MSH6 c.4001+32_4001+35dup, p.?

Reasoning

Per the governing InSiGHT MSH6 VCEP v2.0 specification, BP7 is met for 'A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic).' The variant NM_000179.3:c.4001+32_4001+35dup is an intronic duplication located 32-35 nucleotides 3' of exon 8 (c.4001+32 to c.4001+35), i.e. well beyond +7, and is therefore an intronic variant at or beyond the +7 boundary. BP7_Supporting is met on the positional criterion. This is a positional/descriptive code, not a computational prediction, so it does not double-count the SpliceAI result used in the PP3/BP4 evaluation (neither of which is met). The VCEP rule explicitly notes variants may satisfy both BP7 and BP4, but here BP4 is not met because the SpliceAI delta (0.12) exceeds the <=0.1 BP4 threshold.

Rule

A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.

Evidence considered

• Variant normalization: NM_000179.3:c.4001+32_4001+35dup (intronic duplication, +32 to +35 relative to exon 8, beyond +7)
• InSiGHT MSH6 VCEP v2.0 BP7 rule: intronic variant at or beyond -21/+7 qualifies for BP7_Supporting

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (VCEP) specification for MSH6 v2.0 (cspec, doc 1578294885), which defines PVS1 in three tiers: Very Strong (nonsense/frameshift introducing a PTC <= codon 1341; large genomic alterations of single or multi-exon size; variants at IVS+/-1 or IVS+/-2 where exon skipping or cryptic splice use disrupts the reading frame and is predicted to undergo NMD; or mRNA assays from patient constitutional samples confirming a splicing aberration with no full-length/reference transcript), Strong (initiation codon variants; tandem duplication of >=1 exon with frameshift before the last splice junction; G>non-G at last base of exon; IVS+/-1 or +/-2 variants preserving reading frame in a critical region), and Moderate (nonsense/frameshift with PTC between codons 1342 and 1360). The variant NM_000179.3:c.4001+32_4001+35dup is a 4-bp duplication located deep in intron 9 (positions +32 to +35 downstream of exon 9; ClinVar repeat-unit notation c.4001+27TAAC[3]), i.e. NOT at the canonical splice consensus (canonical_splice_consensus = false per pvs1_variant_assessment). It is not a coding-sequence nonsense/frameshift (protein prediction is NP_000170.1:p.? — no protein-level consequence), not a large genomic (single/multi-exon) alteration, not an IVS+/-1 or IVS+/-2 variant, not an initiation codon variant, and not an exon tandem duplication. SpliceAI predicts no significant splice impact (max delta score 0.12; DS_AG 0.01, DS_AL 0.12, DS_DG 0.01, DS_DL 0.11), and no mRNA/minigene assay evidence exists, which would be the only route to PVS1 for a non-canonical splice-site variant per the VCEP rule. The generic PVS1 scaffold agrees: consequence_class 'unknown', variant_bucket 'other', apply_generic_pvs1_framework = false. Although the gene-level PVS1 gate is eligible (LOF is a known mechanism for MSH6, pvs1_gene_context), the variant does not fall into any PVS1 variant-type bucket, so PVS1 is not met.

Rule

VCEP InSiGHT MSH6 v2.0 PVS1: '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... indicate that the variant allele results in a splicing aberration...' — none of the listed variant-type buckets applies to a deep intronic (IVS9 +32..+35) 4-bp duplication.

Evidence considered

• InSiGHT MSH6 VCEP v2.0 PVS1 rule text (Very Strong/Strong/Moderate buckets) from cspec
• Variant position c.4001+32_4001+35dup (deep intronic, IVS9 +32..+35) from case_summary normalization and ClinVar variant title 'NM_000179.3(MSH6):c.4001+27TAAC[3]'
• pvs1_variant_assessment: canonical_splice_consensus=false, variant_bucket='other', apply_generic_pvs1_framework=false
• pvs1_gene_context: pvs1_gene_gate='eligible' (LOF mechanism supported) — gate passes but no variant-type bucket applies
• SpliceAI: max delta score 0.12 (DS_AL 0.12, DS_DL 0.11) — no predicted splice aberration
• PMID:25741868 Table 3 PVS1 null-variant definition (generic reference definition)

Still missing

• No mRNA/minigene assay data on this variant; if future RNA analysis demonstrated an out-of-frame splicing aberration with NMD from this deep intronic duplication, PVS1 could be revisited (per VCEP rule requiring minigene or independent-laboratory RNA confirmation for non-canonical splice-site variants).

Sources

Reasoning

Under the MSH6 v2.0 VCEP specification, PM2 is met at Supporting strength when the allele frequency is absent/extremely rare (< 0.00002, i.e. < 1 in 50,000 alleles) in the gnomAD v4 dataset. This variant is observed at total AF 9.95616e-05 (159/1597002 alleles) in gnomAD v4.1, approximately 5-fold above the 0.00002 ceiling; the exome-only AF (0.0001031), genome AF (6.60e-5), and every subpopulation AF likewise exceed the ceiling (highest subpopulation: Remaining individuals 0.0003711). The variant is therefore neither absent nor extremely rare and PM2 is not met at any strength. Homozygote count is 0 in gnomAD v4.1 and v2.1, which is noted but does not satisfy PM2 given the frequency. gnomAD v2.1 (total AF 5.88e-5, grpmax FAF 8.6e-5) is consistent with the v4.1 conclusion.

Rule

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

Evidence considered

• gnomad_v4
• gnomad_v2
• cspec

Sources

Reasoning

Governing framework is the ClinGen InSiGHT HCRC/PP MSH6 VCEP specification v2.0 (framework_mode=vcep). For this intronic variant the only applicable PP3 branch is: 'Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023.' SpliceAI lookup for NM_000179.3:c.4001+32_4001+35dup returns max delta score = 0.12 (DS_AL 0.12, DS_DL 0.11, DS_AG 0.01, DS_DG 0.01), which is below the >= 0.2 threshold, so no splice defect is predicted. The HCI-prior branches (>0.68 / >0.81) apply only to missense substitutions and are not applicable to this intronic duplication (variant is not a single-nucleotide missense; HCI-PRIORS-MSH6.txt is a missense-only lookup and no hci_prior value was surfaced). PP3 is therefore not met.

Rule

Missense variant with HCI prior probability for pathogenicity >0.81 (Moderate) OR >0.68 & <=0.81 (Supporting) OR Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023 (Supporting).

Evidence considered

• SpliceAI lookup max delta score 0.12 (DS_AG 0.01, DS_AL 0.12, DS_DG 0.01, DS_DL 0.11) for NM_000179.3:c.4001+32_4001+35dup
• InSiGHT MSH6 VCEP v2.0 PP3 rule: splice-defect branch requires SpliceAI delta score >= 0.2

Still missing

• No RNA/minigene assay in the evidence bundle to functionally confirm splicing outcome; such evidence would be evaluated under BS3/PS3 (functional group), not PP3.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (cspec, doc 1578294885, precedence rank 1), which defines BA1 as gnomAD v4 Grpmax filtering allele frequency >= 0.0022 (0.22%) with the variant excluded as a founder pathogenic variant. For NM_000179.3:c.4001+32_4001+35dup (intron 9 deep-intronic 4-bp duplication), the gnomAD v4.1 joint Grpmax FAF is 0.00020269 (2.03e-4), approximately 10.8-fold below the 0.0022 BA1 threshold. The highest observed subpopulation frequency (Remaining individuals, AF 3.71e-4, 23/61970 alleles) and total AF (9.96e-5, 159/1597002 alleles) are likewise both far below 0.22%. No evidence in the case bundle identifies this variant as a founder pathogenic variant, so the founder-exclusion condition is not the limiting factor. BA1 is not met.

Rule

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

Evidence considered

• gnomad_v4
• cspec

Sources

Reasoning

Under the MSH6 v2.0 VCEP specification, BS1 requires gnomAD v4 Grpmax filtering allele frequency >= 0.00022 and < 0.0022 (0.022-0.22%) with the variant excluded as a founder pathogenic variant. The gnomAD v4.1 joint Grpmax FAF for this variant is 0.00020269, which is just below the 0.00022 lower bound (about 8% short), so BS1 is not met by the strict letter of the rule. However, this is a threshold artifact: the exome-only Grpmax FAF is 0.00030753 and the highest raw subpopulation AF (Remaining individuals) is 0.000371147, both of which exceed the 0.00022 BS1 lower bound; only the joint Grpmax FAF (the metric used in the evidence sentence) falls short. Whether BS1 is met therefore depends on which gnomAD v4 FAF metric/dataset slice is applied, and a routine gnomAD data update could flip the outcome. Human review is requested to confirm the correct metric per the VCEP (joint Grpmax FAF) before finalizing. Homozygote count is 0 in both v4.1 and v2.1. No founder-pathogenic status is documented for this variant in the bundle, so the founder-exclusion condition does not block BS1.

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
• gnomad_v2
• cspec

Still missing

• Confirm the gnomAD v4 metric that the InSiGHT MSH6 VCEP intends by 'Grpmax filtering allele frequency' (joint vs exome-only) given the near-threshold value (joint FAF 2.03e-4 vs lower bound 2.2e-4)

Sources

Reasoning

Per the governing InSiGHT MSH6 VCEP v2.0 specification, for intronic variants BP4 requires: 'SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.' SpliceAI lookup for this variant returns max delta score = 0.12, which exceeds the <= 0.1 threshold, so the SpliceAI branch of BP4 is not met. The alternative BP4 branch (HCI-prior probability of pathogenicity < 0.11) applies only to missense variants and does not apply to this intronic duplication. The max delta of 0.12 falls in the indeterminate band between the VCEP BP4 threshold (<=0.1) and PP3 threshold (>=0.2), so no benign in-silico code can be applied. BP4 is therefore not met.

Rule

Missense variant with HCI-prior probability of pathogenicity <0.11 OR For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.

Evidence considered

• SpliceAI lookup max delta score 0.12 (DS_AG 0.01, DS_AL 0.12, DS_DG 0.01, DS_DL 0.11) for NM_000179.3:c.4001+32_4001+35dup
• InSiGHT MSH6 VCEP v2.0 BP4 rule: intronic variants require SpliceAI delta score <= 0.1

Still missing

• SpliceAI score sits in the 0.1-0.2 indeterminate zone; an mRNA/minigene assay showing no splicing aberration would be needed to support benignity (evaluated under BS3, functional group).

Sources

Reasoning

No de novo evidence of any kind is present in the case bundle. There is no proband-level clinical description, no statement that the variant arose de novo, and no parental testing (maternity/paternity confirmation) or meioses data. The governing InSiGHT MSH6 VCEP v2.0 PS2 rule assigns points per proband (2 points with both parents confirmed and an MMR-deficient Lynch syndrome spectrum tumor, 1 point with both parents confirmed and an LS-spectrum tumor without tumor data, 0.5 points for an assumed de novo variant with parents unconfirmed), so a proband with confirmed de novo status and confirmed parental origin would be required before any strength could be assigned. ClinVar submissions (5 laboratories: 4 Likely benign, 1 Benign; no expert panel) carry no per-criterion evidence and cannot be used to infer de novo status. Per the instruction not to infer criteria from another source's final classification, no PS2 evidence is credited.

Rule

InSiGHT MSH6 VCEP v2.0 PS2: de novo points system - >=4 points = Very Strong; 2-3.5 = Strong; 1-1.5 = Moderate; 0.5 = Supporting (2 points/proband with both parents confirmed + MMR-deficient LS tumor; 1 point/proband with both parents confirmed + LS tumor without tumor data; 0.5 points/proband assumed de novo with parents unconfirmed).

Evidence considered

• InSiGHT MSH6 VCEP v2.0 PS2 rule: 'Proband with a de novo variant with both maternity and paternity confirmed in a case with MMR deficient LS spectrum tumor ... 2 points per proband'; 'assumed de novo variant and maternity and/or paternity unconfirmed ... 0.5 points per proband'; strength thresholds '>=4 de novo points' Very Strong, '2 - 3.5' Strong, '1 - 1.5' Moderate, '0.5' Supporting

Still missing

• Proband de novo status for the variant (tested parents, both biological parents confirmed)
• Maternity and paternity confirmation (or explicit statement of assumption with parents unconfirmed)
• Proband tumor phenotype data (MSI/IHC status or at minimum LS-spectrum tumor type) to determine points per proband per the VCEP PS2 rule
• Any second proband with de novo variant to accumulate >=2 points

Sources

Reasoning

Variant NM_000179.3:c.4001+32_4001+35dup is a deep intronic 4-bp duplication (TAAC[3]) at +32 to +35 of intron 9 (variantvalidator: exon 9i). Governing framework is the ClinGen InSiGHT MSH6 VCEP v2.0 specification (cspec doc 1578294885), which defines PS3 as: Strong = calibrated functional assay functional odds for pathogenicity >18.7; Moderate = odds >4.3 and <=18.7, OR MMR function defect following the MMR functional assay flowchart, OR monoallelic expression with complete loss of expression (<10% of wild-type in cDNA without puromycin, full-length transcript analysed with and without NMD block); Supporting = odds >2.08 and <=4.3. No calibrated functional assay result, MMR function assay, mRNA splicing assay, or allelic-expression assay for this variant exists anywhere in the case evidence bundle (prefetch.json, evidence.json, case_summary.json, literature_pass.json, vcep_materials.json all lack functional assay data; ClinVar submissions report no functional evidence and carry no criterion leads). SpliceAI (max delta = 0.12) is an in silico prediction, not a laboratory functional assay, and cannot satisfy any PS3 strength tier. Because the variant is intronic, protein-based MMR complementation assays (e.g., Drost et al. style) would not be the primary route; RNA-based evidence (splicing aberration or monoallelic expression with NMD-blocked cDNA) would be required. That evidence is absent, so PS3 cannot be assessed. Not 'not_applicable': the VCEP marks PS3 Applicable and the monoallelic-expression branch explicitly contemplates RNA-based assessment applicable to intronic variants.

Rule

InSiGHT MSH6 VCEP v2.0 PS3: Calibrated functional assays with functional odds for pathogenicity >18.7 (Strong); >4.3 and <=18.7, OR MMR function defect following functional assay flowchart, OR monoallelic expression with complete loss of expression (<10% of wild-type in cDNA without puromycin) of the variant allele, full-length transcript analysed with and without NMD block (Moderate); calibrated functional odds >2.08 and <=4.3 (Supporting). Instructions: refer to 'Functional assay SVI documentation (MMR genes)' for calibrated functional assays; the flowchart is a general framework for evaluating assays already performed or from historic publications.

Evidence considered

• cspec
• spliceai
• clinvar
• PMID:10537275

Still missing

• No calibrated functional assay (functional odds for pathogenicity) result for c.4001+32_4001+35dup
• No mRNA assay of patient constitutional RNA (splicing analysis and allelic-imbalance quantitation with and without nonsense-mediated decay inhibition / puromycin) for this variant
• No MMR functional assay (per MMR functional assay flowchart) result for this variant

Sources

Reasoning

PM3 is Applicable in the governing ClinGen InSiGHT MSH6 VCEP v2.0 framework (cspec doc 1578294885): 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 (Aronson et al 2022), scored by points - P/LP in trans = 1.0 point, P/LP phase unknown = 0.5 point, summed across cases (>=4 pts Very Strong, 2-3.5 Strong, 1-1.5 Moderate, 0.5 Supporting). The case bundle contains NO allelic evidence whatsoever: no second MSH6 variant, no phase determination (no parental/offspring testing), no co-occurring P/LP variant, and no CMMRD-consistent patient observation anywhere in review_manifest/case_summary/evidence/prefetch/literature sources. gnomAD v4.1 shows 0 homozygotes in 1,597,002 alleles (AF 9.96e-05) and ClinVar VCV000089502 submissions report no phase/zygosity information (conditions 'not provided'/'not specified'), but population homozygote absence and ClinVar consensus are not substitutes for trans-phase evidence. With 0 points of scored evidence, PM3 cannot be met at any strength; per insufficient-evidence policy the criterion is returned not_assessed. No publication in the literature packet targets PM3 or mentions this variant.

Rule

PM3 (Applicable): '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... Pathogenic/Likely Pathogenic in trans: 1.0 point; Pathogenic/Likely Pathogenic - phase unknown: 0.5 points. Sum all cases with the above evidence to determine the PM3 strength.' Strength thresholds: >=4 points Very Strong; 2-3.5 Strong; 1-1.5 Moderate; =0.5 Supporting.

Evidence considered

• InSiGHT MSH6 VCEP v2.0 PM3 rule: P/LP in trans = 1.0 point; P/LP phase unknown = 0.5 point; sum across cases; 0.5 pt = Supporting, 1-1.5 = Moderate, 2-3.5 = Strong, >=4 = Very Strong
• ClinVar VCV000089502 checked for any trans/cis/phase/compound-heterozygote observation in the 5 germline submissions - none present
• gnomAD v4.1 checked for homozygotes (0/1,597,002 alleles) and population AF (9.96e-05) as context - no biallelic carriers observed

Still missing

• No second MSH6 sequence variant identified in any proband - required for PM3 scoring
• No phase determination (parental or offspring testing) establishing trans versus cis configuration
• No patient with CMMRD-consistent clinical features (Aronson et al 2022 criteria / Table for CMMRD diagnosis) observed to carry this variant with a co-occurring P/LP variant
• No biallelic (homozygous or compound heterozygous) observation of this variant in a proband

Sources

Reasoning

No segregation evidence exists in the case. There are no pedigrees, no co-segregating affected relatives tested for the variant, no meioses, and no Bayes likelihood ratio calculation (the VCEP PP1 rule requires a combined Bayes LR from COOL v3 segregation analysis: >2.08-4.3 Supporting, >4.3-18.7 Moderate, >18.7 in >=2 families Strong). None of the retrieved literature (8 PMIDs, 5 with full text) mentions this variant or provides segregation data for it; a full-text and bundle screen for 'de novo/segregat/proband/pedigree/maternity/paternity/meios' returned zero hits. The ClinVar classification (Likely benign/Benign) cannot be used to infer segregation criteria. PP1 is therefore not assessed pending pedigree data.

Rule

InSiGHT MSH6 VCEP v2.0 PP1: co-segregation with disease, combined Bayes LR >18.7 in >=2 families = Strong; >4.3-18.7 = Moderate; >2.08-4.3 = Supporting (computed via COOL v3, no penetrance/relative-risk files required for MMR genes).

Evidence considered

• InSiGHT MSH6 VCEP v2.0 PP1 rule: 'Co-segregation with disease in pedigree(s) with a combined Bayes Likelihood Ratio >18.7 in >=2 families' (Strong), '>4.3 & <=18.7' (Moderate), '>2.08 & <=4.3' (Supporting); recommended tool COOL v3

Still missing

• Pedigree(s) with affected relatives tested for the variant (>=2 affected family members with genotype/phenotype concordance)
• Number of informative meioses/segregating individuals needed for a COOL v3 Bayes likelihood ratio
• Combined Bayes LR across families (>=2 families required for PP1_Strong per VCEP)
• Phenotype definition for affected status (Lynch syndrome spectrum tumors, age of onset)

Sources

Reasoning

The VCEP MSH6 v2.0 specification defines PP4 via tumor phenotype: 1 CRC/endometrial MSI-H tumor (standard 5-10 marker panel or tumor genome) and/or loss of MMR protein expression consistent with the variant location (Supporting), escalating to Moderate (2 tumors) and Strong (>=3 tumors in >=2 families). However, the consolidated case-evidence bundle contains no tumor phenotype data whatsoever for carriers of c.4001+32_4001+35dup: no MSI status, no MMR immunohistochemistry results, and no tumor genome data. All MSI/MSS/IHC strings in the bundle occur only inside the cspec rule text, not as case observations. With zero phenotype evidence, PP4 cannot be scored and is returned not_assessed pending tumor data.

Rule

1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4.

Still missing

• No tumor MSI status (MSI-H vs MSS) data for proband/family tumors carrying c.4001+32_4001+35dup
• No MMR immunohistochemistry (MSH6 protein expression) data for tumors from carriers of this variant
• No tumor genome/panel MSI results using a standard 5-10 marker panel

Sources

Reasoning

Under the MSH6 v2.0 VCEP specification, BS2 is not a population-frequency criterion: it requires co-occurrence in trans with a known pathogenic sequence variant in the same gene (MSH6) in a patient with colorectal cancer diagnosed after age 45 (or another Lynch syndrome cancer above the median age of onset), with no prior or current evidence of CMMRD clinical manifestations per Aronson et al 2022 ('Table for CMMRD diagnosis.pdf'), and with phase confirmed by testing of parents or offspring. The case-evidence bundle contains no proband or family phenotype data, no report of a second pathogenic MSH6 variant in trans, and no phase-confirmation data, so BS2 cannot be assessed from the available evidence. The CMMRD diagnostic table (vcep_table_for_cmmrd_diagnosis) is available as the reference for the CMMRD-exclusion component but cannot be applied without patient clinical data. Gap: human-curated clinical co-occurrence and phase data required.

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 as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring.

Evidence considered

• cspec
• vcep_table_for_cmmrd_diagnosis

Still missing

• Proband/family phenotype data (CRC or other LS-spectrum cancer and age at diagnosis)
• Evidence of a known pathogenic MSH6 variant in trans with c.4001+32_4001+35dup
• Phase confirmation by parental or offspring testing
• CMMRD clinical manifestations excluded per Aronson et al 2022 (Table for CMMRD diagnosis)

Sources

Reasoning

BS3 under the governing InSiGHT MSH6 VCEP v2.0 framework: Strong = calibrated functional assays with functional odds for pathogenicity <=0.05, OR synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition (abnormal transcripts seen at similar levels in controls count as naturally occurring isoforms); Supporting = calibrated functional odds >0.05 and <=0.48, OR variant-specific proficient function in protein and mRNA-based lab assays per the MMR functional assay flowchart. The variant is a deep intronic duplication (+32_+35 in intron 9), and the BS3_Strong mRNA-aberration branch is the applicable route in principle, but no laboratory mRNA assay (splicing or allelic imbalance, NMD inhibition), no calibrated functional assay, and no protein/mRNA proficient-function assay for this variant is present in the case evidence bundle. SpliceAI max delta score 0.12 is an in silico prediction, not a laboratory assay, and cannot substitute for the required functional/mRNA evidence (it also exceeds the VCEP BP4 splice threshold of <=0.1, so it does not even independently suggest no splicing impact under this framework). ClinVar classifications (4x Likely benign, 1x Benign) were not used to infer BS3 because adjudication rules forbid inferring a criterion from another source's final classification, and submissions carry no functional assertions. Evidence absent, therefore BS3 cannot be assessed.

Rule

InSiGHT MSH6 VCEP v2.0 BS3: Calibrated functional assays with functional odds for Pathogenicity <=0.05, OR synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition; abnormal transcripts identified at similar levels in controls are considered naturally occurring isoforms and not mRNA aberrations (Strong). Calibrated functional odds >0.05 and <=0.48, OR variant-specific proficient function in protein and mRNA-based lab assays per the MMR functional assay flowchart (Supporting).

Evidence considered

• cspec
• spliceai
• clinvar
• PMID:10537275

Still missing

• No mRNA assay with nonsense-mediated decay inhibition (splicing analysis + allelic imbalance) for c.4001+32_4001+35dup
• No calibrated functional assay result (functional odds for pathogenicity) for this variant
• No variant-specific proficient-function protein and mRNA-based lab assay result per the MMR functional assay flowchart

Sources

Reasoning

No non-segregation (lack of co-segregation) evidence is present. There are no pedigrees, no informative meioses showing the variant in unaffected individuals or absence of the variant in affected individuals, and no Bayes likelihood ratio below 1 that would support BS4 (VCEP rule: combined LR <0.05 = Strong; >0.05-0.48 = Supporting). The ClinVar Likely benign/Benign classifications from 5 laboratories are final classifications without published per-criterion evidence and are not used to infer BS4. The criterion is not assessed pending segregation data.

Rule

InSiGHT MSH6 VCEP v2.0 BS4: lack of co-segregation with disease, combined Bayes LR <0.05 = Strong; >0.05-0.48 = Supporting.

Evidence considered

• InSiGHT MSH6 VCEP v2.0 BS4 rule: 'Lack of co-segregation with disease in pedigree(s) with a combined Bayes Likelihood Ratio <0.05' (Strong), '>0.05 & <=0.48' (Supporting)

Still missing

• Pedigree data with tested relatives sufficient to compute a lack-of-co-segregation Bayes LR (e.g., affected relatives who do NOT carry the variant, or carriers who remain unaffected at an age beyond typical onset)
• COOL v3 likelihood ratio calculation showing combined LR <=0.48 (Supporting) or <0.05 (Strong)
• Documented cancer/endoscopy screening status and ages of unaffected carriers to avoid age-dependent penetrance confounders

Sources

Reasoning

The VCEP MSH6 v2.0 specification defines BP5 via tumor phenotype: 2-3 CRC/endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS-spectrum tumors with MMR protein loss inconsistent with the variant gene (Supporting), escalating to Strong for >=4 tumors or >=2 BRAF V600E (CRC) / MLH1-methylated MSI-H tumors, or 1 such tumor at Supporting. The case evidence bundle contains no tumor phenotype data at all for this variant (no MSI/MSS results, no IHC results, no BRAF V600E or MLH1 methylation testing), so none of the BP5 conditions can be evaluated. BP5 is returned not_assessed pending tumor phenotype data.

Rule

2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation. OR 1 BRAF V600E (Colon only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss.

Still missing

• No tumor MSI/MSS status data for tumors from carriers of c.4001+32_4001+35dup
• No MMR IHC results (absence of loss, or loss inconsistent with MSH6) for tumors from carriers
• No BRAF V600E or MLH1 promoter methylation testing results for MSI-H/MLH1-loss tumors

Sources