Variant classification summary

NM_000249.4:c.1039-25T>A

MLH1 · NP_000240.1:p.? · NM_000249.4

GRCh37: chr3:37067103 T>A · GRCh38: chr3:37025612 T>A

Gene: MLH1 Transcript: NM_000249.4

Final call

Benign

BA1 stand-alone benign BP4 supporting BP7 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.?

gnomAD AF

0.00022026736856687676 (v4.1)

ClinVar

Likely benign

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.1039-25T>A is an intronic variant located 25 bases upstream of exon 11 in MLH1.

2

This variant is present in gnomAD v4.1 with a joint grpmax filtering allele frequency of 0.157% (159/721,850 alleles), exceeding the InSiGHT MLH1 VCEP BA1 stand-alone benign threshold of 0.1%.

3

The variant is observed across multiple continental populations with highest frequency in the African/African American population (0.194%), consistent with a common polymorphism rather than a founder pathogenic variant.

4

SpliceAI predicts no splicing impact (max delta score 0.03), supporting application of BP4 (Supporting) for an intronic variant with no predicted splice defect.

5

The variant's intronic position at -25 relative to exon 11 satisfies BP7 (Supporting) under the VCEP rule for variants at or beyond the -21/+7 boundary.

6

The variant has been reported in ClinVar as Likely benign by two clinical laboratories (ClinVar Variation ID 492685), consistent with the population frequency data.

Final determination: Rule17 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 Benign.

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.1039-25T>A is a deep intronic variant located 25 bases upstream of exon 11. It does not affect a canonical splice donor/acceptor site (IVS±1,2). SpliceAI predicts no splicing impact (max delta score 0.03). Under the InSiGHT MLH1 VCEP v2.0, PVS1 is restricted to nonsense/frameshift variants introducing PTC at or before codon 753, large genomic alterations, IVS±1 or ±2 variants, mRNA-confirmed splicing aberrations, or initiation codon variants. This variant does not fall into any of these categories.	spliceai pvs1_variant_assessment pvs1_gene_context cspec
PS1	Not met	PS1 under the MLH1 VCEP requires either (a) a missense substitution encoding the same amino acid change as a VCEP-classified Pathogenic variant, or (b) a variant affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse SpliceAI prediction. This intronic variant encodes no amino acid change and SpliceAI delta is only 0.03 (no predicted splicing defect), so neither condition applies.	spliceai cspec
PS2	Not met	No de novo occurrence data are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP PS2 criterion requires documented de novo events with confirmed maternity and paternity. No publications or ClinVar submissions report de novo observations for this variant.	clinvar
PS3	Not met	No functional data are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP PS3 criterion requires calibrated functional assay data with functional odds for pathogenicity, MMR function defect per the functional assay flowchart, or monoallelic expression data. No such evidence was identified in the InSiGHT functional assay SVI documentation, VCEP pilot variant spreadsheet, ClinVar, or published literature.	cspec
PS4	N/A	PS4 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0.	cspec
PS5	N/A	PS5 is not recognized as a standalone criterion in the ACMG/AMP 2015 framework; the evidence type it represents is subsumed by PS1 and PM5.
PM1	N/A	PM1 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0.	cspec
PM2	Not met	Under the MLH1 VCEP, PM2_Supporting requires an allele frequency < 0.00002 (< 1 in 50,000 alleles) in gnomAD v4. This variant is present in gnomAD v4.1 at an overall AF of 0.00022 (159/721,850 alleles; 0.022%), which exceeds the PM2 threshold.	gnomad_v4
PM5	N/A	PM5 applies only to missense variants with a different missense change at the same amino acid residue classified as Pathogenic or Likely Pathogenic by the VCEP. NM_000249.4:c.1039-25T>A is an intronic variant with no protein consequence.	pm5_candidates cspec
PM6	N/A	PM6 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0.	cspec
PP1	Not met	No co-segregation data are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP PP1 criterion requires co-segregation with disease in pedigrees with a combined Bayes Likelihood Ratio exceeding specified thresholds. No such data were identified in ClinVar or published literature.	clinvar
PP2	N/A	PP2 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0 (missense variant in a gene with low rate of benign missense changes does not apply).	cspec
PP3	Not met	Under the MLH1 VCEP, PP3 for non-canonical splice variants requires a SpliceAI delta score ≥ 0.2 (Supporting) or an HCI prior probability > 0.68 (missense variants only). SpliceAI predicts no splicing impact (max delta 0.03), well below the VCEP threshold. The variant is intronic and has no HCI prior score.	spliceai cspec
PP4	Not met	No tumor phenotype data (MSI status, IHC for MMR proteins) are available for carriers of NM_000249.4:c.1039-25T>A. The MLH1 VCEP PP4 criterion requires MSI-H CRC/endometrial tumors with MMR protein expression loss consistent with the variant location.
PP5	N/A	PP5 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0. ClinVar review status is 'criteria provided, single submitter' (1-star), so the global PP5-at-supporting override for 3-star expert panel submissions does not apply.	cspec clinvar
BA1	Met	Under the MLH1 VCEP, BA1 (Stand Alone) applies when the gnomAD v4 grpmax filtering allele frequency is ≥ 0.001 (0.1%) and the variant is excluded as a founder pathogenic variant. The gnomAD v4.1 joint grpmax FAF is 0.00157 (0.157%), which exceeds the 0.1% threshold. The variant is observed across multiple continental populations (AFR 0.194%, AMR 0.017%, ASJ 0.039%) with no evidence of a founder effect. The ClinVar classification of Likely benign from clinical laboratories is consistent with this being a common polymorphism.	gnomad_v4 clinvar cspec
BS1	Not met	The MLH1 VCEP BS1 threshold is gnomAD v4 grpmax FAF ≥ 0.0001 and < 0.001. The observed grpmax FAF of 0.00157 exceeds the upper bound of the BS1 range and instead meets the higher BA1 threshold. BS1 is superseded by BA1.	gnomad_v4 cspec
BS2	Not met	No co-occurrence data are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP BS2 criterion requires observation in trans with a known pathogenic variant in a patient with colorectal cancer after age 45 without CMMRD features, with confirmed phase.
BS3	Not met	No functional data demonstrating a benign effect are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP BS3 criterion requires calibrated functional assays with odds for pathogenicity ≤ 0.48, or mRNA assays showing no splicing aberration or allelic imbalance with NMD inhibition. SpliceAI predicts no splicing impact (delta 0.03), but this in silico prediction alone does not satisfy the VCEP BS3 requirement for laboratory-based functional evidence.	spliceai cspec
BS4	Not met	No lack-of-segregation data are available for NM_000249.4:c.1039-25T>A. The MLH1 VCEP BS4 criterion requires a combined Bayes Likelihood Ratio < 0.48 from pedigrees showing absence of co-segregation with disease.
BP1	N/A	BP1 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0.	cspec
BP2	N/A	BP2 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0.	cspec
BP4	Met	Under the MLH1 VCEP, BP4_Supporting applies to intronic variants when SpliceAI predicts no splicing impact with a delta score ≤ 0.1 (Walker et al. 2023). SpliceAI analysis of NM_000249.4:c.1039-25T>A yields a maximum delta score of 0.03, indicating no predicted effect on splicing. This criterion is independent of BP7 and both may be applied.	spliceai cspec
BP5	Not met	No tumor phenotype data are available for evaluation under BP5. The MLH1 VCEP BP5 criterion requires CRC/endometrial tumors with MSS and/or no loss of MMR protein expression, or BRAF V600E/MLH1 methylation in MSI-H tumors.
BP6	N/A	BP6 is listed as Not Applicable by the InSiGHT MLH1 VCEP v2.0. ClinVar review status is 'criteria provided, single submitter' (1-star), so the global BP6-at-supporting override for 3-star expert panel submissions does not apply.	cspec clinvar
BP7	Met	Under the MLH1 VCEP, BP7_Supporting applies to intronic variants at or beyond -21/+7 of the exon boundary. NM_000249.4:c.1039-25T>A is located at position -25 relative to exon 11, which is beyond the -21 threshold. SpliceAI also predicts no splicing impact (delta 0.03). Per VCEP guidance, variants may satisfy both BP7 and BP4.	cspec spliceai

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