Classification rationale

PVS1PM2PP5 Pathogenic

MLH1 c.1732-2A>G canonical_splice · exon 15i

PVS1 (Very Strong): disrupts the canonical splice-acceptor AG dinucleotide, predicting exon 16 skipping that shifts the reading frame and triggers nonsense-mediated decay. PM2 (Supporting): variant is absent from gnomAD v4.1. PP5 (Supporting): InSiGHT expert panel classifies the exact variant as Likely pathogenic (ClinVar 3-star). Overall: Pathogenic, per Rule 4 (1 Very Strong + 2 Supporting) of the InSiGHT/ClinGen MLH1 VCEP v2.0 framework.

PVS1 + PM2 + PP5 → Pathogenic

Gene diagram · NM_000249.4 · variants mapped to exon structure

MLH1 NM_000249.4

Fetching transcript structure from UCSC…

Applied criteria · 3 applied · 12 assessed

MetEvidence satisfies this criterion.

Not metEvaluated against available evidence; threshold not reached.

Not assessedApplies in principle, but no evidence was found to evaluate it.

N/ADoesn't apply to this variant type.

Applied · 3

Strength Supporting Moderate Strong Very strong

✓

PVS1 very strong review Pathogenic

Met (Very Strong): disrupts the canonical splice-acceptor AG dinucleotide, predicting exon 16 skipping that shifts the reading frame and triggers nonsense-mediated decay. Flagged for human review: the reported SpliceAI max delta of 0.00 conflicts with expected biology and appears to be a tool artifact.

VariantValidator (prefetch.json) places c.1732-2A>G at the intron 15/exon 16 boundary ('start_exon'/'end_exon' = '15i'), confirming this is the canonical -2 acceptor position of intron 15.InSiGHT/ClinGen MLH1 VCEP Version 2.0 PVS1 rule (case_summary.json / prefetch.json cspec criteria) specifies default Very Strong for 'Variants at IVS±1 or IVS±2 ... where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD', with the caveat 'Not to be combined with PP3'.pvs1_gene_context.json establishes MLH1 germline loss-of-function as an official VCEP-endorsed disease mechanism (pvs1_gate = eligible), satisfying the PVS1 gene-level gate.

cspec ↗spliceai ↗

✓

PM2 supporting Pathogenic

Met (Supporting): absent from gnomAD v4.1, below the required allele-frequency threshold of 0.00002.

The MLH1 InSiGHT VCEP Version 2.0 rule defines PM2 Supporting as absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4.gnomAD v4.1 query for NC_000003.12:g.37047517A>G reports the variant as absent.

cspec ↗gnomad_v4 ↗

✓

PP5 supporting Pathogenic

Met (Supporting): the InSiGHT expert panel classified this exact variant as Likely pathogenic (ClinVar 3-star).

ClinVar identifies the exact transcript-level variant NM_000249.4(MLH1):c.1732-2A>G and an InSiGHT expert-panel classification of Likely pathogenic with three-star review status.The separate ClinVar laboratory submissions were excluded from PP5; only the exact-variant expert-panel assertion was used.ClinVar expert panel classification

clinvar ↗

Assessed · not applied · 3 not met · 9 not assessed

Pathogenic

PS2 Not assessed: no parental testing or other evidence establishes that the variant arose de novo.

PS3 Not assessed: no functional assay of this exact variant is available; a minigene study tested the related c.1732-2A>T change, not this A>G allele.

PM3 Not assessed: no second MLH1 variant, phase information, or CMMRD clinical evidence was available to apply the co-occurrence rule.

PP1 Not assessed: only an unverified statement of familial segregation was available, with no pedigree or informative meioses to quantify.

PP4 Not assessed: tumor findings were reported, but without a primary source or confirmation that MLH1 promoter methylation was excluded.

Benign

BA1 Not met: BA1 requires allele frequency >=0.001, but the variant is absent from gnomAD v4.1.

BS1 Not met: BS1 requires allele frequency 0.0001-0.001, but the variant is absent from gnomAD v4.1.

BS2 Not assessed: no confirmed in-trans co-occurrence with a pathogenic MLH1 variant was available.

BS3 Not assessed: no functional assay demonstrates normal splicing or protein function; the benign no-aberration rule does not apply to canonical splice sites.

BS4 Not assessed: no affected relative tested negative for the variant, so no non-segregation evidence exists.

BP5 Not assessed: insufficient evidence on tumor phenotype, MMR loss, or BRAF/methylation status to apply the inconsistency rule.

BP6 Not met: the expert-panel classification is Likely pathogenic, not benign, so no benign assertion supports BP6.

N/A · 13 PS1 · PS4 · PM1 · PM4 · PM5 · PM6 · PP2 · PP3 · BP1 · BP2 · BP3 · BP4 · BP7

Research & evidence

Population frequency · supports pathogenic

gnomAD v4.1

gnomAD v2.1

v4.1

Absent from gnomAD v4.1.

v2.1

Absent from gnomAD v2.1.

🇨🇦 CA

Absent from gnomAD-Canada v1.0.

Allele frequency by ancestry

three datasets · side by side

gnomAD v4.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v2.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD Canada 🇨🇦

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗

ClinVar

This variant has been reported in ClinVar as Pathogenic (2 clinical laboratories) and as likely pathogenic (1 clinical laboratory) and as Likely pathogenic (1 clinical laboratory) and as Likely pathogenic by International Society for Gastrointestinal Hereditary Tumours (InSiGHT) (expert panel). (ClinVarID = 89861)

ClinVar ↗

In silico

SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). BayesDel score = 0.66.

SpliceAI ↗

Functional No data

No calibrated functional assay or RNA evidence was identified for this variant.

OncoKB ↗

COSMIC

Somatic evidence

COSMIC

This variant has not previously been reported in somatic cancers (COSMIC).

Hotspots

This variant does not lie in a statistically significant cancer hotspot.

COSMIC ↗

Sources & reference links

8Sources

CSpec VCEP

ClinVar

gnomAD v2.1

gnomAD v4.1

gnomAD-Canada

SpliceAI

OncoKB

COSMIC

Triaged references · 8 PMIDs not cited in assessment

16395668 ↗ Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. CLINVAR

24090359 ↗ Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients. CLINVAR

25645574 ↗ ACG clinical guideline: Genetic testing and management of hereditary gastrointestinal cancer syndromes. CLINVAR

26467025 ↗ A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders. CLINVAR

30998989 ↗ Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome. CLINVAR

8571956 ↗ Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16. CLINVAR

9245993 ↗ Reduced frequency of extracolonic cancers in hereditary nonpolyposis colorectal cancer families with monoallelic hMLH1 expression. CLINVAR

24310308 ↗ ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis). CLINVAR