Classification rationale

PVS1PM2PP5 Pathogenic

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

PVS1 (Very Strong): disruption of the canonical splice-acceptor -2 position is predicted to abolish the natural acceptor (SpliceAI max delta 0.996), with exon skipping expected to trigger nonsense-mediated decay. PM2 (Supporting): absent from gnomAD v4.1, below the 0.00002 allele-frequency threshold. PP5 (Supporting): the InSiGHT expert panel classified this variant as Likely pathogenic. Synthesis: PVS1 (Very Strong) with PM2 and PP5 (Supporting) satisfies MLH1 InSiGHT VCEP Rule4 (1 Very Strong + at least 2 Supporting), producing a final classification of Pathogenic.

PVS1 + PM2 + PP5 → Pathogenic

LYFE Sciences is an AI system, and it can make mistakes. Criteria may be applied incorrectly, sources may be misread, and a confident-looking classification can still be wrong. Double-check every criterion and its underlying evidence before relying on any call.

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 -2 position; SpliceAI predicts near-total acceptor loss (max delta 0.996), with exon skipping predicted to trigger nonsense-mediated decay. Caveat for review: the frameshift/NMD outcome was assumed, as RNA confirmation was not available.

InSiGHT MLH1 CSPEC v2.0 PVS1 rule (Very Strong tier): variants at IVS+/-1 or IVS+/-2 where exon skipping or cryptic splice site use disrupts reading frame and is predicted to undergo NMD; not to be combined with PP3 and not for use with a confirmed splice defect from RNA data.VariantValidator exonic position annotation places the variant at '15i' (intron 15/exon 16 boundary) for both GRCh37 and GRCh38, confirming the variant lies at the canonical splice acceptor consensus immediately 5' of an MLH1 exon.SpliceAI lookup (queried at NM_000249.4:c.1732-2A>G) reports DS_AG=0.732, DS_AL=0.996 (acceptor loss), DS_DG=0.0, DS_DL=0.114, with max delta score 0.996, indicating near-certain loss of the natural splice acceptor and no meaningful competing donor-site signal.

cspec ↗spliceai ↗

PM2 supporting Pathogenic

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

The applicable MLH1 InSiGHT VCEP specification (version 2.0) assigns PM2 at Supporting strength for an absent or extremely rare gnomAD v4 allele frequency below 0.00002 (less than 1 in 50,000 alleles).gnomAD v4.1 reports NM_000249.4:c.1732-2A>G (GRCh38 chr3:37047517 A>G) as absent, satisfying the below-0.00002 threshold.

cspec ↗gnomad_v4 ↗

PP5 supporting Pathogenic

Met (Supporting): the InSiGHT expert panel classified this variant as Likely pathogenic.

ClinVar variation 89861 is an exact transcript-HGVS match and includes SCV000106332 from InSiGHT, reviewed by expert panel, with Likely pathogenic significance for Lynch syndrome.ClinVar expert panel classification

clinvar ↗

Assessed · not applied · 2 not met · 10 not assessed

Pathogenic

PS2 Not assessed: no de novo observation or parental test results were available.

PS3 Not assessed: no calibrated functional assay or RNA/cDNA study of this exact variant was available.

PM3 Not assessed: no evidence of a second MLH1 variant or phase information indicating CMMRD was available.

PP1 Not assessed: no pedigree genotypes or segregation data were available.

PP4 Not assessed: an MSI-H tumor with MLH1/PMS2 loss was reported, but no primary tumor report confirmed the MSI method, tumor independence, or methylation exclusion.

Benign

BA1 Not met: absent from gnomAD v4.1, so the BA1 population-frequency threshold cannot be reached.

BS1 Not met: absent from gnomAD v4.1, so the BS1 population-frequency range cannot be reached.

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

BS3 Not assessed: no calibrated functional assay demonstrated normal or proficient MLH1 function for this variant.

BS4 Not assessed: no non-segregation observations or pedigree likelihood data were available.

BP5 Not assessed: no evidence of an alternative molecular basis, such as MLH1 promoter methylation or BRAF V600E-positive tumors, was available.

BP6 Not assessed: no expert-panel benign classification exists; the only panel assertion is InSiGHT Likely pathogenic.

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 possible splice impact for this variant (max delta score = 1.00). 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

23788249 ↗ ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. CLINVAR

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