Variant classification summary

NM_000249.4:c.1344G>T

MLH1 · NP_000240.1:p.(Glu448Asp) · NM_000249.4

GRCh37: chr3:37067433 G>T · GRCh38: chr3:37025942 G>T

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.(Glu448Asp)

gnomAD AF

1.920653468266468e-05 (v4.1)

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.1344G>T (p.Glu448Asp) is a missense variant in exon 12 of the MLH1 gene. It is extremely rare in population databases, with an allele frequency of 1.92e-05 in gnomAD v4.1 (31/1,614,034 alleles, 0 homozygotes), meeting PM2 at supporting strength.

2

Computational evidence strongly predicts a benign effect: the HCI prior probability for pathogenicity is 0.0024 (BP4_Supporting threshold <0.11 met), BayesDel score is -0.059 (benign range), and SpliceAI predicts no splicing impact (max delta = 0.03).

3

No functional data are available for this variant. It is not included in the VCEP calibrated functional assay documentation, and no publication reports variant-specific functional evidence for c.1344G>T.

4

No segregation, de novo, or tumor pathology data are available for this variant. The variant has been reported in ClinVar as Uncertain Significance (VariationID 127612, 1-star review status) by 14 clinical laboratories, with one additional submission as Benign and one as Likely Benign.

5

Applying the InSiGHT VCEP v2.0 combination rules: one pathogenic supporting criterion (PM2_Supporting) and one benign supporting criterion (BP4_Supporting) are met. No criteria at moderate, strong, or very strong strength are met in either direction. This yields a classification of Uncertain Significance.

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 v2.0 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	Not met	NM_000249.4:c.1344G>T is a missense variant (p.Glu448Asp) in exon 12 of MLH1. It does not fall into any PVS1-eligible null-variant category under the InSiGHT VCEP v2.0 rules (nonsense/frameshift, canonical ±1,2 splice, large genomic alteration, initiation codon). PVS1 is not applicable to missense substitutions.	pvs1_variant_assessment cspec
PS1	Not met	No other nucleotide change encoding p.Glu448Asp has been established as Pathogenic by this VCEP. The variant c.1344G>C (also p.E448D) exists but has not been classified as Pathogenic or Likely Pathogenic by the InSiGHT VCEP.	hci_prior cspec
PS2	Not met	No de novo occurrence data are available for this variant. VCEP PS2 requires confirmed de novo status with phased parental testing and point-based scoring; no such evidence exists in the case record.
PS3	Not met	No calibrated functional assay data exist for NM_000249.4:c.1344G>T (p.Glu448Asp). The VCEP Functional Assay SVI documentation (MMR assays spreadsheet) does not contain this variant. No publication reports variant-specific functional evidence. The OncoKB entry for E448D is 'Unknown Oncogenic Effect' with no supporting functional data.	oncokb cspec
PS4	N/A	PS4 is marked as Not Applicable for this VCEP according to the InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specifications v2.0.	cspec
PS5	Not assessed	PS5 is an ACMG criterion for a variant found in a patient with an alternate molecular basis for disease. The InSiGHT VCEP v2.0 does not include PS5 in its criteria specifications, and no alternate molecular basis data are available in the case record.
PM1	N/A	PM1 is marked as Not Applicable for this VCEP according to the InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specifications v2.0. The VCEP does not use domain-level evidence for MLH1.	cspec
PM2	Met	The variant is extremely rare in population databases. In gnomAD v4.1, the overall allele frequency is 1.92e-05 (31/1,614,034 alleles, 0 homozygotes), which is below the VCEP PM2 threshold of <0.00002 (<1 in 50,000 alleles). The variant is absent from gnomAD-Canada v1.0.	gnomad_v4 gnomad_v2 gnomad_canada
PM5	Not met	No different missense change at codon 448 has been classified as Pathogenic or Likely Pathogenic by the InSiGHT VCEP. The VCEP pilot variants spreadsheet contains no MLH1 variant at this codon. Additionally, VCEP PM5 requires PP3 to be supporting for the variant in question, and PP3 is not met (HCI prior = 0.0024).	pm5_candidates cspec
PM6	N/A	PM6 is marked as Not Applicable for this VCEP according to the InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specifications v2.0.	cspec
PP1	Not met	No co-segregation data are available for this variant. VCEP PP1 requires co-segregation with disease in pedigrees with a combined Bayes Likelihood Ratio; no such evidence exists in the case record.
PP2	N/A	PP2 is marked as Not Applicable for this VCEP. The VCEP notes that the missense variant in a gene with low rate of benign missense changes does not apply for MLH1.	cspec
PP3	Not met	The HCI prior probability for pathogenicity is 0.0024, which is far below the VCEP PP3_Supporting threshold of >0.68. SpliceAI predicts no significant splice impact (max delta score = 0.03, below the 0.2 threshold). REVEL score is 0.537 (indeterminate) and BayesDel is -0.059 (benign range), neither of which meets the VCEP PP3 threshold.	hci_prior spliceai revel bayesdel
PP4	Not met	No tumor data (MSI status, immunohistochemistry for MMR proteins) are available for patients carrying this variant. VCEP PP4 requires MSI-H tumors with consistent loss of MMR protein expression; no such evidence exists in the case record.
PP5	N/A	PP5 is marked as Not Applicable for this VCEP as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BA1	Not met	The gnomAD v4.1 grpmax filtering allele frequency is 5.798e-05 (0.0058%), which is far below the VCEP BA1 threshold of >=0.001 (0.1%). The variant is not a common polymorphism.	gnomad_v4
BS1	Not met	The gnomAD v4.1 grpmax filtering allele frequency is 5.798e-05 (0.0058%), which is below the VCEP BS1 threshold of >=0.0001 to <0.001. The variant is too rare to meet BS1.	gnomad_v4
BS2	Not met	No data are available for co-occurrence in trans with a known pathogenic MLH1 variant in a patient with CRC after age 45 without CMMRD features. VCEP BS2 requires phased co-occurrence evidence.
BS3	Not met	No functional data demonstrating a benign effect are available for this variant. The VCEP functional assay SVI documentation does not include c.1344G>T. No publication reports variant-specific functional evidence supporting normal MMR function.	cspec oncokb
BS4	Not met	No segregation data are available to assess lack of co-segregation with disease. VCEP BS4 requires combined Bayes Likelihood Ratio from pedigrees demonstrating absence of segregation.
BP1	N/A	BP1 is marked as Not Applicable for this VCEP according to the InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specifications v2.0.	cspec
BP2	N/A	BP2 is marked as Not Applicable for this VCEP according to the InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specifications v2.0.	cspec
PM3	N/A	PM3 applies to biallelic variants in trans with a pathogenic variant for recessive disorders (CMMRD context for MLH1). No biallelic data are available and this is not a recessive disorder presentation.
PM4	N/A	PM4 applies to protein length changes from in-frame deletions/insertions, stop-loss, or initiation codon variants. This is a missense substitution and does not alter protein length.
BP3	N/A	BP3 applies to in-frame deletions/insertions in repetitive regions; this variant is a single-nucleotide missense substitution.
BP4	Met	The HCI prior probability for pathogenicity is 0.0024, which is below the VCEP BP4_Supporting threshold of <0.11. Multiple in silico tools predict a benign effect: BayesDel score is -0.059 (benign range) and SpliceAI predicts no splicing impact (max delta = 0.03). REVEL score of 0.537 is indeterminate and does not override the HCI prior.	hci_prior bayesdel revel spliceai
BP5	Not met	No tumor data are available to demonstrate microsatellite stability (MSS) or intact MMR protein expression in CRC/endometrial tumors from patients carrying this variant. VCEP BP5 requires tumor evidence of MSS and/or preserved MMR protein expression.
BP6	N/A	BP6 is marked as Not Applicable for this VCEP as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP7	Not met	BP7 applies to synonymous (silent) or intronic variants at or beyond -21/+7 splice positions. NM_000249.4:c.1344G>T is a missense variant (p.Glu448Asp) and does not qualify for BP7.

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