Variant classification summary

NM_000249.4:c.307-1G>A

MLH1 · NP_000240.1:p.? · NM_000249.4

GRCh37: chr3:37045891 G>A · GRCh38: chr3:37004400 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

VUS

PVS1 very strong PM2 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.?

gnomAD AF

ClinVar

Likely pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.307-1G>A is a canonical splice acceptor variant (IVS3-1G>A) in MLH1, predicted to cause exon 4 skipping, frameshift, and nonsense-mediated decay.

2

SpliceAI predicts a severe splicing defect with a max delta score of 0.99 (acceptor loss), consistent with disruption of the canonical splice acceptor.

3

The variant is absent from gnomAD v4.1, v2.1, and gnomAD-Canada, indicating it is extremely rare in the general population.

4

Under the InSiGHT VCEP v2.0.0 for MLH1, the variant qualifies for PVS1_Very_Strong (IVS±1 disrupting reading frame with predicted NMD) and PM2_Supporting (absent from gnomAD v4 with AF <0.00002).

5

The variant has been reported in ClinVar as Likely pathogenic by two clinical laboratories (ClinVar Variation ID: 1727263) and has been observed once in somatic cancers (COSMIC: COSV99212388).

6

No functional studies, co-segregation data, de novo observations, or tumor phenotype data are available for this specific variant in the reviewed literature.

Final determination: No criteria-combination rule matched the adjudicated criteria 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 framework, so the variant remains a Variant of Uncertain Significance pending human review.

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	Met	NM_000249.4:c.307-1G>A disrupts the canonical splice acceptor of intron 3 (IVS3-1G>A). Under the InSiGHT VCEP v2.0.0, variants at IVS±1 or IVS±2 where exon skipping or cryptic splice site use disrupts the reading frame and is predicted to undergo NMD are assigned PVS1_Very_Strong. Skipping of exon 4 (91 bp) would cause a frameshift and premature termination codon predicted to trigger nonsense-mediated decay. SpliceAI supports a severe splicing defect with a max delta score of 0.99 (acceptor loss). PP3 is not applied per VCEP guidance for IVS±1/2 variants receiving PVS1.	cspec spliceai pvs1_variant_assessment pvs1_gene_context
PS1	N/A	Variant is a canonical splice site substitution, not a missense change. The PS1 rule for non-canonical splice nucleotides does not apply to canonical IVS±1/2 positions.	cspec
PS2	Not met	No de novo observations have been reported for this variant. No publications or database entries document confirmed de novo occurrence with maternity and paternity confirmed.
PS3	Not met	No variant-specific functional data were identified. The five publications associated with this case are clinical practice guidelines and policy statements that do not report experimental functional assays for NM_000249.4:c.307-1G>A. The VCEP functional assay documentation spreadsheet does not list this variant in calibrated assays.
PS4	N/A	PS4 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
PS5	N/A	PS5 is not defined in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
PM1	N/A	PM1 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
PM2	Met	The variant is absent from gnomAD v4.1 (0 alleles in the population database). Under the InSiGHT VCEP v2.0.0, an allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 qualifies for PM2_Supporting. The variant is also absent from gnomAD v2.1 and gnomAD-Canada.	gnomad_v4 gnomad_v2 gnomad_canada cspec
PM5	N/A	PM5 applies to missense changes at an amino acid residue where a different pathogenic missense change has been established. This is a canonical splice site variant (not a missense), and the pm5_candidates assessment confirms the variant class is not missense-like. PM5 is not applicable.	cspec pm5_candidates
PM6	N/A	PM6 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
PP1	Not met	No co-segregation data have been reported for this variant. No publications describe pedigree analysis or Bayes likelihood ratio calculations for NM_000249.4:c.307-1G>A.
PP2	N/A	PP2 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification. Additionally, this is a splice site variant, not a missense variant.	cspec
PP3	N/A	The InSiGHT VCEP v2.0.0 specifies that PP3 is not to be combined with PVS1 for IVS±1 or IVS±2 variants. Since this variant (c.307-1G>A, IVS3-1) receives PVS1_Very_Strong, PP3 is excluded per VCEP guidance. Additionally, the SpliceAI PP3 rule applies only to non-canonical splice nucleotides, and this is a canonical splice site.	cspec spliceai
PP4	Not met	No tumor MSI/IHC phenotype data have been reported for patients carrying this variant. PP4 requires MSI-H tumor results and/or loss of MMR protein expression consistent with the variant location. No such data are available in the case record.
PP5	N/A	PP5 is explicitly marked 'Not Applicable for this VCEP' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
BA1	Not met	BA1 requires gnomAD v4 Grpmax filtering allele frequency ≥0.001 (0.1%). The variant is absent from gnomAD v4.1 and all other population databases. Threshold not met.	gnomad_v4 cspec
BS1	Not met	BS1 requires gnomAD v4 allele frequency ≥0.0001 and <0.001 (0.01-0.1%). The variant is absent from gnomAD v4.1. Threshold not met.	gnomad_v4 cspec
BS2	Not met	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant in a patient without CMMRD features. No such data are available.
BS3	Not met	No functional studies demonstrating a benign effect for this variant have been identified. The five publications reviewed are clinical practice guidelines that do not contain experimental functional data.
BS4	Not met	No lack-of-segregation data have been reported. No pedigrees demonstrating absence of co-segregation with disease are available.
BP1	N/A	BP1 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
BP2	N/A	BP2 is explicitly marked 'Not Applicable' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
BP4	Not met	BP4_Supporting requires HCI prior probability <0.11 for missense variants, or SpliceAI delta score ≤0.1 for intronic/synonymous variants. This variant is intronic with SpliceAI max delta 0.99, far exceeding the 0.1 threshold. HCI prior is not available as this is not a missense variant. Both criteria for BP4 are not met.	spliceai cspec
BP5	Not met	No tumor data showing MSS status or normal MMR protein expression have been reported for patients carrying this variant. BP5 requires multiple tumors with MSS and/or normal protein expression to provide benign evidence.
BP6	N/A	BP6 is explicitly marked 'Not Applicable for this VCEP' in the InSiGHT VCEP v2.0.0 specification for MLH1.	cspec
BP7	Not met	BP7 applies to intronic variants at or beyond -21/+7 positions. The variant c.307-1G>A is at position -1 of intron 3, which is within the splice consensus region, not beyond it. The -1 position is a critical splice site nucleotide, not a deep intronic variant eligible for BP7.	cspec

Disclaimer: The content and results provided by LYFE Sciences are for research and educational purposes only and must not be used as a substitute for professional medical judgment, diagnosis, or treatment. Always consult a qualified healthcare professional before making any clinical decisions.