MSH6 c.2975A>G, p.(Glu992Gly)

Reasoning

PM2 Supporting is met under the MSH6 VCEP specification. gnomAD v4.1 reports the variant at total AF 1.91444e-06 (3/1,567,038 alleles), which is below the VCEP threshold of 0.00002 (less than 1 in 50,000 alleles). A single observed homozygote does not prevent PM2 because the specified PM2 rule is based on extreme rarity/allele frequency, while the available data do not establish a benign healthy-adult observation sufficient for BS2.

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 rule assigns PM2 Supporting when the allele frequency is <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports total AF 1.91444e-06 (3 alternate alleles among 1,567,038 alleles), below 0.00002; the highest reported population AF is 3.31246e-05 in the Remaining individuals population, but this is a population-specific observation and does not replace the reported total AF for the VCEP PM2 threshold.
• The gnomAD v4 record reports one homozygote; no individual-level phenotype, age, penetrance, phase, or CMMRD information is provided to convert that observation into BS2 evidence.

Sources

Reasoning

Per the MSH6 VCEP cspec BP4 rule, a missense variant with HCI-prior probability of pathogenicity <0.11 meets BP4_Supporting. The HCI prior lookup table gives c.2975A>G (p.E992G) a probability of 0.0276, below the 0.11 threshold, so BP4_Supporting is met. This is the VCEP-designated computational pathway for this missense variant; the alternate BP4 branch (SpliceAI delta <=0.1 for intronic/synonymous variants) is not applicable since this is a missense coding substitution, though the SpliceAI max delta of 0.002 is directionally consistent (no splice signal).

Rule

cspec BP4 (MSH6 VCEP v2.0)

Evidence considered

• VCEP HCI-PRIORS-MSH6.txt lookup guidance: BP4_Supporting when HCI prior probability for pathogenicity <0.11.
• hci_prior lookup for c.2975A>G (protein p.E992G): probability = 0.0276, which is <0.11, meeting the BP4_Supporting threshold.
• cspec BP4 rule text: 'Missense variant with HCI-prior probability of pathogenicity <0.11 ... OR For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1' — the missense/HCI branch is the applicable one here.

Sources

Reasoning

MSH6 VCEP PM5 requires (a) a different missense change at the same residue (codon 992) previously classified Pathogenic/Likely Pathogenic on the protein level by this VCEP (not due to aberrant splicing), AND (b) PP3 to be met (supporting or greater) for this variant p.Glu992Gly. Neither precondition is satisfied here: the comparator search (pm5_candidates.json) identified zero same-residue candidate variants (same_residue_candidates=0), and the local HCI-prior missense predictor score for c.2975A>G is 0.0276 (well below the VCEP PP3_Supporting threshold of >0.68, and in fact in the BP4_Supporting range of <0.11), so PP3 is not met for this variant. Because the VCEP text explicitly gates PM5 on PP3 being met, PM5 cannot be applied.

Rule

MSH6 VCEP cspec v2.0 PM5

Evidence considered

• MSH6 VCEP v2.0 cspec PM5 rule: 'Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic ... Only use PM5 if PP3 is supporting for the missense change.'
• pm5_candidates.json same-residue comparator harvest returned same_residue_candidates=0, expert_panel_plp_candidates=0, non_expert_majority_plp_candidates=0.
• hci_prior local predictor score for c.2975A>G is 0.0276 (custom_pp2_score 0.029, MAPP score 11.35), below the VCEP PP3_Supporting cutoff of >0.68 and within the BP4_Supporting range (<0.11), so the PP3-met precondition required for PM5 is not satisfied.

Sources

Reasoning

NM_000179.2:c.2975A>G (p.Glu992Gly) is a missense substitution. The governing ClinGen InSiGHT MMR VCEP specification (cspec v2.0) directs PP3 for MSH6 missense variants to be assessed exclusively via the local HCI-prior lookup table (HCI-PRIORS-MSH6.txt / hci_prior source), not via REVEL, BayesDel, or SpliceAI for a coding missense change. The exact coding substitution c.2975A>G was found in the HCI prior table with probability of pathogenicity = 0.0276, well below both the PP3_Moderate threshold (>0.81) and the PP3_Supporting threshold (>0.68-0.81). SpliceAI max delta score is 0.002 (no splicing impact), which is not the applicable in-silico axis for a canonical-position missense variant under this VCEP's PP3 rule (that SpliceAI branch is reserved for non-canonical splice-site nucleotides). REVEL (0.458) and BayesDel (-0.0480668) were retrieved but are not the VCEP-designated predictors for MSH6 PP3/BP4; per pipeline calibration policy BayesDel additionally lacks a verified published threshold and cannot be used regardless. PP3 is therefore not met.

Rule

cspec PP3 (MSH6 VCEP v2.0)

Evidence considered

• VCEP HCI-PRIORS-MSH6.txt lookup guidance (index_guidance_entries): 'Look up the exact coding substitution ... use the HCI prior probability for pathogenicity for PP3/BP4 thresholds: >0.81 = PP3_Moderate, >0.68 and <=0.81 = PP3_Supporting, <0.11 = BP4_Supporting.'
• hci_prior lookup for c.2975A>G (protein p.E992G): probability = 0.0276, mapp_score = 11.35, custom_pp2_score = 0.029 — well below the PP3 supporting threshold of >0.68.
• cspec PP3 rule text: 'Missense variant with HCI prior probability for pathogenicity >0.81' (Moderate) / '>0.68 & <=0.81' (Supporting) OR 'Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2' — the SpliceAI branch does not apply because this is a canonical-position missense substitution, not a non-canonical splice-site variant.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00)' with per-tissue scores DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.002 — confirms no splice signal, consistent with (not independently supportive of) the HCI-based non-met call.

Sources

Reasoning

BA1 is not met. The 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 overall allele frequency of 1.91444e-06 (3/1,567,038 alleles), far below the VCEP threshold. The available record does not identify this variant as a founder pathogenic variant.

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 rule for BA1 requires gnomAD v4 Grpmax filtering allele frequency >=0.0022 (0.22%) and exclusion as a founder pathogenic variant.
• gnomAD v4.1 reports NM_000179.2:c.2975A>G at total AF 1.91444e-06, with 3 alternate alleles among 1,567,038 alleles; this is below 0.0022.
• The gnomAD v4 case record has no founder-pathogenic designation for this variant.

Sources

Reasoning

BS1 is not met. The MSH6 VCEP specifies BS1 Strong for a gnomAD v4 Grpmax filtering allele frequency of at least 0.00022 and below 0.0022 (0.022%-0.22%), with the variant excluded as a founder pathogenic variant. The gnomAD v4.1 overall allele frequency is 1.91444e-06, below the lower threshold, and the available record does not identify a founder pathogenic variant.

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 rule for BS1 Strong requires gnomAD v4 Grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022%-0.22%) and exclusion as a founder pathogenic variant.
• gnomAD v4.1 reports total AF 1.91444e-06 (3/1,567,038 alleles), which is below 0.00022.
• The gnomAD v4 case record has no founder-pathogenic designation for this variant.

Sources

Reasoning

MSH6 VCEP (InSiGHT Hereditary Colorectal Cancer/Polyposis EP v2.0) PS1 rule requires identification of a different nucleotide change producing the same amino acid change (p.Glu992Gly) that has been previously classified Pathogenic by this VCEP (or, for splice-affecting nucleotides, a confirmed Pathogenic splice variant at the same non-canonical position). The dedicated same-residue comparator search (pm5_candidates.json) failed to complete due to an NCBI rate-limit error (HTTP 429) and returned zero candidates of any kind, so it could neither confirm nor exclude a same-amino-acid-change Pathogenic comparator. No other source in the case bundle (ClinVar record, literature pass, evidence.json) reports an alternate codon producing Glu992Gly. Cannot be adjudicated as met or not_met without a completed comparator search.

Rule

MSH6 VCEP cspec v2.0 PS1

Evidence considered

• MSH6 VCEP v2.0 cspec PS1 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).' (Strong) or vs. Likely Pathogenic with normal RNA + PM2_supporting (Moderate).
• pm5_candidates.json (same underlying harvest used for PS1/PM5 comparator search) reports found=false, same_residue_candidates=0, and errors=['HTTP Error 429: Too Many Requests'], indicating the ClinVar comparator search did not complete successfully.
• No alternate-nucleotide comparator producing p.Glu992Gly was identified anywhere else in the case bundle.

Still missing

• Same-residue/same-amino-acid comparator search (pm5_candidates.json) failed with HTTP 429 (rate limited) before returning usable ClinVar comparator variants; needs to be re-run.
• No VCEP-curated list of previously classified MSH6 missense variants at codon 992 was available in this case bundle to manually cross-check.

Sources

Reasoning

No case-specific evidence documents a confirmed de novo occurrence of NM_000179.2:c.2975A>G (p.Glu992Gly), including proband status, testing of both biological parents, or confirmed maternity and paternity. The MSH6 InSiGHT VCEP framework provides a de novo point-based PS2 model, but no de novo points can be assigned from the available evidence.

Rule

≥ 4 de novo points = Very Strong; 2-3.5 de novo points = Strong; 1-1.5 de novo points = Moderate; 0.5 de novo points = Supporting.

Evidence considered

• The governing MSH6 InSiGHT VCEP specification states that PS2 is assigned using de novo points: Very Strong for >=4 points, Strong for 2-3.5 points, Moderate for 1-1.5 points, and Supporting for 0.5 points.
• The consolidated case-evidence bundle contains no documented proband-parent trio result, parental testing result, confirmed de novo observation, or de novo point calculation for this variant.

Still missing

• Proband phenotype and clinical context relevant to the variant are not documented for de novo assessment.
• Parental test results and confirmation of biological parentage are not available.
• No de novo point calculation is possible.

Sources

Reasoning

The MSH6 VCEP (InSiGHT CSPEC v2.0) requires calibrated functional assay data (functional odds for pathogenicity), MMR functional-assay-flowchart results, or monoallelic-expression/NMD RNA data to apply PS3. No variant-specific functional assay data for NM_000179.2:c.2975A>G (p.Glu992Gly) is present anywhere in the case bundle. The literature triage (literature_packet.criteria_index) does not list PS3/functional as a candidate criterion for any of the 8 associated PMIDs, and the case bundle contains no calibrated-assay odds, MSI/IHC functional assay, or RNA/monoallelic-expression result. Two ClinVar submissions (Color Diagnostics SCV000904826, All of Us SCV004839145) explicitly state in their evidence text 'to our knowledge, functional studies have not been reported for this variant,' directly confirming absence of functional assay evidence rather than supplying it.

Rule

MSH6 VCEP (InSiGHT CSPEC v2.0) PS3: Strong if calibrated functional odds for pathogenicity >18.7; Moderate if >4.3 and <=18.7 (or MMR function defect per flowchart, or monoallelic complete loss of expression <10% WT); Supporting if >2.08 and <=4.3.

Evidence considered

• ClinVar submission SCV000904826 (Color Diagnostics) and SCV004839145 (All of Us Research Program) both state verbatim: 'to our knowledge, functional studies have not been reported for this variant', confirming no variant-specific functional assay evidence exists in the reviewed record.
• literature_packet.criteria_index in the case bundle lists candidate criteria (PP3, PP5, PS4) for the 8 triaged PMIDs but none flag PS3/functional-assay content for this variant.
• The InSiGHT MSH6-specific functional assay publications identified in the case bundle (Drost et al. 2019 PMID 30504929; Drost et al. 2020 PMID 31965077; Rath et al. 2022 PMID 36054288; Rayner et al. 2022 PMID 35451539) were not retrieved as full text in this case's publications folder and are not linked to this specific variant anywhere in the bundle, so they cannot be used to ground a PS3 call for c.2975A>G.

Still missing

• No calibrated functional assay (e.g. Drost et al. cell-based MMR functional assay) result reported for this specific variant
• No MMR functional assay flowchart outcome (protein/mRNA proficiency) available
• No monoallelic expression / NMD-block RNA data available

Reasoning

The MSH6 InSiGHT VCEP specifies PM3 for co-occurrence with a known pathogenic or likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD. The framework assigns 1.0 point for a pathogenic/likely pathogenic variant confirmed in trans and 0.5 points when phase is unknown, with strength determined from the summed points. The case evidence bundle contains no affected-proband observation of a second MSH6 pathogenic/likely pathogenic variant, no documented cis/trans phase, and no family or parental/offspring testing establishing phase. Therefore PM3 cannot be applied.

Rule

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

Evidence considered

• The MSH6 VCEP PM3 rule requires co-occurrence with a known pathogenic/likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD; a confirmed in-trans observation contributes 1.0 point and phase-unknown evidence contributes 0.5 points.
• The consolidated case-evidence bundle provides no qualifying second MSH6 variant, affected-proband co-occurrence, or phase information for NM_000179.2:c.2975A>G (p.Glu992Gly).

Still missing

• A documented second MSH6 pathogenic or likely pathogenic variant in the affected proband is needed.
• Parental, offspring, or other valid phasing evidence is needed to establish whether the second variant is in trans or cis.
• Clinical features consistent with CMMRD for the relevant proband must be documented for the VCEP PM3 rule.

Sources

Reasoning

No affected-family segregation data, pedigree, informative meioses, phenotype-confirmed relatives, or Bayes likelihood ratio is provided for NM_000179.2:c.2975A>G. Therefore, PP1 cannot be assigned at Supporting, Moderate, or Strong strength under the MSH6 VCEP thresholds.

Rule

Combined Bayes likelihood ratio >18.7 in >=2 families = Strong; >4.3 and <=18.7 = Moderate; >2.08 and <=4.3 = Supporting.

Evidence considered

• The governing MSH6 InSiGHT VCEP framework requires a combined Bayes likelihood ratio >2.08 and <=4.3 for PP1 Supporting, >4.3 and <=18.7 for PP1 Moderate, and >18.7 in at least two families for PP1 Strong.
• The consolidated case-evidence bundle contains no family segregation observations, pedigree data, or Bayes likelihood ratio for this variant.

Still missing

• No pedigree or family structure is available.
• No genotypes and phenotypes for affected or unaffected relatives are available.
• No informative meiosis count or combined Bayes likelihood ratio is available.

Sources

Reasoning

The MSH6-specific framework permits PP4 based on CRC/endometrial MSI-H tumor evidence or loss of MMR protein expression consistent with the variant location, but the case bundle contains no patient-specific tumor MSI, tumor-genome, or MMR immunohistochemistry result for NM_000179.2:c.2975A>G (p.Glu992Gly). Gene-level descriptions and general guideline statements are insufficient.

Rule

MSH6 InSiGHT Version 2.0 PP4: supporting for 1 qualifying CRC/endometrial MSI-H tumor or tumor genome and/or consistent MMR protein loss; moderate for 2 independent tumors; strong for at least 3 independent tumors in at least 2 families.

Evidence considered

• The MSH6 InSiGHT Version 2.0 PP4 rule requires one or more independent CRC/endometrial MSI-H tumors or tumor-genome evidence and/or loss of MMR protein expression consistent with the variant location; none is documented in the case evidence.

Still missing

• Patient-specific CRC or endometrial tumor MSI-H result using an appropriate marker panel or tumor genome, and/or variant-consistent MSH6 protein loss by immunohistochemistry, is not available.

Sources

Reasoning

BS2 is not assessed. gnomAD v4.1 shows one homozygote, but the MSH6 VCEP BS2 rule requires documented in-trans co-occurrence with a known pathogenic 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), absence of CMMRD manifestations, and phase confirmation through parents or offspring. No such individual-level clinical, phase, or age information is available in the case 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 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 InSiGHT VCEP Version 2.0 BS2 Strong rule requires in-trans co-occurrence with a known pathogenic sequence variant in the same gene in a qualifying older-onset Lynch-spectrum cancer patient, no previous or current evidence of CMMRD, and parental or offspring testing to confirm phase.
• gnomAD v4.1 reports one homozygote for the variant among 1,567,038 alleles; gnomAD population data alone do not provide the clinical phenotype, age, trans configuration, phase confirmation, or CMMRD evaluation required by the VCEP rule.

Still missing

• Individual-level identity and clinical phenotype of the gnomAD homozygote are unavailable.
• No evidence establishes that the homozygote carried a known pathogenic MSH6 variant in trans.
• No parental or offspring testing is available to confirm phase.
• No age-at-diagnosis or adequate healthy-adult/penetrance information is available.
• No assessment of absence of clinical manifestations of constitutional mismatch repair deficiency is available.

Sources

Reasoning

The MSH6 VCEP (InSiGHT CSPEC v2.0) requires calibrated functional assay data (functional odds for pathogenicity <=0.48) or variant-specific proficient function in protein/mRNA-based assays per the MMR functional assay flowchart to apply BS3. No such variant-specific functional data for NM_000179.2:c.2975A>G (p.Glu992Gly) is present in the case bundle. As with PS3, the literature triage does not flag any of the 8 associated PMIDs for functional/PS3-BS3 content, and two ClinVar submissions explicitly note the absence of reported functional studies for this variant.

Rule

MSH6 VCEP (InSiGHT CSPEC v2.0) BS3: Strong if calibrated functional odds for pathogenicity <=0.05, or synonymous/intronic variant with no mRNA aberration under NMD-inhibition assay; Supporting if calibrated functional odds >0.05 and <=0.48, or variant-specific proficient function in protein/mRNA assays per MMR functional assay flowchart.

Evidence considered

• ClinVar submission SCV000904826 (Color Diagnostics) and SCV004839145 (All of Us Research Program) both state verbatim: 'to our knowledge, functional studies have not been reported for this variant', confirming no variant-specific functional assay evidence (proficient or deficient) exists in the reviewed record.
• literature_packet.criteria_index in the case bundle lists candidate criteria (PP3, PP5, PS4) for the 8 triaged PMIDs but none flag BS3/functional-assay content for this variant.

Still missing

• No calibrated functional assay result (odds for pathogenicity) demonstrating proficient/normal function for this variant
• No MMR functional assay flowchart protein/mRNA proficiency result available

Reasoning

No affected-relative non-segregation observations, pedigree, phenotype ascertainment, informative meioses, or combined Bayes likelihood ratio is available. Consequently, lack of co-segregation cannot be established and BS4 cannot be assigned.

Rule

Combined Bayes likelihood ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting.

Evidence considered

• The governing MSH6 InSiGHT VCEP framework requires a combined Bayes likelihood ratio <0.05 for BS4 Strong and >0.05 and <=0.48 for BS4 Supporting.
• The consolidated case-evidence bundle contains no documented lack-of-segregation observation, affected-relative testing, pedigree, or Bayes likelihood ratio for this variant.

Still missing

• No pedigree or family structure is available.
• No affected-relative genotypes and phenotype assessments are available to evaluate non-segregation.
• No combined Bayes likelihood ratio is available; therefore the VCEP BS4 strength thresholds cannot be applied.

Sources

Reasoning

The MSH6-specific framework provides BP5 rules based on multiple patient tumors with MSS and/or no MMR protein loss, discordant MMR loss, or qualifying BRAF V600E/MLH1 methylation findings. The case bundle contains no patient-specific tumor results of these types, so BP5 cannot be assessed.

Rule

MSH6 InSiGHT Version 2.0 BP5: supporting for 2-3 qualifying tumors or 1 qualifying BRAF V600E/MLH1 methylation result; strong for at least 4 qualifying tumors or at least 2 such results.

Evidence considered

• The MSH6 InSiGHT Version 2.0 BP5 rule requires 2 or 3 qualifying tumors for supporting or at least 4 for strong, or specified BRAF V600E/MLH1 methylation findings; none is documented in the case evidence.

Still missing

• Patient-specific qualifying tumor phenotype data for BP5, including tumor count and MSS/MMR protein findings or BRAF V600E/MLH1 methylation results, is not available.

Sources