MSH6 c.2463A>C, p.(Leu821=)

Reasoning

The MSH6 VCEP BP4 rule states that for intronic and synonymous variants, SpliceAI predicting no splicing impact with delta score <=0.1 supports BP4 (per Walker et al 2023). NM_000179.3:c.2463A>C is synonymous (p.(Leu821=)) and SpliceAI predicts a max delta score of 0.001 (DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.0), well below the 0.1 threshold, so BP4_Supporting is met.

Rule

MSH6 VCEP (ClinGen InSiGHT) BP4: For intronic and synonymous variants, SpliceAI predicts no splicing impact with delta score <=0.1 as per Walker et al 2023 (BP4_Supporting). Also applicable to missense variants via HCI-prior <0.11 (not relevant here, variant is synonymous).

Evidence considered

• prefetch.json compact_evidence.spliceai evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).' Raw scores: DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.0, max_delta_score=0.001.
• cspec BP4 rule (MSH6 VCEP v2.0): 'For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <=0.1 as per Walker et al 2023.' Variant qualifies as synonymous with delta well under threshold.

Sources

Reasoning

NM_000179.3:c.2463A>C is a synonymous (silent) variant (p.(Leu821=)) located well within the exon interior (exon spans c.628-c.3172; variant sits 1835 bp from the 5' splice boundary and 709 bp from the 3' splice boundary), far beyond the -21/+7 exonic proximity window used by the MSH6 VCEP BP7 rule. SpliceAI corroborates no splice impact (max delta score = 0.001). The VCEP explicitly notes 'Variants may satisfy both BP7 and BP4,' consistent with applying both here from the same non-splice-impact evidence.

Rule

MSH6 VCEP (ClinGen InSiGHT) BP7: A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic) is BP7_Supporting; may be combined with BP4.

Evidence considered

• case_summary.json compact_evidence.normalization: protein consequence NP_000170.1:p.(Leu821=) confirms synonymous/silent change.
• prefetch.json compact_evidence.spliceai: max_delta_score=0.001 supports absence of cryptic splice disruption at this exonic position.
• cspec BP7 rule (MSH6 VCEP v2.0): 'A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.' Exon boundary data (c.628-c.3172) places the variant far outside the -21/+7 zone.

Sources

Reasoning

The applicable InSiGHT MSH6 VCEP PM2 rule permits Supporting strength only for an absent/extremely rare allele frequency <0.00002 in gnomAD v4. Although the total v4.1 AF is 6.20e-07, the highest observed ancestry-specific AF is 3.38e-05 (1/29,604 Ashkenazi Jewish alleles), exceeding the stated cutoff. PM2 is therefore not met when the ancestry-specific observed frequency is used. This is a singleton threshold artifact: the calculated Grpmax FAF is unavailable, so human review is recommended before relying on the strict non-application of PM2.

Rule

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

Evidence considered

• The InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 PM2 rule is Supporting strength for absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports total AF 6.19593e-07 (1/1,613,962), zero homozygotes, and one Ashkenazi Jewish allele among 29,604 alleles (AF 3.37792e-05); the Grpmax FAF is null.

Still missing

• The gnomAD v4 Grpmax FAF is unavailable for this singleton; a VCEP-confirmed convention for applying PM2 to a raw one-allele ancestry-specific frequency above the cutoff would resolve the threshold-artifact uncertainty.

Sources

Reasoning

NM_000179.3:c.2463A>C is a synonymous variant (NP_000170.1:p.(Leu821=)) located deep within exon 4 (c.628_3172 exon boundaries; variant is 1835 bp from the 5' exon boundary and 709 bp from the 3' exon boundary), not at a non-canonical splice nucleotide. The MSH6 VCEP (ClinGen InSiGHT) PP3 rule only awards PP3 for (a) missense variants via HCI-prior lookup, or (b) predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >=0.2 (Walker et al 2023). This variant is not missense (HCI-prior lookup is inapplicable), and SpliceAI predicts no splice impact (max delta score = 0.001, all DS_AG/AL/DG/DL <=0.001), far below the 0.2 PP3 threshold. No PP3 code applies under the governing VCEP specification.

Rule

MSH6 VCEP (ClinGen InSiGHT) PP3: Missense variant with HCI prior probability >0.81 (Moderate) or >0.68-0.81 (Supporting) as per HCI-PRIORS; OR predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >=0.2 per Walker et al 2023. Neither applies to this synonymous variant with SpliceAI max delta=0.001.

Evidence considered

• prefetch.json compact_evidence.spliceai: DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.0, max_delta_score=0.001 -- no splice impact predicted.
• case_summary.json compact_evidence.normalization: NP_000170.1:p.(Leu821=) confirms synonymous consequence, not missense, so the HCI-prior missense pathway for PP3 does not apply.
• cspec PP3 rule (MSH6 VCEP v2.0): Moderate/Supporting strengths require either HCI-prior >0.68 for missense variants, or SpliceAI delta >=0.2 at a non-canonical splice nucleotide -- neither condition is met here.

Sources

Reasoning

The applicable InSiGHT MSH6 VCEP BA1 rule requires gnomAD v4 Grpmax filtering allele frequency >=0.0022 (0.22%) and exclusion as a founder pathogenic variant. gnomAD v4.1 shows one allele overall (1/1,613,962; total AF 6.20e-07) and its highest observed ancestry-specific AF is 3.38e-05 (1/29,604 Ashkenazi Jewish alleles), both far below 0.0022. BA1 is not met; the founder-exclusion condition cannot alter this threshold failure.

Rule

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

Evidence considered

• The InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 BA1 rule requires gnomAD v4 Grpmax filtering allele frequency >=0.0022 (0.22%) plus exclusion as a founder pathogenic variant.
• gnomAD v4.1 reports one allele in 1,613,962 total alleles (AF 6.19593e-07), zero homozygotes, and a highest observed Ashkenazi Jewish AF of 3.37792e-05 (1/29,604); Grpmax FAF is null.

Still missing

• The gnomAD v4 Grpmax FAF field is unavailable for this singleton; however, the highest observed raw ancestry-specific AF is far below the BA1 threshold, so this does not affect the determination.
• No source documents a separate founder-pathogenic-variant exclusion, but this is not outcome-determinative because the BA1 frequency threshold is not approached.

Sources

Reasoning

The applicable InSiGHT MSH6 VCEP BS1 rule requires gnomAD v4 Grpmax filtering allele frequency >=0.00022 and <0.0022, with exclusion as a founder pathogenic variant. The highest observed gnomAD v4.1 ancestry-specific AF is 3.38e-05 (one Ashkenazi Jewish allele), more than six-fold below the 0.00022 lower bound; total AF is 6.20e-07. BS1 is not met. The null Grpmax FAF is a singleton-data limitation, not a reason to substitute the higher v2.1 Ashkenazi Jewish AF or an unobserved estimate for the v4 rule.

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 InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 BS1 rule requires gnomAD v4 Grpmax filtering allele frequency >=0.00022 and <0.0022 (0.022-0.22%) plus exclusion as a founder pathogenic variant.
• gnomAD v4.1 reports one allele in the Ashkenazi Jewish population (1/29,604; AF 3.37792e-05), zero homozygotes, total AF 6.19593e-07, and no calculated Grpmax FAF.

Still missing

• The gnomAD v4 Grpmax FAF field is unavailable for this singleton; the observed v4 ancestry-specific AF remains below the BS1 lower bound.
• No source documents a separate founder-pathogenic-variant exclusion, but this is not outcome-determinative because the BS1 frequency threshold is not met.

Sources

Reasoning

No proband-level phenotype, parental testing, or confirmed de novo observation is present in the consolidated evidence bundle; de novo points cannot be assigned.

Rule

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

Evidence considered

• The MSH6 InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification applies PS2 by cumulative de novo points: 0.5 Supporting, 1–1.5 Moderate, 2–3.5 Strong, and at least 4 Very Strong.
• The consolidated case evidence contains no individual-level inheritance, parental genotype, or phenotype data for NM_000179.3:c.2463A>C.

Still missing

• Variant testing in both biological parents, including confirmation of maternity and paternity where required.
• Proband phenotype and age-at-diagnosis information sufficient to assess phenotype consistency.
• Number of independent confirmed de novo observations and the corresponding total de novo points.

Sources

Reasoning

MSH6 VCEP (InSiGHT) specifies PS3 based on calibrated functional assay odds-of-pathogenicity (>18.7 Strong, >4.3-<=18.7 Moderate, >2.08-<=4.3 Supporting), MMR functional assay flowchart outcomes, or monoallelic expression/NMD assays showing <10% wild-type allele expression. No such laboratory functional or mRNA/cDNA-based assay results for NM_000179.3:c.2463A>C (p.Leu821=) were found anywhere in the case evidence bundle (prefetch.json, evidence.json, literature_pass.json, vcep_materials.json). The three ClinVar-linked PMIDs (25741868, 25394175, 28492532) are ACMG/AMP guideline and practice-guideline papers, not variant-specific functional studies, and none report a functional assay for this exact variant. SpliceAI in silico delta scores (max 0.001) are a computational splicing prediction, not a laboratory functional/mRNA assay, and are not applicable evidence for PS3 under this VCEP specification.

Still missing

• No calibrated MMR functional assay (e.g. cell-based complementation, in vitro MMR activity) odds-of-pathogenicity value available for this variant
• No patient-derived mRNA/cDNA or NMD-inhibition assay data available to assess monoallelic expression/splicing aberration
• No variant-specific functional-assay publication identified in the literature packet

Reasoning

The MSH6 InSiGHT specification requires co-occurrence with a known pathogenic/likely pathogenic MSH6 variant in a patient with clinical features consistent with constitutional mismatch repair deficiency (CMMRD). No such affected-proband observation, second variant, zygosity, or phase information is present in the case-evidence bundle; therefore no PM3 points can be assigned.

Rule

MSH6 InSiGHT VCEP v2.0 PM3: pathogenic/likely pathogenic variant in trans = 1.0 point; phase unknown = 0.5 points; PM3_Supporting requires 0.5 point, PM3_Moderate 1-1.5 points, PM3_Strong 2-3.5 points, and PM3_VeryStrong at least 4 points.

Evidence considered

• MSH6 InSiGHT VCEP v2.0 PM3 requires co-occurrence with a known pathogenic/likely pathogenic MSH6 variant in a patient with CMMRD-consistent features; a variant in trans contributes 1.0 point and phase-unknown co-occurrence contributes 0.5 points.
• The consolidated case-evidence bundle provides no affected CMMRD proband, second MSH6 variant, co-occurrence, cis/trans result, or phase-testing result for NM_000179.3:c.2463A>C.

Still missing

• Affected-proband records with CMMRD-consistent clinical features.
• The identity and pathogenic/likely pathogenic classification of any second MSH6 variant in the same individual.
• Phase determination by parental or offspring testing when a second variant is identified.

Sources

Reasoning

No pedigree-level genotype/phenotype observations or segregation likelihood ratio are provided; PP1 cannot be evaluated.

Rule

MSH6 InSiGHT VCEP PP1: combined Bayes likelihood ratio >18.7 in ≥2 families = Strong; >4.3 to ≤18.7 = Moderate; >2.08 to ≤4.3 = Supporting.

Evidence considered

• The MSH6 InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification requires a combined co-segregation Bayes likelihood ratio above 2.08 for PP1_Supporting, above 4.3 through 18.7 for PP1_Moderate, and above 18.7 in at least two families for PP1_Strong.
• The consolidated case evidence contains no family segregation data, informative meioses, or calculated likelihood ratio for NM_000179.3:c.2463A>C.

Still missing

• Pedigree data with variant genotypes, disease status, ages, and informative meioses.
• A combined Bayes likelihood ratio from co-segregation analysis, including evidence from at least two families for PP1_Strong.

Sources

Reasoning

No variant-associated CRC/endometrial tumor MSI result or MMR IHC result is present in the consolidated case evidence. Therefore, the VCEP PP4 threshold is not evaluable.

Rule

InSiGHT MSH6 v2.0 PP4: 1 qualifying tumor = Supporting; 2 = Moderate; at least 3 independent tumors in at least 2 families = Strong. For MSH6, IHC consistency requires isolated MSH6 loss.

Evidence considered

• InSiGHT MSH6 v2.0 permits PP4_Supporting for one, PP4_Moderate for two, and PP4_Strong for at least three independent CRC/endometrial MSI-H tumors and/or MMR IHC results consistent with the variant location; no such tumor observations are supplied for this exact variant.

Still missing

• Obtain tumor-level evidence for carriers of NM_000179.3:c.2463A>C: MSI testing by a standard 5-10-marker panel or tumor genome and/or MMR IHC. For MSH6, qualifying IHC loss must be isolated MSH6 loss and consistent with the variant location.

Sources

Reasoning

The applicable InSiGHT MSH6 VCEP BS2 rule is not a general healthy-homozygote rule. It requires a phase-confirmed in-trans co-occurrence with a known pathogenic MSH6 variant in an eligible patient with colorectal cancer after age 45 (or another Lynch syndrome cancer above its median onset age) and no current or prior CMMRD manifestations. No such case, phase, cancer-age, or CMMRD information is present. The zero gnomAD homozygotes do not address this patient-specific requirement.

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), and no previous or current clinical manifestations of CMMRD; phase confirmation requires testing of parents or offspring.

Evidence considered

• The InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 BS2 rule requires phase-confirmed in-trans co-occurrence with a known pathogenic MSH6 variant in a clinically eligible patient without CMMRD manifestations.
• No source in the consolidated case-evidence bundle supplies a qualifying co-occurrence or the required clinical and phase evidence.

Still missing

• A qualifying patient-level observation of this variant in trans with a known pathogenic MSH6 variant is required.
• Parental or offspring testing confirming phase is required.
• The qualifying cancer type and age at diagnosis, and absence of current or prior CMMRD manifestations, must be documented.

Sources

Reasoning

MSH6 VCEP (InSiGHT) specifies BS3 based on calibrated functional assay odds-of-pathogenicity (<=0.05 Strong, >0.05-<=0.48 Supporting), or for synonymous/intronic variants, absence of mRNA aberration (splicing or allelic imbalance) determined by NMD-inhibition laboratory assays. This variant, NM_000179.3:c.2463A>C (p.Leu821=), is synonymous, which is the variant class this BS3 rule specifically targets, but no NMD-inhibition mRNA assay, allelic imbalance study, or calibrated cell-based functional assay result for this exact variant was found in the case evidence bundle. SpliceAI predicts a very low max delta score (0.001), consistent with no predicted splicing impact, but this is a computational prediction relevant to BP7/BP4, not the laboratory-based mRNA assay evidence required by the VCEP for BS3.

Still missing

• No NMD-inhibition mRNA/cDNA assay result available to confirm absence of splicing aberration or allelic imbalance for this synonymous variant
• No calibrated functional assay odds-of-pathogenicity value available for this variant
• No variant-specific functional-assay publication identified in the literature packet

Reasoning

No documented non-segregation observations or combined Bayes likelihood ratio are provided; BS4 cannot be evaluated.

Rule

MSH6 InSiGHT VCEP BS4: combined Bayes likelihood ratio <0.05 = Strong; >0.05 to ≤0.48 = Supporting.

Evidence considered

• The MSH6 InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification assigns BS4_Strong for a combined Bayes likelihood ratio below 0.05 and BS4_Supporting for a ratio above 0.05 through 0.48.
• The consolidated case evidence contains no non-segregation pedigree data or calculated likelihood ratio for NM_000179.3:c.2463A>C.

Still missing

• Pedigree data demonstrating lack of co-segregation, with genotype and disease-status information for informative relatives.
• A combined Bayes likelihood ratio below 0.48 to support BS4, including assessment of age-dependent penetrance and phenocopies.

Sources

Reasoning

No tumor-level MSS result, retained MMR expression, MMR IHC pattern inconsistent with MSH6, BRAF V600E result, or MLH1 methylation result is present for this exact variant. BP5 cannot be evaluated.

Rule

InSiGHT MSH6 v2.0 BP5: 2-3 qualifying tumors = Supporting; at least 4 = Strong; alternatively, 1 qualifying BRAF V600E/MLH1-methylated MSI-H tumor with MLH1 loss = Supporting and at least 2 = Strong.

Evidence considered

• InSiGHT MSH6 v2.0 specifies BP5_Supporting for 2-3 CRC/endometrial tumors with MSS and/or retained MMR expression, or LS-spectrum tumors with MMR protein loss inconsistent with the gene; BP5_Strong requires at least 4 such tumors. It also permits Supporting for one and Strong for at least two qualifying BRAF V600E/MLH1-methylated MSI-H tumors with MLH1 loss. No qualifying tumor data are supplied.

Still missing

• Obtain tumor data for carriers of NM_000179.3:c.2463A>C, including MSI/MSS status, MMR IHC, and where relevant BRAF V600E or MLH1 promoter methylation. The VCEP threshold is 2-3 qualifying tumors for Supporting or at least 4 for Strong; one qualifying BRAF V600E/MLH1-methylated MSI-H tumor with MLH1 loss supports BP5.

Sources