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MLH1
Final classification
VUS
MLH1 c.1558+4C>T · p.?
MLH1

PM2 (Supporting): allele frequency 2.478e-06 in gnomAD v4.1 (4/1,613,954 alleles), below the <0.00002 threshold.

Gene
MLH1
Transcript
NM_000249.4
HGVS · transcript:coding
NM_000249.4:c.1558+4C>T
Consequence
N/A
GRCh38
chr3:37028936 C>T
GRCh37
chr3:37070427 C>T
Basis 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). ▾
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
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
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
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