Variant classification summary

NM_000249.4:c.381-1G>A

MLH1 · NP_000240.1:p.? · NM_000249.4

GRCh37: chr3:37048481 G>A · GRCh38: chr3:37006990 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

Pathogenic

PVS1 very strong PM2 supporting PP4 moderate

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.?

gnomAD AF

ClinVar

Pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.381-1G>A is a canonical splice acceptor variant (IVS4-1) disrupting the invariant AG dinucleotide of MLH1 intron 4, meeting PVS1_Very_Strong under the InSiGHT/ClinGen MLH1 VCEP v2.0.

2

The variant is absent from gnomAD v2.1 and v4.1, meeting PM2_Supporting under the VCEP threshold of allele frequency <0.00002.

3

A patient with this variant, reported by Roth et al. (2016), had two synchronous colorectal cancers showing loss of MLH1 and PMS2 protein expression by immunohistochemistry, meeting PP4_Moderate under the VCEP rule for 2 independent tumors with MMR protein loss consistent with the variant location.

4

This variant was identified as a recurrent mutation in 5 African American families with Lynch syndrome by Guindalini et al. (2015), supporting its enrichment in the Lynch syndrome patient population.

5

ClinVar classifies this variant as Pathogenic (4 clinical laboratories) and Likely pathogenic (1 laboratory); however, the VCEP framework does not permit use of PP5 or BP6, and ClinVar classification alone is not used as an independent criterion.

6

Combining PVS1_Very_Strong (1) + PP4_Moderate (1) under the InSiGHT/ClinGen MLH1 VCEP v2.0 combination rules (Rule 10) yields a classification of Likely Pathogenic.

Final determination: Rule3 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 Pathogenic.

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.381-1G>A is a canonical splice acceptor variant at IVS4-1 (intron 4), disrupting the invariant AG dinucleotide. Under the InSiGHT/ClinGen MLH1 VCEP v2.0, IVS±1 variants where exon skipping disrupts the reading frame and is predicted to undergo NMD qualify for PVS1_Very_Strong. MLH1 loss of function is an established disease mechanism for Lynch syndrome. SpliceAI max delta score of 0.99 confirms a severe splicing impact. Not combined with PP3 per VCEP rules.	cspec spliceai pvs1_variant_assessment pvs1_gene_context
PS1	N/A	PS1 applies to missense variants encoding the same amino acid change as a known pathogenic variant, or to non-canonical splice variants affecting the same nucleotide as a known pathogenic splice variant. c.381-1G>A is a canonical (±1/±2) splice site variant, not a missense substitution, and the VCEP PS1 rule for splice applies only to non-canonical positions.
PS2	Not met	No de novo occurrence data identified for NM_000249.4:c.381-1G>A in the reviewed evidence. VCEP PS2 requires confirmation of both maternity and paternity in a proband with a Lynch syndrome-spectrum tumor; no such data are available in the case materials or literature.
PS3	Not met	No variant-specific functional assay data or calibrated functional odds for NM_000249.4:c.381-1G>A were identified. The VCEP functional assay SVI documentation spreadsheet was searched and this variant was not listed. The variant is a canonical splice site variant; splicing impact is predicted by SpliceAI (delta 0.99) and captured under PVS1, not PS3.	vcep_functional_assay_svi_documentation_mmr
PS4	N/A	PS4 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0.
PS5	N/A	PS5 is not part of the MLH1 VCEP criteria framework (InSiGHT/ClinGen v2.0).
PM1	N/A	PM1 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0.
PM2	Met	NM_000249.4:c.381-1G>A is absent from gnomAD v2.1 (exomes), gnomAD v4.1 (exomes), and gnomAD-Canada v1.0 (genomes). Under the MLH1 VCEP, PM2_Supporting applies when the variant is absent or extremely rare with allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	PM5 requires a missense change at an amino acid residue where a different pathogenic missense change has been observed. c.381-1G>A is a canonical splice variant, not a missense substitution, and has no protein-level comparator candidates.
PM6	N/A	PM6 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0.
PP1	Not met	No co-segregation data with Bayes Likelihood Ratio calculations were identified in the reviewed evidence. The VCEP PP1 rules require combined Bayes Likelihood Ratio >2.08 for supporting strength; no pedigrees with segregation analysis were available.
PP2	N/A	PP2 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0 (missense variant in gene with low rate of benign missense changes does not apply).
PP3	Not met	The VCEP PVS1 rule for canonical (±1/±2) splice variants explicitly states: 'Not to be combined with PP3.' PP3 for splice prediction is reserved for non-canonical splice variants with SpliceAI delta >= 0.2. The splicing impact of this canonical acceptor variant is already fully captured by PVS1_Very_Strong; applying PP3 would constitute double-counting of the same evidence. For missense application, the variant is not a missense change and was not found in the MLH1 HCI prior lookup table.	cspec spliceai
PP4	Met	A patient with this MLH1 variant (reported as IVS4-1G>A) was described by Roth et al. (2016, PMID:27357288) with two synchronous primary colorectal cancers (cecum and rectosigmoid colon), both demonstrating loss of MLH1 and PMS2 protein expression by immunohistochemistry, consistent with the variant location. Under the MLH1 VCEP, 2 independent CRC tumors with loss of MMR protein expression consistent with the variant location qualifies for PP4_Moderate. Independent tumors can be from the same patient/family per VCEP guidance.	PMID:27357288
PP5	N/A	PP5 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0 (this criterion is not for use as recommended by the ClinGen SVI VCEP Review Committee).
BA1	Not met	NM_000249.4:c.381-1G>A is absent from gnomAD v4.1. The VCEP BA1 threshold requires grpmax filtering allele frequency >= 0.001 (0.1%), which is not met.	gnomad_v4
BS1	Not met	Variant is absent from gnomAD v4.1. The VCEP BS1 threshold requires grpmax filtering allele frequency >= 0.0001 and < 0.001 (0.01-0.1%), which is not met.	gnomad_v4
BS2	Not met	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant was identified in the case materials or reviewed literature. VCEP BS2 requires confirmed phase (parental testing) demonstrating co-occurrence in trans with a known pathogenic variant in a patient with CRC after age 45 and no CMMRD features.
BS3	Not met	No functional assay data demonstrating a benign effect for NM_000249.4:c.381-1G>A were identified. The VCEP functional assay SVI documentation spreadsheet was searched and this variant was not listed among calibrated assays.	vcep_functional_assay_svi_documentation_mmr
BS4	Not met	No lack-of-segregation data were identified for NM_000249.4:c.381-1G>A. VCEP BS4 requires pedigrees with combined Bayes Likelihood Ratio <0.05 (strong) or between 0.05 and 0.48 (supporting); no such analysis is available.
BP1	N/A	BP1 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0.
BP2	N/A	BP2 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0 (BS2 is used instead).
BP3	N/A	Skipped: variant is a substitution, not an in-frame deletion/insertion in a repetitive region.
BP4	Not met	BP4_Supporting requires either a missense variant with HCI prior probability <0.11 or an intronic/synonymous variant with SpliceAI delta score <= 0.1. This variant is a canonical splice site variant with SpliceAI max delta score of 0.99, far exceeding the 0.1 threshold. It was not found in the HCI-PRIORS-MLH1 lookup table.	spliceai vcep_hci_priors_mlh1
BP5	Not met	No evidence was identified of tumors with MSS and/or intact MMR protein expression, or BRAF V600E/MLH1 methylation with MSI-H/MLH1 loss, in patients carrying this variant. VCEP BP5 requires tumors showing evidence inconsistent with a pathogenic MMR defect.
BP6	N/A	BP6 is marked Not Applicable by the InSiGHT/ClinGen MLH1 VCEP v2.0 (this criterion is not for use as recommended by the ClinGen SVI VCEP Review Committee).
BP7	Not met	BP7 applies to synonymous or intronic variants at or beyond -21/+7 (i.e., deep intronic or distal exonic positions). NM_000249.4:c.381-1G>A is a canonical splice acceptor variant at position -1, within the splice consensus region, not at or beyond -21/+7. Additionally, the variant is not synonymous/silent.

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