MLH1 c.1759A>G, p.(Met587Val)

Reasoning

The MLH1 VCEP specifies PM2 at Supporting strength for an absent or extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4. The variant is absent from gnomAD v4.1, satisfying this VCEP threshold. The searched VCEP pilot table, VCEP-pilot-variants---MMR.xlsx, contains no entry for c.1759A>G, p.Met587Val, or p.M587V.

VCEP rule

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

Evidence considered

• gnomAD v4.1: variant absent, corresponding to an allele frequency below 0.00002 for application of the VCEP PM2 rule.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1759A>G, p.Met587Val, and p.M587V; no matching row was found.

Still missing

• No matching c.1759A>G, p.Met587Val, or p.M587V entry was found in VCEP-pilot-variants---MMR.xlsx.

Sources

Reasoning

The variant is a predicted missense change, but the searched MLH1 VCEP full-text materials contain no different nucleotide change encoding p.Met587Val/p.M587V that was established by this VCEP as Pathogenic. The exact variant is present in HCI-PRIORS-MLH1.txt as c.1759A>G, p.M587V, but that row supplies predictor values rather than a VCEP pathogenic classification. The alternate protein changes at residue 587 in that table (p.M587L, p.M587K, p.M587T, p.M587R, and p.M587I) likewise are not VCEP classifications and therefore do not satisfy PS1.

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

• HCI-PRIORS-MLH1.txt, row 3863: c.1759A>G, p.M587V, MAPP score 27.32, prior probability 0.5956; the row does not provide a VCEP Pathogenic classification.
• A recursive search of /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/MLH1/fulltext for c.1759A>G, p.Met587Val, and p.M587V found this exact variant only in HCI-PRIORS-MLH1.txt; no VCEP-established Pathogenic same-amino-acid comparator was identified.

Still missing

• No qualifying VCEP-established Pathogenic same-amino-acid comparator was found in the searched MLH1 VCEP full-text materials; the exact c.1759A>G/p.M587V row in HCI-PRIORS-MLH1.txt is a predictor row, not a classification row.

Sources

Reasoning

No different missense change at Met587 was classified by this VCEP as Pathogenic or Likely Pathogenic on the protein level and not due to aberrant splicing. HCI-PRIORS-MLH1.txt lists alternate substitutions at residue 587, including p.M587L, p.M587K, p.M587T, p.M587R, and p.M587I, but provides predictor rows rather than VCEP classifications. In addition, the target HCI prior probability is 0.5956, which does not meet the VCEP PP3 supporting threshold of greater than 0.68 required by the PM5 rule.

VCEP rule

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

Evidence considered

• HCI-PRIORS-MLH1.txt, rows 3862-3870: alternate Met587 substitutions p.M587L, p.M587K, p.M587T, p.M587R, and p.M587I are listed, but the table rows contain predictor values and no VCEP Pathogenic/Likely Pathogenic classification.
• HCI-PRIORS-MLH1.txt, row 3863: target p.M587V prior probability is 0.5956.
• The governing PP3 rule requires an HCI prior probability greater than 0.68 for supporting evidence; 0.5956 does not meet that prerequisite.

Still missing

• No VCEP-classified Pathogenic or Likely Pathogenic missense comparator at Met587 was found in the searched MLH1 VCEP full-text materials; HCI-PRIORS-MLH1.txt provides alternate-residue substitutions but no VCEP classifications.

Sources

Reasoning

The variant is a missense substitution, so the VCEP's missense HCI-prior pathway applies. The governing HCI table contains the exact row for c.1759A>G / p.M587V with HCI prior probability 0.5956. This is below the VCEP PP3 Supporting threshold (>0.68 and <=0.81) and below the PP3 Moderate threshold (>0.81), so PP3 is not met. The alternative SpliceAI PP3 pathway is restricted to non-canonical splicing nucleotides and is not applicable to this coding missense variant; its 0.065 score is therefore not used for PP3.

VCEP rule

Missense variant with HCI prior probability for pathogenicity >0.68 & <=0.81 as per [https://hci-priors.hci.utah.edu/PRIORS](https://hci-priors.hci.utah.edu/PRIORS)

Evidence considered

• HCI-PRIORS-MLH1.txt, row MLH1_03861: c.1759A>G, p.M587V, HCI prior probability of pathogenicity = 0.5956; MAPP score = 27.32; custom PP2 score = 0.488.
• Case normalization identifies NM_000249.4:c.1759A>G as NP_000240.1:p.(Met587Val), a missense variant.
• SpliceAI max delta score = 0.065 was not used because the VCEP's alternative PP3 splice path is for non-canonical splicing nucleotides, not this coding missense situation.

Sources

Reasoning

The MLH1 VCEP defines BA1 as a gnomAD v4 Grpmax filtering allele frequency >=0.001 (0.1%) with the variant excluded as a founder pathogenic variant. This variant is absent from gnomAD v4.1, so the required population-frequency threshold is not reached. The searched VCEP pilot table, VCEP-pilot-variants---MMR.xlsx, contains no entry for c.1759A>G, p.Met587Val, or p.M587V.

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: variant absent; therefore no Grpmax filtering allele frequency reaches the BA1 threshold.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1759A>G, p.Met587Val, and p.M587V; no matching row was found.

Still missing

• No matching c.1759A>G, p.Met587Val, or p.M587V entry was found in VCEP-pilot-variants---MMR.xlsx.

Sources

Reasoning

The MLH1 VCEP defines BS1 as a gnomAD v4 Grpmax filtering allele frequency >=0.0001 and <0.001 (0.01-0.1%) with the variant excluded as a founder pathogenic variant. This variant is absent from gnomAD v4.1, so the required frequency interval is not reached. The searched VCEP pilot table, VCEP-pilot-variants---MMR.xlsx, contains no entry for c.1759A>G, p.Met587Val, or p.M587V.

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: variant absent; therefore its frequency is below the BS1 interval.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1759A>G, p.Met587Val, and p.M587V; no matching row was found.

Still missing

• No matching c.1759A>G, p.Met587Val, or p.M587V entry was found in VCEP-pilot-variants---MMR.xlsx.

Sources

Reasoning

The variant is a missense substitution, so the VCEP's missense HCI-prior pathway applies. The governing HCI table contains the exact row for c.1759A>G / p.M587V with HCI prior probability 0.5956, which is not below the VCEP BP4 threshold of <0.11. Therefore BP4 is not met. The alternative BP4 SpliceAI pathway is restricted to intronic and synonymous variants and does not apply to this missense variant.

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)

Evidence considered

• HCI-PRIORS-MLH1.txt, row MLH1_03861: c.1759A>G, p.M587V, HCI prior probability of pathogenicity = 0.5956; MAPP score = 27.32; custom PP2 score = 0.488.
• Case normalization identifies NM_000249.4:c.1759A>G as NP_000240.1:p.(Met587Val), a missense variant; the VCEP SpliceAI BP4 path is limited to intronic and synonymous variants.

Sources

Reasoning

No case evidence reports that MLH1 NM_000249.4:c.1759A>G (p.Met587Val) is de novo, provides parental genotypes or maternity/paternity confirmation, documents an LS-spectrum tumor in the proband, or assigns de novo points. The VCEP point-based PS2 rule therefore cannot be evaluated.

VCEP rule

≥ 4 _de novo_ points

Evidence considered

• The case materials identify MLH1 NM_000249.4:c.1759A>G (NP_000240.1:p.(Met587Val)) but report no proband-level de novo observation, parental testing, maternity/paternity confirmation, qualifying tumor data, or de novo point calculation.

Still missing

• Proband-level de novo status for NM_000249.4:c.1759A>G.
• Parental genotypes and confirmation of maternity and paternity.
• Qualifying LS-spectrum tumor and MSI/IHC/methylation findings, if applicable.
• VCEP-defined de novo point assignment and total points.

Sources

Reasoning

The MLH1 VCEP permits PS3 from calibrated functional assays using functional odds for pathogenicity >18.7 (Strong), >4.3 and <=18.7 (Moderate), or >2.08 and <=4.3 (Supporting), and from an MMR function defect meeting the functional-assay flowchart. The governing functional-assay documentation lists validated assay classes, controls, and calibration thresholds, but contains no variant-specific entry for c.1759A>G, p.Met587Val, or p.M587V. The flowchart text file contains no variant-specific result. PMID:18206535 reports the variant as a somatic Met587Val substitution detected and confirmed by sequencing in one sporadic gastric carcinoma, explicitly without a functional assay. Therefore no PS3 strength can be assigned from the available evidence.

VCEP rule

Calibrated functional assays with functional odds for pathogenicity > 18.7 ... Calibrated functional assays with functional odds for pathogenicity >4.3 and <= 18.7 ... MMR function defect following functional assay flowchart* ... Calibrated functional odds for pathogenicity >2.08 and <= 4.3

Evidence considered

• CSPEC PS3 rules: calibrated functional assays with functional odds for pathogenicity >18.7 are Strong, >4.3 and <=18.7 are Moderate, and >2.08 and <=4.3 are Supporting; MMR function defect following the functional assay flowchart is a Moderate path.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': approved assay classes are listed with positive and negative controls, validation-control counts, and assay-specific normal/abnormal thresholds; the table contains no row for this variant.
• Functional-assay-flowchart.pdf: searched full extracted text for c.1759A>G, p.Met587Val, and p.M587V; no variant-specific result was present.
• PMID:18206535, Results paragraph 2, Table 2, and Figure 1: Met587Val was found in one sporadic gastric carcinoma and confirmed by bidirectional sequencing; the paper reports no functional assay for this variant.

Still missing

• No entry for c.1759A>G, p.Met587Val, or p.M587V was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.1759A>G, p.Met587Val, or p.M587V was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific result from an approved calibrated MLH1 functional assay, including its functional odds or flowchart-qualified defect classification, is unavailable.

Sources

Reasoning

The MLH1 VCEP PM3 rule requires co-occurrence with a known pathogenic or likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD, with 1.0 point for the other variant in trans or 0.5 points when phase is unknown; MLH1 also must meet PM2_Supporting. The case evidence documents that NM_000249.4:c.1759A>G is absent from gnomAD v4.1, satisfying the VCEP's PM2_Supporting frequency prerequisite, but provides no affected-proband CMMRD features, second MLH1 variant, pathogenicity classification for a second variant, or phase evidence. 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”. For MLH1 variants - the variant has to meet PM2_Supporting criteria. ... 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.

Evidence considered

• ClinGen InSiGHT MLH1 VCEP specification v2.0, PM3 rule: co-occurrence with a known pathogenic/likely pathogenic variant in the same gene in a patient with CMMRD-consistent clinical features; 1.0 point in trans and 0.5 points when phase is unknown; MLH1 requires PM2_Supporting.
• gnomAD v4.1: NM_000249.4:c.1759A>G is absent, satisfying the VCEP PM2_Supporting prerequisite.
• No case evidence identifies a second MLH1 variant, its pathogenicity, affected-proband CMMRD features, or phase.

Still missing

• Affected-proband clinical features consistent with CMMRD are not documented.
• A second known pathogenic or likely pathogenic MLH1 variant is not documented.
• Phase of any second MLH1 variant is not documented; no trans or phase-unknown point can be assigned.

Sources

Reasoning

No affected or unaffected relatives, pedigree, informative meioses, cosegregation observations, or combined Bayes likelihood ratio are reported for MLH1 NM_000249.4:c.1759A>G. The VCEP PP1 strength thresholds therefore cannot be evaluated.

VCEP rule

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

Evidence considered

• The case materials report no affected-relative or unaffected-relative testing, pedigree segregation data, informative meioses, or combined Bayes likelihood ratio for this variant.

Still missing

• Pedigree and individual-level variant and phenotype status for informative relatives.
• Number of informative meioses and family structure.
• Combined Bayes likelihood ratio for cosegregation.

Sources

Reasoning

PP4 is applicable, but the available case evidence does not document a qualifying CRC or endometrial MSI-H tumor, consistent MMR-protein loss, exclusion of MLH1 promoter methylation, or a CMMRD diagnosis. The required VCEP reference files were searched for c.1759A>G, p.Met587Val, and p.M587V; neither file contains a matching variant 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

• The VCEP supporting rule requires 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, with MLH1 promoter methylation excluded.
• The CMMRD scoring table was consulted; no entry for c.1759A>G, p.Met587Val, or p.M587V was found.
• The InSiGHT MMR pilot-variant table was consulted; no entry for c.1759A>G, p.Met587Val, or p.M587V was found.
• The only exact-variant paper finding is one somatic Met587Val mutation in an MSI-H sporadic gastric carcinoma; it does not provide the required germline patient-level CRC/endometrial tumor or MLH1 methylation evidence.

Still missing

• Table-for-CMMRD-diagnosis.pdf searched for c.1759A>G, p.Met587Val, and p.M587V; no matching variant entry found.
• VCEP-pilot-variants---MMR.xlsx searched for c.1759A>G, p.Met587Val, and p.M587V; no matching variant entry found.
• Patient-level tumor MSI/IHC results and MLH1 promoter-methylation status are not available.

Sources

Reasoning

The MLH1 VCEP requires documented trans co-occurrence with a known pathogenic variant in the same gene, an eligible cancer presentation, absence of CMMRD manifestations, and phase confirmation through parents or offspring. The available case evidence contains no qualifying co-occurrence, cancer-age, CMMRD, or phase data. The searched VCEP pilot table, VCEP-pilot-variants---MMR.xlsx, contains no entry for c.1759A>G, p.Met587Val, or p.M587V.

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^b), 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.

Still missing

• No qualifying trans co-occurrence and phase-confirmation evidence is available.
• No matching c.1759A>G, p.Met587Val, or p.M587V entry was found in VCEP-pilot-variants---MMR.xlsx.

Sources

Reasoning

The MLH1 VCEP permits BS3 from calibrated functional assays with functional odds for pathogenicity <=0.05 (Strong) or >0.05 and <=0.48 (Supporting), or from variant-specific proficient function in protein and mRNA-based laboratory assays meeting the MMR functional assay flowchart. The governing functional-assay documentation lists validated assay classes, controls, and thresholds, but contains no variant-specific entry for c.1759A>G, p.Met587Val, or p.M587V. The flowchart text file contains no variant-specific result. PMID:18206535 reports only sequencing-based detection of the variant in a sporadic gastric carcinoma and no functional assay. Thus benign functional evidence cannot be assigned as BS3 Strong or Supporting.

VCEP rule

Calibrated functional assays with functional odds for Pathogenicity <= 0.05 ... Calibrated functional assays with functional odds for Pathogenicity >0.05 & <=0.48 ... Variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart*.

Evidence considered

• CSPEC BS3 rules: calibrated functional assays with functional odds for pathogenicity <=0.05 are Strong and >0.05 and <=0.48 are Supporting; variant-specific proficient function in protein and mRNA-based laboratory assays is an additional Supporting path under the MMR functional assay flowchart.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': approved assay classes are listed with positive and negative controls, validation-control counts, and assay-specific normal/abnormal thresholds; the table contains no row for this variant.
• Functional-assay-flowchart.pdf: searched full extracted text for c.1759A>G, p.Met587Val, and p.M587V; no variant-specific result was present.
• PMID:18206535, Results paragraph 2, Table 2, and Figure 1: the variant was identified and confirmed by sequencing, with no reported protein-function, mRNA-function, or other functional assay.

Still missing

• No entry for c.1759A>G, p.Met587Val, or p.M587V was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.1759A>G, p.Met587Val, or p.M587V was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific proficient-function result from an approved calibrated MLH1 protein or mRNA assay, including functional odds where applicable, is unavailable.

Sources

Reasoning

No pedigree, unaffected carrier, non-segregation observation, or combined Bayes likelihood ratio is reported for MLH1 NM_000249.4:c.1759A>G. The VCEP BS4 strength thresholds therefore cannot be evaluated.

VCEP rule

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

Evidence considered

• The case materials report no non-segregating relatives, pedigree observations, or combined Bayes likelihood ratio for this variant.

Still missing

• Pedigree and individual-level variant and phenotype status for informative relatives.
• Documented unaffected carriers or other lack-of-segregation observations.
• Combined Bayes likelihood ratio for lack of cosegregation.

Sources

Reasoning

BP5 is applicable, but no patient-level evidence documents the VCEP's required benign tumor patterns: 2 or 3 qualifying tumors with MSS and/or no inconsistent MMR-protein loss, or one qualifying BRAF V600E/MLH1-methylated MSI-H tumor with MLH1 loss.

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

• The VCEP BP5 supporting rule requires 2 or 3 tumors with the specified MSS/MMR-protein pattern, or 1 qualifying BRAF V600E/MLH1-methylated MSI-H tumor with MLH1 loss; none is documented.
• PMID:18206535 describes a somatic MSI-H sporadic gastric carcinoma but does not provide the VCEP BP5 tumor pattern or patient-level classification evidence.

Still missing

• Patient-level tumor MSI status, MMR-protein expression, BRAF V600E status, and MLH1 promoter-methylation status are not available.

Sources