MSH6 c.242C>T, p.(Ala81Val)

Reasoning

The variant is a missense substitution, so BP4 is within scope. The governing MSH6 HCI-PRIORS table contains the exact entry c.242C>T / p.A81V with HCI prior probability 0.0035. The MSH6 InSiGHT VCEP directly assigns BP4_Supporting to missense variants with HCI prior probability <0.11; therefore BP4 is met at Supporting strength. REVEL 0.216 does not meet the generic BP4_Supporting threshold <=0.29, and no additional predictor is needed because the governing exact HCI lookup already assigns the criterion. The SpliceAI max delta is 0.32, but the VCEP SpliceAI BP4 alternative applies to intronic and synonymous variants, not this missense variant.

Rule

MSH6 InSiGHT VCEP BP4: missense variant with HCI prior probability <0.11 = Supporting; alternatively, for intronic or synonymous variants, SpliceAI delta <=0.1 = Supporting.

Evidence considered

• The exact HCI-PRIORS-MSH6 table entry for c.242C>T / p.A81V assigns HCI prior probability 0.0035.
• The MSH6 InSiGHT VCEP specifies BP4_Supporting for a missense variant with HCI prior probability <0.11.
• REVEL score is 0.216, which is also below the supplied generic BP4_Supporting cutoff of <=0.29 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997), although the exact VCEP HCI assignment is governing.
• SpliceAI max delta is 0.32; the MSH6 VCEP's BP4 SpliceAI alternative is for intronic and synonymous variants and is not applied to this missense variant.

Sources

Reasoning

The variant is a missense substitution, NM_000179.3:c.242C>T (NP_000170.1:p.(Ala81Val)), in the preferred MSH6 transcript. The MSH6 InSiGHT VCEP PVS1 specification applies to nonsense/frameshift variants, qualifying large genomic alterations, canonical splice-site variants with the specified transcript consequences, and qualifying RNA-confirmed splicing aberrations; it does not apply to this missense variant. No PVS1 null-variant consequence, NMD assessment, exon-skipping event, or protein truncation is established.

Rule

MSH6 InSiGHT VCEP Version 2.0 PVS1 rule: qualifying nonsense/frameshift, large genomic alteration, canonical splice-site, or specified RNA-confirmed splicing consequences; not applicable to a missense substitution.

Evidence considered

• The preferred transcript is NM_000179.3 and the predicted consequence is missense, p.(Ala81Val).
• The MSH6 InSiGHT VCEP PVS1 specification defines PVS1 for qualifying nonsense/frameshift, large genomic alteration, canonical splice, and specified RNA-confirmed splicing consequences; missense variants are not included.
• The case-level PVS1 assessment records no applicable generic null-variant bucket and identifies the consequence as missense.

Sources

Reasoning

The MSH6 VCEP PS1 rule requires a different nucleotide change producing the same amino-acid substitution, established by the VCEP as Pathogenic, or an equivalent splice-nucleotide scenario. The case variant is NM_000179.3:c.242C>T (NP_000170.1:p.(Ala81Val)); the collected ClinVar and literature evidence contains no documented pathogenic comparator for the same amino-acid change, and the available paper extractions do not mention this variant or an applicable same-amino-acid comparator. Therefore PS1 is not met.

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

• The governing MSH6 VCEP defines PS1 as a different nucleotide change encoding the same amino-acid change previously established by the VCEP as Pathogenic, excluding predicted or confirmed splice defects.
• The queried variant is NM_000179.3:c.242C>T, encoding NP_000170.1:p.(Ala81Val).
• The collected ClinVar record and reviewed paper extractions provide no documented VCEP-Pathogenic alternate nucleotide change producing p.Ala81Val; the extracted papers do not explicitly mention c.242C>T or p.(A81V).

Sources

Reasoning

The governing InSiGHT MSH6 Version 2.0 specification defines PM2 Supporting as absent or extremely rare in gnomAD v4, with allele frequency <0.00002 (<1 in 50,000 alleles). gnomAD v4.1 reports total AF 0.000113943645 (164/1,439,308 alleles), grpmax FAF 0.00012624, and highest population AF 0.000145039 in Europeans (non-Finnish); all exceed 0.00002. The non-cancer subset is not the governing source for this VCEP rule.

Rule

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

Evidence considered

• InSiGHT MSH6 Version 2.0 specifies PM2 Supporting for gnomAD v4 allele frequency <0.00002 (<1 in 50,000 alleles).
• gnomAD v4.1 reports total AF 0.000113943645 from 164 of 1,439,308 alleles, grpmax FAF 0.00012624, and highest population AF 0.000145039 in Europeans (non-Finnish), all above 0.00002.
• The VCEP explicitly names gnomAD v4, so the available gnomAD v2.1 and non-cancer subset values do not replace the governing gnomAD v4 assessment.

Sources

Reasoning

The variant is a missense substitution, so PP3 is within scope. The governing InSiGHT MSH6 VCEP specifies PP3_Moderate for an HCI prior probability >0.81 and PP3_Supporting for a probability >0.68 and <=0.81; the exact HCI lookup entry for c.242C>T / p.A81V assigns 0.0035, which does not meet either PP3 threshold. REVEL is 0.216, below the supplied generic PP3_Supporting threshold of 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997). The VCEP's SpliceAI PP3 rule is limited to predicted splice defects at non-canonical splicing nucleotides, whereas this is a coding missense variant; therefore the SpliceAI max delta of 0.32 is not applied to PP3. BayesDel (-0.293941) has no established generic ACMG strength calibration and the MSH6 VCEP does not specify a BayesDel cutoff.

Rule

MSH6 InSiGHT VCEP PP3: HCI prior probability >0.81 = Moderate; >0.68 and <=0.81 = Supporting; alternatively SpliceAI delta >=0.2 only for predicted splice defects at non-canonical splicing nucleotides.

Evidence considered

• The exact HCI-PRIORS-MSH6 table entry for c.242C>T / p.A81V assigns HCI prior probability 0.0035; the governing MSH6 VCEP thresholds are >0.81 for PP3_Moderate and >0.68 to <=0.81 for PP3_Supporting.
• The MSH6 InSiGHT VCEP PP3 SpliceAI alternative is specified for predicted splice defects at non-canonical splicing nucleotides; c.242C>T is a coding missense substitution, so its SpliceAI result is not used for PP3.
• REVEL score is 0.216, below the supplied generic PP3_Supporting cutoff of 0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• BayesDel score is -0.293941; no generic ACMG strength calibration is established and the governing MSH6 VCEP provides no BayesDel cutoff.

Sources

Reasoning

The governing InSiGHT MSH6 Version 2.0 specification requires gnomAD v4 grpmax filtering allele frequency >=0.0022 (0.22%) for BA1, with exclusion of a founder pathogenic variant. gnomAD v4.1 reports a grpmax FAF of 0.00012624 (0.012624%), total AF 0.000113943645, and 164/1,439,308 alleles; this is below the BA1 threshold. No founder pathogenic-variant exception is needed to reach the threshold.

Rule

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

Evidence considered

• InSiGHT MSH6 Version 2.0 specifies BA1 at gnomAD v4 grpmax FAF >=0.0022 (0.22%) and requires exclusion of a founder pathogenic variant.
• gnomAD v4.1 reports grpmax FAF 0.00012624 (0.012624%), total AF 0.000113943645, and 164 of 1,439,308 alleles for NM_000179.3:c.242C>T (p.Ala81Val).

Sources

Reasoning

The governing InSiGHT MSH6 Version 2.0 specification requires gnomAD v4 grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022%-0.22%) for BS1, with exclusion of a founder pathogenic variant. gnomAD v4.1 reports a grpmax FAF of 0.00012624 (0.012624%), which is below the lower BS1 boundary; therefore the criterion 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

• InSiGHT MSH6 Version 2.0 specifies BS1 at gnomAD v4 grpmax FAF >=0.00022 and <0.0022 (0.022%-0.22%), with exclusion of a founder pathogenic variant.
• gnomAD v4.1 reports grpmax FAF 0.00012624 (0.012624%), below 0.00022.

Sources

Reasoning

The MSH6 InSiGHT VCEP PS2 rule is applicable and assigns 2 points per proband when a de novo variant is documented with confirmed maternity and paternity in a proband with an MMR-deficient Lynch-spectrum tumor, 1 point when maternity and paternity are confirmed without tumor MSI/IHC/methylation data, or 0.5 points when de novo status is assumed and one or both parents are unconfirmed. The available case evidence does not document a proband, parental testing, maternity or paternity confirmation, de novo observation, or a variant-specific Lynch-spectrum tumor result for NM_000179.3:c.242C>T (p.Ala81Val); therefore no PS2 points or strength can be assigned.

Rule

Proband with a de novo variant with both maternity and paternity confirmed in a case with MMR deficient LS spectrum tumor receives 2 points; with both maternity and paternity confirmed but no tumor MSI/IHC/methylation data, 1 point; with assumed de novo status and maternity and/or paternity unconfirmed, 0.5 points. PS2 strength is Very Strong for >=4 points, Strong for 2-3.5 points, Moderate for 1-1.5 points, and Supporting for 0.5 points.

Evidence considered

• The MSH6 InSiGHT VCEP Version 2.0 specifies PS2 point assignments of 2, 1, or 0.5 based on confirmed parental relationships, tumor evidence, and assumed de novo status.
• The case evidence contains no documented variant-specific proband de novo finding, parental testing, maternity or paternity confirmation, or Lynch-spectrum tumor MSI/IHC/methylation result.

Still missing

• A documented proband with the variant is needed.
• Parental testing and explicit maternity and paternity confirmation are needed to establish de novo status.
• Variant-specific Lynch-spectrum tumor MSI/IHC and methylation results are needed for the 2-point PS2 category.

Sources

Reasoning

The 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 following its functional-assay flowchart. The available variant-specific literature review identified no assay result for NM_000179.3:c.242C>T (NP_000170.1:p.(Ala81Val)); the reviewed papers either did not mention the variant or provided only general classification methodology. No calibrated functional odds, validated assay result, assay controls, or flowchart-resolvable MMR defect are available, so PS3 cannot be applied.

Rule

Calibrated functional assays with functional odds for pathogenicity > 18.7 (Strong); >4.3 and <=18.7 (Moderate); >2.08 and <=4.3 (Supporting); or MMR function defect following the functional assay flowchart.

Evidence considered

• The MSH6 VCEP PS3 thresholds are calibrated functional odds for pathogenicity >18.7 for strong, >4.3 and <=18.7 for moderate, and >2.08 and <=4.3 for supporting evidence.
• The MSH6 VCEP functional-assay flowchart requires an assay pathway capable of establishing an MMR function defect; no such variant-specific result is documented here.
• The reviewed paper with PMID:26467025 describes a general variant-scoring system and does not report MSH6 c.242C>T or a variant-specific functional assay result.
• The reviewed paper with PMID:27363726 evaluates a clinical history-weighting algorithm and does not report MSH6 c.242C>T or a variant-specific functional assay result.

Still missing

• A validated, variant-specific MSH6 functional assay with appropriate controls and a calibrated functional odds estimate is needed.
• Alternatively, two concordant functional assays sufficient to demonstrate an MMR function defect under the MSH6 VCEP flowchart are needed.

Sources

Reasoning

The ClinGen InSiGHT MSH6 Version 2.0 specification defines PM3 using calibrated biallelic evidence points: 0.5 points for Supporting, 1–1.5 for Moderate, 2–3.5 for Strong, and ≥4 for Very Strong. The available case evidence contains no qualifying affected-proband observation with a second pathogenic or likely pathogenic MSH6 variant, no documented cis/trans phase, and no confirmed biallelic inheritance for NM_000179.3:c.242C>T (p.Ala81Val), so a PM3 point score cannot be assigned.

Rule

MSH6 VCEP PM3: ≥4 points = Very Strong; 2–3.5 points = Strong; 1–1.5 points = Moderate; 0.5 points = Supporting.

Evidence considered

• The MSH6 Version 2.0 VCEP specifies PM3 strengths by biallelic evidence points: 0.5 points for Supporting, 1–1.5 for Moderate, 2–3.5 for Strong, and ≥4 for Very Strong.
• No case-specific affected-proband, second pathogenic-variant, homozygosity, or cis/trans-phase observation was identified for NM_000179.3:c.242C>T (p.Ala81Val).

Still missing

• A documented affected proband with a second pathogenic or likely pathogenic MSH6 variant is absent.
• Phase information establishing the variants are in trans, or evidence of homozygosity, is absent.
• No PM3 point score can therefore be calculated under the MSH6 VCEP framework.

Sources

Reasoning

The governing MSH6 VCEP requires a different missense change at the same amino-acid residue that is Pathogenic or Likely Pathogenic at the protein level and not due to aberrant splicing; PM5 also requires PP3 Supporting for the queried missense variant. The queried residue is Ala81, but the available PM5 comparator artifact reports 0 same-residue candidates among 9 candidates seen and records an HTTP 429 soft failure during harvesting. Because the comparator search was incomplete and no verified same-residue classification is available, PM5 cannot be reliably adjudicated from the collected evidence.

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 the other variant is Likely Pathogenic due to a missense alteration.

Evidence considered

• The governing MSH6 VCEP requires a different missense change at the same amino-acid residue, classified Pathogenic or Likely Pathogenic at the protein level and not due to aberrant splicing; PM5 use also requires PP3 Supporting for the queried missense change.
• The queried variant is NP_000170.1:p.(Ala81Val), affecting residue Ala81.
• The PM5 comparator artifact reports 9 candidates seen but 0 same-residue candidates, with no expert-panel or non-expert-majority Pathogenic/Likely Pathogenic comparator and an HTTP 429 harvesting error.

Still missing

• A complete, independently verified search for alternate missense substitutions at MSH6 residue Ala81 with VCEP Pathogenic or Likely Pathogenic protein-level classification is required.
• The governing PM5 rule requires PP3 Supporting for the queried missense variant; this group does not adjudicate PP3.

Sources

Reasoning

The MSH6 InSiGHT VCEP requires co-segregation to be evaluated using the combined Bayes likelihood ratio, multiplying results across multiple pedigrees. It assigns PP1 Supporting for a combined Bayes likelihood ratio >2.08 and <=4.3, Moderate for >4.3 and <=18.7, and Strong for >18.7 in at least two families. No affected or unaffected relatives, meioses, pedigree information, segregation testing, or calculated Bayes likelihood ratio is documented for NM_000179.3:c.242C>T (p.Ala81Val), so PP1 cannot be assessed.

Rule

Co-segregation with disease in pedigrees has PP1 Supporting strength for a combined Bayes likelihood ratio >2.08 and <=4.3, Moderate for >4.3 and <=18.7, and Strong for >18.7 in at least two families; results from multiple pedigrees are multiplied together.

Evidence considered

• The MSH6 InSiGHT VCEP specifies PP1 Supporting for a combined Bayes likelihood ratio >2.08 and <=4.3, Moderate for >4.3 and <=18.7, and Strong for >18.7 in at least two families.
• The case evidence provides no variant-specific pedigree, affected-relative segregation, unaffected-relative testing, meiosis count, or combined Bayes likelihood ratio.

Still missing

• A pedigree with affected and unaffected relatives is needed.
• Variant testing results for informative relatives and the number of informative meioses are needed.
• A combined Bayes likelihood ratio calculated under the VCEP-recommended approach is needed.

Sources

Reasoning

The governing MSH6 VCEP requires tumor-specific phenotype evidence: 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 for PP4 supporting; two independent tumors for moderate; or at least three independent tumors in at least two families for strong. The available exact-variant ClinVar trail reports observation in ovarian and colorectal cancer but does not document MSI status, IHC protein loss, tumor-genome testing, or the number and independence of qualifying tumors. Therefore the PP4 rule cannot be applied from the available 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; MSI-H tumor with inconsistent protein expression does not meet PP4. Moderate: 2 independent qualifying tumors. Strong: >=3 independent qualifying tumors in >=2 families.

Evidence considered

• The VCEP PP4 supporting rule requires one CRC or endometrial MSI-H tumor with a standard marker panel or tumor genome and/or MMR protein loss consistent with the variant location; the moderate and strong rules require two or at least three qualifying independent tumors, respectively.
• An exact-variant ClinVar submission signal reports c.242C>T in an individual with ovarian cancer and an individual with colorectal cancer, but the available record does not provide the required MSI, tumor-genome, IHC, or qualifying-count details.
• PMID:23047549 describes an ovarian-cancer cohort and does not explicitly mention MSH6 c.242C>T or p.(A81V).

Still missing

• Variant-specific MSI status or tumor-genome results are unavailable.
• Variant-specific MMR immunohistochemistry results consistent with MSH6 loss are unavailable.
• The number, independence, and family distribution of qualifying CRC or endometrial tumors are not documented.

Sources

Reasoning

The governing InSiGHT MSH6 Version 2.0 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-syndrome cancer above its median age of onset, without clinical manifestations of CMMRD; phase must be confirmed through parental or offspring testing. The available population data show zero homozygotes but do not document the required individual-level co-occurrence, phenotype, age, CMMRD assessment, or phase confirmation.

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

• InSiGHT MSH6 Version 2.0 requires BS2 Strong co-occurrence in trans with a known pathogenic variant in the same gene in a qualifying cancer patient, absence of CMMRD manifestations, and phase confirmation through parents or offspring.
• gnomAD v4.1 reports zero homozygotes, but this population summary is not evidence of the VCEP's required individual-level co-occurrence scenario.

Still missing

• Individual-level co-occurrence in trans with a known pathogenic MSH6 sequence variant is not documented.
• Qualifying colorectal or other Lynch-syndrome cancer age, absence of CMMRD manifestations, and parental or offspring phase confirmation are not documented.

Sources

Reasoning

The MSH6 VCEP permits BS3 strong for calibrated functional assays with functional odds for pathogenicity <=0.05, and BS3 supporting for calibrated functional odds >0.05 and <=0.48 or variant-specific proficient function in protein and mRNA-based assays following the MMR functional-assay flowchart. The available variant-specific literature review identified no assay result for NM_000179.3:c.242C>T (NP_000170.1:p.(Ala81Val)); the reviewed papers either did not mention the variant or provided only general classification methodology. No calibrated benign functional odds, validated proficient-function assay, assay controls, or flowchart-resolvable benign result are available, so BS3 cannot be applied.

Rule

Calibrated functional assays with functional odds for pathogenicity <=0.05 (Strong) or >0.05 and <=0.48 (Supporting); alternatively, variant-specific proficient function in protein and mRNA-based assays following the MMR functional assay flowchart.

Evidence considered

• The MSH6 VCEP BS3 thresholds are calibrated functional odds for pathogenicity <=0.05 for strong and >0.05 and <=0.48 for supporting evidence.
• The MSH6 VCEP also allows BS3 supporting for variant-specific proficient protein and mRNA function evaluated through its functional-assay flowchart, but no such result is documented here.
• The reviewed paper with PMID:26467025 describes a general variant-scoring system and does not report MSH6 c.242C>T or a variant-specific benign functional assay result.
• The reviewed paper with PMID:27363726 evaluates a clinical history-weighting algorithm and does not report MSH6 c.242C>T or a variant-specific benign functional assay result.

Still missing

• A validated, variant-specific MSH6 functional assay with appropriate wild-type, deficient, and other relevant controls is needed.
• A calibrated functional odds estimate <=0.48, or variant-specific proficient protein and mRNA function under the MSH6 VCEP flowchart, is needed.

Sources

Reasoning

The MSH6 InSiGHT VCEP assigns BS4 Supporting strength for lack of co-segregation with a combined Bayes likelihood ratio >0.05 and <=0.48, and Strong strength for a combined Bayes likelihood ratio <0.05. No variant-negative affected relatives, discordant pedigree observations, non-segregation testing, or combined Bayes likelihood ratio is documented for NM_000179.3:c.242C>T (p.Ala81Val), so BS4 cannot be assessed.

Rule

Lack of co-segregation with disease has BS4 Strong strength for a combined Bayes likelihood ratio <0.05 and Supporting strength for a combined Bayes likelihood ratio >0.05 and <=0.48.

Evidence considered

• The MSH6 InSiGHT VCEP specifies BS4 Strong for a combined Bayes likelihood ratio <0.05 and Supporting for >0.05 and <=0.48.
• The case evidence provides no variant-specific non-segregation observation, discordant relative testing, pedigree data, or combined Bayes likelihood ratio.

Still missing

• A pedigree containing informative affected or unaffected relatives with discordant variant status is needed.
• Variant testing results for relatives who do not show expected co-segregation are needed.
• A combined Bayes likelihood ratio for lack of co-segregation is needed.

Sources

Reasoning

The governing MSH6 VCEP permits BP5 supporting for 2 or 3 tumors with MSS and/or no loss of MMR protein expression and/or an LS-spectrum tumor with MMR protein loss inconsistent with MSH6, or for one BRAF V600E colon tumor or one MLH1-methylated LS-spectrum tumor with MSI-H/MLH1 loss; BP5 strong requires at least 4 tumors or at least 2 such BRAF/methylation findings. No variant-specific tumor MSS, IHC, BRAF V600E, MLH1 methylation, or qualifying tumor-count evidence is available, so BP5 cannot be assessed.

Rule

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

Evidence considered

• The VCEP BP5 supporting rule requires 2 or 3 qualifying tumors with MSS and/or no MMR protein loss, or one qualifying BRAF V600E or MLH1-methylation finding; the strong rule requires >=4 tumors or >=2 such molecular findings.
• The exact-variant evidence only indicates ovarian and colorectal cancer observations without the tumor molecular details required by the VCEP BP5 rule.

Still missing

• Variant-specific tumor MSS/MSI results and MMR immunohistochemistry are unavailable.
• Variant-specific BRAF V600E or MLH1 methylation results are unavailable.
• The number and independence of potentially qualifying tumors are not documented.

Sources