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 Monogenic Diabetes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for HNF4A Version 2.0.0

Description : ClinGen Monogenic Diabetes Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines

Version : 2.0.0 Release Notes :

Upgrade initiation codon variants from PVS1_Moderate to PVS1_Strong on the basis that next methionine is Met71 and multiple variants not found in gnomAD have been reported in patients meeting diagnostic criteria for MODY (PMID: 23348805)

Monogenic Diabetes VCEP

Released

10/11/2023

00:08:05

Rules for HNF4A

Gene: HNF4A (HGNC:5024) HGNC Name: hepatocyte nuclear factor 4 alpha Transcripts: NM_175914.5 Disease: monogenic diabetes (MONDO:0015967) Mode of Inheritance: Autosomal dominant 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 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 NMD¹ 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 Modification Type: Strength Strong 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. Modification Type: Strength Moderate Supporting Use HNF4A PVS1 decision tree. Apply PVS1_Supporting to nonsense or frameshift variants at c.1258 (G)/p.Gly420 and 3’. Modification Type: Strength Instructions: Per recommendations from the SVI, when RNA analysis demonstrates abnormal splicing from non-canonical splice site variants, apply PS3 instead of PVS1. Not Applicable Comments: Per guidance from ClinGen/SVI, PM2_Supporting + PVS1 is sufficient evidence of a variant being likely pathogenic
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 No change Modification Type: No change Moderate Supporting PS1 may also be used at a supporting level for canonical and non-canonical splicing variants when a different variant at the same nucleotide has been previously classified as pathogenic and the variant being assessed is predicted by SpliceAI to have a similar (SpliceAI score within 10% of the original variant) or greater deleterious impact. Modification Type: Strength 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 Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Modification Type: Gene-specific,Strength Strong Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Modification Type: Gene-specific,Strength Moderate Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Modification Type: Gene-specific,Strength Supporting Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions. Modification Type: Gene-specific,Strength Instructions: To obtain maximum points (“phenotype highly specific for gene”) patient must meet criteria for PP4 (result of ≥50% chance or higher of testing positive for MODY on the MODY Probability calculator (https://www.diabetesgenes.org/mody-probability-calculator/) and negative HNF1A testing). To obtain standard points (“phenotype consistent with gene but not highly specific”), the phenotype of the patient must include diabetes. Probands (and/or family members when assessing segregation for PP1) with evidence of an autoimmune etiology of diabetes and/or absolute or near-absolute insulin deficiency will be excluded when assessing criteria that includes phenotype information. Such evidence includes the following:One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD) (Ref 7,8,9,10). Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL)(Ref 11,12) or urinary C-peptide/creatinine ratio <0.2 nmol/mmol (Ref 8,9) Not Applicable Comments: To obtain maximum points (“phenotype highly specific for gene”) patient must meet criteria for PP4 (result of ≥50% chance or higher of testing positive for MODY on the MODY Probability calculator (https://www.diabetesgenes.org/mody-probability-calculator/) AND have negative HNF4A testing). If patient does not meet PP4 but is noted to have diabetes, use points corresponding to “phenotype consistent with gene but not highly specific”. If patient shows evidence of an autoimmune etiology for their diabetes and/or absolute or near-absolute insulin deficiency (see above), do not apply PS2.
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 Applicable to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing. Modification Type: Gene-specific,Strength Moderate Supporting 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. Modification Type: Gene-specific,Strength Instructions: See list of approved functional studies and guidelines for interpretation of data. Not Applicable Comments: Studies performed on a cell line generated from a patient sample (which will be heterozygous and also contain other variants in the patient’s genome which could modify function) will not count as PS3 but instead will count toward PP4_Moderate. Note that although occurrence in the transactivation domain (codons 281-631, NM_000545.8 has been cited in older publications as evidence for causality, it is known that the transactivation domain is more tolerant to benign missense variation and therefore we will not apply PM1 at any level to variants within this region at this time (PMID: 11272211, 18003757, 23348805).
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 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)⁷,⁸,⁹,¹⁰ Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL)¹¹,¹² or urinary C-peptide/creatinine ratio <0.2 nmol/mmol⁸,⁹ Modification Type: Gene-specific,Strength Moderate 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)⁷,⁸,⁹,¹⁰ Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL)¹¹,¹² or urinary C-peptide/creatinine ratio <0.2 nmol/mmol⁸,⁹ Modification Type: Gene-specific,Strength Supporting Instructions: The phenotype of the patient must include diabetes, with evidence of an autoimmune etiology and/or absolute or near-absolute insulin deficiency (see above) considered as exclusionary. 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). 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 Applicable to amino acids that directly bind DNA and are necessary for Zinc-finger or homodimer formation² 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, Cys93 Modification Type: Gene-specific,Strength Supporting 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 transcription factor binding sites in the promoter (see below for details). Promoter region: c.-132 to c.-151 (HNF6/OC2 and IPF1 binding sites) c.-169 to c.-181 (HNF1A/HNF1B binding sites) DNA binding: codons 37-113 (NM_175914.4:c.175C-339C p.Leu37-Asp113) (While the paper describing the crystal structure of HNF4A² 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) Modification Type: Gene-specific,Strength 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 gnomAD 2.1.1* Popmax FAF ≤ 1:333,000 (≤ 0.000003 or 0.0003%) in gnomAD European Non-Finnish population AND ≤ 2 copies observed in ENF AND ≤ 1 copy in any other founder or non-founder population. *Use gnomAD 2.1.1 exome data unless the region is not covered in the exome, in which case gnomAD 3.1.2 genome data should be used. Modification Type: General recommendation,Gene-specific Instructions: Recommend using as supporting level of evidence (PM2_Supporting) per ClinGen guidance. Per guidance from ClinGen/SVI, PM2_Supporting + PVS1 is sufficient evidence of a variant being likely pathogenic. We recommend investigating the genotype metrics in gnomAD for variants that have been flagged for having failed one or more quality parameters, as it is possible that some of these filtered variants are actually real. The number of filtered alleles can be counted to determine whether PM2_Supporting would be met even if they were genuine calls. If the filtered calls are sufficient in number to not meet PM2_Supporting, then we would not use it. Because it is also possible that these calls are false positives, we would not use filtered variants to support BA1 or BS1. Not Applicable Comments: Per guidance from ClinGen/SVI, PM2_Supporting + PVS1 is sufficient evidence of a variant being likely pathogenic
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 Strong Moderate Supporting Not Applicable
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 For single amino acid deletions, use as supporting level of evidence. Modification Type: Strength Supporting For single amino acid deletions/insertions, use as supporting level of evidence Modification Type: 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 Applicable once two amino acid changes have been classified as pathogenic at the same amino acid residue. Modification Type: Strength Moderate The novel amino acid change must have a Grantham distance greater than or equal to the previously classified pathogenic variant. Modification Type: Strength Supporting 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. 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 Not Applicable Comments: Subsumed by PS2.
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 Use thresholds suggested by Jarvik and Browning⁵ Single Family : ≤ 1/32 (5 meioses) > 1 Family : ≤ 1/16 (4 meioses) Modification Type: General recommendation,Gene-specific Moderate Use thresholds suggested by Jarvik and Browning⁵ Single Family : ≤ 1/16 (4 meioses) > 1 Family : ≤ 1/8 (3 meioses) Modification Type: General recommendation,Gene-specific Supporting Use thresholds suggested by Jarvik and Browning⁵ Single Family : ≤ 1/8 (3 meioses) > 1 Family : ≤ ¼ (2 meioses) Modification Type: General recommendation,Gene-specific Instructions: Variable penetrance and phenocopies complicate co-segregation studies. The presence of type 1 and type 2 diabetes phenocopies and significance of variants in unaffected individuals as defined above will need to be considered. If a family member(s) shows evidence of an autoimmune etiology for their diabetes and/or absolute or near-absolute insulin deficiency (see below), do not include them in PP1 calculation.One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD) (Ref 7,8,9,10). Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL) (Ref 11,12) or urinary C-peptide/creatinine ratio <0.2 nmol/mmol (Ref 8,9). Unaffected family members without the variant under assessment can also be used in segregation analysis(Ref 5). An individual is considered “unaffected” if over age 70 and non-diabetic (based on Exeter work (Ref 13) which shows penetrance of HNF4A-MODY at 98% by age 70). Not Applicable Comments: Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology (see rules).
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: 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.
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 Supporting 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 ⁴,³. Modification Type: General recommendation 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 Phenotype: MODY Probability Calculator result ≥50% chance of testing positive⁶ 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 Modification Type: Gene-specific Supporting MODY Probability Calculator (MPC)⁶ result ≥50% chance of testing positive AND negative HNF1A testing Modification Type: Gene-specific Instructions: MODY probability calculator result of ≥50% chance of testing positive (https://www.diabetesgenes.org/mody-probability-calculator/) AND negative HNF1A genetic analysis, given the similarities in phenotypes between HNF1A-MODY and HNF4A-MODY. Clinical judgement may need to be used when applying this criterion, as the MODY Probability Calculator is not as reliable for non-European ancestry individuals or people diagnosed >35. For example, use of PP4 is acceptable in the absence of HNF1A analysis when the MPC is >50% and the phenotype is specific to HNF4A, for example someone in the family with neonatal hypoglycemia that is responsive to diazoxide or hyperinsulinemic hypoglycemia. If individual was tested due to neonatal hypoglycemia, PP4 can be used if ABCC8 and KCNJ11 testing are negative (no MODY Probability Calculator result required). Certain assumptions can be made in order to use the MODY probability calculator. Specific clinical info about parents not given but lab/literature states “Family history of diabetes”, click “Parent with diabetes” in calculator. If no information about family history of diabetes is provided, run the calculator in both conditions (yes/no) and document whether this makes a difference in overall probability score. Weight/Height/BMI not given but lab/literature states patient is “lean”, enter BMI of 30. HbA1c is not provided, enter 6% and 10% and document whether this makes a difference in overall probability score. Treatment information is not provided, cannot use calculator. Not Applicable Comments: Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology (see rules). Certain assumptions can be made in order to use the MODY probability calculator: * Specific clinical information about parents not given but lab/literature states “Family history of diabetes”: Click “Parent with diabetes” in calculator. * No information about family history of diabetes is provided: Attempt to use the calculator using both possibilities (yes/no). If this makes a difference in the ability to meet the PP4 cutoff (>50%), PP4 cannot be used. * Weight/Height/BMI not given but lab/literature states patient is “lean”: Enter BMI of 30. * HbA1c is not provided: Attempt to use the calculator using values of 6% and 10%. If this makes a difference in the ability to meet the PP4 cutoff (>50%), PP4 cannot be used. * Treatment information is not provided: Cannot use calculator.
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 gnomAD 2.1.1* Popmax Filtering AF ≥ 1:10,000 (≥ 0.01% or 0.0001). *Use gnomAD 2.1.1 exome data unless the region is not covered in the exome, in which case gnomAD 3.1.2 genome data should be used. Modification Type: Gene-specific Very Strong Strong Moderate Supporting Instructions: If there is a Popmax Filtering AF for both exomes and genomes, use the one with the larger denominator. 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 gnomAD 2.1.1* Popmax Filtering AF ≥ 1:30,000 (≥0.0033% or 0.000033). *Use gnomAD 2.1.1 exome data unless the region is not covered in the exome, in which case gnomAD 3.1.2 genome data should be used. Modification Type: Gene-specific Moderate Supporting Instructions: If there is a Popmax Filtering AF for both exomes and genomes, use the one with the larger denominator. 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 Apply to normoglycemic individuals age 70 or older (i.e., genotype positive, phenotype negative) Modification Type: Gene-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 Applicable to non-canonical splice site variants that have RNA and in silico evidence of normal splicing. Modification Type: Gene-specific Moderate Supporting 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. Modification Type: Gene-specific Instructions: To use BS3, 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) it cannot count as BS3. Not Applicable Comments: To use BS3, 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 and other variants in the patient’s genome) it cannot count as BS3.
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 Applicable to family members without variant who have MPC⁶ score ≥50% (i.e., genotype negative, phenotype positive). Modification Type: General recommendation,Gene-specific 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
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 Also applicable when in cis or trans with a likely pathogenic variant. Modification Type: General recommendation 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
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 Supporting 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 0.2³,⁴. Modification Type: General recommendation 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 A variant in another monogenic diabetes gene is Pathogenic/Likely Pathogenic. Modification Type: General recommendation Not Applicable
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 Moderate Supporting Apply BP7 when the predicted change from SpliceAI is below 0.2 AND phyloP100 way < 2.0. Modification Type: Gene-specific Not Applicable

Rules for Combining Criteria

Pathogenic

1 Very Strong (PVS1, PS2_Very Strong) AND ≥ 1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong)

1 Very Strong (PVS1, PS2_Very Strong) AND ≥ 2 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Very Strong (PVS1, PS2_Very Strong) AND 1 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate) AND 1 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Very Strong (PVS1, PS2_Very Strong) AND ≥ 2 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 2 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND ≥ 3 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 2 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 1 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

Likely Pathogenic

1 Very Strong (PVS1, PS2_Very Strong) AND 1 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 1 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 2 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Very Strong (PVS1, PS2_Very Strong) AND ≥ 1 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND ≥ 2 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 3 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

≥ 2 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Moderate (PVS1_Moderate, PS2_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PVS1_Supporting, PS1_Supporting, PS2_Supporting, PS3_Supporting, PM1_Supporting, PM2_Supporting, PM4_Supporting, PM5_Supporting, PP1, PP3, PP4)

Benign

1 Stand Alone (BA1)

≥ 2 Strong (BS1, BS2, BS3, BS4)

Likely Benign

1 Strong (BS1, BS2, BS3, BS4) AND 1 Supporting (BS3_Supporting, BP2, BP4, BP5, BP7)

≥ 2 Supporting (BS3_Supporting, BP2, BP4, BP5, BP7)

Files & Images

PS2 De Novo Points Table: Table for determining strength of PS2 for apparent de novo variants

HNF4A PVS1 Decision Tree: Decision tree for determining strength of PVS1

References

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