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 HNF1A Version 2.0.0

Description : This version specified for the following genes: HNF1A

Version : 2.0.0 Release Notes :

Updates for consistency with MDEP's newly released HNF4A/GCK rules:

Updated PM2_Supporting

Updated BP7

Monogenic Diabetes VCEP

Released

1/11/2023

20:49:33

Rules for HNF1A

Gene: HNF1A (HGNC:11621) HGNC Name: HNF1 homeobox A Transcripts: NM_000545.8 Disease: monogenic diabetes (MONDO:0015967) Mode of Inheritance: Autosomal Dominant

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 HNF1A PVS1 decision tree. 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 (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. PVS1_Strong will be applied to 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” 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. 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 PVS1_Strong for deletions of exon 10 and splicing variants that would predict the skipping of exon 10. 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. 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. Modification Type: Strength Strong Use HNF1A PVS1 decision tree. 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 (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. PVS1_Strong will be applied to 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” 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. 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 PVS1_Strong for deletions of exon 10 and splicing variants that would predict the skipping of exon 10. 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. 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. Modification Type: Strength Moderate Supporting Use HNF1A PVS1 decision tree. 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 (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. PVS1_Strong will be applied to 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” 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. 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 PVS1_Strong for deletions of exon 10 and splicing variants that would predict the skipping of exon 10. 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. 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. Modification Type: Strength Instructions: Use HNF1A PVS1 decision tree. 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” described in PP4 specifications. Modification Type: Gene-specific,Strength Strong Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. Modification Type: Gene-specific,Strength Moderate Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. Modification Type: Gene-specific,Strength Supporting Use SVI recommended point-based system with specifications for “Phenotype Consistency” described in PP4 specifications. Modification Type: Gene-specific,Strength Instructions: Use SVI recommended point-based system with MDEP specifications for “Phenotype Consistency”. 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 Applicable for variants with luciferase assay data (evidence of decreased transactivation (<=40% of wild type) by the Gloyn/Oxford group (Althari et al 2020 https://pubmed.ncbi.nlm.nih.gov/32910913/) Modification Type: Gene-specific,Strength Supporting See list of approved functional studies and guidelines for interpretation of data (below). (1) Luciferase assays for transactivation - "Decreased function" is defined as activity less than 40% of wildtype. Assays should include controls for WT, T2DM-risk, and known MODY variants. For additional specifications and recommendations, please see the HNF1A rules. (2) EMSA for DNA binding - "Decreased function" is defined as activity lesss than 405 of wildtype. 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) (PMID: 11162430, 12574234, 24915262). For additional specifications and recommendations, please see the HNF1A rules. (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 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). 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 Seven or more= 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 (see below). Modification Type: Gene-specific,Strength Moderate 4-6 unrelated occurrences = Moderate. Modification Type: Gene-specific,Strength Supporting Instructions: 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 (see rules). 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 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 Modification Type: Gene-specific,Strength Supporting 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 201-280, NM_000545.8 It 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) 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. 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. 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
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 Browning (PMID: 27236918) 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 Browning (PMID: 27236918) 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 Browning (PMID: 27236918) Single Family : ≤ 1/8 (3 meioses) 1 Family : ≤ ¼ (2 meioses) Modification Type: General recommendation,Gene-specific Instructions: Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology (see rules). 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: 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.
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 above 0.2 (Wai et al. 2020 PMID: 32123317; Jaganathan et al. 2019 PMID: 30661751). 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 MODY Probability Calculator (MPC) result ≥50% chance of testing positive 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 SU (extreme response- hypoglycemia) Low hsCRP in patient with clinical diagnosis of T2DM Biochemical/Molecular phenotypic evidence from patient cell lines Hepatocellular adenomas Modification Type: Gene-specific Supporting MODY Probability Calculator (MPC) result ≥50% chance of testing positive https://www.diabetesgenes.org/mody-probability-calculator/) AND negative HNF4A testing Modification Type: Gene-specific Instructions: Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology (see rules). 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 MAF ≥ 1:10,000 (≥ 0.01% or 0.0001) in gnomAD Popmax Filtering AF. Modification Type: Gene-specific Very Strong Strong Moderate Supporting 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 MAF ≥ 1:30,000 (≥0.0033% or 0.000033) in gnomAD Popmax Filtering AF. Modification Type: Gene-specific Moderate Supporting 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 See HNF1A rules for full list of approved functional studies and guidelines for interpretation of data. (1) Luciferase assays for transactivation - "No functional impact" is defined as ≥ 75% activity of wildtype (2) EMSA for DNA binding - "No functional impact" is defined as ≥ 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. Modification Type: Gene-specific 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 MPC score >50% (i.e., genotype negative, phenotype positive). Modification Type: 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 0.2 (Wai et al. 2020 PMID: 32123317; Jaganathan et al. 2019 PMID: 30661751). 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 other monogenic diabetes gene is P/LP. 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: General recommendation 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 (PS2_Moderate, PS3_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

1 Very Strong (PVS1, PS2_Very Strong) AND 1 Moderate (PS2_Moderate, PS3_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)

≥ 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 (PS2_Moderate, PS3_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 (PS2_Moderate, PS3_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 (PS2_Moderate, PS3_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)

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)

Likely Pathogenic

1 Very Strong (PVS1, PS2_Very Strong) AND 1 Moderate (PS2_Moderate, PS3_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

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)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 1 Moderate (PS2_Moderate, PS3_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

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 (PS2_Moderate, PS3_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

2 Moderate (PS2_Moderate, PS3_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 (PS2_Moderate, PS3_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)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PS4, PM5_Strong, PP1_Strong) AND 2 Moderate (PS2_Moderate, PS3_Moderate, PS4_Moderate, PM1, PM4, PM5, PP1_Moderate, PP4_Moderate)

Benign

≥ 2 Strong (BS1, BS2, BS3, BS4)

1 Stand Alone (BA1)

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)