MLH1 c.350C>T, p.Thr117Met

Reasoning

PS3_Moderate is supported. The exact MLH1 p.T117M variant was tested in the human 293T expression/complementation system described by Trojan et al. (PMID:11781295). The study reported that extracts expressing T117M were MMR deficient, while wild-type MLH1 restored repair to levels comparable with MMR-proficient controls; the study also describes confirmation in a second in-vitro MMR assay. This is a variant-specific MMR function defect that follows the VCEP functional-assay flowchart, but the paper does not provide a calibrated functional odds value, so PS3_Strong cannot be assigned. The VCEP assay documentation was searched for c.350C>T, p.Thr117Met, and p.T117M; no variant-specific pre-assigned code or calibrated odds entry was found in Functional-assay-SVI-documentation-MMR.xlsx or Functional-assay-flowchart.pdf.

VCEP rule

Calibrated functional assays with functional odds for pathogenicity >4.3 and <= 18.7 ... OR MMR function defect following functional assay flowchart* ... OR Variants with monoallelic expression: complete loss of expression (<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block.

Evidence considered

• Trojan et al., Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system (PMID:11781295), Figure 3B/results: the exact T117M construct was expressed in 293T cells and the resulting extracts were reported as MMR deficient; wild-type MLH1 restored repair to 86.5% ± 3.9% in the described assay.
• The paper describes confirmation of the result by testing nucleotide-excision repair in a different in-vitro MMR assay, supporting concordant functional-defect evidence under the VCEP flowchart.
• Functional-assay-SVI-documentation-MMR.xlsx, MMR assays sheet, rows 'Assay (general description)', 'Validation controls P/LP (#)', 'Validation controls B/LB (#)', 'Threshold for abnormal readout', and 'Proposed strength': the VCEP-approved assay documentation identifies calibrated MMR assays and their control/threshold framework, but contains no row keyed to c.350C>T, p.Thr117Met, or p.T117M.
• Functional-assay-flowchart.pdf, page 1, Branch 1: for an in-frame or missense variant with a non-calibrated assay, concordant functional assays leading to a MMR function defect support the deficient-function pathway; no variant-specific row is present.

Still missing

• No variant-specific entry for c.350C>T, p.Thr117Met, or p.T117M was found in Functional-assay-SVI-documentation-MMR.xlsx.
• No variant-specific entry for c.350C>T, p.Thr117Met, or p.T117M was found in Functional-assay-flowchart.pdf.
• PMID:11781295 does not report a calibrated functional odds value, so the VCEP Strong functional-odds threshold cannot be evaluated.
• The scanned PMID:11781295 paper required OCR and its historical assay validation is less directly calibrated than the VCEP-listed assays; human review is retained.

Sources

Reasoning

The MLH1 VCEP PM2 rule assigns supporting evidence when the allele frequency is below 0.00002 in gnomAD v4. gnomAD v4.1 reports MLH1 c.350C>T at AF 7.43483e-06 (12/1,614,024 alleles), with Grpmax FAF 5.53e-06 and no homozygotes; the observed allele frequency is below 0.00002. The VCEP-pilot-variants---MMR.xlsx file was searched for c.350C>T, p.Thr117Met, and p.T117M and contains no exact row; therefore no pre-assigned code overrides the gnomAD result.

VCEP rule

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

Evidence considered

• gnomAD v4.1: MLH1 c.350C>T has AF 7.43483e-06 (12/1,614,024 alleles), Grpmax FAF 5.53e-06, and homozygotes 0.
• The gnomAD v2.1 non-cancer exome result is also rare at AF 4.22208e-06 (1/236,850 alleles; homozygotes 0), but the governing VCEP PM2 rule specifically names gnomAD v4, so the v4 value is decisive.
• The searched VCEP-pilot-variants---MMR.xlsx reference file has no exact row for c.350C>T, p.Thr117Met, or p.T117M; its c.350C>G p.Thr117Arg row was not treated as the same variant.

Still missing

• VCEP-pilot-variants---MMR.xlsx was searched for c.350C>T, p.Thr117Met, and p.T117M; no exact variant row was found.

Sources

Reasoning

The variant is a missense substitution, NM_000249.4:c.350C>T (p.Thr117Met/p.T117M). The governing MLH1 HCI-prior table contains the exact c.350C>T row, with pathogenicity probability 0.893. The VCEP rule assigns PP3_Moderate to a missense variant with HCI prior probability >0.81; therefore PP3 is met at Moderate strength. The directly governing table entry outranks any independent predictor interpretation, and no other computational prediction is counted for PP3.

VCEP rule

Missense variant with HCI prior probability for pathogenicity >0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS)

Evidence considered

• HCI-PRIORS-MLH1.txt, exact row for c.350C>T / p.T117M: pathogenicity probability = 0.893, MAPP score = 22.95, custom PP2 score = 0.988; row identifier MLH1_00283.
• The VCEP CSPEC entry for PP3 assigns Moderate strength when a missense variant has HCI prior probability >0.81.

Sources

Reasoning

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

VCEP rule

Not Applicable

Evidence considered

• MLH1 Version 2.0 CSPEC entry for PP5: Not Applicable.
• ClinVar expert-panel classification was not used as PP5 because the governing VCEP marks PP5 Not Applicable.
• ClinVar expert panel classification

Sources

Reasoning

The VCEP pilot table provides a same-residue comparator, MLH1 c.350C>G (p.Thr117Arg), classified Likely pathogenic and due to a missense alteration, which would support PM5_Supporting rather than PM5. However, the governing PM5 rule requires PP3 at Supporting strength before either PM5 path may be used. The case's VCEP HCI prior for c.350C>T is 0.893; the VCEP PP3 rule places values >0.81 at Moderate, not Supporting. Therefore the required PP3_Supporting prerequisite is not met and PM5 is not met.

VCEP rule

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

Evidence considered

• The VCEP pilot-variant table lists NM_000249.4(MLH1):c.350C>G (p.Thr117Arg) as Likely pathogenic with PM5 and identifies it as a missense comparator at residue 117.
• The HCI-PRIORS-MLH1 table row for c.350C>T / p.T117M reports HCI prior probability 0.893, MAPP score 22.95, and custom PP2 score 0.988.
• Under the VCEP PP3 rule, an HCI prior >0.81 is Moderate, whereas Supporting requires >0.68 and <=0.81; thus the available 0.893 prior does not establish PP3 Supporting, which the PM5 rule requires.

Sources

Reasoning

The MLH1 VCEP BA1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.001 (0.1%) and exclusion as a founder pathogenic variant. gnomAD v4.1 reports a Grpmax FAF of 5.53e-06, with 12/1,614,024 total alleles and no homozygotes; this is below 0.001. No governing-table entry for the exact c.350C>T / p.Thr117Met / p.T117M variant overrides this result.

VCEP rule

GnomAD v4 Grpmax filtering allele frequency ≥ 0.001 (0.1%) and variant is excluded as founder pathogenic variant.

Evidence considered

• gnomAD v4.1: MLH1 c.350C>T has Grpmax FAF 5.53e-06; total allele count 12/1,614,024; homozygotes 0.
• The searched VCEP-pilot-variants---MMR.xlsx reference file has no exact row for c.350C>T, p.Thr117Met, or p.T117M; its c.350C>G p.Thr117Arg row is a different nucleotide substitution and was not used as precedent.

Sources

Reasoning

The MLH1 VCEP BS1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.0001 and below 0.001 (0.01-0.1%) and exclusion as a founder pathogenic variant. gnomAD v4.1 reports a Grpmax FAF of 5.53e-06, below 0.0001, so BS1 is not met. No governing-table entry for the exact c.350C>T / p.Thr117Met / p.T117M variant overrides this result.

VCEP rule

GnomAD v4 Grpmax filtering allele frequency ≥ 0.0001 and < 0.001 (0.01-0.1%) and variant is excluded as founder pathogenic variant.

Evidence considered

• gnomAD v4.1: MLH1 c.350C>T has Grpmax FAF 5.53e-06; total allele count 12/1,614,024; homozygotes 0.
• The searched VCEP-pilot-variants---MMR.xlsx reference file has no exact row for c.350C>T, p.Thr117Met, or p.T117M; its c.350C>G p.Thr117Arg row is a different nucleotide substitution and was not used as precedent.

Sources

Reasoning

BS3 is not met. The available variant-specific functional evidence points to deficient rather than proficient MMR function: T117M was reported as MMR deficient in the human expression/complementation assay. The VCEP BS3 rule requires calibrated functional odds for pathogenicity <= 0.05 or variant-specific proficient function in protein and mRNA-based assays; neither is present, and the observed assay direction is opposite to proficient function. The VCEP assay documentation was searched for c.350C>T, p.Thr117Met, and p.T117M; no variant-specific pre-assigned benign code or calibrated functional-odds entry was found in Functional-assay-SVI-documentation-MMR.xlsx or Functional-assay-flowchart.pdf.

VCEP rule

Calibrated functional assays with functional odds for Pathogenicity <= 0.05 ... OR Variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart*.

Evidence considered

• Trojan et al., Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system (PMID:11781295), Figure 3B/results: extracts expressing T117M were reported as MMR deficient, not proficient.
• Functional-assay-SVI-documentation-MMR.xlsx, MMR assays sheet: the approved assay table defines calibrated functional assay thresholds and proposed PS3/BS3 strengths, but no row is keyed to this variant.
• Functional-assay-flowchart.pdf, page 1: the proficient-function endpoint requires nuclear localization, sensitivity to DNA damage, and no mutator phenotype; the variant-specific report instead describes deficient MMR function.

Still missing

• No variant-specific entry for c.350C>T, p.Thr117Met, or p.T117M was found in Functional-assay-SVI-documentation-MMR.xlsx.
• No variant-specific entry for c.350C>T, p.Thr117Met, or p.T117M was found in Functional-assay-flowchart.pdf.
• No calibrated functional odds for pathogenicity <= 0.05 were reported for this variant.

Sources

Reasoning

The variant is missense, so the applicable BP4 path is the VCEP's HCI-prior condition, which requires a pathogenicity probability <0.11. The exact HCI-PRIORS-MLH1 row for c.350C>T (p.T117M) gives 0.893, so the missense BP4 condition is not met. The alternative BP4 path for SpliceAI delta score <=0.1 applies only to intronic and synonymous variants and cannot be used for this missense variant; the available SpliceAI max delta 0.037 therefore does not establish BP4.

VCEP rule

Missense variant with HCI-prior probability of pathogenicity \<0.11 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS) OR For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score \<= 0.1 as per Walker et al 2023

Evidence considered

• HCI-PRIORS-MLH1.txt, exact row for c.350C>T / p.T117M: pathogenicity probability = 0.893; this fails the VCEP BP4 missense threshold of <0.11.
• SpliceAI evidence reports max delta score = 0.037, but the VCEP BP4 SpliceAI alternative is restricted to intronic and synonymous variants, not this missense substitution.

Sources

Reasoning

PS1 requires a predicted missense substitution encoding the same amino acid change through a different nucleotide change, previously established by this VCEP as Pathogenic, and not a predicted or confirmed splice defect. NM_000249.4:c.350C>T encodes p.(Thr117Met), and the available VCEP pilot table identifies a different substitution at the same residue, p.Thr117Arg, but not an alternate nucleotide change producing p.Thr117Met. The governing VCEP files searched did not provide a qualifying same-amino-acid comparator, so PS1 cannot be determined from the available evidence.

VCEP 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 MLH1 VCEP PS1 rule requires the same amino-acid change from a different nucleotide change and prior VCEP Pathogenic classification, excluding predicted or confirmed splice defects.
• The VCEP pilot-variant table contains NM_000249.4(MLH1):c.350C>G (p.Thr117Arg), classified Likely pathogenic with PM5, PM2_supporting, PP1_moderate, PP3_moderate, and PP4_moderate; it is a different amino-acid change and therefore does not satisfy PS1 for p.Thr117Met.
• The named MMR-Functional-domains.pdf full-text file was searched for c.350C>T, p.Thr117Met, and p.T117M; no matching variant entry was present.

Still missing

• No qualifying alternate-nucleotide p.Thr117Met comparator was found in the available VCEP evidence; the searched MMR-Functional-domains.pdf file contains no c.350C>T, p.Thr117Met, or p.T117M entry.

Sources

Reasoning

The MLH1 VCEP PS2 rule assigns de novo points only when a proband has a documented de novo variant, specified maternity and paternity confirmation, and an LS-spectrum tumor, with two points requiring MMR-deficient tumor data, one point requiring confirmed parentage without tumor data, or 0.5 points for assumed de novo status with unconfirmed parentage. The case materials provide no proband-level parental testing, maternity or paternity confirmation, de novo point assignment, or qualifying tumor evidence for NM_000249.4:c.350C>T; therefore PS2 cannot be assessed.

VCEP rule

≥ 4 _de novo_ points ... 2-3.5 _de novo_ points ... 1-1.5 _de novo_ points ... 0.5 _de novo_ points

Evidence considered

• The case's ClinVar audit contains a general co-segregation signal but does not document de novo status, maternity, paternity, proband-level findings, tumor MSI/IHC/methylation data, or a de novo point total.
• The reviewed variant-specific paper extract for PMID:24362816 states that MLH1 c.350C>T/p.(T117M) is not explicitly mentioned, so it does not provide proband or parental evidence for PS2.

Still missing

• Proband-level report for NM_000249.4:c.350C>T (p.Thr117Met)
• Maternal and paternal testing confirming de novo status
• LS-spectrum tumor diagnosis and MSI/IHC/methylation results
• VCEP de novo point calculation

Sources

Reasoning

The VCEP requires co-occurrence of the MLH1 variant with a known pathogenic or likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD, and assigns points according to phase: 1.0 point for a pathogenic/likely pathogenic variant in trans or 0.5 points when phase is unknown. The available case evidence shows that NM_000249.4:c.350C>T meets the VCEP's PM2-Supporting prerequisite because gnomAD v4.1 grpmax FAF is 5.53e-06, below 0.00002, but it provides no qualifying affected-proband CMMRD observation, second MLH1 pathogenic/likely pathogenic variant, or phase information. Therefore, PM3 cannot be scored.

VCEP 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 - Refer to “Table for CMMRD diagnosis.pdf”.

Evidence considered

• VCEP PM3 rule: co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD is required; phase determines the point assignment.
• The case evidence does not document a qualifying affected proband with CMMRD features, a second known pathogenic/likely pathogenic MLH1 variant, or phase for such a variant.
• gnomAD v4.1: grpmax FAF = 5.53e-06, which is below the VCEP PM2-Supporting threshold of 0.00002; this satisfies the PM3 prerequisite but does not provide PM3 co-occurrence evidence.

Still missing

• Affected-proband clinical features consistent with CMMRD documented using the VCEP-referenced CMMRD criteria.
• A known pathogenic or likely pathogenic variant in the same gene documented in the affected proband.
• Phase of the two MLH1 variants established as in trans or otherwise explicitly characterized.

Sources

Reasoning

The MLH1 VCEP PP1 rule requires a combined pedigree Bayes Likelihood Ratio above 2.08, with moderate evidence above 4.3 through 18.7 and strong evidence above 18.7 in at least two families. The case materials provide no pedigree-level relative genotypes and phenotypes, informative meioses, independent family count, or combined Bayes Likelihood Ratio; therefore PP1 cannot be assessed.

VCEP rule

Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >18.7​ in ≥2 families. ... Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^​f >4.3 & ≤18.7. ... Co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >2.08 & ≤4.3.

Evidence considered

• The ClinVar audit contains general statements referring to co-segregation but no pedigree, relative-level genotype and phenotype observations, family count, or quantitative Bayes Likelihood Ratio.
• The VCEP specifies use of the overall Bayes Factor after combining multiple pedigrees by multiplication; no such pedigree analysis or quantitative result is present.

Still missing

• Pedigree-level segregation data
• Affected and unaffected relatives with variant and phenotype status
• Number of independent families
• Combined Bayes Likelihood Ratio, preferably from COOL v3

Sources

Reasoning

The VCEP PP4 rule requires CRC or endometrial MSI-H tumor evidence or loss of MMR protein expression consistent with the variant, with MLH1 promoter methylation excluded. The case materials provide no patient-level tumor MSI, tumor-genome, MMR-protein-expression, or promoter-methylation results, so no PP4 strength can be assigned. The governing Table-for-CMMRD-diagnosis.pdf was searched for c.350C>T, p.Thr117Met, and p.T117M and had no entry; the governing VCEP-pilot-variants---MMR.xlsx was searched for the same three notations and had no entry.

VCEP rule

1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e 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. For _MLH1_ variants, _MLH1_ promoter methylation is to be excluded in the tumor.

Evidence considered

• MLH1 Version 2.0 PP4 Supporting 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; inconsistent protein expression does not meet PP4; MLH1 promoter methylation must be excluded.
• Table-for-CMMRD-diagnosis.pdf.txt was searched for c.350C>T, p.Thr117Met, and p.T117M; no matching variant entry was found.
• VCEP-pilot-variants---MMR.xlsx.txt was searched for c.350C>T, p.Thr117Met, and p.T117M; no matching variant entry was found.
• No patient-level tumor phenotype or CMMRD scoring data are present in the case evidence.

Still missing

• No patient-level CRC or endometrial MSI-H/tumor-genome result or MMR-protein-expression result, with MLH1 promoter methylation excluded.
• No entry for c.350C>T, p.Thr117Met, or p.T117M in Table-for-CMMRD-diagnosis.pdf.txt.
• No entry for c.350C>T, p.Thr117Met, or p.T117M in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The MLH1 VCEP BS2 rule requires documented co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient meeting specified age and clinical conditions, with phase confirmation by parental or offspring testing. The available case evidence contains no qualifying co-occurrence, phase, age, or CMMRD-exclusion record, so BS2 cannot be evaluated.

VCEP 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^​d), 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

• No qualifying BS2 co-occurrence, phase-confirmation, age-at-cancer, or CMMRD-exclusion evidence was identified in the case evidence.
• The searched VCEP-pilot-variants---MMR.xlsx reference file has no exact row for c.350C>T, p.Thr117Met, or p.T117M.

Still missing

• No qualifying in-trans co-occurrence record, confirmed phase, qualifying cancer age, and CMMRD-exclusion information is available for BS2.
• VCEP-pilot-variants---MMR.xlsx was searched for c.350C>T, p.Thr117Met, and p.T117M; no exact variant row was found.

Sources

Reasoning

The MLH1 VCEP BS4 rule requires a combined pedigree Bayes Likelihood Ratio below 0.05 for strong evidence or above 0.05 and no more than 0.48 for supporting evidence. The case materials provide no affected-relative non-segregation observations, pedigree-level genotypes and phenotypes, informative meioses, or combined Bayes Likelihood Ratio; therefore BS4 cannot be assessed.

VCEP rule

Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f \<0.05​. ... Lack of co-segregation with disease in pedigree(s) with a combined\* Bayes Likelihood Ratio^f >0.05 & ≤0.48.

Evidence considered

• The ClinVar audit mentions co-segregation generally but reports no non-segregating relatives or quantitative segregation analysis, so it cannot establish BS4.
• The VCEP requires a combined Bayes Likelihood Ratio for lack of co-segregation, and no such value is available in the case evidence.

Still missing

• Pedigree-level non-segregation data
• Affected and unaffected relatives with variant and phenotype status
• Number of independent families
• Combined Bayes Likelihood Ratio

Sources

Reasoning

The VCEP BP5 rule requires specified benign tumor findings: at least 2 or 3 qualifying tumors for Supporting, or at least 4 qualifying tumors for Strong, alternatively BRAF V600E or MLH1 methylation findings with MSI-H/MLH1 loss. The case materials provide no patient-level tumor count, MSS/MSI status, MMR-protein-expression result, BRAF V600E result, or MLH1-methylation result, so BP5 cannot be assessed.

VCEP rule

2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d 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.

Evidence considered

• MLH1 Version 2.0 BP5 Supporting rule requires 2 or 3 qualifying tumors or 1 BRAF V600E/MLH1-methylation finding with MSI-H/MLH1 loss.
• No patient-level tumor phenotype or molecular tumor results are present in the case evidence.

Still missing

• No patient-level tumor count or MSS/MSI and MMR-protein-expression findings.
• No BRAF V600E result for a colon tumor and no MLH1-methylation result for an LS-spectrum tumor.

Sources