MSH6 c.1474A>G, p.(Met492Val)

Reasoning

The variant is a missense substitution, NM_000179.3:c.1474A>G (p.Met492Val/p.M492V). The governing MSH6 VCEP specification assigns BP4_Supporting to a missense variant with HCI prior probability <0.11. The exact VCEP HCI prior table entry reports a pathogenicity probability of 0.0086, which meets that threshold. The VCEP-assigned HCI result governs this criterion; REVEL and SpliceAI are not additionally counted.

Rule

MSH6 VCEP BP4: missense HCI prior probability <0.11 = Supporting.

Evidence considered

• The MSH6 VCEP specification defines BP4_Supporting for missense variants with HCI prior probability <0.11.
• The exact HCI-PRIORS-MSH6 entry for c.1474A>G (p.M492V) reports pathogenicity probability 0.0086, which meets the governing BP4 threshold.
• Because the governing VCEP provides an exact pre-assigned HCI prior result, it is applied directly and no additional predictor is combined with it.

Sources

Reasoning

The MSH6 VCEP requires a different nucleotide change encoding the same amino-acid substitution, previously established by the VCEP as Pathogenic, or an equivalent splice-site circumstance. The reviewed variant-specific literature reports only NM_000179.3:c.1474A>G, p.Met492Val itself, and no qualifying alternate nucleotide change encoding p.Met492Val established as Pathogenic was identified. The variant is missense and SpliceAI predicts minimal splice impact (maximum delta 0.014), so the splice alternative does not apply.

Rule

MSH6 VCEP PS1: same amino-acid change from a different nucleotide previously established by this VCEP as Pathogenic, not a splice defect; alternatively, the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse SpliceAI prediction.

Evidence considered

• The MSH6 VCEP PS1 rule requires a different underlying nucleotide change encoding the same amino-acid change, previously established by the VCEP as Pathogenic, or a qualifying non-canonical splice-site circumstance.
• The exact p.Met492Val variant is reported in family 208, a Danish cohort, two HNPCC families, and a cell-free assay paper; these reports concern the target c.1474A>G variant and do not identify a different nucleotide change encoding p.Met492Val that the VCEP established as Pathogenic.
• The variant is a missense substitution and SpliceAI maximum delta is 0.014, below the VCEP's splice-impact pathway threshold of 0.2 for predicted splice defects.

Sources

Reasoning

The MSH6 p.Met492Val variant was tested in a cell-free in vitro mismatch-repair complementation assay using MSH2/MSH6-deficient LoVo nuclear extract and a G·T mismatch substrate. The assay reported the variant as repair proficient, not functionally deficient, and therefore provides no support for PS3. No calibrated functional odds for pathogenicity were reported; consequently, the MSH6 VCEP thresholds (>2.08 supporting, >4.3 moderate, >18.7 strong) cannot be applied.

Rule

Calibrated functional assays with functional odds for pathogenicity >2.08 and <=4.3 support PS3 Supporting; >4.3 and <=18.7 support PS3 Moderate; >18.7 supports PS3 Strong; alternatively, an MMR function defect following the functional assay flowchart supports PS3 Moderate.

Evidence considered

• The MSH6-specific VCEP requires calibrated functional odds for pathogenicity >2.08 for PS3 Supporting, >4.3 and <=18.7 for PS3 Moderate, or >18.7 for PS3 Strong; alternatively, a documented MMR function defect can support PS3 Moderate.
• Drost et al. tested MSH6 M492V (c.1474A>G) in a cell-free in vitro mismatch-repair complementation assay using variant MSH6 with wild-type MSH2 in MSH2/MSH6-deficient LoVo nuclear extract containing a G·T mismatch substrate; the variant was reported as repair proficient.
• A repair-proficient result is directionally inconsistent with a damaging functional result and does not meet the MSH6 VCEP requirements for PS3.

Still missing

• No calibrated functional odds for pathogenicity were reported.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 V2.0 specification requires PM2 Supporting when the allele frequency is <0.00002 (<1 in 50,000 alleles) in gnomAD v4. The gnomAD v4.1 total allele frequency is 0.000101608 (164/1,614,046), and its grpmax filtering allele frequency is 0.00008673; both exceed 0.00002.

Rule

Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset

Evidence considered

• The MSH6 V2.0 VCEP specifies PM2 Supporting for allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports 164/1,614,046 alleles, total allele frequency 0.000101608, grpmax filtering allele frequency 0.00008673, and zero homozygotes; these values exceed 0.00002.

Sources

Reasoning

The variant is a missense substitution, NM_000179.3:c.1474A>G (p.Met492Val/p.M492V). The governing MSH6 VCEP specification assigns PP3_Moderate when the HCI prior probability is >0.81 and PP3_Supporting when it is >0.68 and <=0.81. The exact VCEP HCI prior table entry for c.1474A>G reports a pathogenicity probability of 0.0086, so neither PP3 threshold is met. The VCEP-assigned HCI result governs this criterion; REVEL and SpliceAI are not additionally counted.

Rule

MSH6 VCEP PP3: missense HCI prior probability >0.81 = Moderate; >0.68 and <=0.81 = Supporting.

Evidence considered

• The MSH6 VCEP specification defines PP3_Moderate for missense variants with HCI prior probability >0.81 and PP3_Supporting for probability >0.68 and <=0.81.
• The exact HCI-PRIORS-MSH6 entry for c.1474A>G (p.M492V) reports pathogenicity probability 0.0086, which is below both governing PP3 thresholds.
• Because the governing VCEP provides an exact pre-assigned HCI prior result, it is applied directly and no additional predictor is combined with it.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 V2.0 specification requires gnomAD v4 grpmax filtering allele frequency >=0.0022 (0.22%) for BA1, with exclusion of founder pathogenic variants. The gnomAD v4.1 grpmax filtering allele frequency is 0.00008673 (0.008673%), well below 0.0022; no founder-variant exception is relevant to this frequency result.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.0022 (0.22%) and variant is excluded as founder pathogenic variant.

Evidence considered

• The MSH6 V2.0 VCEP rule specifies BA1 at gnomAD v4 grpmax filtering allele frequency >=0.0022 (0.22%), provided the variant is not a founder pathogenic variant.
• gnomAD v4.1 reports grpmax filtering allele frequency 0.00008673, with total allele frequency 0.000101608 and zero homozygotes.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 V2.0 specification requires gnomAD v4 grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022-0.22%) for BS1, with exclusion of founder pathogenic variants. The gnomAD v4.1 grpmax filtering allele frequency is 0.00008673 (0.008673%), below the lower threshold.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.00022 and < 0.0022 (0.022-0.22%) and variant is excluded as founder pathogenic variant.

Evidence considered

• The MSH6 V2.0 VCEP rule specifies BS1 at gnomAD v4 grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022-0.22%), provided the variant is not a founder pathogenic variant.
• gnomAD v4.1 reports grpmax filtering allele frequency 0.00008673, which is below 0.00022.

Sources

Reasoning

The MSH6-specific specification requires a documented de novo variant in a proband, with maternity and paternity confirmed and an MMR-deficient Lynch-spectrum tumor; the available case evidence does not document de novo status, parental testing, or qualifying tumor findings. Therefore PS2 cannot be assigned.

Rule

MSH6 InSiGHT Version 2.0 PS2: 2 de novo points per proband when de novo status and both maternity and paternity are confirmed with qualifying MMR-deficient Lynch-spectrum tumor evidence; strength is Very Strong at >=4 points, Strong at 2-3.5, Moderate at 1-1.5, and Supporting at 0.5.

Evidence considered

• The MSH6 InSiGHT specification assigns PS2 points only for a proband with a de novo variant and confirmed parental relationships, with the specified Lynch-spectrum tumor evidence; no such case-level documentation is present.
• Published reports identify p.Met492Val in selected Lynch-syndrome families, but they do not establish a de novo event in this proband.

Still missing

• Documented proband-level de novo occurrence is missing.
• Maternity and paternity confirmation are missing.
• Qualifying MMR-deficient Lynch-spectrum tumor data, including MSI/IHC and relevant methylation assessment, are missing.

Sources

Reasoning

The applicable MSH6 InSiGHT VCEP specification limits PM3 to co-occurrence of the assessed variant with a known pathogenic or likely pathogenic variant in the same gene in a patient with clinical features consistent with constitutional mismatch repair deficiency (CMMRD). The case evidence reports MSH6 c.1474A>G (p.Met492Val) in Lynch-syndrome/HNPCC cohorts, but the available reports do not document a second pathogenic/likely pathogenic MSH6 variant, affected-proband CMMRD features, or phase established by parental or offspring testing. Therefore no PM3 points can be assigned: 0 documented in-trans points and 0 documented phase-unknown points.

Rule

MSH6 InSiGHT VCEP v2.0 PM3: co-occurrence with a known pathogenic/likely pathogenic same-gene variant in a patient with CMMRD-consistent clinical features; pathogenic/likely pathogenic in trans = 1.0 point, phase unknown = 0.5 point; sum points across cases; >=4 very strong, 2-3.5 strong, 1-1.5 moderate, and 0.5 supporting.

Evidence considered

• The MSH6 InSiGHT VCEP PM3 rule requires co-occurrence with a known pathogenic/likely pathogenic same-gene variant in a patient with CMMRD-consistent clinical features; a pathogenic/likely pathogenic variant in trans is 1.0 point and phase-unknown co-occurrence is 0.5 point.
• The exact variant was reported as M492V in family 208, but the report gives no second MSH6 pathogenic variant, phase, or CMMRD assessment; MSI, immunohistochemistry, and monoallelic mutation analysis were not performed for that family.
• The exact variant was reported in one Danish Lynch syndrome family as an exon 4 missense alteration of unknown clinical significance, without segregation or second-allele data.
• The exact variant was reported in families H785 and H425 as an unclassified variant; the report does not establish a pathogenic second MSH6 allele or phase.

Still missing

• A documented pathogenic or likely pathogenic variant in the same MSH6 patient is needed.
• CMMRD-compatible clinical features for that patient are needed.
• Parental or offspring testing establishing trans phase is needed for 1.0 PM3 point; otherwise documented phase-unknown co-occurrence could provide 0.5 point.

Sources

Reasoning

The MSH6 VCEP PM5 rule requires a different missense change at the same amino-acid residue classified by the VCEP as Pathogenic or Likely Pathogenic on the protein level and not because of aberrant splicing; PM5 also requires PP3 supporting for the target missense variant. The available case evidence identifies no same-residue comparator at MSH6 codon 492, and the comparator-harvesting record reports zero same-residue candidates after a soft retrieval failure. Therefore a qualifying comparator cannot be established from the available evidence, and PM5 is not assessed rather than applied.

Rule

MSH6 VCEP PM5: same-residue different missense change classified as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing; use only when PP3 supporting is met.

Evidence considered

• The MSH6 VCEP PM5 rule requires a different missense change at the same residue classified by the VCEP as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing; the rule also requires PP3 supporting for the target missense variant.
• The available comparator record contains zero same-residue candidates for MSH6 p.Met492Val and documents an HTTP 429 retrieval failure, so it cannot establish that no comparator exists.
• The reviewed papers report the target M492V variant itself and do not provide a qualifying alternate missense comparator at residue 492.

Still missing

• A reliable search for alternate missense variants at MSH6 codon 492 and their VCEP classifications is required.

Sources

Reasoning

The MSH6-specific PP1 rule requires a combined cosegregation Bayes likelihood ratio greater than 2.08 for Supporting, greater than 4.3 through 18.7 for Moderate, or greater than 18.7 in at least two families for Strong. Although p.Met492Val was reported in families, the available publications provide no affected/unaffected carrier counts, meioses, pedigree-level segregation analysis, or Bayes likelihood ratio for this variant. PP1 therefore cannot be assigned.

Rule

MSH6 InSiGHT Version 2.0 PP1: combined cosegregation Bayes likelihood ratio >18.7 in >=2 families for Strong, >4.3 and <=18.7 for Moderate, and >2.08 and <=4.3 for Supporting.

Evidence considered

• The MSH6 InSiGHT specification sets PP1 thresholds at a combined Bayes likelihood ratio >2.08 and <=4.3 for Supporting, >4.3 and <=18.7 for Moderate, and >18.7 in at least two families for Strong.
• The variant was listed in family 208 in a cohort of 59 U.S. HNPCC families, but the report does not provide segregation analysis for M492V; microsatellite instability, immunohistochemistry, and monoallelic mutation analysis were not performed for family 208.
• The variant was identified in one family in a Danish Lynch syndrome cohort, but the report does not report segregation data for this variant.
• The variant was reported in families H785 and H425, but the report classifies it as an unclassified variant and provides no pedigree-level cosegregation calculation.

Still missing

• Complete pedigrees with phenotype status and variant status for informative relatives are missing.
• The number of informative meioses and a combined Bayes likelihood ratio are missing.
• Independent family-level segregation data are not available.

Sources

Reasoning

The governing MSH6 specification allows PP4 Supporting for 1 CRC or endometrial MSI-H tumor assessed with a standard marker panel or tumor genome, and/or loss of MMR protein expression consistent with the variant location; it allows Moderate for 2 independent qualifying tumors and Strong for at least 3 qualifying tumors in at least 2 families. The exact variant was reported in two families in PMID:22495361, but H785 had normal immunohistochemistry and H425 had normal pMSH6 with absent pPMS2, which is inconsistent with MSH6 loss; PMID:12658575 records MSI and IHC as not performed for family 208. No qualifying MSI-H tumor or MSH6-consistent protein-loss result is established, so the available evidence is insufficient to adjudicate PP4 rather than treating the incomplete tumor data as affirmative or negative evidence.

Rule

Supporting: 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location; Moderate: 2 independent qualifying tumors; Strong: >=3 independent qualifying tumors in >=2 families. MSI-H tumor with inconsistent protein expression does not meet the corresponding PP4 strength.

Evidence considered

• The MSH6 VCEP PP4 rules require 1 qualifying CRC/endometrial MSI-H tumor or MSH6-consistent MMR protein loss for Supporting, 2 independent qualifying tumors for Moderate, or at least 3 independent tumors in at least 2 families for Strong.
• PMID:22495361 reports c.1474A>G/p.Met492Val in families H785 and H425; H785 had normal immunohistochemistry, and H425 had normal pMSH6 with absent pPMS2, an inconsistent protein pattern.
• PMID:12658575 reports M492V in family 208, with microsatellite instability analysis and immunohistochemistry not performed.
• No exact-variant evidence in the reviewed records establishes a qualifying MSI-H tumor or MSH6-consistent protein-loss result.

Still missing

• A qualifying exact-variant-associated CRC or endometrial tumor with standard-panel or genome-defined MSI-H status, or MSH6-consistent protein loss, is not documented.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 V2.0 specification limits BS2 Strong to co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45, or another Lynch-spectrum cancer above its median age of onset, without clinical evidence of CMMRD; phase must be confirmed through parental or offspring testing. The available case evidence does not document such a co-occurrence, cancer-age qualification, CMMRD assessment, or confirmed phase.

Rule

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), and who has no previous or current evidence of clinical manifestations of CMMRD. Confirmation of phase requires testing of parents or offspring.

Evidence considered

• The MSH6 V2.0 VCEP defines BS2 Strong as qualifying in-trans co-occurrence with a known pathogenic variant, an appropriately aged Lynch-spectrum cancer, no CMMRD manifestations, and confirmed phase.
• The available variant-specific reports identify MSH6 p.Met492Val but do not provide the required qualifying in-trans co-occurrence and phase evidence.

Still missing

• A documented known pathogenic MSH6 variant in trans is required.
• The affected individual's qualifying Lynch-spectrum cancer and age criterion must be documented.
• Absence of CMMRD manifestations and parental or offspring phase confirmation must be documented.

Sources

Reasoning

The MSH6 p.Met492Val variant showed repair-proficient activity in one cell-free mismatch-repair assay, which is biologically relevant and supports a benign functional direction. However, the reported assay did not provide calibrated functional odds, and the available evidence does not document the MSH6 VCEP flowchart's required concordant independent assay framework or variant-specific proficient results in both protein- and mRNA-based assays. Therefore BS3 cannot be assigned at Strong or Supporting strength from the available evidence.

Rule

Calibrated functional assays with functional odds for pathogenicity <=0.05 support BS3 Strong; >0.05 and <=0.48 support BS3 Supporting; alternatively, variant-specific proficient function in protein and mRNA-based laboratory assays may support BS3 Supporting under the MMR functional assay flowchart.

Evidence considered

• The MSH6-specific VCEP defines BS3 Strong as calibrated functional odds for pathogenicity <=0.05, or no mRNA aberration for synonymous/intronic variants with appropriate NMD-inhibition testing; BS3 Supporting requires calibrated odds >0.05 and <=0.48 or variant-specific proficient protein and mRNA-based laboratory assays under the MMR functional assay flowchart.
• Drost et al. reported MSH6 M492V (c.1474A>G) as repair proficient in a cell-free in vitro mismatch-repair complementation assay using MSH2/MSH6-deficient LoVo nuclear extract and a G·T mismatch substrate.
• This single protein-level assay is relevant benign-direction functional evidence, but it does not supply calibrated functional odds or establish the VCEP's required concordant protein-plus-mRNA evidence for BS3 assignment.

Still missing

• No calibrated functional odds for pathogenicity were reported.
• No independent concordant functional assay is documented.
• No variant-specific mRNA-based functional result is documented.

Sources

Reasoning

The MSH6-specific BS4 rule requires documented lack of cosegregation with a combined Bayes likelihood ratio below 0.05 for Strong or greater than 0.05 and up to 0.48 for Supporting. The available evidence does not provide affected relatives who lack the variant, a pedigree-level non-segregation analysis, or a Bayes likelihood ratio; absence of reported segregation is not itself evidence of non-segregation. BS4 therefore cannot be assigned.

Rule

MSH6 InSiGHT Version 2.0 BS4: lack of cosegregation with combined Bayes likelihood ratio <0.05 for Strong or >0.05 and <=0.48 for Supporting.

Evidence considered

• The MSH6 InSiGHT specification defines BS4 Strong as a combined Bayes likelihood ratio <0.05 and BS4 Supporting as >0.05 and <=0.48.
• The available reports do not document affected relatives who tested negative for p.Met492Val or provide a non-segregation likelihood ratio; two reports explicitly lack segregation analysis for the variant.

Still missing

• Affected relatives tested negative for the variant are not documented.
• A pedigree-level non-segregation analysis is missing.
• A combined Bayes likelihood ratio for lack of segregation is missing.

Sources

Reasoning

The governing MSH6 specification defines BP5 Supporting as 2 or 3 tumors with MSS and/or no loss of MMR protein expression and/or an LS-spectrum tumor with MMR loss inconsistent with the gene containing the variant; Strong requires at least 4 such tumors, or the specified BRAF V600E/MLH1-methylation alternatives. No numeric likelihood-ratio threshold is part of this MSH6 BP5 rule, and no qualifying set of 2 or more exact-variant-associated tumors is documented. PMID:22495361 reports normal immunohistochemistry in H785 and normal pMSH6 with absent pPMS2 in H425, but does not establish the required BP5 tumor set; PMID:12658575 records the relevant testing as not performed.

Rule

Supporting: 2 or 3 tumors with MSS and/or no loss of MMR protein expression and/or LS-spectrum tumors with MMR loss inconsistent with the gene demonstrating genetic variation; Strong: >=4 such tumors, or >=2 BRAF V600E/MLH1-methylation findings with MSI-H/MLH1 loss or 1 such finding for Supporting.

Evidence considered

• The MSH6 VCEP BP5 rule requires 2 or 3 qualifying tumors for Supporting and at least 4 qualifying tumors for Strong, with specified tumor phenotype and MMR-protein findings.
• PMID:22495361 reports two families with the exact variant, but the available H785 and H425 immunohistochemistry findings do not establish the specified BP5 tumor pattern or a qualifying tumor series.
• PMID:12658575 records MSI and IHC as not performed for the exact-variant family.

Still missing

• At least 2 qualifying exact-variant-associated tumors with MSS/no MMR-protein loss or an allowed inconsistent-loss pattern are not documented.

Sources