MSH6 c.2731C>T, p.(Arg911Ter)

Reasoning

NM_000179.3:c.2731C>T is a nonsense variant producing NP_000170.1:p.(Arg911Ter), an early premature termination codon at codon 911. The applicable InSiGHT/ClinGen MSH6 VCEP specification assigns PVS1 Very Strong to nonsense or frameshift variants introducing a premature termination codon at or before codon 1341 in MSH6. Codon 911 is within that specified range. The variant is represented on the relevant MSH6 RefSeq transcript and no evidence in the case bundle indicates a transcript mismatch, repeat-region exception, noncritical exon, or NMD-escape concern that would require downgrading under the applicable rule.

Rule

MSH6 VCEP PVS1 Very Strong: nonsense/frameshift variant introducing a premature termination codon at or before codon 1341.

Evidence considered

• The MSH6 Version 2.0 VCEP rule specifies PVS1 Very Strong for a nonsense or frameshift variant introducing a premature termination codon at or before codon 1341 in MSH6.
• The case variant is NM_000179.3:c.2731C>T with the predicted protein consequence NP_000170.1:p.(Arg911Ter); codon 911 is below the VCEP cutoff of 1341.
• The ClinVar source record identifies the exact variant as NM_000179.3(MSH6):c.2731C>T (p.Arg911Ter), supporting transcript and protein-consequence relevance without relying on ClinVar's final classification.

Sources

Reasoning

The MSH6-specific InSiGHT VCEP PM2 rule applies at Supporting strength when the allele frequency is less than 0.00002 in gnomAD v4. gnomAD v4.1 reports a total allele frequency of 1.36314e-05 and a Grpmax FAF of 8.78e-06, with zero homozygotes; both are below the VCEP threshold. PM2_Supporting is met.

Rule

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

Evidence considered

• The MSH6 InSiGHT VCEP Version 2.0 specifies PM2_Supporting for an absent or extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports total AF 1.36314e-05 (22/1,613,924 alleles), Grpmax FAF 8.78e-06, and zero observed homozygotes.

Sources

Reasoning

The exact variant NM_000179.3:c.2731C>T (p.Arg911Ter) was reported in two independent endometrial cancer probands whose tumors were MSI-H. This satisfies the MSH6 VCEP PP4_Moderate rule requiring two independent CRC/endometrial MSI-H tumors using a standard marker panel or tumor genome and/or consistent MMR-protein loss.

Rule

ClinGen InSiGHT MSH6 Version 2.0 PP4_Moderate: 2 independent CRC/endometrial MSI-H tumors using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location.

Evidence considered

• The MSH6 VCEP specifies PP4_Moderate for two independent CRC or endometrial MSI-H tumors using a standard 5-10 marker panel or tumor genome and/or MMR-protein loss consistent with the variant location.
• Goodfellow et al. reported the exact C>T substitution at codon 911 (c.2731C>T, Arg→Stop) in endometrial cancer patients 1335 and 1497; both were among the germline MSH6 mutation carriers with MSI-positive cancers, and the paper states that all probands' cancers had high-level MSI.
• Buttin et al. reported the exact R911Z mutation in MSH6 kindreds 1335 and 1497 and stated that all probands' cancers had high-level MSI; the study used MSI testing and MMR-protein immunohistochemistry.

Sources

Reasoning

Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Pathogenic.

Rule

Requested rule: exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification supports PP5 at Supporting strength.

Evidence considered

• The ClinVar record exactly matches NM_000179.3(MSH6):c.2731C>T (p.Arg911Ter) and reports Pathogenic reviewed by expert panel (three stars), with the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) identified as the expert panel.
• Only the exact-variant expert-panel classification was used for PP5; clinical-laboratory submissions, non-expert assertions, and the aggregate label were not independently treated as PP5 evidence.
• ClinVar expert panel classification

Sources

Reasoning

The governing MSH6 InSiGHT VCEP specification assigns PP3_Supporting for a predicted splice defect at a non-canonical splice nucleotide when SpliceAI delta score is >= 0.2. For NM_000179.3:c.2731C>T, SpliceAI reports a maximum delta score of 0.005, so the specified splice-impact threshold is not met. The variant is a nonsense variant, and no calibrated missense PP3 pathway is applicable.

Rule

MSH6 InSiGHT V2.0 PP3_Supporting: predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2.

Evidence considered

• The MSH6 InSiGHT V2.0 specification states that PP3_Supporting may be applied for a predicted splice defect at a non-canonical splicing nucleotide using SpliceAI with delta score >= 0.2.
• SpliceAI scores for NM_000179.3:c.2731C>T are DS_AG 0.001, DS_AL 0.002, DS_DG 0.000, and DS_DL 0.005; maximum delta score is 0.005.
• Variant normalization identifies NM_000179.3:c.2731C>T as NP_000170.1:p.(Arg911Ter), a nonsense rather than missense variant.

Sources

Reasoning

The MSH6-specific InSiGHT VCEP BA1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.0022 (0.22%), with exclusion of a founder pathogenic variant. The gnomAD v4.1 Grpmax FAF is 8.78e-06, far below 0.0022; therefore BA1 is not met.

Rule

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

Evidence considered

• The MSH6 InSiGHT VCEP Version 2.0 specifies BA1 at gnomAD v4 Grpmax filtering allele frequency >=0.0022 (0.22%) and requires exclusion of a founder pathogenic variant.
• gnomAD v4.1 reports this variant with total AF 1.36314e-05 (22/1,613,924 alleles), zero homozygotes, and Grpmax FAF 8.78e-06.

Still missing

• No separate founder-variant determination is documented, but this does not affect the conclusion because the observed Grpmax FAF is far below the BA1 threshold.

Sources

Reasoning

The MSH6-specific InSiGHT VCEP BS1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.00022 and less than 0.0022 (0.022%-0.22%), with exclusion of a founder pathogenic variant. The gnomAD v4.1 Grpmax FAF is 8.78e-06, below 0.00022; therefore BS1 is not met.

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 InSiGHT VCEP Version 2.0 specifies BS1 at gnomAD v4 Grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022%-0.22%) and requires exclusion of a founder pathogenic variant.
• gnomAD v4.1 reports this variant with Grpmax FAF 8.78e-06; the highest listed population AF is 4.80108e-05 in Remaining individuals, also below 0.00022.

Still missing

• No separate founder-variant determination is documented, but this does not affect the conclusion because the observed Grpmax FAF is below the BS1 threshold.

Sources

Reasoning

The MSH6 VCEP BP5 rule requires at least two tumors with MSS and/or no loss of MMR-protein expression or an alternative MLH1/BRAF explanation. No such qualifying evidence was identified for this exact variant. Instead, the exact-variant reports describe MSI-H endometrial tumors, which do not satisfy BP5.

Rule

ClinGen InSiGHT MSH6 Version 2.0 BP5: 2-3 qualifying MSS/no-MMR-loss or inconsistent-MMR-loss tumors, or 1 BRAF V600E/MLH1-methylation finding, for Supporting; 4 or more tumors or 2 alternative-marker findings for Strong.

Evidence considered

• The MSH6 VCEP specifies BP5_Supporting for 2 or 3 qualifying tumors with MSS and/or no MMR-protein loss or one BRAF V600E/MLH1-methylated MSI-H tumor; BP5_Strong requires at least 4 qualifying tumors or at least 2 alternative-marker findings.
• The exact c.2731C>T variant was reported in MSI-positive/high-MSI endometrial tumors rather than qualifying MSS/no-loss tumors. No exact-variant BRAF V600E or MLH1-methylation evidence was identified.

Sources

Reasoning

The MSH6 InSiGHT VCEP specification requires a documented de novo variant in a proband with a Lynch syndrome spectrum tumor, with maternity and paternity confirmation; point values depend on tumor MSI/IHC/methylation data. The case bundle contains no proband-level parental testing, maternity/paternity confirmation, de novo status, or tumor evidence for this variant, so PS2 cannot be assigned.

Rule

InSiGHT MSH6 V2.0 PS2: 2 points per proband for de novo status with confirmed maternity and paternity and an MMR-deficient Lynch syndrome spectrum tumor; 1 point with confirmed maternity and paternity but no tumor data; 0.5 points for assumed de novo status with unconfirmed maternity and/or paternity and no tumor data. Strength: supporting at 0.5 points, moderate at 1-1.5 points, strong at 2-3.5 points, and very strong at >=4 points.

Evidence considered

• The applicable InSiGHT MSH6 V2.0 rule assigns 2 points per proband when both maternity and paternity are confirmed and the Lynch syndrome spectrum tumor is MMR deficient, 1 point when both are confirmed without tumor data, and 0.5 points for an assumed de novo variant with unconfirmed maternity and/or paternity and no tumor data.
• The consolidated case-evidence bundle does not provide case-specific parental testing, de novo documentation, proband phenotype, or tumor MSI/IHC/methylation results for NM_000179.3:c.2731C>T.

Still missing

• Proband-level documentation is needed.
• Maternity and paternity confirmation is needed.
• Constitutional parental genotypes and evidence that the variant is absent in both parents are needed.
• Tumor MSI/IHC and MLH1 methylation results are needed to determine the VCEP point value.

Sources

Reasoning

The applicable ClinGen InSiGHT MSH6 Version 2.0 specification requires calibrated functional odds for pathogenicity greater than 18.7 for PS3_Strong, greater than 4.3 and up to 18.7 or an MMR function defect demonstrated through the specified functional-assay flowchart for PS3_Moderate, and calibrated functional odds greater than 2.08 and up to 4.3 for PS3_Supporting. The case bundle provides no variant-specific functional assay result, calibrated functional odds, protein-function result, or RNA assay result demonstrating an MMR function defect for NM_000179.3:c.2731C>T (p.Arg911Ter). The available ClinVar evidence signal explicitly states that functional studies have not been reported for this variant. Therefore PS3 cannot be applied.

Rule

Calibrated functional assays with functional odds for pathogenicity > 18.7; or calibrated functional assays with functional odds for pathogenicity >4.3 and <= 18.7, or MMR function defect following functional assay flowchart; or calibrated functional odds for pathogenicity >2.08 and <= 4.3.

Evidence considered

• The MSH6 VCEP specification defines PS3_Strong as calibrated functional odds for pathogenicity >18.7, PS3_Moderate as calibrated functional odds >4.3 and <=18.7 or an MMR function defect following the functional assay flowchart, and PS3_Supporting as calibrated functional odds >2.08 and <=4.3.
• The case evidence bundle contains no variant-specific functional assay measurements or calibrated functional odds for NM_000179.3:c.2731C>T (p.Arg911Ter).
• The case bundle's ClinVar evidence signal reports: 'to our knowledge, functional studies have not been reported for this variant.'

Still missing

• Variant-specific validated functional assay data with appropriate positive, negative, and deficient controls are unavailable.
• No calibrated functional odds for pathogenicity are available.
• No variant-specific MMR protein or mRNA assay demonstrating an MMR function defect is available.

Sources

Reasoning

The applicable InSiGHT MSH6 v2.0 rule requires co-occurrence of this variant with a known pathogenic or likely pathogenic variant in MSH6 in a patient with clinical features consistent with constitutional mismatch repair deficiency, scored as 1.0 point when confirmed in trans or 0.5 points when phase is unknown. The reviewed case bundle does not provide an affected proband with a second pathogenic/likely pathogenic MSH6 variant, phase determination, or qualifying CMMRD features for NM_000179.3:c.2731C>T (p.Arg911Ter). Therefore no PM3 points can be assigned from the available evidence.

Rule

Co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD: 1.0 point if in trans; 0.5 points if phase is unknown; sum all qualifying cases. Strength: Supporting = 0.5 points, Moderate = 1-1.5 points, Strong = 2-3.5 points, Very Strong >=4 points.

Evidence considered

• The MSH6 InSiGHT v2.0 specification assigns 1.0 point for a pathogenic/likely pathogenic MSH6 variant confirmed in trans and 0.5 points when phase is unknown; PM3 strength is Supporting at 0.5 points, Moderate at 1-1.5 points, Strong at 2-3.5 points, and Very Strong at >=4 points.
• PMID:15098177 was reviewed because it reports MSH6 mutation kindreds and affected relatives, but the available text does not document a second pathogenic/likely pathogenic MSH6 variant in trans or cis with the reported R911Z mutation, nor does it establish phase or CMMRD features for this variant.

Still missing

• Affected-proband-level documentation of a second pathogenic or likely pathogenic MSH6 variant is absent.
• No parental, offspring, or other testing establishes trans or cis phase.
• No case-level documentation demonstrates clinical features consistent with CMMRD for a PM3 observation.

Sources

Reasoning

The InSiGHT MSH6 V2.0 specification requires cosegregation with disease and assigns strength using a combined Bayes likelihood ratio. The case bundle provides no variant-specific pedigree, carrier status by meiosis, affected/unaffected relative phenotypes, or calculated Bayes likelihood ratio for NM_000179.3:c.2731C>T, so PP1 cannot be assigned.

Rule

InSiGHT MSH6 V2.0 PP1: cosegregation with disease with combined Bayes likelihood ratio >2.08 and <=4.3 (supporting), >4.3 and <=18.7 (moderate), or >18.7 in >=2 families (strong).

Evidence considered

• The applicable InSiGHT MSH6 V2.0 PP1 thresholds are combined Bayes likelihood ratio >2.08 to <=4.3 for supporting, >4.3 to <=18.7 for moderate, and >18.7 in at least two families for strong.
• The consolidated case-evidence bundle contains no variant-specific segregation data or Bayes likelihood ratio for this case.

Still missing

• A variant-specific pedigree with tested relatives is needed.
• Affected and unaffected relative phenotypes and ages or appropriate unaffected follow-up are needed.
• Carrier status and phase across informative meioses are needed.
• A combined Bayes likelihood ratio is needed to select supporting, moderate, or strong strength.

Sources

Reasoning

The MSH6-specific InSiGHT VCEP BS2 rule requires documented 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-syndrome cancer above its median age of onset, without clinical manifestations of CMMRD; phase must be confirmed through parental or offspring testing. The consolidated case-evidence bundle does not provide the required in-trans co-occurrence, phase-confirmation, age, cancer, or CMMRD-exclusion evidence, so BS2 cannot be assessed.

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 another Lynch-syndrome cancer above the median age of onset), without clinical manifestations of CMMRD; phase requires testing of parents or offspring.

Evidence considered

• The MSH6 InSiGHT VCEP Version 2.0 defines BS2 using in-trans co-occurrence with a known pathogenic sequence variant, qualifying cancer age, absence of CMMRD manifestations, and parent/offspring phase confirmation.
• No case-level source in the consolidated evidence bundle documents these BS2 requirements.

Still missing

• A qualifying patient-level co-occurrence in trans with a known pathogenic MSH6 variant is needed.
• Parental or offspring testing confirming phase is needed.
• The qualifying cancer type and age-at-diagnosis, plus evidence excluding CMMRD manifestations, are not documented.

Sources

Reasoning

The applicable ClinGen InSiGHT MSH6 Version 2.0 specification requires calibrated functional odds for pathogenicity <=0.05 for BS3_Strong, or calibrated functional odds >0.05 and <=0.48 or variant-specific proficient function in protein and mRNA-based laboratory assays following the MMR functional assay flowchart for BS3_Supporting. The case bundle provides no variant-specific calibrated functional odds, protein-function assay, mRNA assay, or other validated assay demonstrating proficient function for NM_000179.3:c.2731C>T (p.Arg911Ter). The available ClinVar evidence signal instead states that functional studies have not been reported for this variant. Therefore BS3 cannot be applied.

Rule

Calibrated functional assays with functional odds for pathogenicity <= 0.05; or calibrated functional assays with functional odds for pathogenicity >0.05 and <=0.48, or variant-specific proficient function in protein and mRNA-based laboratory assays as per the MMR functional assay flowchart.

Evidence considered

• The MSH6 VCEP specification defines BS3_Strong as calibrated functional odds for pathogenicity <=0.05 and BS3_Supporting as calibrated functional odds >0.05 and <=0.48 or variant-specific proficient function in protein and mRNA-based laboratory assays following the MMR functional assay flowchart.
• The case evidence bundle contains no variant-specific functional assay measurements, calibrated functional odds, protein assay result, or mRNA assay result demonstrating proficient function for NM_000179.3:c.2731C>T (p.Arg911Ter).
• The case bundle's ClinVar evidence signal reports: 'to our knowledge, functional studies have not been reported for this variant.'

Still missing

• Variant-specific validated functional assay data with appropriate assay controls are unavailable.
• No calibrated functional odds for pathogenicity are available.
• No variant-specific protein and mRNA assay results demonstrating proficient function are available.

Sources

Reasoning

The InSiGHT MSH6 V2.0 specification requires lack of cosegregation and a combined Bayes likelihood ratio. The case bundle contains no variant-specific non-segregation observations, tested non-carrier affected relatives, pedigree data, or Bayes likelihood ratio for NM_000179.3:c.2731C>T, so BS4 cannot be assigned.

Rule

InSiGHT MSH6 V2.0 BS4: lack of cosegregation with combined Bayes likelihood ratio <0.05 (strong) or >0.05 and <=0.48 (supporting).

Evidence considered

• The applicable InSiGHT MSH6 V2.0 BS4 thresholds are a combined Bayes likelihood ratio <0.05 for strong and >0.05 to <=0.48 for supporting.
• The consolidated case-evidence bundle contains no variant-specific non-segregation data or Bayes likelihood ratio for this case.

Still missing

• A variant-specific pedigree with informative affected and unaffected relatives is needed.
• Relative genotypes and disease phenotypes, including assessment for phenocopies and age-related penetrance, are needed.
• A combined Bayes likelihood ratio is needed to determine whether BS4 is supporting or strong.

Sources