual could be a carrier of a pathogenic variant in ACADVL and have another disorder. NA Not Applicable: Autosomal recessive: Do not use. An individual could be carrier of pathogenic variant and have an alternate cause. Therefore, BP5 shouldn’t be used as evidence for benign in this case. Not Applicable: Given the broad spectrum of DICER1-related neoplasms and the General recommendation lack of evidence of other high-penetrance germline variants that could account for such neoplasms (except perhaps for some already low-specificity phenotypes such as Wilms tumor), this rule should not be used at this time. Not Applicable: Variant found in a case with an alternate molecular basis for disease.CCDS VCEP notes for BP5: An individual could be a carrier of a pathogenic variant in GATM and have another disorder Not Applicable: An individual could be a carrier of a pathogenic variant in GAMT and have another disorder. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: While unlikely, it is possible for males with hemophilia to also have a diagnosis of von Willebrand Normandy (or VWD 2N). NA Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. BP5_Strong Not Applicable Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. NA NA NA NA Not Applicable: BP5 is not applicable. In rare circumstances, a patient can carry two pathogenic variants in genes predisposing to hematologic malignancies. Not Applicable: Not applicable Not Applicable: * An individual could be a carrier of a pathogenic variant in GAA and have another disorder.* There is no known alternate molecular basis for deficiency of GAA activity, other than variants in GAA. NA Not Applicable: BP5 is not applicable as individuals have been described with MHS and two pathogenic variants in RYR1. Variant found in a case with an alternate molecular basis for disease. NA NA NA NA NA Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, PALB2 has moderate penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There is no known alternate cause of isolated factor IX deficiency. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There are other genes that can be associated with decreased antithrombin activity levels, such as genes associated with the congenital disorders of glycosylation. NA NA ≥ (-4) Points. Not Applicable: Does not apply. NA NA Not Applicable: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5\_VeryStrong - LR ≤0.00285:1BP5\_Strong - LR ≤0.05:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. ≥ (-4) Points. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5\_VeryStrong – LR ≤0.00285:1BP5\_Strong - LR ≤0.05:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable NA NA Not Applicable ≥ 4 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variationOR≥2 BRAF V600E (CRC only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. Not Applicable Not Applicable Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. Not Applicable NA Not Applicable Not Applicable NA Not Applicable ≥ (-4) Points. ≥ (-4) Points. Not Applicable: Does not apply. NA NA ≥ 4 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variationOR≥2 BRAF V600E (CRC only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. ≥ 4 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variationOR≥2 BRAF V600E (CRC only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. ≥ 4 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variationOR≥2 BRAF V600E (CRC only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. NA No change - use as originally described NA No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: Do not use this rule as an individual can be a carrier of a pathogenic ABCA4 variant while having another molecular etiology for their retinopathy. Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. NA No change - use as originally described Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Secondary deficiencies of related proteins are often seen in LGMD. Not Applicable: Not applicable. Not Applicable: Not applicable. NA NA Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, ATM has low penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. Not Applicable: An individual could be a carrier of a pathogenic variant in ACADVL and have another disorder. NA Not Applicable: Autosomal recessive: Do not use. An individual could be carrier of pathogenic variant and have an alternate cause. Therefore, BP5 shouldn’t be used as evidence for benign in this case. Not Applicable: Given the broad spectrum of DICER1-related neoplasms and the General recommendation lack of evidence of other high-penetrance germline variants that could account for such neoplasms (except perhaps for some already low-specificity phenotypes such as Wilms tumor), this rule should not be used at this time. Not Applicable: Variant found in a case with an alternate molecular basis for disease.CCDS VCEP notes for BP5: An individual could be a carrier of a pathogenic variant in GATM and have another disorder Not Applicable: An individual could be a carrier of a pathogenic variant in GAMT and have another disorder. NA Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* ≥3 cases with alternate molecular basis for disease. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. NA NA NA NA Not Applicable: While unlikely, it is possible for males with hemophilia to also have a diagnosis of von Willebrand Normandy (or VWD 2N). NA Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. BP5_Moderate Not Applicable Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. NA NA NA NA Not Applicable: BP5 is not applicable. In rare circumstances, a patient can carry two pathogenic variants in genes predisposing to hematologic malignancies. Not Applicable: Not applicable Not Applicable: * An individual could be a carrier of a pathogenic variant in GAA and have another disorder.* There is no known alternate molecular basis for deficiency of GAA activity, other than variants in GAA. NA Not Applicable: BP5 is not applicable as individuals have been described with MHS and two pathogenic variants in RYR1. Variant found in a case with an alternate molecular basis for disease. NA NA NA NA NA Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, PALB2 has moderate penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There is no known alternate cause of isolated factor IX deficiency. NA Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There are other genes that can be associated with decreased antithrombin activity levels, such as genes associated with the congenital disorders of glycosylation. NA NA ≥ (-2) Points. Not Applicable: Does not apply. NA NA Not Applicable: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5\_Moderate - LR ≤0.23:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. ≥ (-2) Points. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5\_Moderate - LR ≤0.23:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable NA NA Not Applicable NA Not Applicable Not Applicable Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. Not Applicable NA Not Applicable Not Applicable NA Not Applicable ≥ (-2) Points. ≥ (-2) Points. Not Applicable: Does not apply. NA NA NA NA NA NA No change - use as originally described NA No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: Do not use this rule as an individual can be a carrier of a pathogenic ABCA4 variant while having another molecular etiology for their retinopathy. Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. NA No change - use as originally described Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Secondary deficiencies of related proteins are often seen in LGMD. Not Applicable: Not applicable. Not Applicable: Not applicable. NA NA Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, ATM has low penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. Not Applicable: An individual could be a carrier of a pathogenic variant in ACADVL and have another disorder. NA Not Applicable: Autosomal recessive: Do not use. An individual could be carrier of pathogenic variant and have an alternate cause. Therefore, BP5 shouldn’t be used as evidence for benign in this case. Not Applicable: Given the broad spectrum of DICER1-related neoplasms and the General recommendation lack of evidence of other high-penetrance germline variants that could account for such neoplasms (except perhaps for some already low-specificity phenotypes such as Wilms tumor), this rule should not be used at this time. Not Applicable: Variant found in a case with an alternate molecular basis for disease.CCDS VCEP notes for BP5: An individual could be a carrier of a pathogenic variant in GATM and have another disorder Not Applicable: An individual could be a carrier of a pathogenic variant in GAMT and have another disorder. NA Variant found in a case with an alternate molecular basis for disease.* 2 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 2 cases with alternate molecular basis for disease Variant found in a case with an alternate molecular basis for disease.* 2 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 2 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 2 cases with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. * 2 cases with alternate molecular basis for disease. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. NA NA NA NA Not Applicable: While unlikely, it is possible for males with hemophilia to also have a diagnosis of von Willebrand Normandy (or VWD 2N). NA Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. BP5_Supporting Not Applicable Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Variant found in a case with an alternate molecular basis for disease. Other gene/disorder must be considered highly penetrant AND patient’s personal/family history must demonstrate no overlap between other gene and PTEN. Variant in an autosomal dominant gene found in a patient with an alternate explanation.* Autosomal recessive: Do not use. An individual could be carrier of pathogenic variant and have an alternate cause. Therefore, BP5 shouldn’t be used as evidence for benign in this case.* Autosomal dominant: Can use BP5 as outlined by Richards 2015. * Caveat: consider whether multiple pathogenic autosomal dominant variants could cause a more severe phenotype or whether multigenic inheritance is known to occur (example: Bardet-Biedl syndrome). Applicable as described Per original ACMG/AMP guidelines. Not Applicable: BP5 is not applicable. In rare circumstances, a patient can carry two pathogenic variants in genes predisposing to hematologic malignancies. Not Applicable: Not applicable Not Applicable: * An individual could be a carrier of a pathogenic variant in GAA and have another disorder.* There is no known alternate molecular basis for deficiency of GAA activity, other than variants in GAA. Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: BP5 is not applicable as individuals have been described with MHS and two pathogenic variants in RYR1. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease Variant found in a case with an alternate molecular basis for disease Variant found in a case with an alternate molecular basis for disease Variant found in a case with an alternate molecular basis for disease Mitochondrial DNA variant found in a case with a nuclear DNA-related disease Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, PALB2 has moderate penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. BP5 can be applied for two or more co-occurrences with pathogenic variants in a different gene that fully explained the patient's phenotype, but specific circumstances would need to be met in order for a case to be considered for inclusion. First, the variant in the other gene must be considered highly penetrant, with both the individual's age, tumour type and gender taken into consideration. Additionally, the patient's personal and family history (including up to 2nd degree relatives) should not overlap with features seen in VHL and VHL tumour histologies. As an example, an individual with a personal and family history of pheochromocytoma who harbored a _VHL_ variant in addition to a pathogenic SDHB variant BP5 would not apply, because pheochromocytoma is a known risk in VHL and the _VHL_ variant might have contributed to this individual's pheochromocytoma cancer risk. However, an individual with a personal and family history of chromophobic RCC who was positive for a _VHL_ variant as well as a pathogenic FLCN variant would be considered for BP5 application, as non-clear cell RCC is not associated with VHL disease. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There is no known alternate cause of isolated factor IX deficiency. A second variant in VWF may be considered an alternate molecular basis for disease when that variant is LP/P (as evaluated by the VWD VCEP) and fully explains the phenotype of the patient's reported VWD subtype. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: Do not use this rule as an individual can be a carrier of an unrelated pathogenic variant for a recessive disorder. Not Applicable: This rule code is not recommended for use at this time. There are other genes that can be associated with decreased antithrombin activity levels, such as genes associated with the congenital disorders of glycosylation. A variant in another monogenic diabetes gene is Pathogenic/Likely Pathogenic. A variant in another monogenic diabetes gene is P/LP. ≥ (-1) Point. Not Applicable: Does not apply. Only applicable for an alternate genetic basis of the colorectal polyposis phenotype. A second variant in VWF may be considered an alternate molecular basis for disease when that variant is LP/P (as evaluated by the VWD VCEP) and fully explains the phenotype of the patient's reported VWD subtype. Not Applicable: There is no known alternate molecular basis for deficiency of alpha-L-iduronidase activity, other than variants in IDUA. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5 - LR ≤0.48:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. ≥ (-1) Point. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Use ONLY to capture combined LR against pathogenicity, based on multifactorial likelihood clinical data.BP5 - LR ≤0.48:1Not applicable for co-observation: cases with pathogenic variants in two (or more) different known breast–ovarian cancer risk genes have no specific phenotype. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Co-occurrence with an established pathogenic or likely pathogenic variant for a non-cardiomyopathy related disease does not reduce the likelihood that a variant is independently disease-causing for cardiomyopathy. Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable Variant found in a case with an alternate molecular basis for disease* Caveat: BP5 is not applicable when the phenotypes (ie: trigger type; PMID: 32882399, 11136691, 18373596 or treadmill stress test; PMID: 21699858) indicate _KCNQ1_ as the cause. BP5 is only applicable when the phenotypes match another form of LQTS (caused by a variant in _KCNH2_, _SCN5A_, or _KCNE1_, PMID: 19862833, PMID: 31941373, PMID: 33517668) or when phenotype details are not sufficiently specific.* Note: The rate of cases with multiple suspected deleterious variants in the same or different LQTS-associated genes may be relatively high (5.2% in one cohort, PMID: 24667783). The group will consider this possibility during the pilot. Use with no specification. Not Applicable 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation.OR 1 BRAF V600E (Colon only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. Not Applicable Not Applicable Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. Not Applicable * Two such cases are required to mitigate the VCEP’s concern over relying on other groups’ classifications of pathogenic variants in genes such as _LRBA_. Not Applicable Not Applicable No change - use as originally described Not Applicable ≥ (-1) Point. ≥ (-1) Point. Not Applicable: Does not apply. Apply if a likely pathogenic or pathogenic variant (based on HHT VCEP rules) is found in _ENG_. Apply if a likely pathogenic or pathogenic variant (based on HHT VCEP rules) is found in _ACVRL1_. 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation.OR 1 BRAF V600E (Colon only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation.OR 1 BRAF V600E (Colon only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation.OR 1 BRAF V600E (Colon only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss. Variant found in a case with an alternate molecular basis for disease.At least two such cases are required to mitigate the reliance on assertions of variant pathogenicity in genes outside of the purview of the Antibody Deficiencies VCEP. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Applicable when the variant of interest is identified in a male with an alternative molecular etiology of elevated hyperammonemia including biallelic pathogenic/likely pathogenic variants in autosomal recessive disease or a pathogenic/likely pathogenic variant for autosomal dominant or X-linked disease. Not Applicable: Do not use this rule as an individual can be a carrier of a pathogenic ABCA4 variant while having another molecular etiology for their retinopathy. Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Secondary deficiencies of related proteins are often seen in LGMD. Not Applicable: Not applicable. Not Applicable: Not applicable. A variant in other monogenic diabetes gene is P/LP. No change. Not Applicable: Multiple molecular diagnoses are possible and variants in different genes could have an additive effect. Not Applicable: Do not use: Cases with multiple pathogenic variants have been observed with no noticeable difference in phenotype (e.g. BRCA1 and BRCA2). In addition, ATM has low penetrance and will naturally occur with other pathogenic variants more frequently due to higher tolerance/presence in the general population. Not Applicable: An individual could be a carrier of a pathogenic variant in ACADVL and have another disorder. Variant found in a case with an alternate molecular basis for disease. Not Applicable: Autosomal recessive: Do not use. An individual could be carrier of pathogenic variant and have an alternate cause. Therefore, BP5 shouldn’t be used as evidence for benign in this case. Not Applicable: Given the broad spectrum of DICER1-related neoplasms and the General recommendation lack of evidence of other high-penetrance germline variants that could account for such neoplasms (except perhaps for some already low-specificity phenotypes such as Wilms tumor), this rule should not be used at this time. Not Applicable: Variant found in a case with an alternate molecular basis for disease.CCDS VCEP notes for BP5: An individual could be a carrier of a pathogenic variant in GATM and have another disorder Not Applicable: An individual could be a carrier of a pathogenic variant in GAMT and have another disorder. Variant found in a case with an alternate molecular basis for disease.BP5 applicable as described; the case must have specific features of creatine transporter deficiency, such as low creatine on brain magnetic resonance spectroscopy, or elevated urine creatine in order to apply this criterion. Presence of developmental delay and seizures is not sufficient. Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease.* 1 case with alternate molecular basis for disease. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. ≥ (-1) Point. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Variant found in a case with an alternate molecular basis for disease. Not Applicable: While unlikely, it is possible for males with hemophilia to also have a diagnosis of von Willebrand Normandy (or VWD 2N). Variant found in a case with an alternate molecular basis for disease. Not Applicable: Due to the high genetic heterogeneity and limited phenotypic specificities of retinal dystrophies, this rule should not be used.Additionally, the presence of this variant could simply represent carrier status. PS4_Strong Variant is found in ≥10 unrelated FH cases (FH diagnosis met by validated clinical criteria).Caveat: variant must also meet PM2 Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁸](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYH7/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁹](#pmid_28395867); Oechslin _et al._ 2017[⁶](#pmid_28545618); Hershberger _et al._ 2017[⁵](#pmid_29212902); Ross _et al._ 2020[⁷](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Probands with specificity score 4-15.5 (see text) OR The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls. Fisher Exact or Chi-Squared analysis shows statistical increase in cases over controls, ORAutosomal dominant: ≥15 probands with variant, and variant meets PM2_Supporting. Not Applicable: This criterion is not applicable for PAH. For proband counting, use PM3 criterion. Four - Fifteen families meet HDGC criteria. **PS4:** ≥ 4 probands meeting at least one of the \_RUNX1\_-phenotypic criteria (OR 127.1). ≥ 4-7.5 points Not Applicable: There are no case-control studies for Pompe disease. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. Rule does not apply due to rarity of disorder and lack of appropriate studies. The prevalence of the variant in affected individuals significantly increased compared with the prevalence in controls.* ≥7 MH case points. Probands with a personal or family history of an MH event are awarded 0.5 points, probands with a personal or family history of a positive (MHS) IVCT/CHCT are awarded an additional 0.5 points. Probands with multiple variants in RYR1 classified as VUS, likely pathogenic or pathogenic are not considered. Popmax in gnomAD ≤0.00006. * For variants with popmax MAF gnomAD >0.00006, an odds ratio of ≥18.7 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. Not Applicable Not Applicable Not Applicable Not Applicable Variant present in ≥16 unrelated probands Case-control studies; p-value ≤.05 AND (Odds ratio, hazard ratio, or relative risk ≥3 OR lower 95% CI ≥1.5). **5-15 points the PS4 cut-off and Proband Scoring Tables from a mix of any of the following phenotypes: specific, consistent and nonspecific.** NA 4-7 probands meet criteria described below Appropriate to use code when there are 4-7 probands that meet the laboratory phenotype of the PP4 definition for a specific VWD type 2 phenotype (i.e. – all probands must qualify for a clinical diagnosis of the same VWD type 2 phenotype based on laboratory criteria stated under PP4). NA NA Appropriate to use code when there 4-7 proband points that meet the defined antithrombin deficiency laboratory phenotype. 7 (seven) or more unrelated occurrences = Strong. Variant should meet PM2\_Supporting in order to use PS4 at any level (careful review of gnomAD QC data may be necessary to assess whether variant is real or an artifact, especially if variant is in a polyC region). Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology.* One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD)[⁷](#pmid_21395678),[⁸](#pmid_28701371),[⁹](#pmid_30409810),[¹⁰](#pmid_31704690) * Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (\<200pmol/L or 0.6ng/mL)[¹¹](#pmid_30225972),[¹²](#pmid_23771925) or urinary C-peptide/creatinine ratio \<0.2 nmol/mmol[⁸](#pmid_28701371),[⁹](#pmid_30409810) 7 or more occurrences in unrelated individuals = Strong. ≥5 points. Not Applicable: Does not apply. 4-15.5 phenotype points. For phenotype points curation see **Table 1**. Not Applicable: Use PM3 for proband counting. Not Applicable: There are no case-control studies for MPS1. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Case-control studies; p-value ≤0.05 and OR ≥4 (lower confidence interval excludes 2.0). See Appendix F for details. ≥5 points. {Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁹](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYBPC3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[¹⁰](#pmid_28395867); Oechslin _et al._ 2017[¹¹](#pmid_28545618); Hershberger _et al._ 2017[¹²](#pmid_29212902); Ross _et al._ 2020[¹³](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Case-control studies; p-value ≤0.05 and OR ≥4 (lower confidence interval excludes 2.0). See Appendix F for details. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNI3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNT2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TPM1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/ACTC1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **STRONG** evidence requires the lower bound of the 95% confidence interval (CI) around the odds ratio (OR) estimate to be **≥20**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Not Applicable: CYP1B1 variants cause autosomal recessive disorders associated with glaucoma. The number of probands with the variant will be addressed by PM3. Use of this code requires ≥ 6 probands and PM2\_Supporting must be met. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls* In order to be evaluated for this criterion, the variant must not meet BS1.* PS4 is Met by 6 or more probands.* This code is not considered mutually exclusive with PM3. For example, variants associated with both dominant and recessive cases can meet both PS4 and PM3.* If probands are reported in at least 2 papers, and the author lists from the two papers contain any of the same authors names, definitely the patients are the same, so count only one case for PS4.* If the two lists of authors are totally distinct / different, usually you can count both cases for PS4.* Patients reported as affected must have QTc measurement greater than or equal to 460ms in order to be counted. Diagnosis of long QT syndrome by itself is not sufficient for inclusion. Sum of case scores 4-7.75 points (see instructions below) Not Applicable Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable Not Applicable Not Applicable Not Applicable * In order to be evaluated for this criterion, the variant must not meet BS1 or BA1.* A proband would be excluded for this criterion if found to harbor homozygous or compound heterozygous _LRBA_ variants that are either rare VUS or have been classified Likely Pathogenic or Pathogenic.* PS4 at the default (Strong) level is Met by observation of the variant in at least 4 independent probands.* The probands used for PS4 cannot include the same proband used for PP4.* In order to be counted for this criterion, a proband must meet one of the following two requirements: * To have reported phenotypes that score a minimum of 6 phenotype points, as well as genotyping of the _LRBA_ locus to confirm the absence of biallelic variants in _LRBA_ that have been classified either Likely Pathogenic or Pathogenic. Note: a monoallelic _LRBA_ variant can be tolerated. * **OR** * To have reported phenotypes that score a minimum of 10 phenotype points, in the absence of genotyping of the _LRBA_ locus.* Description of the phenotype point system can be found in the bullet points below or in a more detailed form in the attached table entitled “CTLA4 Phenotype Scoring Table”. The table includes a total of two 4-point criteria, four 2-point criteria, and eleven 1-point criteria:* _Clinical criteria_ * Sinopulmonary findings 4 * Non-infectious gastrointestinal or hepatobiliary disease 4 * Immune-mediated cytopenias 2 * Nonmalignant Lymphoproliferation 2 * Severe, persistent, recurrent viral infections including skin warts 2 * Immune-mediated skin and hair findings 1 * Endocrinopathy 1 * Severe, persistent, recurrent, atypical, opportunistic bacterial, Mycobacterial or fungal infections 1 * Neurological findings 1 * Inflammatory findings (arthritis, vasculitis, recurrent fevers) 1 * Lymphoma 1* _Objective criteria_ * Hypogammaglobulinemia 2 * Lymphopenia 1 * Abnormal TBNK levels 1 * Presence of autoantibodies 1 * Defective antigen-specific immune responses 1 * Histopathology findings of lymphocytic / granulomatous tissue infiltration 1 Not Applicable Not Applicable Prior observation of the variant in >4 unrelated patients with the same phenotype, and its absence in controls. Use of this code requires 5-8 probands, each with retinoschisis. PM2\_Supporting must be met. ≥5 points. ≥5 points. Sum of case scores 4.5-16 points (see instructions below) 4+ probands with phenotype consistent with HHT. 4+ probands with phenotype consistent with HHT. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. * The strength of this code is defined in **Table 4**. Use PS4 at the default level of strength when four or more probands meet the phenotype scoring criteria in **Table 3** and genetic testing requirements described below. Please note that a proband used for PP4 or PP4\_Moderate cannot be included in PS4.* Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3CD-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3CD-related immunodeficiency.* A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (**Table 3**) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the _PIK3R1_ locus in order to be counted toward PS4 * Genome or exome sequencing is acceptable in lieu of a gene panel * For genes associated with autosomal recessive disorders, carrier status is acceptable.* If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.* Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. NEB is associated with Autosomal Recessive disease; PS4 can only be used for case-control studies and not proband counting. Please use PM3 for individual case observations. 8 case observations Specific phenotypes for proband counting include:**A Congenital myopathy panel should be run and negative for other variants (must include BIN1, RYR1, MTM1) AND at least two of these features:*** Presence on Muscle Biopsy of: Oxidative activity and/or radial stranding with spokes on a wheel appearance with centrally nucleated muscle fibers* Distal weakness* Characteristic muscle imaging (See Figure 9 of Saade et al 2019 PMID: 31060725 for example)* Ophthalmoparesis and Ptosis (both of these must be observed to count this as one phenotype criteria)At least 1 point, please see PS4 chart The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.**5+ cases** The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.RYR1 is associated with both autosomal recessive and autosomal dominant disease. If you are curating biallelic observations, please use the AR specifications. For case counting for AD disease, please see the PS4 chart.at least 1 point, please see PS4 chart 4 additional male or female probands observed - must meet PP4 criteria and/or have documented hyperammonemia or metabolic decompensation under physiological stress to be counted Enrichment analysis was performed for all likely bi-allelic _ABCA4_ variants that were published up to December 31, 2020 compared to population matched gnomAD data from [Cornelis et al., 2022](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948157/pdf/main.pdf)¹. This code can be applied to variants with an OR > or = 5, where the CI does not contain 1. Not Applicable Not Applicable: These specifications are only for autosomal recessively inherited ACTA1 variants. Please use PM3 for case counting. There are separate specifications for AD ACTA1 variants. Not Applicable: These specifications are only for autosomal recessively inherited RYR1 variants. Please usePM3 for case counting. There are separate specifications for AD RYR1 variants. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _DYSF_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _SGCB_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _SGCG_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _SGCD_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _CAPN3_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _ANO5_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Use without disease-specific modification if case-control studies are available. While case-control studies could potentially be considered for a few pathogenic variants with high minor allele frequency, the VCEP is unaware of any such studies being conducted for _SGCA_. Any case-control study would require careful selection of an appropriate control population given the potential for late onset and mild disease. Seven or more unrelated occurrences = Strong. Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant is absent from controls (PM2). Strength of evidence is determined by points according to (Table 2B). PS4_Strong = 3.5-15.75 points. ≥ 15 probands from multiple independent studies. Case-control studies; p-value ≤.05 AND (Odds ratio, hazard ratio, or relative risk ≥2 OR lower 95% CI ≥1.5). Not Applicable * If ≥ 4 points. NA ≥4 phenotype points Not Applicable: CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. Not Applicable: The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. * 3 independent male probands with elevated urine creatine/creatinine ratio on one occasion at minimum, in addition to any proband used for PP4.* Variant must meet PM2_Supporting criterion for PS4 to apply. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 5+ observations. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. ≥5 points. Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4-15.5 points** will arrive at **Strong**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 - 15.5 points** will arrive at **Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4-15.5 points** will arrive at **Strong**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 - 15.5 points** will arrive at **Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points 4-7 probands meet criteria described below Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4-15.5 points** will arrive at **Strong**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Not Applicable PS4_Moderate Variant is found in 6-9 unrelated FH cases (FH diagnosis made by validated clinical criteria).Caveat: variant must also meet PM2. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁸](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYH7/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁹](#pmid_28395867); Oechslin _et al._ 2017[⁶](#pmid_28545618); Hershberger _et al._ 2017[⁵](#pmid_29212902); Ross _et al._ 2020[⁷](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Probands with specificity score of 2-3.5 (see text). Autosomal dominant: ≥6 probands with variant, and variant meets PM2_Supporting. Not Applicable: This criterion is not applicable for PAH. For proband counting, use PM3 criterion. Two or three families meet HDGC criteria. **PS4\_Moderate:** 2-3 probands meeting at least one of the \_RUNX1\_-phenotypic criteria (OR 63.5-95.3). 2-3.5 points Not Applicable: There are no case-control studies for Pompe disease. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 2-6 MH case points. Probands with a personal or family history of an MH event are awarded 0.5 points, probands with a personal or family history of a positive (MHS) IVCT/CHCT are awarded an additional 0.5 points. Probands with multiple variants in RYR1 classified as VUS, likely pathogenic or pathogenic are not considered. Popmax in gnomAD ≤0.00006.* For variants with popmax MAF in gnomAD >0.00006, an odds ratio of ≥4.33 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. Not Applicable Not Applicable Not Applicable Not Applicable Variant present in ≥4 unrelated probands NA **2 – 4 points the PS4 cut-off and Proband Scoring Tables from a mix of any of the following phenotypes: specific, consistent and nonspecific.** See instructions below for scoring heterozygous individuals only. Moderate strength applicable for score of 2+ points. 2-3 probands meet criteria described below Appropriate to use code when there are 2-3 probands that meet the laboratory phenotype of the PP4 definition for a specific VWD type 2 phenotype (i.e. – all probands must qualify for a clinical diagnosis of the same VWD type 2 phenotype based on laboratory criteria stated under PP4). See instructions below for scoring heterozygous individuals only. Moderate strength applicable for score of 2+ points. See instructions below for scoring heterozygous individuals only. Moderate strength applicable for score of 2+ points. Appropriate to use code when there 2-3 proband points that meet the defined antithrombin deficiency laboratory phenotype. 4-6 unrelated occurrences = Moderate. Variant should meet PM2\_Supporting in order to use PS4 at any level. Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology.* One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD)[⁷](#pmid_21395678),[⁸](#pmid_28701371),[⁹](#pmid_30409810),[¹⁰](#pmid_31704690)* Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (\<200pmol/L or 0.6ng/mL)[¹¹](#pmid_30225972),[¹²](#pmid_23771925) or urinary C-peptide/creatinine ratio \<0.2 nmol/mmol[⁸](#pmid_28701371),[⁹](#pmid_30409810) 4-6 occurrences in unrelated individuals = Moderate. ≥3 points. Not Applicable: Does not apply. 2-3.5 phenotype points. For phenotype points curation see **Table 1**. Not Applicable: Use PM3 for proband counting. Not Applicable: There are no case-control studies for MPS1. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA ≥3 points. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁹](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYBPC3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[¹⁰](#pmid_28395867); Oechslin _et al._ 2017[¹¹](#pmid_28545618); Hershberger _et al._ 2017[¹²](#pmid_29212902); Ross _et al._ 2020[¹³](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. NA Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNI3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNT2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TPM1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/ACTC1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **MODERATE** evidence requires the lower bound of the 95% CI around the OR to be **≥10**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Not Applicable: CYP1B1 variants cause autosomal recessive disorders associated with glaucoma. The number of probands with the variant will be addressed by PM3. Use of this code at the moderate strength requires 3-5 probands and PM2\_Supporting must be met. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls* In order to be evaluated for this criterion, the variant must not meet BS1.* PS4\_moderate is Met by 3-5 probands.* This code is not considered mutually exclusive with PM3. For example, variants associated with both dominant and recessive cases can meet both PS4 and PM3.* If probands are reported in at least 2 papers, and the author lists from the two papers contain any of the same authors names, definitely the patients are the same, so count only one case for PS4.* If the two lists of authors are totally distinct / different, usually you can count both cases for PS4.* Patients reported as affected must have QTc measurement greater than or equal to 460ms in order to be counted. Diagnosis of long QT syndrome by itself is not sufficient for inclusion. Sum of case scores 2-3.75 points (see instructions below) Not Applicable Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable Not Applicable Not Applicable Not Applicable * PS4\_Moderate is Met by observation of the variant in 2-3 independent probands.* The probands used for PS4\_Moderate cannot include the same proband used for PP4.* In order to be counted for this criterion, a proband must meet one of the following two requirements: * To have reported phenotypes that score a minimum of 6 phenotype points, as well as genotyping of the _LRBA_ locus to confirm the absence of biallelic variants in _LRBA_ that have been classified either Likely Pathogenic or Pathogenic. Note: a monoallelic _LRBA_ variant can be tolerated. * **OR** * To have reported phenotypes that score a minimum of 10 phenotype points, in the absence of genotyping of the _LRBA_ locus.* Description of the phenotype point system can be found in the bullet points below or in a more detailed form in the attached table entitled “CTLA4 Phenotype Scoring Table”. The table includes a total of two 4-point criteria, four 2-point criteria, and eleven 1-point criteria:* _Clinical criteria_ * Sinopulmonary findings 4 * Non-infectious gastrointestinal or hepatobiliary disease 4 * Immune-mediated cytopenias 2 * Nonmalignant Lymphoproliferation 2 * Severe, persistent, recurrent viral infections including skin warts 2 * Immune-mediated skin and hair findings 1 * Endocrinopathy 1 * Severe, persistent, recurrent, atypical, opportunistic bacterial, Mycobacterial or fungal infections 1 * Neurological findings 1 * Inflammatory findings (arthritis, vasculitis, recurrent fevers) 1 * Lymphoma 1* _Objective criteria_ * Hypogammaglobulinemia 2 * Lymphopenia 1 * Abnormal TBNK levels 1 * Presence of autoantibodies 1 * Defective antigen-specific immune responses 1 * Histopathology findings of lymphocytic / granulomatous tissue infiltration 1 Not Applicable Not Applicable Prior observation of the variant in >3 unrelated patients with the same phenotype, and its absence in controls. Use of this code at the moderate strength requires 3-4 probands diagnosed with retinoschisis. PM2\_Supporting must be met. ≥3 points. ≥3 points. Sum of case scores 2.5-4 points (see instructions below) 2-3 probands with phenotype consistent with HHT. 2-3 probands with phenotype consistent with HHT. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. * The strength of this code is defined in **Table 4**. Use PS4\_Moderate when 2-3 probands meet the phenotype scoring criteria in **Table 3** and genetic testing requirements described below. Please note that a proband used for PP4 or PP4\_Moderate cannot be included in PS4\_Moderate.* Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3CD-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3CD-related immunodeficiency.* A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (**Table 3**) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the _PIK3R1_ locus in order to be counted toward PS4 * Genome or exome sequencing is acceptable in lieu of a gene panel * For genes associated with autosomal recessive disorders, carrier status is acceptable.* If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.* Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. NEB is associated with Autosomal Recessive disease; PS4 can only be used for case-control studies and not proband counting. Please use PM3 for individual case observations. 4 case observations 0.5 points, please see PS4 chart 3-4 cases 0.5 points, please see PS4 chart 2 additional male or female probands observed - must meet PP4 criteria and/or have documented hyperammonemia or metabolic decompensation under physiological stress to be counted Enrichment analysis was performed for all likely bi-allelic _ABCA4_ variants that were published up to December 31, 2020 compared to population matched gnomAD data from [Cornelis et al., 2022](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948157/pdf/main.pdf)¹. This code can be applied to variants with an OR greater or equal to 3 and less than 5, where the CI does not contain 1. Not Applicable Not Applicable: These specifications are only for autosomal recessively inherited ACTA1 variants. Please use PM3 for case counting. There are separate specifications for AD ACTA1 variants. Not Applicable: These specifications are only for autosomal recessively inherited RYR1 variants. Please usePM3 for case counting. There are separate specifications for AD RYR1 variants. NA NA NA NA NA NA NA 4-6 unrelated occurrences = Moderate. Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant is absent from controls (PM2). Strength of evidence is determined by points according to (Table 2B). PS4_Moderate = 1.5-3.25 points. ≥ 6 probands from multiple independent studies. NA Not Applicable * If 2-3.5 points. NA 2 – 3.5 phenotype points Not Applicable: CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. Not Applicable: The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. * 2 independent male probands with elevated urine creatine/creatinine ratio on one occasion at minimum, in addition to any proband used for PP4.* Variant must meet PM2_Supporting criterion for PS4 to apply. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* 3-4 observations. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. ≥3 points. Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2-3.5 points** will arrive at **Moderate**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 - 3.5 points** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2-3.5 points** will arrive at **Moderate**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 - 3.5 points** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points 2-3 probands meet criteria described below Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2-3.5 points** will arrive at **Moderate**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Not Applicable PS4_Supporting Variant is found in 2-5 unrelated FH cases (FH diagnosis made by validated clinical criteria).Caveat: variant must also meet PM2. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁸](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYH7/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁹](#pmid_28395867); Oechslin _et al._ 2017[⁶](#pmid_28545618); Hershberger _et al._ 2017[⁵](#pmid_29212902); Ross _et al._ 2020[⁷](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Phenotype specific for disease with single genetic etiology. Proband(s) with specificity score of 1-1.5 (see text). Autosomal dominant: ≥2 probands with variant, and variant meets PM2_Supporting. Not Applicable: This criterion is not applicable for PAH. For proband counting, use PM3 criterion. One family meets HDGC criteria. **PS4\_Supporting:** 1 proband meeting at least one of the \_RUNX1\_-phenotypic criteria (OR 31.8). 1-1.5 points Not Applicable: There are no case-control studies for Pompe disease. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA The prevalence of the variant in affected individuals is significantly increased compared withthe prevalence in controls.* 1 MH case point. Probands with a personal or family history of an MH event are awarded 0.5 points, probands with a personal or family history of a positive (MHS) IVCT/CHCT are awarded an additional 0.5 points. Probands with multiple variants in RYR1 classified as VUS, likely pathogenic or pathogenic are not considered. Popmax in gnomAD ≤0.00006.For variants with popmax MAF in gnomAD >0.00006, an odds ratio of ≥2.08 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. Not Applicable Not Applicable Not Applicable Not Applicable Variant present in 2 unrelated probands in different top-level haplogroups NA **1 point the PS4 cut-off and Proband Scoring Tables from a mix of any of the following phenotypes: specific, consistent and nonspecific.** See instructions below for scoring heterozygous individuals only. Supporting strength applicable for scores ranging from 1-1.75 1 proband meets criteria described below Appropriate to use code when there is 1 proband that meets the laboratory phenotype of the PP4 definition for a specific VWD type 2 phenotype. See instructions below for scoring heterozygous individuals only. Supporting strength applicable for scores ranging from 1-1.75 See instructions below for scoring heterozygous individuals only. Supporting strength applicable for scores ranging from 1-1.75 Appropriate to use code when there is 1 proband point that meets the defined antithrombin deficiency laboratory phenotype. NA NA ≥1 points. Not Applicable: Does not apply. 1-1.5 phenotype point. For phenotype points curation see **Table 1**. Not Applicable: Use PM3 for proband counting. Not Applicable: There are no case-control studies for MPS1. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA ≥1 points. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁹](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYBPC3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[¹⁰](#pmid_28395867); Oechslin _et al._ 2017[¹¹](#pmid_28545618); Hershberger _et al._ 2017[¹²](#pmid_29212902); Ross _et al._ 2020[¹³](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. NA Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNI3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TNNT2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/TPM1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/ACTC1/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL2/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Currently few well-designed case-control studies have been performed for inherited cardiomyopathies. Until such studies become available, comparative analyses can be undertaken using case data (e.g., internal and/or published cohorts) and control data from population-level cohorts (e.g., gnomAD). Cohorts used in these analyses should meet the following criteria: 1. The cases have a clinical diagnosis of the specified disorder or related phenotype (e.g., all cases have HCM or another relevant phenotype\*). * When assessing cases, it's important to consider how likely another potential cause of the phenotype has been excluded. This includes considering the presence of other variants in relevant genes (particularly those likely to be contributing to phenotype) and the extent of testing performed (i.e., single gene sequencing, panel testing, whole exome/genome sequencing).2. The controls should not be derived from study populations that might be enriched for the specified disorder.3. The denominator of the cohorts must be available (e.g., variant detected in 5 out of 3,500 cases and 1 out of 60,000 controls).4. The cohorts do not include closely related individuals (i.e., family members are not included in the case counts).5. The cohorts do not overlap with other cohorts being used in the analysis (i.e., cases are not being counted more than once).6. The population diversity of the case and control cohorts are broadly similar.7. Consider the size of the case cohort — larger cohorts are likely to provide more accurate estimates of variant frequency; therefore, it may be preferable to use data from the largest available case series for case-control analyses (e.g., Walsh _et al._ 2017[⁵](#pmid_27532257), [DECIPHER](https://www.deciphergenomics.org/gene/MYL3/patient-overlap/snvs)).To account for limitations that arise when performing unmatched case-control analyses, the following stringent OR threshold is recommended:* **SUPPORTING** evidence requires the lower bound of the 95% CI around the OR to be **≥5**A PS4 calculator is available at [www.cardiodb.org](https://www.cardiodb.org/ps4_calculator/ps4_calculator.html).If multiple cohorts are available, the final ORs and associated CIs need to be harmonized across all cohorts to determine the final level (e.g., if 2 large cohorts have an OR of ~6 and a third small cohort has an OR of 11, application at a SUPPORTING level should be considered). **\*RELEVANT PHENOTYPES:**1. Cases of HCM and RCM may be combined as they are considered part of the same disease spectrum. 2. For the eight genes covered by these guidelines, the combination of probands with other phenotypes should be reviewed by a clinical expert to determine if grouping is appropriate. 3. Additional considerations for LVNC and end-stage HCM: * Due to the current debate about whether isolated LVNC represents a true disease entity or variation of typical cardiac morphology (Anderson _et al._ 2017[⁶](#pmid_28395867); Oechslin _et al._ 2017[⁷](#pmid_28545618); Hershberger _et al._ 2017[⁸](#pmid_29212902); Ross _et al._ 2020[⁹](#pmid_31143950)), individuals with isolated LVNC should NOT be added to proband or segregation counts (including individuals with isolated LVNC in a family with other cardiomyopathies).HCM and DCM have distinct mechanisms of disease and therefore pathogenetic variants are not anticipated to cause both primary phenotypes. While occurrence in both phenotypes may initially be considered as evidence against pathogenicity, end-stage HCM can present similarly to DCM. Careful consideration is needed before including DCM or related phenotypes in case or segregation data for primarily HCM variants. Not Applicable: CYP1B1 variants cause autosomal recessive disorders associated with glaucoma. The number of probands with the variant will be addressed by PM3. Use of this code at the supporting strength requires ≥ 2 probands and PM2\_Supporting must be met.OR**If PS4 Met at the PS4\_Supporting level by 1 proband (when a separate unrelated proband has been used for PP4):**This variant has been reported in at least 1 proband meeting one of the PS4 requirements of a male with some functional vision impairment by age 30 and/or decreased or absent ERG responses, or a female with functional visual abnormality and documentation of a male relative affected with retinitis pigmentosa, as well as a second apparently unrelated proband previously used for the PP4 code. The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls* In order to be evaluated for this criterion, the variant must not meet BS1.* PS4\_supporting is Met by 2 independent observations of the variant in probands.* This code is not considered mutually exclusive with PM3. For example, variants associated with both dominant and recessive cases can meet both PS4 and PM3.* If probands are reported in at least 2 papers, and the author lists from the two papers contain any of the same authors names, definitely the patients are the same, so count only one case for PS4.* If the two lists of authors are totally distinct / different, usually you can count both cases for PS4.* Patients reported as affected must have QTc measurement greater than or equal to 460ms in order to be counted. Diagnosis of long QT syndrome by itself is not sufficient for inclusion. Sum of case scores 1-1.75 points (see instructions below) Not Applicable Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable Not Applicable Not Applicable Not Applicable * PS4\_Supporting is Met by 1 observation of the variant in a proband.* The proband used for PS4\_Supporting cannot be the same proband used for PP4.* In order to be counted for this criterion, a proband must meet one of the following two requirements: * To have reported phenotypes that score a minimum of 6 phenotype points, as well as genotyping of the _LRBA_ locus to confirm the absence of biallelic variants in _LRBA_ that have been classified either Likely Pathogenic or Pathogenic. Note: a monoallelic _LRBA_ variant can be tolerated. * **OR** * To have reported phenotypes that score a minimum of 10 phenotype points, in the absence of genotyping of the _LRBA_ locus.* Description of the phenotype point system can be found in the bullet points below or in a more detailed form in the attached table entitled “CTLA4 Phenotype Scoring Table”. The table includes a total of two 4-point criteria, four 2-point criteria, and eleven 1-point criteria:* _Clinical criteria_ * Sinopulmonary findings 4 * Non-infectious gastrointestinal or hepatobiliary disease 4 * Immune-mediated cytopenias 2 * Nonmalignant Lymphoproliferation 2 * Severe, persistent, recurrent viral infections including skin warts 2 * Immune-mediated skin and hair findings 1 * Endocrinopathy 1 * Severe, persistent, recurrent, atypical, opportunistic bacterial, Mycobacterial or fungal infections 1 * Neurological findings 1 * Inflammatory findings (arthritis, vasculitis, recurrent fevers) 1 * Lymphoma 1* _Objective criteria_ * Hypogammaglobulinemia 2 * Lymphopenia 1 * Abnormal TBNK levels 1 * Presence of autoantibodies 1 * Defective antigen-specific immune responses 1 * Histopathology findings of lymphocytic / granulomatous tissue infiltration 1 Not Applicable Not Applicable Prior observation of the variant in >1 unrelated patients with the same phenotype, and its absence in controls. Use of this code at the supporting strength requires 1 proband diagnosed with retinoschisis, and a second proband answering the PP4 requirements. PM2\_Supporting must be met.Or 2 probands, each with retinoschisis. PM2\_Supporting must be met. ≥1 points. ≥1 points. Sum of case scores 1-2 points (see instructions below) 1 proband with phenotype consistent with HHT. 1 proband with phenotype consistent with HHT. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. * The strength of this code is defined in **Table 4**. Use PS4\_Supporting when one proband meets the phenotype scoring criteria in **Table 3** and genetic testing requirements. Please note that a proband used for PP4 or PP4\_Moderate cannot be included in PS4\_Supporting.* Point strength has been determined based on a survey of clinical experts, with the goal of quantifying the degree to which a proband’s phenotypes are specific to PIK3CD-related immunodeficiency. Phenotypes have been grouped into categories such as “respiratory findings” or “gastrointestinal disease” to prioritize the diversity of systems affected. Probands receive full points for at least one reported feature in the category. Points per category have been tailored to reward those considered most characteristic of and prevalent within patients with PIK3CD-related immunodeficiency.* A proband must reach greater than or equal to 6 points in the phenotype scoring criteria above (**Table 3**) and, at minimum, a primary immunodeficiency or antibody gene testing panel must have identified no likely pathogenic or pathogenic variants in the _PIK3R1_ locus in order to be counted toward PS4 * Genome or exome sequencing is acceptable in lieu of a gene panel * For genes associated with autosomal recessive disorders, carrier status is acceptable.* If no gene testing panel was performed, additional phenotypic features (reaching greater than or equal to 10 points) are required to count the proband toward PS4.* Scoring of probands with previous genetic testing should be prioritized, particularly in the event of scoring historical or rare probands/variants.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. NEB is associated with Autosomal Recessive disease; PS4 can only be used for case-control studies and not proband counting. Please use PM3 for individual case observations. 2 case observations 0.25 points, please see PS4 chart 2 cases 0.25 points, please see PS4 chart One additional male or female proband observed - must meet PP4 criteria and/or have documented hyperammonemia or metabolic decompensation under physiological stress to be counted NA Not Applicable Not Applicable: These specifications are only for autosomal recessively inherited ACTA1 variants. Please use PM3 for case counting. There are separate specifications for AD ACTA1 variants. Not Applicable: These specifications are only for autosomal recessively inherited RYR1 variants. Please usePM3 for case counting. There are separate specifications for AD RYR1 variants. NA NA NA NA NA NA NA NA Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant is absent from controls (PM2). Strength of evidence is determined by points according to (Table 2B). PS4_Supporting = 0.5 – 1.25 points. ≥ 2 probands from multiple independent studies. NA Not Applicable * If 1-1.5 points. NA 1 – 1.5 phenotype points Not Applicable: CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. Not Applicable: The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. * One independent male proband in addition to any proband used for PP4.* Variant must meet PM2\_Supporting criterion for PS4 to apply. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.* Use for 2nd independent occurrence. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 points. ≥1 point. ≥1 points. ≥1 points. Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1-1.5 points** will arrive at **Supporting**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 - 1.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1-1.5 points** will arrive at **Supporting**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 - 1.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points 1 proband meets criteria described below Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1-1.5 points** will arrive at **Supporting**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Not Applicable BP4_Supporting REVEL score ≤0.5 (missense variants), or no predicted impact to splicing using MaxEntScan (see Fig. 2 for suggested thresholds). As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹⁴](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. Multiple lines of computational evidence suggest no impact on gene or gene product. * Splicing variants: Concordance of SpliceAl and VarSeak* Missense variants: REVEL scores \< 0.5 Computational evidence suggests no impact; REVEL score ≤0.15 or no impact to splicing in MaxEntScan. Applicable as described in Pejaver et al.* REVEL score of 0.183 - 0.290 for missense variants* In frame deletion or insertion predicted benign by PROVEAN, MutationTaster, and MutPred-InDel* No predicted impact on splicing by SpliceAI (score \<0.1) Splicing predictions only. At least three in silico splicing predictors in agreement (SpliceAI, MaxEntScan, SSF, GeneSplicer, HSF, TraP, varSEAK). **For missense variants:****BP4:** REVEL score \< 0.50 AND SpliceAI ≤ 0.20**For synonymous and Intronic variants:****BP4:** SpliceAI ≤ 0.20 **Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants):_BayesDel \< 0.16 and > -0.008 irrespective of aGVGD score (except C65, this case do not apply BP4) AND no predicted differences in splicing (SpliceAI \< 0.2)**Single amino acid inframe deletions** _(See single aa BayesDel spreadsheet):_BayesDel score \< 0.16 AND no predicted splicing impact (Splice AI \< 0.2)**Silent or Intronic Variants (outside ± 1,2 positions):**SpliceAI ≤ 0.1 Multiple lines of computational evidence suggest no impact on gene or gene product. * REVEL score <0.5 for missense variants. * In frame deletion or insertion predicted benign by PROVEAN, MutationTaster, and MutPred-InDel. * No predicted impact on splicing by SpliceAI (score <0.2) REVEL score of < 0.25 Computational evidence suggest no impact on gene or gene product, REVEL score of <0.5 Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant. No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively Agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively. Will also utilize POLG pathogenicity prediction server if/when live again (PMID: 28480171); both tools (REVEL and server) will have to be in agreement to score No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 f or PP3 and BP4, respectively Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, etc) * Missense: Do not use. * Splicing: No predicted impact via splicing (SpliceAI **≤0.1**). Do not apply for missense variants. Due to the lack of benign variants, and the drop in classification accuracy for benign VHL variants, missense predictors should not be used to assign the BP4 evidence code. BP4 can be applied to assess lack of splicing impact, with concordance of Splice AI (≤0.1) and VarSeak (Class 1 or Class 2). For a missense variant apply when REVEL score is less than or equal to 0.290, based on recommendations of Pejaver et al., 2022 (PMID: 36413997) AND SpliceAI score is zero.OR for a synonymous or intronic variant apply when SpliceAI score is zero. This code can be applied for variants reaching a REVEL score of 0.3 or below AND a Splice AI score of less than or equal to 0.1. Use for missense variants that have a REVEL score of less than or equal to 0.290 AND SpliceAI cutoff of \<0.1. Use SpliceAI cutoff of \<0.1 for other variant types. For a missense variant apply when REVEL score is less than or equal to 0.290, based on recommendations of Pejaver et al., 2022 (PMID: 36413997) AND SpliceAI score is zero.OR for a synonymous or intronic variant apply when SpliceAI score is zero. For a missense variant apply when REVEL score is less than or = to 0.290, based on recommendations of Pejaver et al., 2022 (PMID: 36413997) AND SpliceAI score is zero.OR for a synonymous or intronic variant apply when SpliceAI score is zero. Use this code for missense variants with a REVEL score of \< or equal to 0.30 and no evidence of a potential splicing effect using the Splice AI prediction tool (less than or equal to 0.1), or for non-canonical intronic variants with no evidence of a potential splicing effect using the Splice AI prediction tool. Use a REVEL score of ≤0.15 as supportive evidence of no predicted impact on the gene or gene product. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply BP4 when the predicted change is below 0.2[³](#pmid_30661751),[⁴](#pmid_32123317). Use a REVEL score of ≤0.15 as supportive evidence of no predicted impact on the gene or gene product. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply BP4 when the predicted change is below 0.2[⁹](#pmid_32123317),[¹⁰](#pmid_30661751). For missense variants: REVEL ≤0.3. Not Applicable: Does not apply. **Missense variants:** BP4 is not applicable.**Synonymous (silent) or intronic variants**: Multiple in silico splicing predictors suggest no impact on gene or gene product. Use for missense variants that have a REVEL score of less than or equal to 0.290 AND SpliceAI cutoff of \<0.1. Use SpliceAI cutoff of \<0.1 for other variant types. * Missense changes with a REVEL score \< 0.290 (threshold based on Pejaver et al; however, BP4 will only be applied at the supporting level). * For in frame insertions and deletions, use PROVEAN ([http://provean.jcvi.org/seq_submit.php](http://provean.jcvi.org/seq_submit.php) ; score >-2.5), Mutation Taster ([http://www.mutationtaster.org/](http://www.mutationtaster.org/) ; count if “polymorphism”, and MutPred-Indel ([http://mutpredindel.cs.indiana.edu/](http://mutpredindel.cs.indiana.edu/) ; score \<0.5. Apply BP4 if all predictors indicate that the variant is benign.* For non-canonical splice site variants, use SpliceAI ([https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) ) to assess the impact of variants that are not +/-1 or 2 canonical splice site variants. This criterion can be applied as PP3 (Splicing) for SpliceAI scores ≤0.1 (based on Walker et al, PMID: 37352859). * If there is any evidence for possible creation of a cryptic splice site, this criterion should not be applied. Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤0.15 AND SpliceAI ≤0.1).Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1).Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA1 RING aa 2-101; BRCA1 coiled-coil aa 1391-1424; BRCA1 BRCT repeats aa 1650-1857. See Specifications Figure1A and Appendix J for details. For missense variants: REVEL ≤0.3. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis _et al._ 2016[¹⁷](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[²](#pmid_30661751) is recommended for evaluation of predicted splice impacts. Missense or in-frame insertion, deletion or delins variants inside a (potentially) clinically important functional domain, and no predicted impact via protein change or splicing (BayesDel no-AF score ≤ 0.18 AND SpliceAI ≤0.1).Silent variant inside a (potentially) clinically important functional domain, if no predicted impact via splicing (SpliceAI ≤0.1).Intronic variants outside of the native donor and acceptor splice sites (i.e. not +/- 1,2 positions) AND no predicted impact via splicing (SpliceAI ≤0.1).As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure1A and Appendix J for details. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹³](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹⁴](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. As many _in silico_ algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Meta-predictors, such as REVEL, are preferred over multiple individual predictors.Use of REVEL (Ioannidis et al. 2016[¹²](#pmid_27666373)) is recommended at thresholds of **≤0.40 for BP4**.Clinical judgment is needed if any individual algorithms or conservation data are contradictory to REVEL data.Positive predictive value for benign/no impact predictions is generally higher than for pathogenic/impact predictions.[SpliceAI](https://spliceailookup.broadinstitute.org)[¹³](#pmid_30661751) is recommended for evaluation of predicted splice impacts. For missense variants: SpliceAI ≤ 0.1 AND REVEL score of 0.184-0.290For all other variants (not meeting PVS1 or PM4) located outside of donor/acceptor ±1,2 dinucleotide positions, when splicing assay is not available: SpliceAI ≤ 0.1 Applies to:Missense variants: REVEL between 0.184 and 0.290Synonymous variants or noncoding variants: SpliceAI ≤ 0.1 Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.) * REVEL and SpliceAI are the preferred predictors* Apply this code for a missense variant if the REVEL score is \<0.25 AND no splicing defect is predicted according to SpliceAI.* Use SpliceAI to look for a possible splicing defect. Apply this code if delta score for donor gain, donor loss, acceptor gain, or acceptor loss is \<0.1 (in addition to REVEL score \<0.25).* Apply this code for a synonymous or intronic variant if delta score for donor gain, donor loss, acceptor gain, or acceptor loss is \<0.1 (PMID: 37352859). •Supporting evidence can be applied for a REVEL score of \<0.290 based on recommendations of Pejaver et al., 2022 (PMID: 36413997).•Also applicable to synonymous or intronic variants not predicted to impact splicing by SpliceAI ∆ score ≤0.1 Not Applicable Missense variant with HCI-prior probability of pathogenicity \<0.11 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS)ORFor intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score \<= 0.1 as per Walker et al 2023 Not Applicable Not Applicable For a missense variant use REVEL, requires a score between 0.183 - 0.290. In addition, highest SpliceAI delta score should also be below cutoff of 0.1.For a silent / intronic variant outside the designated splice region (conservatively at or beyond positions +7/-21) and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon, BP4 can be met if the highest of the four delta scores within SpliceAI is below the cutoff of ≤0.1. See RPE65-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Please note that BP7 can be met as well. Not Applicable * Met at the default (BP4) level by a missense variant with REVEL score \<0.25, CADD PHRED score \<20, and SpliceAI Δ score \<0.1.* The above requirement for agreement between two _in silico_ tools was reached following a pilot study of suspected pathogenic and suspected benign variants (assembled from ClinVar submissions and published assertions of pathogenicity). Some suspected pathogenic _CTLA4_ variants fall in REVEL’s benign range while some suspected benign _CTLA4_ variants fall in CADD’s pathogenic range. All false positive and false negative predictions in the test set could be avoided by requiring that both tools agree in order for PP3 or BP4 to be Met.* Applicable to both synonymous variants and all intronic variants outside the +/-1,2 dinucleotide positions not predicted to impact splicing by SpliceAI (SpliceAI Δ score \<0.1, PMID: 37352859). Not Applicable Not Applicable Two out of three REVEL/AlphaMissense/CADD predictor scores must meet the thresholds for supporting as specified in Pejaver 2022 PMID: 36413997 (CADD) and Bergquist et al 2025 PMID: 40084623 (AlphaMissense, REVEL). CADD ≤22.7, AlphaMissense ≤0.169, REVEL ≤0.29. The criterion can also be used for non-canonical splice variants if SpliceAI ≤0.1; can be applied in conjunction with BP7. For synonymous variants, CADD ≤22.7 is sufficient since REVEL and AlphaMissense are not applicable. Applies to:Missense variants: REVEL between 0.184 and 0.290 with SpliceAI ≤ 0.1or Synonymous variants or noncoding variants: SpliceAI ≤ 0.1 For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. Not Applicable: Does not apply. * For missense variants: REVEL score ≤0.15 and SpliceAI score ≤0.1.* For synonymous and intronic variants: SpliceAI score ≤0.1. * For missense variants: REVEL score ≤0.15 and SpliceAI score ≤0.1.* For synonymous and intronic variants: SpliceAI score ≤0.1. Missense variant with HCI-prior probability of pathogenicity \<0.11 as per https://hci-priors.hci.utah.edu/PRIORSORFor intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score \<= 0.1 as per Walker et al 2023. Missense variant with HCI-prior probability of pathogenicity \<0.11 as per https://hci-priors.hci.utah.edu/PRIORSORFor intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score \<= 0.1 as per Walker et al 2023. Missense variant with “MAPP/PP2 Prior P” score \<0.11 from [http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique](http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique) ORFor intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score \<= 0.1 as per Walker et al 2023. * Met at the default (BP4) level by a missense variant with REVEL score less than or equal to 0.290 and CADD PHRED score less than or equal to 22.7.* SpliceAI Δ scores should be checked for missense variants as well. Variants with a predicted impact on splicing (SpliceAI Δ score greater than or equal to 0.2) should not be evaluated using these specifications, but rather using alternative specifications for autosomal recessive disease with a loss-of-function mechanism, unless functional evidence indicates a gain-of-function effect. The current specifications are written for a PIK3CD gain-of-function disease mechanism that has so far not been reported in association with variants that disrupt splicing.* Met at the default (BP4) level by a synonymous or intronic variant not predicted to impact splicing by SpliceAI (SpliceAI Δ score \<0.1, PMID: 37352859).* Higher strength levels of BP4\_Moderate or BP4\_Strong have not been recommended due to pilot data indicating poor performance and significant discordance between REVEL and CADD predictions for established benign and gain-of-function PIK3CD variants. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Missense variants - REVEL score >0.183 and ≤0.290 Splice region and intronic variants - spliceAI delta score \<0.1Synonymous variants – spliceAI delta score \<0.1 if variant is not within the first or last three nucleotides of an exon. This code is applicable for missense variants with a REVEL score between 0.184-0.290 or for synonymous or indel variants with a CADD score between 17.4-20 (PMID: 40225145). This code is also applicable for intronic variants where BP7 is not applicable and the SpliceAI score is \< or = 0.1. For a missense variant use REVEL, requires a score between 0.183 - 0.290. In addition, highest SpliceAI delta score should also be below cutoff of 0.1.For a silent / intronic variant outside the designated splice region (conservatively at or beyond positions +7/-21) and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon, BP4 can be met if the highest of the four delta scores within SpliceAI is below the cutoff of ≤0.1. See GUCY2D-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Please note that BP7 can be met as well. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.BP4 is met if the REVEL score ≤ 0.15 or if the variant is not predicted to impact splicing using SpliceAI. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. For missense variants, use REVEL with a score ≤0.1 AND SpliceAI with a score ≤0.05.For variants that may affect splicing, use SpliceAI with a score ≤0.05. Splice AI scores can be calculated here: [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/)For any variant with RNA or other experimental data indicating no impact on splicing, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Use a REVEL score of ≤0.15 as supportive evidence of no predicted impact on the gene or gene product. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply BP4 when the predicted change is below 0.2 [⁸](#pmid_32123317),[⁹](#pmid_30661751). Award BP4 for a synonymous, intronic positions (except canonical splice sites) or non-coding variants in the UTRs, if two out of three of the splicing prediction tools predicted no impact on splicing function.Not applicable for any variant type except for synonymous, intronic positions (except canonical splice sites) and non-coding variants in the UTRs,. This criterion can be applied when two of three splicing prediction tools predict no splicing change. The splicing prediction tools used are: varSEAK, spliceAI and MaxEntScan. * For missense variants: REVEL score of 0.184-0.290* For all other variants located outside of donor/acceptor ±1,2 dinucleotide positions, when splicing assay is not available: SpliceAI ≤ 0.1 * Missense: REVEL score **≤.249*** Splicing: No predicted impact via splicing (SpliceAI **≤0.1**). * Missense changes with a REVEL score \<0.5 will meet BP4.* For in-frame deletions and insertions, use Mutation Taster. * For non-canonical splice site variants, use Splice AI. Based on data from Jaganathan et al., 2019 (PMID: 30661751), Houdayer et al., 2012 (PMID: 22505045), Tang et al., 2016 (PMID: 27313609), and Walker et al., (2023) PMID:37352859, BP4 can be applied if there is a Δ Score ≤ 0.1.* Do not apply this rule if there is evidence for creation of a cryptic splice site.* Can be used with BP7 code. * Recommended prediction program for missense variants: REVEL. Use 0.326 as a discriminatory cut-off value.* Recommended prediction programs for splice variants: GeneSplicer, MaxEntscan, and NNSPLICE. The outcome of all 3 prediction programs need to be in concordance. Computational evidence suggests no impact; REVEL score ≤0.15 or no impact to splicing in MaxEntScan.* Use REVEL, award BP4 if score is 0.15 or lower. Make sure to also check MAXENTSCAN to rule out the creation of a cryptic splice site. For missense variants, REVEL score \< 0.500 and agreement in splicing predictors that no splicing effects are predicted. For synonymous/intronic/non-coding variants concordance of MaxEntScan and SpliceAI. * REVEL score \<0.29 for missense variants (based on guidance from Pejaver et al, 2022, PMID: 36413997).* In frame deletion or insertion predicted benign by MutPredIndel and MutationTaster.* No predicted impact on splicing by SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) , based on a score \<0.1 (as indicated in PMID: 37352859, Table 1 and Figure 4). * REVEL score \<0.29 for missense variants (based on guidance from Pejaver et al, 2022, PMID: 36413997).* In frame deletion or insertion predicted benign by PROVEAN and MutationTaster.* No predicted impact on splicing by SpliceAI, [https://spliceailookup.broadinstitute.org/](https://spliceailookup.broadinstitute.org/) , based on a score \<0.1 (as indicated in PMID: 37352859, Table 1 and Figure 4). * REVEL score \<0.2 for missense variants* In frame deletion or insertion predicted benign by PROVEAN, MutPred indel, and MutationTaster.* No predicted impact on splicing by SpliceAI and varSEAK. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.)* For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc).* For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. * For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. * For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)* For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc).* For missense variants use REVEL with a score ≤ 0.290.* For splice site variants use SpliceAI with a score ≤ 0.1. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. For missense variants: REVEL ≤0.3. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. This code can be applied for variants reaching a REVEL score of 0.3 or below AND a Splice AI score of less than or equal to 0.1. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. For a missense variant use REVEL, requires a score between 0.183 - 0.290. In addition, highest SpliceAI delta score should also be below cutoff of 0.1.For a silent / intronic variant outside the designated splice region (conservatively at or beyond positions +7/-21) and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon, BP4 can be met if the highest of the four delta scores within SpliceAI is below the cutoff of ≤0.1. See AIPL1-specific PVS1 Decision Tree **part (a)** for SpliceAI flowchart.* Please note that BP7 can be met as well. PS1_Strong Missense variant at the same codon as a variant classified pathogenic (by these guidelines), and predicts the same amino acid change.Caveat: there is no in silico predicted splicing impact for either variant. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change OR different variant at same nucleotide position as a pathogenic splicing variant, where in silico models predict impact equal to or greater than the known pathogenic variant. Same amino acid change as an established pathogenic variant; ORsplice variants at same nucleotide and with similar impact prediction as previously reported pathogenic variant.* Established variant must meet criteria for pathogenicity by the HL specifications * Can also use PS1 for splice variants located in the splice consensus sequence, at the same nucleotide position as a previously reported pathogenic variant * Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T* No additional hearing loss specifications for missense variants. Follow recommendations as outlined in Richard 2015 and/or the Sequence Variant Interpretation working group within ClinGen.* Caveat (from ACMG/AMP guidelines): Assess the possibility that the variant may act directly through the DNA change (e.g. through splicing disruption as assessed by at least computational analysis) instead of through the amino acid change) Same predicted splicing impact as a previously classified (likely) pathogenic variantApplicable as described in Walker et al. (PMID: 36865205) Not Applicable: Not applicable for CDH1. **PS1:** Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.**For splice site variants:** **PS1\_Variable:** Follow recommendations from the ClinGen SVI Splicing Subgroup (Walker et al., 2023, PMID: 37352859 [⁵](#PMID_37352859)) Can be applied to variants asserted as Pathogenic following the _TP53_ VCEP’s specifications. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Use with no specification. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change * Previously established pathogenic variant must reach a classification of pathogenic without PS1 Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change Same amino acid change as a previously established pathogenic variant regardless of nucleotide change Same amino acid change as a previously established pathogenic variant regardless of nucleotide change Same amino acid change as a previously established pathogenic variant regardless of nucleotide change Applied per original ACMG/AMP guidelines Use PALB2 PS1 Splicing table Applied only to variants with interpretation by the VHL VCEP or by a variant with pathogenicity established using VHL VCEP specifications. Use as originally specified, but the comparison variant must reach a pathogenic classification using these rule specifications in order to apply code. This evidence code can be applied when there is 1 pathogenic variant or 2 likely pathogenic variants at the same residue based on _F9_ gene rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect.**OR**When two or more variants are share the same predicted splicing effect and one comparison splicing variant reaches a pathogenic classification or 2 comparison variants reach a likely pathogenic classification using the Coagulation Factor Deficiency VCEP specifications modified from Walker, et al 2023 (PMID: 37352859). Use with no specification except comparison variant must be classified as pathogenic using rules from the VWD VCEP. Use as originally specified, but the comparison variant must reach a pathogenic classification using the these rule specifications in order to apply code. Use as originally specified, but the comparison variant must reach a pathogenic classification using the these rule specifications in order to apply code. Use with no specification except comparison variant must be classified as pathogenic using _SERPINC1_ rule specifications from the Thrombosis VCEP. Applicable for a same amino acid change if the previously established variant is classified as pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. Applicable for a same amino acid change if the previously established variant is classified as pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ NA The previously established variant was classified as Pathogenic according to the APC-specific modifications.This criterion can be applied to both missense and splice variants in _APC_. **Missense variants:** when the variant under assessment results in the same amino acid change as previously established Pathogenic variant(s). There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other variants leading to the same missense change at these positions meet PS1\_Moderate. No missense variant has been classified as Pathogenic based on current evidence. **Splice variants**: when the variant under assessment affects splicing at the same nucleotide as a previously established Pathogenic variant. The splice prediction must be above defined thresholds (see instructions) or similar to the previously established variant by multiple _in silico_ predictors. Use with no specification except comparison variant must be classified as pathogenic using rules from the VWD VCEP. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Splice region variants following Table 3 in Walker et al (PMID: 37352859). Apply **PS1**, for predicted **missense** substitutions, where a previously classified **pathogenic** variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI≤0.1)).Apply **PS1**, for exonic and intronic variants with same predicted impact on **splicing**, as a previously classified **pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant. See Specifications Table 5 and Appendix E, J and K for details. Same amino acid change as a previously established pathogenic variant in _LZTR1_ regardless of nucleotide change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[⁶](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. Apply **PS1**, for predicted **missense** substitutions, where a previously classified **pathogenic** variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI≤0.1)).Apply **PS1**, for exonic and intronic variants with same predicted impact on **splicing,** as a previously classified **pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant. See Specifications Table 5 and Appendix E, J and K for details. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. No cardiomyopathy specifications. Apply as outlined by Richards _et al_. 2015[¹](#pmid_25741868).Example of when rule should NOT be applied. NM\_000256.3(_MYBPC3_): c.2308G>A (p.Asp770Asn) has an established impact on splicing leading to nonsense mediated decay (NMD) and should not be used to provide evidence for other variants observed to result in the same amino acid change. For missense variants that do not affect splicing (SpliceAI ≤ 0.2): same amino acid change as a previously established pathogenic variant.For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.[⁶](#PMID_37352859) Same amino acid change as a previously established Pathogenic variant regardless of nucleotide change.* Comparison variant must have been evaluated by the X-linked IRD VCEP using these rules and established as a pathogenic variant and classified as such.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other.Same predicted splicing impact as a previously classified Pathogenic variant.* Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the same position that has been designated Pathogenic.* Used in conjunction with PVS1 for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and that have a comparable variant within the same splice donor/acceptor +/-1,2 dinucleotide that has been designated Pathogenic. Specific combinations are found in \_RPGR\_-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.* The comparison variant must reach a pathogenic classification using these rule specifications, and must have reached pathogenic without the use of the PS1 code.* SpliceAI must be used to ensure that the comparison variant is not causing a splicing defect. Applicable for a same amino acid change if previously established variant is classified as pathogenic by SCID VCEP specifications for _FOXN1._Can also be applied for variants with the same predicted splicing event as a known Pathogenic variant (as classified by the SCID VCEP specifications for _FOXN1_), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: [37352859](https://pubmed.ncbi.nlm.nih.gov/37352859)) for determining when PS1 should be applied at PS1\_Strong, \_Moderate, or \_Supporting. It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _ADA._ A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect).ORVariants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _DCLRE1C._ It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _IL7R._ Same amino acid change as a previously established Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other.Same predicted splicing impact as a previously classified Pathogenic variant. * Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the **same position** that has been designated **Pathogenic**.* Used in conjunction with PVS1 for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and that have a comparable variant within the same splice donor/acceptor +/-1,2 dinucleotide that has been designated Pathogenic.Specific combinations are found in RPE65-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _JAK3._ * Use PS1 for missense variants when other variant was classified pathogenic by VCEP standards without using PS1.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Splicing predictions (by SpliceAI) should remain the same for WT and both mutant alleles.* Use at the default (PS1) level for splicing variants in combination with **PP3** if located **outside** the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant **at the same position** that has been classified **pathogenic** by VCEP standards (PMID: 37352859).* Use at the default (PS1) level for splicing variants in combination with **PVS1** if located **within** the splice donor/acceptor +/-1,2 dinucleotide positions with a comparable variant **within the same splice donor/acceptor +/-1,2 dinucleotide** that has been classified **pathogenic** by VCEP standards (PMID: 37352859). It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _RAG1._ It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.Applicable if the previously established variant is classified as **pathogenic** by SCID VCEP specifications for _RAG2._ Same amino acid change as a previously established _**pathogenic**_ variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established **Pathogenic** variant. Comparison variant must have been evaluated by the X-linked IRD VCEP using these rules and established as a Pathogenic variant and classified as such. For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other.When evaluating splicing impact, as a previously classified Pathogenic variant, * Use in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and have a comparable nucleotide variant at the same position that has been designated **Pathogenic**.* Used in conjunction with PVS1 for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and that have a comparable variant within the same splice donor/acceptor +/-1,2 dinucleotide that has been designated **Pathogenic**.Specific combinations are found in RS1-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ Same amino acid change as a previously established pathogenic variant in _PPP1CB_ regardless of nucleotide change. Strength modification depending upon classification of previously established variant (pathogenic vs. likely pathogenic).Previously established variant must be classified using the SCID VCEP specifications for _IL2RG._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect).ORVariants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect).ORVariants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect).ORVariants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. * Use at the default level (PS1) for missense variants when other variant was classified as Pathogenic for autosomal dominant PIK3CD gain-of-function-related disease by Antibody Deficiencies VCEP specifications for _PIK3CD_ without using PS1.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Neither change should be predicted to affect splicing (SpliceAI Δ score \<0.2). Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Applicable when the previously reported variant is classified as Pathogenic using these OTC specifications. Comparison missense variant must reach a pathogenic classification using the _ABCA4_ VCEP specifications. Do not apply if the comparison variant is suspected to cause a splicing defect via SpliceAI or other splice predictor.**OR**Comparison splicing variant must reach a pathogenic classification using the _ABCA4_ VCEP specifications and both variants must share the same predicted splicing effect. **OR**See Walker, et al 2023 (PMID: 37352859) Figure 5 to use this rule code for variants with RNA sequencing data. Same amino acid change as a previously established Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification.Same predicted splicing impact as a previously classified Pathogenic variant. * Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the **same position** that has been designated **Pathogenic**.* Used in conjunction with PVS1 for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and that have a comparable variant within the same splice donor/acceptor +/-1,2 dinucleotide that has been designated Pathogenic.Specific combinations are found in GUCY2D-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Strong for 1 pathogenic or 2 likely pathogenic variants resulting in the same amino acid change. The likely pathogenic or pathogenic variant(s) must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant(s) resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". Applicable for a same amino acid change if the previously established variant is classified as pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances.[³](#pmid_37352859) No change. Same amino acid change as previously established pathogenic variant * Use for missense changes as long as splicing is ruled-out for both alterations. * Use ATM PS1 Splicing table for splicing variants with similar predictions or observations of splice defect. * Same amino acid change as a previously established pathogenic variant classified using the ACADVL specifications without application of PS1, regardless of nucleotide change OR Same amino acid change as ≥2 previously established likely pathogenic variants classified using the ACADVL specifications without application of PS1, regardless of nucleotide change. * Caveat (from ACMG/AMP guidelines): Assess the possibility that the variant may act directly through the DNA change (e.g. through splicing disruption as assessed by at least computational analysis) instead of through the amino acid change.* Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al., (2023) PMID: 37352859). * PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al., (2023) PMID: 37352859. * Beware of changes that impact splicing rather than the amino acid. Splicing predictions should remain the same for WT and mutant alleles.* Original variant should be pathogenic according to the (modified) ACMG guidelines for variant classification. Same amino acid change as an established pathogenic variant; OR splice variants at same nucleotide and with similar impact prediction as previously reported pathogenic variant.* Established variant must meet criteria for pathogenicity by the HL specifications.* Can also use PS1 for splice variants located in the splice consensus sequence, at the same nucleotide position as a previously reported pathogenic variant. * Example: c.105+1G>C is known to be pathogenic, can use PS1 for c.105+1G>T.* No additional hearing loss specifications for missense variants. Follow recommendations as outlined in Richard 2015 and/or the Sequence Variant Interpretation working group within ClinGen.* Caveat (from ACMG/AMP guidelines): Assess the possibility that the variant may act directly through the DNA change (e.g. through splicing disruption as assessed by at least computational analysis) instead of through the amino acid change). For same AA change, must confirm there is no difference in splicing using RNA data or in-silico modeling data (concordance of MaxEntScan and SpliceAI). For non-canonical intronic splicing variants at same nucleotide should have equal or worse splicing impact.This rule code can only be used to compare variants asserted as pathogenic by the ClinGen DICER1 VCEP. Likely pathogenic changes do not apply. * This criterion is applicable as is for any variant resulting in the same amino acid change as a previously established pathogenic variant regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.* PS1 may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). * This criterion is applicable for any variant resulting in the same amino acid change as a variant that has been previously established as pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.* PS1 may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). PS1 is applicable as described. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant in _SHOC2_ regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous pathogenic residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _BRAF_ and _RAF1._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _SOS1_ and _SOS2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _SOS1_ and _SOS2._ Same amino acid change as a previously established pathogenic variant in _PTPN11_ regardless of nucleotide change. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _MAP2K1_ and _MAP2K2_. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _MAP2K1_ and _MAP2K2._ Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _BRAF_ and _RAF1_. Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.* **\>1** Identical amino acid change in paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023). * PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.* **\>1** Identical amino acid change in paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023). * PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.* **\>1** Identical amino acid change in paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.* **\>1** Identical amino acid change in paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). This evidence code can be applied when there is 1 pathogenic variant or 2 likely pathogenic variants at the same residue based on _F8_ gene rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect.**OR**When two or more variants are share the same predicted splicing effect and one comparison splicing variant reaches a pathogenic classification or 2 comparison variants reach a likely pathogenic classification using the Coagulation Factor Deficiency VCEP specifications modified from Walker, et al 2023 (PMID: 37352859). Same amino acid change as a previously established **Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same amino acid change as a previously established Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification.Same predicted splicing impact as a previously classified variant. * Refer to the AIPL1-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023) for specific combinations. BS4_Strong Lack of segregation in ≥2 index case families (unrelated), when data is available for ≥2 informative meioses in each family.Caveat: must be ≥1 unaffected relative (LDL-C <50th centile) who is positive for the variant. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Lack of segregation in affected members of two or more families. Non-segregation with disease.* Phenotype+/genotype- * Strong evidence for benign. * Be cautious when using this as the possibility for phenocopy is high. The hearing loss phenotype should be consistent within the family to consider it a non-segregation, though intra-familial variability has been reported. Factors to consider are: * Age of onset (ie. congenital/early childhood vs. adult onset). * Hearing loss prevalence increases significantly with age. A congenital hearing loss in a child and a late onset hearing loss in a grandparent would not be a consistent phenotype. * Severity (ie - mild vs. profound). * Minor differences may exist among family members. * Keep in mind that progression in older individuals may account for a discrepancy between individuals. * Sex -based differences (infertility, genes on X chromosomes) * Audiogram shape. * May not be completely consistent among family members even with same etiology.* Genotype+/phenotype- * Confounding variables to applying this rule: Age-related/sex-related penetrance, variable expressivity, etc. * If the gene is associated with later onset and individual with the non-segregation is beyond the expected age that the hearing loss would occur, consider applying BS4_Supporting * Recommend only using for fully penetrant genes (typically genes associated with AR hearing loss). * Must be confident that patient is truly unaffected and a hearing loss is not missed or subclinical. Be cautious if only phenotyping was newborn hearing screening. Diagnostic audiometric testing (auditory brainstem response (ABR) or audiogram should be required). * Any evidence for reduced penetrance, do not use BS4 Applicable as described Per original ACMG/AMP guidelines. **BS4:** Applicable when observed in ≥ 2 informative meioses. Lack of segregation in affected family members (i.e. family members diagnosed with LFS-associated cancers as described in Table of LFS Cancers and Points for PS2 and PP1 Code Application). Not Applicable Variant not detected in an affected family member. Not Applicable: BS4 is not applicable. Phenotype for MHS is routinely determined based on the vitro contraction test (IVCT) that has a false positive rate of approximately 6% (PP1) or the caffeine-halothane contracture test (CHCT). As the phenotype in individuals who have not experienced an MH crisis cannot be reliably determined BS4 is not utilized. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected and/or treated members of a family. Lack of segregation in affected and/or treated members of a family. Lack of segregation in affected and/or treated members of a family. Lack of segregation in affected and/or treated members of a family. Lack of segregation in affected members of a family and/or segregation of disease in paternal family members LOD ≤ -1.28 or Bayes Factor (LR) ≤.053:1 Lack of segregation is seen in affected members of ≥2 families Variant not tracking in an affected family member. This evidence code can be used when a _F9_ variant is observed in a male with a family history of hemophilia B and has a normal factor IX activity level. Appropriate to use when two or more relatives have the phenotype consistent with VWD type 2 without harboring the variant identified in other affected family members. Additionally, there is not another established cause of type 2 VWD (e.g. - there are not multiple type 2 VWD diagnoses) segregating in the family. Variant not tracking in an affected family member. Variant not tracking in an affected family member. Appropriate to use when the variant is found not to segregate in a minimum of four relatives with abnormal antithrombin activity levels \[\< 0.8 IU/mL (or below the lower limit of a laboratory’s assays reference range)\] within the same family, **OR** the variant does not segregate in 2 or more families. Non-segregation defined by having abnormal antithrombin activity levels without _SERPINC1_ variant of interest. Applicable to family members without variant who have MPC⁶ score ≥50% (i.e., genotype negative, phenotype positive). Applicable to family members without variant who meet PP4 criteria (HbA1C 5.6 – 7.6% (38-60 mmol/mol) (if given multiple results, use maximum value) AND Fasting glucose 5.5-8 mmol/L (100-144 mg/dL)) Requires only one informative meiosis. Can be applied without additional specifications. To apply the BS4 criteria, it is sufficient to have one affected family member without the segregation of the variant. Affected member without the variant must score at least 1 phenotype point or at least two affected members without the variant must each score at least 0.5 phenotype points (see **Table 1**). Appropriate to use when two or more relatives have the phenotype consistent with VWD type 2 without harboring the variant identified in other affected family members. Additionally, there is not another established cause of type 2 VWD (e.g. - there are not multiple type 2 VWD diagnoses) segregating in the family. Non-segregation with disease in a family i.e. variant is absent in an affected individual. Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4 - LR ≤0.05:1BS4\_VeryStrong – LR ≤0.00285:1 Requires only one informative meiosis. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4 - LR ≤0.05:1BS4\_VeryStrong – LR ≤0.00285:1 Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Any non-segregations should be carefully evaluated to rule out a phenocopy or the presence of a second disease-causing variant before considering it as conflicting or benign evidence. 1. The presence of “phenocopies” (e.g., athlete’s heart, hypertensive heart disease, ischemic cardiomyopathy, alcoholic cardiomyopathy, diabetic cardiomyopathy) can mimic non-segregation (i.e., lack of segregation) among affected individuals. 2. Families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent ‘non-segregation’.Because of these possibilities, **multiple (≥2) non-segregations** that are highly unlikely to be phenocopies or due to alternate variants (e.g., those without a possible alternate cause) **are required to apply this rule**. A higher number of non-segregations is necessary for instances where alternative causes are possible (e.g., non-segregation in a sibling with childhood onset cardiomyopathy versus a grandparent with hypertension and HCM).Careful consideration of the above points is required when using this data as conflicting evidence, especially when overall evidence supports likely pathogenic or pathogenic. Non-segregations with PCG, ASD or JOAG. Use caution and consider phenotypes of the affected segregations, age at diagnosis and pedigree structure before applying to non-segregations with JOAG or POAG. For maternally inherited variants in _RPGR_, a variant present in a clinically verified (documented with a normal eye examination with functional studies including a normal ERG) unaffected male over age 30 could be used to establish this code. Inheritance is complicated in consanguineous families and in dual diagnoses situations so panel testing of unaffected members required to confirm presence of the variant. Lack of segregation in affected members of a family * BS4 is met by absence of the variant in 2 or more affected family members* The family member must have QTc greater than or equal to 480ms or Schwartz score >3 or syncope.* Please use the European Society of Cardiology guidelines for 2022 (PMID: 36017572, Table 10) to calculate a modified Schwartz score. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Can be applied without additional specifications. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05. Can be applied without additional specifications. Can be applied without additional specifications. Lack of segregation in affected members of a family.* One or both variants are absent in a similarly affected family member. Can be applied without additional specifications. * Due to incomplete penetrance (45-70%), BS4 will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting lack of segregation.* Lack of segregation should be identified in more than 1 affected family member. Can be applied without additional specifications. Can be applied without additional specifications. Lack of variant segregation in affected members of a family. NA Requires only one informative meiosis. Requires only one informative meiosis. Can be applied without additional specifications. Lack of segregation in affected members of a family. Lack of segregation in affected members of a family. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05. \*For multiple pedigrees, results are combined. Recommended segregation analysis tool: COOL (COsegregation OnLine) v2 [http://fengbj-laboratory.org/cool2/manual.html](http://fengbj-laboratory.org/cool2/manual.html) * Caution in case the phenotype is not highly specific. The patients lacking segregation must reach 6 or more points of the phenotypic scoring system described in **Table 4.*** Lack of segregation should be identified in more than 1 affected family member to meet BS4 at the default level of strength. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. NA This code is applicable when there are two affected family members without the variant of interest. Lack of segregation in affected members of a family.* One or both variants are absent in a similarly affected family member. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Use as is. One affected individual (genotype-, phenotype+) is sufficient for BS4. Do not apply for genotype+, phenotype- individuals, as LGMD is characterized by variable expressivity and late onset is not uncommon. Applicable to family members without variant who have MPC score >50% (i.e., genotype negative, phenotype positive). Not Applicable: Not applicable as these are de novo, germline mosaic or post-zygotic mutations. Not Applicable: The presence of phenocopies, the reduced age-related penetrance and the possibility that more than one pathogenic variant can contribute to the phenotype observed in families make non-segregation difficult to assess in the context of MYOC and POAG. Not Applicable: AD Condition: Co-segregation analysis in low penetrance genes can lead to false positive results (PMID 32773770)AR Condition: Informative instances of lack of co-segregation in A-T families are too rare to be considered for weight at this time and can also be considered for BP2 if biallelic unaffected patients are observed in an A-T family. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group * Caution is warranted when the phenotype is not highly specific. Lack of segregation should then be clear in >1 affected family member. Non-segregation with disease.* Phenotype+/genotype- * Strong evidence for benign. * Be cautious when using this as the possibility for phenocopy is high. The hearing loss phenotype should be consistent within the family to consider it a non-segregation, though intra-familial variability has been reported. Factors to consider are: * Age of onset (ie. congenital/early childhood vs. adult onset) * Hearing loss prevalence increases significantly with age. A congenital hearing loss in a child and a late onset hearing loss in a grandparent would not be a consistent phenotype. * Severity (ie - mild vs. profound) * Minor differences may exist among family members. * Keep in mind that progression in older individuals may account for a discrepancy between individuals. * Audiogram shape * May not be completely consistent among family members even with same etiology.- Genotype+/phenotype-* Confounding variables to applying this rule: Age-related penetrance, variable expressivity, etc.* If the gene is associated with later onset and individual with the non-segregation is beyond the expected age that the hearing loss would occur, consider applying BS4_Supporting.* Recommend only using for fully penetrant genes (typically genes associated with AR hearing loss)o Must be confident that patient is truly unaffected and a hearing loss is not missed or subclinical. Be cautious if only phenotyping was newborn hearing screening. Diagnostic audiometric testing (auditory brainstem response (ABR) or audiogram should be required).* Any evidence for reduced penetrance, do not use BS4. Family members should be phenotype-positive (must be high- or moderatespecificity phenotype; see phenotype table), genotype-negative 1st, 2nd, or 3rd degree relatives of the proband. Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. Lack of segregation in affected members of a family. Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families* Need to confirm that the family member is ‘affected with a neurodevelopmental phenotype consistent with the gene’ at a minimum. Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families Lack of segregation in affected members of a family.* Absent in a similarly affected family member, when seen in two or more families. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Requires only one informative meiosis. Not Applicable: Reduced penetrance, variable expressivity and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies This evidence code can be used when a _F8_ variant is observed in a male with a family history of hemophilia A and has a normal factor VIII activity level. Not Applicable: Reduced penetrance, variable expressivity and phenocopies Lack of segregation in affected members of a family.* One or both variants are absent in a similarly affected family member. PM5_Strong Missense variant at a codon with ≥2 missense variants classified pathogenic (by these guidelines), and predicts a different amino acid change. NA NA Missense change at same codon as two different pathogenic missense variants.* Located at an amino acid residue with known pathogenic variation (at least 2 other variants at the same site meet pathogenic criteria for based on independent data)* Caveat: Assess whether the variants in question could have an impact at the DNA level, such as through splicing impacts. NA NA **PM5\_Strong:** Missense change at an AA residue where ≥ 2 different missense changes which have been determined to be pathogenic before (after accounting for Grantham scores).Caveats:\*Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20.\*The nonsense/frameshift variants before c.98 only affect one of the _RUNX1_ functional transcript. PVS1 is also not appliable in this region based on the _RUNX1_ PVS1 decision tree.\*PM5 cannot be used if PM1 was applied at any strength level. Missense variant at an amino acid residue where ≥2 different missense variants previously determined to be pathogenic according to the _TP53_ VCEP’s specifications have been seen before. NA NA NA Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. NA NA NA NA NA NA NA NA NA NA NA NA NA Applicable once two amino acid changes have been classified as pathogenic at the same amino acid residue. Applicable once two amino acid changes have been classified as pathogenic at the same amino acid residue. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. Not Applicable: Does not apply. NA NA NA Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. ≥2 different \[likely\] pathogenic residue changes at the same codon observed in ≥5 probands. NA Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. NA NA NA NA NA NA Same residue as 2 previously established pathogenic variants (in which case both variants must be assessed independently of PM5). NA NA PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA NA ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. PM5 may be applied at a Strong level of evidence for any nonsense variant with 4+ points from informative variants (see below point table). PM5\_Strong should be downgraded to PM5\_Moderate if PVS1 is applied at any strength. **Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. ≥2 different missense changes at same codon have been determined to be likely pathogenic or pathogenic based on HHT VCEP rules. ≥2 different missense changes at same codon have been determined to be likely pathogenic or pathogenic based on HHT VCEP rules. NA NA NA NA No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA NA NA No change - use as originally described No change - use as originally described Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Strong for 2 pathogenic or 3 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Applicable once two amino acid changes have been classified as pathogenic at the same amino acid residue NA Same residue as 2 previously established pathogenic variants or 1 previously established pathogenic and 2 likely pathogenic variants or 4 previously established likely pathogenic variants (all assessed independently of PM5) NA * See PM5 table. Note: Cannot be applied with PM1, apply criteria with the highest strength. If both are applicable at the same strength, apply PM5 as it is amino acid specific. NA Missense change at same codon as two different pathogenic missense variants.Located at an amino acid residue with known pathogenic variation (at least 2 other variants at the same site meet pathogenic criteria for based on independent data).* Caveat: Assess whether the variants in question could have an impact at the DNA level, such as through splicing impacts. NA NA NA NA Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* ≥2 different missense changes affecting the amino acid residue.* Do not apply PM1 in these situations. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands. Greater than or equal to 2 known pathogenic variants at same site as novel change (within the same gene). Greater than or equal to 2 known pathogenic variants at same site as novel change (within the same gene).Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Greater than or equal to 2 known pathogenic variants at same site as novel change (within the same gene).Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Greater than or equal to 2 known pathogenic variants at same site as novel change (within the same gene).Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. NA This should say greater than or equal to 2 known pathogenic variants at same site as novel change. NA PM5_Moderate Missense variant at the same codon as a variant classified pathogenic (by these guidelines), and predicts a different amino acid change. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. Missense change at an amino acid residue where a different missense change determined to be pathogenic or likely pathogenic has been seen before. In addition, variant being interrogated must have BLOSUM62 score equal to or less than the known variant. Missense change at same codon as another pathogenic missense variant.No changes. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group. Applicable as described. NA **PM5:** Missense change at an AA residue where a different missense change which has been determined to be pathogenic before (after accounting for Grantham scores).Caveats:\*Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20.\*The nonsense/frameshift variants before c.98 only affect one of the _RUNX1_ functional transcript. PVS1 is also not appliable in this region based on the _RUNX1_ PVS1 decision tree.\*PM5 cannot be used if PM1 was applied at any strength level. Missense variant at an amino acid residue where 1 different missense variant previously determined to be pathogenic according to the _TP53_ VCEP’s specifications has been seen before. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Use with no specification. Missense change at an amino acid residue where a different missense varaint previously determined to be pathogenic * Previously established pathogenic variant must reach a classification of pathogenicity without PM5 * Grantham score for alternate pathogenic variant must be less than for variant being assessed Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Applied per original ACMG/AMP guidelines (protein-coding genes) NA Pathogenicity of prior variant is established by interpretation of the VHL VCEP or variants with pathogenicity established using VHL VCEP specifications. The Grantham distance should be used to compare variants. The variant under consideration must be equal or a larger distance than the classified pathogenic variant (Grantham, 1974, Table 2 [¹⁶](#pmid_4843792) ). Splice metapredictors should be used to ensure the variant is not predicted to have an effect on splicing. Use as originally specified, but the comparison variant must reach a pathogenic classification using the these rule specifications in order to apply code. This evidence code can be applied when there is 1 pathogenic variant or 2 likely pathogenic variants at the same residue based on _F9_ rule specification from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect. Use code when previously reported variant reaches a pathogenic classification using the VWD Type 2 rule specifications. Previously reported variant can be associated with a different type of VWD.Code may also be applied when two previously reported variants reach a likely pathogenic classification using the VWD Type 2 rule specifications. Previously reported variants can be associated with a different type of VWD. Use as originally specified, but the comparison variant must reach a pathogenic classification using these rule specifications in order to apply code. Use as originally specified, but the comparison variant must reach a pathogenic classification using these rule specifications in order to apply code. Use code when previously reported variant reaches a pathogenic classification using the _SERPINC1_ rule specifications from the Thrombosis VCEP. The novel amino acid change must have a Grantham distance greater than or equal to the previously classified pathogenic variant. The novel amino acid change must have a Grantham distance greater than or equal to the previously classified pathogenic variant. 1 \[likely\] pathogenic residue change at the same codon. Not Applicable: Does not apply. The reported missense variant was determined to be Pathogenic according to the APC-specific modifications.There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other different missense variants at these positions meet PM5\_supporting. No missense variant has been classified as Pathogenic based on current evidence. Grantham´s distance of the variant under assessment must have an equal or higher score than the reported variant \[Reference 3\]. Use code when previously reported variant reaches a pathogenic classification using the VWD Type 2 rule specifications. Previously reported variant can be associated with a different type of VWD.Code may also be applied when two previously reported variants reach a likely pathogenic classification using the VWD Type 2 rule specifications. Previously reported variants can be associated with a different type of VWD. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Stop loss variant if another stop loss variant has been determined to be pathogenic. Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. 1 \[likely\] pathogenic residue change at the same codon. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. This criterion can be used at MODERATE if a different missense variant at the same codon has been classified as _pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to SUPPORTING if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. If both are applicable at MODERATE weight, use of PM5 is most appropriate since it is variant specific. Same residue as previously established pathogenic variant (assessed independently of PM5) or 2 previously established likely pathogenic variants (in which case both variants must be assessed independently of PM5). Must have 2 comparison variants reaching pathogenic classification using these specifications. \- The novel change must not affect splicing (SpliceAI ≤ 0.2), must meet PP3, and have a Grantham score equal to or greater than the previously published variants. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Caveat: Residue must be highly conserved across all 5 human KCNQ paralogues in order to be considered for PM5. To confirm conservation, curators should access paralogue data and regions of alignment and non-alignment between KCNQ1 and KCNQ2, KCNQ3, KCNQ4, and KCNQ5 at the site below: [https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1](https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1).* Poor conservation (ineligibility for PM5) is defined whenever 1 or more KCNQ’s show a different amino acid at the position. * Comparison variants must have a Pathogenic or Likely Pathogenic classification reached using these VCEP-specified rules without using PM5.* Variants at any codon where a likely benign / benign variant has been classified by the group are not eligible for PM5.* SpliceAI must be used to examine both variants for similar predicted effect or lack of effect on splicing (SpliceAI Δ score \<0.2).* Not used at the PM5\_Strong level. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5\_Supporting if other variant is Likely Pathogenic due to a missense alteration. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. * Do not apply for any variant that meets BS1 or BA1.* Not mutually exclusive with PM1.* Comparison variant must have a Pathogenic or Likely Pathogenic classification reached using these VCEP-specified rules without using PM5.* SpliceAI must be used to examine both variants for similar predicted effect or lack of effect on splicing (SpliceAI Δ score \<0.2). Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Novel missense change at an amino acid residue where a different missense change determined to be _**pathogenic**_ has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Also applicable for variants affecting the same splice site as a confirmed splice variant with similar or worse splicing in silico predictions Same residue as a previously established pathogenic variant classified using these specifications (assessed independently of PM5) . The novel change must not affect splicing (SpliceAI ≤ 0.2), must meet PP3, and have a Grantham score equal or greater than the previously published variants. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. PM5 may also be applied at a Moderate level of evidence for any nonsense variant with 2+ points from informative variants (see below point table). PM5\_Moderate may not be combined with PVS1\_VeryStrong (should be downgraded to PM5\_Supporting if PVS1\_VeryStrong is applied).**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. A different missense change at same codon has been determined to be likely pathogenic or pathogenic based on HHT VCEP rules. A different missense change at same codon has been determined to be likely pathogenic or pathogenic based on HHT VCEP rules. Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5\_Supporting if other variant is Likely Pathogenic due to a missense alteration. Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5\_Supporting if other variant is Likely Pathogenic due to a missense alteration. Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5\_Supporting if other variant is Likely Pathogenic due to a missense alteration. * Use at the default strength level (PM5) for missense variants when other variant was classified as Pathogenic for autosomal dominant PIK3CD-related disease by Antibody Deficiencies VCEP specifications for _PIK3CD_ without using PM5.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Neither change should be predicted to affect splicing (SpliceAI Δ score ≤0.2).* Do not apply at codon where any benign variants are known.* The variant being evaluated must have a Grantham distance value greater than or equal to that of the known pathogenic variant, indicating that the variant residue is equally chemically different or more chemically different than the known pathogenic residue in comparison to the wild type residue. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. PM5 is applicable as described if the variant under review occurs at the same amino acid as a variant classified Pathogenic using these OTC specifications. Comparison variant must reach a pathogenic classification using the _ABCA4_ VCEP specifications. Do not apply if the comparison variant is suspected to cause a splicing defect via SpliceAI or other splice predictor. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification.* SpliceAI scores for both variants should be in the same category (\<0.1, between 0.1 and 0.2, >0.2) Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Moderate for 1 pathogenic or 2 likely pathogenic variants resulting in different amino acid changes at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. The novel amino acid change must have a Grantham distance greater than or equal to the previously classified pathogenic variant. No change. Same residue as a previously established pathogenic variant (assessed independently of PM5) or 2 previously established likely pathogenic variants (both assessed independently of PM5) NA * See PM5 table. Note: Cannot be applied with PM1, apply criteria with the highest strength. If both are applicable at the same strength, apply PM5 as it is amino acid specific. * Two likely pathogenic variants at the same codon * One pathogenic variant at the same codon * Add caveat: Use argument with caution when the original missense variant created a cysteine especially in a cbEGFlike domain (cfr PM1_strong) as this may increase thepathogenicity level of this variant improperly.* Add caveat: original variant should be pathogenic according to the (modified) ACMG guidelines for variant classification. Missense change at same codon as another pathogenic missense variant.No changes. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group. Missense variant under evaluation should have equal or worse Grantham score. Splicing should be ruled out with either RNA data or agreement in splicing predictors (MaxEntScan and SpliceAI) that show no splicing effects. The other variant must be interpreted as pathogenic by the ClinGen DICER1 VCEP. Likely pathogenic changes do not apply. This rule cannot be applied in combination with PM1 or PS1. If the pathogenicity of another missense change at the same amino acid residue is unknown, determine its pathogenicity using these specifications in order to determine if this criterion can be used. If the variant is pathogenic, use PM5. If the variant is likely pathogenic, use PM5\_Supporting. * This criterion is applicable for any variant resulting in a different amino acid change, at the same amino acid position, as a variant that has been previously established as pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing. * If the variant is likely pathogenic, use PM5\_Supporting. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Applicable to all genes as written.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. 1 \[likely\] pathogenic residue change at the same codon. Novel missense change at an amino acid residue in the same gene where a different missense variant was determined to be Pathogenic. * Novel missense change at an amino acid residue where a different missense change determined to be Pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. * Novel missense change at an amino acid residue where a different missense change determined to be Pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. * Novel missense change at an amino acid residue where a different missense change determined to be Pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. This evidence code can be applied when there is 1 pathogenic variant or 2 likely pathogenic variants at the same residue based on the _F8_ rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect. * Novel missense change at an amino acid residue where a different missense change determined to be **Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Pathogenic classification using this rule specification. BS2_Strong Variant is identified in ≥3 heterozygous or ≥1 homozygous well-phenotyped, untreated, normolipidemic adults (unrelated). Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Observed in the homozygous state in a healthy or PHTS-unaffected individual. One observation if homozygous status confirmed, two if not confirmed. To be applied at supporting evidence level if BS1 is also applied. Observation of variant (biallelic with known pathogenic variant for recessive) in controls inconsistent with disease penetrance.* Advise caution when using this rule, since most of hearing loss is autosomal recessive, and autosomal dominant hearing loss could display reduced penetrance or variable expression.* However, if biallelic observations in controls are inconsistent with disease penetrance, this may be applicable. Only to be used when variant is observed in the homozygous state in a healthy adult. Variant seen in ≥10 individuals w/o GC, DGC, gSRC tumors, or LBC & whose families do not suggest HDGC. Not Applicable: MM-VCEP notes: BS2 is not applicable since FPD/AML patients display incomplete penetrance and the average age of onset of hematologic malignancies is 33 years. ≥ 8 unrelated females who have reached at least 60 years of age without cancer. These individuals all must have come from a single source (single lab, database, etc). Cases cannot be counted across sources. Individuals with a diagnosis of sarcoma ≥ 61 years of age should not be counted towards the BS2 total. Observed in the homozygous state in a healthy adult. * Homozygous individual of any age with normal GAA activity. >1 homozygote who is unaffected proven with at least aggregometry. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age. * Two or more variant positive individuals with a negative IVCT/CHCT test Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy, untreated, adult individual in the homozygous state Observed in at least two healthy male adults. Note: Individual’s phenotype is well-characterized (not just seen in database of presumed healthy individuals) AND/OR ≥16 hemizygotes in gnomAD Observed in a healthy adult individual in the homozygous state AND/OR Normal mtDNA content (1. Must be performed in muscle and/or liver; blood, fibroblast, and buccal not acceptable; 2. Must be performed in children only - defined as <18 years old; 3. A normal level is defined as >50%.) Observed in a healthy adult individual in the homozygous state Observed at a higher heteroplasmy in a healthy adult individual, especially in healthy maternal family members, than in same tissue tested in an affected individual BS2 **≥ 4** pointsSee Fanconi Anemia BS2 tables for approach to assign points per proband. VHL is not highly penetrant _at an early age._ BS2 can be applied if: There are at least 3 individuals, all >=65yo, unaffected, harboring the same variant, _**with full phenotyping and screening**_ for the absence of VHL-related cancers. Use this rule with 1 or more homozygotes who are unaffected (proven with aggregometry OR flow cytometry AND normal platelet count AND normal platelet size). This evidence code can be used when a _F9_ variant is observed in a male with a normal factor IX activity level (at least >40% IU or as defined by laboratory cut off). Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. Use this rule with 1 or more homozygotes who are unaffected (proven with aggregometry OR flow cytometry AND normal platelet count AND normal platelet size). Use this rule with 1 or more homozygotes who are unaffected (proven with aggregometry OR flow cytometry AND normal platelet count AND normal platelet size). This evidence code is available when the variant is identified in 2 or more heterozygotes or 1 or more homozygotes with normal antithrombin levels \[> 0.8 IU/mL (or above the lower limit of a laboratory’s assays reference range)\]. Apply to normoglycemic individuals age 70 or older (i.e., genotype positive, phenotype negative) We expect to see hyperglycemia at birth in an individual with \_GCK\_-MODY and therefore consider an individual unaffected if euglycemic in childhood or adulthood. Since individuals typically do not present with symptoms of diabetes, evidence that someone is “nondiabetic” is insufficient; fasting glucose must be tested and found to be within normal limits (\<100 mg/dl / 5.5 mmol/L). \-4 Points. BS2\_Strong: Observed in >=3 (3 or more) homozygotes in gnomAD. ≥ 10 points for healthy individuals **OR** ≥ 2 times in homozygous state.A **healthy individual** worth 1 point is defined by:Age ≥ 50 years \+ Less than 5 adenomatous polyps in a colonoscopy \+ Absence of features in Table 1**OR**Age ≥ 50 years \+ Colorectal cancer/polyposis was not the indication for testingA **healthy individual** worth 0.5 points is defined by keywords including control, non-cancer, normal, unaffected population. Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. BS2 can be applied if there is clear documentation that an individual of any age is either homozygous for the variant, or has the variant confirmed in trans with a pathogenic or likely pathogenic variant and has IDUA activity in the unaffected range as documented by standard diagnostic laboratory-based activity determination. Values for IDUA activity and the reference range for the laboratory must be provided.Note: Patients with late onset MPS1 can present late in life (5^th\-6^th decade), can have mild symptoms, and may remain undiagnosed. Therefore, it is possible that individuals who are homozygous for hypomorphic IDUA variants could be present in population databases. Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2 = ≥ 4 points \-4 Points. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2 = ≥ 4 points Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: CYP1B1 variants can have an incomplete penetrance and late age of onset. Adults with known pathogenic homozygous CYP1B1 variants who had a normal eye examination have been reported. Only count males over age 30 with a documented eye examination with functional studies (normal ERG or FAF). Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.* Not applicable due to incomplete penetrance.* Please note that hearing loss and other phenotypes are not completely penetrant in homozygotes (PMID: 23392653, [https://www.ncbi.nlm.nih.gov/books/NBK1405/#jln.Summary](https://www.ncbi.nlm.nih.gov/books/NBK1405/#jln.Summary)). Not Applicable: Does not apply due to reduced penetrance. NA Co-occurrence _in trans_ with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^d), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. NA NA Variant is present in ≥ 3 homozygotes without any features of the phenotype. This rule only applies to individuals found in the literature who have been well-phenotyped and are unaffected by age 40.Presence in databases such as gnomAD are not considered. NA Not Applicable: Not applicable due to incomplete penetrance. NA NA Observed in ≥3 homozygotes in gnomAD controls or reported in the literature (healthy adult individuals). Only count males over age 30 with a documented eye exam without retinoschisis.BS2\_strong for variants observed in at least 3 unaffected males. \-4 Points. \-4 Points. BS2\_Strong: Observed in >=3 (3 or more) hemizygotes in gnomAD. Not Applicable: Full penetrance at an early age is not observed in HHT. Not Applicable: Full penetrance at an early age is not observed in HHT. Co-occurrence _in trans_ with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^d), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. Co-occurrence _in trans_ with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^d), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. Co-occurrence _in trans_ with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^d), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. Not Applicable: Does not apply due to incomplete penetrance and variable expressivity of disease. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. The variant is observed in >5 (female) homozygotes or 5 (male) hemizygotes in the most current version of gnomAD available at the time of curation. Not Applicable: This code is not applicable as the age of onset is variable and penetrance is known to be incomplete for some variants. Variant is present in ≥ 3 homozygotes without any features of the phenotype. This rule applies to individuals found in the literature who have been well-phenotyped and are unaffected by age 40. Alternatively, this strength can be applied if the variant is present in ≥ 6 homozygotes in gnomAD v.4.1.0 or later. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late onset is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Apply to normoglycemic individuals age 70 or older (i.e., genotype positive, phenotype negative) Award BS2 if ≥3 homozygotes present in gnomAD or ≥3 heterozygous in well phenotyped family members.Clinical laboratories are encouraged to accumulate more than 2 (≥3) instances of well phenotyped family members before applying this strong criterion. To be considered for this point, the variant should be either germline (most common), or somatic in a relevant tissue. Homozygous occurrences in gnomAD or ExAC can also be counted for this point (≥3). Not Applicable: MYOC variants have an incomplete penetrance and late age of onset. Not Applicable: Do not use: ATM has incomplete penetrance. Not Applicable Not Applicable Observation of variant (biallelic with known pathogenic variant for recessive) in controls inconsistent with disease penetrance.* Advise caution when using this rule, since most of hearing loss is autosomal recessive, and autosomal dominant hearing loss could display reduced penetrance or variable expression.* However, if biallelic observations in controls are inconsistent with disease penetrance, this may be applicable. Ensure age of the unaffected individual is appropriate. MYO15A and OTOF are expected to cause congenital or childhood onset hearing loss. Therefore, an adult (ie >18 years) may be an appropriate individual to consider application of this criteria. Please see additional considerations listed under BS4 “Genotype+/phenotype-”. 40+ unrelated females from a single source are tumor-free through age 50 (caveat: ratio of BS2-eligible females to PS4-eligible probands must be ≥ 40:1) OR 2+ observations of homozygosity in healthy individualsOR 1+ observation(s) of homozygosity in a healthy individual with status confirmed by parental testing. Observed in the homozygous state in a healthy adult. Observed in the homozygous state in a healthy adult, or confirmed in trans with a variant that has been classified as pathogenic by the CCDS VCEP using these criteria. The variant is observed in ≥2 hemizygotes in gnomAD, or one or more hemizygous male(s) or homozygous female(s) who have documented normal urine creatine/creatine ratio. Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related or unrelated) heterozygotes Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related or unrelated) hemizygotes Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related or unrelated) heterozygotes or hemizygotes Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related or unrelated) heterozygotes. Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related or unrelated) heterozygotes or hemizygotes. Observed in the heterozygous/hemizygous state in a healthy adult.* 4 unaffected (related and maternally inherited or unrelated) heterozygotes \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. \-4 Points. Observed in a healthy adult individual. Observed in a healthy adult individual with full penetrance expected at an early age. Observed in a healthy adult individual. Observed in a healthy adult individual with full penetrance expected at an early age. This evidence code can be used when a _F8_ variant is observed in a male with a normal factor VIII activity level (at least >40% IU or as defined by laboratory cut off) using a one stage and/or a chromogenic assay. Observed in a healthy adult individual. Variant is present in ≥ 3 homozygotes without any features of the phenotype. This rule applies to individuals found in the literature who have been well-phenotyped and are unaffected by age 40. Alternatively, this strength can be applied if the variant is present in ≥ 6 homozygotes in gnomAD v.4.1.0 or later. BP6_Very Strong Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. BP6_Strong Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. BP6_Supporting Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. BP6_Moderate Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Not Applicable: This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PM6_Moderate See PS2 above. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Assumed _de novo,_ but without confirmation of paternity and maternity, in proband with the disease and no family history. See PS2 above Not Applicable One patient meets the HDGC individual phenotype criteria w/o parental confirmation _**RUNX1**_ **Specification:**Following the SVI guidance, assumed _de novo_ _RUNX1_ variants will be scored at the third tier of the point-based system with maximum allowable value of 1 point contributing to overall score:**PM6**: ≥ 4 assumed _de novo_ occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: See explanation for PS2. Use proposed SVI point recommendations. * Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 2-3 points Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity Assumed de novo, but without confirmation of paternity and maternity Assumed de novo, but without confirmation of paternity and maternity Assumed de novo, but without confirmation of paternity and maternity Assumed de novo, but without confirmation of maternity (maternal testing done by targeted variant analysis and/or targeted gene sequencing) Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase See PS2 evidence code for scoring and phenotypes. Assumed _de novo_ receives half the points as compared to maternity and paternity confirmed _de novo_. If paternity and maternity are not confirmed, score as the PM6 code. PM6 can receive “VeryStrong” strength. For example, if there are >4 de novo probands with Danish Criteria and none have paternity confirmed, this can receive PM6\_VeryStrong. Note: the VCI as of Nov 2022 does not allow PM6\_VeryStrong. Instead apply the PS2 evidence code and increase the strength to “VeryStrong” with a note that paternity and/or maternity is not confirmed. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Subsumed by PS2. Not Applicable: Subsumed in PS2. 1 Point. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 1-1.5 _de novo_ scores. For curation of _de novo_ score see **Tables 1** and **2**. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: See PS2. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 1 Point. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[⁷](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Refer to SVI guidance on number/combination of cases required based on phenotype specificity[²](#url_c73e109e-b916-5a72-b7b1-1762446f3c11).For most cardiomyopathies, it is recommended to default to “phenotype consistent with gene but not highly specific”. Clinical judgment is required for shifting to a higher or lower phenotypic consistency. See PS2 for additional considerations. Not Applicable: Refer to PS2 Not Applicable: See PS2 for de novo data. Assumed de novo, but without confirmation of paternity and maternity.* For PM6, maternity and paternity are not confirmed but assumed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years).* The _de novo_ variant in question must be coding or flanking.* The PM6 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).![](https://lh6.googleusercontent.com/4dOzwji0rs6_gSczQOWVPcEOk-W6h4Ey-wBCZTtqRdLJizyKdB6AU08BvSlBsOQ0HOkPeRFn1xEL6ofBU7fc1B0awCfBVfKQYdboYFwdPZmyf1zH0vBaH4AE-eenxPhcIlpuAXes9kp9vlJQ9f-kcA) Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Please see PS2 Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). * The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system: * (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”. * (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”. * (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Confirmation of maternity and paternity is required. Not Applicable: See PS2 for de novo data. 1 Point. 1 Point. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: De novo occurrences without confirmation of paternity and maternity can still be counted under the PS2 code, using the point system shown in Tables 1 and 2. Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity. Use PS2/PM6 criteria described under PS2. Occurrences of de novo and presumed de novo are summed for a final PS2/PM6 evidence strength. Not Applicable: Use the PS2 code for all (including non-confirmed paternity) de novo variants. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Assumed de novo, but without confirmation of paternity and maternity. Assumed de novo, but without confirmation of paternity and maternity. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Subsumed by PS2. Not Applicable: This point is addressed according to PS2 and will not be used. Not Applicable: Refer to PS2 Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase Assumed de novo, but without confirmation of paternity and maternity. 1 point. See PS2 above. Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: Assumed de novo but without confirmation of paternity and maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with GAMT deficiency, to our knowledge. Furthermore, the observation that a variant in GAMT has arisen de novo does not support its causality because GAMT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GAMT, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Variant identified as de novo in an affected male with the mother negative for the variant but maternity not confirmed. Confirmed de novo without confirmation of paternity and maternity. Confirmed de novo without confirmation of paternity and maternity.* 1 occurrence of PM6 Confirmed de novo without confirmation of paternity and maternity.* 1 occurrence of PM6. Confirmed de novo without confirmation of paternity and maternity.* 1 occurrence of PM6. Confirmed de novo without confirmation of paternity and maternity.* 1 occurrence of PM6. Assumed de novo without confirmation of paternity and maternity.* 1 occurrence of PM6. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Dravet\*: 1 pointsGenetic Epilepsy with Febrile Seizures Plus: 0.5 pointsDevelopmental and Epileptic Encephalopathy: 0.5 pointsHemiplegic migraine: 0.25 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Developmental and Epileptic Encephalopathy: 0.5 pointsOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. BP1_Very Strong Not Applicable Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: This rule is not applicable to PTEN. Not Applicable Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: BP1 is not applicable for RUNX1, because both truncating and missense variants cause FPD/AML. Not Applicable: This rule code does not apply to these genes, as truncating variants account for only a portion of disease causing variants. Not Applicable: Does not apply. All types of variants cause Pompe disease. NA Not Applicable: BP1 is not applicable. MH is caused primarily by missense variants in RYR1. NA Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: Most variants in protein-coding mtDNA genes are not truncating, but rather missense variants. Even if truncating variants were more common, this would not preclude missense variants from also causing a loss of protein function. NA Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for F9 gene. Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: This rule code does not apply to the SERPINC1 gene, as missense and truncating variants account for disease. Not Applicable Not Applicable NA Not Applicable: Does not apply. NA Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Does not apply. All types of variants cause MPS1. NA Not Applicable: Not applicable, both missense and truncating variants can cause disease. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. NA Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: Both truncating and missense CYP1B1 variants are causative. Not Applicable NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Does not apply. DCLRE1C missense variants are a known mechanism of disease. Not Applicable: Does not apply. IL7R missense variants are a known mechanism of disease. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. JAK3 missense variants are a known mechanism of disease. Not Applicable: Not applicable, as pathogenic CTLA4 variants are not limited to truncating variants, but can be missense as well. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Both LOF and missense variants are known to cause PAH. Not Applicable NA NA Not Applicable: Does not apply. IL2RG missense variants are a known mechanism of disease. Not Applicable: Missense variants commonly seen in HHT genes. Not Applicable: Missense variants commonly seen in HHT genes Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable Not Applicable: Both missense and truncating variants in NEB are disease-causing. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in DNM2 are disease-causing. Not Applicable: Both missense and truncating variants in MTM1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable. Pathogenic missense variants have been documented for OTC deficiency. Not Applicable: Rule code does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for GUCY2D. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable Not Applicable: Not applicable as LOF is not the disease mechanism. Not Applicable: Both truncating and missense MYOC variants are causative. Not Applicable: Do not use: Missense pathogenic variants are known for ATM Not Applicable: This rule does not apply. There are known pathogenic missense variants in ACADVL. Not Applicable Not Applicable Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable: Does not apply. All types of variants cause GAMT-D. Not Applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for F8 gene. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for AIPL1. BP1_Strong Not Applicable Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: This rule is not applicable to PTEN. Not Applicable Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: BP1 is not applicable for RUNX1, because both truncating and missense variants cause FPD/AML. Not Applicable: This rule code does not apply to these genes, as truncating variants account for only a portion of disease causing variants. Not Applicable: Does not apply. All types of variants cause Pompe disease. NA Not Applicable: BP1 is not applicable. MH is caused primarily by missense variants in RYR1. Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: Most variants in protein-coding mtDNA genes are not truncating, but rather missense variants. Even if truncating variants were more common, this would not preclude missense variants from also causing a loss of protein function. NA Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for F9 gene. Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: This rule code does not apply to the SERPINC1 gene, as missense and truncating variants account for disease. Not Applicable Not Applicable NA Not Applicable: Does not apply. NA Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Does not apply. All types of variants cause MPS1. **Apply BP1\_Strong** for silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI ≤0.1). As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA1 RING aa 2-101; BRCA1 coiled-coil aa 1391-1424; BRCA1 BRCT repeats aa 1650-1857. See Specifications Figure1A and Appendix J for details. Not Applicable: Not applicable, both missense and truncating variants can cause disease. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. **Apply BP1\_Strong** for silent substitution, missense or in-frame insertion, deletion or delins variants outside a (potentially) clinically important functional domain AND no splicing predicted (SpliceAI ≤0.1). As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure1A and Appendix J for details. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: Both truncating and missense CYP1B1 variants are causative. Not Applicable NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Does not apply. DCLRE1C missense variants are a known mechanism of disease. Not Applicable: Does not apply. IL7R missense variants are a known mechanism of disease. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. JAK3 missense variants are a known mechanism of disease. Not Applicable: Not applicable, as pathogenic CTLA4 variants are not limited to truncating variants, but can be missense as well. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Both LOF and missense variants are known to cause PAH. Not Applicable NA NA Not Applicable: Does not apply. IL2RG missense variants are a known mechanism of disease. Not Applicable: Missense variants commonly seen in HHT genes. Not Applicable: Missense variants commonly seen in HHT genes Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable Not Applicable: Both missense and truncating variants in NEB are disease-causing. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in DNM2 are disease-causing. Not Applicable: Both missense and truncating variants in MTM1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable. Pathogenic missense variants have been documented for OTC deficiency. Not Applicable: Rule code does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for GUCY2D. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable Not Applicable: Not applicable as LOF is not the disease mechanism. Not Applicable: Both truncating and missense MYOC variants are causative. Not Applicable: Do not use: Missense pathogenic variants are known for ATM Not Applicable: This rule does not apply. There are known pathogenic missense variants in ACADVL. Not Applicable Not Applicable Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable: Does not apply. All types of variants cause GAMT-D. Not Applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for F8 gene. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for AIPL1. BP1_Moderate Not Applicable Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: This rule is not applicable to PTEN. Not Applicable Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: BP1 is not applicable for RUNX1, because both truncating and missense variants cause FPD/AML. Not Applicable: This rule code does not apply to these genes, as truncating variants account for only a portion of disease causing variants. Not Applicable: Does not apply. All types of variants cause Pompe disease. NA Not Applicable: BP1 is not applicable. MH is caused primarily by missense variants in RYR1. Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: Most variants in protein-coding mtDNA genes are not truncating, but rather missense variants. Even if truncating variants were more common, this would not preclude missense variants from also causing a loss of protein function. NA Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for F9 gene. Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: This rule code does not apply to the SERPINC1 gene, as missense and truncating variants account for disease. Not Applicable Not Applicable NA Not Applicable: Does not apply. NA Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Does not apply. All types of variants cause MPS1. NA Not Applicable: Not applicable, both missense and truncating variants can cause disease. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. NA Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: Both truncating and missense CYP1B1 variants are causative. Not Applicable NA Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Does not apply. DCLRE1C missense variants are a known mechanism of disease. Not Applicable: Does not apply. IL7R missense variants are a known mechanism of disease. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. JAK3 missense variants are a known mechanism of disease. Not Applicable: Not applicable, as pathogenic CTLA4 variants are not limited to truncating variants, but can be missense as well. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Both LOF and missense variants are known to cause PAH. Not Applicable NA NA Not Applicable: Does not apply. IL2RG missense variants are a known mechanism of disease. Not Applicable: Missense variants commonly seen in HHT genes. Not Applicable: Missense variants commonly seen in HHT genes Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable Not Applicable: Both missense and truncating variants in NEB are disease-causing. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in DNM2 are disease-causing. Not Applicable: Both missense and truncating variants in MTM1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable. Pathogenic missense variants have been documented for OTC deficiency. Not Applicable: Rule code does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for GUCY2D. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable Not Applicable: Not applicable as LOF is not the disease mechanism. Not Applicable: Both truncating and missense MYOC variants are causative. Not Applicable: Do not use: Missense pathogenic variants are known for ATM Not Applicable: This rule does not apply. There are known pathogenic missense variants in ACADVL. Not Applicable Not Applicable Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable: Does not apply. All types of variants cause GAMT-D. Not Applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for F8 gene. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for AIPL1. BP1_Supporting Not Applicable Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: This rule is not applicable to PTEN. Not Applicable Not Applicable Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: BP1 is not applicable for RUNX1, because both truncating and missense variants cause FPD/AML. Not Applicable: This rule code does not apply to these genes, as truncating variants account for only a portion of disease causing variants. Not Applicable: Does not apply. All types of variants cause Pompe disease. Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: BP1 is not applicable. MH is caused primarily by missense variants in RYR1. Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable Not Applicable Not Applicable Not Applicable Not Applicable: Most variants in protein-coding mtDNA genes are not truncating, but rather missense variants. Even if truncating variants were more common, this would not preclude missense variants from also causing a loss of protein function. Apply to all missense variants. Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for F9 gene. Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Rule does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: This rule code does not apply to the SERPINC1 gene, as missense and truncating variants account for disease. Not Applicable Not Applicable This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. Not Applicable: Does not apply. BP1 is applicable to APC with the exception of missense variants located in the first 15-amino acid repeat of the β-catenin binding domain (codon 1021-1035). Not Applicable: The VWF gene is not constrained for missense variation (gnomAD). Not Applicable: Does not apply. All types of variants cause MPS1. NA Not Applicable: Not applicable, both missense and truncating variants can cause disease. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. NA Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: For the current genes where null variants are a known mechanism, pathogenic missense variants have also been reported. Not Applicable: Both truncating and missense CYP1B1 variants are causative. Not Applicable Missense variant in a gene for which primarily truncating variants are known to cause disease* Not applicable, as pathogenic _KCNQ1_ variants are not limited to truncating variants, but can be missense as well. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Does not apply. DCLRE1C missense variants are a known mechanism of disease. Not Applicable: Does not apply. IL7R missense variants are a known mechanism of disease. Not Applicable: Not applicable for RPE65. Not Applicable: Does not apply. JAK3 missense variants are a known mechanism of disease. Not Applicable: Not applicable, as pathogenic CTLA4 variants are not limited to truncating variants, but can be missense as well. Not Applicable: Does not apply. Not Applicable: Does not apply. Not Applicable: Both LOF and missense variants are known to cause PAH. Not Applicable This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. Not Applicable: Does not apply. IL2RG missense variants are a known mechanism of disease. Not Applicable: Missense variants commonly seen in HHT genes. Not Applicable: Missense variants commonly seen in HHT genes Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable: Missense variant in a gene where only loss of function causes disease is not applicable. Not Applicable Not Applicable: Both missense and truncating variants in NEB are disease-causing. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in DNM2 are disease-causing. Not Applicable: Both missense and truncating variants in MTM1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable. Pathogenic missense variants have been documented for OTC deficiency. Not Applicable: Rule code does not apply as truncating variants do not predominate and missense variants are a known cause of disease. Not Applicable: Not applicable for GUCY2D. Not Applicable: Both missense and truncating variants in ACTA1 are disease-causing. Not Applicable: Both missense and truncating variants in RYR1 are disease-causing. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable: Not applicable as missense variants are also known to cause disease. Not Applicable Not Applicable: Not applicable as LOF is not the disease mechanism. Not Applicable: Both truncating and missense MYOC variants are causative. Not Applicable: Do not use: Missense pathogenic variants are known for ATM Not Applicable: This rule does not apply. There are known pathogenic missense variants in ACADVL. Not Applicable Not Applicable Not Applicable: This rule code does not apply to this gene, as truncating variants account for only a portion of disease-causing variants. Not Applicable: Missense variant in a gene for which primarily truncating variants are known to cause disease. Not Applicable: Does not apply. All types of variants cause GAMT-D. Not Applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for F8 gene. Not Applicable: Missense variants are common cause of disease. Not Applicable: Not applicable for AIPL1. PM4_Moderate In-frame deletion/insertions smaller than one whole exon, or in-frame whole-exon duplications not considered in any PVS1 criteria.Caveat: variant must also meet PM2. Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Applies to in-frame insertions or deletions impacting at least one residue in a catalytic motif (see PM1), and variants causing protein extension. Protein length change due to an in-frame deletion or insertion that are not located in repetitive regions.* No changes. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group. Applicable as described Only apply to stop-loss variantsVariant example: CDH1 c.2647T>C (p.Ter883Glnext*29). In-frame deletion/insertion impacting at least one of the following amino acid residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Not Applicable: Not applicable Protein length changes due to in-frame deletions/insertions in a nonrepeat region or stop-loss variants. * In frame deletion/insertions of two or more amino acids but less than one exon. Use with no specification. Not Applicable: PM4 is not applicable. The majority of RYR1 variants that are causative for MHS are missense variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants Applied per original ACMG/AMP guidelines Not Applicable: Do not use:● In-frame deletions/insertions that are not already PVS1-eligible: no information is available to justify the application of this rule. ● Missense and small in-frame indels: not yet confirmed as a mechanism of disease for PALB2.● Stop-loss: lack of data on stop-loss variants. In-frame insertion / deletions in the B and alpha domains and stop-loss variants adding significant additional amino acids to VHL [¹⁵](#pmid_20560986) cites multiple pathogenic cases and experimental evidence of stop loss extensions in VHL that are associated with Type 2A VHL disease). The functional domains are: Beta (β) domain (AA 63 - 155, Nuclear Export), Alpha (ɑ) domain (AA 156-192, Elongin C binding), and Second Beta (β) domain (AA 193-204). PM4 does not apply to in-frame indels prior to codon 54 that do not alter the Met54 VHL p19 codon and beyond. Use with no specification. Use code with no specification. Use with no specification for type 2A and 2M. This rule code is not applicable to variants associated with type 2B disease, since type 2B is only associated with gain of function variants. Use with no specification. Use with no specification. No specification For deletions/insertions of more than one amino acid in a non-repeat region, use as moderate level of evidence. For deletions/insertions of more than one amino acid in a non-repeat region, use as moderate level of evidence. No known repetitive areas in gene. Use as described. * Defined to include insertions/duplications of 6 or more nucleotides increasing the distance between the TATA box (spanning n.-32 to n.-24) and the transcription start site (n.4). Not Applicable Use with no specification. Stop loss variants, and in frame deletion/insertions of two or more amino acids but less than one exon. Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). No known repetitive areas in gene. Use as described. Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Strength of rule should be carefully considered and may require downgrading to SUPPORTING based on the predicted impact of the variant, including the size of the deletion/insertion, its location, and conservation of the region. For genes where PVS1 is not applicable (i.e., where there is no evidence that pLOF variants cause disease), consider using this rule at MODERATE or SUPPORTING strength for truncating variants that do NOT undergo nonsense mediated decay (NMD). Stop loss variants are not a known disease mechanism, therefore PM4 does not apply to that variant type Protein length changes in exons 1-14, due to in-frame deletions/insertions in a non-repeat region. Or, in ORF15 outside of the repetitive region in amino acids 585 to 1078, due to in-frame deletions/insertions in a non-repeat region. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants* This code is mutually exclusive with PVS1 (PMID: 30192042) and PP3 (in order not to double-count _in silico_ predictor data) but can be used together with PM1.* In-frame insertions or deletions of any size will meet PM4 at the moderate (default) level, due to the greater importance of the location of the variant rather than the size. Additional requirement that when applied to deletion variants, the deleted region must contain a known pathogenic or likely pathogenic variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of ≥2 amino acids that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acids adjacent to at least one conserved residue (PhyloP>2.0). When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. * This code is mutually exclusive with PVS1 (PMID: 30192042) and PP3 (in order not to double-count _in silico_ predictor data) but can be used together with PM1.* Met at default strength (PM4) by an in-frame deletion resulting in a protein length change greater than or equal to 2 amino acids, if at least one of the deleted nucleotides is highly conserved (PhyloP score greater than or equal to 2.0) and if SpliceAI score is \<0.2.* Met at default strength (PM4) by an in-frame insertion resulting in a protein length change greater than or equal to 2 amino acids, if at least one of the adjacent amino acids is highly conserved (PhyloP score greater than or equal to 2.0) and if SpliceAI score is \<0.2.* The region of the protein affected by the variant is key to consider. If variants within this region occur in healthy populations, the region is polymorphic. Incomplete penetrance should also be considered as a caveat for polymorphism. When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **pathogenic** or **likely pathogenic** variant that is not predicted/observed to alter splicing. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. In-frame deletion/insertions smaller than one whole exon, in a non-repetitive region, or stop-loss variants. Variant must not be considered in any PVS1 criteria. Variant must also meet PM2. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. Additional requirement that deletion variants must contain a known **pathogenic variant, likely pathogenic variant, or variant of uncertain significance** that is not predicted/observed to alter splicing in order to apply PM4, with the strength of evidence dependent upon the classification of the variant contained within the deletion. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: This criterion does not apply since the mechanism of disease is the Gain of function caused by missense mutations. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. PM4 is applicable as described for in-frame loss or gain of ≥1 amino acid but less than an entire exon. For in-frame deletions or insertions ≥1 exon, defer to PVS1 flowchart. This code can be applied for >1 amino acid deletion/insertion or a stop loss variant, or >1 nucleotide with PhyloP ≥7.367 (https://www.medrxiv.org/content/10.1101/2023.04.24.23288782v1). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of ≥2 amino acids that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acids adjacent to at least one conserved residue (PhyloP>2.0). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. Use as is, regardless of the length of the in-frame insertion or deletion. For deletions/insertions of more than one amino acid in a non-repeat region, use as moderate level of evidence. Not Applicable: Although there have been reported in-frame deletion/insertions in these genes which cause the overgrowth phenotype, they are exceptionally rare. Most insertion/deletions are associated with a LoF disease mechanism and so this point will still not be used even though we recognize that it is possible that a variant is an in-frame indel that results in a GoF mechanism. In-frame del/ins, stop-loss variants and truncating variants involving >10% of the protein and located within the conserved olfactomedin domain (AA 246-502). Use for stop-loss variants. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group. * Add caveat: cannot be applied simultaneously with PVS1 (at any strength level) Protein length change due to an in-frame deletion or insertion that are not located in repetitive regions.No changes. Follow recommendations as outlined in ACMG/AMP guidelines and/or Sequence Variant Interpretation working group. In-frame indels with a residue within the RNase IIIb domain (p.Y1682 – p.S1846). CCDS VCEP notes: Stop loss variants in GATM have not been reported, as far as we are aware. GATM specifications: Use this rule “as is” for in frame deletions and insertions of 2 or more amino acids, but downgrade to PM4\_Supporting for single amino acid deletions and insertions. GAMT specifications: Use this rule “as is” for stop loss variant, and in frame deletions and insertions of 2 or more amino acids, but downgrade to PM4\_Supporting for single amino acid deletions and insertions. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Do not use for in-frame deletions/insertions in CDKL5 C-terminus (exons 19-21, or after p.P904) **(when using the NM\_003159.2 transcript)**. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Do not use PM4 for in-frame deletions/insertions in the Histidine-rich region (p.37-p.57), Proline- and Glutamine-rich region (p.58-p.86) and Proline-rich region (p.105-p.112). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Do not use PM4 for in-frame deletions/insertions in the Proline-rich region of gene (p.381-p.405) Protein length changes due to in-frame deletions/insertions in a non-repeat region. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. No known repetitive areas in gene. Use as described. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Use code with no specification. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of ≥2 amino acids that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acids adjacent to at least one conserved residue (PhyloP>2.0). BS2_Very Strong NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. NA NA NA NA Not Applicable: MM-VCEP notes: BS2 is not applicable since FPD/AML patients display incomplete penetrance and the average age of onset of hematologic malignancies is 33 years. NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA NA NA NA NA NA NA NA Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA NA NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: CYP1B1 variants can have an incomplete penetrance and late age of onset. Adults with known pathogenic homozygous CYP1B1 variants who had a normal eye examination have been reported. NA NA Not Applicable: Does not apply due to reduced penetrance. NA NA NA NA NA NA Not Applicable: Not applicable due to incomplete penetrance. NA NA NA NA NA NA NA Not Applicable: Full penetrance at an early age is not observed in HHT. Not Applicable: Full penetrance at an early age is not observed in HHT. NA NA NA Not Applicable: Does not apply due to incomplete penetrance and variable expressivity of disease. NA NA NA NA NA NA Not Applicable: This code is not applicable as the age of onset is variable and penetrance is known to be incomplete for some variants. NA NA NA Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late onset is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. NA NA Not Applicable: MYOC variants have an incomplete penetrance and late age of onset. Not Applicable: Do not use: ATM has incomplete penetrance. Not Applicable Not Applicable NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS2_Moderate NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. NA NA NA NA Not Applicable: MM-VCEP notes: BS2 is not applicable since FPD/AML patients display incomplete penetrance and the average age of onset of hematologic malignancies is 33 years. 4-7 unrelated females who have reached at least 60 years of age without cancer. These individuals all must have come from a single source (single lab, database, etc). Cases cannot be counted across sources. Individuals with a diagnosis of sarcoma ≥ 61 years of age should not be counted towards the BS2 total. NA NA Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age. * One variant positive individual with a negative IVCT/CHCT test Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. NA NA NA NA NA BS2\_Moderate = **2** pointsSee Fanconi Anemia BS2 tables for approach to assign points per proband. NA NA NA Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA NA NA NA NA NA NA NA Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2\_Moderate = 2 points NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2\_Moderate = 2 points Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: CYP1B1 variants can have an incomplete penetrance and late age of onset. Adults with known pathogenic homozygous CYP1B1 variants who had a normal eye examination have been reported. NA NA Not Applicable: Does not apply due to reduced penetrance. NA NA NA NA NA NA Not Applicable: Not applicable due to incomplete penetrance. NA NA Observed in ≥2 homozygotes in gnomAD controls or reported in the literature (healthy adult individuals). NA NA NA NA Not Applicable: Full penetrance at an early age is not observed in HHT. Not Applicable: Full penetrance at an early age is not observed in HHT. NA NA NA Not Applicable: Does not apply due to incomplete penetrance and variable expressivity of disease. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: This code is not applicable as the age of onset is variable and penetrance is known to be incomplete for some variants. NA No change - use as originally described No change - use as originally described Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late onset is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. NA NA Not Applicable: MYOC variants have an incomplete penetrance and late age of onset. Not Applicable: Do not use: ATM has incomplete penetrance. Not Applicable Not Applicable NA NA NA NA NA NA NA NA NA NA Observed in the heterozygous/hemizygous state in a healthy adult.* 3 unaffected (related and maternally inherited or unrelated) heterozygotes NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS2_Supporting NA Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Two homozygous observations with no clinical data provided, or meets criteria for BS2 but BS1 is also applied. NA NA Variant seen in ≥3 individuals w/o GC, DGC, SRC tumors, or LBC & whose families do not suggest HDGC. Not Applicable: MM-VCEP notes: BS2 is not applicable since FPD/AML patients display incomplete penetrance and the average age of onset of hematologic malignancies is 33 years. 2-3 unrelated females who have reached at least 60 years of age without cancer. These individuals all must have come from a single source (single lab, database, etc). Cases cannot be counted across sources. Individuals with a diagnosis of sarcoma ≥ 61 years of age should not be counted towards the BS2 total. NA NA NA Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age. NA Observed in 4-15 hemizygotes in gnomAD AND/OR Pyruvate radioactive enzyme assay showing normal (defined as >3rd percentile of controls) for PDC, activated and normal ratios (PDC/E3 and/or PDC/CS) in fibroblasts with no evidence of skewed X-inactivation in fibroblasts. Lack of COX negative fibers in muscle (children and adults) Normal laboratory values (specific labs outlined in PP4) Observed at a higher heteroplasmy in a healthy adult individual, especially in healthy maternal family members, than in different tissue(s) tested in an affected individual BS2\_Supporting = **1** pointSee Fanconi Anemia BS2 tables for approach to assign points per proband. VHL is not highly penetrant _at an early age._ BS2\_Supporting can be applied if: At least 3 individuals, all >=65yo, unaffected, harboring the same variant, _**lacking full phenotyping and screening**_, with no noted VHL-related cancers NA NA Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA NA This evidence code is available when the variant is identified in 1 heterozygote with normal antithrombin levels \[> 0.8 IU/mL (or above the lower limit of a laboratory’s assays reference range)\]. NA NA \-1 Point. BS2\_Supporting: Can be applied at Supporting level of evidence if observed at least 2 homozygotes in gnomAD. ≥ 3 points for healthy individuals.A **healthy individual** worth 1 point is defined by:Age ≥ 50 years \+ Less than 5 adenomatous polyps in a colonoscopy \+ Absence of features in Table 1**OR**Age ≥ 50 years \+ Colorectal cancer/polyposis was not the indication for testingA **healthy individual** worth 0.5 points is defined by keywords including control, non-cancer, normal, unaffected population. Not Applicable: Not applicable due to the incomplete penetrance seen in VWD. NA Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2\_Supporting = 1 points \-1 Point. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Applied in absence of features of recessive disease, namely Fanconi Anemia phenotype. See **Specifications Table 8** for additional stipulations, and approach to assign points per proband, and final BS2 code assignment based on the sum of BS2-related points. See Appendix H for additional details.BS2\_Supporting = 1 points Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: Inherited cardiomyopathies generally display reduced penetrance, variable expressivity, and adult-onset. Not Applicable: CYP1B1 variants can have an incomplete penetrance and late age of onset. Adults with known pathogenic homozygous CYP1B1 variants who had a normal eye examination have been reported. NA NA Not Applicable: Does not apply due to reduced penetrance. Only to be used when the variant is observed in the homozygous state in a healthy adult. NA Only to be used when the variant is observed in the homozygous state in a healthy adult. Only to be used when the variant is observed in the homozygous state in a healthy adult. NA Only to be used when the variant is observed in the homozygous state in a healthy adult. Not Applicable: Not applicable due to incomplete penetrance. Only to be used when the variant is observed in the homozygous state in a healthy adult. Only to be used when the variant is observed in the homozygous state in a healthy adult. Observed in ≥1 homozygote in gnomAD controls or reported in the literature (healthy adult individuals). NA \-1 Point. \-1 Point. BS2\_Supporting: Can be applied at Supporting level of evidence if observed at least 2 hemizygotes in gnomAD. Not Applicable: Full penetrance at an early age is not observed in HHT. Not Applicable: Full penetrance at an early age is not observed in HHT. NA NA NA Not Applicable: Does not apply due to incomplete penetrance and variable expressivity of disease. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: This code is not applicable as the age of onset is variable and penetrance is known to be incomplete for some variants. Variant is present in ≥ 3 homozygotes in gnomAD v.4.1.0 or later. No change - use as originally described No change - use as originally described Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late onset is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. Not Applicable: Not applicable as LGMD is characterized by variable expressivity and late-onset LGMD is not uncommon. NA NA Not Applicable: MYOC variants have an incomplete penetrance and late age of onset. Not Applicable: Do not use: ATM has incomplete penetrance. Not Applicable Not Applicable NA 10+ unrelated females from a single source are tumor-free through age 50 (caveat: ratio of BS2-eligible females to PS4-eligible probands must be ≥ 10:1) OR 2+ observations of homozygosity in individuals lacking clinical information NA NA NA Observed in the heterozygous/hemizygous state in a healthy adult.* 1 unaffected (related or unrelated) heterozygote Observed in the heterozygous/hemizygous state in a healthy adult.* 1 unaffected (related or unrelated) hemizygote Observed in the heterozygous/hemizygous state in a healthy adult.* 1 unaffected (related or unrelated) heterozygote or hemizygote Observed in the heterozygous/hemizygous state in a healthy adult.* 1 unaffected (related or unrelated) heterozygote Observed in the heterozygous/hemizygous state in a healthy adult.* 1 unaffected (related or unrelated) heterozygote or hemizygote Observed in the heterozygous/hemizygous state in a healthy adult.* 2 unaffected (related and maternally inherited or unrelated) heterozygotes \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. \-1 Point. NA NA NA NA NA NA Variant is present in ≥ 3 homozygotes in gnomAD v.4.1.0 or later. PM3_Very Strong NA Not Applicable: It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: This rule is not applicable to PTEN. 4 points awarded from tables 7a and 7bExample: Detected in trans in ≥4 probands with a pathogenic variant (recessive). Applicable as described in SVI recommendations for in trans criterion Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes:FPD/AML is inherited in an autosomal dominant manner, thus PM3 is not applicable. Not Applicable: This rule does not apply to TP53/Li-Fraumeni syndrome. Detected in trans with a pathogenic variant. Points-based system. See main specifications document. Use proposed SVI point recommendations. Both variants must be classified using ITGA2B/ITGB3 Rule Specifications. Not Applicable: PM3 is not applicable. MHS is inherited as an autosomal dominant trait with reduced penetrance. For recessive disorders, detected in trans with a pathogenic variantNote: This requires testing of parents (or offspring) to determine phase. NA Not Applicable NA NA Not Applicable: mtDNA variants are maternally inherited and not inherited in an autosomal recessive manner. PM3\_VeryStrong **≥ 8** pointsSee Fanconi Anemia PM3 tables for approach to assign points per proband. Not Applicable: Autosomal dominant. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications. Not Applicable: Not applicable for the F9 gene. Not Applicable: These are dominant conditions, so this rule code does not apply. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications, with the exception that a 22q11.2 deletion in trans ([https://www.ncbi.nlm.nih.gov/books/NBK1523/](https://www.ncbi.nlm.nih.gov/books/NBK1523/)) may be automatically scored 1pt with confirmation that deletion includes the _GP1BB_ gene. Use proposed SVI point recommendations (see below). Both variants must be classified using these rule specifications. Not Applicable: Variants in this gene are being curated as a dominant condition, so this rule code does not apply. Not Applicable Use SVI-recommended point-based system. Not Applicable: Not applicable. Use ClinGen SVI recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for RMRP. Not Applicable Use SVI recommended point system for this code for probands with a VWD type 2N diagnosis. Total of 4 points required. Detected in trans with a pathogenic variant. Points system based on [guidance](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. NA ≥4 points. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. NA Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. ≥ 4.0 points Not Applicable For recessive disorders, detected in _trans_ with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase* Can be used for variants seen in patients with Jervell and Lange-Nielsen syndrome, if the phenotype includes both long QT interval and congenital deafness.* This code is not considered mutually exclusive with PS4. For example, variants associated with both recessive and dominant cases can meet both PM3 and PS4.* Use the ClinGen SVI recommendations to determine evidence weight for this rule code.* Both variants must be classified using these rule specifications.![](https://lh6.googleusercontent.com/k8TLTROZpnhrx_KFdTguCHToaXwLEnf9Kn9MqXkNr-AdlbStJ_8aGhhXI2u2CZhCuDWJY0FrfVsfOmSdd5Wu8rfHx7gxvGoj7u_j8uqLzURiWrcj8OZOZipYCcQfCkeZCWcJIsqtKnY8) Sum of case scores ≥8 points (see instructions below) Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _ADA._ ≥ 4 points Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _DCLRE1C._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _IL7R._ Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* ≥4 points from Table 1 Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _JAK3._ Not Applicable: Not applicable, as this code is specific to recessive disorders. Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG1._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG2._ Not Applicable: PAH is autosomal dominant. Not Applicable Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Does not apply. Not Applicable: HHT is autosomal dominant disorder. Not Applicable: HHT is autosomal dominant disorder. ≥ 4 points ≥ 4 points ≥ 4 points Not Applicable: This criterion does not apply since the model of inheritance is autosomal dominant. 4.0 points per the PM3 chart Not Applicable: Biallelic cases should not be counted using the ACTA1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Biallelic case counts should not be used for DNM2. Not Applicable: Biallelic case counts should not be used for MTM1. Not Applicable: Biallelic cases should not be counted using the RYR1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Not applicable, as OTC is an X-linked gene and biallelic females are sufficiently rare Use proposed SVI point recommendations. Both variants must be classified using _ABCA4_ Rule Specifications. Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* ≥4 points from Table 1 4.0 points per the PM3 chart 4.0 points per the PM3 chart Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). ≥ 4 pts is required to apply PM3 at Very Strong. Not Applicable Not Applicable: Not applicable since disease-causing variants are heterozygous. Not Applicable: MYOC variants have an autosomal dominant mode of inheritance. PM3\_VeryStrong **≥ 8** pointsSee ATM PM3/BP2 table for approach to assign points per proband. * Details of the cDNA change must be used to describe any variants used as evidence for PM3. If the variant is described only as an amino acid change, this is not sufficient. Probands must also meet PP4 criteria to be counted.* If more than one case has the same genotype and the variants are not confirmed in trans, then only one case should be used for assigning points to avoid overcounting evidence if the variants are actually in cis and hence inherited together in multiple individuals or potentially counting the same case twice. If the variants are confirmed to be in trans, more than one individual with the same genotype can be counted as long as the reports do not represent the same case.* These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign.* For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases.* See PM3 table * PM3 score ≥ 4.0 Not Applicable 4 points awarded from tables 7a and 7b.Example: Detected in trans in ≥4 probands with a pathogenic variant (recessive). Not Applicable: Autosomal dominant. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. Not Applicable: SLC6A8 is an X-linked gene, therefore PM3 is not applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: SCN1A is associated with autosomal dominant inheritance. Not Applicable: SCN2A is associated with autosomal dominant inheritance. Not Applicable: SCN3A is associated with autosomal dominant inheritance. Not Applicable: SCN8A is associated with autosomal dominant inheritance. Not Applicable: Not applicable for the F8 gene. For recessive disorders, points-based system based on confirmation of phase and classification of other variant. Total of **4.0 points** will arrive at **Very Strong**. * Classification of other variant is Pathogenic/Likely Pathogenic * Confirmed in trans: 1 point * Phase unknown: 0.5 (P) or 0.25 (LP) points* Homozygous occurrence (max point 1.0) * Confirmed in trans: 0.5 points* Classification of other variant is Uncertain Significance * Confirmed in trans: 0.25 points * Phase unknown: 0 points Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* ≥4 points from Table 1 PM3_Strong NA Not Applicable: It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: This rule is not applicable to PTEN. 2 points awarded from tables 7a and 7bExample: Detected in trans in 2 probands with a pathogenic variant (recessive). Applicable as described in SVI recommendations for in trans criterion Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes:FPD/AML is inherited in an autosomal dominant manner, thus PM3 is not applicable. Not Applicable: This rule does not apply to TP53/Li-Fraumeni syndrome. Detected in trans with a pathogenic variant. Points-based system. See main specifications document. Use proposed SVI point recommendations. Both variants must be classified using ITGA2B/ITGB3 Rule Specifications. Not Applicable: PM3 is not applicable. MHS is inherited as an autosomal dominant trait with reduced penetrance. For recessive disorders, detected in trans with a pathogenic variantNote: This requires testing of parents (or offspring) to determine phase. NA Not Applicable NA NA Not Applicable: mtDNA variants are maternally inherited and not inherited in an autosomal recessive manner. PM3\_Strong **≥ 4** pointsSee Fanconi Anemia PM3 tables for approach to assign points per proband. Not Applicable: Autosomal dominant. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications. Not Applicable: Not applicable for the F9 gene. Not Applicable: These are dominant conditions, so this rule code does not apply. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications, with the exception that a 22q11.2 deletion in trans ([https://www.ncbi.nlm.nih.gov/books/NBK1523/](https://www.ncbi.nlm.nih.gov/books/NBK1523/)) may be automatically scored 1pt with confirmation that deletion includes the _GP1BB_ gene. Use proposed SVI point recommendations (see below). Both variants must be classified using these rule specifications. Not Applicable: Variants in this gene are being curated as a dominant condition, so this rule code does not apply. Not Applicable Use SVI-recommended point-based system. Not Applicable: Not applicable. Use ClinGen SVI recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for RMRP. Not Applicable Use SVI recommended point system for this code for probands with a VWD type 2N diagnosis. Total of 2 points required. Detected in trans with a pathogenic variant. Points system based on [guidance](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene.Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3\_Strong = ≥4 points ≥2 points. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene. Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3\_Strong = ≥4 points Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. ≥ 2.0 points Not Applicable For recessive disorders, detected in _trans_ with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase* Can be used for variants seen in patients with Jervell and Lange-Nielsen syndrome, if the phenotype includes both long QT interval and congenital deafness.* This code is not considered mutually exclusive with PS4. For example, variants associated with both recessive and dominant cases can meet both PM3 and PS4.* Use the ClinGen SVI recommendations to determine evidence weight for this rule code.* Both variants must be classified using these rule specifications. Sum of case scores 4-7.75 points (see instructions below) Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _ADA._ 2 - 3.5 points Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _DCLRE1C._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _IL7R._ Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 2 to 3.75 points from Table 1 Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _JAK3._ Not Applicable: Not applicable, as this code is specific to recessive disorders. Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG1._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG2._ Not Applicable: PAH is autosomal dominant. Not Applicable Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Does not apply. Not Applicable: HHT is autosomal dominant disorder. Not Applicable: HHT is autosomal dominant disorder. 2 - 3.5 points 2 - 3.5 points 2 - 3.5 points Not Applicable: This criterion does not apply since the model of inheritance is autosomal dominant. 2.0 points per the PM3 chart Not Applicable: Biallelic cases should not be counted using the ACTA1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Biallelic case counts should not be used for DNM2. Not Applicable: Biallelic case counts should not be used for MTM1. Not Applicable: Biallelic cases should not be counted using the RYR1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Not applicable, as OTC is an X-linked gene and biallelic females are sufficiently rare Use proposed SVI point recommendations. Both variants must be classified using _ABCA4_ Rule Specifications. Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 2 to 3.75 points from Table 1 2.0 points per the PM3 chart 2.0 points per the PM3 chart Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Strong should be applied for ≥ 2 pts but \< 4 pts. Not Applicable Not Applicable: Not applicable since disease-causing variants are heterozygous. Not Applicable: MYOC variants have an autosomal dominant mode of inheritance. PM3\_Strong = **4** pointsSee ATM PM3/BP2 table for approach to assign points per proband. * Details of the cDNA change must be used to describe any variants used as evidence for PM3. If the variant is described only as an amino acid change, this is not sufficient. Probands must also meet PP4 criteria to be counted.* If more than one case has the same genotype and the variants are not confirmed in trans, then only one case should be used for assigning points to avoid overcounting evidence if the variants are actually in cis and hence inherited together in multiple individuals or potentially counting the same case twice. If the variants are confirmed to be in trans, more than one individual with the same genotype can be counted as long as the reports do not represent the same case.* These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign.* For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases.* See PM3 table * PM3 score ≥ 2.0 \< 4.0 Not Applicable 2 points awarded from tables 7a and 7b.Example: Detected in trans in 2 probands with a pathogenic variant (recessive). Not Applicable: Autosomal dominant. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. Not Applicable: SLC6A8 is an X-linked gene, therefore PM3 is not applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: SCN1A is associated with autosomal dominant inheritance. Not Applicable: SCN2A is associated with autosomal dominant inheritance. Not Applicable: SCN3A is associated with autosomal dominant inheritance. Not Applicable: SCN8A is associated with autosomal dominant inheritance. Not Applicable: Not applicable for the F8 gene. For recessive disorders, points-based system based on confirmation of phase and classification of other variant. Total of **2.0 points** will arrive at **Strong**. * Classification of other variant is Pathogenic/Likely Pathogenic * Confirmed in trans: 1 point * Phase unknown: 0.5 (P) or 0.25 (LP) points* Homozygous occurrence (max point 1.0) * Confirmed in trans: 0.5 points* Classification of other variant is Uncertain Significance * Confirmed in trans: 0.25 points * Phase unknown: 0 points Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 2 to 3.75 points from Table 1 PM3_Supporting NA Not Applicable: It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: This rule is not applicable to PTEN. 0.5 points awarded from tables 7a and 7bExamples: Two variants that meet PM2_Supporting detected in trans; ORa homozygous variant meeting PM2_Supporting. Applicable as described in SVI recommendations for in trans criterion Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes:FPD/AML is inherited in an autosomal dominant manner, thus PM3 is not applicable. Not Applicable: This rule does not apply to TP53/Li-Fraumeni syndrome. Detected in trans with a pathogenic variant. Points-based system. See main specifications document. Use proposed SVI point recommendations. Both variants must be classified using ITGA2B/ITGB3 Rule Specifications. Not Applicable: PM3 is not applicable. MHS is inherited as an autosomal dominant trait with reduced penetrance. For recessive disorders, detected in trans with a pathogenic variantNote: This requires testing of parents (or offspring) to determine phase. NA Not Applicable NA NA Not Applicable: mtDNA variants are maternally inherited and not inherited in an autosomal recessive manner. PM3\_Supporting = **1** pointSee Fanconi Anemia PM3 tables for approach to assign points per proband. Not Applicable: Autosomal dominant. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications. Not Applicable: Not applicable for the F9 gene. Not Applicable: These are dominant conditions, so this rule code does not apply. Use proposed SVI point recommendations. Both variants must be classified using these rule specifications. Use proposed SVI point recommendations (see below). Both variants must be classified using these rule specifications. Not Applicable: Variants in this gene are being curated as a dominant condition, so this rule code does not apply. Not Applicable Use SVI-recommended point-based system. Not Applicable: Not applicable. Use ClinGen SVI recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for RMRP. Not Applicable Use SVI recommended point system for this code for probands with a VWD type 2N diagnosis. Total of 0.5 points required. Detected in trans with a pathogenic variant. Points system based on [guidance](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) from the ClinGen Sequence Variant Interpretation Working Group. See Appendix 4 for the Lysosomal Diseases VCEP's specification of PM3. Note that points will NOT be applied for any variants of uncertain significance confirmed in trans, due to the high number of pseudodeficiency variants in IDUA. These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene.Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3\_Supporting = 1 point ≥0.5 points. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Apply for patient with phenotype consistent with BRCA1- or BRCA2-related Fanconi Anemia (FA), and co-occurrent variants in the same gene.Phenotype is considered consistent with BRCA1- or BRCA2-related FA if:(i) Increased chromosome breakage (DEB, MMC, or spontaneous) and at least one clinical feature indicative of BRCA1/2-related FA, categorized under: physical features, pathology and laboratory findings, cancer diagnosis _≤5yr_.(ii) Result unknown for chromosome breakage, and at least two clinical features indicative of BRCA1/2-related FA under at least two of the three categories: physical features, pathology and laboratory findings, cancer diagnosis ≤5yr.See **Specifications Table 6** for approach to assign points per proband, and final PM3 code assignment based on the sum of PM3-related points. Also see Appendix H for additional details.PM3\_Supporting = 1 point Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. Not Applicable: While compound heterozygosity leading to a more severe phenotype has been documented, this rule was designed for traditional recessive inheritance. It is acknowledged that there is increasing evidence supporting that some of these genes/variants may also be recessive (e.g., MYL2, MYL3), but addressing those edge cases was outside the scope of this current guideline. ≥ 0.5 points Not Applicable For recessive disorders, detected in _trans_ with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase* Can be used for variants seen in patients with Jervell and Lange-Nielsen syndrome, if the phenotype includes both long QT interval and congenital deafness.* This code is not considered mutually exclusive with PS4. For example, variants associated with both recessive and dominant cases can meet both PM3 and PS4.* Use the ClinGen SVI recommendations to determine evidence weight for this rule code.* Both variants must be classified using these rule specifications. Sum of case scores 1-1.75 points (see instructions below) Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _ADA._ \= 0.5 points Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _DCLRE1C._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _IL7R._ Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 0.5 to 0.75 points from Table 1 Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _JAK3._ Not Applicable: Not applicable, as this code is specific to recessive disorders. Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG1._ Use ClinGen SVI adapted recommendations for _in trans_ criterion with the additional requirement that the co-occurring variant must be classified using the SCID VCEP specifications for _RAG2._ Not Applicable: PAH is autosomal dominant. Not Applicable Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Does not apply. Not Applicable: HHT is autosomal dominant disorder. Not Applicable: HHT is autosomal dominant disorder. \= 0.5 points \= 0.5 points \= 0.5 points Not Applicable: This criterion does not apply since the model of inheritance is autosomal dominant. 0.5 points per the PM3 chart Not Applicable: Biallelic cases should not be counted using the ACTA1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Biallelic case counts should not be used for DNM2. Not Applicable: Biallelic case counts should not be used for MTM1. Not Applicable: Biallelic cases should not be counted using the RYR1 autosomal dominant specifications. Please see the autosomal recessive specifications for use of PM3. Not Applicable: Not applicable, as OTC is an X-linked gene and biallelic females are sufficiently rare Use proposed SVI point recommendations. Both variants must be classified using _ABCA4_ Rule Specifications. Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 0.5 to 0.75 points from Table 1 0.5 points per the PM3 chart 0.5 points per the PM3 chart Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Use the SVI Working Group’s recommended point system to determine PM3 strength (see supplementary file “PM3 table”). PM3\_Supporting should be applied for ≥ 0.5 pts but \< 1 pt. Not Applicable Not Applicable: Not applicable since disease-causing variants are heterozygous. Not Applicable: MYOC variants have an autosomal dominant mode of inheritance. PM3\_Supporting = **1** pointSee ATM PM3/BP2 table for approach to assign points per proband. * Details of the cDNA change must be used to describe any variants used as evidence for PM3. If the variant is described only as an amino acid change, this is not sufficient. Probands must also meet PP4 criteria to be counted.* If more than one case has the same genotype and the variants are not confirmed in trans, then only one case should be used for assigning points to avoid overcounting evidence if the variants are actually in cis and hence inherited together in multiple individuals or potentially counting the same case twice. If the variants are confirmed to be in trans, more than one individual with the same genotype can be counted as long as the reports do not represent the same case.* These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign.* For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases.* See PM3 table * PM3 score ≥ 0.5 \< 1.0 Not Applicable 0.5 points awarded from tables 7a and 7bExamples: Two variants that meet PM2_Supporting detected in trans; ORa homozygous variant meeting PM2_Supporting. Not Applicable: Autosomal dominant. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. * Follow SVI guidance for PM3 ([https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf)).* Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous. Not Applicable: SLC6A8 is an X-linked gene, therefore PM3 is not applicable Not Applicable: Not applicable for TCF4. Not Applicable: Not applicable for SLC9A6. Not Applicable: Not applicable for CDKL5. Not Applicable: Not applicable for FOXG1. Not Applicable: Not applicable for MECP2. Not Applicable: Not applicable for UBE3A. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: SCN1A is associated with autosomal dominant inheritance. Not Applicable: SCN2A is associated with autosomal dominant inheritance. Not Applicable: SCN3A is associated with autosomal dominant inheritance. Not Applicable: SCN8A is associated with autosomal dominant inheritance. Not Applicable: Not applicable for the F8 gene. For recessive disorders, points-based system based on confirmation of phase and classification of other variant. Total of **0.5 points** will arrive at **Supporting**. * Classification of other variant is Pathogenic/Likely Pathogenic * Confirmed in trans: 1 point * Phase unknown: 0.5 (P) or 0.25 (LP) points* Homozygous occurrence (max point 1.0) * Confirmed in trans: 0.5 points* Classification of other variant is Uncertain Significance * Confirmed in trans: 0.25 points * Phase unknown: 0 points Points are calculated using Table 1 from [https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf](https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf) (or attached file “PM3 Tables”)* 0.5 to 0.75 points from Table 1 PVS1_Supporting NA Not Applicable: Not applicable for MYH7. NA See PVS1 flowchart for PVS1_Supporting variants in gene where LOF is a known mechanism of disease. NA NA NA NA NA NA Not Applicable: PVS1 is not applicable. MHS is due to gain of function variants in RYR1. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.Caveats: * Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). * Use caution interpreting LOF variants at the extreme 3’ end of a gene. * Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. * Use caution in the presence of multiple transcripts. NA NA NA NA Assessment of small deletions, nonsense, and frameshift variants in protein-coding genes should follow established guidelines (Abou Tayoun et al., 2018) Use PALB2 PVS1 Decision Tree NA Use _GP1BA_ modified decision tree as per SVI WG. Per Coagulation Factor Deficiency VCEP/SVI PVS1 decision tree. Not Applicable: VWD type 2 is defined by qualitative defects in the VWF protein and not caused by null variants. Use _GP1BB_ modified decision tree as per SVI WG. Use _GP9_ modified decision tree as per SVI WG. Use decision tree as per SVI WG with specified “regions critical to protein function”. Use _HNF4A_ PVS1 decision tree.Apply PVS1\_Supporting to nonsense or frameshift variants at c.1258 (G)/p.Gly420 and 3’. Use _GCK_ PVS1 decision tree.* Apply PVS1\_Supporting to initiation codon variants given MDEP has only reviewed one variant and classified as VUS (c.3G>A, PVS1\_Supporting + PM2\_Supporting; one case submitted, dx.53 and no other info provided to lab). The next methionine is at codon 8 and there are no variants classified as pathogenic 5' of p.Met8. * Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable: Not applicable. Not Applicable: Does not apply. Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (**Figure 1**) \[Reference 1\]. Not Applicable: VWD type 2N is caused by qualitative protein defects and not null variants. NA Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Null variant in a gene where loss of function is a known mechanism of disease. NA Null variant (nonsense, frameshift, splice site (donor/acceptor +/−1,2), initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Apply at appropriate strength according to PVS1 flowchart, which considers knowledge of clinically important functional domains. See Specifications Table 4 and Appendix D for details.Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect _as measured by effect on mRNA transcript profile (mRNA assay only)._ Apply as PVS1 (RNA) at appropriate strength. See Specifications Figure1B and Appendix E for details. Not Applicable: Not currently applicable to TNNI3. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TNNT2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to TPM1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to ACTC1. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL2. See PM4 for truncating variants that do NOT undergo NMD. Not Applicable: Not currently applicable to MYL3. See PM4 for truncating variants that do NOT undergo NMD. Applies to:* Deletions removing exon 1 only* Duplications (≥1 exon and completely contained within the gene) disrupting the reading frame after the haem-binding domain (aa4934-Ter) AND presumed in tandem PVS1\_Supporting designation for initiation codon variants without upstream pathogenic variants of closest potential in-frame start codon. Apply PVS1\_Supporting for truncating variants between codons 621 and 676, for which nonsense-mediated decay is not predicted, and the SAD is retained. As a result, these distal variants may still yield functional channels. Please note that although one variant with good evidence of pathogenicity has been found within this region, (NM\_000218.3(KCNQ1):c.1893del (p.Arg632fs)) it appears to exert a pathogenic effect through a dominant negative (mistrafficking) mechanism rather than true null mechanism (PMID: 22739119).[https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1](https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1) NA Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)). NA NA NA NA NA * Apply PVS1\_Supporting for initiation codon variants in accordance with Tayoun et al (PMID: 30192042). There are no known alternative start codons and the closest potential in-frame start codon is at codon 38. No disease-causing missense variants have yet been identified in the N-terminal region between codons 1 and 37. NA NA NA NA Not Applicable: Not applicable. Not Applicable: Not applicable. Use ClinGen SVI recommendations for loss of function criterion (Tayoun et al., 2018 (PMID: 30192042)) NA NA NA NA NA Not Applicable: Does not apply given gain of function disease mechanism. See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant alpha-actinopathy caused by variants in ACTA1. Not Applicable: Loss of function is not a mechanism of disease for DNM2-related AD Centronuclear myopathy See PVS1 flowchart Not Applicable: Loss of function is not a mechanism of disease for autosomal dominant RYR1-related myopathy. NA NA NA See PVS1 flowchart See PVS1 flowchart Please see attached _DYSF_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCB_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCG_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCD_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _CAPN3_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _ANO5_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Please see attached _SGCA_ PVS1 flowchart. In addition, for any variant with RNA/splicing data, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “experimental splice data”. Use HNF1A PVS1 decision tree.* Apply PVS1\_Supporting levels for nonsense variants occurring 3’ of c.1803 (p.601) and frameshift variants occurring 3’ of c.1854 (p.618) as there is limited evidence of patients with MODY phenotype at this time.Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable: LOF and/or haploinsufficiency have not been clearly identified as disease mechanisms underlying brain malformations related to these genes, so in general this rule is not applicable. The disease mechanism for these genes is gain of function (GOF). Not Applicable: MYOC variants cause JOAG/POAG through a gain of function (GoF) disease mechanism and not loss of function (LoF). Truncating variants in exon 3 are expected to be pathogenic because they escape nonsense-mediated decay. Use ATM PVS1 Decision Tree Loss of function is a known mechanism for VLCAD Deficiency. The specifications below are based on published guidance for assigning strength of evidence for PVS1 (Abou Tayoun et al., (2018) PMID: 30192042). There are multiple transcripts for ACADVL. The major isoform, NM\_000018.4, encodes a 655 amino acid precursor protein that contains a 40 amino acid N-terminal target sequence that is removed during uptake (Aoyama et al., (1995) PMID: 7668252). In a joint project between NCBI and EMBL-EBI (MANE), NM\_000018.4 was designated as the most relevant transcript. Nonsense or Frameshift:* Use caution when interpreting LOF variants at the 3’ end of the gene.* NMD is not predicted if the variant is in the last exon (exon 20) or in the last 50 nucleotides of the penultimate exon (exon 19). * Canonical Splice Site (+1, +2, -1, -2): All donor/acceptor sites follow the GT/AG rule, except for the donor splice site of intron 8, which begins with GC. PVS1 should not be applied for variants in the splice donor site of intron 8 since the impact of GC donor splice sites is not well understood. For +1 or +2 GT donor splice site variants, the exon immediately 5’ of the variant is predicted to be skipped. For -1 or -2 AG acceptor splice site variants, the exon immediately 3’ of the variants is predicted to be skipped.* Initiation codon: The next in-frame methionine is at position 6 (on transcript NM\_000018). However, the first 40 amino acids comprise the leader sequence in the precursor peptide and are important for proper localization of the protein (Aoyama et al., (1995) PMID: 7668252). Therefore, initiator codon variants will meet PVS1\_Strong.* Well-established \_in vitro\_ or \_in vivo\_ functional studies supportive of a damaging effect \_as measured by effect on mRNA transcript profile (mRNA assay only).\_ Apply as PVS1 (RNA) at appropriate strength. * See ACADVL PVS1 decision tree; cannot be combined with PM1 NA See PVS1 flowchart for PVS1_Supporting variants in gene where LOF is a known mechanism of disease. NA NA NA NA Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Supporting is applicable for initiation codon variants in _TCF4._ Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Supporting is applicable for initiation codon variants in _SLC9A6_ Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Supporting is applicable for initiation codon variants in _CDKL5._ Null variant in a gene where loss of function is a known mechanism of disease.* PVS1\_Supporting is applicable for initiation codon variants in _FOXG1._ NA NA Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: LOF and/or haploinsufficiency has not been clearly identified as disease mechanisms for these genes relative to the RASopathy spectrum phenotype, therefore in general this rule is not applicable. Note that PTPN11 is currently the only gene with a confirmed association to another non-RASopathy disorder due to LOF alleles. Variants in PTPN11 with predicted LOF should not be evaluated by these RASopathy specific criteria, but should defer to non-adjusted criteria. Given that some historical LOF variants (e.g. canonical splice sites) could potentially result in a gain of function, users should assess using these criteria and non-adjusted criteria to identify the highest likelihood of pathogenicity for all associated diseases. We recommend that the ClinGen Dosage Sensitivity Map Status (http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml) be reviewed for any new apparently LOF disease associations prior to classification assessment. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Not Applicable: Not applicable. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. NA Follow SVI guidance per workflow in Tayoun et al (2018), included as “PVS1 Decision Tree”. NA PM5_Supporting NA This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. NA NA Applicable when the different missense change is likely pathogenic. PM5_supporting is applicable to nonsense and frameshift variants that are predicted/proved to undergo NMD or located upstream of the last known pathogenic truncating variant. Site-specific recommendations for the application of PM5_Supporting for canonical splicing variants. **PM5\_Supporting**: Missense change at an AA residue where a different missense change which has been determined to be likely pathogenic before (after accounting for Grantham scores).**PM5\_Supporting** is also applied to nonsense/frameshift variants that are downstream of c.98 (in transcript NM\_001754.4).Caveats:\*Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20.\*The nonsense/frameshift variants before c.98 only affect one of the _RUNX1_ functional transcript. PVS1 is also not appliable in this region based on the _RUNX1_ PVS1 decision tree.\*PM5 cannot be used if PM1 was applied at any strength level. Missense variant at an amino acid residue where 1 different missense variant previously determined to be likely pathogenic according to the _TP53_ VCEP’s specifications has been seen before. **The previously seen likely pathogenic variant must have clinical data that demonstrates pathogenicity (i.e. PS2, PS4, PP1) in order for it to count towards PM5\_Supporting code application.** Missense change at an amino acid residue where a different missense change determined to be 'likely pathogenic' has been seen before. Use at adjusted strength. Missense change at an amino acid residue where a different missense variant previously determined to be pathogenic. * Previously established likely pathogenic variant can be considered supporting evidence, must reach a classification of likely pathogenic without PM5.* Grantham score for alternate likely pathogenic variant must be less than for variant being assessed. Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.Example: Arg156His is pathogenic; now you observe Arg156Cys.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. NA NA NA NA Same nucleotide position as previously established pathogenic variant in a rRNA/tRNA * Apply to frameshifting or truncating variants with premature termination codons upstream of p.Tyr1183.* Apply to splice variants as with premature termination codons upstream of p.Tyr1183 where PVS1\_VS(RNA) is applied based on high quality observed splicing impact and must be NMD prone. NA Use as originally specified, but the comparison variant must reach a likely pathogenic classification using the these rule specifications in order to apply code. This evidence code can be applied when there is 1 likely pathogenic variant at the same residue based on _F9_ rule specifications Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect. A “highly suspicious” VUS is defined as a variant that is 1 supporting code away from reaching a likely pathogenic classification. Use code when previously reported variant reaches a likely pathogenic classification using the VWD Type 2 rule specifications. Previously reported variant can be associated with a different type of VWD. Use as originally specified, but the comparison variant must reach a likely pathogenic classification using these rule specifications in order to apply code. Use as originally specified, but the comparison variant must reach a likely pathogenic classification using these rule specifications in order to apply code. Use code when previously reported variant reaches a likely pathogenic classification using the _SERPINC1_ rule specifications from the Thrombosis VCEP. Apply if the previously classified amino acid change is likely pathogenic (rather than pathogenic) or if the previously classified variant is pathogenic but has a greater Grantham distance than the novel variant. Apply if the previously classified amino acid change is likely pathogenic (rather than pathogenic) or if the previously classified variant is pathogenic but has a greater Grantham distance. NA Not Applicable: Does not apply. The reported missense variant was determined to be Likely Pathogenic according to the APC-specific modifications.There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other different missense variants at these positions meet PM5\_supporting. No missense variant has been classified as Pathogenic based on current evidence. Grantham´s distance of the variant under assessment must have an equal or higher score than the reported variant \[Reference 3\]. Use code when previously reported variant reaches a likely pathogenic classification using the VWD Type 2 rule specifications. Previously reported variant can be associated with a different type of VWD. Missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before.Stop loss variant if another stop loss variant has been determined to be likely pathogenic. Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. NA This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. Protein termination codon (PTC) variant in an exon where a different proven pathogenic PTC variant has been seen before. Use to justify additional weight for PTC variants annotated as PVS1. See Specifications Table 4 for PM5\_PTC code strengths applicable per exon. See Appendix D for additional details. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. This criterion can be considered at SUPPORTING if a different missense variant at the same codon has been classified as _likely pathogenic_ using these modified guidelines without application of PM5.The impact of the amino acid change being evaluated needs to be compared to the impact of the amino acid change that is established as likely pathogenic (e.g., a change of Ala to His is less severe than Ala to Cys change). Consider reducing the strength of this rule to NOT APPLICABLE if the predicted impact is not expected to be equivalent or more severe.PM5 should not be combined with PM1. The one with the higher strength should be applied, but if both are applicable at SUPPORTING weight, use of PM5 is most appropriate since it is variant specific. Same residue as previously established likely pathogenic variant (assessed independently of PM5). Same residue as a previously established likely pathogenic variant (assessed independently of PM5).\- The novel change must not affect splicing (SpliceAI ≤ 0.2), must meet PP3, and have a Grantham score equal to or greater than the previously published variants. NA Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Missense change at an amino acid residue where a different missense change was classified as Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5\_Supporting if PP3 is supporting for the missense change. Use PM5 if other variant is Pathogenic due to a missense alteration. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2 Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. Novel missense change at an amino acid residue where a different missense change determined to be _**likely pathogenic**_ has been seen before. Same residue as a previously established likely pathogenic variant classified using these specifications (assessed independently of PM5) . The novel change must not affect splicing (SpliceAI ≤ 0.2), must meet PP3, and have a Grantham score equal or greater than the previously published variants. NA NA Applicable at default strength (PM5) if previously established variant is classified as pathogenic or at reduced strength of PM5\_Supporting if previously established variant is classified as likely pathogenic. Also applicable to a nonsense variant with 1 point from an informative variant (see point table). Informative variants must also be classified by these rule specifications.**Notes**: The informative variant must be classified by the SCID VCEP specifications and may not be the same variant used to meet “+1 pathogenic variant downstream” on the PVS1 flowchart. If negative points are calculated, the curator should not apply PM5 and should reconsider if PVS1 is applicable for the VUA. The VUA must be sufficiently rare, meet PM2\_Supporting, to apply this point system. If the informative variant is a frameshift or nonsense variant, it must reach classification as Pathogenic or Likely Pathogenic without use of PM5 and without use of only PVS1 plus PM2. NA NA Missense change at an amino acid residue where a different missense change was classified as Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5\_Supporting if PP3 is supporting for the missense change. Use PM5 if other variant is Pathogenic due to a missense alteration. Missense change at an amino acid residue where a different missense change was classified as Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5\_Supporting if PP3 is supporting for the missense change. Use PM5 if other variant is Pathogenic due to a missense alteration. Missense change at an amino acid residue where a different missense change was classified as Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5\_Supporting if PP3 is supporting for the missense change. Use PM5 if other variant is Pathogenic due to a missense alteration. * Use at PM5\_Supporting strength for missense variants when other variant was classified as Likely Pathogenic for autosomal dominant PIK3CD-related disease by Antibody Deficiencies VCEP specifications for _PIK3CD_ without using PM5.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Neither change should be predicted to affect splicing (SpliceAI Δ score \<0.2).* Do not apply at codon where any benign variants are known.* The variant being evaluated must have a Grantham distance value greater than or equal to that of the known likely pathogenic variant, indicating that the variant residue is equally chemically different or more chemically different than the known likely pathogenic residue in comparison to the wild type residue. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described PM5 is applicable as described if the variant under review occurs at the same amino acid as a variant classified Likely Pathogenic using these OTC specifications. Comparison variant must reach a likely pathogenic classification using the _ABCA4_ VCEP specifications. Do not apply if the comparison variant is suspected to cause a splicing defect via SpliceAI or other splice predictor. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification.* SpliceAI scores for both variants should be in the same category (\<0.1, between 0.1 and 0.2, >0.2) No change - use as originally described No change - use as originally described Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply only for missense variants for which the amino acid change is the expected mechanism of disease. For the missense variant under curation and the variant(s) resulting in a different amino acid change, exclude likely splice effects (SpliceAI score \<0.5 or experimental evidence of normal splicing). The REVEL score for the missense variant under curation should be >0.7. Missense changes at the same residue must be classified according to LGMD VCEP specifications, and no benign missense variation should be present at the residue. Do not apply for missense variants encoded by the first or last 3 nucleotides of an exon unless a splice effect has been ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. Apply at Supporting for 1 likely pathogenic variant resulting in a different amino acid change at the same residue as the variant under curation.PM5 can potentially be applied to multiple amino acid changes at the same residue as long as the variant classification that determines the strength level does not depend on PM5 application. Apply if the previously classified amino acid change is likely pathogenic (rather than pathogenic), or if the previously classified variant is pathogenic but has a greater Grantham distance. NA Same residue as a previously established likely pathogenic variant (assessed independently of PM5) * Apply to frameshifting or truncating variants with premature termination codons upstream of p.Arg3047.* Apply to splice variants as with premature termination codons upstream of p.Arg3047 where PVS1\_VS(RNA) is applied based on high quality observed splicing impact and must be NMD prone. * See PM5 table. Note: Cannot be applied with PM1, apply criteria with the highest strength. If both are applicable at the same strength, apply PM5 as it is amino acid specific. * One likely pathogenic variant at the same codon NA NA NA Missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before. * This criterion is applicable for any variant resulting in a different amino acid change, at the same amino acid position, as a variant that has been previously established as likely pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing. * If the variant is likely pathogenic, use PM5. Missense change at an amino acid residue where a different missense change determined to be likely pathogenic has been seen before. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA * Novel missense change at an amino acid residue where a different missense change determined to be **Likely Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. * \>1 Non-Identical aa change in paralogous gene(s) where a different missense change determined to be **Pathogenic** or **Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A)Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. * Novel missense change at an amino acid residue where a different missense change determined to be **Likely Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. * \>1 Non-Identical aa change in paralogous gene(s) where a different missense change determined to be **Pathogenic**or **Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A)Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. * Novel missense change at an amino acid residue where a different missense change determined to be **Likely Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. * \>1 Non-Identical aa change in paralogous gene(s) where a different missense change determined to be **Pathogenic**or **Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A)Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. * Novel missense change at an amino acid residue where a different missense change determined to be **Likely Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. * \>1 Non-Identical aa change in paralogous gene(s) where a different missense change determined to be **Pathogenic**or **Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A)Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. This evidence code can be applied when there is 1 likely pathogenic variant at the same residue based on the _F8_ rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect. * Novel missense change at an amino acid residue where a different missense change determined to be **Likely Pathogenic** has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification. BS3_Moderate NA See PS3 specifications. NA NA NA NA NA NA NA NA Well-established functional studies show no damaging effect on protein function * Three or more independent ex vivo studies, NO significant release of Ca2+ in response to agonist Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. Not Applicable Not Applicable Not Applicable Not Applicable NA Not Applicable: Do not use: ● Protein functional studies (BS3) See PS3 for details ● RNA functional studies (Use BP7_Variable(RNA)) NA NA NA Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. NA NA Not Applicable: There are no available assays or model organisms that can recapitulate disease, and in vitro studies cannot dependably rule out pathogenicity. NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. NA Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. See PS3 specifications. NA See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. See PS3 specifications. Not Applicable: Given that normal protein abundance and stability does not rule out impact on enzymatic activity, and that normal enzymatic activity for one substrate is not indicative of other substrates, the Glaucoma VCEP decided to not apply BS3. Not Applicable Well-established _in vitro_ or _in vivo_ functional studies show no damaging effect on protein function or splicing.Please refer to the linked table below for strength level specifications:[https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1](https://docs.google.com/presentation/d/1cdlToGFzYGRbyaTmseiIDQx9AlCKNPfJ/edit#slide=id.p1)Caveats:1. Conflicting evidence from different papers - no points2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 13. No more than 2 pieces of evidence can be counted from the same paper4. Same finding in 2 papers from the same group – count 15. Electrophysiology result can only meet BS3 if the variant is co-expressed with KCNE1 and if the variant current magnitude is within the normal range defined by the paper and is NOT statistically significantly different from the normal control.6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for BS3 are:(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)(2) Automated patch-clamp (e.g. PMID: 30571187)(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) (4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)RNA or Protein Metabolism assays approved by the VCEP for BS3 are:(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034) (6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)* Functional assays are described at the following link: [https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446](https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446) Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. * BS3 will be applied at the moderate level of strength for an normal result in an approved _in vitro_ assay with a minimum of 11 total pathogenic and benign variant controls (classified using these same specifications, as recommended in PMID: 31892348). Example assays with sufficient control variants have not yet been found in published reports at the time of these specifications, but are anticipated in the future as additional studies are published. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: The many secretion assays have not tested enough benign variants to meet the threshold in PMID 31892348 Supplemental Table 1 or Supplemental Table 2. NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. NA NA NA NA NA * BS3 will be applied at the moderate level of strength for an normal result in an approved _in vitro_ assay with a minimum of 11 total pathogenic and benign variant controls (classified using these same specifications, as recommended in PMID: 31892348). Please note that an example assay with sufficient control variants has been identified in the literature, but was recommended for BS3\_Supporting instead based on OddsPath ratio (PMID: 40543502). The two assays from BS3\_Supporting may be stacked to reach a Moderate Strength Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an MTM1 variant because of the numerous possible functions of myotubularin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. BS3\_Moderate is applicable when growth of the variant strain is ≥80% when compared to strains containing the wildtype OTC gene. NA NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. NA NA Applies to variants showing solubility or secretion in functional assays for studies with OddsPath \<0.23 as per the SVI recommendations. Use when a variant rescues both an ATM specifc feature (e.g. phosphorylation of ATM-specific targets) AND radiosensitivity. Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic ID/ASD, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. NA Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Not applicable for splicing effects (replaced by BP7_Strong (RNA)). PM2_Supporting NA The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹¹](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. Absent in population* Databases present at \<0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population. If multiple alleles are present within any subpopulation, allele frequency in that subpopulation must be \<0.00002 (0.002%). Absent/Rare in population databases (absent or ≤0.00007 (0.007%) for autosomal recessive, ≤0.00002 (0.002%) for autosomal dominant).* Background: Rarity or absence in the general population is not robust evidence for pathogenicity, particularly for autosomal recessive disorders. However, the ACMG/AMP Guidelines were devised in such a way that absence or rarity were considered moderate evidence towards pathogenicity, and the framework requires multiple pieces of evidence to classify a variant as likely pathogenic or pathogenic. * Threshold \<0.0002 (0.02%)The 0.0002 cutoff is based on disease frequency of 1:12,000 and the most common PAH pathogenic variant, R408W, the ExAC frequency is 0.0006594 (ExAC MAF: 0.001109 74/66718 European Non-Finnish) and gnomAD overall: 0.0009056 (gnomAD MAF: 0.001728 219/126,700 European Non-Finnish). ≤ One out of 100,000 alleles in gnomAD cohort; if present in ≥2 individuals within a subpopulation, must be present in ≤ One out of 50,000 alleles. **PM2\_Supporting:** Minor allele frequency ≤ 0.00005 with at least 2000 alleles tested around and 20x coverage at the position.Caveat: \*We recommend evaluating PM2\_supporting using the GrpMax FAF when it is available in gnomAD v4.1.0. If a GrpMax FAF value is not available, we recommend requiring that all subpopulations meet the PM2\_supporting threshold. This rule should be applied at supporting level. Variant should have an allele frequency of less than 0.00003 (0.003%) in gnomAD or another large sequenced population. If multiple alleles are present within any genetic ancestry group, allele frequency in that group must be \<0.00004 (0.004%). Genetic ancestry groups influenced by founder effects (such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”) should be ignored. If the variant being assessed does not meet any population rule codes (PM2, BA1, BS1) **AND** has a total allele frequency >0.00003 with no single genetic ancestry group having multiple alleles with a frequency >0.00004, curators should recalculate the total allele frequency based on the number of alleles with variant allele fraction (VAF) >0.35 to assess whether PM2 may be met after excluding the low VAF alleles which are likely to represent clonal hematopoiesis of indeterminant potential (CHIP) contamination in the database. This can be done by visualizing the “allele balance” for heterozygotes under the genotype quality metrics for a given variant. By hovering over the histogram bars, the number of variant carriers for each bar between 0.35 and 0.65 can be totaled and this can be used to revise the allele count to determine the allele frequency that can be used to assess if PM2\_Supporting can be met.In general, the most recent version of gnomAD should be used when available; however, other population databases or earlier versions of gnomAD may be utilized if they are able to provide information the curator deems necessary for optimal variant classification (e.g, they would provide superior information for a particular variant type; have a larger sample size; or better representation for certain subpopulations, etc.) NA Prevalence <1/10,000 (<0.0001) alleles in gnomAD. Not Applicable: PM2 is not used alone. Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.Caveat: Population data for indels may be poorly called by next generation sequencing. NA NA NA NA Frequency <0.00002 (0.002%, 1/50,000) from controls Frequency ≤ 1/300,000 (**0.000333%**) in gnomAD v4 dataset PM2\_Supporting can be applied for variants either absent from gnomAD or with \<= 0.00000156 (0.000156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4 release). If no GroupMax Filtering Allele Frequency is calculated (ex. due to a single variant present), PM2\_Supporting may also be applied. gnomAD MAF of less than or equal to 0.0001114. Variant must be absent in males in population databases, such as gnomAD. Use code for variants with a popmax MAF of \<0.0001 in gnomAD. gnomAD MAF of less than or equal to 0.00006517. gnomAD MAF of less than or equal to 0.0000329. Use code for variants with a popmax MAF of \<0.00002 in gnomAD. gnomAD Grpmax FAF ≤ 1:333,000 (≤ 0.000003 or 0.0003%) gnomAD Grpmax FAF ≤ 1:333,000 (≤ 0.000003 or 0.0003%) The variant must be absent from controls (gnomAD). Absent in population databases (or at extremely low frequency if recessive).* Downgraded to PM2\_Supporting.* gnomAD popmax filtering allele frequency \<0.0000447The applicability of PM2 to suspected founder variants with allele frequencies exceeding the PM2 threshold will be evaluated on a case-by-case basis by the VCEP. Rare in controls, defined by an allele frequency ≤ 0.0003% (0.000003) if the allele count is > 1 OR by an allele frequency \< 0.001% (0.00001) if the allele count is ≤ 1. Use code for variants with a popmax MAF of \<0.005 in gnomAD. This criterion will be applied at the supporting level based on [guidance](https://www.clinicalgenome.org/site/assets/files/5182/pm2_-_svi_recommendation_-_approved_sept2020.pdf) from the ClinGen Sequence Variant Interpretation Working Group.Minor allele frequency \<0.025% (0.00025) in any continental population with >2000 alleles in the most recent version of gnomAD (version # will be stated in the written summary).Variants may be observed in the homozygous state because MPS1 can present in adulthood, and some variants may be hypomorphic. However, the presence and number of homozygotes should be noted. Absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer). Region around the variant must have an average read depth ≥25. See Appendix G for details. The variant must be absent from controls (gnomAD). For variants in _LZTR1_, PM2\_P ≤0.0025% may be applied to support AR disease. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹⁵](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. Absent from controls in an outbred population, from gnomAD v2.1 (non-cancer, exome only subset) and gnomAD v3.1 (non-cancer). Region around the variant must have an average read depth ≥25. See Appendix G for details. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹¹](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹¹](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹⁰](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹⁰](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹⁰](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. The values used to calculate the PM2 thresholds were derived from studies in Northern European populations that have been relatively well-characterized with regards to disease prevalence and variant spectrum. These thresholds can be applied to any population where disease prevalence is considered comparable (1/500 or lower), where the most frequent pathogenic variant accounts for no more than 2% of cases (e.g., has an allele frequency of ≤0.02 in cases based on the upper bound of 95% CI), and where the penetrance of a pathogenic variant is expected to be at least 50% (Kelly _et al._ 2018[¹⁰](#pmid_29300372)).A threshold of **≤0.00004** in the subpopulation with the highest frequency when using the upper bound of the 95% CI activates this rule.1. Alternatively, this is equivalent to the variant NOT being observed more than once (≤1 allele) in gnomAD v.2.1.1 in one of the non-founder populations (e.g., absence required from the Other and Ashkenazi Jewish subpopulations).2. Applying a threshold of ≤0.00004 (upper bound of 95% CI of the allele frequency in gnomAD) is equivalent to the variant being seen in a single subpopulation and that subpopulation meets any of the following: * **Allele Count (AC) in Allele Number (AN)** * ≤1 in ≥120,000 * ≤2 in ≥160,000 * ≤3 in ≥195,000 * ≤4 in ≥230,000gnomAD is the preferred database for this calculation, but currently only displays the filtering allele frequency (FAF), which is equivalent to a lower bound estimate of the 95% CI, when the upper bound is what is needed.* Confidence interval tools, such as [Confit-de-MAF](https://www.genecalculators.net/confit-de-maf.html), can be used to determine the upper bound of the 95% CI of the observed allele frequency.Due to current technical limitations of next generation sequencing technologies, minor allele frequencies for complex variants (e.g., large indels) may not be accurately represented in population databases.Caution should be used when a variant is only identified, or over-represented, in one of the smaller gnomAD populations, as the gnomAD allele frequencies may not accurately represent the true population frequency.Population databases may contain affected or pre-symptomatic individuals for diseases with reduced penetrance/variable onset. Allele frequency ≤ 0.0005 in population databases. Allele frequency in males ≤ 0.00005 (≤5x10^-5) in population databases.* Highest allele frequency in a subpopulation should be used to assess this. Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.* Supporting level only* Maximum allele frequency in gnomAD (in one of the 5 continental populations; African/African-American, East Asian, European non-Finnish, Latino/Admixed-American, or South Asian) \<0.00001 (0.001%) gnomAD Grpmax filtering allele frequency ≤0.00002412 gnomAD popmax filtering allele frequency \<0.0001742* An additional requirement is that **no homozygotes** have been observed in gnomAD. Absent/extremely rare allele frequency \<0.00002 (\<1 in 50,000 alleles ) in gnomAD v4 dataset gnomAD popmax filtering allele frequency \<0.00003266 * An additional requirement is that **no homozygotes** have been observed in gnomAD. gnomAD popmax filtering allele frequency \<0.00004129.* An additional requirement is that **no homozygotes** have been observed in gnomAD. Absent/rare from controls in an ethnically-matched cohort population sample.* Used if the gnomAD PopMax Filtering Allele Frequency (FAF) is ≤ 2.0 x 10^-4. gnomAD popmax filtering allele frequency \<0.000115* An additional requirement is that **no homozygotes** have been observed in gnomAD. * Supporting level only* Met for total allele frequency lower than 1.43 x 10^-7 (0.000000143) across all populations in gnomAD v4.1.0.* Threshold is based on the experts’ estimate of CTLA-4 insufficiency prevalence of 1/200,000 – 1/1,000,000 people and 45-70% penetrance. The lower end of the prevalence estimate (1 in 1,000,000) and the higher end of the penetrance estimate (70%) were used for this calculation. Allelic heterogeneity of 1 and genetic heterogeneity of 1 were also assumed for the calculation. gnomAD popmax filtering allele frequency \<0.000102 * An additional requirement is that **no homozygotes** have been observed in gnomAD. gnomAD popmax filtering allele frequency \<0.0000588 * An additional requirement is that **no homozygotes** have been observed in gnomAD. Present at \<0.01% among gnomAD controls, using the subpopulation with the highest frequency and at least 1,000 allele counts. Caveat: Population data for indels may be poorly called by next generation sequencing. At low frequency in males in population databases. Use \<2.0x10^-6 for cut off. This is defined relative to the BA1 cutoff. The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). Strength modification based on an abnormal result in at least one approved _in vitro_ assay. \<6 total alleles in gnomAD or \<0.00004 (0.004%) in gnomAD subpopulations. \<6 total alleles in gnomAD or \<0.00004 (0.004%) in gnomAD subpopulations. Absent/extremely rare allele frequency \<0.00002 (\<1 in 50,000 alleles ) in gnomAD v4 dataset Absent/extremely rare allele frequency \<0.00002 (\<1 in 50,000 alleles ) in gnomAD v4 dataset Absent/extremely rare allele frequency \<0.00002 (\<1 in 50,000 alleles ) in gnomAD v4 dataset * Downgraded to PM2\_Supporting* Applicable to variants with a total allele frequency \<0.00000132 across all populations in gnomAD v4.1.0.* Maximum credible population allele frequency threshold determined using Whiffin/Ware calculator ([https://www.cardiodb.org/allelefrequencyapp/](https://www.cardiodb.org/allelefrequencyapp/)) and the following estimated parameters (with the prevalence estimated for autosomal dominant PIK3CD-related immune disease): * Prevalence: 1 in 4000 (a conservative estimate for primary immunodeficiency diseases from PMID: 17577648, PMID: 23201919) * Allelic heterogeneity: 1 * Genetic heterogeneity: 1 * Penetrance: 0.95 (a conservative estimate based on multiple reports of incomplete but nearly complete penetrance in PMID: 27555459, PMID: 36749229, PMID: 37390899) PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is ≤ 0.0000559. 1 allele is allowed. PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is absent (1 allele allowed) for autosomal dominant PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is absent (1 allele allowed) PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is absent (1 observation allowed in females only) PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is absent (1 allele allowed) for autosomal dominant Applicable for variants in OTC with Grpmax Filtering Allele Frequency \<0.000015 (0.0015%) AND ≤1 homo- or hemizygote in the most current version of gnomAD available at the time of curation. Rationale: The most common pathogenic variant in population databases is p.Arg40Cys, which is associated with late onset OTC Deficiency (PMID: 23209112, 7860066, 11260212, others) and present in 17 heterozygotes and 6 hemizygotes in gnomAD(v4.0.0) (Mino Allele frequency=0.001586% in European populations). Other commonly reported pathogenic variants (p.Arg277Trp, p.Arg141Gln, p.Arg141Ter) are rare or absent in population databases, therefore a threshold of 0.0015% is set for PM2\_Supporting. Total MAF \<0.0001 in gnomAD. Absent/rare from controls in an ethnically-matched cohort population sample.* Used if the gnomAD total allele frequency is ≤ 4.0 x 10^-4. PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is ≤ 0.000005 for autosomal recessive PM2\_Supporting may be applied if the minor allele frequency in population databases of at least 2000 alleles is ≤ 0.00000697 for autosomal recessive Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.0001. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.00009. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.00009. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.00009. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.0001. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.0001. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). Apply if the Grpmax variant allele frequency / upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequency in gnomAD is \<0.00009. Do not use data for which the variant does not pass quality control filters. * If only 1 or 2 variant alleles are present in the Grpmax population, use the Grpmax variant allele frequency* If at least 3 variant alleles are present in the Grpmax population, use the upper bound of the 95% confidence interval (95% CI) of the Grpmax variant allele frequencyGrpmax refers to the gnomAD subpopulation with the highest variant allele frequency. Use large, non-bottlenecked genetic ancestry groups for the Grpmax; avoid using the Amish, Ashkenazi Jewish, European Finnish, and Remaining Individuals groups as well as the genomes-only data for the Middle Eastern group.The upper bound of the 95% CI must be calculated using variant allele numbers and counts from gnomAD. Confidence interval tools, such as Confit-de-MAF ([https://www.genecalculators.net/confit-de-maf.html](https://www.genecalculators.net/confit-de-maf.html)), can be used.Use the gnomAD version with the largest allele number. For larger deletions or duplications that may not be well represented in gnomAD (e.g., single- or multi-exon events), also confirm the variant is not common in gnomAD SVs, gnomAD CNVs, or the Database of Genomic Variants (DGV) ([https://dgv.tcag.ca/dgv/app/home](https://dgv.tcag.ca/dgv/app/home)). gnomAD Grpmax FAF ≤ 1:333,000 (≤ 0.000003 or 0.0003%) Absent/rare from controls in an ethnically-matched cohort population sample ( ≥1). Allele frequency ≤ 0.0001 in population databases. Frequency **≤.001%** in gnomAD v4 datasetIf n=1 in a single sub population, that is sufficiently rare and PM2\_supporting would apply. Variants with a highest population minor allele frequency (MAF) \<0.001 (0.1%) in any continental population with >2000 alleles in gnomAD will meet PM2\_supporting.* Calculated using the Prevalence of 1:100,000, Allelic Contribution of 0.2, Genetic Contribution of 1, and Penetrance of 0.75 to allow for mild VLCADD that may develop in adulthood. This was multiplied by 1.5 to account for mildly pathogenic variants being present in carriers within the population databases.* It is acceptable for an ACADVL variant to be present in controls because VLCAD deficiency is a recessive condition. It is also possible for homozygous ACADVL variants to be present in population databases due to later onset of the condition. If homozygous variants are present, the number should be noted and discussed with an expert. * Threshold: <5.0E-6 (<0.0005%).* Use the highest ethnic population allele frequency.* Caveat: PVS1 + PM2_Supportive may reach Likely Pathogenic.* Caveat: Do not use Finnish, Ashkenazi Jewish, or “Other” populations in gnomAD.* Minimum amount of studied alleles should be 2000. Absent/Rare in population databases (absent or ≤0.00007 (0.007%) for autosomal recessive). Allele frequency \<0.000005 across gnomAD with no more than one allele in any subpopulation and at least 20x coverage. **Allele frequency \<0.000055 (\<0.0055%) in all populations in gnomAD.**CCDS VCEP notes: It is acceptable for a GATM variant to be present in controls, if heterozygous, because AGAT-D is a recessive disorder. Homozygotes should not be seen in a population database, such as gnomAD, because the penetrance of this condition in individuals with biallelic pathogenic variants is expected to be 100%. GATM specifications:* All subpopulations in gnomAD must have a maximum allele frequency less than 0.000055 (based on the prevalence of the most common suspected pathogenic variants, c.484+1G>T and p.Arg169Ter) (see Appendix 4). Use the current version recommended by SVI; version number will be stated in classification summary.* Note – PM2 will NOT be used at moderate strength; PM2 will only be applied as a Supporting criterion.* If homozygotes are observed, the variant will meet BS2 (assuming 100% penetrance for an individual with 2 pathogenic variants in trans). **Allele frequency \<0.0004 (\<0.04%) in all populations in gnomAD.**It is acceptable for a GAMT variant to be present in controls, if heterozygous, because GAMT-D is a recessive disorder. Homozygotes should not be seen in a population database, such as gnomAD, because the penetrance of this condition in individuals with biallelic pathogenic variants is expected to be 100% and the condition is expected to present with severe symptoms early in life.GAMT specifications:* All subpopulations in gnomAD v4.0 must have a maximum allele frequency less than 0.0004 (the highest population minor allele frequency of the most common pathogenic GAMT variant, c.327G>A, in gnomAD). Any variant with a frequency below this cutoff will meet PM2\_Supporting.* If homozygotes are observed, or variant is confirmed in trans with a known pathogenic variant, the variant will meet BS2 (assuming 100% penetrance for an individual with 2 pathogenic variants in trans). Absent/rare from controls in an ethnically-matched cohort population sample. Threshold: \<0.00002 (0.002%) AND 0 hemizygotes in gnomAD. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. Absent/rare from controls in an ethnically-matched cohort population sample.* Use if absent, zero observations in control databases. The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). The variant must be absent from controls (gnomAD). One or fewer alleles, if a minimum of 10,000 alleles assessed in population databases, such as the Genome Aggregation Database (gnomAD). Caveat: Population data for indels may be poorly called by next generation sequencing. One or fewer alleles, if a minimum of 10,000 alleles assessed in population databases, such as the Genome Aggregation Database (gnomAD). Caveat: Population data for indels may be poorly called by next generation sequencing. One or fewer alleles, if a minimum of 10,000 alleles assessed in population databases, such as the Genome Aggregation Database (gnomAD). Caveat: Population data for indels may be poorly called by next generation sequencing. One or fewer alleles, if a minimum of 10,000 alleles assessed in population databases, such as the Genome Aggregation Database (gnomAD). Caveat: Population data for indels may be poorly called by next generation sequencing. Variant must be absent in males in population databases, such as gnomAD. One or fewer alleles, if a minimum of 10,000 alleles assessed in population databases, such as the Genome Aggregation Database (gnomAD). Caveat: Population data for indels may be poorly called by next generation sequencing. Absent/rare from controls in an ethnically-matched cohort population sample.* Used if the gnomAD total allele frequency is ≤ 4.0 x 10^-4. PP4_Very Strong NA Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: PTEN EP Commentary: Phenotype specificity has been incorporated into the rule specifications for PS4 Use 2. NA NA Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The FPD/AML phenotype is rather unspecific and can be caused by a number of other inherited predisposition syndromes, somatic mutations or environmental factors that are insufficient to meet the original ACMG/AMP rule PP4. NA NA NA Not Applicable: PP4 is not applicable, variants in CACNA1S also result in MHS. NA NA NA NA NA NA Not Applicable: Do not use for AD disorder as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.For AR disorder, use PM3 for specific phenotype considerations Not Applicable: Combine with PS4 to avoid double counting probands. NA NA NA NA NA NA NA NA Not Applicable: Not applicable, see PS4. NA Not Applicable NA NA NA Not Applicable: PP4 is not applicable due to genetic heterogeneity. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. NA Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: The phenotype associated with CYP1B1 variants is not highly specific and there is genetic heterogeneity. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: PAH does not have a single genetic etiology. NA Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. NA NA NA NA NA NA NA NA NA Not Applicable: PP4 is factored into the strength of PS4. See case counting specifications above. NA Not Applicable: PP4 is factored into the weight of PS4 case counting and should not be applied separately. NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not applicable since this criterion is accounted for under PS4. Not Applicable: The phenotype associated with MYOC variants is not highly specific and there is genetic heterogeneity. Not Applicable: Autosomal Dominant: do not use as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.Autosomal Recessive: do not use as a separate line of evidence. Such evidence is built into the Ataxia Telangiectasia PM3|BP2 table NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 NA Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 NA PP4_Strong NA Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: PTEN EP Commentary: Phenotype specificity has been incorporated into the rule specifications for PS4 Use 2. NA NA Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The FPD/AML phenotype is rather unspecific and can be caused by a number of other inherited predisposition syndromes, somatic mutations or environmental factors that are insufficient to meet the original ACMG/AMP rule PP4. NA NA Proband with clinical diagnosis of GT based on the presence of mucocutaneous bleeding and appropriate lab abnormalities. Full sequencing of both genes is required at this strength Not Applicable: PP4 is not applicable, variants in CACNA1S also result in MHS. Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. NA NA NA NA NA Not Applicable: Do not use for AD disorder as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.For AR disorder, use PM3 for specific phenotype considerations Not Applicable: Combine with PS4 to avoid double counting probands. NA NA NA NA NA NA NA NA Not Applicable: Not applicable, see PS4. NA Not Applicable NA NA Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4\_Strong – LR ≥18.7:1PP4\_Very Strong – LR ≥350:1See Specifications Table7 and Appendix B for details. Not Applicable: PP4 is not applicable due to genetic heterogeneity. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4\_Strong – LR ≥18.7:1PP4\_Very Strong – LR ≥350:1See Specifications Table7 and Appendix B for details. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: The phenotype associated with CYP1B1 variants is not highly specific and there is genetic heterogeneity. NA NA NA A patient score of ≥ 6 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Strong. For _MLH1_ variants, _MLH1_ promoter methylation is to be excluded in the tumors. Independent tumors can be from the same patient/family. A patient score of ≥ 6 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). A patient score of ≥ 6 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). NA A patient score of ≥ 6 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). NA A patient score of ≥ 4 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). A patient score of ≥ 4 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). Not Applicable: PAH does not have a single genetic etiology. NA Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. A patient score of ≥ 6 points¹. ¹CNV (Copy number variation) testing is required to consider PP4\_Strong in order to certify that the variant in question is the causative for the phenotype and not one CNV event corrected by gene therapy and not identified previously (see instructions below). NA NA ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Strong. Independent tumors can be from the same patient/family. ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Strong. Independent tumors can be from the same patient/family. ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Strong. Independent tumors can be from the same patient/family. NA NA PP4 follows the published SVI guidance from Biesecker et al 2023 (PMID:38103548). For ACTA1, a conservative estimate of the diagnostic yield is 33%, which corresponds to +2 points and a moderate strength in Table 2 of this guidance. However, if the proband meets PP4\_Moderate criteria and has had a comprehensive myopathy panel, exome, or genome testing that is negative for all other causes of myopathy, PP4 can be applied at strong, per the SVI guidance. The combination of PP1 and PP4 is capped at strong. Not Applicable: PP4 is factored into the strength of PS4. See case counting specifications above. NA Not Applicable: PP4 is factored into the weight of PS4 case counting and should not be applied separately. NA NA NA PP4 follows the published SVI guidance from Biesecker et al 2023 (PMID:38103548). For ACTA1, a conservative estimate of the diagnostic yield is 33%, which corresponds to +2 points and a moderate strength in Table 2 of this guidance. However, if the proband meets PP4\_Moderate criteria and has had a comprehensive myopathy panel, exome, or genome testing that is negative for all other causes of myopathy, PP4 can be applied at strong, per the SVI guidance. The combination of PP1 and PP4 is capped at strong. NA Use the PP4 table (see supplementary file “PP4 table DYSF”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table SGCB”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table SGCG”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table SGCD”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). Use the PP4 table (see supplementary file “PP4 table CAPN3”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). NA Use the PP4 table (see supplementary file “PP4 table SGCA”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). NA Not Applicable: Not applicable since this criterion is accounted for under PS4. Not Applicable: The phenotype associated with MYOC variants is not highly specific and there is genetic heterogeneity. Not Applicable: Autosomal Dominant: do not use as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.Autosomal Recessive: do not use as a separate line of evidence. Such evidence is built into the Ataxia Telangiectasia PM3|BP2 table NA NA NA NA 4 or more points based on any combination of the following. Two or more data types are required to meet Strong: • Low urine guanidinoacetate with or without low or low normal creatine (1 point) • Low plasma guanidinoacetate with or without low or low normal creatine (2 points) • Significantly decreased creatine peak in brain magnetic resonance spectroscopy (3 points) • AGAT enzyme activity \<5% of normal (3 points)\* Variant must meet PM2\_Supporting for PP4 to apply at any strength. \* For PP4 to be applied at strong, full GATM gene sequencing, including all coding exons and intron/exon boundaries, must have been carried out. If not, consider downgrading. 4 points based on any combination of the following. Two or more data types are required to apply PP4\_Strong:* Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).* Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).* Significantly decreased creatine peak in brain magnetic resonance spectroscopy with or without visible guanidinoacetate peak (3 points).* GAMT enzyme activity \<5% of normal (3 points).* Variant must meet PM2\_Supporting for PP4 to apply at any strength.* For PP4 to be applied at strong, full GAMT gene sequencing, including all coding exons and intron/exon boundaries, must have been carried out. If not, consider downgrading. 4 or more points based on combinations of the following. * Elevated urine creatine/creatinine ratio on one occasion (1 point)* Elevated urine creatine/creatinine ratio on more than one occasion (2 points)* Significantly decreased creatine peak, with absent guanidinoacetate peak, if reported (3 points)* Deficient creatine uptake in cultured fibroblasts (\<10% of normal with \<125uM creatine) (3 points)Additional specifications:* Two or more data types are required for PP4\_Strong.* An individual used to assign PP4, at any weight, cannot be also included for PS4 count. If multiple unrelated probands with the variant have been identified, it is recommended that the case with the highest PP4 points is assigned the appropriate weight for PP4, and the other cases are used for PS4.* Variant must meet PM2\_Supporting for PP4 to apply at any strength.* For PP4 to be applied at strong, full SLC6A8 gene sequencing, including all coding exons and intron/exon boundaries, must have been carried out. If not, consider downgrading. NA NA NA NA NA NA Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 NA Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 NA PP4_Moderate NA Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: PTEN EP Commentary: Phenotype specificity has been incorporated into the rule specifications for PS4 Use 2. NA Plasma phenylalanine concentration persistently above 120 µmol/L (2mg/dL), and either normal urine pterins and normal DHPR activity, or sequencing of genes in the BH4 cofactor metabolism pathway to exclude a defect of BH4 cofactor metabolism. Not Applicable: Not applicable for CDH1. Not Applicable: MM-VCEP notes: The FPD/AML phenotype is rather unspecific and can be caused by a number of other inherited predisposition syndromes, somatic mutations or environmental factors that are insufficient to meet the original ACMG/AMP rule PP4. At least 2 independent observations of the variant with VAF 5-25%. Phenotype specific for disease with single genetic etiology. * Points-based system. See main specifications document Proband with clinical diagnosis of GT based on the presence of mucocutaneous bleeding and appropriate lab abnormalities. Not Applicable: PP4 is not applicable, variants in CACNA1S also result in MHS. Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. NA NA 1. Mitochondrial DNA depletion showing ≤ 20% of controls, OR 2. Multiple mitochondrial DNA deletions (NOTE:depletion and/or deletion analysis must be performed in muscle and/or liver; other tissues such as blood, fibroblast, and buccal are not acceptable; Must be performed in child, as defined as <18 years old) Note: For both scenarios 1 and 2, will only apply if other mtDNA maintenance disorders have been excluded (exome sequencing or comprehensive panel-based testing) Individual has abnormally high urinary ethylmalonic acid AND one of the following: (1) All of the following symptoms present: -Acrocyanosis -Petechiae -Chronic diarrhea -Developmental delay (2) ≥3 or more of the following biochemical studies: -Abnormally high blood C4-Acylcarnitine esters -Abnormally high blood C5-acylcarnitine -Abnormally high plasma thiosulphate -Abnormally low cytochrome oxidase activity in skeletal muscle (without evidence of other complexes decreased) NA Not Applicable: Do not use for AD disorder as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.For AR disorder, use PM3 for specific phenotype considerations Not Applicable: Combine with PS4 to avoid double counting probands. Must meet both criteria:1)Proband with platelet aggregation study absent for ristocetin and present for all other agonists OR flow cytometry or Western blot less than 10% expression of GPIba.2)Proband must have full sequencing of all three BSS genes (_GP1BA, GP1BB_ and _GP9_) and deletion/duplication analysis. Proband must meet hemophilia B phenotype criteria AND have full gene sequencing and deletion/duplication analysis. The patient must have a clinical phenotype of excessive mucocutaneous bleeding and required laboratory values to use the PP4 rule code at the moderate strength. See Table 2A for required and consistent laboratory values. Must meet both criteria:1)Proband with platelet aggregation study absent for ristocetin and present for all other agonists OR flow cytometry or Western blot less than 10% expression of GPIba 2)Proband must have full sequencing of all three BSS genes (_GP1BA, GP1BB_ and _GP9_) and deletion/duplication analysis. Must meet both criteria: 1. Proband with platelet aggregation study absent for ristocetin and present for all other agonists OR flow cytometry or Western blot less than 10% expression of GPIba 2. Proband must have full sequencing of all three BSS genes (_GP1BA, GP1BB_ and _GP9_) and deletion/duplication analysis. NA Phenotype: MODY Probability Calculator result ≥50% chance of testing positive[⁶](#url_187f5e4a-1941-5c2d-9c9f-1dc999cc5454) AND negative _HNF1A_ testing AND presence of at least one additional feature characteristic of \__HNF4A_\_-MODY:   * Antibody negative and/or persistent C-peptide after five years following T1DM diagnosis  * Personal or family history of persistent neonatal hypoglycemia * Personal or family history of large for gestational age (LGA) infants or macrosomia in the absence of sufficient maternal hyperglycemia * Response to low-dose SU (extreme response- hypoglycemia) * Biochemical/Molecular phenotypic evidence from patient cell lines * Fanconi phenotype in conjunction with c.187C>T p.R63W HbA1C 5.6 – 7.6% (38-60 mmol/mol) (if given multiple results, use maximum value) AND fasting plasma glucose always (FPG) 5.5-8 mmol/L (100-144 mg/dL) AND presence of any of the following additional features:* Pediatric patient (prepubertal or \<10 years) (picked up in the absence of symptoms, either incidentally during workup for an unrelated indication or during routine screening)* Multiple values (=persistent) of mild fasting hyperglycemia in PP4 range * Either (≥ 1 HbA1c + ≥ 2 FPG) OR (≥ 2 HbA1c + ≥ 1 FPG) * Only 1 HbA1c and 1 FPG in range but they are at least 6 months apart * Patient in literature is described as having a multi-year history of impaired fasting glucose (IFG)* OGTT (oral glucose tolerance test) with 2-hour increment \<3 mmol/L (54 mg/dl) * Antibody negative * Macrosomia in normoglycemic offspring of hyperglycemic gestational parent* Low birthweight in hyperglycemic offspring of hyperglycemic gestational parent. * Three-generation, dominant family history of diabetes or hyperglycemia (in a family not used for PP1) Not Applicable: Not applicable, see PS4. A patient score of ≥2 points (see instructions below). Not Applicable The patient must have a clinical phenotype of excessive mucocutaneous bleeding and required laboratory values to use the PP4 rule code, including a low factor VIII activity level and evidence of decreased VWF:FVIII binding.Additional consistent information should be noted but is not required, including either normal or low VWF:Ag, normal high molecular weight multimers, and sequencing with duplication/deletion analysis of the F8 gene. 3 or more of the following criteria are met:* Deficient IDUA activity, within the affected range (usually non-detectable or stated to be in the affected range by lab) in fibroblasts, leukocytes, or plasma. * Do not count if one or more pseudodeficiency variants are reported to be present, or if the result was obtained on newborn screen without confirmatory enzyme testing.* Enzyme replacement therapy results in a significant reduction in urine GAGs (either total, or dermatan or heparan sulfate).* Elevated urinary and/or blood GAGs expressed as either total GAGs, specific GAG (heparan sulfate, dermatan sulfate, or endogenous biomarker) stated to be consistent with MPS I. * An individual with clinical features specific to MPS I; At minimum at least 2 of the following: dysostosis multiplex, hepatosplenomegaly, arthropathy, corneal involvement, valvular thickening; AND/OR case reported within the context of a larger clinically-diagnosed MPS I cohort, when published by groups with demonstrated experience (as determined at the discretion of the VCEP) in lysosomal disorders and the clinical care of affected individuals. When detailed clinical information is limited, confidence in the phenotypic classification is strengthened when the report originates from groups with established familiarity in the diagnosis and longitudinal management of MPS I.* Bone marrow transplant results in a significant reduction in urine GAGs (either total, or dermatan or heparan sulfate). Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4\_Moderate – LR ≥4.3:1See Specifications Table7 and Appendix B for details. Not Applicable: PP4 is not applicable due to genetic heterogeneity. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Breast cancer is very common and has a high degree of genetic heterogeneity (caused by pathogenic variants in numerous genes). Use ONLY to capture combined LR towards pathogenicity, based on multifactorial likelihood clinical data.PP4\_Moderate – LR ≥4.3:1See Specifications Table7 and Appendix B for details. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: Inherited cardiomyopathies have high locus heterogeneity as well as non-genetic etiologies. Not Applicable: The phenotype associated with CYP1B1 variants is not highly specific and there is genetic heterogeneity. \> 8 phenotype points required; two specific criteria must be met NA Patient score of ≥2 points (see instructions below) A patient score of 2-\<6 points (see instructions below). 2 independent CRC/Endometrial MSI-H tumors using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Moderate. For _MLH1_ variants, _MLH1_ promoter methylation is to be excluded in the tumors. Independent tumors can be from the same patient/family. A patient score of 2-\<6 points (see instructions below). A patient score of 2-\<6 points (see instructions below). * 8 phenotype points or more are required to use this code at the moderate strength for a single proband (see points list below).* Additionally, at least one **specific** criterion must be met (see below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. A patient score of 2-\<6 points (see instructions below). * In order to be counted for PP4\_Moderate, a proband must; * score at least 10 phenotype points in the PS4 counting rubric * AND * have been genotyped using a method able to detect _LRBA_ variants without biallelic variants found AND * have experimental data from the patient's cells showing a functional defect in the CTLA4 pathway such as defective CD80 and/or CD86 transendocytosis, defective Treg suppression of T cell proliferation, or reduced CTLA4 cell surface expression.* Patient cell-based assays approved for PP4\_Moderate are; * (1) CD80-GFP transendocytosis to CTLA4-expressing patient Tregs or memory CD4+ T cells (i.e. PMID: 25329329, PMID: 34111452, PMID: 28159733) * (2) Total expression of CTLA4 by patient Tregs and other T cells (i.e. PMID: 25213377, PMID: 25329329) * (3) Cell surface expression of CTLA4 by patient B cells or T cells (i.e. PMID: 25213377, PMID: 25329329, PMID: 34111452, PMID: 28159733)* This proband cannot be used for the PS4 code.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. A patient score of ≥2-\<4 points (see instructions below). A patient score of ≥2-\<4 points (see instructions below). Not Applicable: PAH does not have a single genetic etiology. A male proband diagnosed with retinoschisis by the age of 13 with visual acuity impairment, showing schisis, and having retinal detachment. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. A patient score of ≥2.5-\<6 points (see instructions below). Patient's phenotype meets consensus clinical diagnostic (Curaçao) criteria for HHT, and sequencing and large deletion/duplication analysis was performed for both _ENG_ and _ACVRL1_ with any other identified variants ruled out. Patient's phenotype meets consensus clinical diagnostic (Curaçao) criteria for HHT, and sequencing and large deletion/duplication analysis was performed for both _ENG_ and _ACVRL1_ with any other identified variants ruled out. 2 independent CRC/Endometrial MSI-H tumors using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Moderate. Independent tumors can be from the same patient/family. 2 independent CRC/Endometrial MSI-H tumors using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Moderate. 2 independent CRC/Endometrial MSI-H tumors using a standard panel of 5-10 markers^e or tumor genome **and/or** loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4\_Moderate. * PP4\_Moderate is met when a proband scores greater than or equal to 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out a variant in the _PIK3R1_ locus AND has patient cell-based functional assays directly showing abnormally high activity of the disease-relevant PI3K delta pathway.* Patient cell-based functional experiments generally isolate T cells from affected and unaffected patients and often perform stimulation by anti-CD3 and anti-CD28 antibodies, followed by assessment of PI3K delta pathway function using western blotting or flow cytometry in combination with phospho-specific antibodies. These methods detect levels of AKT phosphorylation at Ser473 (PMID: 24136356, PMID: 28414062, PMID: 28428270) or Thr308 (PMID: 24165795) and/or S6 phosphorylation at Ser235/Ser236 (PMID: 28428270) or Ser240/Ser244 (PMID: 28414062). Phosphorylation may be upregulated by 1.2-fold to 7.5-fold in cells from the affected patient relative to the healthy control, and in some assays may be shown to be abrogated by PI3K-specific inhibitor (PMID: 28428270).* Please note that a proband used for PP4\_Moderate cannot be included in PS4.* In order to be evaluated for this criterion, the variant must not meet BS1 or BA1. NA PP4 follows the published SVI guidance from Biesecker et al 2023 (PMID:38103548). For ACTA1, a conservative estimate of the diagnostic yield is 33%, which corresponds to +2 points and a moderate strength in Table 2 of this guidance. PP4\_Moderate is met with the presence of any of these features**Presence on Muscle Biopsy of:*** Accumulated thin filaments* Intranuclear rods* Cores, fiber type disproportion* Zebra bodies Not Applicable: PP4 is factored into the strength of PS4. See case counting specifications above. NA Not Applicable: PP4 is factored into the weight of PS4 case counting and should not be applied separately. Proband phenotype points total ≥1.5 points This code is applicable when a single proband can be awarded 8 or more phenotype points. * 8 phenotype points or more are required to use this code at the moderate strength for a single proband (see points list below).* Additionally, at least one **specific** criterion must be met (see below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. PP4 follows the published SVI guidance from Biesecker et al 2023 (PMID:38103548). For ACTA1, a conservative estimate of the diagnostic yield is 33%, which corresponds to +2 points and a moderate strength in Table 2 of this guidance. PP4\_Moderate is met with the presence of any of these features**Presence on Muscle Biopsy of:*** Accumulated thin filaments* Intranuclear rods* Cores, fiber type disproportion* Zebra bodies NA Use the PP4 table (see supplementary file “PP4 table DYSF”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. Use the PP4 table (see supplementary file “PP4 table SGCB”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. Use the PP4 table (see supplementary file “PP4 table SGCG”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. Use the PP4 table (see supplementary file “PP4 table SGCD”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. Use the PP4 table (see supplementary file “PP4 table CAPN3”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. NA Use the PP4 table (see supplementary file “PP4 table SGCA”) to determine the appropriate PP4 strength level. Apply PP4 only once, for a patient meeting the highest possible strength level. When applied together, PP1 and PP4 cannot exceed 5 Bayesian pts (Supporting + Strong or Moderate + Moderate). If PP1\_Moderate is applied and the criteria for PP4\_Strong are also met, a downgraded PP4\_Moderate can be applied. MODY Probability Calculator (MPC) result ≥50% chance of testing positive [https://www.diabetesgenes.org/mody-probability-calculator/](https://www.diabetesgenes.org/mody-probability-calculator/)) AND negative HNF4A testing AND presence of at least one additional feature characteristic of HNF1A-MODY:* Antibody negative and/or persistent C-peptide after five years post-T1DM diagnosis* Response to low-dose sulfonyurea (SU) (extreme response- hypoglycemia)* Low hsCRP in patient with clinical diagnosis of T2DM* Biochemical/Molecular phenotypic evidence from patient cell lines* Hepatocellular adenomas Not Applicable: Not applicable since this criterion is accounted for under PS4. Not Applicable: The phenotype associated with MYOC variants is not highly specific and there is genetic heterogeneity. Not Applicable: Autosomal Dominant: do not use as breast cancer is a disease with multiple genetic etiology (genetic heterogeneity) and there are no features that can readily distinguish hereditary from sporadic causes.Autosomal Recessive: do not use as a separate line of evidence. Such evidence is built into the Ataxia Telangiectasia PM3|BP2 table * Abnormal tests that are consistent with VLCAD deficiency include deficient VLCAD enzyme activity in patient cells (leukocytes, fibroblasts, liver, heart, or skeletal muscle, or amniocytes), abnormal C14:1 acylcarnitine values from newborn screening (NBS), and abnormal acylcarnitine values from follow-up plasma analysis. * 2 points (See PP4 Table) NA NA NA 3 points based on any combination of the following. Two or more data types are recommended to reach moderate: • Low urine guanidinoacetate with or without low or low normal creatine (1 point) • Low plasma guanidinoacetate with or without low or low normal creatine (2 points) • Significantly decreased creatine peak in brain magnetic resonance spectroscopy (3 points) • AGAT enzyme activity \<5% of normal (3 points)Variant must meet PM2\_Supporting for PP4 to apply at any strength. 3 points based on any combination of the following. Two or more data types are recommended to apply PP4\_Moderate:* Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).* Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).* Significantly decreased creatine peak in brain magnetic resonance spectroscopy with or without visible guanidinoacetate peak (3 points).* GAMT enzyme activity \<5% of normal (3 points).* Variant must meet PM2\_Supporting for PP4 to apply at any strength. 3 or more points based on: * Elevated urine creatine/creatinine ratio on one occasion (1 point)* Elevated urine creatine/creatinine ratio on more than one occasion (2 points)* Significantly decreased creatine peak, with absent guanidinoacetate peak, if reported (3 points)* Deficient creatine uptake in cultured fibroblasts (\<10% of normal with \<125uM creatine) (3 points)Additional specifications:* Two or more data types are recommended for PP4\_Moderate.* An individual used to assign PP4, at any weight, cannot be also included for PS4 count. If multiple unrelated probands with the variant have been identified, it is recommended that the case with the highest PP4 points is assigned the appropriate weight for PP4, and the other cases are used for PS4.* Variant must meet PM2\_Supporting for PP4 to apply at any strength. NA NA NA NA NA NA Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Not applicable, see PS4. Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 Proband must meet hemophilia A phenotype criteria AND have full gene sequencing and deletion/duplication analysis. Not Applicable: Phenotypic specificity incorporated into PS2, PM6, PS4 * 8 phenotype points or more are required to use this code at the moderate strength for a single proband (see points list below).* Additionally, at least one **specific** criterion must be met (see below).* Do not include a proband with a suspected diagnosis of more than one retinal dystrophy. PS2_Very Strong NA NA Two proven OR four assumed OR one proven + two assumed de novo observations in a patient with the disease and no family history. 4 points per tables 5a and 5b:Examples: 2 proven de novo occurrences; OR 1 proven + 2 assumed de novo occurrences; OR4 assumed de novo occurrences. NA ≥Two patients meet the HDGC individual phenotype criteria w/ parental confirmation. NA ≥ 8 points Not Applicable: De novo variants are rarely reported in GAA (PMIDs 7981676, 27142047). The occurrence of de novo variants in GAA is not a mechanism of disease for Pompe disease, and the observation that a variant in GAA has arisen de novo does not support its causality. Any de novo variants will be assessed based on the variant type, functional evidence, and in trans data as described in these guidelines. NA Each proven de novo case, 2 points, each assumed de novo case, 1 point, ≥8 points De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. NA NA NA NA De novo (maternity confirmed or identical full mtDNA sequence) in a patient with the disease and no family history; with weighting per ClinGen SVI guidance Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase● Autosomal Dominant Disease: Do not use-Informative de novo occurrences have not yet been observed for autosomal dominant disease. As breast cancer is relatively common and occurs frequently as an apparently sporadic event, de novo is unlikely to ever be informative unless specific features of PALB2-related cancer predisposition are identified.● Autosomal Recessive Disease: Do not use - de novo occurrences are too rare to be informative at this time. In addition, in a biallelic state, de novo occurrences have an exceedingly low probability of being able to be confirmed as in trans because parental testing (and identification of one variant in each parent) is typically required without the use of long-range technologies. A single proband cannot be very strong evidence, but multiple probands can be combined to reach very strong (4+ points). Use proposed SVI point recommendations. See further instruction below. Total of 4 points. Use the SVI recommendations for de novo cases; 4 points. Use de novo guidance below to determine point value. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 4 points. Use proposed SVI point recommendations. See further instruction below. Total of 4 points. Use proposed SVI point recommendations. See further instruction below. Total of 4 points. Use proposed SVI point recommendations for “Phenotype highly specific for gene.” See de novo rule code guidance attached. Required 4 points. Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Use SVI-recommended point-based system with specifications for “Phenotype Consistency” per instructions. 4 Points. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). ≥ 4 _de novo_ scores. For curation of _de novo_ score see **Tables 1** and **2**. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 4 points. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 4 Points. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA ≥ 4.0 points Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 3 “Phenotype consistent with gene but not hightly specific and high genetic heterogeneity.” Confirmed _de novo_ with confirmed maternity worth 0.5 points per proband. Total 4.0 points to use at the very strong strength. _De novo_ (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.* For PS2, both maternity and paternity must be confirmed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years). Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, _etc._ can contribute to non-maternity. Cases and parents genotyped by trio whole exome sequencing are considered to have confirmed maternity and paternity.* The _de novo_ variant in question must be coding or flanking.* The PS2 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).![](https://lh6.googleusercontent.com/4dOzwji0rs6_gSczQOWVPcEOk-W6h4Ey-wBCZTtqRdLJizyKdB6AU08BvSlBsOQ0HOkPeRFn1xEL6ofBU7fc1B0awCfBVfKQYdboYFwdPZmyf1zH0vBaH4AE-eenxPhcIlpuAXes9kp9vlJQ9f-kcA) Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). ≥ 4 _de novo_ points Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). Use option 3 “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” (maximum 0.5 points/proband)* Total of 4 or more points required for Very Strong level Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:* (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.* (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.* (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). NA Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 1 “Phenotype highly specific for gene.” Confirmed _de novo_ with confirmed maternity worth 2.0 points per proband. Assumed _de novo_ with assumed maternity worth 1.0 points per proband. Total 4.0 points to use at the very strong strength. 4 Points. 4 Points. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. NA NA ≥ 4 _de novo_ points ≥ 4 _de novo_ points ≥ 4 _de novo_ points * Use PS2\_VeryStrong when there are four or more _de novo_ occurrences according to the attached Recommendation for determining the appropriate ACMG/AMP evidence strength level for de novo occurrences (Table 2).* The “phenotypic consistency” used on the attached Table 1 (Points awarded per _de novo_ occurrence) will be chosen for each proband by the number of phenotype points scored by the proband on the attached Phenotype scoring criteria per affected individual (Table 3):1. If the proband scores at least 4 and \<6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.2. If the proband scores at least 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.3. If the proband scores at least 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”.* PS2\_VeryStrong can only be applied if the variant does not meet BA or BS1. NA No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Use ClinGen Sequence Variant Interpretation (SVI) Review Committee recommended scoring system, attached “PS2.PM6\_Scoring”. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific” for probands with Stargardt disease. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” for probands with other ABCA4-related retinopathies (e.g. - retinitis pigmentosa, cone-rod dystrophy, etc.). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 4 or more points required for Very Strong level No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. NA NA Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase Not Applicable Four points 4 points per tables 5a and 5b:Examples: 2 proven de novo occurrences; OR1 proven + 2 assumed de novo occurrences; OR4 assumed de novo occurrences. ≥4 de novo points Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity. NA De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2.* Evidence from literature must be fully evaluated to support independent events. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2.* Evidence from literature must be fully evaluated to support independent events. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2.* Evidence from literature must be fully evaluated to support independent events. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2. De novo (maternity and paternity confirmed) in a patient with the disease and no family history.* ≥2 independent occurrences of PS2.* ≥2 independent occurrences of PM6 and one occurrence of PS2.* Evidence from literature must be fully evaluated to support independent events. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. 4 Points. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 points** will arrive at **Very Strong**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 points** will arrive at **Very Strong**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use the SVI recommendations for de novo cases; 4 points. Use de novo guidance below to determine point value. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **4 points** will arrive at **Very Strong**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 4 or more points required for Very Strong level PM6_Very Strong NA NA Two proven OR four assumed OR one proven + two assumed de novo observations in a patient with the disease and no family history. NA Not Applicable >Four patients meet the HDGC individual phenotype criteria w/o parental confirmation. NA Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: See explanation for PS2. Use proposed SVI point recommendations.* Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, ≥8 points Assumed de novo, but without confirmation of paternity and maternity. NA NA NA NA Assumed de novo, but without confirmation of maternity (maternal testing done by targeted variant analysis and/or targeted gene sequencing) Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Subsumed by PS2. Not Applicable: Subsumed in PS2. NA NA NA Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: See PS2. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA Not Applicable: Refer to PS2 Not Applicable: See PS2 for de novo data. NA NA NA Not Applicable: Please see PS2 NA NA Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. NA NA NA NA Not Applicable: Confirmation of maternity and paternity is required. Not Applicable: See PS2 for de novo data. NA NA NA Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: De novo occurrences without confirmation of paternity and maternity can still be counted under the PS2 code, using the point system shown in Tables 1 and 2. NA NA NA NA NA NA Not Applicable: Use the PS2 code for all (including non-confirmed paternity) de novo variants. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. NA NA Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Subsumed by PS2. Not Applicable: This point is addressed according to PS2 and will not be used. Not Applicable: Refer to PS2 Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA 4 points NA Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: Assumed de novo but without confirmation of paternity and maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with GAMT deficiency, to our knowledge. Furthermore, the observation that a variant in GAMT has arisen de novo does not support its causality because GAMT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GAMT, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. NA Confirmed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. Confirmed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. * Evidence from literature must be fully evaluated to support independent events. Assumed de novo without confirmation of paternity and maternity.* ≥4 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. NA Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. PM6_Strong NA NA Two probands with presumed _de novo_ occurrence (maternity/ paternity not confirmed) with the disease and no family history.* May also be used for a proband with presumed de novo occurrence for an individual with a highly specific phenotype (meets criteria to count towards PS4) NA Not Applicable ≥Two patients meet the HDGC individual phenotype criteria w/o parental confirmation. NA Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: See explanation for PS2. Use proposed SVI point recommendations. * Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 4-7 points Assumed de novo, but without confirmation of paternity and maternity. De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history De novo in a patient with the disease and no family history Assumed de novo, but without confirmation of maternity (maternal testing done by targeted variant analysis and/or targeted gene sequencing) Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Subsumed by PS2. Not Applicable: Subsumed in PS2. 2 Points. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 2-3.5 _de novo_ scores. For curation of _de novo_ score see **Tables 1** and **2**. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: See PS2. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 2 Points. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA Not Applicable: Refer to PS2 Not Applicable: See PS2 for de novo data. Assumed de novo, but without confirmation of paternity and maternity.* For PM6, maternity and paternity are not confirmed but assumed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years).* The _de novo_ variant in question must be coding or flanking.* The PM6 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).![](https://lh6.googleusercontent.com/4dOzwji0rs6_gSczQOWVPcEOk-W6h4Ey-wBCZTtqRdLJizyKdB6AU08BvSlBsOQ0HOkPeRFn1xEL6ofBU7fc1B0awCfBVfKQYdboYFwdPZmyf1zH0vBaH4AE-eenxPhcIlpuAXes9kp9vlJQ9f-kcA) Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Please see PS2 Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). * The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system: * (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”. * (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”. * (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Confirmation of maternity and paternity is required. Not Applicable: See PS2 for de novo data. 2 Points. 2 Points. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: De novo occurrences without confirmation of paternity and maternity can still be counted under the PS2 code, using the point system shown in Tables 1 and 2. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Use PS2/PM6 criteria described under PS2. Occurrences of de novo and presumed de novo are summed for a final PS2/PM6 evidence strength. Not Applicable: Use the PS2 code for all (including non-confirmed paternity) de novo variants. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. No change - use as originally described No change - use as originally described Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Subsumed by PS2. Not Applicable: This point is addressed according to PS2 and will not be used. Not Applicable: Refer to PS2 Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA 2-3 points. NA Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: Assumed de novo but without confirmation of paternity and maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with GAMT deficiency, to our knowledge. Furthermore, the observation that a variant in GAMT has arisen de novo does not support its causality because GAMT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GAMT, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. NA Confirmed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6. Confirmed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6.* Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6.* Evidence from literature must be fully evaluated to support independent events. Confirmed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6.* Evidence from literature must be fully evaluated to support independent events. Assumed de novo without confirmation of paternity and maternity.* ≥2 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. 2 Points. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Total of **4 points** will arrive at **Very Strong**. Dravet\*: 1 pointsGenetic Epilepsy with Febrile Seizures Plus: 0.5 pointsDevelopmental and Epileptic Encephalopathy: 0.5 pointsHemiplegic migraine: 0.25 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Total of **4 points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Total of **4 points** will arrive at **Very Strong**. Developmental and Epileptic Encephalopathy: 0.5 pointsOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Total of **4 points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **2 points** will arrive at **Strong**. Total of **4 points** will arrive at **Very Strong**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. BS1_Supporting NA NA Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%). MAF of ≥0.0007 (0.07%) for autosomal recessive. No BS1_Supporting criteria for autosomal dominant. NA NA NA NA NA NA NA Allele frequency is greater than expected for disorder. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Filter allele frequency (FAF) is above 0.002% (FAF > 0.00002) and less than or equal to 0.01% (FAF ≤ 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. NA NA Filter allele frequency (FAF) is above 0.002% (FAF > 0.00002) and less than or equal to 0.01% (FAF ≤ 0.0001) in gnomAD v2.1 (non-cancer, exome only subset) and/or gnomAD v3.1 (non-cancer), non-founder population(s). See Appendix G for details. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA >0.08% to 0.2% (based on gnomAD Popmax FAF). >0.08% to 0.2% (based on gnomAD Popmax FAF). NA NA NA NA NA NA NA NA NA NA Grpmax allele frequency cutoff of greater than 0.00163 in gnomAD. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MAF of ≥0.0007 (0.07%) for autosomal recessive. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS4_Supporting NA NA Lack of segregation in affected members of one family. NA NA NA NA NA Not Applicable NA Not Applicable: BS4 is not applicable. Phenotype for MHS is routinely determined based on the vitro contraction test (IVCT) that has a false positive rate of approximately 6% (PP1) or the caffeine-halothane contracture test (CHCT). As the phenotype in individuals who have not experienced an MH crisis cannot be reliably determined BS4 is not utilized. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. NA NA NA NA NA LOD ≤-.32 or Bayes Factor (LR) ≤.48 Lack of segregation is seen in 1 family. NA NA Appropriate to use when only one relative has the phenotype consistent with VWD type 2 without harboring the variant identified in other affected family members. Additionally, there is not another established cause of type 2 VWD (e.g. - there are not multiple type 2 VWD diagnoses) segregating in the family. NA NA Appropriate to use when the variant is found not to segregate in a minimum of two relatives with abnormal antithrombin activity levels \[\< 0.8 IU/mL (or below the lower limit of a laboratory’s assays reference range)\] within the same family. NA NA NA NA Affected member without the variant must score at least 0.5 phenotype points (see **Table 1**). Appropriate to use when only one relative has the phenotype consistent with VWD type 2 without harboring the variant identified in other affected family members. Additionally, there is not another established cause of type 2 VWD (e.g. - there are not multiple type 2 VWD diagnoses) segregating in the family. NA Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4\_Supporting - LR ≤0.48:1 NA NA Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4\_Supporting - LR ≤0.48:1 NA NA NA NA NA NA NA NA Lack of segregation in affected members of a family* BS4\_supporting is met by absence of the variant in 1 affected family member* The family member must have QTc greater than or equal to 480ms or Schwartz score >3 or syncope.* Please use the European Society of Cardiology guidelines for 2022 (PMID: 36017572, Table 10) to calculate a modified Schwartz score. NA NA Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48. NA NA NA NA * Due to incomplete penetrance (45-70%), BS4\_Supporting will require each family member to reach at least 6 points in the PS4 counting rubric in order to be considered affected for the purpose of counting lack of segregation.* If only 1 affected family member lacks segregation of the genotype, downgrade to BS4\_Supporting. NA NA NA Paternal inheritance is inconsistent with this gene. For RS1, unaffected males over age 10 who have been examined could be used to establish this code if they have no schisis and good acuity. NA NA NA NA NA Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48. Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48. \*with multiple pedigrees, results are combined. Recommended segregation analysis tool: COOL (COsegregation OnLine) v2 [http://fengbj-laboratory.org/cool2/manual.html](http://fengbj-laboratory.org/cool2/manual.html) * Caution in case the phenotype is not highly specific. The patient lacking segregation must reach 6 or more points of the phenotypic scoring system described in **Table 4.*** BS4\_Supporting is met if only 1 affected family member lacks segregation of the genotype. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Presence of the variant under review in an unaffected male of a family if the age of the unaffected male is at or above the age of onset for other affected males in the family. NA NA No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA NA Not Applicable: Not applicable as these are de novo, germline mosaic or post-zygotic mutations. Not Applicable: The presence of phenocopies, the reduced age-related penetrance and the possibility that more than one pathogenic variant can contribute to the phenotype observed in families make non-segregation difficult to assess in the context of MYOC and POAG. Not Applicable: AD Condition: Co-segregation analysis in low penetrance genes can lead to false positive results (PMID 32773770)AR Condition: Informative instances of lack of co-segregation in A-T families are too rare to be considered for weight at this time and can also be considered for BP2 if biallelic unaffected patients are observed in an A-T family. NA NA NA NA Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. NA Lack of segregation in affected members of a family.* Absent in a similarly affected family member Lack of segregation in affected members of a family.* Absent in a similarly affected family member Lack of segregation in affected members of a family.* Absent in a similarly affected family member Lack of segregation in affected members of a family.* Absent in a similarly affected family member Lack of segregation in affected members of a family.* Absent in a similarly affected family member Lack of segregation in affected members of a family.* Absent in a similarly affected family member. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies NA PS4_Very Strong NA NA Probands with specificity score ≥16 (see text). NA Not Applicable: This criterion is not applicable for PAH. For proband counting, use PM3 criterion. ≥Sixteen families meet HDGC criteria. NA ≥ 8 points Not Applicable: There are no case-control studies for Pompe disease. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA NA The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. Not Applicable Not Applicable Not Applicable Not Applicable NA NA **16+ points the PS4 cut-off and Proband Scoring Tables from a mix of any of the following phenotypes: specific, consistent and nonspecific.** NA ≥8 probands meet criteria described below Appropriate to use code when there are 8 or more probands that meet the laboratory phenotype of the PP4 definition for a specific VWD type 2 phenotype (i.e. – all probands must qualify for a clinical diagnosis of the same VWD type 2 phenotype based on laboratory criteria stated under PP4). NA NA Appropriate to use code when there 8 or more proband points that meet the defined antithrombin deficiency laboratory phenotype. NA NA NA Not Applicable: Does not apply. ≥ 16 phenotype points. For phenotype points curation see **Table 1**. Not Applicable: Use PM3 for proband counting. Not Applicable: There are no case-control studies for MPS1. As this is a recessive disorder, the prevalence of the variant in affected individuals may not be increased compared to controls (who could be heterozygous carriers). The number of patients with the variant will be addressed by the PM3 evidence code. NA NA NA NA NA NA NA NA NA NA Not Applicable: CYP1B1 variants cause autosomal recessive disorders associated with glaucoma. The number of probands with the variant will be addressed by PM3. Use of this code requires ≥ 8 probands and PM2\_Supporting must be met. NA Sum of case scores ≥8 points (see instructions below) Not Applicable Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable Not Applicable Not Applicable Not Applicable NA Not Applicable Not Applicable NA Use of this code requires 9 or more probands, each with retinoschisis. PM2\_Supporting must be met. NA NA Sum of case scores >16 points (see instructions below) NA NA Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. Not Applicable: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting. NA NA NA NA NA NA 8 additional male or female probands observed - must meet PP4 criteria and/or have documented hyperammonemia or metabolic decompensation under physiological stress to be counted NA Not Applicable Not Applicable: These specifications are only for autosomal recessively inherited ACTA1 variants. Please use PM3 for case counting. There are separate specifications for AD ACTA1 variants. Not Applicable: These specifications are only for autosomal recessively inherited RYR1 variants. Please usePM3 for case counting. There are separate specifications for AD RYR1 variants. NA NA NA NA NA NA NA NA Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant meets criteria for (PM2). Strength of evidence is determined by points according to (Table 2B). PS4\_VeryStrong ≥ 16 points. For PS4, for cases reported in the literature, we recommend assigning each one to the SINGLE category below that is associated with the highest point value (Table 2A). The total score obtained for all reported cases with a particular variant will determine the strength of PS4 assigned according to the scale (Table 2B)^*._PS4\_VeryStrong ≥ 16 points._^*Applicable if the variant is absent/rare from controls according to PM2 to ensure the variant is not simply present due to beinging common in the general population. NA NA Not Applicable NA Fisher Exact or Chi-Squared analysis shows statistical increase in cases over controls. NA Not Applicable: CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. Not Applicable: The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.CCDS VCEP notes for PS4:This rule is typically used for autosomal dominant disorders, with PM3 used as a case-counting mechanism for autosomal recessive conditions. * 4 independent male probands with elevated urine creatine/creatinine ratio on one occasion at minimum, in addition to any proband used for PP4.* Variant must meet PM2_Supporting criterion for PS4 to apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **16+ points** will arrive at **Very Strong**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **16+ points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **16+ points** will arrive at **Very Strong**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Present in multiple unrelated patients with consistent phenotypes and absent in controls. Points based system for each unrelated proband determined by phenotypic specificity. Total of **16+ points** will arrive at **Very Strong**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points ≥8 probands meet criteria described below Present in in multiple unrelated patients with consistent phenotype. Points based system for each unrelated proband determined by phenotypic specificity. Total of **16+ points** will arrive at **Very Strong**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 points Not Applicable PS2_Moderate NA NA NA 1 point per tables 5a and 5b:Examples: 1 proven de novo occurrence (phenotype consistent but not specific to gene); OR1 assumed de novo occurrence; OR 2 assumed de novo occurrences (phenotype/gene not specific). NA NA **PS2\_Moderate:** ≥ 2 proven _de novo_ occurrences (both maternity and paternity confirmed) in patients with FPD/AML phenotype. 2-3 points Not Applicable: De novo variants are rarely reported in GAA (PMIDs 7981676, 27142047). The occurrence of de novo variants in GAA is not a mechanism of disease for Pompe disease, and the observation that a variant in GAA has arisen de novo does not support its causality. Any de novo variants will be assessed based on the variant type, functional evidence, and in trans data as described in these guidelines. Use proposed SVI point recommendations. * Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 2-3 points De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. NA NA NA NA De novo (maternity confirmed or identical full mtDNA sequence) in a patient with the disease and no family history; with weighting per ClinGen SVI guidance Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase● Autosomal Dominant Disease: Do not use-Informative de novo occurrences have not yet been observed for autosomal dominant disease. As breast cancer is relatively common and occurs frequently as an apparently sporadic event, de novo is unlikely to ever be informative unless specific features of PALB2-related cancer predisposition are identified.● Autosomal Recessive Disease: Do not use - de novo occurrences are too rare to be informative at this time. In addition, in a biallelic state, de novo occurrences have an exceedingly low probability of being able to be confirmed as in trans because parental testing (and identification of one variant in each parent) is typically required without the use of long-range technologies. Phenotype consistent but not highly specific. Ex. VHL spectrum cancer without family history or strong indication of VHL phenotype. (≥1 but less than 2 _de novo_ points) Use proposed SVI point recommendations. See further instruction below. Total of 1 point. Use the SVI recommendations for de novo cases; 1 point. Use de novo guidance below to determine point value. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 1 point. Use proposed SVI point recommendations. See further instruction below. Total of 1 point. Use proposed SVI point recommendations. See further instruction below. Total of 1 point. Use proposed SVI point recommendations for “Phenotype highly specific for gene.” See de novo rule code guidance attached. Required 1 point. Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Use SVI-recommended point-based system with specifications for “Phenotype Consistency” per instructions. 1 Point. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 1-1.5 _de novo_ score. For curation of _de novo_ score see **Tables 1** and **2**. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. Use **“Phenotype highly specific for gene”** phenotype consistency if the proband meets **PP4\_Moderate criteria**. See Table 1 attached. Required 1 point. Variant occurs de novo in an affected individual, and the biological relationship of the parent without the variant is not confirmed. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 1 Point. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA ≥ 1.0 points Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 3 “Phenotype consistent with gene but not hightly specific and high genetic heterogeneity.” Confirmed _de novo_ with confirmed maternity worth 0.5 points per proband. Assumed de novo with assumed maternity worth 0.25 points per proband. Total 1.0 points to use at the moderate strength. _De novo_ (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.* For PS2, both maternity and paternity must be confirmed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years). Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, _etc._ can contribute to non-maternity. Cases and parents genotyped by trio whole exome sequencing are considered to have confirmed maternity and paternity.* The _de novo_ variant in question must be coding or flanking.* The PS2 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 1-1.5 _de novo_ points Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). Use option 3 “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” (maximum 0.5 points/proband)* Total of 1.00 - 1.75 points required for Moderate level Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:* (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.* (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.* (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). NA Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 1 “Phenotype highly specific for gene.” Confirmed _de novo_ with confirmed maternity worth 2.0 points per proband. Assumed _de novo_ with assumed maternity worth 1.0 points per proband. Total 1.0 points to use at the moderate strength. 1 Point. 1 Point. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. NA NA 1 - 1.5 _de novo_ points 1 - 1.5 _de novo_ points 1 - 1.5 _de novo_ points * Use PS2\_Moderate when there is one de novo occurrence according to the attached Recommendation for determining the appropriate ACMG/AMP evidence strength level for de novo occurrences (Table 2).* The “phenotypic consistency” used on the attached Table 1 (Points awarded per _de novo_ occurrence) will be chosen for each proband by the number of phenotype points scored by the proband on the attached Phenotype scoring criteria per affected individual (Table 3):1. If the proband scores at least 4 and \<6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.2. If the proband scores at least 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.3. If the proband scores at least 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”.* PS2\_Moderate can only be applied if the variant does not meet BA or BS1. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Use SVI recommended scoring system, attached “PS2.PM6\_Scoring”. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific” for probands with Stargardt disease. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” for probands with other ABCA4-related retinopathies (e.g. - retinitis pigmentosa, cone-rod dystrophy, etc.). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 1.00 - 1.75 points required for Moderate level No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. Award the PS2_Moderate point if Criteria 1 is fulfilled, OR if parents are not available but Criteria 2 is fulfilled. * ≥2 confirmed de novo in POAG* Or 1 confirmed de novo in JOAG* Or ≥2 assumed _de novo_ in JOAG Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase Not Applicable One point 1 point per tables 5a and 5b:Examples: 1 proven de novo occurrence (phenotype consistent but not specific to gene); OR1 assumed de novo occurrence; OR 2 assumed de novo occurrences (phenotype/gene not specific). ≥1 but less than 2 de novo points Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity. Newly hemizygous male with the variant identified de novo in the mother with no family history of other affected males. NA NA NA NA NA NA 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. 1 Point. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use the SVI recommendations for de novo cases; 1 point. Use de novo guidance below to determine point value. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **1 point** will arrive at **Moderate**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See PS2-Table 1 within PS2/PM6 file). * Total of 1.00 - 1.75 points required for Moderate level PS2_Supporting NA NA NA 0.5 points per tables 5a and 5b:Example: 1 assumed de novo occurrence (phenotype/gene not specific). NA NA **PS2\_Supporting**: 1 proven _de novo_ occurrence (both maternity and paternity confirmed) in a patient with FPD/AML phenotype. 1 point Not Applicable: De novo variants are rarely reported in GAA (PMIDs 7981676, 27142047). The occurrence of de novo variants in GAA is not a mechanism of disease for Pompe disease, and the observation that a variant in GAA has arisen de novo does not support its causality. Any de novo variants will be assessed based on the variant type, functional evidence, and in trans data as described in these guidelines. NA Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 1 point De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. NA NA NA NA De novo (maternity confirmed or identical full mtDNA sequence) in a patient with the disease and no family history; with weighting per ClinGen SVI guidance Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase● Autosomal Dominant Disease: Do not use-Informative de novo occurrences have not yet been observed for autosomal dominant disease. As breast cancer is relatively common and occurs frequently as an apparently sporadic event, de novo is unlikely to ever be informative unless specific features of PALB2-related cancer predisposition are identified.● Autosomal Recessive Disease: Do not use - de novo occurrences are too rare to be informative at this time. In addition, in a biallelic state, de novo occurrences have an exceedingly low probability of being able to be confirmed as in trans because parental testing (and identification of one variant in each parent) is typically required without the use of long-range technologies. Phenotype consistent but not highly specific (≥0.5 but less than 1 _de novo_ points) Ex. subject included in a VHL cohort, but specific information on tumor types is not provided. Use proposed SVI point recommendations. See further instruction below. Total of 0.5 point. Use the SVI recommendations for de novo cases; 0.5 point. Use de novo guidance below to determine point value. Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. If the proband meets PP4\_Moderate criteria, use a moderate or higher evidence weight (see above). See Table 1 attached. Required 0.5 point. NA Use proposed SVI point recommendations. See further instruction below. Total of 0.5 point. Use proposed SVI point recommendations for “Phenotype highly specific for gene.” See de novo rule code guidance attached. Required 0.5 point. Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Use SVI-recommended point-based system with specifications for “Phenotype Consistency” per instructions. NA Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). NA Use proposed SVI point recommendations for **“Phenotype consistent with gene but not highly specific”** if the proband meets **PP4 criteria**. If the proband meets PP4\_Moderate criteria, use a moderate or higher evidence weight (see above). See Table 1 attached. Required 0.5 point. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA ≥ 0.5 points Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 3 “Phenotype consistent with gene but not hightly specific and high genetic heterogeneity.” Confirmed _de novo_ with confirmed maternity worth 0.5 points per proband. Assumed de novo with assumed maternity worth 0.25 points per proband. Total 0.5 points to use at the supporting strength. _De novo_ (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.* For PS2, both maternity and paternity must be confirmed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years). Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, _etc._ can contribute to non-maternity. Cases and parents genotyped by trio whole exome sequencing are considered to have confirmed maternity and paternity.* The _de novo_ variant in question must be coding or flanking.* The PS2 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 0.5 _de novo_ points Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See Table 1 within PS2/PM6 file). Use option 3 “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” (maximum 0.5 points/proband)* Total of 0.50 - 0.75 points required for Supporting level Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system:* (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.* (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.* (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). NA Use SVI point scale for counting cases (see Table 1 on PS2\_PM6 table), use option 1 “Phenotype highly specific for gene.” Confirmed _de novo_ with confirmed maternity worth 2.0 points per proband. Assumed _de novo_ with assumed maternity worth 1.0 points per proband. Total 0.5 points to use at the supporting strength. NA NA Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. NA NA 0.5 _de novo_ points 0.5 _de novo_ points 0.5 _de novo_ points * Use PS2\_Supporting when there is one de novo occurrence according to the attached Recommendation for determining the appropriate ACMG/AMP evidence strength level for de novo occurrences (Table 2).* The “phenotypic consistency” used on the attached Table 1 (Points awarded per _de novo_ occurrence) will be chosen for each proband by the number of phenotype points scored by the proband on the attached Phenotype scoring criteria per affected individual (Table 3):1. If the proband scores at least 4 and \<6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”.2. If the proband scores at least 6 phenotype points in the PS4 counting rubric but lacks genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”.3. If the proband scores at least 10 phenotype points in the PS4 counting rubric AND has genotyping to rule out variants in the _PIK3R1_ locus, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”.* PS2\_Supporting can only be applied if the variant does not meet BA or BS1. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Use SVI recommended scoring system, attached “PS2.PM6\_Scoring”. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific” for probands with Stargardt disease. Use proposed SVI point recommendations for “Phenotype consistent with gene but not highly specific and high genetic heterogeneity” for probands with other ABCA4-related retinopathies (e.g. - retinitis pigmentosa, cone-rod dystrophy, etc.). De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See Table 1 within PS2/PM6 file). * Total of 0.50 - 0.75 points required for Supporting level No change - use as originally described No change - use as originally described Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Apply for confirmed _de novo_ occurrence in a proband meeting the criteria for PP4 (Supporting). Maternity and paternity should be confirmed by trio WES/WGS or other testing. Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. NA * 1 confirmed de novo in POAG* Or ≥2 assumed _de novo_ in POAG* Or 1 assumed _de novo_ in JOAG Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase Not Applicable 0.5 points 0.5 points per tables 5a and 5b:Example: 1 assumed de novo occurrence (phenotype/gene not specific). ≥0.5 but less than 1 de novo points Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, and so on, can contribute to non-maternity. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Dravet\*: 2 pointsGenetic Epilepsy with Febrile Seizures Plus: 1 pointDevelopmental and Epileptic Encephalopathy: 1 pointHemiplegic migraine: 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Developmental and Epileptic Encephalopathy: 1 pointOther phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 1 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Use the SVI recommendations for de novo cases; 0.5 point. Use de novo guidance below to determine point value. De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 1 pointOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.5 pointsNote: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. De novo (**both** maternity and paternity confirmed) in a patient with the disease and no family history.* Use ClinGen SVI's recommendation for assigning weight to the PS2/PM6 codes (See Table 1 within PS2/PM6 file). * Total of 0.50 - 0.75 points required for Supporting level BP7_Strong NA NA NA NA Applicable as described by Walker et al. (PMID: 36865205). NA NA A (synonymous) silent or intronic variant for which RNA splicing assay data demonstrates no splicing aberration, as per recommendations from Walker et al., 2023 (PMID: 37352859). NA NA NA A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. NA NA NA NA NA BP7\_Strong(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. NA NA Applicable for variants that have no observable splicing impact with RNA sequencing and/or minigene assay and a SpliceAI score of less than or equal to 0.1. NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA Experimental evidence (RT-PCR, RNA Seq, minigene) shows that the variant does not impact splicing. BP7\_Strong (RNA) will be used only under strict circumstances in which it is clear that the allele with the variant is expressed at the normal level to avoid counting a “normal” result because the allele with the variant is absent due to nonsense-mediated decay (Walker et al, PMID: 37352859). Well-established in vitro or in vivo functional studies shows no damaging effect on protein function _as measured by effect on mRNA transcript profile – mRNA assay only._ Apply as BP7\_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain. Missense variants located inside a (potentially) clinically important functional domain must meet BS3 to be eligible for BP7\_Strong (RNA). See Specifications Figure1B and Appendix E for details.As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA1 RING aa 2-101; BRCA1 coiled-coil aa 1391-1424; BRCA1 BRCT repeats aa 1650-1857. See Specifications Figure1A and Appendix J for details. NA NA Well-established in vitro or in vivo functional studies shows no damaging effect on protein function _as measured by effect on mRNA transcript profile – mRNA assay only._ Apply as BP7\_Strong (RNA) for intronic and silent variants, as well as missense/in-frame variants located outside a (potentially) clinically important functional domain. Missense variants located inside a (potentially) clinically important functional domain must meet BS3 to be eligible for BP7\_Strong (RNA). See Specifications Figure1B and Appendix E for details.As justified in the appendices, (potentially) clinically important functional domains are defined as: BRCA2 PALB2 binding domain aa 10-40; BRCA2 DNA binding aa 2481-3186. See Specifications Figure1A and Appendix J for details. NA NA NA NA NA NA NA NA NA NA NA NA NA NA BP7\_Strong (RNA) Used to designate capture of splicing data (not BS3). See RPE65-specific PVS1 Decision Tree, file attached, for weighting and combining with other codes. NA NA NA NA If BP7 is met and negative RNA splicing assay data is available, then apply BP7\_strong. Acceptable splicing assays should have positive and negative controls, preferably from patients and matched unaffected individuals. Note that splicing assay results may be tissue-sensitive. Applies to splicing assay data demonstrating a variant is not associated with aberrantly spliced transcript(s) relative to transcript profiles in controls. See RS1 PVS1 Decision Tree. As per Walker et al, 2023 (PMID 37352859) guidance. NA NA NA NA NA NA NA NA NA NA NA NA NA NA Applicable for potential splicing or intronic variants with experimental evidence demonstrating no splicing effect of the variant under review (BP7\_strong (RNA). NA BP7\_Strong (RNA) Used to designate capture of splicing data (not BS3). See GUCY2D-specific PVS1 Decision Tree, file attached, for weighting and combining with other codes. NA NA For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. For any variant that has been experimentally shown to have no splice impact, follow the SVI Working Group’s recommendations (Walker et al. 2023; PMID: 37352859). See supplementary file “Experimental splice data”. Apply BP7\_Strong if a splicing assay shows no effect on splicing and a protein impact can be ruled out. NA NA NA BP7\_Strong(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. BP7\_Strong (RNA) in vitro evidence of no splicing impact for intronic or synonymous variants irrespective of position and predicted impact on splicing. NA NA NA Experimental evidence, such as RT-PCR, shows no impact on splicing. Follow the decision tree outlined in Figure 5, Walker et al, 2023, PMID: 37352859. Note that splicing may appear normal in compound heterozygous patients with one allele that is degraded by nonsense-mediated decay as the results of a frameshift and premature termination codon due to splicing defect. Therefore. caution must be used in asessing the data prior to applying this code. Experimental evidence, such as RT-PCR, shows no impact on splicing. Follow the decision tree outlined in Figure 5, Walker et al, 2023, PMID: 37352859. Note that splicing may appear normal in compound heterozygous patients if the splicing defect generates a transcript that is degraded by nonsense-mediated decay. Therefore, caution must be used when assessing the data prior to applying this code. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Applicable for variants that have no observable splicing impact with RNA sequencing and/or minigene assay and a SpliceAI score of less than or equal to 0.1. NA BP7\_Strong (RNA) Used to designate capture of splicing data (not BS3). See AIPL1-specific PVS1 Decision Tree, file attached, for weighting and combining with other codes. PM6_Supporting NA NA NA NA Not Applicable NA _**RUNX1**_ **Specification:**Following the SVI guidance, assumed _de novo_ _RUNX1_ variants will be scored at the third tier of the point-based system with maximum allowable value of 1 point contributing to overall score:**PM6\_Supporting**: 2 or 3 assumed _de novo_ occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: See explanation for PS2. Use proposed SVI point recommendations. * Only applicable when proband has a known pathogenic or likely pathogenic variant with the de novo variant Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 1 point Assumed de novo, but without confirmation of paternity and maternity. NA NA NA NA Assumed de novo, but without confirmation of maternity (maternal testing done by targeted variant analysis and/or targeted gene sequencing) Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use PS2 for de novo cases in lieu of this rule code. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: Subsumed by PS2. Not Applicable: Subsumed in PS2. 0.5 Points. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). 0.5 _de novo_ scores. For curation of _de novo_ score see **Tables 1** and **2**. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Not Applicable: See PS2. Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. 0.5 Points. NA Not Applicable: BRCA1/2-related cancers occur relatively commonly. No information to calibrate the predictive capacity of de novo occurrences. NA NA NA NA NA NA Not Applicable: Refer to PS2 Not Applicable: See PS2 for de novo data. Assumed de novo, but without confirmation of paternity and maternity.* For PM6, maternity and paternity are not confirmed but assumed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years).* The _de novo_ variant in question must be coding or flanking.* The PM6 strength level depends on the clinical phenotype specificity and number of probands as defined in Table 1.* When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.* When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose \_KCNQ1\_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).![](https://lh6.googleusercontent.com/4dOzwji0rs6_gSczQOWVPcEOk-W6h4Ey-wBCZTtqRdLJizyKdB6AU08BvSlBsOQ0HOkPeRFn1xEL6ofBU7fc1B0awCfBVfKQYdboYFwdPZmyf1zH0vBaH4AE-eenxPhcIlpuAXes9kp9vlJQ9f-kcA) Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Please see PS2 Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). * The “phenotypic consistency” used on the SVI point-counting table below will be chosen for each proband by the number of phenotype points scored by the proband on the PS4 scoring system: * (A) If the proband scores greater than or equal to 4 and \<6 phenotype points in the PS4 counting rubric, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific and high genetic heterogeneity”. * (B) If the proband scores 6 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype consistent with gene but not highly specific”. * (C) If the proband scores 10 or more phenotype points in the PS4 counting rubric AND is not known to harbor biallelic _LRBA_ variants, use the number of _de novo_ points corresponding to “Phenotype highly specific for gene”. Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Use ClinGen SVI recommendations for _de novo_ criteria (see instructions below). Not Applicable: Confirmation of maternity and paternity is required. Not Applicable: See PS2 for de novo data. 0.5 Points. 0.5 Points. Use ClinGen SVI recommendations for _de novo_ criteria ([https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf](https://clinicalgenome.org/site/assets/files/3461/svi_proposal_for_de_novo_criteria_v1_1.pdf)).Phenotypic consistency determined using points-based system defined in PP4. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: De novo variants are rare in HHT. De novo variants should be confirmed not presumed for HHT. Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: Please see PS2 Not Applicable: De novo occurrences without confirmation of paternity and maternity can still be counted under the PS2 code, using the point system shown in Tables 1 and 2. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Use PS2/PM6 criteria described under PS2. Occurrences of de novo and presumed de novo are summed for a final PS2/PM6 evidence strength. Not Applicable: Use the PS2 code for all (including non-confirmed paternity) de novo variants. Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. No change - use as originally described No change - use as originally described Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Not applicable. See PS2. Not Applicable: Subsumed by PS2. Not Applicable: This point is addressed according to PS2 and will not be used. Not Applicable: Refer to PS2 Not Applicable: Do not use for AD or AR disease: Informative de novo occurrences have not yet been observed and de novo AR conditions are unlikely to be informed by phase NA 0.5 points. NA Not Applicable: Combined with PS2. Use PS2 instead of PM6. Not Applicable: CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with AGAT deficiency, to our knowledge. Furthermore, the observation that a variant in GATM has arisen de novo does not support its causality because AGAT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GATM, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. Not Applicable: Assumed de novo but without confirmation of paternity and maternity.CCDS VCEP notes for PS2 and PM6: De novo variants have not been reported in patients with GAMT deficiency, to our knowledge. Furthermore, the observation that a variant in GAMT has arisen de novo does not support its causality because GAMT deficiency is an autosomal recessive disorder. The occurrence of de novo variants is more supportive in autosomal dominant and X-linked disorders. Any de novo variants in GAMT, should they be observed, will be assessed based on the variant type, functional evidence, and in trans data as described. NA NA NA NA NA NA NA 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. 0.5 Points. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Dravet\*: 1 pointsGenetic Epilepsy with Febrile Seizures Plus: 0.5 pointsDevelopmental and Epileptic Encephalopathy: 0.5 pointsHemiplegic migraine: 0.25 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Developmental and Epileptic Encephalopathy: 0.5 pointsOther phenotypes not consistent w/neurodevelopmental disorder: 0 points Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. * Complex Neurodevelopmental Disorder: 0.5 points* Other phenotypes not consistent w/neurodevelopmental disorder: 0 points Not Applicable: This rule code is combined with PS2. Please combined assumed de novo cases with confirmed de novo cases and apply PS2 at the appropriate weight. Assumed de novo, but without confirmation of paternity and maternity. Points based system for each unrelated proband determined by phenotypic specificity. Total of **0.5 points** will arrive at **Supporting**. Genetic Epilepsy with Febrile Seizures Plus (GEFS+): 0.5 pointsOther epilepsy types or syndromes not included above, with or without associated neurodevelopmental features: 0.25 points Not Applicable: Use the PS2 code in lieu of using this code for de novo variants. PP3_Strong NA NA NA NA * Applicable as described in Pejaver et al (PMID: 36413997): REVEL score ≥0.932 for missense variants* PP3 + PM1 should not exceed Strong NA NA NA NA NA NA Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA For missense variants: REVEL score of ≥ 0.932 Applies to missense variants with a REVEL score above 0.932, that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2). This criteria is met at the strong level. NA NA NA NA NA NA NA NA NA NA NA NA Applies to missense variants with a REVEL score above 0.931, that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2). This criteria is met at the strong level. NA NA NA NA NA NA NA NA NA NA NA NA NA NA Missense variants - REVEL score ≥0.932 NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: This criterion is not applicable since these variants are GOF, and traditional mutation pathogenicity prediction algorithms focus on LOF mechanisms. Use of this criterion can be revisited if there emerges additional published experience with predictive algorithms specifically designed to detect gain of function mutations. REVEL score of ≥ 0.932 NA NA NA NA NA NA Missense variant with a REVEL score equal to or >0.932 (based on guidance from Pejaver et al, 2022, PMID: 36413997). NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA * For a missense variant use REVEL, requires a score of ≥0.093* Splice variants use PP3 only at Supporting level. PP3_Moderate NA NA NA NA Applicable as described in Pejaver et al (PMID: 36413997): REVEL score 0.773 - 0.932 for missense variants Variants affecting the same splice site as a well-characterized variant with similar or worse in silico/ RNA predictions. NA **Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_aGVGD Class C65 and BayesDel score ≥ 0.16 NA NA Multiple lines of computational evidence support a deleterious effect on the gene or gene product * Use REVEL score of >0.85 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant. NA NA NA NA NA NA NA REVEL score of ≥0.773 based on recommendations of Pejaver et al., 2022 (PMID: 36413997). NA NA REVEL score of ≥0.773 based on recommendations of Pejaver et al., 2022 (PMID: 36413997). REVEL score of ≥0.773 based on recommendations of Pejaver et al., 2022 (PMID: 36413997). NA NA NA NA NA NA NA Any missense changes with a REVEL score >0.773 will meet PP3\_Moderate (Based on Pejaver et al, PMID: 3641399; Note that the VCEP has chosen not to apply PP3 at strong). NA NA NA NA NA NA NA NA NA NA For missense variants: REVEL score of 0.773-0.931 Applies to missense variants with a REVEL score between 0.773 and 0.931, that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2). This criteria is met at the moderate level. NA Moderate evidence can be applied for a REVEL score of ≥0.932, downgraded from the recommendation of Strong in Pejaver et al., 2022 (PMID: 36413997). NA Missense variant with HCI prior probability for pathogenicity >0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) NA NA * For a missense variant use REVEL, requires a score of ≥ 0.774.* Splice variants use PP3 only at Supporting level. NA NA NA NA NA Applies to missense variants with a REVEL score between 0.773 and 0.931, that are not predicted to disrupt splicing (with a SpliceAI score less than 0.2). This criteria is met at the moderate level. NA NA NA NA NA Missense variant with HCI prior probability for pathogenicity >0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) Missense variant with HCI prior probability for pathogenicity >0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) Missense variant with “MAPP/PP2 Prior P” score >0.81 from [http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique](http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique) NA NA NA NA NA NA Missense variants - REVEL score ≥0.773 and \<0.932 This code is applicable for missense variants with a REVEL score of >0.772 or for synonymous or indel variants with a CADD score of greater or equal to 28.1 (https://www.medrxiv.org/content/10.1101/2023.04.24.23288782v1). This code is also applicable for variant with a SpliceAI score of greater than or equal to 0.8. * For a missense variant use REVEL, requires a score of ≥ 0.774.* Splice variants use PP3 only at Supporting level. NA NA NA NA NA NA NA NA NA NA Not Applicable: This criterion is not applicable since these variants are GOF, and traditional mutation pathogenicity prediction algorithms focus on LOF mechanisms. Use of this criterion can be revisited if there emerges additional published experience with predictive algorithms specifically designed to detect gain of function mutations. REVEL score of 0.773-0.931 NA NA NA NA NA Missense variant with a REVEL score >0.773 (based on guidance from Pejaver et al, 2022, PMID: 36413997). Missense variant with a REVEL score 0.773-0.932 (based on guidance from Pejaver et al, 2022, PMID: 36413997). NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong NA Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool, with the following stipulations:1. Strength should be capped at Moderate, and 2. limit the combination of PP3 and PM1 to reach no higher than strong * For a missense variant use REVEL, requires a score of 0.774 - 0.092* Splice variants use PP3 only at Supporting level. BP4_Strong NA NA NA NA Applicable as described in Pejaver et al. NA NA NA NA NA NA Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA For missense variants: SpliceAI ≤ 0.1 AND REVEL score of ≤ 0.016 Stand Alone criteria applies to:Missense variants: REVEL ≤ 0.003Synonymous variants or noncoding variants: SpliceAI ≤ 0.1Strong criteria applies to:Missense variants: REVEL between 0.004 and 0.016Synonymous variants or noncoding variants: SpliceAI ≤ 0.1 NA NA Not Applicable NA Not Applicable Not Applicable NA Not Applicable NA Not Applicable Not Applicable NA **Stand Alone** criteria applies to:Missense variants: REVEL ≤ 0.003 with SpliceAI ≤ 0.1**Strong criteria** applies to:Missense variants: REVEL between 0.004 and 0.016 with SpliceAI ≤ 0.1 NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA Missense variants - REVEL score ≤0.016 NA NA NA NA NA NA NA NA NA NA NA NA NA REVEL score of ≤ 0.016 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BP4_Moderate NA NA NA NA Applicable as described in Pejaver et al. NA NA **Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants):_BayesDel ≤ -0.008 irrespective of aGVGD score (except C65, in this case do not apply BP4\_Moderate) AND no predicted differences in splicing (SpliceAI \< 0.2) NA NA NA Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA For missense variants: SpliceAI ≤ 0.1 AND REVEL score of 0.017-0.183 Applies to:Missense variants: REVEL between 0.017 and 0.183Synonymous variants or noncoding variants: SpliceAI ≤ 0.1 NA NA Not Applicable NA Not Applicable Not Applicable For a missense variant use REVEL, requires a score of ≤0.183. In addition, highest SpliceAI delta score should also be below cutoff of 0.1. Not Applicable NA Not Applicable Not Applicable NA Applies to:Missense variants: REVEL between 0.017 and 0.183 with SpliceAI ≤ 0.1 NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA Missense variants - REVEL score >0.016 and ≤0.183 This code is applicable for missense variants with a REVEL score of \<0.184 or for synonymous or indel variants with a CADD score of less than or equal to 17.3 (PMID: 40225145). For a missense variant use REVEL, requires a score of ≤0.183. In addition, highest SpliceAI delta score should also be below cutoff of 0.1. NA NA NA NA NA NA NA NA NA NA NA REVEL score of 0.017-0.183 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)) using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. NA Follow ClinGen’s recommendations ([PMID: 36413997](https://pubmed.ncbi.nlm.nih.gov/36413997/)), using REVEL as the computational tool. For a missense variant use REVEL, requires a score of ≤0.183. In addition, highest SpliceAI delta score should also be below cutoff of 0.1. BP2_Very Strong NA NA NA NA Not Applicable NA NA Not Applicable: Not applicable NA NA NA NA NA Not Applicable NA NA NA Not Applicable: Do not use: See Fanconi Anemia BS2 table NA NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). NA NA NA NA NA NA Not Applicable: Does not apply. NA Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). Not Applicable Not Applicable: Applied only in the context of BS2. NA NA Not Applicable: Applied only in the context of BS2. NA NA NA NA NA NA Not Applicable: Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. Not Applicable: X-linked gene. NA NA Not Applicable Not Applicable: BS2 is used instead. Not Applicable Not Applicable NA Not Applicable Not Applicable: Do not use this criterion.BP2 would be more applicable to a very large, polymorphic gene, while it’s rarely going to come up for a short gene like CTLA4.Also, while biallelic cases have not yet been found, they exist for other IEI genes. Not Applicable Not Applicable NA Not Applicable: For this X-linked gene, only variants in cis with a pathogenic variant in RS1 in an affected male could be used, but these variants could have combined effect. The VCEP does not use this code. NA NA Not Applicable: Does not apply. NA NA Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BP2 is considered not applicable, as the field at present does not understand all of the potential allelic mechanisms associated with PIK3CD variants, so that the possibility of diverse combinatorial variant effects cannot be excluded. This has been described for other inborn errors of immunity genes. NA NA NA NA NA NA Not Applicable: Rule code does not apply because some modifier gene variants are in cis with known pathogenic variants. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Biallelic variants (either compound heterozygotes or homozygotes) have been reported (with variable phenotype) and are not incompatible with life. Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. NA NA NA NA NA NA NA Not Applicable NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. NA NA BP2_Strong NA NA NA NA Not Applicable Variant observed in trans w/known pathogenic variant (phase confirmed) OR observed in the homozygous state in individual w/o personal &/or family history of DGC, LBC, or SRC tumors. NA Not Applicable: Not applicable NA NA NA Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. NA Not Applicable NA NA NA Not Applicable: Do not use: See Fanconi Anemia BS2 table i) -variant observed in trans with a known pathogenic variant (phase confirmed), in the absence of congenital polycythemia (clinical manifestations or molecular)ii) -OR observed in the homozygous state in an individual without personal &/or family history of Von Hippel-Lindau disease or congenital polycythemiaiii) -OR observed _in cis_ or with unknown phase with three or more different pathogenic _VHL_ variants NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). NA NA NA NA NA ≥ (-4) Points. Not Applicable: Does not apply. NA Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). Not Applicable Not Applicable: Applied only in the context of BS2. ≥ (-4) Points. NA Not Applicable: Applied only in the context of BS2. NA NA NA NA NA NA Not Applicable: Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. Not Applicable: X-linked gene. NA NA Not Applicable Not Applicable: BS2 is used instead. Not Applicable Not Applicable NA Not Applicable Not Applicable: Do not use this criterion.BP2 would be more applicable to a very large, polymorphic gene, while it’s rarely going to come up for a short gene like CTLA4.Also, while biallelic cases have not yet been found, they exist for other IEI genes. Not Applicable Not Applicable NA Not Applicable: For this X-linked gene, only variants in cis with a pathogenic variant in RS1 in an affected male could be used, but these variants could have combined effect. The VCEP does not use this code. ≥ (-4) Points. ≥ (-4) Points. Not Applicable: Does not apply. NA NA Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BP2 is considered not applicable, as the field at present does not understand all of the potential allelic mechanisms associated with PIK3CD variants, so that the possibility of diverse combinatorial variant effects cannot be excluded. This has been described for other inborn errors of immunity genes. No change - use as originally described NA No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: Rule code does not apply because some modifier gene variants are in cis with known pathogenic variants. NA NA No change - use as originally described NA NA NA NA NA NA NA NA NA Not Applicable: Biallelic variants (either compound heterozygotes or homozygotes) have been reported (with variable phenotype) and are not incompatible with life. Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. BP2\_Strong **≤ -4** pointsSee ATM PM3/BP2 table for approach to assign points per proband. NA NA NA NA NA NA Not Applicable NA NA NA NA NA NA ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. ≥ (-4) Points. NA NA NA NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. NA NA BP2_Moderate NA NA NA NA Not Applicable NA NA Not Applicable: Not applicable NA NA NA Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. NA Not Applicable NA NA NA Not Applicable: Do not use: See Fanconi Anemia BS2 table NA NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). NA NA NA NA NA ≥ (-2) Points. Not Applicable: Does not apply. NA Not Applicable: Do not use due to potential of variant being associated with VWD 2N (recessive disease). Not Applicable Not Applicable: Applied only in the context of BS2. ≥ (-2) Points. NA Not Applicable: Applied only in the context of BS2. NA NA NA NA NA NA Not Applicable: Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. Not Applicable: X-linked gene. NA NA Not Applicable Not Applicable: BS2 is used instead. Not Applicable Not Applicable NA Not Applicable Not Applicable: Do not use this criterion.BP2 would be more applicable to a very large, polymorphic gene, while it’s rarely going to come up for a short gene like CTLA4.Also, while biallelic cases have not yet been found, they exist for other IEI genes. Not Applicable Not Applicable NA Not Applicable: For this X-linked gene, only variants in cis with a pathogenic variant in RS1 in an affected male could be used, but these variants could have combined effect. The VCEP does not use this code. ≥ (-2) Points. ≥ (-2) Points. Not Applicable: Does not apply. NA NA Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BS2 is used instead. Not Applicable: BP2 is considered not applicable, as the field at present does not understand all of the potential allelic mechanisms associated with PIK3CD variants, so that the possibility of diverse combinatorial variant effects cannot be excluded. This has been described for other inborn errors of immunity genes. No change - use as originally described NA No change - use as originally described No change - use as originally described No change - use as originally described NA Not Applicable: Rule code does not apply because some modifier gene variants are in cis with known pathogenic variants. NA NA No change - use as originally described NA NA NA NA NA NA NA NA NA Not Applicable: Biallelic variants (either compound heterozygotes or homozygotes) have been reported (with variable phenotype) and are not incompatible with life. Two missense variants in cis could act synergistically or the effect of a variant occurring after a truncating variant may not be predicted. BP2\_Moderate = **\-2** pointsSee ATM PM3/BP2 table for approach to assign points per proband. NA NA NA NA NA NA Not Applicable NA NA NA NA NA NA ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. ≥ (-2) Points. NA NA NA NA Not Applicable: Not being used at this time. There are reports of males with hemophilia having two suspicious pathogenic variants. NA NA PM1_Very Strong NA NA NA NA NA Not Applicable: Not applicable for CDH1. NA NA NA NA NA NA Not Applicable NA Not Applicable Not Applicable Not Applicable Not Applicable: Do not use: Missense pathogenic variation in PALB2 is not yet confirmed as a mechanism of disease. NA NA NA Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Rule does not apply due to gene being polymorphic. NA NA NA NA NA Not Applicable Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Not Applicable: Not applicable at this time. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For TPM1, there is evidence for gene-level enrichment of rare missense variants (see PP2 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For ACTC1, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL2, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL3, there is insufficient evidence for regional enrichment of rare missense variants. NA Not Applicable: See PM2_Supporting. NA NA Not Applicable Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: Does not apply. No known missense variation hot spots in the DCLRE1C gene have been described. See PVS1 for the note about a known hotspot for DCLRE1C deletion variants. Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608). Not Applicable NA NA NA NA NA NA NA NA Not Applicable: Not applicable for PPP1CB. NA NA NA Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: The PM1 code has been considered but does not apply to PIK3CD variant curation for this disease entity. Not Applicable: There are no defined hotspots or critical functional domains in NEB at this time. Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. Not Applicable: There are no defined hotspots or critical functional domains in DNM2 at this time. Not Applicable: There are no defined hotspots or critical functional domains in MTM1 at this time. NA NA Not Applicable: This code is not applicable as there is benign variation throughout the ABCA4 gene. NA Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. NA Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. NA NA Not Applicable: MYOC has no mutational hot spot and benign variants are present though the well-characterised olfactomedin domain in exon 3. Not Applicable: Do not use: Benign and pathogenic variants are known to occur within the same domains and germline mutational hotspots are not well defined at this time NA NA NA NA Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Not applicable NA Not Applicable: Not applicable for SLC9A6. NA NA NA NA Not Applicable: Not applicable. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Currently, insufficient numbers of pathogenic variants have been reported in SCN1B to calculate “mutational hotspots”. SCN1B does not belong to a gene family to utilize PERs. Not Applicable: Functional domains have not been definitively identified. PM1_Strong NA NA NA NA NA Not Applicable: Not applicable for CDH1. **PM1\_strong:** Variant affecting one of the following 13 AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. NA NA NA NA Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. Not Applicable NA Not Applicable Not Applicable Not Applicable Not Applicable: Do not use: Missense pathogenic variation in PALB2 is not yet confirmed as a mechanism of disease. NA NA This code can be used for variants affecting any of the 3 catalytic residues (H267, D315 or S411) and 2 activation residues (R191-A192 and R226-V227) in the _F9_ gene (PMID: 12554099). Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Rule does not apply due to gene being polymorphic. NA NA NA NA * Defined to include insertions/duplications between the TATA box (spanning n.-32 to n.-24) and the transcription start site (n.4). Caveat: All variants should be sufficiently rare - variant does not have to meet PM2 specification criteria but variant should not meet BS1/BA1 criteria (unless a suspected founder variant). The applicability of PM1 to suspected founder variants exceeding the BS1/BA1 threshold will be evaluated on a case-by-case basis by the VCEP. Not Applicable Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Not Applicable: Not applicable at this time. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For TPM1, there is evidence for gene-level enrichment of rare missense variants (see PP2 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For ACTC1, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL2, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL3, there is insufficient evidence for regional enrichment of rare missense variants. NA Not Applicable: See PM2_Supporting. NA NA Not Applicable Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: Does not apply. No known missense variation hot spots in the DCLRE1C gene have been described. See PVS1 for the note about a known hotspot for DCLRE1C deletion variants. Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608). Not Applicable NA NA NA NA NA Variant changes a _critical_ amino acid. Extracellular domain: p.Cys34, Cys60, Cys66, Cys84, Cys94, Cys99, Cys116, Cys117, Cys118, Cys123. Kinase domain: p.Gly210, Gly212, Lys230, Glu/Asn245, Asp333, Asn338, Asp351, Gly353 Glu386, Asp405, Gly410, Arg491. KD heterodimerization: p.Asp485, Gln486, Asp487, Ala488, Arg489, Ala490, Arg491, Leu492.Note that Gly182Asp and Met186Val have been demonstrated _non-critical/not necessary_ for kinase activity based on a luciferase assay (PMID: 18321866) (apply BS3). We note that Glu503Asp has demonstrated lack of of effect on canonical signaling in one assay, this has not been replicated in an independent assay; in the absence of further investigation, this variant cannot be conclusively designated non-critical (do not apply BS3). While tested variants in the extracellular domain (p.Gln42Arg, Gly47Asn, Gln82His, Thr102Ala, Ser107Pro) have limited or no effect on canonical signaling, studies have indicated that they may play a role in disruption of non-canonical/SMAD-independent pathways (PMIDs: 14583445 and 16002577). In the absence of further investigation, these variants cannot be conclusively designated non-critical (do not apply BS3). Applies to variants in amino acids p.Cys40, p.Cys59, p.Cys63, p.Cys110, p.Cys142, p.Cys219, and p.Cys223 which form disulfide bridges required for structure of the retinoschisin monomer, dimers, or octamers. NA Not Applicable: Not applicable for PPP1CB. Defined to include IL2RG-specific hot spots and functional domains. NA NA Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: The PM1 code has been considered but does not apply to PIK3CD variant curation for this disease entity. Not Applicable: There are no defined hotspots or critical functional domains in NEB at this time. Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. Not Applicable: There are no defined hotspots or critical functional domains in DNM2 at this time. Not Applicable: There are no defined hotspots or critical functional domains in MTM1 at this time. NA NA Not Applicable: This code is not applicable as there is benign variation throughout the ABCA4 gene. NA Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. NA Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. NA NA Not Applicable: MYOC has no mutational hot spot and benign variants are present though the well-characterised olfactomedin domain in exon 3. Not Applicable: Do not use: Benign and pathogenic variants are known to occur within the same domains and germline mutational hotspots are not well defined at this time NA * Cys residues in cbEGF-like domains* Add caveat: PM5/PS1 should not be used when this argument applies. NA NA Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Not applicable NA Not Applicable: Not applicable for SLC9A6. NA NA NA NA Not Applicable: Not applicable. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA This code can be applied at the strong level for variants involving the following residues: R391-S392, R759-S760, E1701-Q1705, R1708-S1709, Y1683, Y1689, Y737, Y742. Not Applicable: Currently, insufficient numbers of pathogenic variants have been reported in SCN1B to calculate “mutational hotspots”. SCN1B does not belong to a gene family to utilize PERs. Not Applicable: Functional domains have not been definitively identified. PM1_Supporting NA NA NA NA NA Not Applicable: Not applicable for CDH1. **PM1\_Supporting**: Variant affecting one of the other AA residues 89-204 within the RHD. Missense variants seen in cancerhotspots.org with 2-9 somatic occurrences for the same amino acid change. NA NA Located in a mutational hot spot and/or critical and well established functional domain.* Residues 1-552 (N-terminal region) and 2,101-2,458 (central region), if PS1/PM5 applicable then PM1 should be used at supporting * Residues 4,631-4,991 (C-terminal region). Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. Not Applicable NA Not Applicable Not Applicable Not Applicable Not Applicable: Do not use: Missense pathogenic variation in PALB2 is not yet confirmed as a mechanism of disease. Putative missense variants seen in somatic databases, having \<10 instances for the same AA in cancerhotspots.org. See the table of Germline and Somatic Hotspots. NA NA Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Rule does not apply due to gene being polymorphic. NA This criterion can be used for missense variants in well-conserved regions within the DNA and ligand-binding domains. It can also be used for variants within certain conserved transcription factor binding sites in the promoter (see below for details).* Promoter region: * c.-132 to c.-141 (HNF6/OC2 binding site) * c.-143 to c.-149, c.-151 (PDX1 (formerly IPF1) binding site) * c.-169, c.-174, c.-176, and c.-177 (HNF1A/HNF1B binding site)* DNA binding: * codons 37-113 (NM\_175914.4:c.175C-339C p.Leu37-Asp113) (While the paper describing the crystal structure of _HNF4A_[²](#pmid_18829458) shows the sequence as amino acids 33-113, amino acids 33-36 do not bind DNA and the conserved sequence starts as Leu37.)* Ligand binding: * codons 180-220 and 300-350 * (NM\_175914.4:c.538G-658G p.Ala180-Val220) * (NM\_175914.4:c.898T-1048G p.Tyr300-Glu350) NA NA NA Not Applicable Not Applicable: Rule does not apply due to benign variation being present throughout the gene. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). Not Applicable: Not applicable at this time. NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA Applicable to missense variants in _TNNT2_ in the specific regions listed below (Walsh _et al._ 2019[¹⁰](#pmid_30696458)). 1. Transcripts ENST00000367318 with codons 79-1792. ENST00000656932.1 and NM\_001276345.2 with codons 89-189Data from HCM case cohorts was used to derive these cluster regions. Therefore, this rule should NOT be applied when additional evidence for the variant supports that the variant causes a phenotype other than HCM (e.g., variant seen in multiple DCM cases).Enrichment was not observed for DCM in any genes.Rule should NOT be combined with PM5 because presence of pathogenic variants in the same codon/region were used to determine clustering and would be double-counting evidence. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For TPM1, there is evidence for gene-level enrichment of rare missense variants (see PP2 specifications). Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For ACTC1, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL2, there is insufficient evidence for regional enrichment of rare missense variants. Not Applicable: Application of this rule takes into consideration empirical data quantifying levels of rare missense variant enrichment in HCM referral cohorts compared to population-based cohorts (Walsh et al. 2019 PMID:30696458). For MYL3, there is insufficient evidence for regional enrichment of rare missense variants. Missense variants and in-frame indels in the G helix (aa253-282) or the I helix (339-365). Not Applicable: See PM2_Supporting. NA NA Not Applicable Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: Does not apply. No known missense variation hot spots in the DCLRE1C gene have been described. See PVS1 for the note about a known hotspot for DCLRE1C deletion variants. Note: Exons 1-3 and exons 1-4 have been reported as a hot spot for deletion variants as a result of homologous recombination of the wild-type DCLRE1C gene with a DCLRE1C pseudogene (PMID: 19953608). Not Applicable NA NA NA Strength is dependent upon the location of the variant within specific functional domains (PMID: 26996199):* PM1\_Supporting: missense variant located elsewhere in the **core domain** (amino acids 387-1011) Strength is dependent upon the location of the variant within specific functional domains (PMID: 26996199):* PM1\_Supporting: missense variant located in the **core domain** (amino acids 1-383); NA NA NA Not Applicable: Not applicable for PPP1CB. NA NA NA Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data). Not Applicable: The PM1 code has been considered but does not apply to PIK3CD variant curation for this disease entity. Not Applicable: There are no defined hotspots or critical functional domains in NEB at this time. Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. Not Applicable: There are no defined hotspots or critical functional domains in DNM2 at this time. Not Applicable: There are no defined hotspots or critical functional domains in MTM1 at this time. NA NA Not Applicable: This code is not applicable as there is benign variation throughout the ABCA4 gene. * Met by variants not explicitly listed above, encoding missense substitutions between positions p.873-951. This region forms part of the guanylate cyclase catalytic domain and exhibits intolerance to missense variation [¹⁴](#pmid_31704230).* Met by variants in the 6 bp CRX binding site located at position 17:8002593-8002598 (hg38) [¹⁸](#pmid_36084042) Not Applicable: There are no defined hotspots or critical functional domains in ACTA1 at this time. NA Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Not Applicable: Not applicable at this time. Use for defined regions in the DNA binding and dimerization domains.* Dimerization: codons 1-32, NM\_000545.8* Subset of DNA binding domains: codons 107-174 and 201-280It can also be used for variants within certain transcription factor binding sites of the promoter:* –c.-187 to c.-195 (AP1 binding site)* –c.-209 to c.-227 (Overlapping HNF3 & NF-Y sites)* –c.-238 to c.-259 (HNF1A binding site)* –c.-276 to c.-288 (HNF4A binding site) Residues affecting critical functional domains provided in Table 4 for each gene. Not Applicable: MYOC has no mutational hot spot and benign variants are present though the well-characterised olfactomedin domain in exon 3. Not Applicable: Do not use: Benign and pathogenic variants are known to occur within the same domains and germline mutational hotspots are not well defined at this time NA NA NA Putative missense variants at residues in the RNase IIIb domain (p.Y1682 – p.S1846), besides the metal ion-binding residues (see PM1). Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. Not Applicable: Not applicable NA Not Applicable: Not applicable for SLC9A6. NA NA NA NA Not Applicable: Not applicable. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Currently, insufficient numbers of pathogenic variants have been reported in SCN1B to calculate “mutational hotspots”. SCN1B does not belong to a gene family to utilize PERs. Not Applicable: Functional domains have not been definitively identified. PS1_Very Strong NA NA NA NA NA Not Applicable: Not applicable for CDH1. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PS1_Moderate NA NA NA NA NA Not Applicable: Not applicable for CDH1. **PS1\_Moderate:** Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change.**For splice site variants:** **PS1\_Variable:** Follow recommendations from the ClinGen SVI Splicing Subgroup (Walker et al., 2023, PMID: 37352859 [⁵](#PMID_37352859)) Can be applied to variants asserted as Likely Pathogenic following the _TP53_ VCEP’s specifications. NA NA Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change.* Previously established likely pathogenic variant must reach a classification of likely pathogenic without PS1. Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. NA NA NA NA NA Use PALB2 PS1 Splicing table NA Use as originally specified, but the comparison variant must reach a likely pathogenic classification using these rule specifications in order to apply code. This evidence code can be applied when there is 1 likely pathogenic variants at the same residue based on _F9_ gene rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggest a splicing defect.**OR**When the comparison variant shares the same predicted splicing effect and the comparison splicing variant reaches a likely pathogenic classification using the Coagulation Factor Deficiency VCEP specifications based on Walker, et al 2023 (PMID: 37352859). Use with no specification except comparison variant must be classified as likely pathogenic using rules from the VWD VCEP. Use as originally specified, but the comparison variant must reach a likely pathogenic classification using the these rule specifications in order to apply code. Use as originally specified, but the comparison variant must reach a likely pathogenic classification using the these rule specifications in order to apply code. Use with no specification except comparison variant must be classified as likely pathogenic using _SERPINC1_ rule specifications from the Thrombosis VCEP. Applicable for a same amino acid change if the previously established variant is classified as likely pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. Applicable for a same amino acid change if the previously established variant is classified as likely pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. NA NA The previously established variant was classified as Likely Pathogenic according to the APC-specific modifications.This criterion can be applied to both missense and splice variants in _APC_. **Missense variants:** when the variant under assessment results in the same amino acid change as previously established Likely Pathogenic variant(s). There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other variants leading to the same missense change at these positions meet PS1\_Moderate. No missense variant has been classified as Pathogenic based on current evidence. **Splice variants**: when the variant under assessment affects splicing at the same nucleotide as a previously established Likely Pathogenic variant. The splice prediction must be above defined thresholds (see instructions) or similar to the previously established variant by multiple _in silico_ predictors. Use with no specification except comparison variant must be classified as likely pathogenic using rules from the VWD VCEP. Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change. Splice region variants following Table 3 in Walker et al (PMID: 37352859). Apply **PS1\_Moderate**, for predicted **missense** substitutions, where previously classified **likely pathogenic** variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI≤0.1)).Apply **PS1\_Moderate**, for exonic and intronic variants with same predicted impact on **splicing**, as a previously classified **(likely) pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant.See Specifications Table 5 and Appendix E, J and K for details. NA NA Apply **PS1\_Moderate**, for predicted **missense** substitutions, where a previously classified **likely pathogenic** variant is considered to act via protein change (no confirmed or predicted effect on mRNA splicing (SpliceAI≤0.1)).Apply **PS1\_Moderate**, for exonic and intronic variants with same predicted impact on **splicing**, as a previously classified **(likely) pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant.See Specifications Table 5 and Appendix E, J and K for details. NA NA NA NA NA NA For missense variants that do not affect splicing (SpliceAI ≤ 0.2): same amino acid change as a previously established likely pathogenic variant.For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.[⁶](#PMID_37352859) Same amino acid change as a previously established Likely Pathogenic variant regardless of nucleotide change.* Comparison variant must have been evaluated by the X-linked IRD VCEP using these rules and established as likely pathogenic variant and classified as such.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other.Same predicted splicing impact as a previously classified Pathogenic variant.* Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the same position that has been designated Likely Pathogenic.* Used in conjunction with PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and that have a comparable variant within the same splice donor/acceptor motif (but outside of the +/-1,2 dinucleotide) that has been designated Pathogenic. Specific combinations are found in \_RPGR\_-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). * PS1 is met at the PS1\_Moderate level if the comparison variant reaches a likely pathogenic classification using these rule specifications, without the use of the PS1 code.* This code can also be met at the PS1\_Moderate level when the corresponding variant in the paralogous KCNQ2 gene meets criteria to be classified as Pathogenic by the KCNQ Channel Brain Disorders Variant Curation Expert Panel specifications. Curators should access paralogue data and regions of alignment and non-alignment between KCNQ1 and KCNQ2 at the site below: [https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1.](https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1.)* The corresponding variant in the paralogous gene must substitute the same amino acid as the variant being considered, not a different amino acid.* An example is that the NM\_000218.3(KCNQ1):c.430A>G (p.Thr144Ala) has a paralogous variant in NM\_172107.4(KCNQ2):c.340A>G (p.Thr114Ala). If the latter variant in _KCNQ2_ is classified Pathogenic in the future by the KCNQ Channel Brain Disorders Variant Curation Expert Panel specifications, the paralogous p.Thr144Ala variant in _KCNQ1_ will meet PS1\_Moderate.* The paralogous variant must have reached Pathogenic classification without the use of the PS1 code.* While this paralogue-based strategy has been approved for _KCNQ2_ only, this criterion may eventually apply to the _KCNQ3_, _KCNQ4_, and _KCNQ5_ genes too, following future specifications and variant classifications by the KCNQ Channel Brain Disorders Variant Curation Expert Panel. Applicable for a same amino acid change if previously established variant is classified as likely pathogenic by SCID VCEP specifications for _FOXN1._Can also be applied for variants with the same predicted splicing event as a known (Likely) Pathogenic variant (as classified by the SCID VCEP specifications for _FOXN1_), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: [37352859](https://pubmed.ncbi.nlm.nih.gov/37352859)) for determining when PS1 should be applied at PS1\_Strong, \_Moderate, or \_Supporting. It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _ADA._ A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change as a previously established Likely Pathogenic missense variant with normal RNA result\*, and PM2\_supporting is met.\*Otherwise, if the previously established Likely Pathogenic missense variant truly is a splice defect, the new missense variant also has to be investigated on a functional level for RNA splicing.ORVariants affecting the same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _DCLRE1C._ It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _IL7R._ Same amino acid change as a previously established Likely Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification.* For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other.Same predicted splicing impact as previously classified Likely Pathogenic variant.* Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the **same position** that has been designated **Likely Pathogenic**.* Used in conjunction with PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and have a comparable variant **within the same splice donor/acceptor motif** (but outside the +/-1,2 dinucleotide) that has been designated **Pathogenic**.Specific combinations are found in RPE65-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _JAK3._ * Use PS1\_Moderate for missense variants when the other variant was classified likely pathogenic by VCEP standards without using PS1.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Splicing predictions (by SpliceAI) should remain the same for WT and both mutant alleles.* Use at the PS1\_Moderate level for splicing variants in combination with **PP3** if located **outside** the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 with a comparable nucleotide variant **at the same position** that has been classified **likely pathogenic** by VCEP standards (PMID: 37352859).* Use at the PS1\_Moderate level for splicing variants in combination with **PP3** if located **outside** the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 with a comparable variant **within the same splice region** that has been classified **pathogenic** by VCEP standards (PMID: 37352859).* Use at the PS1\_Moderate level for splicing variants in combination with **PVS1 (at reduced strength)** if located **within** the splice donor/acceptor +/-1,2 dinucleotide positions with a comparable variant **within the same splice donor/acceptor motif (but outside the +/-1,2 dinucleotide)** that has been classified **pathogenic** by VCEP standards (PMID: 37352859). It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _RAG1._ It can also be applied for splice variants at the same nucleotide and with similar impact prediction as previously reported pathogenic variant (if the predicted impact is equal to or greater than the known pathogenic variant per in silico splicing tool SpliceAI). - Example: c.105+1G>C is known to be likely pathogenic, can use PS1 for c.105+1G>TApplicable if the previously established variant is classified as **likely pathogenic** by SCID VCEP specifications for _RAG2._ Same amino acid change as a previously established _**likely pathogenic**_ variant regardless of nucleotide change. For splice variants outside splice donor/acceptor ± 1,2 dinucleotide positions but residing in the same splice donor motif (last 3 bases of the exon and intronic positions +3 to +6) or acceptor motif (first base of the exon and intronic positions -20 to -3) as a previously established _**pathogenic**_ variant. The splice motif coordinates are as defined by Walker et al 2023 (PMID: 37352859). Same amino acid change as a previously established **(Likely) Pathogenic** variant. Comparison variant must have been evaluated by the X-linked IRD VCEP using these rules and established as a Likely Pathogenic variant and classified as such. For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other. When evaluating splicing impact, with a previously classified Likely Pathogenic or Pathogenic variant, * Use in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and have a comparable nucleotide variant at the same position that has been designated **Likely Pathogenic**.* Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and have a comparable nucleotide variant within the same splice region with the same predicted impact that has been designated **Pathogenic**.* Use in conjunction with PVS1 at any strength for variants located within splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and a comparable nucleotide variant within the same splice motif region but outside the donor/acceptor +/-1,2 dinucleotide positions with the same predicted impact that has been designated **Pathogenic**.Specific combinations are found in RS1-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). NA NA Strength modification depending upon classification of previously established variant (pathogenic vs. likely pathogenic).Previously established variant must be classified using the SCID VCEP specifications for _IL2RG._ NA NA A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change as a previously established Likely Pathogenic missense variant with normal RNA result\*, and PM2\_supporting is met.\*Otherwise, if the previously established Likely Pathogenic missense variant truly is a splice defect, the new missense variant also has to be investigated on a functional level for RNA splicing.ORVariants affecting the same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change as a previously established Likely Pathogenic missense variant with normal RNA result\*, and PM2\_supporting is met.\*Otherwise, if the previously established Likely Pathogenic missense variant truly is a splice defect, the new missense variant also has to be investigated on a functional level for RNA splicing.ORVariants affecting the same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change as a previously established Likely Pathogenic missense variant with normal RNA result\*, and PM2\_supporting is met.\*Otherwise, if the previously established Likely pathogenic missense variant truly is a splice defect, the new missense variant also has to be investigated on a functional level for RNA splicing.ORVariants affecting the same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. * Use at the PS1\_Moderate level for missense variants when other variant was classified as Likely Pathogenic for autosomal dominant PIK3CD gain-of-function-related disease by Antibody Deficiencies VCEP specifications for _PIK3CD_ without using PS1.* Beware of changes that impact splicing rather than the amino acid (based on RNA data or splicing predictors). Neither change should be predicted to affect splicing (SpliceAI Δ score \<0.2). No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described Applicable when the previously reported variant is classified as Likely Pathogenic using these OTC specifications. Comparison variant must reach a likely pathogenic classification using the _ABCA4_ VCEP specifications. Do not apply if the comparison variant is suspected to cause a splicing defect via SpliceAI or other splice predictor.**OR**Comparison splicing variant must reach a likely pathogenic classification using the _ABCA4_ VCEP specifications and both variants must share the same predicted splicing effect. **OR**See Walker, et al 2023 (PMID: 37352859) Figure 5 to use this rule code for variants with RNA sequencing data. Same amino acid change as a previously established Likely Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification.Same predicted splicing impact as previously classified Likely Pathogenic variant.* Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant at the **same position** that has been designated **Likely Pathogenic**.* Used in conjunction with PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and have a comparable variant **within the same splice donor/acceptor motif** (but outside the +/-1,2 dinucleotide) that has been designated **Pathogenic**.Specific combinations are found in GUCY2D-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). No change - use as originally described No change - use as originally described For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For missense variants for which the amino acid change is the expected mechanism of disease, apply at Moderate for 1 likely pathogenic variant resulting in the same amino acid change. The likely pathogenic variant must have been classified using LGMD VCEP specifications, and potential splice effects must be excluded for the missense variant under curation and the variant resulting in the same amino acid change (SpliceAI score ≤0.10 or experimental evidence of normal splicing). PS1 can potentially be applied to multiple nucleotide changes at the same residue as long as the variant classification that determines the strength level does not depend on PS1 application.For missense variants encoded by the first or last 3 nucleotides of an exon, PS1 should be considered only in the context of altered splicing (see below), unless a splice effect has been experimentally ruled out for the variant under curation and the variant(s) resulting in the same amino acid change. For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". Applicable for a same amino acid change if the previously established variant is classified as likely pathogenic by ClinGen MDEP specifications.PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances.[³](#pmid_37352859) NA Same amino acid change as a previously established likely pathogenic variant Use ATM PS1 Splicing table for splicing variants with similar predictions or observations of splice defect. * PS1\_Moderate: Same amino acid change as a previously established likely pathogenic variant classified using the ACADVL specifications without application of PS1, regardless of nucleotide change. * Caveat (from ACMG/AMP guidelines): Assess the possibility that the variant may act directly through the DNA change (e.g. through splicing disruption as assessed by at least computational analysis) instead of through the amino acid change.* Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al., (2023) PMID: 37352859). * PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker et al., (2023) PMID: 37352859. NA NA NA * This criterion is applicable as for any variant resulting in the same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.* PS1\_Moderate may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). * This criterion is applicable for any variant resulting in the same amino acid change as a variant that has been previously established as likely pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.* If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.* PS1\_Moderate may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Likely Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Likely Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Likely Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* Same amino acid change as a previously established **Likely Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). This evidence code can be applied when there is 1 likely pathogenic variants at the same residue based on _F8_ gene rule specifications from the Coagulation Factor Deficiency VCEP and where _in silico_ predictors do not suggesting a splicing defect.**OR**When the comparison variant shares the same predicted splicing effect and the comparison splicing variant reaches a likely pathogenic classification using the Coagulation Factor Deficiency VCEP specifications based on Walker, et al 2023 (PMID: 37352859). Same amino acid change as a previously established **Likely Pathogenic** variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same amino acid change as a previously established Likely Pathogenic variant regardless of nucleotide change.* Must have one comparison variant that reaches a Likely Pathogenic classification using this rule specification.Same predicted splicing impact as previously classified variant.* Refer to the AIPL1-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023) for specific combinations. PS1_Supporting NA NA NA NA NA Not Applicable: Not applicable for CDH1. NA NA NA NA NA Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. NA NA NA NA NA Use PALB2 PS1 Splicing table NA NA NA NA NA NA NA PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances. NA Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.* Downgraded to PS1\_Supporting.* Applicable if a different nucleotide change at the same nucleotide position has been previously classified as pathogenic or likely pathogenic.* Cannot be applied if a different nucleotide change at the same position has been previously classified as benign or likely benign.* Previously established variants must be classified by SCID VCEP specifications for _RMRP_. NA NA Splice region variants following Table 3 in Walker et al (PMID: 37352859). Apply **PS1\_Supporting**, for exonic and intronic variants with same predicted impact on **splicing,** as a previously classified **(likely) pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant.See Specifications Table 5 and Appendix E, J and K for details. NA NA Apply **PS1**, for exonic and intronic variants with same predicted impact on **splicing**, as a previously classified **(likely) pathogenic** variant. Vary weight depending on relative positions, and confidence in classification of the reference variant.See Specifications Table 5 and Appendix E, J and K for details. NA NA NA NA NA NA For variants that affect splicing (SpliceAI > 0.2), refer to Table 3.[⁶](#PMID_37352859) * Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant within the same motif that has been designated Likely Pathogenic.* Used in conjunction with PVS1 or PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and have a comparable Likely Pathogenic or Pathogenic variant either within the same splice site donor/acceptor motif, outside the +/-1,2 dinucleotide, or at the +/-1,2 dinucleotide.Specific combinations are found in \_RPGR\_-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). NA Can be applied for variants with the same predicted splicing event as a known (Likely) Pathogenic variant (as classified by the SCID VCEP specifications for _FOXN1_), only when the strength of the prediction for the variant under assessment is of similar or higher strength than the strength of the prediction for the comparison (Likely) Pathogenic variant (i.e., per in silico splicing tool SpliceAI). See attached instructions (from Table 2 of PMID: [37352859](https://pubmed.ncbi.nlm.nih.gov/37352859)) for determining when PS1 should be applied at PS1\_Strong, \_Moderate, or \_Supporting. NA NA NA NA * Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant within the **same motif** that has been designated **Likely Pathogenic**.* Used in conjunction with PVS1 or PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and have a comparable Likely Pathogenic or Pathogenic variant either within the same splice site donor/acceptor motif, outside the +/-1,2 dinucleotide, or at the +/-1,2 dinucleotide. Specific combinations are found in RPE65-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). NA * Use at the PS1\_Supporting level for splicing variants in combination with **PP3** if located **outside** the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 with a comparable nucleotide variant **within the same motif** that has been classified l**ikely pathogenic** by VCEP standards (PMID: 37352859).* Use at the PS1\_Supporting level in combination with **PVS1** for splicing variants **within** splice donor/acceptor +/-1,2 dinucleotide positions with a variant at the **same nucleotide position** or **within the same donor/acceptor +/-1,2 dinucleotide** with the same predicted impact that has been classified **pathogenic** by VCEP standards (PMID: 37352859).* Use at the PS1\_Supporting level in combination with **PVS1** for splicing variants **within** splice donor/acceptor +/-1,2 dinucleotide positions with a variant **within the same splice region, but outside the donor/acceptor +/-1,2 dinucleotide**, with the same predicted impact that has been classified **pathogenic or likely pathogenic** by VCEP standards (PMID: 37352859).* Use at the PS1\_Supporting level in combination with **PVS1 (at reduced strength)** for splicing variants **within** splice donor/acceptor +/-1,2 dinucleotide positions with a variant **within the same splice region, but outside the donor/acceptor +/-1,2 dinucleotide**, with the same predicted impact that has been classified **likely pathogenic** by VCEP standards (PMID: 37352859). NA NA For splice variants outside splice donor/acceptor ± 1,2 dinucleotide positions, same splice donor motif (last 3 bases of the exon and intronic positions +3 to +6) or acceptor motif (first base of the exon and intronic positions -20 to -3) as a previously established _**likely**_ _**pathogenic**_ variant. The splice motif coordinates are as defined by Walker et al 2023 (PMID: 37352859). Same amino acid change as a previously established **(Likely) Pathogenic** variant. Comparison variant must have been evaluated by the X-linked IRD VCEP using these rules and established as a Likely Pathogenic variant and classified as such. For assessing same amino acid changes, SpliceAI scores for both variants should be within 10% of each other. When evaluating splicing impact, with a previously classified Likely Pathogenic variant, * Use in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and have a comparable nucleotide variant variant within the same splice motif region but outside the donor/acceptor +/-1,2 dinucleotide positions with the same predicted impact that has been designated **Likely Pathogenic**.* Used in conjunction with PVS1 for variants located within the splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and have a comparable nucleotide variant at the same splice donor/acceptor +/-1,2 dinucleotide position with the same predicted impact that has been designated **Pathogenic**.* Use in conjunction with PVS1 for variants located within splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and a comparable nucleotide variant within the same splice motif region but outside the donor/acceptor +/-1,2 dinucleotide positions with the same predicted impact that has been designated either **Likely Pathogenic** or **Pathogenic**.* Use in conjunction with PVS1 at any strength for variants located within splice donor/acceptor +/-1,2 dinucleotide positions that have a SpliceAI score ≥0.2 and a comparable nucleotide variant within the same splice motif region but outside the donor/acceptor +/-1,2 dinucleotide positions with the same predicted impact that has been designated **Likely** **Pathogenic**.Specific combinations are found in RS1-specific PVS1 Decision Tree part (b) (Table 2 from Walker 2023). NA NA NA NA NA NA NA NA NA No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA See Walker, et al 2023 (PMID: 37352859) Figure 5 to use this rule code for variants with RNA sequencing data. * Used in conjunction with PP3 for variants located outside the splice donor/acceptor +/-1,2 dinucleotide positions that have a splice AI score ≥0.2 and have a comparable nucleotide variant within the **same motif** that has been designated **Likely Pathogenic**.* Used in conjunction with PVS1 or PVS1\_(reduced strength) for variants located at the splice donor/acceptor +/-1,2 dinucleotide positions and have a comparable Likely Pathogenic or Pathogenic variant either within the same splice site donor/acceptor motif, outside the +/-1,2 dinucleotide, or at the +/-1,2 dinucleotide. Specific combinations are found in GUCY2D-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023). No change - use as originally described No change - use as originally described For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". For variants for which the nucleotide change is the expected mechanism of disease (altered splicing), follow SVI Working Group recommendations (Walker et al. 2023; PMID: 37352859), as outlined in supplementary file “PS1 splicing". PS1 can also be applied for canonical and non-canoncial splicing variants that have a SpliceAI score within 10% of the original variant, or a greater predicted deleterious impact than the comparision (likely) pathogenic variant. See Table 2 from [PMID: 37352859](https://pmc.ncbi.nlm.nih.gov/articles/PMC10357475/table/tbl2/) for determining when PS1 should be applied at the Strong, Moderate, or Supporting level in these instances.[³](#pmid_37352859) NA NA Use ATM PS1 Splicing table for splicing variants with similar predictions or observations of splice defect. * Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al., (2023) PMID: 37352859). * PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker et al., (2023) PMID: 37352859. NA NA NA PS1\_Supporting may be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). PS1\_Supporting may be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859). NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* A single identical amino acid change in a paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* A single identical amino acid change in a paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A). See Paralogous Gene Table for corresponding amino acid positions.Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* A single identical amino acid change in a paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A).Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same/identical amino acid change as previously reported (Caveat: beware of changes that impact splicing rather than at the amino acid/protein level).* A single identical amino acid change in a paralogous gene previously established as **Pathogenic or Likely Pathogenic**, including NDD genes with equivalent constraint scores (SCN1A, SCN2A, SCN3A, SCN8A).Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). NA Same predicted impact on splicing as previously classified variant (Refer to Table 2 in Walker et al, 2023).* PS1 can be applied at varying strengths for splice variants, in conjunction with either PP3 or PVS1. PS1 strength depends on location of the variant under assessment (within or outside the +/- 1,2 dinucleotide positions) and the location of the previously classified variant (within or outside the +/- 1,2 dinucleotide position). Specific combinations are outlined in Table 2 in Walker, et al (2023) PMID: 37352859, also provided as a supplement ("PS1\_Variants impacting splicing"). Same predicted splicing impact as previously classified variant.* Refer to the AIPL1-specific PVS1 Decision Tree **part (b)** (Table 2 from Walker 2023) for specific combinations. PM4_Strong NA NA NA NA NA NA **For in-frame/indel variants:****PM4\_strong:** In-frame deletion/insertion impacting at least one of the following AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204.**For stop loss variants:****PM4:** Stop-loss variant causing a protein extension. Not Applicable: Not applicable NA NA Not Applicable: PM4 is not applicable. The majority of RYR1 variants that are causative for MHS are missense variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. NA NA NA NA NA Not Applicable: Do not use:● In-frame deletions/insertions that are not already PVS1-eligible: no information is available to justify the application of this rule. ● Missense and small in-frame indels: not yet confirmed as a mechanism of disease for PALB2.● Stop-loss: lack of data on stop-loss variants. NA NA NA NA NA NA NA NA NA NA NA Not Applicable NA NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA NA NA NA NA NA Use for stop loss variants in aa 1153. These variants will produce a 38 amino acid extension which was shown to have a deleterious effect (PMID:33805381, variant in c.3457). NA NA NA Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: This criterion does not apply since the mechanism of disease is the Gain of function caused by missense mutations. In-frame deletions due to the repetitive nature of NEB, particularly in exon 55, are deleterious and pathogenic (Anderson 2004 PMID:15221447, Lehtokari 2009 PMID:19232495). No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA NA NA No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA NA Not Applicable: Although there have been reported in-frame deletion/insertions in these genes which cause the overgrowth phenotype, they are exceptionally rare. Most insertion/deletions are associated with a LoF disease mechanism and so this point will still not be used even though we recognize that it is possible that a variant is an in-frame indel that results in a GoF mechanism. NA NA NA NA NA NA NA NA NA NA NA NA NA Protein length changes due to stop-loss variants.* PM4\_Strong is applicable to stop-loss variants in _MECP2_, as several stop loss variants in this gene has been described in affected individuals.[²](#PMID_11469283) Protein length changes due to stop-loss variants.* PM4\_Strong is applicable to stop-loss variants in UBE3A, as several stop loss variants in this gene has been described in affected individuals.[⁴](#PMID_25212744) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PM4_Supporting NA NA NA NA NA NA **PM4\_Supporting:**In-frame deletion/insertion impacting at least one of the other AA residues 89-204 within the RHD. Not Applicable: Not applicable Protein length changes due to in-frame deletions/insertions in a nonrepeat region or stop-loss variants. * In frame deletion/insertions of one amino acid. NA Not Applicable: PM4 is not applicable. The majority of RYR1 variants that are causative for MHS are missense variants. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. NA NA NA NA NA Not Applicable: Do not use:● In-frame deletions/insertions that are not already PVS1-eligible: no information is available to justify the application of this rule. ● Missense and small in-frame indels: not yet confirmed as a mechanism of disease for PALB2.● Stop-loss: lack of data on stop-loss variants. NA NA NA NA NA NA NA For single amino acid deletions/insertions, use as supporting level of evidence Apply at the Supporting level when there is an insertion or deletion of a single amino acid in a non-repeat region. NA NA Not Applicable NA In frame deletion/insertion of one amino acid. Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA NA NA NA NA NA NA NA Additional requirement that when applied to deletion variants, the deleted region must contain a known VUS variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of 1 amino acid that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acid adjacent to at least one conserved residue (PhyloP>2.0). When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. NA When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. When applied to deletion variants, the deleted region must contain a known **VUS** variant that is not predicted/observed to alter splicing. NA NA NA NA Additional requirement that deletion variants must contain a known **pathogenic variant, likely pathogenic variant, or variant of uncertain significance** that is not predicted/observed to alter splicing in order to apply PM4, with the strength of evidence dependent upon the classification of the variant contained within the deletion. NA NA Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: This criterion does not apply since the mechanism of disease is the Gain of function caused by missense mutations. No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA This code can be applied for 1 amino acid deletion/insertion or a stop loss variant, or 1 nucleotide with PhyloP ≥7.367 (https://www.medrxiv.org/content/10.1101/2023.04.24.23288782v1). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of 1 amino acid that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acid adjacent to at least one conserved residue (PhyloP>2.0). No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA For single amino acid deletions/insertions, use as supporting level of evidence Not Applicable: Although there have been reported in-frame deletion/insertions in these genes which cause the overgrowth phenotype, they are exceptionally rare. Most insertion/deletions are associated with a LoF disease mechanism and so this point will still not be used even though we recognize that it is possible that a variant is an in-frame indel that results in a GoF mechanism. In-frame del/ins, stop-loss variants and truncating variants involving ≤10% of the protein and located within the conserved olfactomedin domain (AA 246-502). NA NA NA NA In-frame indels outside of the RNase IIIb domain (p.Y1682 – p.S1846) and repeat regions (p.D606-p.D609; p.E1418-p.E1420; p.E1422-p.E1425). Downgrade to PM4\_Supporting for in frame deletion/insertion of a single amino acid. Downgrade to PM4\_Supporting for single amino acid deletions and insertions. NA Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domains for each gene). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (\< 3 amino acid residues). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domains for each gene). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domains for each gene). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domains for each gene). Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Smaller in-frame events (\< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domain for this gene). NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.* Protein length change of 1 amino acid that leads to loss of at least one conserved residue (PhyloP>2.0) or insertion of new amino acid adjacent to at least one conserved residue (PhyloP>2.0). PM4_Very Strong NA NA NA NA NA NA NA Not Applicable: Not applicable NA NA Not Applicable: PM4 is not applicable. The majority of RYR1 variants that are causative for MHS are missense variants. NA NA NA NA NA NA Not Applicable: Do not use:● In-frame deletions/insertions that are not already PVS1-eligible: no information is available to justify the application of this rule. ● Missense and small in-frame indels: not yet confirmed as a mechanism of disease for PALB2.● Stop-loss: lack of data on stop-loss variants. NA NA NA NA NA NA NA NA NA NA NA Not Applicable NA NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA Not Applicable: Considered as component of bioinformatic analysis (PP3/BP4). NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: Protein length change from an in-frame variant is not used due to lack of evidence. Not Applicable: This criterion does not apply since the mechanism of disease is the Gain of function caused by missense mutations. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Although there have been reported in-frame deletion/insertions in these genes which cause the overgrowth phenotype, they are exceptionally rare. Most insertion/deletions are associated with a LoF disease mechanism and so this point will still not be used even though we recognize that it is possible that a variant is an in-frame indel that results in a GoF mechanism. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS4_Very Strong NA NA NA NA NA NA NA NA Not Applicable NA Not Applicable: BS4 is not applicable. Phenotype for MHS is routinely determined based on the vitro contraction test (IVCT) that has a false positive rate of approximately 6% (PP1) or the caffeine-halothane contracture test (CHCT). As the phenotype in individuals who have not experienced an MH crisis cannot be reliably determined BS4 is not utilized. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not applicable as these are de novo, germline mosaic or post-zygotic mutations. Not Applicable: The presence of phenocopies, the reduced age-related penetrance and the possibility that more than one pathogenic variant can contribute to the phenotype observed in families make non-segregation difficult to assess in the context of MYOC and POAG. Not Applicable: AD Condition: Co-segregation analysis in low penetrance genes can lead to false positive results (PMID 32773770)AR Condition: Informative instances of lack of co-segregation in A-T families are too rare to be considered for weight at this time and can also be considered for BP2 if biallelic unaffected patients are observed in an A-T family. NA NA NA NA Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies NA BS4_Moderate NA NA NA NA NA NA NA NA Not Applicable NA Not Applicable: BS4 is not applicable. Phenotype for MHS is routinely determined based on the vitro contraction test (IVCT) that has a false positive rate of approximately 6% (PP1) or the caffeine-halothane contracture test (CHCT). As the phenotype in individuals who have not experienced an MH crisis cannot be reliably determined BS4 is not utilized. Lack of segregation in affected members of a family.Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. NA NA NA NA NA LOD ≤ -.64 or Bayes Factor (LR) ≤.23 NA NA NA NA NA NA NA NA NA NA NA NA NA NA Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4\_Moderate - LR ≤0.23:1 NA NA Lack of segregation in affected members of a family, as measured by a quantitative co-segregation analysis method. See Appendix I for details.Apply weight as per Bayes Score:BS4\_Moderate - LR ≤0.23:1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described No change - use as originally described NA NA NA No change - use as originally described No change - use as originally described NA NA NA NA NA NA NA NA Not Applicable: Not applicable as these are de novo, germline mosaic or post-zygotic mutations. Not Applicable: The presence of phenocopies, the reduced age-related penetrance and the possibility that more than one pathogenic variant can contribute to the phenotype observed in families make non-segregation difficult to assess in the context of MYOC and POAG. Not Applicable: AD Condition: Co-segregation analysis in low penetrance genes can lead to false positive results (PMID 32773770)AR Condition: Informative instances of lack of co-segregation in A-T families are too rare to be considered for weight at this time and can also be considered for BP2 if biallelic unaffected patients are observed in an A-T family. NA NA NA NA Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. Not Applicable: Lack of segregation in a family. Caveat: The presence of phenocopies for common phenotypes. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies Not Applicable: Reduced penetrance and phenocopies NA Not Applicable: Reduced penetrance, variable expressivity and phenocopies NA PP1_Very Strong NA NA NA NA NA NA NA NA Not Applicable: Sib-ships large enough to meet this criterion are extremely rare. In addition, because GAA is the only gene involved in Pompe disease, all patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Not applicable since disease-causing variants are germline mosaic, de novo or mosaic. NA NA NA NA NA NA Not Applicable: CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GATM is the only gene involved in AGAT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. Not Applicable: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.CCDS VCEP notes for PP1: Sibships large enough to use meet this criterion are extremely rare. In addition, because GAMT is the only gene involved in GAMT-D, ALL patients are expected to be bi-allelic, regardless of whether the pathogenic variants can be, or have been, detected. A variant under assessment may not be the true pathogenic variant but instead in linkage disequilibrium with an unidentified pathogenic variant. For this reason, this criterion does not facilitate assessment of pathogenicity. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PM2_Very Strong NA NA NA NA NA NA NA NA NA NA Not Applicable: PM2 is not used alone. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PM2_Strong NA NA NA NA NA NA NA NA NA NA Not Applicable: PM2 is not used alone. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS1_Moderate NA NA NA NA NA NA NA NA NA NA NA Allele frequency is greater than expected for disorder. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BP7_Moderate NA NA NA NA NA NA NA NA NA NA NA A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. NA NA NA NA NA BP7\_Moderate(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Rule code is applicable to intronic (outside of canonical -21 to +7) or synonymous variant when Splice AI prediction is \<0.1 (PMID: 40225145). This code does not apply when conflicting minigene or other functional data is available. NA NA NA NA NA NA NA NA NA NA NA NA NA BP7\_Moderate(RNA): Observed lack of aberrant RNA defect for silent substitutions and intronic variants. Variable weight applied depending on curator discretion of assay quality. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PS3_Very Strong NA NA NA NA NA NA NA NA NA NA NA Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. NA Not Applicable Not Applicable NA NA Not Applicable: ● Protein: Do not use: Lack of known positive controls ● RNA: Do not use: See code PVS1_Variable(RNA) NA NA NA NA NA NA NA NA NA NA NA **RNA assays** show1. a premature stop codon OR2. inframe skipping of exon 13 or 14**AND** the absence of full-length transcript. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for PPP1CB at this time. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA * Functional evidence from non-patient derived material with only a single variant best reflects the variant-level data. Apply patient-derived evidence in PP4.* Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart).* Enzyme activity assays, total protein production, protein stability, dimer formation and transcript production are valid assays to consider for PS3. Apply criteria at the level determined by validation parameters (see PS3 BS3 flowchart). OddsPath analysis sufficient for very strong application. NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. PS3 is not applicable for SHOC2 at this time. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS3_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable Not Applicable Not Applicable Not Applicable NA Not Applicable: Do not use: ● Protein functional studies (BS3) See PS3 for details ● RNA functional studies (Use BP7_Variable(RNA)) NA NA NA Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. NA NA Not Applicable: There are no available assays or model organisms that can recapitulate disease, and in vitro studies cannot dependably rule out pathogenicity. NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. NA Not Applicable: There are no available assays that can clearly and dependably show no damaging protein effects. NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. NA NA NA NA NA NA NA NA Not Applicable: Given that normal protein abundance and stability does not rule out impact on enzymatic activity, and that normal enzymatic activity for one substrate is not indicative of other substrates, the Glaucoma VCEP decided to not apply BS3. Not Applicable NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. Not Applicable: There is not a well-established functional study which can rule out all damaging effects on protein function. NA Not Applicable: The many secretion assays have not tested enough benign variants to meet the threshold in PMID 31892348 Supplemental Table 1 or Supplemental Table 2. NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Does not apply. NA NA NA NA NA NA NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an MTM1 variant because of the numerous possible functions of myotubularin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. NA NA NA Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an ACTA1 variant because of the numerous possible functions of Actin; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: The VCEP has decided that lack of demonstrated effect in a functional assay should not count against the pathogenicity of an RYR1 variant because of the numerous possible functions of the ryanodine receptor; therefore all specified functional assays will only be used as evidence for pathogenicity. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since the muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. Not Applicable: Not applicable. Since muscle disease mechanisms are complex, it is not feasible at this time to exclude all pathogenic functional abnormalities through available assays. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Approved functional studies are available for each individual gene in the supplemental material. Additional functional studies can be submitted to the expert panel for approval. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic ID/ASD, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. NA Not Applicable: Cellular electrophysiology (voltage clamp recording): Values indicating “no impact on channel function” have not been sufficiently characterized to date. Additionally, one cannot exclude non-electrophysiological defects such as mis-localization in a neuron based solely on heterologous expression studies. This can be re-assessed by the EP over time and as benign variants are functionally characterized in the future.Animal Models: Lack of an epilepsy phenotype in an animal model is insufficient to support benignity of a variant. Additionally, some non-epilepsy co-morbidities, such as behavioral characteristics that may mimic intellectual disability and/or autism spectrum disorder, are still being established and could support pathogenicity. This can be re-assessed by the EP over time. Not Applicable: Not applicable for splicing effects (replaced by BP7_Strong (RNA)). BP7_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PM5_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BP4_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable NA Not Applicable Not Applicable NA Not Applicable NA Not Applicable Not Applicable NA NA NA NA Not Applicable: Does not apply. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PP3_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Not Applicable: This criterion is not applicable since these variants are GOF, and traditional mutation pathogenicity prediction algorithms focus on LOF mechanisms. Use of this criterion can be revisited if there emerges additional published experience with predictive algorithms specifically designed to detect gain of function mutations. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BS1_Very Strong NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MAF of ≥0.003 (0.3%) for autosomal recessive. Likely benign, provided there is no conflicting evidence. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA