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 InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH6 Version 1.0.0

Description : ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH6 Version 1.0.0

Version : 1.0.0 Release Notes :

Initial release

InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP

Released

8/9/2024

03:13:58

Rules for MSH6

Gene: MSH6 (HGNC:7329) HGNC Name: mutS homolog 6 Transcripts: NM_000179.3 Disease: Lynch syndrome (MONDO:0005835) Mode of Inheritance: Autosomal dominant inheritance mismatch repair cancer syndrome 1 (MONDO:0010159) 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 Nonsense/frameshift variant introducing Premature Termination Codon (PTC)^a ≤ codon 1341 in MSH6. Refer to Appendix for details. OR Large genomic alterations^a of single or multi-exon size. OR Variants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). If exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b then use PVS1_Strong. If exon skipping or use of a cryptic splice site disrupts reading frame and is NOT predicted to undergo NMD then use PVS1_Moderate. OR Variants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript) leading to premature stop codon or in-frame deletion disrupting a functional domain^b or protein conformation. Splicing aberration must be confirmed in a minigene assay or an additional RNA assay from an independent laboratory if it is a non-canonical splice site variant. Modification Type: General recommendation Strong Variants in the initiation codon of MSH6. OR Presumed by default in tandem duplication of ≥1 exon resulting in a frameshift before the last splice junction. This rule does not apply for variants that involve the UTR (i.e. exon 1 or last exon) and whole gene duplications. OR G>non-G at last base of exon if first 6 bases of the intron are not GTRRGT. If confirmed to cause a splice defect, then PVS1 should be used instead. OR Variants at IVS±1 or IVS±2^a,c where exon skipping or use of a cryptic splice site preserves reading frame and the altered region is critical to protein function^b. Not to be combined with PP3 and not to be used for a confirmed splice defect (see PVS1 for variants where patient mRNA assays indicate splicing aberration). Modification Type: General recommendation Moderate Nonsense/frameshift variant introducing premature termination codon between codons 1342 & 1360 in MSH6. Refer to Appendix for details. Modification Type: Gene-specific Supporting 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 A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect). OR Variants affecting the same non-canonical splice nucleotide as a confirmed pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. Modification Type: General recommendation Moderate A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change as a previously established Likely Pathogenic missense variant with normal RNA result, and PM2_supporting is met. Otherwise, if the previously established Likely pathogenic missense variant truly is a splice defect, the new missense variant also has to be investigated on a functional level for RNA splicing. Modification Type: General recommendation Supporting Instructions: SpliceAI masked score option should be checked on. 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 ≥ 4 de novo points Modification Type: Disease-specific Strong 2 or 3 de novo points Modification Type: Disease-specific Moderate 1 de novo point Modification Type: Disease-specific Supporting 0.5 de novo points Modification Type: Disease-specific Instructions: Proband with a de novo variant with both maternity and paternity confirmed in a case with MMR deficient LS spectrum tumor* (i.e. MSI/IHC consistent with affected gene, with no MLH1 methylation in tumor tissue, with the exception of MLH1 constitutional promoter methylation. If there is no tumor data, see PS2_Moderate). Refer to Appendix for protein expression consistent with variant location. 2 points per proband OR Proband with a de novo variant with both maternity and paternity confirmed in a case with LS spectrum tumor* (with no tumor data for MSI/IHC/methylation, otherwise see PS2). 1 point per proband OR Proband with assumed de novo variant and maternity and/or paternity unconfirmed with LS spectrum tumor* (No tumor data for MSI/IHC/methylation). 0.5 points per proband *Lynch Syndrome (LS) tumors include: colorectal/colon/rectal, endometrial, ovarian, small bowel/small intestine, renal pelvis, ureter, and stomach/gastric carcinomas, sebaceous skin tumors (adenomas and carcinomas), gliomas. 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 Calibrated functional assays with functional odds for Pathogenicity > 18.7 Modification Type: General recommendation Moderate Calibrated functional assays with functional odds for pathogenicity >4.3 and <= 18.7 OR MMR function defect following functional assay flowchart* OR Variants with monoallelic expression: complete loss of expression (<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. Modification Type: General recommendation Supporting Calibrated functional odds for Pathogenicity >2.08 and <= 4.3 Modification Type: General recommendation Instructions: Refer to file ‘Functional assay SVI documentation (MMR genes)’ for calibrated functional assays. *The functional assay flowchart is a general framework for evaluating functional assays that were already performed, or from historic publications, not for prospective studies on variants. The information describing these assays are generic. The VCEP recommends use of the calibrated assays for prospective testing. 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 Not Applicable Comments: Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting.
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 Supporting Not Applicable Comments: There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data).
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/extremely rare (<1 in 50,000 alleles) in gnomAD v4 dataset Modification Type: General recommendation 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 ≥ 4 points Modification Type: Disease-specific Strong ≥ 2 and < 4 points Modification Type: Disease-specific Moderate ≥1 and < 2 points Modification Type: Disease-specific Supporting = 0.5 points Modification Type: Disease-specific Instructions: Co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD as per Aronson et al 2022 - Refer to “Table for CMMRD diagnosis.pdf”. For MLH1 variants - the variant has to meet PM2_Supporting criteria. Classification/zygosity of other variant: Pathogenic/Likely Pathogenic in trans: 1.0 point; Pathogenic/Likely Pathogenic - phase unknown: 0.5 points Sum all cases with the above evidence to determine the PM3 strength. 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 Supporting Not Applicable Comments: Protein length change from an in-frame variant is not used due to lack of evidence.
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 was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5_Supporting if other variant is Likely Pathogenic due to a missense alteration. Modification Type: General recommendation Supporting Missense change at an amino acid residue where a different missense change was classified as Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5_Supporting if PP3 is supporting for the missense change. Use PM5 if other variant is Pathogenic due to a missense alteration. Modification Type: General recommendation 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: Proband with assumed de novo variant and maternity and/or paternity unconfirmed with LS spectrum tumor* (No tumor data for MSI/IHC/methylation). 0.5 points per proband Multiple probands meeting PS2/PS2_moderate and PM6/PM6_Strong criteria can be combined to increase evidence strength - apply PS2 or PS2_VeryStrong for these criteria combinations. *Lynch Syndrome (LS) tumors include: colorectal/colon/rectal, endometrial, ovarian, small bowel/small intestine, renal pelvis, ureter, and stomach/gastric carcinomas, sebaceous skin tumors (adenomas and carcinomas), gliomas. Not Applicable Comments: Please see 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 Co-segregation with disease in pedigree(s) with a combined* Bayes Likelihood Ratio^h >18.7 in ≥2 families. Modification Type: General recommendation Moderate Co-segregation with disease in pedigree(s) with a combined* Bayes Likelihood Ratio^h >4.3 & ≤18.7. Modification Type: General recommendation Supporting Co-segregation with disease in pedigree(s) with a combined* Bayes Likelihood Ratio^h >2.08 & ≤4.3. Modification Type: General recommendation Instructions: *For multiple pedigrees, results are combined by multiplying together. Recommended segregation analysis tool: COOL(COsegregation OnLine) v3 https://fenglab.chpc.utah.edu/cool3/manual.html Copy the example pedigree format and complete the fields to build the pedigree in text format. Refer to online manual for cancer types to enter into pedigree. Click on the 'Analysis' tab to view the webform for pedigree file upload and enter appropriate parameters for population and allele frequency. Penetrance file and relative risk file are not required for MMR genes. Use the 'overall Bayes Factor' to determine evidence strength. 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: Missense variant in a gene with low rate of benign missense changes does not apply.
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 Missense variant with HCI prior probability for pathogenicity >0.81 as per https://hci-priors.hci.utah.edu/PRIORS Modification Type: General recommendation Supporting Missense variant with HCI prior probability for pathogenicity >0.68 & ≤0.81 as per https://hci-priors.hci.utah.edu/PRIORS OR Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. Modification Type: General recommendation Instructions: SpliceAI masked score option should be checked on. For HCI-PRIORS, ensure correct gene is selected from the tabs, and enter the nucleotide number in either HGVS position or HG38 genomic co-ordinate and click ‘view’. The output shows 3 substitutions at the nucleotide location, with probability based on splicing and protein predictions. Ensure the ‘applicable prior’ is used that corresponds to the variant under review. 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 ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families using a standard panel of 5-10 markers^g or tumor genome and/or loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4_Strong. Independent tumors can be from the same patient/family. Modification Type: Disease-specific Moderate 2 independent CRC/Endometrial MSI-H tumors using a standard panel of 5-10 markers^g or tumor genome and/or loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4_Moderate. Modification Type: Disease-specific Supporting 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^g or tumor genome and/or loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4. Modification Type: Disease-specific Instructions: ^gStandard MSI markers panel: BAT25, BAT26, BAT40, BAT34, D5S346, D17S250, ACTC, D18S55, D10S197, MYCL; D2S123, D18S69; NR21, NR24, NR27 Protein Expression and consistency with variant location: IHC evidence should be consistent with the variant gene and the protein that is tested and take into account the MutSα and MutLα heterodimers: MLH1 and PMS2 loss is consistent with an MLH1 pathogenic variant, MSH2 and MSH6 loss is consistent with an MSH2 pathogenic variant, MSH6 loss is consistent with an MSH6 pathogenic variant, and PMS2 loss is consistent with a PMS2 pathogenic variant. 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 GnomAD v4 Grpmax filtering allele frequency ≥ 0.0022 (0.22%) and variant is excluded as founder pathogenic variant. 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 GnomAD v4 Grpmax filtering allele frequency ≥ 0.00022 and < 0.0022 (0.022-0.22%) and variant is excluded as founder pathogenic variant. 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 Co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^f), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. 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 Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05 OR Synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. Modification Type: General recommendation Moderate Supporting Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48 OR Variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart. Modification Type: General recommendation Instructions: Refer to file ‘Functional assay SVI documentation (MMR genes)’ for calibrated functional assays. *The functional assay flowchart is a general framework for evaluating functional assays that were already performed, or from historic publications, not for prospective studies on variants. The information describing these assays are generic. The VCEP recommends use of the calibrated assays for prospective testing. 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 co-segregation with disease in pedigree(s) with a combined* Bayes Likelihood Ratio^h <0.05. Modification Type: General recommendation Moderate Supporting Lack of co-segregation with disease in pedigree(s) with a combined* Bayes Likelihood Ratio^h >0.05 & ≤0.48. Modification Type: General recommendation Instructions: *For multiple pedigrees, results are combined by multiplying together. Recommended segregation analysis tool: COOL(COsegregation OnLine) v3 https://fenglab.chpc.utah.edu/cool3/manual.html Copy the example pedigree format and complete the fields to build the pedigree in text format. Refer to online manual for cancer types to enter into pedigree. Click on the 'Analysis' tab to view the webform for pedigree file upload and enter appropriate parameters for population and allele frequency. Penetrance file and relative risk file are not required for MMR genes. Use the 'overall Bayes Factor' to determine evidence strength. 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: Missense variant in a gene where only loss of function causes disease is 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 Not Applicable Comments: BS2 is used instead.
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: In-frame deletions/insertions in a repetitive region without a known function is not used.
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 Missense variant with HCI-prior probability of pathogenicity <0.11 as per https://hci-priors.hci.utah.edu/PRIORS OR For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023. Modification Type: General recommendation Instructions: SpliceAI masked score option should be checked on. For HCI-PRIORS, ensure correct gene is selected from the tabs, and enter the nucleotide number in either HGVS position or HG38 genomic co-ordinate and click ‘view’. The output shows 3 substitutions at the nucleotide location, with probability based on splicing and protein predictions. Ensure the ‘applicable prior’ is used that corresponds to the variant under review. 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 ≥ 4 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^f with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation OR ≥2 BRAF V600E (CRC only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss. Modification Type: Disease-specific Moderate Supporting 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^f with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation. OR 1 BRAF V600E (Colon only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss. Modification Type: Disease-specific 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 A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4. Modification Type: General recommendation Not Applicable

Rules for Combining Criteria

Pathogenic

1 Very Strong (PVS1) AND ≥ 1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong)

1 Very Strong (PVS1) AND ≥ 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Very Strong (PVS1) AND ≥ 2 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 2 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND ≥ 3 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Very Strong (PVS1) AND ≥ 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

Likely Pathogenic

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND 1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND ≥ 2 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

≥ 3 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 2 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate) AND ≥ 4 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

1 Strong (PVS1_Strong, PS1, PS2, PS3, PP1_Strong, PP4_Strong) AND 2 Moderate (PVS1_Moderate, PS1_Moderate, PS2_Moderate, PS3_Moderate, PM3, PM5, PM6, PP1_Moderate, PP3_Moderate, PP4_Moderate)

1 Very Strong (PVS1) AND 1 Supporting (PS3_Supporting, PM2_Supporting, PM5_Supporting, PP1, PP3, PP4)

Benign

≥ 2 Strong (BS1, BS2, BS3, BS4, BP5_Strong)

1 Stand Alone (BA1)

Likely Benign

1 Strong (BS1, BS2, BS3, BS4, BP5_Strong) AND 1 Supporting (BS3_Supporting, BS4_Supporting, BP4, BP5, BP7)

≥ 2 Supporting (BS3_Supporting, BS4_Supporting, BP4, BP5, BP7)

Files & Images

Appendix: Appendix

MMR functional domains: MMR functional domains

VCEP pilot variants - MMR: VCEP pilot variants - MMR

MMR Functional assay flowchart: Flowchart demonstrating the interpretation of functional assay data

Functional assay SVI documentation: Functional assay SVI documentation (MMR genes)

Table for CMMRD diagnosis: Scoring system for aiding CMMRD diagnosis

MMR PVS1 Decision Tree: PVS1 Decision Tree for MMR genes

References

1. Belman S Parsons MT et al. Considerations in assessing germline variant pathogenicity using cosegregation analysis. Genet Med (2020) 22 (12) p. 2052-2059. 10.1038/s41436-020-0920-4 32773770

2. Aronson M Colas C et al. Diagnostic criteria for constitutional mismatch repair deficiency (CMMRD): recommendations from the international consensus working group. J Med Genet (2022) 59 (4) p. 318-327. 10.1136/jmedgenet-2020-107627 33622763

3. Li S Qian D et al. Tumour characteristics provide evidence for germline mismatch repair missense variant pathogenicity. J Med Genet (2020) 57 (1) p. 62-69. 10.1136/jmedgenet-2019-106096 31391288

4. Canson DM Dumenil T et al. The splicing effect of variants at branchpoint elements in cancer genes. Genet Med (2022) 24 (2) p. 398-409. 10.1016/j.gim.2021.09.020 34906448

5. Cyr JL Brown GD et al. The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex. Mol Carcinog (2012) 51 (8) p. 647-58. 10.1002/mc.20838 21837758

6. Drost M Tiersma Y et al. A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome. Genet Med (2019) 21 (7) p. 1486-1496. 10.1038/s41436-018-0372-2 30504929

7. Drost M Tiersma Y et al. Two integrated and highly predictive functional analysis-based procedures for the classification of MSH6 variants in Lynch syndrome. Genet Med (2020) 22 (5) p. 847-856. 10.1038/s41436-019-0736-2 31965077

8. Rath A Radecki AA et al. A calibrated cell-based functional assay to aid classification of MLH1 DNA mismatch repair gene variants. Hum Mutat (2022) 43 (12) p. 2295-2307. 10.1002/humu.24462 36054288

9. Rayner E Tiersma Y et al. Predictive functional assay-based classification of PMS2 variants in Lynch syndrome. Hum Mutat (2022) 43 (9) p. 1249-1258. 10.1002/humu.24387 35451539

10. Tavtigian SV Greenblatt MS et al. Modeling the ACMG/AMP variant classification guidelines as a Bayesian classification framework. Genet Med (2018) 20 (9) p. 1054-1060. 10.1038/gim.2017.210 29300386

11. Abou Tayoun AN Pesaran T et al. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum Mutat (2018) 39 (11) p. 1517-1524. 10.1002/humu.23626 30192042

12. Thompson BA Walters R et al. Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation. Front Genet (2020) 11 p. 798. 10.3389/fgene.2020.00798 32849802

13. Whiffin N Minikel E et al. Using high-resolution variant frequencies to empower clinical genome interpretation. Genet Med (2017) 19 (10) p. 1151-1158. 10.1038/gim.2017.26 28518168

14. Walker LC Hoya M et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet (2023) 110 (7) p. 1046-1067. 10.1016/j.ajhg.2023.06.002 37352859