Variant classification summary

NM_000249.4:c.1637A>G

MLH1 · NP_000240.1:p.(Lys546Arg) · NM_000249.4

GRCh37: chr3:37081755 A>G · GRCh38: chr3:37040264 A>G

Gene: MLH1 Transcript: NM_000249.4

Final call

PM2 supporting BP4 supporting benign

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Lys546Arg)

gnomAD AF

3.7178498930498515e-06 (v4.1)

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

HCI prior probability for pathogenicity is 0.0023, meeting BP4_Supporting per InSiGHT VCEP v2.0.0 (threshold <0.11). REVEL (0.469) and BayesDel (-0.0407) are consistent with a benign in silico profile.

2

Extremely rare in gnomAD v4.1 (grpmax FAF = 1.24e-06; 6/1,613,836 alleles, 0 homozygotes), meeting PM2_Supporting per InSiGHT VCEP v2.0.0 (threshold <0.00002). Absent from gnomAD-Canada v1.0.

3

This variant has been reported in ClinVar (Variation ID: 127617) with submissions of Uncertain Significance (10 clinical laboratories), Benign (1), and Likely Benign (1); no expert panel classification is available.

4

NM_000249.4:c.1637A>G (p.Lys546Arg) was observed in 1 of 711 patients with hereditary breast cancer in a Russian case-control study (PMID:32547938); no variant-specific MSI, IHC, or functional data were reported.

Final determination: Rule31 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 Uncertain Significance - Conflicting Evidence.

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	Missense variant p.Lys546Arg; not a null variant (nonsense, frameshift, canonical splice, initiation codon, or exon-level deletion). Does not meet any PVS1 rule under the InSiGHT MLH1 VCEP v2.0.0.
PS1	Not met	No different underlying nucleotide change encoding the same amino acid substitution (Lys546Arg) has been established as Pathogenic or Likely Pathogenic by the InSiGHT VCEP.
PS2	Not met	No de novo observation reported in any reviewed literature. No publication identified a proband with confirmed maternity and paternity and a MMR-deficient LS-spectrum tumor carrying this variant.
PS3	Not met	This variant is not listed in the VCEP calibrated functional assay documentation (Functional-assay-SVI-documentation-MMR.xlsx) and no variant-specific functional data demonstrating a damaging effect on MMR function were identified in any reviewed publication.
PS4	N/A	VCEP rules: PS4 is not utilized for MMR variant classification using proband counting, due to the availability of tumor IHC data for variant classification via PP4.
PS5	N/A	PS5 is not defined or used by the InSiGHT MLH1 VCEP (version 2.0.0). This criterion does not appear in the CSPEC criteria set for this gene.
PM1	N/A	VCEP rules: there are no recognized mutational hot spots in MMR genes that can be used for classification purposes.
PM2	Met	Extremely rare in gnomAD v4.1: grpmax filtering allele frequency = 1.24e-06, which is below the VCEP PM2_Supporting threshold of <0.00002 (<1 in 50,000 alleles). Observed in 6/1,613,836 alleles (0 heterozygotes), with highest subpopulation frequency in African/African American (AF = 1.34e-05). Absent from gnomAD-Canada v1.0.	gnomad_v4
PM5	Not met	No different missense variant at amino acid residue Lys546 has been classified as Pathogenic or Likely Pathogenic by the InSiGHT VCEP. Additionally, PM5 requires PP3 to be supporting, which is not met (HCI prior = 0.0023).
PM6	N/A	VCEP rules: PM6 (assumed de novo without confirmation of maternity and paternity) is superseded by PS2 in this framework and is explicitly marked Not Applicable.
PP1	Not met	No co-segregation data available for this variant in any reviewed publication. No pedigree analysis with Bayes Likelihood Ratio could be performed.
PP2	N/A	VCEP rules: PP2 (missense variant in a gene with low rate of benign missense changes) does not apply for MLH1.
PP3	Not met	HCI prior probability for pathogenicity = 0.0023, well below the VCEP PP3_Supporting threshold of >0.68. The SpliceAI-based PP3 rule (delta ≥0.2) applies only to non-canonical splice site variants and does not apply to this exonic missense variant. REVEL score = 0.469 and BayesDel score = -0.0407 are not informative in either direction.	hci_prior
PP4	Not met	No MSI-H tumor data or MMR protein IHC loss data reported for this specific variant in any reviewed publication. The variant was observed in a breast cancer case-control study (PMID:32547938) but individual variant-level MSI/IHC results were not reported.
PP5	N/A	VCEP rules: PP5 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BA1	Not met	gnomAD v4 grpmax filtering allele frequency = 1.24e-06, far below the VCEP BA1 threshold of ≥0.001 (0.1%).	gnomad_v4
BS1	Not met	gnomAD v4 grpmax filtering allele frequency = 1.24e-06, below the VCEP BS1 threshold of ≥0.0001 (0.01%).	gnomad_v4
BS2	Not met	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant in a patient meeting the specified clinical criteria (CRC after age 45 or other LS cancer above median onset age, without CMMRD features).
BS3	Not met	This variant is not listed in the VCEP calibrated functional assay documentation and no variant-specific functional data demonstrating proficient MMR function were identified in any reviewed publication.
BS4	Not met	No co-segregation analysis data available for this variant. No pedigree data demonstrating lack of segregation with disease could be assessed.
BP1	N/A	VCEP rules: BP1 (missense variant in a gene where only loss of function causes disease) does not apply for MLH1.
BP2	N/A	VCEP rules: BP2 is not used; BS2 is used instead for trans co-occurrence assessment.
BP3	N/A	In-frame deletions/insertions in repetitive regions not applicable for a missense substitution variant.
BP4	Met	HCI prior probability for pathogenicity = 0.0023, which is below the VCEP BP4_Supporting threshold of <0.11. Multiple in silico predictors support a benign effect: REVEL = 0.469 (not predictive of pathogenicity), BayesDel = -0.0407 (slightly benign-leaning).	hci_prior revel bayesdel
BP5	Not met	No tumor data demonstrating MSS status, lack of MMR protein loss, BRAF V600E mutation, or MLH1 promoter methylation were reported for this variant in any reviewed publication.
BP6	N/A	VCEP rules: BP6 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BP7	N/A	BP7 applies only to synonymous (silent) or intronic variants at or beyond -21/+7; this is a missense substitution variant.

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