Criteria Specification Registry

Criteria Specification (CSpec) Registry is intended to provide access to the Criteria Specifications used and applied by ClinGen Variant Curation Expert Panels and biocurators in the classification of variants.

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Criteria Specification

ClinGen Leber Congenital Amaurosis/early onset Retinal Dystrophy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RPE65 Version 1.0.0

Description : LCA/eoRD Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RPE65

Version : 1.0.0

Leber Congenital Amaurosis/early onset Retinal Dystrophy VCEP

Released

10/24/2023

07:58:03

Rules for RPE65

Gene: RPE65 (HGNC:10294) HGNC Name: retinoid isomerohydrolase RPE65 Transcripts: NM_000329.3 Disease: RPE65-related recessive retinopathy (MONDO:0100368) Mode of Inheritance: Autosomal recessive inheritance

Criteria & Strength Specifications
PVS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Null variant in a gene where loss of function is a known mechanism of disease. Use as defined by ClinGen SVI working group ¹⁶ and as updated by the ClinGen SVI Splicing Subgroup ¹⁷ 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. Modification Type: Gene-specific Strong Null variant in a gene where loss of function is a known mechanism of disease. Use as defined by ClinGen SVI working group ¹⁶ and as updated by the ClinGen SVI Splicing Subgroup ¹⁷ 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. Modification Type: Gene-specific Moderate Supporting Instructions: See attached RPE65-specific PVS1 Decision Tree file, which has been modified from Walker 2023 ¹⁷ Not Applicable
PS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong 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). Modification Type: Gene-specific Moderate 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). Modification Type: Gene-specific,Strength Supporting 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). Modification Type: Gene-specific,Strength Instructions: For assessing same amino acid changes, SpliceAI must be used to ensure comparison variant is not causing a splicing defect (score ≤0.1) RPE65-specific PVS1 Decision Tree for scoring splicing variants is based on the recommendations of the ClinGen SVI Splicing Subgroup published in Walker et al. 2023 ¹⁷ and is attached. Not Applicable
PS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong 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 Modification Type: Gene-specific Strong 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 Modification Type: Gene-specific Moderate 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 Modification Type: Gene-specific Supporting 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 Modification Type: Gene-specific Instructions: Use point table from SVI Recommendation for De Novo Criteria (PS2 & PM6) - Version 1.1 ¹ , file attached Individuals must have 2 variants to consider scoring one for de novo. To determine the appropriate strength level to apply for de novo occurrence(s), each proband with a de novo variant is awarded a point value based upon phenotypic consistency and confirmed or assumed parental relationships (PS2/PM6 file -Table 1). The combined point value of all de novo occurrences is then compared to Table 2 to determine the applicable evidence strength level. For “Phenotypic consistency” category, use option 3: "Phenotype consistent with gene but not highly specific and high genetic heterogeneity" Note that all probands being considered for any pathogenic phenotype codes (i.e. – PP1, PP4, PM3, PM6, PS2) at any strength must have the following phenotype characteristics: Absent or severely decreased rod electroretinogram response OR congenital night blindness/nyctalopia OR a diagnosis of Leber congenital amaurosis/eoRD. Not Applicable
PS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate Supporting 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. Modification Type: Gene-specific,Strength Instructions: Studies included in attached table: Jin et al. 2005⁶ Jin et al. 2016¹⁰ Philp et al. 2009³ Li et al. 2014⁴ Li et al. 2015⁵ Redmond et al. 2005¹⁸ Lorenz et al. 2008⁸ Bereta et al. 2008² Takahashi et al. 2006¹⁹ Chen et al. 2006⁷ Nikolaeva et al. 2011⁹ Not Applicable
PS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate Supporting Instructions: Detailed phenotype not needed. Need to confirm patient is ‘affected with a neurodevelopmental phenotype consistent with the gene’ at a minimum. Patient can be published OR an internal case OR observed at an outside lab (i.e. via ClinVar) OR described in the reputable databases [LOVD (UBE3A), RettBASE (MECP2, CDKL5, FOXG1)]. However independent case has to be confirmed to be a different patient than yours (compare gender/age). Do not use this criterion for variants where BS1 is applied or where PM2 does not apply. Not Applicable
PM1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. Stand Alone Very Strong Strong Moderate 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 site¹⁶. Met by variants encoding missense substitutions between Ala107 and Gly125, which are known to mediate localization to the ER membrane¹⁵. Modification Type: Gene-specific Supporting Instructions: Because these sites have been identified based on well-established functional domains rather than based on a known cluster of pathogenic or likely pathogenic variants, this criterion is not mutually exclusive with PM5. Missense variants that meet BS1 or BA1 should not be eligible to meet this criterion. Not Applicable
PM2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate Supporting 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. Modification Type: Disease-specific,Strength Instructions: gnomAD computes computes a threshold filter allele frequency (PopMax FAF) for a variant based on the allele count in the population with the highest allele frequency. Cutoff value of 2.0x10^-4 is set between the FAF of the most common pathogenic RPE65 variant (1.6x10^-4) and the Whiffin-Ware calculation of 8x10^-3 for the maximum credible population allele frequency for the disease. This rule should not be applied if variant would otherwise meet criteria for a benign classification, as rarity of the variant should not outweigh other types of benign evidence. Not Applicable
PM3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase. Stand Alone Very Strong Points are calculated using Table 1 from 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 Modification Type: Disease-specific Strong Points are calculated using Table 1 from 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 Modification Type: Disease-specific Moderate Points are calculated using Table 1 from 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 Modification Type: Disease-specific Supporting Points are calculated using Table 1 from 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 Modification Type: Disease-specific Instructions: Use SVI recommendations found in https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf (or attached file “PM3 Tables”) To determine the appropriate strength level to apply for in trans occurrence(s), each proband is awarded a point value based upon phasing of the two variants in question (confirmed in trans versus unknown) and classification of the variant on the other allele (Table 1). The combined point value of all proband occurrences is then summed and compared to Table 2 to determine the applicable evidence strength level. Both variants must be classified using these rule specifications. Note that all probands being considered for any pathogenic phenotype codes (i.e. – PP1, PP4, PM3, PM6, PS2) at any strength must have the following phenotype characteristics: Absent or severely decreased rod electroretinogram response OR congenital night blindness/nyctalopia OR a diagnosis of Leber congenital amaurosis/eoRD. Not Applicable Comments: Not applicable for UBE3A.
PM4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Stand Alone Very Strong Strong Moderate 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). Modification Type: Gene-specific Supporting 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). Modification Type: Gene-specific,Strength Not Applicable
PM5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate 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. Modification Type: None Supporting 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. Modification Type: Strength Not Applicable
PM6 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Assumed de novo, but without confirmation of paternity and maternity. Stand Alone Very Strong Strong Moderate Supporting Instructions: Use point table from SVI Recommendation for De Novo Criteria (PS2 & PM6) - Version 1.1 ¹, file attached To determine the appropriate strength level to apply for de novo occurrence(s), each proband with a de novo variant is awarded a point value based upon phenotypic consistency and confirmed or assumed parental relationships (Table 1). The combined point value of all de novo occurrences is then compared to Table 2 to determine the applicable evidence strength level. For “Phenotypic consistency” category, use option 3: "Phenotype consistent with gene but not highly specific and high genetic heterogeneity" Note that all probands being considered for any pathogenic phenotype codes (i.e. – PP1, PP4, PM3, PM6, PS2) at any strength must have the following phenotype characteristics: Absent or severely decreased rod electroretinogram response OR congenital night blindness/nyctalopia OR a diagnosis of Leber congenital amaurosis/eoRD. Not Applicable Comments: Use the PS2 code in lieu of using this code for de novo variants.
PP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong 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 Modification Type: Disease-specific,Strength Moderate 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 Modification Type: Disease-specific,Strength Supporting 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 Modification Type: Disease-specific,Strength Instructions: Note that all probands being considered for any pathogenic phenotype codes (i.e. – PP1, PP4, PM3, PM6, PS2) at any strength must have the following phenotype characteristics: Absent or severely decreased rod electroretinogram response OR congenital night blindness/nyctalopia OR a diagnosis of Leber congenital amaurosis/eoRD. Not Applicable
PP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: Not applicable for RPE65.
PP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate For a missense variant use REVEL, requires a score of ≥ 0.774. Splice variants use PP3 only at Supporting level. Modification Type: Gene-specific,Strength Supporting 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. Modification Type: Gene-specific Instructions: PP3 should not be used to evaluate variants at canonical splice sites. For non-canonical sites, if SpliceAI score is ≥0.2, apply PP3 (splicing) instead. REVEL outperformed other in silico prediction models for this gene (RPE65) using 161 assumed pathogenic variants, 19 assumed benign variants and 17 different in silico predictors (courtesy of R. Chen Lab at Baylor College of Medicine). See attached file “PP3 performance of multiple prediction models” Score ranges are based on calculations found in the publication of the SVI Working Group “Calibration of computational tools for missense variant pathogenicity classification and ClinGen recommendations for PP3/BP4 criteria” ¹¹ Not Applicable
PP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. Stand Alone Very Strong Strong Moderate 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. Modification Type: Disease-specific,Strength Supporting 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. Modification Type: Disease-specific Instructions: A point system was developed to determine when there is enough information about a proband’s phenotype to qualify for use of this code (see below). Review eligible phenotype characteristics and add up points for each finding. This code can be used for a single proband. A proband must have two RPE65 variants to consider applying PP4 (phase is not considered here). Required for use of PP4 (0.5 points each) Absent or severely decreased rod electroretinogram (ERG) responses -OR- Congenital night blindness/nyctalopia -OR- Clinical diagnosis of Leber congenital amaurosis Specific RPE65 Phenotype Findings List (2 points each, 8 points for successful, documented gene therapy) Absence or minimal autofluorescence White/yellow dots on color photography of the type seen in fundus albipunctatus in the context of severe retinal dysfunction Previous exome, genome or 100+ retinal dystrophy gene panel testing that did not provide an alternative explanation for visual impairment. Participation in a gene therapy trial or study with strict inclusion criteria and subsequent positive results (2 pts) - OR - Significant, documented improvement of FST or other measure of dark-adapted vision after treatment with Luxturna or other RPE65 gene therapy. Supporting information required from treating clinician if sufficient detail is not included in published report (8 pts). Consistent with RPE65 Phenotype Findings List (0.5 or 1 point each) Optic disc drusen (0.5) Optic nerve pallor (0.5) Pigmentary retinopathy with attenuated vessels (0.5) Poor pupillary light response (0.5) Posterior subcapsular cataract (0.5) RPE mottling (0.5) Macular atrophy (0.5) Symptomatic onset between birth and age five years (1) OCT is preserved with respect to vision loss (1) Decreased peripheral vision (1) Abnormal color vision or evidence of cone involvement on ERG (1) Decreased central visual acuity (1) Nystagmus (1) Light staring (1) Not Applicable
PP5
Original ACMG Summary Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Stand Alone Use gnomAD PopMax FAF if available, cutoff of ≥8 x 10^-3 Use large population databases (i.e. gnomAD). Modification Type: Disease-specific Very Strong Strong Moderate Supporting Instructions: The maximum credible population allele frequency for the disease, based on the Whiffen-Ware calculator, is 8.16 x 10 ^-3. This assumes a population frequency of 1 in 3000 individuals, genetic heterogeneity = 20%, penetrance of 100%, allele heterogeneity of 1. Not Applicable
BS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is greater than expected for disorder. Stand Alone Very Strong Strong Allele frequency greater than expected for disorder. Use gnomAD PopMax FAF if available. Allele frequency of between 8x10^-3 and 8x10^-4 Modification Type: Disease-specific Moderate Supporting Instructions: The BS1 value was derived by decreasing the BA1 cutoff (> 8.0 x 10^-3) by one order of magnitude. Not Applicable
BS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong 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. Modification Type: Disease-specific Moderate Supporting Not Applicable
BS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. Stand Alone Very Strong Strong Moderate Supporting 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. Modification Type: Gene-specific,Strength Instructions: Studies included in attached table: Jin et al. 2005⁶ Jin et al. 2016¹⁰ Philp et al. 2009³ Li et al. 2014⁴ Li et al. 2015⁵ Redmond et al. 2005¹⁸ Lorenz et al. 2008⁸ Bereta et al. 2008² Takahashi et al. 2006¹⁹ Chen et al. 2006⁷ Nikolaeva et al. 2011⁹ Not Applicable
BS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Lack of segregation in affected members of a family. One or both variants are absent in a similarly affected family member. Modification Type: Clarification Moderate Supporting Not Applicable
BP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene for which primarily truncating variants are known to cause disease. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: Not applicable for RPE65.
BP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate Supporting 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. Modification Type: Disease-specific Not Applicable
BP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary In frame-deletions/insertions in a repetitive region without a known function. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: Not applicable for RPE65.
BP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Moderate 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. Modification Type: Gene-specific,Strength Supporting 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. Modification Type: Gene-specific Instructions: Only applicable if both the REVEL and SpliceAI scores are below cutoffs. Not Applicable
BP5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Variant found in a case with an alternate molecular basis for disease. Stand Alone Very Strong Strong Moderate Supporting Not Applicable Comments: 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.
BP6
Original ACMG Summary Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong 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. Modification Type: Disease-specific Moderate Supporting 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 Modification Type: Gene-specific Instructions: BP4 and BP7 can be added unless variant is in an excluded region. Evolutionary conservation is not considered informative for application of this code. Not Applicable

Rules for Combining Criteria

Pathogenic

1 Very Strong (PVS1, PS2_Very Strong, PM3_Very Strong) AND ≥ 1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong)

1 Very Strong (PVS1, PS2_Very Strong, PM3_Very Strong) AND ≥ 2 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Very Strong (PVS1, PS2_Very Strong, PM3_Very Strong) AND 1 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND 1 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Very Strong (PVS1, PS2_Very Strong, PM3_Very Strong) AND ≥ 2 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 2 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND ≥ 3 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND 2 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND 1 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

Likely Pathogenic

1 Very Strong (PVS1, PS2_Very Strong, PM3_Very Strong) AND 1 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND 1 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND ≥ 2 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 3 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

2 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS1_Supporting, PS2_Supporting, PS3_Supporting, PM2_Supporting, PM3_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PM3_Strong, PP1_Strong) AND 2 Moderate (PS1_Moderate, PS2_Moderate, PM1, PM3, PM4, PM5, PP1_Moderate, PP3_Moderate, PP4_Moderate)

Benign

≥ 2 Strong (BS1, BS2, BS4, BP7_Strong)

Likely Benign

1 Strong (BS1, BS2, BS4, BP7_Strong) AND 1 Supporting (BS3_Supporting, BP2, BP4, BP7)

≥ 2 Supporting (BS3_Supporting, BP2, BP4, BP7)

Files & Images

PP3 performance of multiple prediction models: Modelling done at Baylor College of Medicine by Dr. Rui Chen for RPE65, REVEL performs the best

PS2 PM6 Tables: PS2/PM6: Table 1 - points awarded per de novo occurrence, Table 2 - evidence strength levels

PM3 Tables: PM3: Table 1 - points awarded per in trans occurrence, Table 2 - evidence strength levels

RPE65 Rule combination rules: The point system of Tavtigian et al. is being used for all classifications. Table 2 describes the points value for each evidence code and Table 3 specifies the point-based classification categories.

PS3 Approved functional assays: Description of all functional assays to be used for consideration of the PS3 rule. August 10, 2023.

RPE65-specific PVS1 Decision Tree: Document describes RPE65-specific PVS1 decision tree and other modified codes based on Walker 2023. Sub-parts (a) through (c) include detailed descriptions of rules for variants that are likely to be spliceogenic or for which effects on splicing have been considered.

RPE65 Pilot Variants, Sept.11, 2023: Results from Pilot study of RPE65 rules

References

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