MSH6 c.1569_1572del, p.(Tyr524ValfsTer46)

Reasoning

NM_000179.3:c.1569_1572del is a 4-bp deletion in MSH6 exon 4 (c.628-3172) that shifts the reading frame and introduces a premature termination codon at codon 569 (NP_000170.1:p.(Tyr524ValfsTer46); predicted protein ends at residue 569, c.1705-1707). NM_000179.3 is the MANE Select transcript and the transcript designated by the governing specification, so the transcript is biologically relevant. The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 v2.0 (cspec), whose PVS1 Very Strong rule states: 'Nonsense/frameshift variant introducing Premature Termination Codon (PTC) <= codon 1341 in MSH6.' A PTC at codon 569 is far upstream of codon 1341, so full very-strong strength applies. The InSiGHT MMR PVS1 decision tree reaches PVS1 for nonsense/frameshift variants predicted to undergo NMD at <= codon 1317 in MSH6, and also for truncations within the region critical to protein function at <= codon 1341; codon 569 satisfies both arms. NMD is predicted rather than escaped: the PTC lies in exon 4 and ~2295 nucleotides upstream of the last exon-exon junction (final exon begins at c.4002), far beyond the ~50-55 nt window associated with NMD escape. No downgrade rule applies - this is a coding frameshift, not a canonical IVS+/-1,2 splice variant (so the splice-specific arms of the decision tree and the PP3 non-stacking caveat are not engaged; SpliceAI max delta 0.087 is below the 0.2 supporting cutoff), the truncation is not in the last exon, it does not escape NMD, it does not lie in the codon 1342-1360 region that the MMR decision tree restricts to PVS1_Moderate, and the affected region shows no population loss-of-function enrichment (gnomAD v4.1: 1/1,614,208 alleles, AF 6.19e-07, no homozygotes). MSH6 germline loss of function is an established disease mechanism for Lynch syndrome in this framework. PVS1 is therefore met at full, un-downgraded very-strong strength. ClinVar and OncoKB classifications were not used to reach this call.

Rule

InSiGHT MSH6 v2.0 PVS1 Very Strong: nonsense/frameshift variant introducing a PTC <= codon 1341 in MSH6 (no downgrade rule triggered).

Evidence considered

• Variant NM_000179.3:c.1569_1572del is a 4-bp deletion in MSH6 exon 4 (c.628-3172) causing an out-of-frame frameshift with a premature termination codon at codon 569 (NP_000170.1:p.(Tyr524ValfsTer46)); the predicted protein terminates at residue 569 (c.1705-1707).
• NM_000179.3 is the MANE Select transcript and the transcript specified by the governing MSH6 specification (raw_rule_origin preferredTranscript = NM_000179.3), so it is the clinically relevant transcript.
• Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification to the ACMG/AMP guidelines for MSH6, Version 2.0 (doc 1578294885), which takes precedence over generic ACMG/AMP defaults.
• The MSH6 v2.0 PVS1 Very Strong rule reads: 'Nonsense/frameshift variant introducing Premature Termination Codon (PTC) <= codon 1341 in MSH6.' A PTC at codon 569 is 772 codons upstream of this boundary, so full very-strong strength is retained.
• The InSiGHT MMR PVS1 decision tree assigns PVS1 to nonsense/frameshift variants predicted to undergo NMD at <= codon 1317 in MSH6 and to truncations within the region critical to protein function at <= codon 1341; codon 569 satisfies both arms. The decision tree limits PVS1_Moderate to the codon 1342-1360 interval, which this variant does not enter.
• NMD is predicted, not escaped: the PTC lies in exon 4 and approximately 2295 nucleotides upstream of the last exon-exon junction (final exon begins at c.4002), far beyond the ~50-55 nt threshold at which a PTC escapes NMD.
• No named SVI/VCEP downgrade rule applies: the variant is a coding frameshift (not a canonical IVS+/-1,2 splice variant, so the splice arms of the decision tree and the 'do not combine with PP3' caveat are not engaged; SpliceAI max delta is 0.087, below the 0.2 supporting cutoff), the PTC is not in the last exon, it does not escape NMD, and it does not fall in the codon 1342-1360 unknown-function interval.
• MSH6 germline loss of function is an established disease mechanism in this framework (Lynch syndrome / mismatch repair cancer syndrome 1); the gene-level PVS1 gate is eligible.
• The affected region is not enriched for population loss-of-function variation: gnomAD v4.1 lists a single allele (1/1,614,208; AF 6.19e-07; no homozygotes), and the variant is absent from gnomAD v2.1.
• ClinVar (Pathogenic, multiple submitters) and OncoKB (biological effect: likely loss-of-function) were read only as variant-level consequence context; neither classification was used to infer PVS1.
• PMID:25741868 (ACMG/AMP 2015) defines the null-variant/PVS1 concept applied here; PMID:24362816 (InSiGHT VIC) documents that truncating MMR variants are treated as assumed pathogenic, with the caveat that MSH6 truncations may show lower penetrance.

Sources

Reasoning

The MSH6 VCEP v2.0 (cspec) sets PM2 at an absent/extremely rare allele frequency < 0.00002 (< 1 in 50,000 alleles) in gnomAD v4, applied at Supporting strength. The variant is present in gnomAD v4.1 at a total allele frequency of 6.19e-07 (1/1,614,208 alleles; exome AF 6.84e-07; 0 homozygotes) with a highest subpopulation frequency of 8.47e-07 in European (non-Finnish), i.e. roughly 32-fold below the 0.00002 cutoff, and it is absent from gnomAD v2.1 (exomes) and gnomAD-Canada v1.0. This satisfies the VCEP's rarity threshold. The exact-match ClinVar criterion-lead submission SCV002154851 (Labcorp/Invitae) likewise applied PM2 on the basis that the variant is 'not present in population databases (gnomad no frequency)'. PM2 is met at Supporting strength, consistent with the VCEP rule strength and the ClinGen SVI recommendation to apply PM2 at Supporting.

Rule

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

Evidence considered

• MSH6 VCEP v2.0 (cspec) PM2 rule: Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset (applied strength: Supporting).
• gnomAD v4.1: total AF 6.19499e-07 (AC=1, AN=1,614,208, homozygotes=0); exome AF 6.84053e-07 (AC=1, AN=1,461,876).
• gnomAD v4.1 highest subpopulation: European (non-Finnish) AF 8.47435e-07 (AC=1, AN=1,180,032, homozygotes=0).
• gnomAD v2.1 (exomes) and gnomAD-Canada v1.0: variant absent.
• ClinVar submission SCV002154851 (Labcorp Genetics/Invitae) records PM2 with the sentence 'this variant is not present in population databases (gnomad no frequency)' for the exact-match variant.

Sources

Reasoning

The governing specification (ClinGen InSiGHT MSH6 v2.0) defines PS1 as a predicted missense substitution that encodes the same amino acid change as a different nucleotide change previously established by this VCEP as Pathogenic (or a non-canonical splice nucleotide with similar/worse SpliceAI). The case variant NM_000179.3:c.1569_1572del is a frameshift, NP_000170.1:p.(Tyr524ValfsTer46), not a missense substitution, so the same-amino-acid-change premise cannot be satisfied and no same-change comparator missense variant exists. PS1 is therefore not met.

Rule

CSPEC MSH6 v2.0 PS1: same amino-acid change via a different nucleotide change (missense) established as Pathogenic/Likely Pathogenic by this VCEP.

Evidence considered

• ClinGen InSiGHT MSH6 v2.0 PS1 rule requires a predicted missense substitution encoding the same amino acid change as a VCEP-established Pathogenic (or Likely Pathogenic) missense variant.
• Case variant is a frameshift, p.(Tyr524ValfsTer46); it is not a missense substitution, so PS1's core precondition is absent regardless of comparator data.

Sources

Reasoning

ClinGen InSiGHT MSH6 v2.0 PM5 requires a missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic or Likely Pathogenic on the protein level (and only with supporting PP3). The case variant is a frameshift, NM_000179.3:c.1569_1572del / p.(Tyr524ValfsTer46); there is no missense residue context, and the case's own PM5 comparator collection (pm5_candidates) is empty with eligible_for_classic_pm5_search = false because the consequence class is frameshift, not missense. PM5 is therefore not met.

Rule

CSPEC MSH6 v2.0 PM5: missense change at a residue with a VCEP-classified pathogenic missense; only with PP3 supporting.

Evidence considered

• CSPEC MSH6 v2.0 PM5 requires a missense change at an amino acid residue where a different missense change was VCEP-classified Pathogenic (PM5) or Likely Pathogenic (PM5_Supporting).
• pm5_candidates.json: eligible_for_classic_pm5_search = false, total_candidates_seen = 0, reason 'variant consequence class (frameshift) is not missense'.
• No same-residue missense comparator exists for a frameshift allele.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 v2.0 (cspec), which sets BA1 (Benign Stand Alone) at a gnomAD v4 Grpmax filtering allele frequency >= 0.0022 (0.22%) for a variant excluded as a founder pathogenic variant. This variant (NM_000179.3:c.1569_1572del, MSH6, p.Tyr524ValfsTer46) is present in gnomAD v4.1 at a total allele frequency of 6.19e-07 (1/1,614,208 alleles; exome-only AF 6.84e-07 from 1/1,461,876; genome count 0) with 0 homozygotes, and its highest observed subpopulation frequency is European (non-Finnish) at 8.47e-07 (1/1,180,032 alleles, 0 homozygotes). It is absent from gnomAD v2.1 and from gnomAD-Canada v1.0. Every available frequency (total 6.19e-07, popmax 8.47e-07, exome 6.84e-07) is more than 2,500-fold below the 0.0022 stand-alone threshold, and there is no evidence of a founder effect relevant to this variant. The reported Grpmax filtering allele frequency field was null, but a filtering frequency cannot exceed the underlying allele frequency, which is already orders of magnitude below threshold, so the verdict is unaffected. 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

• MSH6 VCEP v2.0 (cspec) BA1 rule: GnomAD v4 Grpmax filtering allele frequency >= 0.0022 (0.22%) and variant excluded as founder pathogenic variant.
• gnomAD v4.1: total AF 6.19499e-07 (AC=1, AN=1,614,208, homozygotes=0); exome AF 6.84053e-07 (AC=1, AN=1,461,876); genome AC=0, AN=0.
• gnomAD v4.1 highest subpopulation: European (non-Finnish), AF 8.47435e-07 (AC=1, AN=1,180,032, homozygotes=0).
• gnomAD v2.1 (exomes): variant absent.
• gnomAD-Canada v1.0: variant absent.

Sources

Reasoning

The MSH6 VCEP v2.0 (cspec) sets BS1 (Benign Strong) at a gnomAD v4 Grpmax filtering allele frequency >= 0.00022 and < 0.0022 (0.022-0.22%) for a variant excluded as a founder pathogenic variant. The variant's gnomAD v4.1 total allele frequency is 6.19e-07 (1/1,614,208 alleles; exome 6.84e-07; 0 homozygotes) and its highest subpopulation frequency is European (non-Finnish) 8.47e-07 (1/1,180,032); it is absent from gnomAD v2.1 and gnomAD-Canada v1.0. All observed frequencies fall roughly 260-fold or more below the lower BS1 bound of 0.00022, so the variant does not reach the BS1 range. The Grpmax filtering allele frequency field was reported as null, but no filtering frequency can exceed the underlying allele frequency, which is already far below the threshold. 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

• MSH6 VCEP v2.0 (cspec) BS1 rule: GnomAD v4 Grpmax filtering allele frequency >= 0.00022 and < 0.0022 (0.022-0.22%) and variant excluded as founder pathogenic variant.
• gnomAD v4.1: total AF 6.19499e-07 (AC=1, AN=1,614,208, homozygotes=0); highest subpopulation European (non-Finnish) AF 8.47435e-07 (AC=1, AN=1,180,032).
• gnomAD v2.1 (exomes) and gnomAD-Canada v1.0: variant absent.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885), which marks PS2 Applicable and grades it on a curated per-proband de novo point total: Very Strong at >=4 points, Strong at 2-3.5 points, Moderate at 1-1.5 points and Supporting at 0.5 point. The MSH6 VCEP assigns 2 points per proband for a confirmed de novo variant (maternity and paternity both confirmed) in a case with an MMR-deficient LS-spectrum tumour, 1 point for a confirmed de novo in a case with an LS-spectrum tumour but no MSI/IHC/methylation data, and 0.5 point for an assumed de novo with maternity and/or paternity unconfirmed. This case is a variant-level review: it contains no proband record, no parental/trio testing, no tumour MMR (MSI/IHC/methylation) data and no statement of de novo origin anywhere in the evidence bundle, the ClinVar submissions (VCV001386337; Labcorp, Myriad, Ambry) or the fetched literature. Because no de novo point total can be computed, PS2 cannot be evaluated and is recorded as not assessed rather than not met.

Rule

MSH6 VCEP v2.0 PS2: >=4 de novo points = Very Strong; 2-3.5 = Strong; 1-1.5 = Moderate; 0.5 = Supporting. Per-proband points: 2 (confirmed de novo plus MMR-deficient LS-spectrum tumour), 1 (confirmed de novo plus LS-spectrum tumour without MSI/IHC/methylation), 0.5 (assumed de novo, parentage unconfirmed).

Evidence considered

• Reviewed the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885): PS2 is Applicable and graded on a curated de novo point total - >=4 points Very Strong, 2-3.5 Strong, 1-1.5 Moderate, 0.5 Supporting.
• MSH6 VCEP v2.0 de novo point rules: 2 points per proband for a confirmed de novo (maternity and paternity confirmed) in a case with an MMR-deficient LS-spectrum tumour; 1 point for a confirmed de novo with an LS-spectrum tumour but no MSI/IHC/methylation data; 0.5 point for an assumed de novo with maternity and/or paternity unconfirmed. LS-spectrum tumours are defined by the VCEP as colorectal/colon/rectal, endometrial, ovarian, small bowel/small intestine, renal pelvis, ureter, stomach/gastric, sebaceous skin and glioma.
• Searched the case bundle, the ClinVar record (VCV001386337) and the fetched literature for a de novo assertion, a proband record or parental testing: none is present. The three ClinVar submitters (Labcorp, Myriad, Ambry) report classifications only, with no de novo or family data.
• PMID:25741868 supplies the generic ACMG/AMP 2015 PS2 concept (de novo with confirmed maternity and paternity in a patient with the disease and no family history), which is the origin of the criterion the MSH6 VCEP re-parameterises into its point scale.
• No criterion was inferred from any source's final classification; the ClinVar Pathogenic labels were not treated as de novo evidence.

Still missing

• No proband-level clinical record for this variant (curation is variant-level only).
• No trio/parental testing to establish maternity and paternity and confirm a de novo occurrence.
• No tumour MSI/IHC/methylation data for any carrier, which the MSH6 VCEP requires to choose the 2-point (MMR-deficient tumour) versus 1-point (no tumour data) de novo score.
• No explicit statement of de novo origin in any ClinVar submission or fetched paper.

Sources

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 v2.0. Its PS3 rule assigns strong evidence at calibrated functional odds for pathogenicity >18.7, moderate evidence at >4.3 and <=18.7 (or an MMR-function defect following the MMR functional-assay flowchart, or complete loss of monoallelic expression <10% of wild-type without puromycin), and supporting evidence at >2.08 and <=4.3. This variant is a frameshift, NP_000170.1:p.(Tyr524ValfsTer46), and the VCEP functional-assay flowchart is scoped to in-frame indel/missense variants and to splice-site/silent/intronic variants; nonsense and frameshift truncating variants are routed to the separate VCEP PVS1 decision tree. No variant-specific calibrated functional-odds result, mammalian MMR activity result, protein-expression/stability result, or mRNA/allelic-expression result for c.1569_1572del was located in the case evidence or in the VCEP functional-assay materials, and all six extracted functional-theme publications were checked and none mentions this variant. With no variant-specific functional measurement, PS3 cannot be assessed.

Rule

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 by the functional-assay flowchart (Moderate), >2.08 and <=4.3 (Supporting).

Evidence considered

• The governing MSH6 VCEP (InSiGHT Hereditary Colorectal Cancer/Polyposis specification v2.0) assigns PS3 at calibrated functional odds for pathogenicity >18.7 (Strong), >4.3 and <=18.7 (Moderate), or >2.08 and <=4.3 (Supporting); an MMR-function defect following the MMR functional-assay flowchart, or complete loss of monoallelic expression (<10% of wild-type in cDNA without puromycin), also qualifies at Moderate strength.
• This variant is a frameshift, NP_000170.1:p.(Tyr524ValfsTer46) (genomic NC_000002.11:g.48026691_48026694del); the VCEP functional-assay flowchart is scoped to in-frame indel/missense and to splice-site/silent/intronic variants, while nonsense/frameshift truncating variants are handled by the VCEP PVS1 decision tree.
• No variant-specific calibrated functional-odds result, mammalian MMR activity assay, protein-expression/stability result, or mRNA/allelic-expression result for c.1569_1572del was present in the case evidence or in the VCEP functional-assay materials.
• All six extracted functional-theme publications were checked and each reports variant_mentioned = false for c.1569_1572del / p.(Tyr524ValfsTer46); their gene-level observations were therefore not transferred to this variant as PS3 evidence.

Still missing

• No variant-specific calibrated MSH6 functional-odds-for-pathogenicity result is available for c.1569_1572del (p.Tyr524ValfsTer46); the VCEP PS3 thresholds (>18.7 / >4.3 / >2.08) therefore cannot be applied.
• No variant-specific MMR activity assay, protein-expression/stability result, or mRNA/monoallelic-expression (NMD-block) result for this variant is reported in the case evidence or in the VCEP functional-assay materials.
• The VCEP functional-assay flowchart and the Functional-assay-SVI-documentation-MMR.xlsx contain no entry for c.1569_1572del, p.Tyr524ValfsTer46, or p.Y524Vfs*46.
• This frameshift's predicted loss of function is adjudicated under the VCEP PVS1 decision tree rather than the functional-assay PS3 path.

Sources

Reasoning

The governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 (version 2.0) redefines PM3 as co-occurrence with a known pathogenic or likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with constitutional mismatch repair deficiency (CMMRD) as per Aronson et al. 2022. Points are summed across qualifying observations: 1.0 point when the second variant is confirmed in trans, 0.5 points when its phase is unknown; the total gives PM3_Supporting (0.5 point), PM3 (1-1.5 points), PM3_Strong (2-3.5 points) or PM3_VeryStrong (>=4 points). This criterion operates in MSH6's autosomal-recessive context, mismatch repair cancer syndrome 1 / CMMRD (MONDO:0010159), while the gene's dominant role is Lynch syndrome (MONDO:0005835). The case variant NM_000179.3:c.1569_1572del (p.Tyr524ValfsTer46) is a frameshift documented in ClinVar (Variation ID 1386337 / VCV001386337) under dominant Lynch-syndrome-spectrum conditions (Hereditary cancer-predisposing syndrome, Hereditary nonpolyposis colorectal neoplasms, Lynch syndrome 5), with no CMMRD or biallelic report. No source in the case records a second pathogenic or likely pathogenic MSH6 allele, an affected proband with CMMRD-consistent clinical features, or any cis/trans phase determination; the six full-text papers available do not mention this variant. With no co-occurring pathogenic/likely pathogenic allele there is no PM3 point to score and therefore no strength can be assigned, so PM3 is not assessed rather than met or excluded.

Rule

ClinGen InSiGHT MSH6 v2.0 PM3: co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with CMMRD-consistent clinical features (Aronson et al. 2022); 1.0 point if in trans, 0.5 points if phase unknown; Supporting = 0.5 point, Moderate = 1-1.5 points, Strong = 2-3.5 points, Very Strong = >=4 points.

Evidence considered

• ClinGen InSiGHT MSH6 v2.0 PM3 instruction: co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD per Aronson et al. 2022; 1.0 point if in trans, 0.5 points if phase unknown; strength = Supporting (0.5 point), Moderate (1-1.5 points), Strong (2-3.5 points), Very Strong (>=4 points).
• MSH6 inheritance context from the framework: Lynch syndrome (MONDO:0005835) is autosomal dominant; mismatch repair cancer syndrome 1 / CMMRD (MONDO:0010159) is autosomal recessive and is the context in which VCEP PM3 is applied.
• ClinVar record for this variant (Variation ID 1386337 / VCV001386337) lists the conditions Hereditary cancer-predisposing syndrome, Hereditary nonpolyposis colorectal neoplasms and Lynch syndrome 5 - the dominant LS spectrum, with 3 clinical laboratories reporting Pathogenic and 1 Likely pathogenic, and no CMMRD or biallelic observation.
• No case source documents a second pathogenic/likely pathogenic MSH6 allele, an affected proband with CMMRD features, or any cis/trans phase result for c.1569_1572del; the six full-text papers reviewed (PMID:24362816, PMID:1651234, PMID:22810696, PMID:24755471, PMID:9111312, PMID:25741868) do not mention this variant and provide no phase or co-occurrence data.

Still missing

• No co-occurring pathogenic or likely pathogenic MSH6 variant and no cis/trans phase determination are documented for c.1569_1572del.
• No affected-proband record with clinical features meeting CMMRD criteria (Aronson et al. 2022) is available for this allele.
• Biallelic (CMMRD) status is untested; the variant is reported only in the dominant Lynch-syndrome context in ClinVar.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885), which marks PP1 Applicable and grades co-segregation on a combined Bayes likelihood ratio across pedigrees: Strong if >18.7 in >=2 families, Moderate if >4.3 and <=18.7, and Supporting if >2.08 and <=4.3; the specification states that results from multiple pedigrees are multiplied together and recommends the COOL (Cosegregation OnLine) v3 tool using its 'overall Bayes Factor'. Evaluating PP1 therefore requires a pedigree with genotyped affected and unaffected relatives from which a cosegregation likelihood ratio can be computed. This case provides none: it is a variant-level review with no proband, no pedigree, no meiosis count and no genotyped relatives, and none of the fetched papers reports this variant in a family. With no segregation data in either direction, no likelihood ratio can be derived and PP1 is recorded as not assessed rather than not met.

Rule

MSH6 VCEP v2.0 PP1: combined Bayes likelihood ratio >18.7 in >=2 families = Strong; >4.3 and <=18.7 = Moderate; >2.08 and <=4.3 = Supporting.

Evidence considered

• Reviewed the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885): PP1 is Applicable and graded on a combined Bayes likelihood ratio - Strong >18.7 in >=2 families, Moderate >4.3 and <=18.7, Supporting >2.08 and <=4.3; results across pedigrees are multiplied and the recommended analysis tool is COOL (Cosegregation OnLine) v3 evaluated by the 'overall Bayes Factor'.
• Searched the case bundle, the ClinVar record and the fetched literature for a genotyped pedigree or any co-segregation statement for this variant: none exists; the assessment is variant-level.
• PMID:24362816 (InSiGHT 5-tier classification of 2,360 MMR variants) reports that MSH6 deleterious variants have lower penetrance and later age of onset and that gene-specific criteria, explicitly including segregation odds, were recommended for MSH6; this provides the rationale for the VCEP's Bayes-likelihood-ratio PP1 thresholds. It reports no data on c.1569_1572del.
• PMID:25741868 supplies the generic ACMG/AMP 2015 PP1 definition (co-segregation with disease in multiple affected family members), which the MSH6 VCEP re-parameterises.
• No criterion was inferred from any source's final classification.

Still missing

• No family pedigree with genotyped affected and unaffected relatives for c.1569_1572del.
• No meiosis count and no combined cosegregation Bayes likelihood ratio.
• No segregation reported in any ClinVar submission or fetched paper.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 v2.0 specification does define PP4, with count-based thresholds: Strong = '≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families' using a standard 5-10 marker panel or tumor genome and/or loss of MMR protein expression consistent with the variant location; Moderate = '2 independent CRC/Endometrial MSI-H tumors'; Supporting = '1 CRC/Endometrial MSI-H tumor'. Writing the Supporting threshold explicitly as an inequality with the observed value substituted: number of documented qualifying MSI-H/MMR-loss tumors (0 documented) ≥ 1? cannot be answered because no tumor testing is reported, so this is an evidence gap rather than a failed comparison. The case evidence bundle contains no tumor MSI status (MSI-H/MSS), no MMR immunohistochemistry (MLH1/PMS2/MSH2/MSH6) result, and no tumor gene-panel data for the proband or family, and the ClinVar submission audit likewise carries no phenotype or tumor fields. PP4 therefore cannot be evaluated and is returned not_assessed.

Rule

CSPEC MSH6 v2.0 PP4: ≥3 (Strong) / 2 (Moderate) / 1 (Supporting) independent CRC or endometrial MSI-H tumors with standard MSI panel or tumor genome and/or loss of MMR protein expression consistent with the variant location.

Evidence considered

• ClinGen InSiGHT MSH6 v2.0 PP4 thresholds quoted literally: Strong '≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families'; Moderate '2 independent CRC/Endometrial MSI-H tumors'; Supporting '1 CRC/Endometrial MSI-H tumor', each requiring a standard 5-10 marker MSI panel 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 at the corresponding strength.
• The case evidence (case_summary, evidence.json, literature_pass, ClinVar submission audit) contains no tumor MSI or MMR-IHC data for this variant; a targeted search of the case output for MSI-H/MSS, tumor, endometrial, colorectal, and phenotype/HPO terms returned no records.
• ClinVar criterion-lead submission SCV002154851 (Labcorp) cites PMID 18269114 and PMID 24362816, neither of which reports tumor MSI/IHC data for this exact variant.

Still missing

• No tumor MSI status (MSI-H vs MSS) and no MMR protein immunohistochemistry (MLH1, PMS2, MSH2, MSH6) results are available for the proband or any family member.
• No tumor-genome MSI classification or number of independent CRC/endometrial tumors is documented, so the PP4 count thresholds (≥3 / 2 / 1) cannot be applied.

Sources

Reasoning

The MSH6 VCEP v2.0 (cspec) defines BS2 as co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or another Lynch-syndrome-spectrum cancer above its median age of onset), in the absence of any previous or current clinical evidence of CMMRD per Aronson et al. 2022, with phase confirmed by testing of parents or offspring. This criterion requires proband-level genotype, phase, and phenotype information. Neither the case evidence bundle, the four exact-match ClinVar submissions (3 Pathogenic, 1 Likely pathogenic), the ClinVar criterion-lead submission (SCV002154851), nor the reviewed publication extracts report a second MSH6 pathogenic variant in trans, parental/offspring phase testing, or a co-occurring CRC/CMMRD phenotype for this variant. There is therefore insufficient evidence to assess BS2, and it is recorded as not_assessed rather than met or not met.

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), without CMMRD, with phase confirmed by testing of parents or offspring.

Evidence considered

• MSH6 VCEP v2.0 (cspec) BS2 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), without previous or current evidence of clinical manifestations of CMMRD (Aronson et al. 2022); confirmation of phase requires testing of parents or offspring.
• ClinVar exact-match submissions for this variant (SCV004188255 Myriad, SCV005032810 Ambry, SCV002154851 Labcorp/Invitae, SCV007911333 Blueprint) provide classifications only, with no reported trans co-occurrence, phase, or CMMRD-relevant phenotype.
• Reviewed publication extracts for the fetched PMIDs do not report a co-occurring pathogenic MSH6 variant in this patient.

Still missing

• No proband genotype/phase information: absence of data on a second (trans) pathogenic MSH6 variant in the same patient.
• No phase confirmation (parental or offspring testing) reported for any co-occurring variant.
• No clinical phenotype (colorectal cancer after age 45, other LS-spectrum cancer, or CMMRD features) reported for a carrier of this variant.

Sources

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MSH6 v2.0. Its BS3 rule assigns strong evidence at calibrated functional odds for pathogenicity <=0.05 (or for synonymous/intronic variants with no mRNA aberration on NMD-inhibited assays), and supporting evidence at >0.05 and <=0.48, or for variant-specific proficient function in protein- and mRNA-based laboratory assays following the MMR functional-assay flowchart. This variant is a frameshift, NP_000170.1:p.(Tyr524ValfsTer46), and the VCEP functional-assay flowchart is scoped to in-frame indel/missense variants and to splice-site/silent/intronic variants; nonsense and frameshift truncating variants are routed to the separate VCEP PVS1 decision tree. No variant-specific calibrated functional-odds result or variant-specific proficient-function result for c.1569_1572del was located in the case evidence or in the VCEP functional-assay materials, and all six extracted functional-theme publications were checked and none mentions this variant. With no variant-specific functional measurement, BS3 cannot be assessed.

Rule

MSH6 VCEP v2.0 BS3: calibrated functional assays with functional odds for pathogenicity <=0.05 (Strong); >0.05 and <=0.48, or variant-specific proficient function by protein/mRNA assays per the MMR functional-assay flowchart (Supporting).

Evidence considered

• The governing MSH6 VCEP (InSiGHT Hereditary Colorectal Cancer/Polyposis specification v2.0) assigns BS3 at calibrated functional odds for pathogenicity <=0.05 (Strong), or variant-specific proficient function in protein- and mRNA-based laboratory assays following the MMR functional-assay flowchart, and at >0.05 and <=0.48 (Supporting).
• The variant is a frameshift, NP_000170.1:p.(Tyr524ValfsTer46); the functional-assay flowchart path used for BS3 covers in-frame indel/missense and splice/silent/intronic variants, and frameshift truncating variants are handled by the VCEP PVS1 decision tree instead.
• No variant-specific calibrated functional-odds result or variant-specific protein/mRNA proficient-function result for c.1569_1572del was present in the case evidence or in the VCEP functional-assay materials.
• All six extracted functional-theme publications were checked and each reports variant_mentioned = false for c.1569_1572del / p.(Tyr524ValfsTer46); their gene-level observations were therefore not transferred to this variant as BS3 evidence.

Still missing

• No variant-specific calibrated MSH6 functional-odds-for-pathogenicity result is available for c.1569_1572del (p.Tyr524ValfsTer46); the VCEP BS3 thresholds (<=0.05 / <=0.48) therefore cannot be applied.
• No variant-specific protein- and mRNA-based proficient-function assay result (per the MMR functional-assay flowchart) is reported for this variant in the case evidence or in the VCEP functional-assay materials.
• The VCEP functional-assay flowchart and the Functional-assay-SVI-documentation-MMR.xlsx contain no entry for c.1569_1572del, p.Tyr524ValfsTer46, or p.Y524Vfs*46.
• A frameshift truncation is not a variant class producing normal protein/mRNA function, but benign functional classification still requires a variant-specific assay result, which is absent.

Sources

Reasoning

Under the governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885), BS4 is Applicable and graded on the same combined Bayes likelihood ratio as PP1 but in the opposite direction: Strong if <0.05, and Supporting if >0.05 and <=0.48. Evaluating it requires a pedigree in which the variant demonstrably fails to track with disease, i.e. genotyped affected relatives who lack the variant or unaffected carriers, together with the specification's caveats that phenocopies for common phenotypes such as cancer can mimic lack of segregation and that a family may carry more than one pathogenic variant. This case contains no proband, no pedigree and no genotyped relatives, so no non-segregation likelihood ratio can be derived. The ClinVar submissions carry classifications only, and no fetched paper reports this variant in a family. BS4 is recorded as not assessed rather than met.

Rule

MSH6 VCEP v2.0 BS4: combined Bayes likelihood ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting.

Evidence considered

• Reviewed the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification for MSH6 v2.0 (CSPEC doc 1578294885): BS4 is Applicable and graded on a combined Bayes likelihood ratio - Strong <0.05, Supporting >0.05 and <=0.48; the accompanying caveats note that phenocopies for common phenotypes (cancer) can mimic lack of segregation and that families may have more than one pathogenic variant contributing to the disorder.
• Searched the case bundle, the ClinVar record and the fetched literature for genotyped affected non-carriers or any non-segregation statement for this variant: none exists; the assessment is variant-level.
• PMID:25741868 supplies the generic ACMG/AMP 2015 BS4 definition (lack of segregation in affected family members), which the MSH6 VCEP re-parameterises.
• No criterion was inferred from any source's final classification.

Still missing

• No genotyped multiply-affected family in which c.1569_1572del fails to segregate with disease.
• No genotype data for relatives of any carrier and no meiosis count in either direction.
• No non-segregation statement in any ClinVar submission or fetched paper.

Sources

Reasoning

The governing ClinGen InSiGHT MSH6 v2.0 specification does define BP5, with count-based thresholds: Strong = '≥4 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) inconsistent with the gene demonstrating genetic variation', OR '≥2 BRAF V600E (CRC only)/MLH1 methylation with MSI-H/MLH1 loss'; Supporting = '2 or 3 tumors' meeting the MSS/no-loss/inconsistent-expression condition, OR '1 BRAF V600E/MLH1 methylation with MSI-H/MLH1 loss'. Writing the Supporting threshold explicitly as an inequality with the observed value substituted: number of documented qualifying (MSS / no MMR loss / inconsistent expression) tumors (0 documented) ≥ 2? cannot be answered because no tumor MSI, MMR-IHC, BRAF V600E, or MLH1 methylation data are reported for this case. This is an evidence gap, not a failed comparison, so BP5 is returned not_assessed.

Rule

CSPEC MSH6 v2.0 BP5: ≥4 (Strong) / 2-3 (Supporting) tumors with MSS and/or no MMR protein loss and/or MMR loss inconsistent with the variant gene, or BRAF V600E/MLH1 methylation with MSI-H/MLH1 loss.

Evidence considered

• ClinGen InSiGHT MSH6 v2.0 BP5 thresholds quoted literally: Strong '≥4 tumors ... MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with loss of MMR protein(s) inconsistent with the gene demonstrating genetic variation' OR '≥2 BRAF V600E (CRC only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss'; Supporting '2 or 3 tumors ...' OR '1 BRAF V600E (Colon only)/MLH1 methylation with MSI-H/MLH1 loss'.
• The case evidence bundle and the ClinVar submission audit contain no tumor MSI, MMR-IHC, BRAF V600E, or MLH1 methylation data for this variant, so no benign tumor-phenotype evidence is documented.
• A targeted search of the case output for MSI-H/MSS, tumor, endometrial, colorectal, and phenotype terms returned no records.

Still missing

• No tumor MSS/MSI status, MMR protein immunohistochemistry, BRAF V600E, or MLH1 promoter methylation results are available for this case.
• Because no tumor data exist, neither the BP5 Strong (≥4 tumors; or ≥2 BRAF V600E/MLH1-methylation) nor the BP5 Supporting (2 or 3 tumors; or 1 BRAF V600E/MLH1-methylation) threshold can be applied.

Sources