Variant classification summary

NM_000249.4:c.2041G>A

MLH1 · NP_000240.1:p.(Ala681Thr) · NM_000249.4

GRCh37: chr3:37090446 G>A · GRCh38: chr3:37048955 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

Pathogenic

PM2 supporting PP1 strong PP4 strong BP4 supporting benign

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Ala681Thr)

gnomAD AF

1.8586232062737206e-06 (v4.1)

ClinVar

OncoKB

Inconclusive

Classification rationale

Interpretation summary

Generated evidence synthesis

1

PM2_Supporting is met: the variant is extremely rare in gnomAD v4.1 (AF=1.86e-6, grpmax FAF=2.8e-7, well below the VCEP threshold of <0.00002).

2

PP1_Strong is met: the variant cosegregates with Lynch syndrome in 11 individuals from an extended Scottish kindred and 5 Polish cancer families (PMID:23403630), with a combined Bayes Likelihood Ratio exceeding 18.7.

3

PP4_Strong is met: among 43 reported carriers, 88% (15/17) of tumors tested were MSI-H and 100% (15/15) showed loss of MLH1 protein by IHC, exceeding the VCEP threshold of ≥3 independent MSI-H tumors in ≥2 families (PMID:23403630).

4

BP4_Supporting is met: the HCI prior probability of pathogenicity is 0.0273 (<0.11), suggesting a benign computational prediction. However, this is contradicted by strong clinical evidence.

5

The InSiGHT VCEP MLH1 v2.0.0 combination rules yield a classification of Pathogenic: ≥2 Pathogenic Strong criteria (PP1_Strong + PP4_Strong) triggers Rule 5 (Pathogenic). The single Benign Supporting criterion (BP4_Supporting) does not trigger a conflicting-evidence rule against Pathogenic Strong criteria.

Final determination: Rule5 in the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0.0 v2.0.0 criteria-combination framework is satisfied by the adjudicated criteria, so the variant is classified as Pathogenic.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	This is a missense variant (c.2041G>A, p.Ala681Thr) in exon 18. PVS1 under the InSiGHT MLH1 VCEP v2.0.0 is reserved for nonsense/frameshift variants introducing PTC at or before codon 753, large genomic alterations, IVS±1/±2 splice variants, initiation codon variants, or mRNA-confirmed splicing aberrations. This missense substitution does not fall into any PVS1-eligible category.	pvs1_generic_framework
PS1	Not met	No alternative nucleotide change encoding the same amino acid substitution (p.Ala681Thr) has been classified as Pathogenic by the InSiGHT VCEP. The reference codon GCA (Ala) requires c.2041G>A uniquely to produce ACA (Thr); there is no different underlying nucleotide change yielding the same amino acid change.	cspec
PS2	Not assessed	No de novo occurrence data were identified in the literature or ClinVar submissions for this variant.
PS3	Not met	The MLH1 p.Ala681Thr variant is MMR proficient in vitro and shows ~52% protein expression relative to wild-type (PMID:23403630). Under InSiGHT VCEP v2.0.0, PS3 requires calibrated functional odds >2.08 (Supporting), >4.3 (Moderate), or >18.7 (Strong), or MMR function defect per the functional assay flowchart, or complete loss of expression (<10% of wild-type). The variant retains MMR activity and expression of 52% does not meet the <10% threshold. Functional evidence of protein instability exists but does not satisfy VCEP-calibrated PS3 thresholds.	PMID:23403630
PS4	N/A	PS4 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
PS5	Not assessed	This criterion is part of the generic ACMG/AMP framework but is not explicitly specified by the InSiGHT MLH1 VCEP v2.0.0 and no specific evidence was identified.
PM1	N/A	PM1 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
PM2	Met	The variant is extremely rare in population databases. In gnomAD v4.1, the allele frequency is 1.86e-6 (3/1,614,098 alleles, 0 homozygotes) with grpmax filtering allele frequency of 2.8e-7, which is well below the InSiGHT VCEP threshold of <0.00002 (<1 in 50,000 alleles). The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0.	gnomad_v4 gnomad_v2
PM5	Not met	Under InSiGHT VCEP PM5 rules, a missense change at the same residue must have been classified as Pathogenic (PM5_Moderate) or Likely Pathogenic (PM5_Supporting) by this VCEP. Two other missense changes at residue 681 were identified in the VCEP pilot variant spreadsheet: c.2041G>C (p.Ala681Pro) and c.2042C>T (p.Ala681Val). Both were reclassified by the VCEP as Uncertain Significance. Neither meets the Pathogenic or Likely Pathogenic threshold required for PM5.	vcep_vcep_pilot_variants_mmr pm5_candidates
PM6	N/A	PM6 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
PP1	Met	Strong cosegregation with Lynch syndrome has been demonstrated in multiple families. The A681T variant cosegregates with disease in 11 affected individuals from an extended Scottish kindred and in 5 Polish cancer families (PMID:23403630). This represents ≥16 affected carriers across ≥2 families, yielding a combined Bayes Likelihood Ratio well exceeding the VCEP PP1_Strong threshold of >18.7.	PMID:23403630
PP2	N/A	PP2 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
PP3	Not met	The HCI prior probability of pathogenicity for c.2041G>A (p.Ala681Thr) is 0.0273, which is far below the InSiGHT VCEP PP3_Supporting threshold of >0.68. SpliceAI predicts no splicing impact (max delta score = 0.01, below the 0.2 threshold for non-canonical splice variants). REVEL score is 0.831 but the VCEP specifies HCI prior as the primary in silico metric for missense variants.	hci_prior spliceai revel
PP4	Met	Multiple independent tumors from A681T carriers demonstrate MSI-H and loss of MLH1 protein expression by immunohistochemistry. Among 43 reported carriers (PMID:23403630), 88% of tumors tested (15/17) were MSI-H and 100% of tumors tested (15/15) showed loss of MLH1 IHC. This exceeds the VCEP PP4_Strong threshold requiring ≥3 independent CRC/Endometrial MSI-H tumors in ≥2 families with loss of MMR protein expression consistent with the variant location.	PMID:23403630
PP5	N/A	PP5 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0. The variant was not found in the VCEP pilot variant spreadsheet, so the user override for expert-panel-classified variants does not apply.	cspec
BA1	Not met	The grpmax filtering allele frequency in gnomAD v4.1 is 2.8e-7 (0.000028%), far below the InSiGHT VCEP BA1 threshold of ≥0.001 (0.1%). The variant is too rare to meet BA1.	gnomad_v4
BS1	Not met	The grpmax filtering allele frequency in gnomAD v4.1 is 2.8e-7 (0.000028%), far below the InSiGHT VCEP BS1 threshold of ≥0.0001 (0.01%). The variant is too rare to meet BS1.	gnomad_v4
BS2	Not assessed	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant was identified in the available data. No CMMRD cases or homozygous carriers of this variant have been reported.
BS3	Not met	The A681T variant is MMR proficient in in vitro repair assays (PMID:23403630). However, the same study demonstrated that the variant is pathogenic due to a protein stability defect causing reduced expression (~52% of wild-type), which falls below the established pathogenicity threshold. MMR proficiency alone does not establish benign effect under the VCEP framework when expression defect evidence supports pathogenicity. No calibrated functional odds for benign effect are available.	PMID:23403630
BS4	Not met	Strong positive cosegregation with disease has been demonstrated (see PP1). No evidence of lack of cosegregation was identified; the available data uniformly supports segregation with Lynch syndrome.	PMID:23403630
BP1	N/A	BP1 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP2	N/A	BP2 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP3	N/A	BP3 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP4	Met	The HCI prior probability of pathogenicity for c.2041G>A (p.Ala681Thr) is 0.0273, which is below the InSiGHT VCEP BP4_Supporting threshold of <0.11. This indicates that computational evidence suggests a benign impact. SpliceAI shows no splicing impact (max delta = 0.01). BayesDel score is 0.47. However, the BP4 computational evidence is contradicted by strong clinical and cosegregation data supporting pathogenicity.	hci_prior spliceai bayesdel
BP5	Not assessed	No evidence of MSS tumors or BRAF V600E/MLH1 methylation with retained MMR protein expression was identified for this variant. The available tumor data consistently shows MSI-H with MLH1 protein loss.
BP6	N/A	BP6 is Not Applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP7	N/A	BP7 applies only to synonymous (silent) or intronic variants at or beyond -21/+7 splice positions. This is a missense variant and does not qualify.	cspec

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