MSH6 c.914C>A, p.Thr305Asn

Reasoning

The MSH6 VCEP defines PM2 Supporting as an allele frequency below 0.00002 in the gnomAD v4 dataset. gnomAD v4.1 reports this variant at total AF 6.195203425699686e-07 (1/1,614,152 alleles), with zero homozygotes; this is below 0.00002. The governing VCEP pilot file was searched for c.914C>A, p.Thr305Asn, and p.T305N and contains no matching entry or pre-assigned code for this variant.

VCEP rule

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

Evidence considered

• gnomAD v4.1 record for NM_000179.3:c.914C>A: total AF 6.195203425699686e-07 (1/1,614,152 alleles), total allele count 1, and homozygotes 0.
• The governing VCEP pilot file VCEP-pilot-variants---MMR.xlsx.txt was searched for c.914C>A, p.Thr305Asn, and p.T305N; no matching row or pre-assigned code was found.

Still missing

• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The variant is a missense substitution, and the governing VCEP rule directly assigns BP4_Supporting when the exact HCI prior probability of pathogenicity is <0.11. The HCI-PRIORS-MSH6 row for c.914C>A / p.T305N reports 0.0264, which is below 0.11. The alternative SpliceAI BP4 path for intronic and synonymous variants is not needed and does not govern this missense path.

VCEP rule

Missense variant with HCI-prior probability of pathogenicity <0.11 as per https://hci-priors.hci.utah.edu/PRIORS

Evidence considered

• HCI-PRIORS-MSH6.txt, exact row for exon 4 c.914C>A / p.T305N: HCI prior probability of pathogenicity = 0.0264; MAPP score = 10.82; custom PP2 score = 0.044; row identifier MSH6_01987.
• The exact HCI prior value 0.0264 satisfies the VCEP BP4 missense threshold of <0.11.

Sources

Reasoning

The variant is a predicted missense substitution encoding p.Thr305Asn (p.T305N), but searches of the governing VCEP fulltext files for c.914C>A, p.Thr305Asn, and p.T305N found only the exact variant in HCI-PRIORS-MSH6, not a different nucleotide change encoding the same amino-acid change previously established by this VCEP as Pathogenic. The exact HCI row reports c.914C>A/p.T305N and therefore is not a qualifying PS1 comparator. No qualifying same-amino-acid pathogenic comparator was identified.

VCEP rule

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).

Evidence considered

• CSPEC MSH6 v2.0 PS1 rule requires a predicted missense substitution encoding the same amino-acid change through a different nucleotide change, previously established by this VCEP as Pathogenic and not a splice defect.
• HCI-PRIORS-MSH6.txt.txt, row MSH6_01987: c.914C>A, p.T305N, HCI prior probability 0.0264; this is the indexed variant itself, not a different-nucleotide comparator.
• Searches for c.914C>A, p.Thr305Asn, and p.T305N in the governing MSH6 fulltext directory returned no qualifying alternate-nucleotide same-amino-acid entry.

Still missing

• No qualifying same-amino-acid comparator was found in the searched VCEP fulltext files: MMR-Functional-domains.pdf.txt and HCI-PRIORS-MSH6.txt.txt.

Sources

Reasoning

The variant is missense at residue 305, but no different missense variant at the same residue satisfying the VCEP PM5 comparator requirement was identified. The collected PM5 candidate summary reports zero same-residue candidates, and the governing rule requires a different missense change at the residue classified by this VCEP as Pathogenic or Likely Pathogenic at the protein level and not because of aberrant splicing; the additional PP3-supporting condition is also not established within this group.

VCEP rule

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.

Evidence considered

• CSPEC MSH6 v2.0 PM5 Moderate rule requires a different missense change at the same amino-acid residue classified by this VCEP as Pathogenic at the protein level and not due to aberrant splicing; PM5 requires PP3-supporting for the reviewed missense change.
• pm5_candidates.json summary: classic_same_residue_missense mode, total candidates seen 10, same_residue_candidates 0, expert_panel_plp_candidates 0, and non_expert_majority_plp_candidates 0.
• HCI-PRIORS-MSH6.txt.txt, row MSH6_01987, contains the exact variant c.914C>A/p.T305N rather than a different missense substitution at residue 305.

Still missing

• No qualifying same-residue comparator was found in pm5_candidates.json or the searched VCEP fulltext files: MMR-Functional-domains.pdf.txt and HCI-PRIORS-MSH6.txt.txt.

Sources

Reasoning

The variant is a missense substitution, so the governing HCI-prior missense path applies. The exact HCI-PRIORS-MSH6 row for c.914C>A / p.T305N reports an HCI prior probability of pathogenicity of 0.0264. This is not >0.81 for PP3_Moderate and not >0.68 and <=0.81 for PP3_Supporting; the VCEP's alternative SpliceAI PP3 path is limited to predicted splice defects at non-canonical splicing nucleotides and is not applicable to this missense coding substitution.

VCEP rule

Missense variant with HCI prior probability for pathogenicity >0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) ... Missense variant with HCI prior probability for pathogenicity >0.68 & <=0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](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.

Evidence considered

• HCI-PRIORS-MSH6.txt, exact row for exon 4 c.914C>A / p.T305N: HCI prior probability of pathogenicity = 0.0264; MAPP score = 10.82; custom PP2 score = 0.044; row identifier MSH6_01987.
• The missense HCI prior value 0.0264 is below both VCEP PP3 thresholds: >0.81 for Moderate and >0.68 and <=0.81 for Supporting.
• The VCEP SpliceAI PP3 alternative is not used because this is a missense variant rather than a non-canonical splicing-nucleotide variant.

Sources

Reasoning

The case evidence contains no proband-level de novo observation, parental testing, maternity/paternity confirmation, or de novo point assignment for NM_000179.3:c.914C>A (p.Thr305Asn). Therefore the VCEP's point-based PS2 rule cannot be evaluated.

VCEP rule

≥ 4 _de novo_ points ... 2 - 3.5 _de novo_ points ... 1 - 1.5 _de novo_ points ... 0.5 _de novo_ points

Evidence considered

• Case evidence contains no proband, parental-testing, maternity/paternity, or de novo-segregation result for NM_000179.3:c.914C>A.
• The two fetched papers were variant-negative and provided no de novo or family evidence for c.914C>A/p.Thr305Asn.

Still missing

• Documented proband de novo status for c.914C>A
• Testing confirming both biological parents and the variant absent in them
• VCEP de novo point assignment, if applicable

Sources

Reasoning

PS3 is applicable to this missense variant under the MSH6 VCEP specification. The governing functional-assay documentation identifies approved calibrated MMR assays, including the Drost et al. CIMRA assay (PMIDs 30504929 and 31965077), and records validation controls and assay readout thresholds, but it contains no result for c.914C>A, p.Thr305Asn, or p.T305N. The functional-assay flowchart directs missense variants without predicted splicing impact to a calibrated functional assay and requires quantitative multifactorial analysis or conversion of a functional likelihood ratio to an ACMG/AMP evidence weight when such an assay is available. The case evidence contains no variant-specific functional assay result, functional odds, functional likelihood ratio, or concordant assay results. Therefore, the VCEP PS3 thresholds cannot be evaluated and PS3 is not_assessed.

VCEP rule

PS3

Evidence considered

• MSH6 VCEP v2.0 PS3 rule: calibrated functional assays with functional odds forpathogenicity > 18.7 support PS3 Strong; functional odds >4.3 and <=18.7, or an MMR function defect following the functional assay flowchart, support PS3 Moderate; calibrated functional odds >2.08 and <=4.3 support PS3 Supporting.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays', row 'PMID': approved calibrated assays include 30504929/31965077; the table records 3 MSH6 P/LP validation controls and 4 MSH6 B/LB validation controls for that assay grouping, with abnormal MSH6/PMS2 readout <18%, but no result for this variant.
• Functional-assay-flowchart.pdf, page 1: for an in-frame or missense variant without predicted splicing impact, a calibrated functional assay is required; if conducted, quantitative multifactorial analysis or conversion of the functional LR to an ACMG/AMP evidence weight is used.
• Exact-variant searches for c.914C>A, p.Thr305Asn, and p.T305N returned no match in either governing functional-assay file.

Still missing

• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific calibrated functional assay result or functional odds/likelihood ratio is required to assess PS3 strength.

Sources

Reasoning

The InSiGHT MSH6 VCEP applies PM3 to co-occurrence of the variant with a known pathogenic/likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD. The available case evidence does not document an affected proband with CMMRD features, a second pathogenic/likely pathogenic MSH6 variant, or confirmed phase. Therefore, no PM3 points can be assigned and PM3 cannot be assessed.

VCEP rule

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”. ... 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.

Evidence considered

• The case evidence identifies NM_000179.3:c.914C>A (p.Thr305Asn) but contains no documented CMMRD clinical assessment, pathogenic/likely pathogenic MSH6 allele in the same patient, or phase result.
• No affected-proband, parent/offspring testing, trans/cis determination, or qualifying co-occurrence observation was available for PM3 evaluation.

Still missing

• Affected-proband clinical features consistent with CMMRD are not documented.
• A pathogenic/likely pathogenic second MSH6 variant is not documented.
• Phase between this variant and any second MSH6 variant is not documented.

Sources

Reasoning

The case evidence provides no pedigree, affected or unaffected relatives, meioses, segregation observations, or combined Bayes likelihood ratio for NM_000179.3:c.914C>A. The VCEP PP1 thresholds therefore cannot be evaluated.

VCEP rule

Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7​ in ≥2 families. ... Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^​f >4.3 & ≤18.7. ... Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3.

Evidence considered

• No family pedigree, informative meioses, affected-relative testing, unaffected-relative testing, or segregation likelihood ratio is present in the case evidence.
• The fetched papers do not mention MSH6 c.914C>A or p.Thr305Asn and provide no segregation data for this variant.

Still missing

• Pedigree and phenotype status of tested relatives
• Variant results in affected and unaffected relatives
• Combined Bayes likelihood ratio and number of informative families

Sources

Reasoning

The VCEP PP4 rule requires one or more CRC or endometrial MSI-H tumors, tumor-genome evidence, and/or loss of MMR protein expression consistent with the variant location. The available case evidence does not report a qualifying tumor, MSI result, tumor-genome result, or MSH6 protein-expression result, so PP4 cannot be assessed. The required VCEP files were searched for c.914C>A, p.Thr305Asn, and p.T305N: Table-for-CMMRD-diagnosis.pdf contains only the general CMMRD scoring table and no variant entry, and VCEP-pilot-variants---MMR.xlsx contains no entry for this variant; neither file supplies qualifying patient phenotype evidence.

VCEP rule

CRC/endometrial MSI-H tumors or tumor-genome evidence and/or loss of MMR protein expression consistent with the variant location.

Evidence considered

• The MSH6 VCEP PP4 rule requires 1, 2, or at least 3 independent CRC/endometrial MSI-H tumors using a standard 5-10-marker panel or tumor genome and/or loss of MMR protein expression consistent with the variant location.
• Table-for-CMMRD-diagnosis.pdf.txt was searched for c.914C>A, p.Thr305Asn, and p.T305N; it contains general CMMRD scoring criteria, including the >=3-point CMMRD threshold, but no entry for this variant or patient-specific phenotype.
• VCEP-pilot-variants---MMR.xlsx.txt was searched for c.914C>A, p.Thr305Asn, and p.T305N; it contains precedent rows for other MMR variants but no entry for this variant.
• The case evidence does not provide a qualifying CRC/endometrial MSI-H tumor, tumor-genome result, or MSH6 protein-expression result.

Still missing

• Patient-specific CRC or endometrial tumor histology and MSI-H testing are not available.
• Patient-specific tumor-genome evidence and MSH6 protein-expression results consistent with the variant location are not available.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in Table-for-CMMRD-diagnosis.pdf.txt.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The MSH6 VCEP requires the gnomAD v4 Grpmax filtering allele frequency to be at least 0.0022 and exclusion of a founder pathogenic variant. The available gnomAD v4 record reports total AF 6.195203425699686e-07 and highest observed South Asian AF 1.0979358805445763e-05, but grpmax_faf is null; therefore the required Grpmax value cannot be evaluated. The VCEP pilot file was searched for c.914C>A, p.Thr305Asn, and p.T305N and contains no matching entry.

VCEP rule

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

Evidence considered

• gnomAD v4.1 record for NM_000179.3:c.914C>A: total AF 6.195203425699686e-07 (1/1,614,152 alleles), highest observed South Asian AF 1.0979358805445763e-05 (1/91,080 alleles), homozygotes 0, and grpmax_faf null.
• The governing VCEP pilot file VCEP-pilot-variants---MMR.xlsx.txt was searched for c.914C>A, p.Thr305Asn, and p.T305N; no matching row was found.

Still missing

• gnomAD v4 grpmax_faf is unavailable for this variant; the VCEP BA1 threshold cannot be directly evaluated.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The MSH6 VCEP requires the gnomAD v4 Grpmax filtering allele frequency to be at least 0.00022 and below 0.0022, with the variant excluded as a founder pathogenic variant. The available gnomAD v4 record reports total AF 6.195203425699686e-07 and highest observed South Asian AF 1.0979358805445763e-05, but grpmax_faf is null; therefore the required Grpmax value cannot be evaluated. The VCEP pilot file was searched for c.914C>A, p.Thr305Asn, and p.T305N and contains no matching entry.

VCEP 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

• gnomAD v4.1 record for NM_000179.3:c.914C>A: total AF 6.195203425699686e-07 (1/1,614,152 alleles), highest observed South Asian AF 1.0979358805445763e-05 (1/91,080 alleles), homozygotes 0, and grpmax_faf null.
• The governing VCEP pilot file VCEP-pilot-variants---MMR.xlsx.txt was searched for c.914C>A, p.Thr305Asn, and p.T305N; no matching row was found.

Still missing

• gnomAD v4 grpmax_faf is unavailable for this variant; the VCEP BS1 interval cannot be directly evaluated.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The MSH6 VCEP requires documented co-occurrence in trans with a known pathogenic variant in the same gene in a patient meeting the specified cancer-age condition, absence of CMMRD manifestations, and parental or offspring testing to confirm phase. The available case evidence contains no such co-occurrence, phase, cancer-age, CMMRD, or parental/offspring data; gnomAD reports zero homozygotes but that does not evaluate this rule.

VCEP 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^​d​), 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.

Evidence considered

• The governing VCEP rule requires in-trans co-occurrence, a colorectal cancer after age 45 or another Lynch-syndrome cancer above its median onset age, no CMMRD manifestations, and phase confirmation by testing parents or offspring; none is documented in the available case evidence.
• gnomAD v4.1 reports homozygotes = 0, but homozygote absence is not the VCEP's BS2 co-occurrence-and-phase rule.

Still missing

• Qualifying in-trans co-occurrence with a known pathogenic MSH6 variant is not documented.
• Cancer age, absence of CMMRD manifestations, and parental or offspring phase confirmation are not documented.

Sources

Reasoning

BS3 is applicable to this missense variant under the MSH6 VCEP specification. The governing functional-assay documentation identifies approved calibrated assays and their validation controls, including normal and abnormal readout thresholds, but contains no result for c.914C>A, p.Thr305Asn, or p.T305N. The functional-assay flowchart provides a route to proficient function only when appropriate concordant assay evidence is available, and the VCEP requires calibrated functional odds for the calibrated BS3 pathways. The case evidence contains no variant-specific proficient-function result, functional odds, functional likelihood ratio, or concordant assay results. Therefore, the VCEP BS3 thresholds cannot be evaluated and BS3 is not_assessed.

VCEP rule

BS3

Evidence considered

• MSH6 VCEP v2.0 BS3 rule: calibrated functional assays with functional odds for Pathogenicity <= 0.05 support BS3 Strong; calibrated functional assays with 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, support BS3 Supporting.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays', row 'PMID': approved calibrated assays include 30504929/31965077; the table records positive and negative validation controls and assay readout thresholds, but no result for this variant.
• Functional-assay-flowchart.pdf, page 1: proficient function for missense variants requires appropriate functional assay evidence, including concordant assay results and, where applicable, nuclear localization, sensitivity to DNA damage, and no mutator phenotype.
• Exact-variant searches for c.914C>A, p.Thr305Asn, and p.T305N returned no match in either governing functional-assay file.

Still missing

• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.914C>A, p.Thr305Asn, or p.T305N was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific calibrated functional assay result demonstrating proficient function, or a calibrated functional odds/likelihood ratio, is required to assess BS3 strength.

Sources

Reasoning

The case evidence provides no pedigree, non-segregating affected or unaffected relatives, meioses, or combined Bayes likelihood ratio. Consequently, neither VCEP BS4 threshold can be evaluated.

VCEP rule

Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05​. ... Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48.

Evidence considered

• No unaffected or affected relative testing, non-segregation observation, pedigree, or segregation likelihood ratio is present in the case evidence.
• The fetched papers do not mention MSH6 c.914C>A or p.Thr305Asn and provide no non-segregation data for this variant.

Still missing

• Documented non-segregation in an informative pedigree
• Phenotype and variant status of tested relatives
• Combined Bayes likelihood ratio

Sources

Reasoning

The VCEP BP5 rule requires 2 or 3 tumors for Supporting or at least 4 tumors for Strong with MSS and/or no loss of MMR protein expression, or specified BRAF V600E or MLH1-methylation findings. No patient-specific tumor phenotype, MMR protein-expression, BRAF V600E, or MLH1-methylation evidence is available, so BP5 cannot be assessed.

VCEP rule

2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d 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. ... ​≥ 4 tumors: CRC/Endometrial tumors with MSS ​and/or​ no ​loss of MMR protein expression​ ​and/or LS spectrum tumors^d 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.

Evidence considered

• The VCEP BP5 Supporting path requires 2 or 3 qualifying tumors or 1 BRAF V600E/MLH1-methylation finding; the Strong path requires at least 4 qualifying tumors or at least 2 BRAF V600E/MLH1-methylation findings.
• The available case evidence contains no patient-specific tumor count, MSS result, MMR protein-expression result, BRAF V600E result, or MLH1-methylation result.

Still missing

• Patient-specific tumor count, MSS status, and MMR protein-expression results are not available.
• Patient-specific BRAF V600E or MLH1-methylation results are not available.

Sources