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NM_000249.4:c.1558+4C>T
p.? · MLH1
ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0 · v2.0
0%
complete
Final classification
VUS
PM2BP4
MLH1
c.1558+4C>T
p.?
Gene context NCBI Gene ↗ OncoKB ↗

MLH1 encodes a DNA mismatch repair protein that, together with partners such as PMS2, corrects errors that arise during DNA replication. Loss of this repair function leads to the accumulation of mutations, particularly in repetitive microsatellite sequences, and tumors with defective MLH1 typically show high microsatellite instability. Germline mutations in MLH1 cause Lynch syndrome (hereditary non-polyposis colorectal cancer), which strongly predisposes to colorectal, endometrial, ovarian, and other cancers, while biallelic mutations cause constitutional mismatch repair deficiency (CMMRD). Because it normally protects against cancer by maintaining genomic stability, MLH1 acts as a tumor suppressor, and mismatch-repair-deficient tumors often respond well to immunotherapy.

This variant

MLH1 is a DNA mismatch repair gene whose loss causes Lynch syndrome and microsatellite-unstable tumors. This intronic splice-site variant is a VUS because the only available evidence points in conflicting directions - an extremely low population frequency (pathogenic-supporting) versus no predicted splicing impact (benign-supporting) - and no functional, tumor, or family data exists to resolve it. A VUS means current evidence neither establishes nor excludes Lynch syndrome risk for carriers.

Transcript
NM_000249.4
HGVS · transcript:coding
NM_000249.4:c.1558+4C>T
GRCh38
chr3:37028936 C>T
GRCh37
chr3:37070427 C>T
VUS: only PM2_Supporting (gnomAD AF 2.48e-06 < 0.00002) and BP4_Supporting (SpliceAI max delta 0.00 <= 0.1) are met, and one benign-supporting plus one pathogenic-supporting criterion maps to Uncertain Significance (Rule31).
Classification rationale
PM2 BP4 VUS
MLH1 c.1558+4C>T

PM2 (Supporting): allele frequency 2.478e-06 in gnomAD v4.1 (4/1,613,954 alleles), below the <0.00002 threshold. BP4 (Supporting): SpliceAI max delta 0.00 is at or below the <=0.1 threshold, predicting no splicing impact for this intronic variant. Overall: VUS (Uncertain Significance - Conflicting Evidence), per Rule31 requiring at least one benign-supporting and one pathogenic-supporting criterion.

PM2 + BP4 → VUS
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 · 2 applied · 15 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 · 2
Strength Supporting Moderate Strong Very strong
PM2 supporting Pathogenic
Met (Supporting): gnomAD v4.1 total allele frequency 2.478e-06 (4/1,613,954 alleles) is below the <0.00002 threshold.
gnomAD v4.1 (gnomad_v4): total AF 2.478e-06 (4/1,613,954 alleles; <1 in 50,000), joint grpmax FAF 4.43e-06, homozygotes 0; AFR subpopulation AF 2.667e-05 (2/74,988)gnomAD v2.1 (gnomad_v2): total AF 7.958e-06 (2/251,334 alleles), homozygotes 0gnomAD-Canada v1.0 (gnomad_canada): variant absent
gnomad_v4 ↗gnomad_v2 ↗gnomad_canada ↗cspec ↗
BP4 supporting Benign
Met (Supporting): SpliceAI max delta 0.00 is at or below the <=0.1 BP4 threshold, predicting no splicing impact.
SpliceAI (SpliceAI Lookup, Broad Institute) for NM_000249.4:c.1558+4C>T: max_delta_score = 0.00; DS_AG = 0.00, DS_AL = 0.00, DS_DG = 0.00, DS_DL = 0.00.InSiGHT MLH1 VCEP v2.0 (cspec doc 1564688410) BP4 rule: 'For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023' — threshold 0.1 named by the governing VCEP specification; max delta 0.00 <= 0.1, BP4 Supporting met.
spliceai ↗cspec ↗
Assessed · not applied · 7 not met · 8 not assessed
Pathogenic
PVS1 Not met: intronic +4 variant with no predicted protein consequence and SpliceAI max delta 0.00, so no loss-of-function mechanism is established.
PS1 Not met: no Pathogenic or Likely Pathogenic variant at the same +4 nucleotide exists, and SpliceAI max delta is 0.00.
PS2 Not assessed: no parental testing or reported de novo occurrence is available to score de novo evidence.
PS3 Not assessed: no calibrated functional assay, MMR defect, or mRNA expression data exists for this variant.
PM3 Not assessed: no co-occurrence with a pathogenic MLH1 variant in trans or of unknown phase is reported, so no PM3 points can be assigned.
PP1 Not assessed: no pedigrees or carrier/meioses counts exist to compute a co-segregation Bayes likelihood ratio.
PP3 Not met: SpliceAI max delta 0.00 is well below the >=0.2 PP3 threshold, so no splice defect is predicted.
PP4 Not assessed: no MSI, MMR IHC, or MLH1 promoter methylation data is available for any carrier.
Benign
BA1 Not met: gnomAD v4.1 grpmax filtering AF 4.43e-06 is ~226-fold below the >=0.001 BA1 threshold.
BS1 Not met: gnomAD v4.1 grpmax filtering AF 4.43e-06 is below the 0.0001 lower bound of the BS1 window.
BS2 Not met: no in-trans co-occurrence with a pathogenic MLH1 variant is reported, and gnomAD shows zero homozygotes.
BS3 Not assessed: no mRNA (with NMD inhibition) or calibrated functional assay data exists; in silico SpliceAI predictions do not satisfy BS3.
BS4 Not assessed: no pedigrees or non-segregation observations exist to compute a likelihood ratio.
BP5 Not assessed: no MSI, MMR IHC, BRAF V600E, or MLH1 promoter methylation data exists to show an alternate molecular basis.
BP7 Not met: at donor position +4, the variant is not "at or beyond +7", so the BP7 positional requirement fails.
N/A · 11 PS4 · PM1 · PM4 · PM5 · PM6 · PP2 · PP5 · BP1 · BP2 · BP3 · BP6
Research & evidence
Population frequency
gnomAD v4.1 screenshot
gnomAD v4.1
gnomAD v2.1 screenshot
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 2.47839e-06; MAF= 0.00025%, 4/1613954 alleles, homozygotes = 0) and has highest observed frequency in the African/African American population (AF= 2.66709e-05; MAF= 0.00267%, 2/74988 alleles, homozygotes = 0); grpmax FAF= 4.43e-06.
v2.1
This variant is present in gnomAD v2.1 (AF= 7.95754e-06; MAF= 0.00080%, 2/251334 alleles, homozygotes = 0) and has highest observed frequency in the African/African American population (AF= 6.15233e-05; MAF= 0.00615%, 1/16254 alleles, homozygotes = 0).
🇨🇦 CA
Absent from gnomAD-Canada v1.0.
Allele frequency by ancestry
three datasets · side by side
gnomAD v4.1
0.00025% · 4 / 1,613,954
0 hom · FAF 0.00044%
African/African American
2 / 74,988
0.0027%
European (non-Finnish)
2 / 1,179,960
0.00017%
+ 8 not observed (Remaining individuals, Admixed American, European (Finnish), Amish, East Asian, Middle Eastern, South Asian, Ashkenazi Jewish)
gnomAD v2.1
0.0008% · 2 / 251,334
0 hom
African/African American
1 / 16,254
0.0062%
European (non-Finnish)
1 / 113,638
0.00088%
+ 6 not observed (Admixed American, Ashkenazi Jewish, East Asian, European (Finnish), Remaining individuals, South Asian)
gnomAD Canada 🇨🇦
Absent · 0 / ?
0 hom
Not observed in any ancestry group.
gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗
ClinVar screenshot
ClinVar
This variant has been reported in ClinVar as Uncertain significance (3 clinical laboratories) and as Likely benign (3 clinical laboratories). (ClinVarID = 187689)
ClinVar ↗
SpliceAI screenshot
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).
SpliceAI ↗
Functional No data
No calibrated functional assay or RNA evidence was identified for this variant.
OncoKB ↗
COSMIC screenshot
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 · 9 PMIDs not cited in assessment
25645574 ↗ ACG clinical guideline: Genetic testing and management of hereditary gastrointestinal cancer syndromes. CLINVAR
25741868 ↗ Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. CLINVAR
26467025 ↗ A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders. CLINVAR
29887214 ↗ Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes. CLINVAR
31672839 ↗ Management of patients with increased risk for familial pancreatic cancer: updated recommendations from the International Cancer of the Pancreas Screening (CAPS) Consortium. CLINVAR
20301390 ↗ Lynch Syndrome. CLINVAR
25394175 ↗ A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. CLINVAR
26389505 ↗ Genetics of Colorectal Cancer (PDQ®): Health Professional Version. CLINVAR
28492532 ↗ Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. CLINVAR