MSH6 c.3926C>A, p.(Pro1309Gln)

Reasoning

The governing MSH6 InSiGHT VCEP permits PM2 at Supporting strength when the allele frequency is absent or extremely rare (<0.00002, <1 in 50,000 alleles) in gnomAD v4. NM_000179.2:c.3926C>A (p.Pro1309Gln) is absent from gnomAD v4.1, satisfying the specified PM2 Supporting rule.

Rule

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

Evidence considered

• The MSH6 VCEP PM2 rule is absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in the gnomAD v4 dataset.
• NM_000179.2:c.3926C>A (MSH6 p.Pro1309Gln) was reported absent from gnomAD v4.1, meeting the VCEP's absent/extremely rare population-frequency requirement.

Sources

Reasoning

NM_000179.2:c.3926C>A (p.Pro1309Gln) is a missense variant. Per the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification v2.0 for MSH6, PP3 is applied via the gene-specific HCI-PRIORS lookup: HCI prior probability of pathogenicity >0.81 = PP3_Moderate; >0.68 and <=0.81 = PP3_Supporting. The local HCI-PRIORS-MSH6 lookup for this exact coding substitution (c.3926C>A / p.P1309Q, dbid MSH6_08617) returns a prior probability of 0.823, which exceeds the 0.81 threshold, meeting PP3 at Moderate strength per the VCEP's own pre-assigned rule. SpliceAI predicts no splice impact (max delta = 0.00) for this variant, but the SpliceAI-based PP3 clause in this VCEP's rule only applies to non-canonical splice nucleotide variants, not to standard missense substitutions, so it is not invoked here; the missense (HCI-prior) path governs.

Rule

MSH6 InSiGHT VCEP CSpec v2.0 PP3: Missense variant with HCI prior probability for pathogenicity >0.81 = PP3_Moderate.

Evidence considered

• VCEP PP3 rule (MSH6 InSiGHT CSpec v2.0): 'Missense variant with HCI prior probability for pathogenicity >0.81 as per https://hci-priors.hci.utah.edu/PRIORS' assigns PP3 at Moderate strength.
• HCI-PRIORS-MSH6 local lookup table entry for c.3926C>A (p.P1309Q, dbid MSH6_08617): hci_prior_probability = 0.823, which is >0.81, meeting the VCEP's PP3_Moderate threshold.
• SpliceAI lookup for NM_000179.2:c.3926C>A shows max delta score = 0.00 (no significant predicted splice impact); this variant is a missense substitution, not a non-canonical splice nucleotide variant, so the SpliceAI-based PP3 clause of the VCEP rule does not apply and only the HCI-prior missense path is used.

Sources

Reasoning

The governing MSH6 InSiGHT 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. The variant is absent from gnomAD v4.1, so the BA1 frequency threshold 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 VCEP BA1 rule is gnomAD v4 Grpmax filtering allele frequency ≥0.0022 (0.22%), with exclusion of founder pathogenic variants.
• NM_000179.2:c.3926C>A (MSH6 p.Pro1309Gln) was reported absent from gnomAD v4.1; therefore its observed frequency is below the BA1 threshold rather than at or above it.

Sources

Reasoning

The governing MSH6 InSiGHT VCEP specifies BS1 Strong when the gnomAD v4 Grpmax filtering allele frequency is at least 0.00022 and below 0.0022 (0.022–0.22%) and the variant is not a founder pathogenic variant. The variant is absent from gnomAD v4.1, so this frequency interval 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 VCEP BS1 rule is gnomAD v4 Grpmax filtering allele frequency ≥0.00022 and <0.0022 (0.022–0.22%), with exclusion of founder pathogenic variants.
• NM_000179.2:c.3926C>A (MSH6 p.Pro1309Gln) was reported absent from gnomAD v4.1; no allele frequency in the BS1 interval was observed.

Sources

Reasoning

The VCEP-specific BP4 rule requires HCI prior probability <0.11 for missense variants (or SpliceAI delta <=0.1 for intronic/synonymous variants). This variant is missense with an HCI prior probability of 0.823, far above the 0.11 BP4 threshold, so BP4 does not apply via the missense path. The SpliceAI intronic/synonymous clause is not applicable to this missense substitution.

Rule

MSH6 InSiGHT VCEP CSpec v2.0 BP4: Missense variant with HCI prior probability <0.11.

Evidence considered

• VCEP BP4 rule (MSH6 InSiGHT CSpec v2.0): '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 as per Walker et al 2023.'
• HCI-PRIORS-MSH6 lookup for c.3926C>A gives hci_prior_probability = 0.823, well above the 0.11 BP4 threshold, so the missense BP4 path is not met.
• This variant is missense, not intronic or synonymous, so the SpliceAI-based BP4 clause of the VCEP rule is not applicable.

Sources

Reasoning

MSH6 VCEP (ClinGen InSiGHT Hereditary CRC/Polyposis EP, v2.0) defines PS1 as a predicted missense substitution encoding the same amino acid change (p.Pro1309Gln) via a different nucleotide change previously established by this VCEP as Pathogenic (Strong) or Likely Pathogenic with normal RNA result plus PM2_supporting (Moderate). No comparator variant encoding the identical p.(Pro1309Gln) change via an alternate codon was identified in the case bundle; the PM5/PS1 comparator harvest for this residue returned zero same-residue candidates and failed with an HTTP 429 rate-limit error during ClinVar querying, so absence of a same-amino-acid comparator cannot be confirmed with confidence.

Rule

PS1_Strong: same amino acid change via alternate nucleotide, VCEP-classified Pathogenic (not splice). PS1_Moderate: same amino acid change via alternate nucleotide as VCEP Likely Pathogenic missense with normal RNA and PM2_supporting met.

Evidence considered

• cspec v2.0 PS1 rule: same amino acid change via different nucleotide change, previously VCEP-classified Pathogenic (Strong) or Likely Pathogenic with normal RNA + PM2_supporting (Moderate).
• pm5_candidates.json same-residue comparator harvest for P1309 found 0 same-residue candidates out of 7 total candidates seen, and the harvest itself failed softly with error 'HTTP Error 429: Too Many Requests', meaning the search was incomplete rather than a confirmed negative.

Still missing

• Same-residue/same-amino-acid comparator search (pm5_candidates.json) failed with HTTP 429 (Too Many Requests) before completing; a clean re-query of ClinVar for other codon changes producing p.Pro1309Gln classified Pathogenic/Likely Pathogenic by this VCEP is needed to confirm PS1 does not apply.

Sources

Reasoning

The MSH6 InSiGHT VCEP framework makes PS2 applicable and requires documented de novo status with maternity/paternity confirmation plus the specified Lynch-spectrum tumor context. The case-evidence bundle contains no proband-level de novo observation, parental testing, maternity/paternity confirmation, or qualifying tumor evidence for NM_000179.2:c.3926C>A; therefore PS2 cannot be assigned.

Rule

MSH6 InSiGHT VCEP PS2: >=4 de novo points = Very Strong; 2-3.5 points = Strong; 1-1.5 points = Moderate; 0.5 points = Supporting.

Evidence considered

• The MSH6 InSiGHT VCEP PS2 rule requires 2 de novo points for a proband with confirmed maternity and paternity and an MMR-deficient Lynch-spectrum tumor, 1 point for confirmed maternity and paternity without tumor data, or 0.5 points for assumed de novo status with unconfirmed maternity and/or paternity and a Lynch-spectrum tumor.
• The consolidated case-evidence bundle provides no case-specific proband, parental testing, de novo, or tumor findings for this criterion.

Still missing

• Proband-level documentation that the variant is de novo.
• Maternity and paternity testing results.
• Lynch-spectrum tumor diagnosis and, if applicable, MSI/IHC/methylation results.
• Number of eligible probands and de novo points, if any.

Sources

Reasoning

The InSiGHT MMR VCEP specification (cspec) defines PS3 using calibrated functional-assay odds of pathogenicity (Strong >18.7, Moderate >4.3-18.7, Supporting >2.08-4.3), MMR function-defect-per-flowchart, or monoallelic expression loss (<10% WT). No variant-specific functional assay data (cell-based MMR activity/complementation assay, splicing/mRNA assay, or monoallelic expression study) for NM_000179.2:c.3926C>A (p.Pro1309Gln) is present anywhere in the case bundle. The VCEP-supplied Functional-assay-flowchart.pdf and Functional-assay-SVI-documentation-MMR.xlsx are generic calibration/methodology references only, containing no variant-specific results, and the sole literature hit (PMID:28492532, Sherloc) is tagged case_observation/PS4 in this case's literature triage, not a functional assay report, and was not retrieved as containing assay data for this variant. No in silico predictor substitutes for PS3 under this VCEP (PS3 requires wet-lab functional assay evidence, distinct from PP3's HCI-prior pathway). Cannot assess PS3 without a published or laboratory functional assay result for this specific variant.

Evidence considered

• cspec PS3 rule (InSiGHT MMR VCEP v2.0): Strong >18.7, Moderate >4.3-18.7 (or MMR function defect per flowchart, or monoallelic <10% WT expression), Supporting >2.08-4.3 functional odds for pathogenicity
• Case bundle contains only generic VCEP reference materials (Functional-assay-flowchart.pdf, Functional-assay-SVI-documentation-MMR.xlsx) with no variant-specific assay results extracted for this variant
• PMID:28492532 (Sherloc) is triaged in this case's literature_packet under theme case_observation with candidate_criteria BAS/PS4, not functional assay data; not opened for PS3 purposes as it was not flagged as containing functional evidence for this variant

Still missing

• No calibrated MMR functional assay (e.g. cell-based complementation/MMR activity assay) result reported for c.3926C>A / p.Pro1309Gln
• No mRNA/splicing or monoallelic expression assay data for this variant
• No functional-odds-for-pathogenicity value available to apply the VCEP PS3 strength tiers

Reasoning

The MSH6 InSiGHT VCEP framework makes PM3 applicable 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 case evidence bundle provides no affected-proband observation, second pathogenic or likely pathogenic MSH6 variant, phase determination, or phase-unknown co-occurrence observation. Therefore no PM3 points can be assigned and the criterion cannot be assessed 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; pathogenic/likely pathogenic in trans = 1.0 point, phase unknown = 0.5 points; sum all cases to determine PM3 strength.

Evidence considered

• The governing MSH6 InSiGHT VCEP rule assigns 1.0 PM3 point for a pathogenic/likely pathogenic variant in trans and 0.5 points when phase is unknown, with PM3 strength thresholds of supporting at 0.5 points, moderate at 1-1.5 points, strong at 2-3.5 points, and very strong at 4 or more points.
• The case bundle contains no report of a second MSH6 pathogenic/likely pathogenic variant, affected-proband CMMRD features, or phase information for NM_000179.2:c.3926C>A (p.Pro1309Gln).

Still missing

• Documented clinical features consistent with CMMRD in the affected proband
• A second known pathogenic or likely pathogenic MSH6 sequence variant in the same proband
• Parental or offspring testing establishing the variants are in trans, or an explicitly documented phase-unknown co-occurrence
• Evidence that the second variant is pathogenic or likely pathogenic independent of its source classification

Sources

Reasoning

MSH6 VCEP PM5 requires a different missense change at the same residue (P1309) previously classified Pathogenic (PM5_Moderate) or Likely Pathogenic (PM5_Supporting) on the protein level (not aberrant splicing), and additionally requires that PP3 be met (supporting or greater) for the current variant before PM5 can be applied. The dedicated same-residue comparator harvest (pm5_candidates.json) returned 0 same-residue candidates among 7 total candidates seen, but the underlying ClinVar query failed with an HTTP 429 rate-limit error, so this cannot be treated as a confirmed absence of comparator variants at codon 1309.

Rule

PM5_Moderate: different missense at same residue, VCEP Pathogenic on protein level, PP3 met. PM5_Supporting: different missense at same residue, VCEP Likely Pathogenic on protein level, PP3 met.

Evidence considered

• cspec v2.0 PM5 rule: different missense change at same residue previously VCEP-classified Pathogenic (Moderate) or Likely Pathogenic (Supporting) on protein level, not aberrant splicing; PM5 usage is gated on PP3 being met for the variant under review.
• pm5_candidates.json: residue P1309 (reference aa Pro, position 1309), same_residue_candidates = 0 of 7 total candidates seen, but harvest recorded error 'HTTP Error 429: Too Many Requests', flagged by the tool itself as a soft failure requiring manual review rather than automatic PM5 exclusion.

Still missing

• Same-residue comparator search failed with HTTP 429 before completion; needs a successful re-query of ClinVar/literature for other missense substitutions at P1309 with VCEP Pathogenic/Likely Pathogenic classification.
• Even if a comparator surfaces, VCEP rule requires PP3 to be met (supporting) for c.3926C>A before PM5 can be applied; that dependency should be checked once PP3 status from another group is finalized.

Sources

Reasoning

The MSH6 InSiGHT VCEP framework makes PP1 applicable and requires a combined segregation Bayes likelihood ratio. The case-evidence bundle contains no affected-relative genotypes, pedigree, meioses, segregation counts, or calculated Bayes likelihood ratio for NM_000179.2:c.3926C>A; therefore PP1 cannot be assigned at any strength.

Rule

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

Evidence considered

• The MSH6 InSiGHT VCEP PP1 rule uses the combined segregation Bayes likelihood ratio: >18.7 in at least 2 families for Strong, >4.3 and <=18.7 for Moderate, and >2.08 and <=4.3 for Supporting.
• The consolidated case-evidence bundle provides no case-specific pedigree, relative testing, meiosis, segregation, or Bayes likelihood ratio data for this variant.

Still missing

• Pedigree with phenotype status and relationship for informative relatives.
• Genotypes for the variant in affected and unaffected relatives.
• Informative meioses and penetrance/phenotype assumptions as needed for analysis.
• Combined segregation Bayes likelihood ratio.

Sources

Reasoning

PP4 is applicable under the MSH6 VCEP framework, but the case bundle provides no patient-level tumor MSI or MMR immunohistochemistry results. Therefore, the required phenotype-specific evidence cannot be assessed.

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. Moderate: 2 independent qualifying tumors. Strong: at least 3 independent qualifying tumors in at least 2 families.

Evidence considered

• The MSH6 VCEP PP4 rule requires one or more qualifying CRC/endometrial MSI-H tumors using a standard MSI panel or tumor genome and/or MMR protein loss consistent with the variant location; the bundle contains no such patient-level tumor results.
• No publication was retained for this group because the only linked full text does not mention the exact variant NM_000179.2:c.3926C>A or p.Pro1309Gln.

Still missing

• Patient-level CRC or endometrial tumor MSI status using a qualifying marker panel or tumor genome is not provided.
• Patient-level MMR protein immunohistochemistry results, including whether MSH6 loss is consistent with the variant location, are not provided.
• The number of independent qualifying tumors and families is not provided.

Sources

Reasoning

The MSH6 VCEP BS2 rule requires documented in-trans co-occurrence 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 case bundle contains no qualifying individual-level co-occurrence, phase, age, or CMMRD assessment.

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 VCEP BS2 rule requires in-trans co-occurrence with a known pathogenic variant in the same gene, qualifying Lynch syndrome cancer age criteria, no CMMRD manifestations, and phase confirmation by parents or offspring.
• The available case evidence reports population absence and does not provide a qualifying patient-level co-occurrence or phase analysis.

Still missing

• No documented patient-level in-trans co-occurrence with a known pathogenic MSH6 variant.
• No confirmed phase from parental or offspring testing.
• No qualifying cancer type and age-of-onset documentation.
• No assessment excluding prior or current clinical manifestations of CMMRD.

Sources

Reasoning

The InSiGHT MMR VCEP specification (cspec) defines BS3 using calibrated functional-assay odds of pathogenicity (Strong <=0.05, Supporting >0.05-0.48) or variant-specific proficient function in protein/mRNA-based assays per the MMR functional assay flowchart (for missense variants; the synonymous/intronic-NMD pathway does not apply, as this is a missense substitution). No variant-specific functional assay data of any kind for NM_000179.2:c.3926C>A (p.Pro1309Gln) is present in the case bundle. Without a functional odds value or a documented proficient-function assay result for this exact variant, BS3 cannot be evaluated.

Evidence considered

• cspec BS3 rule (InSiGHT MMR VCEP v2.0): Strong functional odds for pathogenicity <=0.05, Supporting >0.05-0.48, or variant-specific proficient function in protein/mRNA-based assays per MMR functional assay flowchart
• Case bundle contains only generic VCEP reference materials, with no variant-specific functional assay result located for this variant

Still missing

• No calibrated functional assay result (functional odds <=0.48) demonstrating proficient MMR function for c.3926C>A / p.Pro1309Gln
• No protein- or mRNA-based lab assay result per the MMR functional assay flowchart for this variant

Reasoning

The MSH6 InSiGHT VCEP framework makes BS4 applicable and requires documented lack of co-segregation with a combined Bayes likelihood ratio. The case-evidence bundle contains no non-segregation observations, pedigree, relative genotypes, informative meioses, or calculated Bayes likelihood ratio for NM_000179.2:c.3926C>A; therefore BS4 cannot be assigned.

Rule

MSH6 InSiGHT VCEP BS4: combined non-segregation Bayes likelihood ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting.

Evidence considered

• The MSH6 InSiGHT VCEP BS4 rule uses the combined non-segregation Bayes likelihood ratio: <0.05 for Strong and >0.05 and <=0.48 for Supporting, with cautions concerning phenocopies and alternative pathogenic variants.
• The consolidated case-evidence bundle provides no case-specific non-segregation, pedigree, relative testing, meiosis, or Bayes likelihood ratio data for this variant.

Still missing

• Pedigree with phenotype status and relationship for informative relatives.
• Variant genotypes in affected and unaffected relatives.
• Documented non-segregation observations and assessment of phenocopy or alternative-variant explanations.
• Combined non-segregation Bayes likelihood ratio.

Sources

Reasoning

BP5 is applicable under the MSH6 VCEP framework, but the case bundle contains no patient-level tumor data establishing an alternate molecular basis, such as MSS/no MMR protein loss, inconsistent MMR protein loss, BRAF V600E, or MLH1 methylation.

Rule

Supporting: 2 or 3 qualifying tumors with MSS and/or no MMR protein loss and/or inconsistent MMR protein loss, or 1 qualifying BRAF V600E/MLH1 methylation result. Strong: at least 4 qualifying tumors or at least 2 qualifying BRAF V600E/MLH1 methylation results.

Evidence considered

• The MSH6 VCEP BP5 rule requires 2-3 qualifying tumors for supporting strength or at least 4 qualifying tumors for strong strength, with specified MSS/IHC-inconsistent findings, or qualifying BRAF V600E/MLH1 methylation evidence; none is documented in the case bundle.
• The absence of the variant from COSMIC is not evidence of an alternate molecular basis for this patient's disease and was not used for BP5.

Still missing

• Patient-level tumor MSI/MSS results and MMR protein expression results are not provided.
• Patient-level BRAF V600E or MLH1 methylation results, if relevant, are not provided.
• The number of qualifying tumors is not provided.

Sources