MSH6 c.1019T>C, p.(Phe340Ser)

Reasoning

The variant is a missense substitution, and the governing MSH6 HCI-PRIORS-MSH6 table contains the exact c.1019T>C / p.F340S entry with HCI prior probability 0.0019. The MSH6 VCEP directly assigns BP4 supporting when the HCI prior probability is <0.11; 0.0019 satisfies that rule. This governing assignment takes precedence over generic predictor thresholds. SpliceAI is not used for missense BP4, and BayesDel has no applicable MSH6 cutoff in the governing specification.

Rule

Missense variant with HCI-prior probability of pathogenicity <0.11, as specified by the MSH6 VCEP.

Evidence considered

• The governing MSH6 HCI-PRIORS-MSH6 table was searched for c.1019T>C, p.Phe340Ser, and p.F340S and contains the exact entry with HCI prior probability 0.0019.
• The MSH6 VCEP specification assigns BP4 supporting to a missense variant with HCI prior probability <0.11; the observed value 0.0019 satisfies this criterion.
• Because this is a missense variant, SpliceAI no-impact evidence is not used for BP4; the governing HCI assignment is applied directly.

Sources

Reasoning

Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Likely benign.

Rule

BP6 supporting: exact-variant ClinVar expert-panel classification of Benign or Likely benign.

Evidence considered

• ClinVar records the exact NM_000179.3:c.1019T>C (p.Phe340Ser) variant as Likely benign, reviewed by the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) expert panel with three stars.
• BP6 was applied only from this exact-variant expert-panel assertion; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions were excluded.
• ClinVar expert panel classification

Sources

Reasoning

The applicable InSiGHT MSH6 specification requires a different nucleotide change producing the same amino-acid substitution that this VCEP established as Pathogenic, or a qualifying splice comparison. The reviewed evidence concerns NM_000179.3:c.1019T>C, p.Phe340Ser itself; no different nucleotide change producing p.Phe340Ser established as Pathogenic by the VCEP was identified. The exact variant was reported as likely benign by the expert panel, and the relevant papers do not provide a qualifying PS1 comparator.

Rule

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).

Evidence considered

• The MSH6 VCEP PS1 rule requires the same amino-acid change from a different nucleotide change and prior VCEP establishment as Pathogenic, excluding predicted or confirmed splice defects.
• The reviewed records and papers identify the target as c.1019T>C, p.Phe340Ser; they do not identify a different nucleotide change producing p.Phe340Ser that was established as Pathogenic by the VCEP.
• The exact variant is reported as Likely benign by the InSiGHT expert panel, which is not qualifying PS1 evidence for a different nucleotide comparator.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP specifies PM2_Supporting for an allele frequency <0.00002 in the gnomAD v4 dataset. The default all-comers gnomAD v4.1 allele frequency is 9.10722e-05 (147/1,614,104 alleles), with zero homozygotes, which exceeds the VCEP cutoff. The gnomAD v2.1 and v3.1 non-cancer subset frequencies are also above 0.00002, so PM2 is not met.

Rule

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

Evidence considered

• The InSiGHT MSH6 VCEP Version 2.0 defines PM2_Supporting as absent or extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.
• gnomAD v4.1 reports 147 alternate alleles among 1,614,104 alleles, AF 9.10722e-05, and zero homozygotes; this is above 0.00002.
• The non-cancer subsets also exceed the VCEP cutoff: gnomAD v2.1 non-cancer exomes AF 3.80087e-05 and gnomAD v3.1 non-cancer genomes AF 6.0819e-05.
• Plaschke et al. reported the variant absent from 246 healthy-control chromosomes, but that historical small control observation does not satisfy the VCEP's required gnomAD v4 threshold.

Sources

Reasoning

The MSH6 VCEP requires a combined cosegregation Bayes likelihood ratio >2.08 for PP1 Supporting, >4.3 and ≤18.7 for Moderate, or >18.7 in at least two families for Strong. For this exact variant, the multifactorial analysis reports a segregation likelihood ratio of 1.000, which is below the VCEP Supporting threshold; the source also reports no family history, no siblings, and no descendants in the reported patient. Thus PP1 is not met.

Rule

MSH6 InSiGHT VCEP v2.0 PP1: combined Bayes likelihood ratio >2.08–≤4.3 = Supporting; >4.3–≤18.7 = Moderate; >18.7 in ≥2 families = Strong.

Evidence considered

• The MSH6 VCEP PP1 thresholds are combined Bayes likelihood ratio >2.08 and ≤4.3 for Supporting, >4.3 and ≤18.7 for Moderate, and >18.7 in ≥2 families for Strong.
• For MSH6 c.1019T>C p.(Phe340Ser), the multifactorial likelihood table reports a segregation likelihood ratio of 1.000.
• The original case report describes patient KN18 as having no family history and no siblings or descendants, providing no additional informative pedigree for cosegregation.

Still missing

• No informative affected or unaffected relatives with genotypes are available to establish additional cosegregation

Sources

Reasoning

The variant is a missense substitution, so the applicable computational path is the missense path. The governing MSH6 HCI lookup contains c.1019T>C / p.F340S with HCI prior probability 0.0019. Under the MSH6 VCEP specification, PP3 requires an HCI prior probability >0.68 for supporting or >0.81 for moderate; 0.0019 meets neither threshold. SpliceAI is not used to support or refute PP3 for this missense variant, and the available REVEL score of 0.54 does not provide a governing VCEP PP3 assignment.

Rule

Missense variant with HCI prior probability for pathogenicity >0.81 for PP3_Moderate, or >0.68 and <=0.81 for PP3_Supporting, as specified by the MSH6 VCEP.

Evidence considered

• VariantValidator identifies NM_000179.3:c.1019T>C as the missense variant NP_000170.1:p.(Phe340Ser) / p.(F340S).
• The governing MSH6 HCI-PRIORS-MSH6 table was searched for c.1019T>C, p.Phe340Ser, and p.F340S and contains the exact entry with HCI prior probability 0.0019.
• The MSH6 VCEP specification assigns PP3 supporting at HCI prior probability >0.68 and PP3 moderate at >0.81; the observed value 0.0019 meets neither threshold.
• REVEL is available at 0.54, but no governing MSH6 PP3 code is assigned from that score; SpliceAI is not used for missense PP3/BP4 evaluation.

Sources

Reasoning

The MSH6 VCEP requires at least one CRC or endometrial MSI-H tumor assessed with a standard 5-10-marker panel or tumor genome, and/or MMR-protein loss consistent with the variant location. The exact-variant report describes only MSI at BAT40, not MSI-H by a standard panel, and immunostaining was positive for hMSH6, hMSH2, and hMLH1 rather than showing consistent MMR-protein loss. Thus the PP4 supporting rule is not met.

Rule

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

Evidence considered

• The MSH6 VCEP supporting rule requires 1 CRC or endometrial MSI-H tumor using a standard 5-10-marker panel or tumor genome and/or MMR-protein loss consistent with the variant location; inconsistent protein expression does not meet PP4.
• The exact-variant report describes a colorectal tumor with MSI at BAT40 only and positive immunostaining for hMSH2, hMLH1, and hMSH6; it does not document MSI-H by the specified standard panel or consistent loss of MSH6 protein.

Sources

Reasoning

The exact-variant ClinVar record has an InSiGHT expert-panel classification of Likely benign, not Pathogenic or Likely pathogenic. The available ordinary laboratory submissions and literature classifications do not qualify for PP5, so no pathogenic expert-panel assertion supports this criterion.

Rule

PP5 supports only an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic; ordinary laboratory submissions, aggregate labels, and non-expert assertions do not qualify.

Evidence considered

• ClinVar identifies the exact NM_000179.3:c.1019T>C (p.Phe340Ser) variant as Likely benign with a three-star InSiGHT expert-panel review; no exact-variant Pathogenic or Likely pathogenic expert-panel classification is present.
• Non-expert laboratory submissions, aggregate labels, and computational or publication classifications were not used to trigger PP5.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP specifies BA1 when gnomAD v4 Grpmax filtering allele frequency is >=0.0022, provided the variant is not a founder pathogenic variant. The default all-comers gnomAD v4 Grpmax filtering allele frequency is 9.857e-05 (joint), far below 0.0022; the available non-cancer values are also below this threshold (gnomAD v2.1 non-cancer exomes AF 3.80087e-05 and gnomAD v3.1 non-cancer genomes AF 6.0819e-05). Therefore BA1 is not met. No founder-pathogenic designation is documented, but that does not alter the frequency result.

Rule

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

Evidence considered

• The InSiGHT MSH6 VCEP Version 2.0 defines BA1 as gnomAD v4 Grpmax filtering allele frequency >=0.0022 (0.22%), excluding founder pathogenic variants.
• gnomAD v4.1 reports joint Grpmax filtering allele frequency 9.857e-05 for 147/1,614,104 alleles, with zero homozygotes.
• The non-cancer subsets do not approach the BA1 threshold: gnomAD v2.1 non-cancer exomes AF 3.80087e-05 and gnomAD v3.1 non-cancer genomes AF 6.0819e-05.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP specifies BS1_Strong when gnomAD v4 Grpmax filtering allele frequency is >=0.00022 and <0.0022, provided the variant is not a founder pathogenic variant. The all-comers gnomAD v4 joint Grpmax filtering allele frequency is 9.857e-05, below the lower bound of 0.00022; the non-cancer subset values are likewise below that bound. BS1 is therefore not met.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.00022 and < 0.0022 (0.022-0.22%) and variant is excluded as founder pathogenic variant.

Evidence considered

• The InSiGHT MSH6 VCEP Version 2.0 defines BS1_Strong as gnomAD v4 Grpmax filtering allele frequency >=0.00022 and <0.0022, excluding founder pathogenic variants.
• gnomAD v4.1 reports joint Grpmax filtering allele frequency 9.857e-05 for this variant, below 0.00022.
• The non-cancer subset observations are also below the VCEP BS1 lower bound: gnomAD v2.1 non-cancer exomes AF 3.80087e-05 and gnomAD v3.1 non-cancer genomes AF 6.0819e-05.

Sources

Reasoning

The MSH6 VCEP requires a combined cosegregation Bayes likelihood ratio <0.05 for BS4 Strong or >0.05 and ≤0.48 for BS4 Supporting. The exact-variant multifactorial analysis reports a segregation likelihood ratio of 1.000, not a likelihood ratio within either BS4 range, and the reported case provides no informative non-segregation pedigree. Therefore BS4 is not met.

Rule

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

Evidence considered

• The MSH6 VCEP BS4 thresholds are combined Bayes likelihood ratio <0.05 for Strong and >0.05 and ≤0.48 for Supporting.
• For MSH6 c.1019T>C p.(Phe340Ser), the multifactorial likelihood table reports a segregation likelihood ratio of 1.000, which falls outside both BS4 ranges.
• The original case report describes no family history, siblings, or descendants, so it does not provide an informative non-segregation pedigree.

Still missing

• No informative unaffected relatives carrying the variant or affected relatives lacking the variant are documented

Sources

Reasoning

The MSH6 VCEP BP5 rule requires two or three qualifying tumors for supporting evidence: CRC or endometrial tumors with MSS and/or no MMR-protein loss, or LS-spectrum tumors with MMR-protein loss inconsistent with MSH6; alternatively, one BRAF V600E or MLH1-methylated MSI-H tumor under the specified conditions. The exact-variant report provides one colorectal tumor with MSI at BAT40 only and positive MSH6 staining, not a qualifying MSS/no-loss or alternative-molecular-basis pattern. No BRAF V600E or MLH1 methylation evidence is reported.

Rule

Supporting: 2 or 3 qualifying tumors with MSS and/or no MMR-protein loss and/or inconsistent MMR loss, or 1 BRAF V600E/MLH1-methylated tumor with MSI-H and MLH1 loss.

Evidence considered

• The MSH6 VCEP supporting BP5 rule requires 2 or 3 qualifying tumors with MSS and/or no MMR-protein loss, or one BRAF V600E/MLH1-methylated tumor with MSI-H and MLH1 loss.
• The exact-variant report describes one colorectal tumor with MSI at BAT40 only and positive hMSH6 immunostaining; it reports no BRAF V600E or MLH1 methylation result.

Sources

Reasoning

The MSH6 VCEP specifies PS2 by de novo point count: 0.5 points is Supporting, 1–1.5 Moderate, 2–3.5 Strong, and at least 4 Very Strong. The available case and literature evidence document no trio testing, parental genotypes, confirmed de novo observation, maternity/paternity confirmation, or other de novo point-bearing observation for c.1019T>C (p.Phe340Ser), so PS2 cannot be assessed.

Rule

MSH6 InSiGHT VCEP v2.0 PS2: ≥4 de novo points = Very Strong; 2–3.5 = Strong; 1–1.5 = Moderate; 0.5 = Supporting.

Evidence considered

• The MSH6 VCEP PS2 rule assigns Supporting strength at 0.5 de novo points, Moderate at 1–1.5, Strong at 2–3.5, and Very Strong at ≥4 points.
• No de novo event, parental genotype result, or confirmed trio observation is reported in the available case evidence for c.1019T>C (p.Phe340Ser).

Still missing

• Trio or parental testing demonstrating the variant is absent in both biological parents
• Confirmation of maternity and paternity if applying a confirmed de novo point model

Sources

Reasoning

The governing InSiGHT MSH6 VCEP requires calibrated functional odds greater than 18.7 for PS3_Strong, greater than 4.3 and up to 18.7 for PS3_Moderate, or greater than 2.08 and up to 4.3 for PS3_Supporting. For p.Phe340Ser, the available variant-specific publication reports an in-vitro "Wild type" result but does not provide calibrated functional odds or sufficient documentation of a validated protein-function assay with the assay controls and calibration required by the VCEP functional-assay framework. The reported result therefore cannot establish a PS3 strength.

Rule

Calibrated functional assays with functional odds for pathogenicity >18.7 = PS3_Strong; >4.3 and <=18.7 = PS3_Moderate; >2.08 and <=4.3 = PS3_Supporting.

Evidence considered

• The MSH6 VCEP specifies PS3_Strong for calibrated functional odds for pathogenicity >18.7, PS3_Moderate for >4.3 to <=18.7, and PS3_Supporting for >2.08 to <=4.3.
• The variant-specific report lists MSH6 c.1019T>C p.(Phe340Ser) with an in-vitro result of "Wild type," but does not report calibrated functional odds or sufficient protein-assay validation and control details to assign PS3.

Still missing

• A calibrated MSH6 functional assay with quantitative functional odds is not available.
• A validated protein-function assay with documented wild-type, deficient, and pathogenic controls is not available for this variant.

Sources

Reasoning

The MSH6 InSiGHT VCEP specifies PM3 by point-counting observations of the variant in affected individuals with a pathogenic or likely pathogenic MSH6 variant in trans, with strengths of Supporting at 0.5 points, Moderate at 1–1.5 points, Strong at 2–3.5 points, and Very Strong at ≥4 points. The available exact-variant report describes one patient with MSH6 p.Phe340Ser and tumor loss of the wild-type allele, but does not document a second pathogenic MSH6 variant, confirmed trans phase, or a qualifying biallelic affected-proband observation. Therefore PM3 cannot be assigned from the available evidence.

Rule

MSH6 InSiGHT VCEP PM3: ≥4 points Very Strong; 2–3.5 points Strong; 1–1.5 points Moderate; 0.5 points Supporting.

Evidence considered

• The MSH6 InSiGHT VCEP PM3 rule assigns strength from point counts: 0.5 points Supporting, 1–1.5 Moderate, 2–3.5 Strong, and ≥4 Very Strong.
• The exact-variant report describes F340S in one 75-year-old woman with colorectal cancer and reports loss of the wild-type allele in the tumor, but does not report a second pathogenic MSH6 variant or confirmed trans phase.
• Tumor loss of the wild-type allele is not, by itself, evidence that the germline variant was observed in trans with a pathogenic MSH6 allele in an affected proband.

Still missing

• A qualifying affected individual with MSH6 p.Phe340Ser and a pathogenic or likely pathogenic MSH6 variant in trans is not documented.
• Confirmed phase and the number of independent qualifying observations are unavailable.

Sources

Reasoning

The governing MSH6 PM5 rule requires a different missense change at the same amino-acid residue classified by this VCEP as Pathogenic or Likely Pathogenic on the protein level and not because of aberrant splicing; it also requires PP3 Supporting for the target missense change. The target is p.Phe340Ser, but the collected same-residue comparator search found no usable candidates, and the reviewed paper extracts identify the target variant rather than a qualifying alternate missense change at residue 340. Therefore PM5 cannot be adjudicated from the available evidence.

Rule

Missense change at an amino acid residue where a different missense change was classified as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change.

Evidence considered

• The MSH6 VCEP PM5 rule requires a different missense change at the same residue classified by the VCEP as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing; PM5 also requires PP3 Supporting for the target missense change.
• The collected PM5 candidate record reports zero same-residue candidates and no expert-panel or non-expert majority Pathogenic/Likely Pathogenic comparator candidates, with retrieval limited by an HTTP 429 error.
• The reviewed papers mention or evaluate the target p.Phe340Ser but do not provide a qualifying alternate missense comparator at residue 340.

Still missing

• A curated same-residue search for alternate MSH6 missense variants at p.Phe340 with VCEP Pathogenic or Likely Pathogenic classification is still needed.

Sources

Reasoning

The MSH6 VCEP requires confirmed 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-spectrum cancer above the median age of onset, plus no evidence of CMMRD and parental or offspring confirmation of phase. The reviewed case evidence provides no qualifying co-occurrence, phase confirmation, or CMMRD assessment, so BS2 cannot be adjudicated.

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 another Lynch-spectrum cancer above the median age of onset), without evidence of CMMRD; phase requires parental or offspring testing.

Evidence considered

• The InSiGHT MSH6 VCEP BS2 rule requires confirmed in-trans co-occurrence with a known pathogenic sequence variant in the same gene, qualifying cancer age or tumor context, no CMMRD manifestations, and phase confirmation through parents or offspring.
• No qualifying co-occurrence, phase, or CMMRD evidence is available in the reviewed case materials.

Still missing

• A documented patient with this variant and a known pathogenic MSH6 sequence variant in confirmed trans phase.
• Qualifying cancer age or Lynch-spectrum cancer context and documented absence of CMMRD manifestations.
• Parental or offspring testing confirming phase.

Sources

Reasoning

The governing InSiGHT MSH6 VCEP requires calibrated functional odds for pathogenicity <=0.05 for BS3_Strong, or >0.05 and <=0.48 for BS3_Supporting; alternatively, variant-specific proficient function must be demonstrated in protein and mRNA-based assays according to the MMR functional-assay flowchart. The available report lists an in-vitro "Wild type" result for p.Phe340Ser, but the extracted assay context indicates an in-vitro splicing analysis rather than a documented calibrated protein-function assay, and it does not establish the concordant protein and mRNA evidence or controls required by the flowchart. Therefore BS3 cannot be assigned.

Rule

Calibrated functional assays with functional odds for pathogenicity <=0.05 = BS3_Strong; >0.05 and <=0.48 = BS3_Supporting; alternatively, variant-specific proficient function in protein and mRNA-based assays as per the MMR functional assay flowchart.

Evidence considered

• The MSH6 VCEP specifies BS3_Strong for calibrated functional odds for pathogenicity <=0.05 and BS3_Supporting for >0.05 and <=0.48, or variant-specific proficient function in protein and mRNA-based assays following the MMR functional-assay flowchart.
• The variant-specific report lists p.Phe340Ser as "Wild type" in its in-vitro column, but the available methods describe in-vitro splicing analysis and do not document calibrated protein-function results, quantitative functional odds, or the concordant assay controls required to apply BS3.

Still missing

• No calibrated functional odds for pathogenicity are reported for p.Phe340Ser.
• A documented variant-specific protein assay with appropriate wild-type, deficient, and pathogenic controls is not available.
• The available in-vitro result does not establish the VCEP flowchart requirement for concordant proficient protein and mRNA function.

Sources