Variant classification summary

NM_000249.3:c.1433A>G

MLH1 · NP_000240.1:p.(Asp478Gly) · NM_000249.3

GRCh37: chr3:37070298 A>G · GRCh38: chr3:37028807 A>G

Gene: MLH1 Transcript: NM_000249.3

Final call

PM2 supporting BP4 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.3

Protein

NP_000240.1:p.(Asp478Gly)

gnomAD AF

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

PM2_supporting met: variant is absent from gnomAD v4.1, meeting the VCEP allele frequency threshold of <0.00002 (<1 in 50,000 alleles).

2

BP4_supporting met: HCI prior probability for pathogenicity is 0.0146 (<0.11), consistent with a benign in silico prediction. REVEL score 0.604 and BayesDel 0.161 also favor a benign interpretation.

3

PP3 not met: HCI prior 0.0146 does not reach the PP3 supporting threshold of >0.68. SpliceAI delta 0.00 does not indicate splicing impact.

4

PS3 not met: no variant-specific functional data from calibrated MMR assays available. OncoKB reports Unknown Oncogenic Effect.

5

PVS1 not applicable: this is a missense variant and does not meet any VCEP PVS1 rule criteria.

6

Overall, one supporting pathogenic criterion (PM2) and one supporting benign criterion (BP4) are met, yielding insufficient evidence for classification. Defaults to Uncertain Significance per ACMG/AMP 2015 framework.

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 substitution (c.1433A>G, p.Asp478Gly); does not meet any VCEP PVS1 rule criteria, which are restricted to nonsense/frameshift variants introducing PTC at or before codon 753, large genomic alterations, IVS±1/±2 splice variants, initiation codon variants, or confirmed mRNA splicing aberrations.	pvs1_gene_context pvs1_variant_assessment
PS1	Not met	No different nucleotide change encoding the same amino acid substitution (p.Asp478Gly) has been classified as Pathogenic or Likely Pathogenic by the InSiGHT MMR VCEP. The VCEP pilot variants spreadsheet does not list any variant at codon 478.	vcep_vcep_pilot_variants_mmr
PS2	Not assessed	No de novo observations reported for this variant. VCEP PS2 requires de novo points based on confirmed parentage.
PS3	Not met	No variant-specific functional data from calibrated MMR assays exists. OncoKB reports Unknown Oncogenic Effect with no reviewed functional evidence. The VCEP pilot variants spreadsheet does not include this variant, and no functional odds for pathogenicity are available from the calibrated assay documentation.	oncokb vcep_functional_assay_svi_documentation_mmr vcep_vcep_pilot_variants_mmr
PS4	N/A	PS4 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
PS5	N/A	PS5 is not included in the InSiGHT MMR VCEP specification (version 2.0.0) criteria list.	cspec
PM1	N/A	PM1 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
PM2	Met	This variant is absent from gnomAD v4.1, meeting the VCEP PM2_supporting threshold of allele frequency <0.00002 (<1 in 50,000 alleles). It is also absent from gnomAD v2.1 and gnomAD-Canada v1.0.	gnomad_v4 gnomad_v2 gnomad_canada
PM3	N/A	PM3 requires observation in trans with a pathogenic variant; no such data has been provided for this case. Trivially not applicable per case skip directive.
PM4	N/A	PM4 applies only to non-frameshift in-frame deletions/insertions and stop-loss variants; this is a missense substitution. Trivially not applicable.
PM5	Not met	No pathogenic or likely pathogenic missense variant at the same amino acid residue (Asp478) has been classified by the InSiGHT MMR VCEP. The PM5 candidate search found zero same-residue comparator variants. VCEP PM5 requires an existing P/LP missense classification at the same residue by this VCEP and supporting PP3 evidence.	pm5_candidates vcep_vcep_pilot_variants_mmr
PM6	N/A	PM6 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
PP1	Not assessed	No co-segregation data available for this variant. VCEP PP1 requires Bayes likelihood ratios from pedigrees with combined analysis.
PP2	N/A	PP2 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
PP3	Not met	The HCI prior probability for pathogenicity is 0.0146, which does not meet the VCEP PP3 thresholds (>0.68 for supporting, >0.81 for moderate). SpliceAI predicts no splicing impact (max delta score 0.00), which does not meet the ≥0.2 threshold for non-canonical splice site variants. REVEL score is 0.604 and BayesDel is 0.161, but the VCEP PP3 rules rely on HCI priors and SpliceAI.	hci_prior spliceai revel bayesdel
PP4	Not assessed	No tumor MSI or IHC data available. VCEP PP4 requires specific counts of MSI-H CRC/endometrial tumors with loss of MMR protein expression consistent with the variant location, with MLH1 promoter methylation excluded.
PP5	N/A	PP5 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0), per ClinGen SVI VCEP Review Committee recommendation.	cspec
BA1	Not met	This variant is absent from gnomAD v4.1, which does not meet the VCEP BA1 threshold of gnomAD v4 Grpmax filtering allele frequency ≥0.001 (0.1%).	gnomad_v4
BS1	Not met	This variant is absent from gnomAD v4.1, which does not meet the VCEP BS1 threshold of gnomAD v4 Grpmax filtering allele frequency ≥0.0001 and <0.001 (0.01-0.1%).	gnomad_v4
BS2	Not assessed	No data on co-occurrence in trans with a known pathogenic variant in the same gene in a CRC patient after age 45 without CMMRD features.
BS3	Not met	No functional data from calibrated MMR assays demonstrating proficient function exists for this variant. VCEP BS3 requires calibrated functional odds for pathogenicity ≤0.05 (strong) or >0.05 and ≤0.48 (supporting), or variant-specific proficient function per the MMR assay flowchart.	vcep_functional_assay_svi_documentation_mmr oncokb
BS4	Not assessed	No co-segregation data available. VCEP BS4 requires lack of co-segregation with disease in pedigrees with combined Bayes likelihood ratio.
BP1	N/A	BP1 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
BP2	N/A	BP2 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
BP3	N/A	BP3 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0).	cspec
BP4	Met	The HCI prior probability for pathogenicity is 0.0146, which is <0.11, meeting the VCEP BP4_Supporting threshold for missense variants. Multiple in silico tools predict a benign impact: REVEL score 0.604 (below typical pathogenic threshold), BayesDel 0.161, and SpliceAI max delta 0.00 (no predicted splicing effect).	hci_prior spliceai revel bayesdel
BP5	Not assessed	No tumor phenotype data available. VCEP BP5 requires specific counts of CRC/endometrial tumors with MSS and/or no loss of MMR protein expression, or BRAF V600E/MLH1 methylation in relevant tumors.
BP6	N/A	BP6 is marked Not Applicable by the InSiGHT MMR VCEP specification (version 2.0.0), per ClinGen SVI VCEP Review Committee recommendation.	cspec
BP7	N/A	BP7 applies only to synonymous (silent) and intronic variants at or beyond -21/+7. This is a missense substitution (c.1433A>G, p.Asp478Gly) and is not eligible.

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