Five-step scale from benign to pathogenic. This variant is likely benign. Select a step to read what it means.
This variantScenario
BenignPathogenic
Likely benign.A likely benign classification under the recorded framework.
In plain language
Gene-level background from the recorded interpretation.
Words with a dotted underline have a short definition. Hover or tap them.
About MSH6
MSH6 encodes a protein in the DNA mismatch repair system, which fixes errors made during DNA replication. Partnering with MSH2, it forms a complex that recognizes and helps correct mismatched DNA bases, keeping the genetic code stable. Inherited mutations in MSH6 cause Lynch syndrome (hereditary nonpolyposis colorectal cancer), raising the risk of colorectal, endometrial, ovarian, and other cancers, while mutations in both copies lead to constitutional mismatch repair deficiency. Because faulty mismatch repair drives tumor development and produces microsatellite instability, MSH6 acts as a tumor suppressor, and cancers with such repair defects often respond well to immune checkpoint inhibitor therapy.
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Criteria may be applied incorrectly, sources may be misread, and a
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Double-check every criterion and its underlying evidence
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01The reasoning
How it was classified
The recorded framework is ClinGen MSH6 VCEP v2.0. The criteria and strengths shown below come from the saved assessment; this page does not recalculate the classification.
Recorded applied criteria
The applied criteria and their strengths are shown as assessed under the stated framework.
Met (supporting): gnomAD v4.1 total allele frequency 0.00192% is below the 0.002% (<1 in 50,000) threshold. Flagged for human review: grpmax FAF and AMR subpopulation frequencies exceed the threshold, leaving essentially no margin.
Benign criteria applied · 2
BP4Supporting
BP4 · supporting
Met (supporting): HCI prior probability of pathogenicity 0.1095 is below the 0.11 BP4 threshold. Flagged for human review: the value sits only 0.0005 below the cutoff and should be re-confirmed against HCI PRIORS.
BP6Supporting
BP6 · supporting
Met (supporting benign): the InSiGHT expert panel classified this variant Likely Benign (ClinVar 3-star expert-panel review).
Recorded classification: Likely benign
Likely Benign: VCEP Rule19 (>=2 Benign.Supporting) is satisfied by BP4 (HCI prior 0.1095 < 0.11) and BP6 (InSiGHT 3-star Likely Benign); the lone pathogenic-direction criterion, PM2 supporting, is insufficient.
02The sources
Evidence at a glance
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1
gnomAD v2.1
Population frequency
31 / 1,613,978 alleles
gnomAD v4.1: 31 / 1,613,978 alleles. Recorded AF 1.92072e-05. Recorded homozygote count 0.
gnomAD v2.1
This variant is present in gnomAD v2.1 (AF= 3.18547e-05; MAF= 0.00319%, 8/251140 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 0.000115761; MAF= 0.01158%, 4/34554 alleles, homozygotes = 0); grpmax FAF= 3.896e-05.
OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. MSH6, a DNA mismatch repair protein, is frequently mutated in colorectal, small bowel, and endometrial cancers.
ACMG/AMP criteria under ClinGen MSH6 VCEP v2.0, with the data and sources behind each one.
28 criteria with recorded statuses: 3 met · 10 not met · 6 not assessed · 9 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH6 Version 2.0 (cspec, precedence rank 1); used in preference to generic ACMG/AMP.
NM_000179.3:c.1403G>A is a single-nucleotide missense substitution predicted to encode p.(Arg468His) with no change in protein length (Mutalyzer reference and predicted proteins identical in length).
PVS1 not met: the variant is not a nonsense/frameshift (no PTC), not a large single/multi-exon deletion, not an IVS+/-1/2 or last-base-of-exon variant, not an initiation-codon or tandem-duplication variant, and SpliceAI predicts no splicing impact (max delta = 0.02); no mRNA assay evidence of a splicing aberration exists in the case bundle. None of the VCEP PVS1 branches (Very Strong/Strong/Moderate) is satisfied.
PM4 not_applicable: the VCEP declares the criterion Not Applicable ('Protein length change from an in-frame variant is not used due to lack of evidence'), and the variant does not change protein length in any case.
BP3 not_applicable: the VCEP declares the criterion Not Applicable ('In-frame deletions/insertions in a repetitive region without a known function is not used'), and the variant is a missense substitution, not an in-frame repeat-region indel.
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSpec doc 1578294885); generic ACMG/AMP is not used for this group because an applicable VCEP specification exists.
PS1 not met: codon 468 encodes Arg (CGC/CGT) and c.1403G>A is the only single-nucleotide substitution that yields His at this residue, so no 'same amino acid change, different nucleotide' variant exists; the only known p.Arg468His allele is this variant, classified Likely benign by the InSiGHT VCEP (ClinVar VCV000089192, SCV000107856); c.1403G>A is not a splice nucleotide (SpliceAI max delta 0.02), so the PS1 splice-nucleotide limb also fails.
PM5 not met: the VCEP PM5 rule requires a different missense at residue 468 classified Pathogenic (or Likely Pathogenic for PM5_Supporting) by this VCEP, and only if PP3 is supporting for this variant. No same-residue comparator was found in any collected source (pm5_candidates.json: 0 same-residue candidates; harvest rate-limited HTTP 429), and the PP3 gate independently fails because the HCI prior probability for c.1403G>A is 0.1095, below the VCEP PP3_Supporting threshold of >0.68.
PM1 not applicable: the VCEP explicitly declares PM1 Not Applicable ('no recognized mutational hot spots' usable for classification; pathogenic variants are generalized over all MMR domains). Cancer Hotspots found no statistically significant hotspot at MSH6 R468; OncoKB lists R468H as Unknown Oncogenic Effect.
PP2 not applicable: the VCEP explicitly declares PP2 Not Applicable.
BP1 not applicable: the VCEP explicitly declares BP1 Not Applicable; missense is an established disease mechanism in MSH6.
No criterion in this group is met. The variant's ClinVar Likely-benign expert-panel status is consistent with the absence of any pathogenic same-residue/same-amino-acid comparator but was not itself used as criterion evidence.
Hotspot/domain status: Cancer Hotspots - no row for MSH6 R468; COSMIC records 4 somatic observations of the same variant (COSV52276785), which does not establish a germline hotspot.
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH6 v2.0 (cspec doc 1578294885) was used for all four criteria in this group; generic ACMG was not used because this gene-specific specification exists and governs.
PS2 not_assessed: no de novo occurrence or parental testing data for c.1403G>A exists in the case bundle; under the VCEP de novo points scheme (0.5-2 points per proband) no points can be awarded.
PM6 not_applicable: the InSiGHT MSH6 VCEP v2.0 declares PM6 Not Applicable and folds assumed-de-novo evidence into the PS2 points system.
PP1 not_assessed: no segregation data. The only full text reporting this variant (PMID:18033691, Barnetson 2008) found c.1403G>A in a single CRC proband (1/932 cases, 0/1008 controls, MSS tumor, normal MMR IHC) with category 'Undefined', score 1, and no segregation points; no BLR is computable. Family studies cited by ClinVar (PMID:17854147, PMID:18067074) lack full text in this bundle.
BS4 not_assessed: no documented non-segregation (no affected relative tested negative, no lack-of-co-segregation BLR) exists in the bundle; absence of data is not scored as BS4.
The ClinVar record (VCV000089192) with 13 usable submissions and an InSiGHT expert-panel 'Likely benign' classification was consulted only to confirm that no de novo/segregation fields are present; the panel's final classification was not used as criterion evidence.
Conclusion for this group: no segregation or de novo evidence is available for NM_000179.3:c.1403G>A; PS2, PP1, and BS4 are not assessed (insufficient evidence), PM6 is not applicable under the governing VCEP.
Functional group adjudication for MSH6 c.1403G>A (p.Arg468His) under the ClinGen InSiGHT MSH6 VCEP v2.0 specification (cspec doc 1578294885); generic ACMG not used because an applicable VCEP exists.
PS3: not met. All variant-specific functional evidence in the case bundle is proficiency-oriented: four independent clinical laboratories report MMR activity similar to wild type / no damaging effect / MMR proficiency, including testing in the VCEP-calibrated CIMRA assay (Drost et al. 2020). No calibrated FOP above 2.08, no flowchart-conformant MMR defect, and no monoallelic expression loss are documented. SpliceAI max delta 0.02 adds no splicing-impact support.
BS3: not assessed (insufficient evidence, not contradicted). The reported variant-specific proficient MMR function (4 independent labs, calibrated CIMRA) supports the BS3_Supporting direction, but the VCEP rules require either a calibrated FOP (<=0.48 supporting, <=0.05 strong) or flowchart-conformant proficient function in protein AND mRNA-based lab assays. The FOP is not retrievable from this case (primary papers PMID 28531214/31965077 absent from source registry) and no mRNA/cDNA assay evidence exists for this variant; SpliceAI in silico cannot fill the RNA-assay requirement.
If primary CIMRA data confirm repair activity >=100% of WT with FOP <=0.05, BS3_Strong would apply; if FOP falls in 0.05-0.48 (or an mRNA-based assay shows normal transcripts), BS3_Supporting would apply. Human review with the Drost et al. 2020 supplementary data is required.
No final classification was inferred from any submitter's or the InSiGHT expert panel's ClinVar classification (SCV000107856 'Likely benign'); only reported experimental findings from the submission audit were used.
PS4: not applicable - InSiGHT MSH6 VCEP v2.0 explicitly declares PS4 Not Applicable; no variant-specific case-control study exists in the bundle.
PP4: not assessed - VCEP PP4 requires tumor MSI-H and/or MMR-protein-loss data for carriers; no tumor phenotype data for c.1403G>A is available in the evidence bundle (variant absent from all machine-readable full texts; PMID:17854147/18067074 image-only).
PP5: not met - the only exact-variant ClinVar expert-panel classification (InSiGHT, 3-star) is Likely benign (benign direction, supports BP6); no expert-panel Pathogenic/Likely pathogenic classification exists.
BP5: not assessed - VCEP BP5 requires tumor MSS/retained-MMR-protein or BRAF V600E/MLH1-methylation data for carriers; no such data is available in the evidence bundle.
BP6: met at Supporting strength - exact-variant ClinVar expert-panel (InSiGHT, 3-star, reviewed by expert panel) classification is Likely benign; applied per the global 3-star expert-panel policy despite the VCEP 'Not Applicable' flag.
Net phenotype/prevalence direction for this variant is benign (BP6 supporting from the InSiGHT expert panel); PP4/BP5 remain open pending tumor phenotype data.
PM3: not_met - 0 points; no in-trans or phase-unknown co-occurrence with a known P/LP MSH6 variant and no biallelic/CMMRD observations in any reviewed source.
BP2: not_applicable - declared Not Applicable by the InSiGHT MSH6 VCEP v2.0 specification; BS2 is used instead in this framework.
Allelic evidence contributes no pathogenic weight for c.1403G>A: monoallelic presence in gnomAD v4 (31 alleles, 0 homozygotes) and a single MSS, IHC-normal, 'Undefined'-category CRC case (PMID:18033691) are the only allelic-relevant observations.
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 VCEP v2.0 (cspec doc 1578294885, precedence rank 1). Gene-specific PP3/BP4 rules for missense variants are based on the HCI prior lookup table (vcep_hci_priors_msh6); generic ACMG computational-evidence rules are not applied because an applicable specification exists.
PP3 not met (high confidence): HCI prior 0.1095 is far below the 0.68 supporting threshold, and SpliceAI max delta 0.02 is below the 0.2 splice-defect threshold. BayesDel is uncalibrated (not_available) and REVEL is outside the VCEP missense PP3 path.
BP4 met at supporting strength (medium confidence): HCI prior 0.1095 < 0.11 satisfies the VCEP BP4 supporting rule. The value is within 0.0005 of the cutoff, so human confirmation against the HCI PRIORS resource is requested (needs_human_review = true).
BP7 not applicable: c.1403G>A is a missense variant (p.Arg468His), not a synonymous or intronic variant, so the BP7 variant-type gate fails.
No literature review was required for this group: literature_pass.json indexes only PP5/PS4 case-observation papers with no PP3/BP4 relevance, and all in silico evidence used here is variant-specific lookup data (HCI prior, SpliceAI) plus the VCEP rule text. No pipeline source code was consulted or cited.
Consistency note: ClinVar 3-star InSiGHT expert panel classification (Likely benign) is consistent with BP4 supporting, but was not used as evidence for any criterion in this group (never infer a criterion from another source's final classification).
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MSH6 v2.0 (cspec doc 1578294885) was used for BA1, BS1, BS2 and PM2; generic ACMG/AMP was not needed because this VCEP specification exists and defines all four criteria.
BA1 not met: gnomAD v4.1 grpmax FAF 2.256e-05 (0.0023%) is ~100-fold below the 0.0022 (0.22%) stand-alone threshold; variant is not a founder pathogenic variant.
BS1 not met: gnomAD v4.1 grpmax FAF 2.256e-05 (0.0023%) is below the 0.00022 (0.022%) BS1 lower bound; even the highest subpopulation AF (AMR 0.0067%) is below it.
PM2_Supporting met (borderline): gnomAD v4.1 total AF 1.92072e-05 (0.00192%; 31/1,613,978 alleles, 0 homozygotes) is < 0.00002 (1 in 50,000). Borderline because grpmax FAF (2.256e-05), AMR subpopulation AF (6.669e-05) and gnomAD v2.1 AF (3.185e-05) all exceed 0.00002; flagged for human review.
BS2 not met: no phase-confirmed in-trans co-occurrence with a known pathogenic MSH6 variant in a CRC patient >45 years without CMMRD is documented; 0 homozygotes in gnomAD v4.1; the single carrier case in PMID:18033691 had no second MSH6 variant (MSS tumor, normal MMR IHC).
No criterion was inferred from any other source's final classification (e.g., the ClinVar InSiGHT expert-panel Likely benign label was not used as evidence).
No criteria match.
Met · 3
PM2SupportingMet (supporting): gnomAD v4.1 total allele frequency 0.00192% is below the 0.002% (<1 in 50,000) threshold. Flagged for human review: grpmax FAF and AMR subpopulation frequencies exceed the threshold, leaving essentially no margin.
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Reasoning
PM2 per InSiGHT MSH6 v2.0 is met at Supporting strength when the allele frequency in gnomAD v4 is < 0.00002 (<1 in 50,000 alleles). gnomAD v4.1 total AF = 1.92072e-05 (0.00192%; 31/1,613,978 alleles, 0 homozygotes), which is below 0.00002. The rule text specifies 'allele frequency ... in gnomAD v4 dataset' (unlike BA1/BS1, which explicitly specify Grpmax FAF). BORDERLINE: the gnomAD v4.1 grpmax FAF (2.256e-05, 0.00226%) and the AMR subpopulation AF (6.669e-05, 0.0067%) both exceed 0.00002, and gnomAD v2.1 total AF (3.185e-05, 0.00319%) also exceeds the threshold, so the call rests on the v4.1 total AF metric with essentially no margin. Flagged for human review to confirm the intended metric. PMID:18033691 independently shows 0/1008 unaffected controls carrying the variant, corroborating extreme rarity.
Rule
Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset.
Evidence considered
gnomAD v4.1 total AF = 1.92072e-05 (0.00192%) < 0.00002, 31/1,613,978 alleles, homozygotes = 0; grpmax FAF = 2.256e-05; gnomAD v2.1 total AF = 3.18547e-05 (8/251,140, homozygotes = 0); PMID:18033691 control frequency 0/1008
Still missing
Confirm the intended PM2 metric: gnomAD v4.1 total AF (1.92072e-05) meets <0.00002, but grpmax FAF (2.256e-05), AMR subpopulation AF (6.669e-05) and gnomAD v2.1 total AF (3.185e-05) all exceed the threshold; VCEP rule text refers to 'allele frequency' without specifying FAF.
BP4SupportingMet (supporting): HCI prior probability of pathogenicity 0.1095 is below the 0.11 BP4 threshold. Flagged for human review: the value sits only 0.0005 below the cutoff and should be re-confirmed against HCI PRIORS.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 BP4 rule (cspec doc 1578294885) states: missense variant with HCI-prior probability of pathogenicity <0.11 (https://hci-priors.hci.utah.edu/PRIORS) = BP4_Supporting. The gene-specific HCI prior lookup (vcep_hci_priors_msh6 / hci_prior; dbid MSH6_03076, model source PMID 22949387) returns a prior probability of 0.1095 for c.1403G>A, which is below 0.11, so BP4 is met at supporting strength. This is a near-threshold call: 0.1095 lies only 0.0005 below the 0.11 cutoff, so the value should be re-confirmed against the HCI PRIORS resource (hci-priors.hci.utah.edu) before final classification; hence needs_human_review is set. The alternative BP4 splice path (SpliceAI delta <= 0.1 per Walker et al 2023) is restricted to intronic and synonymous variants and was not used for this missense variant; SpliceAI max delta 0.02 is recorded as corroborating context only and is not double-counted. BayesDel (0.392893) and REVEL (0.801) were not used (uncalibrated / outside VCEP missense path, respectively).
Rule
BP4 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): Missense variant with HCI-prior probability of pathogenicity <0.11 as per https://hci-priors.hci.utah.edu/PRIORS OR (intronic/synonymous variants only) SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.
Evidence considered
HCI prior probability for MSH6 c.1403G>A = 0.1095, which is <0.11 and therefore meets BP4 Supporting per InSiGHT MSH6 VCEP v2.0 rule (cspec doc 1578294885; PRIORS at hci-priors.hci.utah.edu; underlying model PMID 22949387 Thompson et al. 2013). Margin to threshold is 0.0005 - near-boundary, flagged for human review.
SpliceAI max delta score = 0.02 (<=0.1) would satisfy the BP4 splice-impact path per Walker et al. 2023 (PMID 37352859), but that path is restricted to intronic and synonymous variants per the VCEP rule; noted as context only, not counted for this missense variant.
Still missing
HCI prior 0.1095 is within 0.0005 of the 0.11 BP4 threshold; confirm the value against the HCI PRIORS website (hci-priors.hci.utah.edu/PRIORS) for MSH6 c.1403G>A before finalizing BP4 supporting.
BP6SupportingMet (supporting benign): the InSiGHT expert panel classified this variant Likely Benign (ClinVar 3-star expert-panel review).
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Reasoning
Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Likely benign.
Rule
Global policy: BP6 at supporting strength for exact-variant ClinVar expert-panel (3-star) classification Benign/Likely benign; VCEP NA flag ignored
Evidence considered
clinvar: VCV000089192 classified 'Likely benign' by International Society for Gastrointestinal Hereditary Tumours (InSiGHT) expert panel; review_status 'reviewed by expert panel', review_stars 3; SCV000107856, date_last_evaluated 2013-09-05; VCV date_last_updated 2026-04-13
ClinVar expert panel classification
Still missing
The InSiGHT expert-panel rationale/codes for this classification are not visible in the fetched record (submission from 2013 predates the current VCEP v2.0 specification); the classification itself is current in ClinVar (3 stars, reviewed by expert panel)
PVS1Not met: c.1403G>A is a missense substitution with no protein length change and no splicing impact (SpliceAI max delta 0.02).
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Reasoning
Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 specification (cspec), which applies PVS1. Its PVS1 Very Strong branch requires a nonsense/frameshift variant introducing a premature termination codon (PTC) at or before codon 1341, a large single/multi-exon genomic alteration, an IVS+/-1 or +/-2 variant with frame-disrupting exon skipping/cryptic splice predicted to undergo NMD, or an mRNA-assay-confirmed splicing aberration producing no full-length reference transcript; Strong branch covers initiation-codon, tandem-duplication, and last-base-of-exon (G>non-G, intron not GTRRGT) variants; Moderate branch covers nonsense/frameshift PTCs between codons 1342 and 1360. NM_000179.3:c.1403G>A is a single-nucleotide missense substitution predicted to encode p.(Arg468His) with no change in protein length (Mutalyzer reference and predicted proteins identical in length; position 468 in both). It is not a nonsense/frameshift variant, not a large genomic alteration, not located at a canonical splice consensus position (canonical_splice_consensus = false), and not an initiation-codon or tandem-duplication event. SpliceAI predicts no splicing impact (max delta score = 0.02; DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02). No mRNA/minigene assay data for this variant are present in the case bundle, and the only retrieved full text mentioning the variant (PMID:18033691) reports it as a missense with no mRNA/splicing analysis performed for it (mRNA analysis in that study was restricted to putative splice-site mutations). None of the VCEP PVS1 branches is satisfied; therefore PVS1 is not met. Gene-level LOF is an established disease mechanism for MSH6 (pvs1_gene_context: lof_mechanism_supported = true), but this does not alter the variant-level assessment.
Rule
PVS1 (InSiGHT MSH6 VCEP v2.0, cspec): 'Nonsense/frameshift variant introducing Premature Termination Codon (PTC) <= codon 1341 in MSH6. OR Large genomic alterations of single or multi-exon size. OR Variants at IVS+/-1 or IVS+/-2 where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD. OR Variants where mRNA assays using RNA derived from patient constitutional biological samples indicate that the variant allele results in a splicing aberration (with evidence that the variant allele produces no full-length/reference transcript)...' with Strong branch (initiation codon; tandem duplication; G>non-G at last base of exon if first 6 bases of intron are not GTRRGT) and Moderate branch (nonsense/frameshift PTC between codons 1342 & 1360).
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 PVS1 rule - Very Strong requires nonsense/frameshift PTC <= codon 1341, large single/multi-exon alterations, IVS+/-1/2 variants with frame-disrupting splice and NMD, or mRNA-confirmed splicing aberration with no full-length transcript; Strong covers initiation codon, tandem duplication, last-base-of-exon G>non-G; Moderate covers PTC codons 1342-1360.
spliceai: max delta score = 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02) - no predicted splicing impact for c.1403G>A.
pvs1_variant_assessment: consequence_class = missense; canonical_splice_consensus = false; variant_bucket = other - variant does not fall into any generic PVS1 null-variant bucket.
pvs1_gene_context: lof_mechanism_supported = true for MSH6 (gene gate eligible) - LOF mechanism confirmed but variant is not a null variant.
clinvar: variant title 'NM_000179.3(MSH6):c.1403G>A (p.Arg468His)' used only for variant-consequence annotation (missense); no ClinVar classification used.
PMID:18033691: Barnetson et al. 2008 reports c.1403G>A p.R468H as a germline missense variant (1 CRC case, 0/1008 controls, MSS tumor, normal MLH1/MSH2/MSH6 IHC) with no mRNA/splicing aberration data for this variant - consistent with PVS1 not met.
Still missing
No patient-derived mRNA/minigene splicing assay data are available in the case bundle; not required for PVS1 adjudication given the missense consequence with no predicted splice impact, but it is the only evidence type that could conceivably change this assessment.
PS1Not met: no alternative nucleotide change producing p.Arg468His has been classified pathogenic; the only such allele, c.1403G>A itself, is InSiGHT Likely Benign.
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Reasoning
Variant NM_000179.3(MSH6):c.1403G>A encodes p.(Arg468His). PS1 per the governing InSiGHT MSH6 VCEP v2.0 requires (a) a predicted missense producing the SAME amino acid change via a DIFFERENT underlying nucleotide change previously established by this VCEP as Pathogenic, or (b) a variant at the same non-canonical splice nucleotide as a confirmed Pathogenic/Likely Pathogenic splice variant. Limb (a): the reference codon at position 468 is an Arg codon (CGC/CGT); a G>A change at c.1403 is the only single-nucleotide substitution that produces His at this residue, so no alternative nucleotide change yielding p.Arg468His exists, and the only known p.Arg468His allele is c.1403G>A itself, which the InSiGHT VCEP classified Likely benign (ClinVar VCV000089192, SCV000107856, reviewed by expert panel, 2013-09-05) - it is not a previously established Pathogenic variant. Limb (b): c.1403G>A is a deep exonic missense (exon 4 of 10), not a splice nucleotide; SpliceAI predicts no significant splicing impact (max delta score 0.02), and no Pathogenic/LP splice variant at this nucleotide exists. PS1 is therefore not met. Note the PS1_Moderate path (same AA change as an established LP missense + PM2_supporting) is also unavailable because no LP same-amino-acid-change variant exists.
Rule
InSiGHT MSH6 VCEP v2.0 PS1 (Pathogenic Strong): 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); PS1_Moderate: same amino acid change as a previously established Likely Pathogenic missense variant with normal RNA result, with PM2_supporting met; or variants affecting the same non-canonical splice nucleotide as a confirmed/LP splice variant.
Evidence considered
InSiGHT MSH6 VCEP v2.0 PS1 rule text (CSpec doc 1578294885): same amino-acid change via different nucleotide established Pathogenic by this VCEP, or same non-canonical splice nucleotide as Pathogenic/LP splice variant
ClinVar VCV000089192: only p.Arg468His allele is c.1403G>A itself, Likely benign by InSiGHT expert panel; no Pathogenic/LP same-amino-acid comparator
SpliceAI: max delta score 0.02 for c.1403G>A - no splice impact; c.1403 is not a splice nucleotide
pm5_candidates.json: no same-residue comparator variants harvested
PMID:18033691 (Barnetson 2008): c.1403G>A p.R468H classified Benign - same variant, no pathogenic same-AA comparator
PS3Not met: four independent laboratory assays report proficient mismatch repair activity similar to wild type, contradicting a damaging effect.
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Reasoning
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis EP MSH6 v2.0 (cspec doc 1578294885, framework_mode=vcep). PS3 requires calibrated functional assays with functional odds for pathogenicity (FOP) >2.08 (Supporting), >4.3-<=18.7 OR an MMR function defect per the functional assay flowchart OR monoallelic complete loss of expression (<10% of WT in cDNA without puromycin) (Moderate), or FOP >18.7 (Strong). No damaging-effect functional evidence exists for c.1403G>A (p.Arg468His). Four independent ClinVar clinical-laboratory submissions (Quest SCV007111877, Color SCV000685190, GeneDx SCV001819075, Catalan ICO SCV006324660) report variant-specific functional assay results showing MMR proficiency: 'no damaging effect on mismatch repair activity', 'mismatch repair activity similar to wild type', 'MMR proficiency in a site-directed mutagenesis assay', and presence of the variant in the VCEP-calibrated cell-free in vitro MMR activity (CIMRA) assay of Drost et al. The calibrated CIMRA (Drost et al., PMID 30504929/31965077) is the VCEP-approved MSH6 assay (normal readout >=100% of WT repair for MSH6; abnormal <18%). No calibrated FOP value, no flowchart-conformant MMR defect, and no monoallelic expression loss are reported anywhere in the case bundle. SpliceAI max delta 0.02 indicates no splicing impact. All available functional evidence therefore contradicts a damaging effect and PS3 is not met; confidence is medium because the primary assay papers (PMID 28531214, 31965077) are not in the source registry and only secondary submission-reported statements were available.
Rule
VCEP MSH6 v2.0 PS3: Strong = calibrated FOP >18.7; Moderate = calibrated FOP >4.3 and <=18.7 OR MMR function defect following functional assay flowchart OR monoallelic expression with complete loss of expression (<10% of WT in cDNA without puromycin); Supporting = calibrated FOP >2.08 and <=4.3. No damaging-effect functional evidence for this variant; not met.
Evidence considered
ClinVar submission audit (SCV007111877, Quest Diagnostics): 'functional studies report this variant has no damaging effect on mismatch repair activity (pmid: 28531214 (2017), 31965077 (2020))' — no damaging effect, PS3 direction refuted
ClinVar submission audit (SCV000685190, Color Health): 'functional studies have shown that this variant to have mismatch repair activity similar to wild type (pmid: 31965077)' — proficient MMR activity
ClinVar submission audit (SCV001819075, GeneDx): 'published functional studies demonstrate mmr proficiency in a site-directed mutagenesis a[sic-truncated]' — proficient function
ClinVar submission audit (SCV006324660, Catalan Institute of Oncology): 'this variant is also found in a clinically calibrated functional assay, the cell-free in vitro mmr activity (cimra) assay from drost et al[sic-truncated]' — tested in the VCEP-calibrated CIMRA assay
VCEP Functional-assay-flowchart.pdf (OCR): calibrated assay path converts functional LR to ACMG/AMP evidence weight; MMR defect path requires flowchart-conformant deficient function
SpliceAI lookup: max delta score 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02) — no predicted splicing impact (in silico only)
PMID:18033691 (Barnetson 2008) Table 1: c.1403G>A p.R468H — in vitro MMR assay column blank (no variant-specific functional assay performed in this study)
Still missing
Primary functional assay data not in source registry: exact CIMRA repair activity / FOP for c.1403G>A from Drost et al. 2020 (PMID 31965077) and the Houlleberghs 2017 assay (PMID 28531214) could not be verified directly; a damaging-effect result in the primary data would reopen PS3.
No variant-specific mRNA/cDNA assay (with and without NMD inhibition) data in the case bundle for c.1403G>A.
PM3Not met: no in-trans or biallelic co-occurrence with a pathogenic MSH6 variant is documented; gnomAD reports 0 homozygotes.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 specification (cspec) applies a point system to PM3: co-occurrence with a known pathogenic/likely pathogenic (P/LP) sequence variant in the same gene in a patient with clinical features consistent with CMMRD as per Aronson et al 2022 (vcep_table_for_cmmrd_diagnosis). Points: P/LP in trans = 1.0; P/LP phase unknown = 0.5; >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting. No source in this case documents c.1403G>A co-occurring in trans (or with unknown phase) with a known P/LP MSH6 variant, and no biallelic observation exists: gnomAD v4.1 reports 31/1,613,978 alleles (AF 1.92072e-05) with 0 homozygotes; none of the 15 ClinVar submissions (13 usable) report co-occurrence, phase, or zygosity data; the only clinical observation in the available full texts (PMID:18033691) is a single CRC case with an MSS tumor and normal MLH1/MSH2/MSH6 IHC, classified 'Undefined' by multifactorial analysis, with no second MMR variant, no parental/offspring phase testing, and no CMMRD features; PMID:19389263 and PMID:21153778 mention p.R468H only in in silico prediction tables. PM3 therefore accrues 0 points. Note: the InSiGHT expert-panel 'Likely benign' ClinVar classification is recorded as context only and was NOT used to reach this determination (no inference from another source's final classification).
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. Classification/zygosity of other variant: P/LP in trans = 1.0 point; P/LP phase unknown = 0.5 points; sum all cases. >=4 points = Very Strong; 2-3.5 = Strong; 1-1.5 = Moderate; 0.5 = Supporting. (InSiGHT MSH6 VCEP v2.0)
Evidence considered
cspec: PM3 rule (InSiGHT MSH6 VCEP v2.0): P/LP in trans = 1.0 point; P/LP phase unknown = 0.5 points; >=4 points Very Strong, 2-3.5 Strong, 1-1.5 Moderate, 0.5 Supporting; requires CMMRD-consistent clinical features per Aronson et al 2022
clinvar: 15 submissions, 13 usable for classification; none report co-occurrence, phase, or biallelic status for c.1403G>A; InSiGHT expert panel Likely benign (2013, context only, not used as evidence)
PMID:18033691: Table 1 - c.1403G>A p.R468H observed in 1/932 CRC cases, MSS tumor, normal MMR IHC (1/1/1), effect size 8.01 (95% CI 0.16-405.27), category 'Undefined', 0/1008 controls; no second MMR variant, no phase data, no CMMRD features
PMID:19389263: in silico nsSNP analysis lists rs41295268 A/G p.R468H (SIFT Tolerant, PolyPhen Possibly damaging); no clinical or allelic data
PMID:21153778: in silico prioritization table lists p.R468H; no clinical or allelic data
Still missing
No proband genotype or pedigree data (parental/offspring phase testing) are provided in the case bundle; a documented in-trans or phase-unknown co-occurrence with a known P/LP MSH6 variant in a CMMRD-consistent patient would change this result.
No biallelic/CMMRD case reports of c.1403G>A were identified in the reviewed ClinVar submission trail or the available literature full texts.
PM5Not met: no pathogenic missense at residue 468 exists, and the PP3 gate fails (HCI prior 0.1095 is below the 0.68 supporting threshold).
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Reasoning
PM5 per the InSiGHT MSH6 VCEP v2.0 requires (a) a different missense change at amino acid residue 468 classified Pathogenic (PM5) or Likely Pathogenic (PM5_Supporting) by this VCEP on the protein level and not due to aberrant splicing, AND (b) PP3 supporting for the variant under evaluation. Limb (a): no different missense at residue 468 (e.g., p.Arg468Cys/Leu/Pro/Ser) appears in any collected evidence - pm5_candidates.json harvested 0 same-residue candidates (6 candidates seen overall), the ClinVar record VCV000089192 contains no other residue-468 variant, and none of the reviewed papers report one (PMID:18033691, PMID:19389263, PMID:21153778 describe only p.Arg468His itself). Limb (b) is independently dispositive: the VCEP operationalizes PP3 via the HCI prior probability of pathogenicity, and PP3_Supporting requires a prior >0.68; the HCI prior for c.1403G>A is 0.1095, so PP3 is not supporting and the VCEP rule states PM5 may only be used if PP3 is supporting. PM5 is therefore not met regardless of comparator availability. The systematic same-residue ClinVar enumeration was rate-limited (HTTP 429), so this gap is recorded; it does not change the verdict given the failed PP3 gate.
Rule
InSiGHT MSH6 VCEP v2.0 PM5 (Pathogenic Moderate): 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; PM5_Supporting if the other variant is Likely Pathogenic due to a missense alteration.
Evidence considered
InSiGHT MSH6 VCEP v2.0 PM5 rule: different missense at the same residue classified Pathogenic (PM5) or Likely Pathogenic (PM5_Supporting) by this VCEP; only use PM5 if PP3 is supporting for the missense change
pm5_candidates.json: total candidates seen 6, same_residue_candidates 0; harvest errored with HTTP 429
ClinVar VCV000089192: no other missense variant at residue 468 present in the record
HCI prior lookup (source_registry hci_prior): probability 0.1095 for c.1403G>A - PP3_Supporting requires >0.68, so the PM5 PP3 gate is not met
PMID:18033691, PMID:19389263, PMID:21153778: only p.Arg468His (the variant under evaluation) is described; no different residue-468 missense reported
Still missing
Systematic enumeration of other missense variants at MSH6 residue 468 (ClinVar query 'MSH6[GENE] AND R468[VARNAME]' etc.) was not completed because the pm5_candidates harvest failed with HTTP 429 (rate limit); 0 same-residue candidates were harvested. Re-run the comparator search if the PP3 gate were ever met (HCI prior for c.1403G>A = 0.1095, far below the >0.68 PP3_Supporting threshold).
PP3Not met: the HCI prior probability of pathogenicity is 0.1095, far below the 0.68 supporting threshold, and SpliceAI max delta 0.02 is below 0.2.
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Reasoning
Governing framework is the ClinGen InSiGHT MSH6 VCEP v2.0 (cspec doc 1578294885), whose PP3 rule for missense variants is: HCI prior probability of pathogenicity >0.81 = PP3_Moderate, >0.68 and <=0.81 = PP3_Supporting (https://hci-priors.hci.utah.edu/PRIORS), OR predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 (Walker et al 2023). The gene-specific HCI prior lookup (vcep_hci_priors_msh6 / hci_prior) returns a prior probability of 0.1095 for c.1403G>A (p.R468H, dbid MSH6_03076; underlying model Thompson et al. 2013, PMID 22949387), which is far below the 0.68 supporting threshold, so the missense PP3 path is not met. The splice path is also not met: SpliceAI max delta score is 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02), below the 0.2 threshold. BayesDel score 0.392893 is treated as not_available (no verified published calibration threshold available to this pipeline). REVEL score 0.801 was not applied because the governing MSH6 VCEP specifies HCI prior as the sole missense PP3/BP4 predictor for this gene; generic SVI REVEL thresholds (Pejaver et al. 2022, PMID 36413997) are only used when no applicable VCEP specification exists. PP3 is therefore not met on both available sub-paths with high confidence.
Rule
PP3 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): Missense variant with HCI prior probability for pathogenicity >0.81 = Moderate; >0.68 & <=0.81 = Supporting (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 prior probability for MSH6 c.1403G>A = 0.1095 (LOVD MSH6 priors lookup, dbid MSH6_03076; model source PMID 22949387 Thompson et al. 2013); InSiGHT MSH6 VCEP v2.0 PP3 rule requires >0.68 (Supporting) or >0.81 (Moderate) per cspec doc 1578294885 -> not met.
SpliceAI max delta score = 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02); PP3 splice path requires delta >= 0.2 at non-canonical splicing nucleotides per Walker et al. 2023 (PMID 37352859), as adopted by MSH6 VCEP v2.0 -> not met.
REVEL score 0.801 (local REVEL v1.3 lookup): not applied - governing MSH6 VCEP v2.0 specifies HCI prior as the missense PP3/BP4 predictor; generic SVI REVEL thresholds (Pejaver et al. 2022, PMID 36413997) apply only when no VCEP specification exists.
BayesDel score 0.392893 (local BayesDel lookup): treated as not_available - no verified published calibration threshold available to this pipeline, so it cannot support PP3/BP4.
PP5Not met: the only expert-panel classification for this variant is InSiGHT Likely Benign, which supports the benign direction (BP6), not PP5.
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Reasoning
PP5 is applied in this pipeline only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic (ClinVar 3-star 'reviewed by expert panel'; the InSiGHT MSH6 VCEP v2.0 'Not Applicable' flag for PP5 is ignored per the global 3-star expert-panel policy). For this exact variant (ClinVar VCV000089192, NM_000179.3(MSH6):c.1403G>A p.Arg468His), the only expert-panel classification is 'Likely benign' (International Society for Gastrointestinal Hereditary Tumours, InSiGHT; SCV000107856, evaluated 2013-09-05, review status 'reviewed by expert panel'). A benign-direction expert-panel classification cannot support PP5 and is instead handled under BP6. No expert-panel Pathogenic/Likely pathogenic classification exists for this variant, so PP5 is not met. Ordinary laboratory submissions (7x Likely benign, 3x VUS, 1x Benign, etc.) and the aggregate ClinVar label were deliberately not used for PP5.
Rule
Global policy: PP5 at supporting strength only for exact-variant ClinVar expert-panel (3-star) classification Pathogenic/Likely pathogenic; VCEP NA flag ignored. Here the expert-panel classification is Likely benign, so PP5 is not met.
Evidence considered
clinvar: VCV000089192 expert_panels = {Likely benign: International Society for Gastrointestinal Hereditary Tumours (InSiGHT)}; review status 'reviewed by expert panel' (3 stars) - benign direction, no Pathogenic/Likely pathogenic expert-panel classification exists for this variant
BA1Not met: gnomAD v4.1 grpmax FAF 0.00226% is roughly 100-fold below the 0.22% BA1 threshold.
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Reasoning
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel MSH6 v2.0 (cspec doc 1578294885). BA1 requires gnomAD v4 Grpmax filtering allele frequency >= 0.0022 (0.22%) and the variant excluded as a founder pathogenic variant. Observed gnomAD v4.1 grpmax FAF = 2.256e-05 (0.00226%; 31/1,613,978 alleles, 0 homozygotes), which is ~100-fold below the 0.22% threshold. The variant is not a known founder pathogenic variant in MSH6, and no source suggests founder status. BA1 is not met.
Rule
GnomAD v4 Grpmax filtering allele frequency ≥ 0.0022 (0.22%) and variant is excluded as founder pathogenic variant.
BS1Not met: gnomAD v4.1 grpmax FAF 0.00226% is about 10-fold below the 0.022% BS1 threshold.
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Reasoning
BS1 per InSiGHT MSH6 v2.0 requires gnomAD v4 Grpmax FAF >= 0.00022 and < 0.0022 (0.022-0.22%) with the variant excluded as a founder pathogenic variant. Observed gnomAD v4.1 grpmax FAF = 2.256e-05 (0.00226%), ~10-fold below the 0.022% lower bound. Even the highest subpopulation frequency (AMR 6.669e-05, 0.0067%) is below the threshold. Control frequency data from PMID:18033691 (0/1008 Scottish controls) are likewise far below 0.022%. BS1 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
gnomAD v4.1 grpmax FAF = 2.256e-05 (0.00226%); highest subpopulation AF AMR = 6.669e-05; VCEP MSH6 v2.0 BS1 threshold range 0.00022-0.0022; PMID:18033691 control frequency 0/1008 for c.1403G>A
BS2Not met: no in-trans co-occurrence with a pathogenic MSH6 variant is documented; gnomAD reports 0 homozygotes.
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Reasoning
BS2 per InSiGHT MSH6 v2.0 requires co-occurrence of the variant in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or another LS cancer above the median age of onset) with no clinical manifestations of CMMRD, with phase confirmed by testing of parents or offspring. No such observation is present in the case bundle or the reviewed literature: no compound-heterozygous/in-trans case involving c.1403G>A is reported; gnomAD v4.1 and v2.1 show 0 homozygotes (31 and 8 alleles respectively, all heterozygous); the single CRC carrier in PMID:18033691 (932 population-based cases <55y) had no reported second MSH6 variant, an MSS tumor and normal MMR IHC (MLH1/MSH2/MSH6 all retained). Hampel et al. (PMID:18809606), reviewed as a candidate case series, does not mention this variant. BS2 is not met on the available 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
PMID:18033691 single carrier case: no second MSH6 variant reported, MSS tumor, IHC 1/1/1; gnomAD v4.1 homozygotes = 0 (31/1,613,978 alleles); gnomAD v2.1 homozygotes = 0 (8/251,140 alleles); no in-trans observation in bundle
Still missing
No phase-confirmed in-trans co-occurrence of c.1403G>A with a known pathogenic MSH6 variant (CRC >45 years, no CMMRD) is documented in any available source; case-level co-occurrence data (e.g., LOVD/InSiGHT database entries) are not present in this case bundle.
PS2Not assessed: no de novo occurrence or parental testing data were available for this variant.
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Reasoning
Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MSH6 v2.0 (cspec doc 1578294885), which scores de novo evidence via a points system (2 points/proband with maternity AND paternity confirmed plus MMR-deficient LS-spectrum tumor; 1 point/proband with both parents confirmed plus LS-spectrum tumor without tumor data; 0.5 point/proband for assumed de novo with maternity and/or paternity unconfirmed; >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting). The case evidence bundle contains NO de novo occurrence data for c.1403G>A: no proband is reported as de novo, no parental (maternity/paternity) testing results are present in the ClinVar submissions (VCV000089192), and no available full text describes de novo occurrence or parental testing for this variant. Therefore no de novo points can be awarded and PS2 cannot be scored; it is not_assessed rather than not_met because the absence reflects missing data, not a documented negative.
InSiGHT MSH6 VCEP v2.0 PS2 rule (de novo points system) retrieved from cspec doc 1578294885
ClinVar VCV000089192 (15 submissions incl. InSiGHT expert panel): submission payloads contain no de novo or parental testing fields
PMID:18033691 (Barnetson 2008) full text: variant reported in a single CRC proband; no parental testing or de novo testing described
Still missing
No de novo occurrence (proband + unaffected parents) reported for c.1403G>A in any source in the case bundle
No maternity/paternity confirmation results available for any proband carrying this variant
Full text of ClinVar-cited family studies PMID:17854147 and PMID:18067074 (Chinese HNPCC pedigrees) was not available in this case bundle, so any de novo data they may contain could not be evaluated
PP1Not assessed: no co-segregation or pedigree data were available to compute a segregation likelihood ratio.
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Reasoning
Governing framework: InSiGHT MSH6 VCEP v2.0 PP1 is scored from a combined Bayes Likelihood Ratio (BLR) of co-segregation (Supporting BLR >2.08 and <=4.3; Moderate >4.3 and <=18.7; Strong >18.7 in >=2 families; results multiplied across pedigrees, recommended tool COOL v3). No pedigree/meiosis data for c.1403G>A exists in the case bundle, so no BLR can be computed. The only full text that reports this variant, PMID:18033691 (Barnetson 2008), found it in 1/932 population-based CRC cases, 0/1008 controls, tumor MSS with normal MLH1/MSH2/MSH6 immunohistochemistry, and classified the variant 'Undefined' with score 1 and zero segregation points - no family members were reported as tested, so no co-segregation is documented. ClinVar-cited Chinese HNPCC family studies (PMID:17854147, PMID:18067074) could plausibly carry segregation data but their full texts were not available in this bundle. PP1 is therefore not_assessed due to insufficient evidence.
Rule
PP1 (Pathogenic Supporting default): co-segregation with disease in pedigree(s) with combined Bayes Likelihood Ratio (multiplied across pedigrees, COOL v3) >18.7 in >=2 families = Strong; >4.3 and <=18.7 = Moderate; >2.08 and <=4.3 = Supporting.
Evidence considered
InSiGHT MSH6 VCEP v2.0 PP1 rule (Bayes likelihood ratio thresholds) retrieved from cspec doc 1578294885
PMID:18033691 (Barnetson 2008) Table 1: c.1403G>A p.R468H in 1 case, 0/1008 controls, MSS tumor, 'Undefined' category, no segregation points earned
PMID:18809606 (Hampel 2008) full text: variant c.1403G>A absent from the reported MSH6 mutation list - no segregation data applicable
ClinVar VCV000089192: submissions carry no segregation/co-segregation data
Still missing
No pedigree/meiosis data available to compute a Bayes likelihood ratio for c.1403G>A
PMID:18033691 reports the variant in a single proband with no segregation testing of relatives (category 'Undefined', score 1, 0 segregation points) - no informative meioses
Full text of PMID:17854147 and PMID:18067074 (family-based MSH6 studies cited by ClinVar) not available in this case bundle; potential segregation data not extractable
No other source in the bundle (ClinVar submissions, prefetch, evidence) contains segregation information for this variant
PP4Not assessed: no tumor MSI or MMR immunohistochemistry data were available for carriers of this variant.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 PP4 rule requires at least 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 (Supporting), escalating to Moderate (2 tumors) and Strong (>=3 independent tumors); an MSI-H tumor with inconsistent protein expression does not meet PP4. No tumor MSI status or MMR immunohistochemistry data for carriers of c.1403G>A is available anywhere in the evidence bundle, and none of the machine-readable full texts (PMID:15604628, 18033691, 18809606, 19389263, 21153778) mentions the variant. The Chinese HNPCC family reports that are most likely to describe this variant (PMID:17854147, PMID:18067074) are available only as image (PNG) copies in this case and could not be machine-verified. Evidence is insufficient to apply the VCEP PP4 rule; the criterion is therefore not assessed.
Rule
InSiGHT MSH6 VCEP v2.0 PP4: Supporting = 1 CRC/Endometrial MSI-H tumor (standard 5-10 marker panel or tumor genome) and/or loss of MMR protein expression consistent with variant location; Moderate = 2 tumors; Strong = >=3 independent tumors in >=2 families
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 PP4 rule text (>=1 CRC/endometrial MSI-H tumor and/or loss of MMR protein expression consistent with variant location; inconsistent protein expression does not meet PP4)
PMID:18033691: full text searched for c.1403G>A/p.Arg468His; variant absent, no variant-specific MSI/IHC data
PMID:18809606: full text searched for c.1403G>A/p.Arg468His; variant absent, no variant-specific MSI/IHC data
Still missing
No tumor phenotype data (MSI status and/or MMR protein IHC) for c.1403G>A carriers is present in the evidence bundle
PMID:17854147 and PMID:18067074 (Chinese HNPCC family studies cited by ClinVar submissions) may contain variant-specific tumor phenotype/segregation data but are only available as image files in this case and could not be read
BS3Not assessed: assays indicate proficient MMR activity, but the calibrated FOP values and mRNA-based assay evidence the VCEP requires were unavailable.
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Reasoning
Governing framework: ClinGen InSiGHT MSH6 VCEP v2.0. BS3_Strong = calibrated FOP <=0.05 (or synonymous/intronic with no mRNA aberration under NMD inhibition — not applicable, this is a missense variant). BS3_Supporting = calibrated FOP >0.05 and <=0.48 OR 'Variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart'. Evidence present: four independent clinical labs report variant-specific proficient MMR function for p.Arg468His — activity similar to wild type (Color, PMID 31965077), no damaging effect on MMR activity (Quest, PMID 28531214/31965077), MMR proficiency in a site-directed mutagenesis assay (GeneDx), and testing in the VCEP-calibrated CIMRA assay of Drost et al. (Catalan ICO); the calibrated CIMRA is VCEP-approved for MSH6 with normal readout >=100% of WT. This is a strong supportive signal for the BS3_Supporting direction. However, the VCEP rules cannot be formally met with the case-bundle evidence: (1) the variant-specific FOP (calibrated odds) is not available — the primary papers (PMID 28531214, 31965077) are absent from the source registry, so neither the BS3_Strong (FOP <=0.05) band nor the FOP band of BS3_Supporting (>0.05-<=0.48) can be applied; (2) the flowchart-based path explicitly requires proficient function in protein AND mRNA-based lab assays, and no mRNA/cDNA-based assay (with/without NMD inhibition, allelic imbalance/full-length transcript) exists for this variant in the case bundle — SpliceAI (max delta 0.02) is in silico only and cannot substitute for a laboratory RNA assay. Evidence is therefore insufficient to formally meet BS3; status not_assessed pending primary data retrieval and RNA-based assay evidence.
Rule
VCEP MSH6 v2.0 BS3: Strong = calibrated functional assays with FOP <=0.05 OR synonymous/intronic variants with no mRNA aberration under NMD inhibition (N/A for missense); Supporting = calibrated FOP >0.05 and <=0.48 OR variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart. FOP unavailable and mRNA-based assay evidence absent; not formally met — not_assessed.
Evidence considered
ClinVar submission audit (SCV007111877, Quest): 'functional studies report this variant has no damaging effect on mismatch repair activity (pmid: 28531214 (2017), 31965077 (2020))' — proficient function, BS3 direction
ClinVar submission audit (SCV000685190, Color): 'functional studies have shown that this variant to have mismatch repair activity similar to wild type (pmid: 31965077)' — proficient function, BS3 direction
ClinVar submission audit (SCV001819075, GeneDx): 'published functional studies demonstrate mmr proficiency in a site-directed mutagenesis a[sic-truncated]' — proficient function, BS3 direction
ClinVar submission audit (SCV006324660, Catalan ICO): 'this variant is also found in a clinically calibrated functional assay, the cell-free in vitro mmr activity (cimra) assay from drost et al[sic-truncated]' — variant present in VCEP-calibrated CIMRA dataset, BS3 direction
VCEP Functional-assay-SVI-documentation-MMR.xlsx: CIMRA approved as calibrated assay with proposed strengths including BS3 and BS3_Supporting; MSH6 normal threshold >=100% WT, abnormal <18%; controls and validation (3 P/LP, 4 B/LB MSH6) met
VCEP Functional-assay-flowchart.pdf (OCR): BS3-consistent proficient function requires protein-based (mammalian MMR activity) AND mRNA-based (patient-derived RNA, NMD-inhibited) assay evidence per flowchart; calibrated path requires FOP
SpliceAI: max delta score 0.02 — no predicted splicing impact; used only to orient the flowchart branch, not as RNA assay evidence
PMID:18033691 (Barnetson 2008) Table 1: c.1403G>A p.R468H has blank in vitro MMR column (no variant-specific functional assay performed); tumor MSS with normal MLH1/MSH2/MSH6 IHC is tumor phenotype, not a BS3 functional assay
Still missing
Variant-specific calibrated FOP (or raw CIMRA repair % of WT) for c.1403G>A from Drost et al. 2020 (Genet Med 22:847-856, PMID 31965077 — the VCEP-calibrated MSH6 CIMRA) is not in the source registry; required to apply BS3_Strong (FOP <=0.05) or the BS3_Supporting FOP band (>0.05-<=0.48).
No mRNA/cDNA-based assay (full-length transcript with and without puromycin / NMD inhibition) for c.1403G>A in the case bundle; the flowchart-based BS3_Supporting path requires proficient function in protein AND mRNA-based lab assays.
ClinVar submission sentences for GeneDx (SCV001819075) and Catalan ICO (SCV006324660) are truncated in the audit extraction; full assertion text should be re-pulled for exact CIMRA wording and any assay quantitative readout.
BS4Not assessed: no documented non-segregation exists to compute a lack-of-segregation likelihood ratio.
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Reasoning
Governing framework: InSiGHT MSH6 VCEP v2.0 BS4 is scored from lack of co-segregation (Strong: combined Bayes Likelihood Ratio <0.05; Supporting: BLR >0.05 and <=0.48). The case bundle contains no documented non-segregation for c.1403G>A: no affected relative tested negative for the variant, no pedigree with calculable lack-of-segregation BLR. PMID:18033691 (Barnetson 2008) reports the variant in a single proband without any segregation testing and classified it 'Undefined' (not Benign), i.e. the study did not apply lack-of-segregation evidence to this variant. Absence of non-segregation data cannot be scored as BS4 (which requires positive evidence of non-segregation), so the criterion is not_assessed rather than not_met. The ClinVar InSiGHT expert-panel 'Likely benign' label was not used as evidence for BS4 (final classifications of other sources are not criterion evidence).
Rule
BS4 (Benign Strong default): lack of co-segregation with disease in pedigree(s) with combined Bayes Likelihood Ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting.
Evidence considered
InSiGHT MSH6 VCEP v2.0 BS4 rule (BLR <0.05 = Strong; >0.05 and <=0.48 = Supporting) retrieved from cspec doc 1578294885
PMID:18033691 (Barnetson 2008): no non-segregation reported for c.1403G>A; variant category 'Undefined' (not Benign) - lack-of-segregation evidence was not applied
ClinVar VCV000089192: submissions carry no segregation data; expert-panel Likely benign label not used as criterion evidence
Still missing
No non-segregation events documented (no affected family members tested negative for c.1403G>A in any available source)
No lack-of-co-segregation Bayes likelihood ratio computable from available data
Full text of PMID:17854147 and PMID:18067074 (family-based MSH6 studies cited by ClinVar) not available in this case bundle; potential non-segregation data not extractable
BP5Not assessed: no tumor MSI/MSS or MMR immunohistochemistry data were available to count MSS tumors in carriers.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 BP5 rule requires 2-3 tumors with MSS and/or no loss of MMR protein expression and/or LS-spectrum tumors with MMR protein loss inconsistent with the variant's gene (Supporting), >=4 such tumors (Strong), or >=2 BRAF V600E (CRC) / MLH1 methylation (LS-spectrum tumor) events with MSI-H/MLH1 loss. No tumor phenotype data (MSI/MSS status, MMR IHC, BRAF/MLH1 methylation) for c.1403G>A is present in the evidence bundle, and no machine-readable full text mentions the variant, so the number of MSS/no-protein-loss tumors in carriers cannot be established. Evidence is insufficient to apply the VCEP BP5 rule; the criterion is therefore not assessed.
Rule
InSiGHT MSH6 VCEP v2.0 BP5: Supporting = 2-3 CRC/Endometrial tumors MSS and/or no MMR-protein loss and/or LS-spectrum tumors with inconsistent protein loss (or 1 BRAF V600E/MLH1 methylation event); Strong = >=4 tumors or >=2 BRAF V600E/MLH1 methylation events
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 BP5 rule text (Supporting = 2-3 tumors with MSS and/or no loss of MMR protein expression and/or inconsistent MMR protein loss; Strong = >=4 tumors; BRAF V600E/MLH1 methylation alternatives)
PMID:18033691: full text searched for c.1403G>A/p.Arg468His; variant absent, no variant-specific MSS/IHC data
PMID:18809606: full text searched for c.1403G>A/p.Arg468His; variant absent, no variant-specific MSS/IHC data
Still missing
No tumor phenotype data (MSS status, retained MMR protein expression, BRAF V600E/MLH1 methylation) for c.1403G>A carriers is present in the evidence bundle
PMID:17854147 and PMID:18067074 may contain variant-specific tumor data but are only available as image files in this case and could not be read
Not applicable · 9· recorded as not applicableShow reasons ▾Hide reasons ▴PS4VCEPNot applicable: the InSiGHT MSH6 VCEP does not use PS4; phenotype specificity is captured by PP4 and population enrichment by BS1/BA1.
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Reasoning
The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 v2.0 (CSPEC doc 1578294885), which explicitly declares PS4 'Not Applicable': phenotype specificity for Lynch syndrome is captured instead by PP4 (tumor MSI/MMR-protein-expression) and population enrichment by BS1/BA1 frequency rules. Consistent with this, no case-control enrichment study for this exact variant (NM_000179.3:c.1403G>A, p.Arg468His) is present in the evidence bundle; the only population-based CRC studies with full text (PMID:18033691, PMID:18809606) do not report the variant. PS4 is therefore not used under the VCEP framework.
VCEP rule
InSiGHT MSH6 VCEP v2.0 (cspec): PS4 applicability = Not Applicable
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 declares PS4 Not Applicable (phenotype specificity handled by PP4; frequency by BS1/BA1)
PMID:18033691: population-based CRC study (932 probands, MSI/IHC assessed) reviewed; variant c.1403G>A not reported
No variant-specific case-control study (cases vs controls allele counts) exists for c.1403G>A in the evidence bundle; even under generic ACMG/AMP this would be a gap, but the VCEP declares PS4 not applicable regardless.
PM1VCEPNot applicable: the MSH6 VCEP recognizes no mutational hotspots usable for classification in this gene.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 explicitly marks PM1 as Not Applicable: 'There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains (unpublished data).' Consistent with this, the Cancer Hotspots lookup for MSH6 R468 returned no statistically significant hotspot row, and OncoKB lists MSH6 R468H as Unknown Oncogenic Effect with no variant-specific reviewed evidence. Generic ACMG PM1 is not used because an applicable VCEP specification exists. PM1 is therefore not applicable.
VCEP rule
InSiGHT MSH6 VCEP v2.0: PM1 = Not Applicable (no recognized mutational hot spots; pathogenic variants generalized over all MMR domains).
Evidence considered
InSiGHT MSH6 VCEP v2.0 (CSpec doc 1578294885): PM1 applicability = Not Applicable, with explicit statement that no recognized mutational hotspots exist and pathogenic variants are distributed across all MMR functional domains
Cancer Hotspots (via evidence.json hotspots lookup): no statistically significant hotspot row for MSH6 R468
OncoKB MSH6/R468H: Unknown Oncogenic Effect, no variant-specific reviewed evidence
VCEP MMR-Functional-domains.pdf: domain schematic exists but VCEP does not use domain position for PM1
PM4VCEPNot applicable: the MSH6 VCEP does not use protein-length-change evidence, and this missense does not alter protein length.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 specification declares PM4 Not Applicable: 'Protein length change from an in-frame variant is not used due to lack of evidence.' Independently, the variant does not alter protein length: NM_000179.3:c.1403G>A is a single-nucleotide missense substitution encoding p.(Arg468His) with reference and predicted proteins of identical length (Mutalyzer). PM4 therefore cannot be applied under the governing framework, and no generic ACMG fallback is warranted because a VCEP specification exists and is in force.
VCEP rule
PM4 (InSiGHT MSH6 VCEP v2.0, cspec): criterion declared 'Not applicable' - 'Protein length change from an in-frame variant is not used due to lack of evidence.'
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 - PM4 applicability 'Not applicable'; instructionsToUse 'Protein length change from an in-frame variant is not used due to lack of evidence.'
PMID:18033691: reports the variant as missense substitution p.R468H - no protein length change described, consistent with PM4 not applicable.
PM6VCEPNot applicable: assumed-de-novo evidence is scored within the PS2 points system under the MSH6 VCEP, not as PM6.
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Reasoning
The governing ClinGen InSiGHT MSH6 VCEP v2.0 specification declares PM6 'Not applicable' (applicability: Not applicable). Assumed-de-novo evidence with unconfirmed maternity/paternity is consolidated into the PS2 points system (0.5 points per proband) rather than scored as PM6. Because a gene-specific specification governs this case, generic ACMG/AMP PM6 must not be applied. No further adjudication is possible or required.
VCEP rule
PM6: Not Applicable per InSiGHT MSH6 VCEP v2.0; assumed de novo evidence is captured within the PS2 points system (0.5 points per proband with unconfirmed parents).
PP2VCEPNot applicable: the MSH6 VCEP explicitly does not use PP2.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 explicitly marks PP2 as Not Applicable. Because an applicable VCEP specification exists, generic ACMG/AMP PP2 (low rate of benign missense variation and missense as a common disease mechanism) is not applied. PP2 is therefore not applicable.
BP1VCEPNot applicable: missense is an established disease mechanism in MSH6, and the VCEP does not use BP1.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 explicitly marks BP1 as Not Applicable. Generic ACMG BP1 (missense in a gene where only truncating variants cause disease) does not apply because missense is an established, common disease mechanism in MSH6 and the VCEP maintains extensive missense-specific rules (PP3/HCI priors, PS3/BS3 functional assays, PM5, PS1). BP1 is therefore not applicable.
BP2VCEPNot applicable: the MSH6 VCEP uses BS2 instead of BP2 for in-trans co-occurrence evidence.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 specification (cspec) declares BP2 'Not applicable' with the instruction 'BS2 is used instead'; in-trans co-occurrence with a known pathogenic variant in a monoallelic Lynch syndrome patient without CMMRD features is handled by BS2 in this framework, and BP2 is therefore not usable for MSH6. Per the framework-first rule, generic ACMG BP2 is not applied. Independently, even under the generic ACMG/AMP definition (observed in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern), no cis- or trans-observation of c.1403G>A with a pathogenic variant is documented in any reviewed source: the only clinical report (PMID:18033691) describes a single heterozygous carrier with no second variant and no phase testing.
VCEP rule
BP2: Not applicable per InSiGHT MSH6 VCEP v2.0 ('BS2 is used instead' for in-trans co-occurrence in monoallelic Lynch syndrome without CMMRD features).
Evidence considered
cspec: BP2 applicability 'Not applicable'; instructionsToUse 'BS2 is used instead'
PMID:18033691: single heterozygous CRC observation of c.1403G>A; no cis/trans data (no second variant, no phase testing)
BP3VCEPNot applicable: the VCEP does not use BP3, and this missense is not an in-frame indel in a repetitive region.
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Reasoning
The governing InSiGHT MSH6 VCEP v2.0 specification declares BP3 Not Applicable: 'In-frame deletions/insertions in a repetitive region without a known function is not used.' Independently, the variant is a missense substitution (c.1403G>A, p.Arg468His), not an in-frame deletion/insertion within a repetitive region, so it could not satisfy BP3 even under the generic ACMG definition. No generic ACMG fallback is warranted because a VCEP specification exists and is in force.
VCEP rule
BP3 (InSiGHT MSH6 VCEP v2.0, cspec): criterion declared 'Not applicable' - 'In-frame deletions/insertions in a repetitive region without a known function is not used.'
Evidence considered
cspec: InSiGHT MSH6 VCEP v2.0 - BP3 applicability 'Not applicable'; instructionsToUse 'In-frame deletions/insertions in a repetitive region without a known function is not used.'
PMID:18033691: confirms the variant is a single-nucleotide missense substitution, not an in-frame indel in a repeat region.
BP7N/ANot applicable: BP7 applies only to synonymous or intronic variants, and c.1403G>A is a missense substitution.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 BP7 rule (cspec doc 1578294885) applies only to 'a synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic)'. NM_000179.3:c.1403G>A is a missense substitution (p.(Arg468His), exon 4; canonical splice consensus = false per pvs1_variant_assessment), so the variant-type gate of BP7 is not met and the criterion is not applicable. SpliceAI max delta 0.02 does not rescue BP7 because that consideration is confined to synonymous/intronic variants under this VCEP.
Rule
BP7 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.
Evidence considered
VCEP BP7 rule (cspec doc 1578294885) applies to synonymous (silent) or intronic variants at or beyond -21/+7; c.1403G>A is missense p.(Arg468His) -> criterion not applicable.
SpliceAI max delta score = 0.02 (local lookup) - irrelevant to BP7 because the variant type gate (synonymous/intronic) fails.
AF 1.9e-05 · 31 / 1,613,978 alleles · 0 hom · FAF 2.3e-05
Admixed American4 / 59,976
6.7e-05
South Asian4 / 91,086
4.4e-05
East Asian1 / 44,900
2.2e-05
European (non-Finnish)21 / 1,180,028
1.8e-05
European (Finnish)1 / 63,998
1.6e-05
0 alternate alleles recorded in 5 other groups: Remaining individuals, Amish, Middle Eastern, Ashkenazi Jewish, African/African American.
gnomAD v2.1
AF 3.2e-05 · 8 / 251,140 alleles · 0 hom · FAF 3.9e-05
Admixed American4 / 34,554
0.00012
South Asian2 / 30,616
6.5e-05
European (non-Finnish)2 / 113,514
1.8e-05
0 alternate alleles recorded in 5 other groups: African/African American, Ashkenazi Jewish, East Asian, European (Finnish), Remaining individuals.
Statements
gnomAD v4.1
This variant is present in gnomAD v4.1 (AF= 1.92072e-05; MAF= 0.00192%, 31/1613978 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 6.66933e-05; MAF= 0.00667%, 4/59976 alleles, homozygotes = 0); grpmax FAF= 2.256e-05.
gnomAD v2.1
This variant is present in gnomAD v2.1 (AF= 3.18547e-05; MAF= 0.00319%, 8/251140 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 0.000115761; MAF= 0.01158%, 4/34554 alleles, homozygotes = 0); grpmax FAF= 3.896e-05.
gnomAD-Canada
Not available in gnomAD-Canada v1.0.
SpliceAI and Pangolin
SpliceAI acceptor lossat +9 bp
0.000
SpliceAI acceptor gainat +452 bp
0.000
SpliceAI donor lossat +203 bp
0.020
SpliceAI donor gainat -119 bp
0.010
Pangolin splice lossmagnitude shown
-0.020
Pangolin splice gain
0.020
Other predictors
REVEL
0.801
BayesDel
0.393
How the predictions were used
BP4SupportingMet (supporting): HCI prior probability of pathogenicity 0.1095 is below the 0.11 BP4 threshold. Flagged for human review: the value sits only 0.0005 below the cutoff and should be re-confirmed against HCI PRIORS.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 BP4 rule (cspec doc 1578294885) states: missense variant with HCI-prior probability of pathogenicity <0.11 (https://hci-priors.hci.utah.edu/PRIORS) = BP4_Supporting. The gene-specific HCI prior lookup (vcep_hci_priors_msh6 / hci_prior; dbid MSH6_03076, model source PMID 22949387) returns a prior probability of 0.1095 for c.1403G>A, which is below 0.11, so BP4 is met at supporting strength. This is a near-threshold call: 0.1095 lies only 0.0005 below the 0.11 cutoff, so the value should be re-confirmed against the HCI PRIORS resource (hci-priors.hci.utah.edu) before final classification; hence needs_human_review is set. The alternative BP4 splice path (SpliceAI delta <= 0.1 per Walker et al 2023) is restricted to intronic and synonymous variants and was not used for this missense variant; SpliceAI max delta 0.02 is recorded as corroborating context only and is not double-counted. BayesDel (0.392893) and REVEL (0.801) were not used (uncalibrated / outside VCEP missense path, respectively).
Rule
BP4 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): Missense variant with HCI-prior probability of pathogenicity <0.11 as per https://hci-priors.hci.utah.edu/PRIORS OR (intronic/synonymous variants only) SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.
Evidence considered
HCI prior probability for MSH6 c.1403G>A = 0.1095, which is <0.11 and therefore meets BP4 Supporting per InSiGHT MSH6 VCEP v2.0 rule (cspec doc 1578294885; PRIORS at hci-priors.hci.utah.edu; underlying model PMID 22949387 Thompson et al. 2013). Margin to threshold is 0.0005 - near-boundary, flagged for human review.
SpliceAI max delta score = 0.02 (<=0.1) would satisfy the BP4 splice-impact path per Walker et al. 2023 (PMID 37352859), but that path is restricted to intronic and synonymous variants per the VCEP rule; noted as context only, not counted for this missense variant.
Still missing
HCI prior 0.1095 is within 0.0005 of the 0.11 BP4 threshold; confirm the value against the HCI PRIORS website (hci-priors.hci.utah.edu/PRIORS) for MSH6 c.1403G>A before finalizing BP4 supporting.
PP3Not met: the HCI prior probability of pathogenicity is 0.1095, far below the 0.68 supporting threshold, and SpliceAI max delta 0.02 is below 0.2.
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Reasoning
Governing framework is the ClinGen InSiGHT MSH6 VCEP v2.0 (cspec doc 1578294885), whose PP3 rule for missense variants is: HCI prior probability of pathogenicity >0.81 = PP3_Moderate, >0.68 and <=0.81 = PP3_Supporting (https://hci-priors.hci.utah.edu/PRIORS), OR predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 (Walker et al 2023). The gene-specific HCI prior lookup (vcep_hci_priors_msh6 / hci_prior) returns a prior probability of 0.1095 for c.1403G>A (p.R468H, dbid MSH6_03076; underlying model Thompson et al. 2013, PMID 22949387), which is far below the 0.68 supporting threshold, so the missense PP3 path is not met. The splice path is also not met: SpliceAI max delta score is 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02), below the 0.2 threshold. BayesDel score 0.392893 is treated as not_available (no verified published calibration threshold available to this pipeline). REVEL score 0.801 was not applied because the governing MSH6 VCEP specifies HCI prior as the sole missense PP3/BP4 predictor for this gene; generic SVI REVEL thresholds (Pejaver et al. 2022, PMID 36413997) are only used when no applicable VCEP specification exists. PP3 is therefore not met on both available sub-paths with high confidence.
Rule
PP3 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): Missense variant with HCI prior probability for pathogenicity >0.81 = Moderate; >0.68 & <=0.81 = Supporting (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 prior probability for MSH6 c.1403G>A = 0.1095 (LOVD MSH6 priors lookup, dbid MSH6_03076; model source PMID 22949387 Thompson et al. 2013); InSiGHT MSH6 VCEP v2.0 PP3 rule requires >0.68 (Supporting) or >0.81 (Moderate) per cspec doc 1578294885 -> not met.
SpliceAI max delta score = 0.02 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.01, DS_DL 0.02); PP3 splice path requires delta >= 0.2 at non-canonical splicing nucleotides per Walker et al. 2023 (PMID 37352859), as adopted by MSH6 VCEP v2.0 -> not met.
REVEL score 0.801 (local REVEL v1.3 lookup): not applied - governing MSH6 VCEP v2.0 specifies HCI prior as the missense PP3/BP4 predictor; generic SVI REVEL thresholds (Pejaver et al. 2022, PMID 36413997) apply only when no VCEP specification exists.
BayesDel score 0.392893 (local BayesDel lookup): treated as not_available - no verified published calibration threshold available to this pipeline, so it cannot support PP3/BP4.
BP7N/ANot applicable: BP7 applies only to synonymous or intronic variants, and c.1403G>A is a missense substitution.
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Reasoning
The InSiGHT MSH6 VCEP v2.0 BP7 rule (cspec doc 1578294885) applies only to 'a synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic)'. NM_000179.3:c.1403G>A is a missense substitution (p.(Arg468His), exon 4; canonical splice consensus = false per pvs1_variant_assessment), so the variant-type gate of BP7 is not met and the criterion is not applicable. SpliceAI max delta 0.02 does not rescue BP7 because that consideration is confined to synonymous/intronic variants under this VCEP.
Rule
BP7 (InSiGHT MSH6 VCEP v2.0, cspec doc 1578294885): A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.
Evidence considered
VCEP BP7 rule (cspec doc 1578294885) applies to synonymous (silent) or intronic variants at or beyond -21/+7; c.1403G>A is missense p.(Arg468His) -> criterion not applicable.
SpliceAI max delta score = 0.02 (local lookup) - irrelevant to BP7 because the variant type gate (synonymous/intronic) fails.
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.02). REVEL score = 0.801. BayesDel score = 0.392893. HCI prior probability for pathogenicity = 0.1095. MAPP score = 14.41. Custom PP2 score = 0.246.
8 paper records listed. 1 cited in the classification.
Cited in the classification
Classification of ambiguous mutations in DNA mismatch repair genes identified in a population-based study of colorectal cancer.
Barnetson RA et al. · 2008 Mar
Barnetson et al. (Hum Mutat 2008) report c.1403G>A p.R468H as one of the MSH6 germline variants identified in a population-based study of 932 colorectal cancer patients diagnosed before age 55. In Table 1, the variant is shown in 1 case (case frequency 0.0011) and 0/1008 controls; the single assessed tumor was microsatellite stable (MSS) with normal MLH1, MSH2, and MSH6 immunohistochemical staining. The study's mRNA analysis was performed only for putative splice-site mutations, and no RNA/splicing analysis, in-frame length change, or null-consequence data are reported for this variant. The paper's own pathogenicity category for this variant was not used to infer any criterion in this group. For the consequence/LOF group this source confirms the variant is a missense substitution with no reported splicing aberration and no protein length change, consistent with PVS1 not met and PM4/BP3 not applicable. Note: in the text extraction, '>' is rendered as '4' (e.g., 'c.1403G4A'); quotes preserve the extracted text.
Context Population-based cohort of 932 colorectal cancer patients diagnosed at age <55 years; germline sequencing of MLH1, MSH2, and MSH6; variant allele frequency determined in control DNA samples; tumor MSI (5-marker panel) and MMR protein IHC; mRNA analysis restricted to putative splice-site mutations; bioinformatic protein-function predictions.
Other recorded publications
Genetic cancer risk assessment and counseling: recommendations of the national s
Feasibility of screening for Lynch syndrome among patients with colorectal cancer.
Hampel H et al. · 2008
Hampel et al. 2008 (J Clin Oncol 26(35):5783-5788) evaluated feasibility of routine Lynch syndrome screening among newly diagnosed CRC patients at Ohio State University. The germline MSH6 mutations reported (c.3939_3957dup19 p.A1320SfsX5, c.3261dupC p.F1088LfsX5, c.3920_3923dupATCT p.P1309SfsX11) do not include c.1403G>A p.R468H; full-text search for '1403', 'Arg468', and 'R468' returned zero matches. This source therefore contains no de novo or segregation data applicable to this variant and was ruled out for this group's criteria.
Table 2 (germline mutations identified), extracted full text lines 486-506; full-text search for '1403', 'Arg468', 'R468' returned zero matches; the only 'de novo' hit (line 832) is a reference citation (J Med Genet 37:646-652, 2000), not variant-specific data
Investigation on the role of nsSNPs in HNPCC genes--a bioinformatics approach.
Doss CG et al. · 2009
In silico nsSNP analysis of HNPCC genes (Doss & Sethumadhavan 2009), Table 1, lists MSH6 rs41295268 A/G p.R468H with SIFT tolerance index 0.54 (Tolerant) and PolyPhen PSIC score 1.954 (Possibly damaging). This is computational prediction for the same variant under evaluation; the paper provides no clinical classification and reports no other variant at residue 468.
Prioritization of candidate SNPs in colon cancer using bioinformatics tools: an alternative approach for a cancer biologist.
George Priya Doss C et al. · 2010
Follow-up bioinformatics prioritization study of colon cancer candidate SNPs (George Priya Doss et al. 2010) lists the same MSH6 SNP rs41295268 A/G p.R468H with SIFT 0.54 (category IV) and PolyPhen 1.954 (B). Only the variant under evaluation is described; no other residue-468 variant is reported.
The evidence statements recorded by the interpretation pipeline. They have not been reinterpreted by the page.
gnomAD v4.1
This variant is present in gnomAD v4.1 (AF= 1.92072e-05; MAF= 0.00192%, 31/1613978 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 6.66933e-05; MAF= 0.00667%, 4/59976 alleles, homozygotes = 0); grpmax FAF= 2.256e-05.
gnomAD v2.1
This variant is present in gnomAD v2.1 (AF= 3.18547e-05; MAF= 0.00319%, 8/251140 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 0.000115761; MAF= 0.01158%, 4/34554 alleles, homozygotes = 0); grpmax FAF= 3.896e-05.
gnomAD-Canada
Not available in gnomAD-Canada v1.0.
ClinVar
This variant has been reported in ClinVar as Likely benign (7 clinical laboratories) and as Uncertain significance (3 clinical laboratories) and as Benign (1 clinical laboratory) and as Uncertain Significance (1 clinical laboratory) and as Likely Benign (1 clinical laboratory) and as Likely benign by International Society for Gastrointestinal Hereditary Tumours (InSiGHT) (expert panel). (ClinVarID = 89192)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.02). REVEL score = 0.801. BayesDel score = 0.392893. HCI prior probability for pathogenicity = 0.1095. MAPP score = 14.41. Custom PP2 score = 0.246.
Functional
OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. MSH6, a DNA mismatch repair protein, is frequently mutated in colorectal, small bowel, and endometrial cancers.
Somatic
This variant does not lie in a statistically significant hotspot. This variant has previously been reported in somatic cancers (COSMIC; COSV52276785, n = 4 times).
Cancer Hotspots
This variant does not lie in a statistically significant hotspot.
This variant was interpreted by pipeline 7.1.0.
The current version is 11.0.07.
Re-running queues a fresh interpretation; the result replaces this page when complete.
Re-runs are not free. Blank draws on today's public interpretation allowance; with a code, it uses one of that code's uses.