MSH6 c.2294G>C, p.(Cys765Ser)

Reasoning

The governing InSiGHT/ClinGen MSH6 VCEP specifies PM2 at an allele frequency below 0.00002 (less than 1 in 50,000 alleles) in the gnomAD v4 dataset. gnomAD v4.1 reports this variant at total AF 6.195364628185191e-07, with 1 alternate allele among 1,614,110 alleles and zero homozygotes; the highest population AF is 8.47450445335209e-07 in the European non-Finnish population, also with zero homozygotes. The variant is therefore extremely rare under the VCEP threshold, meeting PM2 at Supporting strength.

Rule

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

Evidence considered

• The MSH6 VCEP specifies PM2 at allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports total AF 6.195364628185191e-07, based on 1 alternate allele among 1,614,110 alleles, with zero homozygotes.
• The highest observed population AF is 8.47450445335209e-07 in the European non-Finnish population, with zero homozygotes, which remains below 0.00002.

Sources

Reasoning

The variant is a missense substitution, so PP3 is within scope. The governing MSH6 VCEP specification assigns missense PP3 using the HCI prior lookup: moderate requires a prior probability >0.81 and supporting requires >0.68 and <=0.81. The exact HCI-PRIORS-MSH6 entry for c.2294G>C / p.C765S / p.C765S reports MAPP/PP2 prior probability 0.4345, below the supporting threshold; therefore PP3 is not met. The table also reports a custom PP2 score of 0.690, but that is not the governing HCI prior probability and was not substituted for it. The generic REVEL result (0.921) was not used because the MSH6 VCEP-specific missense HCI-prior rule governs this criterion.

Evidence considered

• The ClinGen InSiGHT MSH6 VCEP Version 2.0 specification defines missense PP3 as HCI prior probability >0.81 for moderate or >0.68 and <=0.81 for supporting.
• The exact entry in /Users/gs_agent/LYFESCI_HERMES_v9/vcep_db/MSH6/fulltext/HCI-PRIORS-MSH6.txt.txt was found for c.2294G>C and p.C765S; its table columns identify Custom_PP2_score 0.690, MAPP_score 14.04, and MAPP/PP2_Prior 0.4345.
• The case evidence records the variant as missense, with the exact HCI prior probability 0.4345 and a REVEL score of 0.921; the VCEP-specific HCI-prior rule takes precedence over generic REVEL thresholds.

Sources

Reasoning

The governing InSiGHT/ClinGen MSH6 VCEP specifies BA1 when the gnomAD v4 grpmax filtering allele frequency is at least 0.0022 (0.22%) and the variant is not a founder pathogenic variant. gnomAD v4.1 reports an all-comers total allele frequency of 6.195364628185191e-07 (1/1,614,110 alleles), with the highest observed population frequency 8.47450445335209e-07 in the European non-Finnish population (1/1,180,010 alleles); no grpmax value is reported, but all observed frequencies are far below 0.0022. BA1 is therefore 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 VCEP specifies BA1 at gnomAD v4 grpmax filtering allele frequency >=0.0022 (0.22%), excluding founder pathogenic variants.
• gnomAD v4.1 reports total AF 6.195364628185191e-07 from 1 alternate allele among 1,614,110 alleles, and the highest population AF is 8.47450445335209e-07 in the European non-Finnish population from 1,180,010 alleles.
• No gnomAD v4 grpmax value is reported for this variant; the available total and population frequencies are each substantially below the VCEP BA1 cutoff.

Sources

Reasoning

The governing InSiGHT/ClinGen MSH6 VCEP specifies BS1 at a gnomAD v4 grpmax filtering allele frequency of at least 0.00022 and below 0.0022 (0.022-0.22%), provided the variant is not a founder pathogenic variant. gnomAD v4.1 reports a total allele frequency of 6.195364628185191e-07 and a highest observed population frequency of 8.47450445335209e-07; both are below 0.00022. BS1 is therefore 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 VCEP specifies BS1 for gnomAD v4 grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022-0.22%), excluding founder pathogenic variants.
• gnomAD v4.1 reports total AF 6.195364628185191e-07 and highest population AF 8.47450445335209e-07 in the European non-Finnish population.
• The observed frequencies are below 0.00022, so the VCEP BS1 interval is not reached.

Sources

Reasoning

The variant is a missense substitution, so BP4 is within scope. The governing MSH6 VCEP specification assigns missense BP4 when the HCI prior probability is <0.11. The exact HCI-PRIORS-MSH6 entry for c.2294G>C / p.C765S reports an HCI prior probability of 0.4345, which is not below 0.11; therefore BP4 is not met. The SpliceAI max delta score of 0.002 and generic REVEL score of 0.921 were not used to override the governing VCEP-specific HCI-prior missense rule.

Evidence considered

• The ClinGen InSiGHT MSH6 VCEP Version 2.0 specification defines missense BP4 as HCI prior probability <0.11.
• The exact entry in /Users/gs_agent/LYFESCI_HERMES_v9/vcep_db/MSH6/fulltext/HCI-PRIORS-MSH6.txt.txt was found for c.2294G>C and p.C765S, with MAPP/PP2 prior probability 0.4345.
• The case evidence records SpliceAI max delta 0.002 and REVEL 0.921, but neither is used to replace the governing VCEP-specific HCI-prior assessment for this missense variant.

Sources

Reasoning

The governing MSH6 VCEP defines PS1 for a missense substitution producing the same amino-acid change as a different nucleotide change previously established by the VCEP as Pathogenic, or for certain non-canonical splice substitutions. The case variant is missense p.Cys765Ser, but the available comparator collection contains no validated alternate-nucleotide p.Cys765Ser established as VCEP Pathogenic, and no splice-variant scenario applies. Because the comparator search reported a soft retrieval failure, absence of a qualifying PS1 comparator cannot be treated as conclusive.

Rule

MSH6 VCEP PS1: same amino-acid change from a different nucleotide change previously established by this VCEP as Pathogenic; specified splice alternative not applicable.

Evidence considered

• The MSH6 VCEP PS1 rule requires the same amino-acid change from a different nucleotide change previously established by the VCEP as Pathogenic, or a specified non-canonical splice comparison.
• The variant is a missense substitution, NM_000179.3:c.2294G>C, p.Cys765Ser; the collected comparator search reported zero same-residue candidates and an HTTP 429 retrieval error.

Still missing

• Confirm through a complete VCEP/ClinVar review whether an alternate-nucleotide MSH6 variant causing p.Cys765Ser is VCEP-Pathogenic.

Sources

Reasoning

The MSH6 InSiGHT VCEP specifies PS2 using de novo points: 2 points per proband when both maternity and paternity are confirmed and an MMR-deficient Lynch-spectrum tumor is documented; 1 point per proband when both parents are confirmed without tumor data; and 0.5 points per proband when maternity and/or paternity are unconfirmed without tumor data. The available case evidence does not document a proband-level de novo observation, parental testing, maternity or paternity confirmation, or an eligible Lynch-spectrum tumor with the required molecular characterization. Therefore, no PS2 points can be assigned.

Rule

MSH6 InSiGHT VCEP v2.0 PS2: 2 de novo points per proband with confirmed maternity and paternity plus an MMR-deficient Lynch-spectrum tumor; 1 point with confirmed maternity and paternity but no tumor data; 0.5 points when maternity and/or paternity are unconfirmed without tumor data; >=4 points PS2_VeryStrong, 2-3.5 PS2_Strong, 1-1.5 PS2_Moderate, and 0.5 PS2_Supporting.

Evidence considered

• The MSH6 InSiGHT VCEP PS2 rule assigns 2 points per proband for confirmed maternity and paternity with an MMR-deficient Lynch-spectrum tumor, 1 point when both parents are confirmed without tumor data, and 0.5 points when maternity and/or paternity are unconfirmed without tumor data.
• The reviewed case evidence contains no documented proband-level parental testing, de novo finding, maternity or paternity confirmation, or qualifying tumor molecular results for NM_000179.3:c.2294G>C.

Still missing

• Documented proband-level testing of the variant in the child and both biological parents.
• Confirmation of maternity and paternity, if available.
• Documentation of an eligible Lynch-spectrum tumor and MSI/IHC/methylation results, if available.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP permits PS3 for calibrated functional assays with functional odds for pathogenicity >18.7 (strong), >4.3 and <=18.7 (moderate), or >2.08 and <=4.3 (supporting), and for a demonstrated MMR function defect under its functional-assay flowchart. The available case evidence identifies this as the missense variant NM_000179.3:c.2294G>C (p.Cys765Ser), but contains no variant-specific calibrated assay result, functional-odds estimate, MMR activity result, protein-expression result, or patient-derived functional assay result. General MSH6 functional-assay publications and VCEP assay documentation establish the applicable framework but do not provide evidence for this variant; therefore PS3 cannot be assigned.

Rule

Calibrated functional assays with functional odds for pathogenicity >18.7 (strong), >4.3 and <=18.7 (moderate), or >2.08 and <=4.3 (supporting); moderate may also be supported by an MMR function defect following the functional assay flowchart.

Evidence considered

• The MSH6 Version 2.0 VCEP specifies PS3 strong at functional odds for pathogenicity >18.7, moderate at >4.3 and <=18.7, and supporting at >2.08 and <=4.3.
• The MSH6 functional-assay flowchart requires a calibrated assay or concordant validated functional evidence demonstrating an MMR function defect before assigning PS3.
• Case-specific records identify NM_000179.3:c.2294G>C (p.Cys765Ser) but provide no variant-specific functional assay measurement or assay-derived classification result.

Still missing

• Variant-specific calibrated functional assay data or a validated MMR function-defect result is required.
• If using calibrated evidence, the functional odds for pathogenicity must be reported to select the VCEP strength tier: >2.08, >4.3, or >18.7.

Sources

Reasoning

The applicable MSH6 InSiGHT VCEP specification assigns PM3 strength by points (supporting at 0.5 points, moderate at 1–1.5, strong at 2–3.5, and very strong at ≥4 points). The available case evidence documents no affected proband with a second pathogenic or likely pathogenic MSH6 variant, no confirmed trans or cis phase, and no informative biallelic inheritance observation; therefore no PM3 points can be assigned.

Rule

PM3: ≥4 points = Very Strong; 2–3.5 points = Strong; 1–1.5 points = Moderate; =0.5 points = Supporting.

Evidence considered

• The MSH6 VCEP specification defines PM3 using a point system: supporting at 0.5 points, moderate at 1–1.5 points, strong at 2–3.5 points, and very strong at ≥4 points.
• The reviewed case materials contain no documented affected-proband observation, second pathogenic or likely pathogenic MSH6 allele, or confirmed trans/cis phase for NM_000179.3:c.2294G>C.
• MSH6 has both autosomal dominant Lynch syndrome and autosomal recessive constitutional mismatch repair deficiency contexts, so PM3 requires explicit biallelic phase and inheritance evidence rather than inference from the gene alone.

Still missing

• Affected-proband observation involving this variant and a second pathogenic or likely pathogenic MSH6 variant
• Confirmed trans or cis phase between the variants
• Inheritance or family data sufficient to establish biallelic disease

Sources

Reasoning

The governing MSH6 VCEP permits PM5 for a missense change at a residue where a different missense change was classified by the VCEP as Pathogenic, or PM5_Supporting when the comparator is Likely Pathogenic, with the specified dependence on PP3. The target is p.Cys765Ser, but the collected same-residue comparator set contains zero candidates. The collection also records an HTTP 429 retrieval failure, so the absence of a comparator is not sufficiently complete to conclude that PM5 is not met.

Rule

MSH6 VCEP PM5: different missense change at the same residue classified by this VCEP as Pathogenic or Likely Pathogenic on the protein level, not due to aberrant splicing; use only with target PP3 support.

Evidence considered

• The MSH6 VCEP PM5 rule requires a different missense substitution at the same amino-acid residue classified by the VCEP as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing; it also requires PP3 support for the target missense variant.
• The collected PM5 candidate artifact reports zero same-residue candidates and an HTTP 429 retrieval error, so comparator absence is incomplete.

Still missing

• Complete a validated search for alternate missense substitutions at MSH6 residue Cys765 and verify each comparator's VCEP classification and mechanism.

Sources

Reasoning

The MSH6 InSiGHT VCEP requires a combined Bayes likelihood ratio from cosegregation: greater than 2.08 and up to 4.3 supports PP1, greater than 4.3 and up to 18.7 supports PP1_Moderate, and greater than 18.7 in at least two families supports PP1_Strong. No affected-relative pedigree, informative meioses, segregation counts, or combined Bayes likelihood ratio is documented for this variant. Therefore, PP1 cannot be assessed.

Rule

MSH6 InSiGHT VCEP v2.0 PP1: combined Bayes likelihood ratio >2.08 and <=4.3 is Supporting; >4.3 and <=18.7 is Moderate; >18.7 in >=2 families is Strong.

Evidence considered

• The MSH6 InSiGHT VCEP 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 evidence.
• The reviewed case evidence contains no informative pedigree, affected-relative segregation results, meioses, or combined Bayes likelihood ratio for NM_000179.3:c.2294G>C.

Still missing

• A pedigree identifying tested affected and unaffected relatives.
• Variant status and phenotype status for each informative relative.
• A combined Bayes likelihood ratio, ideally calculated using the VCEP-recommended segregation method.

Sources

Reasoning

The applicable MSH6 InSiGHT specification provides PP4 Supporting for one CRC or endometrial MSI-H tumor, using a standard 5–10-marker panel or tumor genome, and/or loss of MMR protein expression consistent with the variant location; PP4 Moderate requires two independent tumors and PP4 Strong requires at least three. The case materials provide no patient-specific MSI result, tumor-genome result, MMR immunohistochemistry result, or count of qualifying independent tumors for NM_000179.3:c.2294G>C, so PP4 cannot be assessed.

Rule

PP4 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 tumors; Strong: ≥3 independent tumors.

Evidence considered

• The MSH6 InSiGHT PP4 rule requires one or more CRC/endometrial MSI-H tumors and/or consistent MMR protein loss, with higher strengths requiring two or at least three independent tumors.

Still missing

• Patient-specific tumor phenotype data: MSI status or tumor-genome result, MMR protein expression, and number of independent qualifying CRC/endometrial tumors.

Sources

Reasoning

The governing MSH6 VCEP requires BS2 evidence of confirmed 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 constitutional mismatch repair deficiency; phase must be confirmed through parental or offspring testing. The available case materials provide no qualifying co-occurrence, phase, unaffected-homozygote, penetrance, or age-at-diagnosis evidence for this variant. BS2 cannot be assessed from the available evidence.

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 prior or current clinical manifestations of CMMRD; phase requires parental or offspring testing.

Evidence considered

• The MSH6 VCEP BS2 rule requires in-trans co-occurrence with a known pathogenic sequence variant, qualifying cancer age, absence of CMMRD manifestations, and phase confirmation through parents or offspring.
• No qualifying BS2 co-occurrence, phase, or clinical-context record is present in the available case evidence.

Still missing

• Variant-specific documentation of co-occurrence in trans with a known pathogenic MSH6 sequence variant.
• Confirmation of phase through parental or offspring testing.
• Required clinical context: cancer type and age above the applicable Lynch-syndrome median, with no CMMRD manifestations.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP permits BS3 strong for calibrated functional assays with functional odds for pathogenicity <=0.05, or for synonymous/intronic variants with no mRNA aberration after appropriate testing; it permits BS3 supporting for calibrated functional odds >0.05 and <=0.48 or variant-specific proficient protein- and mRNA-based assay results under the MMR flowchart. This is a missense variant, NM_000179.3:c.2294G>C (p.Cys765Ser), and the available case evidence contains no variant-specific calibrated assay result, functional-odds estimate, proficient protein assay, mRNA assay, or validated benign functional finding. Therefore BS3 cannot be assigned.

Rule

Calibrated functional assays with functional odds for pathogenicity <=0.05 (strong) or >0.05 and <=0.48 (supporting); supporting may also be based on variant-specific proficient protein and mRNA assays under the MMR functional-assay flowchart.

Evidence considered

• The MSH6 Version 2.0 VCEP specifies BS3 strong at functional odds for pathogenicity <=0.05 and BS3 supporting at >0.05 and <=0.48.
• The VCEP also allows BS3 supporting for variant-specific proficient protein and mRNA-based laboratory assays performed according to the MMR functional-assay flowchart.
• Case-specific records identify NM_000179.3:c.2294G>C (p.Cys765Ser) but provide no variant-specific proficient assay result, calibrated functional odds, or benign functional finding.

Still missing

• Variant-specific calibrated functional assay data or concordant proficient protein and mRNA assay results is required.
• If using calibrated evidence, the functional odds for pathogenicity must be reported to distinguish BS3 strong (<=0.05) from BS3 supporting (>0.05 and <=0.48).

Sources

Reasoning

The MSH6 InSiGHT VCEP requires a combined Bayes likelihood ratio below 0.48 for lack of cosegregation: below 0.05 supports BS4_Strong, while greater than 0.05 and up to 0.48 supports BS4_Supporting. No pedigree, unaffected or affected relative testing, informative non-segregation observation, or combined Bayes likelihood ratio is documented. Therefore, BS4 cannot be assessed.

Rule

MSH6 InSiGHT VCEP v2.0 BS4: combined Bayes likelihood ratio <0.05 is Strong; >0.05 and <=0.48 is Supporting.

Evidence considered

• The MSH6 InSiGHT VCEP BS4 thresholds are a combined Bayes likelihood ratio <0.05 for strong evidence and >0.05 to <=0.48 for supporting evidence.
• The reviewed case evidence contains no informative non-segregation observations, pedigree data, relative testing results, or combined Bayes likelihood ratio for NM_000179.3:c.2294G>C.

Still missing

• A pedigree identifying tested relatives and their disease or unaffected status.
• Variant testing results for informative relatives who do not show expected cosegregation.
• A combined Bayes likelihood ratio for the non-segregation evidence.

Sources

Reasoning

The applicable MSH6 InSiGHT specification defines BP5 Supporting as two or three tumors with MSS and/or no MMR-protein loss and/or an LS-spectrum tumor with MMR-protein loss inconsistent with MSH6, or one BRAF V600E/MLH1-methylation finding in the specified tumor context; Strong requires at least four qualifying tumors or at least two BRAF V600E/MLH1-methylation findings. No patient-specific tumor pathology, MSI/MSS result, MMR-protein expression, BRAF V600E result, MLH1 methylation result, or qualifying tumor count is available, so BP5 cannot be assessed.

Rule

BP5 Supporting: 2 or 3 qualifying tumors, or 1 BRAF V600E/MLH1-methylation finding in the specified context; Strong: ≥4 qualifying tumors or ≥2 such findings.

Evidence considered

• The MSH6 InSiGHT BP5 rule requires two or three qualifying tumors or one specified BRAF V600E/MLH1-methylation finding for Supporting, and at least four qualifying tumors or two specified findings for Strong.

Still missing

• Patient-specific tumor pathology data and qualifying tumor count, including MSS/MSI, MMR-protein expression, BRAF V600E, or MLH1 methylation where applicable.

Sources