NM_000249.4:c.1039-25T>A is an intronic variant located 25 bases upstream of exon 11 in MLH1. This variant is present in gnomAD v4.1 with a joint grpmax filtering allele frequency of 0.157% (159/721,850 alleles), exceeding the InSiGHT MLH1 VCEP BA1 stand-alone benign threshold of 0.1%.1 The variant is observed across multiple continental populations with highest frequency in the African/African American population (0.194%), consistent with a common polymorphism rather than a founder pathogenic variant.2 SpliceAI predicts no splicing impact (max delta score 0.03), supporting application of BP4 (Supporting) for an intronic variant with no predicted splice defect.3 The variant's intronic position at -25 relative to exon 11 satisfies BP7 (Supporting) under the VCEP rule for variants at or beyond the -21/+7 boundary.4 The variant has been reported in ClinVar as Likely benign by two clinical laboratories (ClinVar Variation ID 492685), consistent with the population frequency data.5
MLH1
Final classification
Benign
MLH1 c.1039-25T>A · p.?
MLH1
NM_000249.4:c.1039-25T>A is an intronic variant located 25 bases upstream of exon 11 in MLH1.
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: matched Rule17 (1 Benign.Stand Alone) with applied criteria: BA1 stand-alone benign, BP4 supporting, BP7 supporting; maps to Benign.
Classification rationale
BA1BP4BP7
Benign
MLH1 c.1039-25T>A
BA1 + BP4 + BP7
→
Benign
Gene diagram
· NM_000249.4 · variants mapped to exon structure
MLH1
NM_000249.4
Fetching transcript structure from UCSC…
Applied criteria · 3 applied · 13 assessed
Applied · 3
Strength
Supporting
Moderate
Strong
Very strong
✓
BA1
stand-alone
Benign
Under the MLH1 VCEP, BA1 (Stand Alone) applies when the gnomAD v4 grpmax filtering allele frequency is ≥ 0.001 (0.1%) and the variant is excluded as a founder pathogenic variant. The gnomAD v4.1 joint grpmax FAF is 0.00157 (0.157%), which exceeds the 0.1% threshold. The variant is observed across multiple continental populations (AFR 0.194%, AMR 0.017%, ASJ 0.039%) with no evidence of a founder effect. The ClinVar classification of Likely benign from clinical laboratories is consistent with this being a common polymorphism.
gnomAD v4.1 joint grpmax FAF 0.00157 (0.157%) ≥ VCEP BA1 threshold of 0.001Observed in multiple populations: AFR (69/35582
✓
BP4
supporting
Benign
Under the MLH1 VCEP, BP4_Supporting applies to intronic variants when SpliceAI predicts no splicing impact with a delta score ≤ 0.1 (Walker et al. 2023). SpliceAI analysis of NM_000249.4:c.1039-25T>A yields a maximum delta score of 0.03, indicating no predicted effect on splicing. This criterion is independent of BP7 and both may be applied.
SpliceAI max delta 0.03 ≤ 0.1 — predicts no splicing impactVCEP BP4 rule for intronic variants: SpliceAI delta ≤ 0.1
✓
BP7
supporting
Benign
Under the MLH1 VCEP, BP7_Supporting applies to intronic variants at or beyond -21/+7 of the exon boundary. NM_000249.4:c.1039-25T>A is located at position -25 relative to exon 11, which is beyond the -21 threshold. SpliceAI also predicts no splicing impact (delta 0.03). Per VCEP guidance, variants may satisfy both BP7 and BP4.
Intronic variant at c.1039-25 (position -25)beyond the -21 thresholdSpliceAI delta 0.03 — no predicted splicing impact
Assessed · not applied
Pathogenic
PVS1
NM_000249.4:c.1039-25T>A is a deep intronic variant located 25 bases upstream of exon 11.
PS1
PS1 under the MLH1 VCEP requires either (a) a missense substitution encoding the same amino acid change as a VCEP-classified Pathogenic variant, or (b) a variant affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse SpliceAI prediction.
PS2
No de novo occurrence data are available for NM_000249.4:c.1039-25T>A.
PS3
No functional data are available for NM_000249.4:c.1039-25T>A.
PM2
Under the MLH1 VCEP, PM2_Supporting requires an allele frequency < 0.00002 (< 1 in 50,000 alleles) in gnomAD v4.
PP1
No co-segregation data are available for NM_000249.4:c.1039-25T>A.
PP3
Under the MLH1 VCEP, PP3 for non-canonical splice variants requires a SpliceAI delta score ≥ 0.2 (Supporting) or an HCI prior probability > 0.68 (missense variants only).
PP4
No tumor phenotype data (MSI status, IHC for MMR proteins) are available for carriers of NM_000249.4:c.1039-25T>A.
Benign
BS1
The MLH1 VCEP BS1 threshold is gnomAD v4 grpmax FAF ≥ 0.0001 and < 0.001.
BS2
No co-occurrence data are available for NM_000249.4:c.1039-25T>A.
BS3
No functional data demonstrating a benign effect are available for NM_000249.4:c.1039-25T>A.
BS4
No lack-of-segregation data are available for NM_000249.4:c.1039-25T>A.
BP5
No tumor phenotype data are available for evaluation under BP5.
N/A · 9
PS4 · PM1 · PM5 · PM6 · PP2 · PP5 · BP1 · BP2 · BP6
Research & evidence
Population frequency · supports benign
gnomAD v4.1
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 0.000220267; MAF= 0.02203%, 159/721850 alleles, homozygotes = 0) and has highest observed frequency in the African/African American population (AF= 0.00193918; MAF= 0.19392%, 69/35582 alleles, homozygotes = 0); grpmax FAF= 0.00157149.
v2.1
This variant is present in gnomAD v2.1 (AF= 0.000921359; MAF= 0.09214%, 34/36902 alleles, homozygotes = 0) and has highest observed frequency in the African/African American population (AF= 0.00301887; MAF= 0.30189%, 16/5300 alleles, homozygotes = 0); grpmax FAF= 0.00198078.
🇨🇦 CA
This variant is present in gnomAD-Canada v1.0 (AF= 0.00040830611292580493, 7/17144 alleles, homozygotes = 0).
Allele frequency by ancestry
three datasets · side by side
gnomAD v4.1
0.022%
· 159 / 721,850
0 hom · FAF 0.16%
0 hom · FAF 0.16%
African/African American 69 / 35,582 |
0.19% |
South Asian 28 / 22,538 |
0.12% |
Ashkenazi Jewish 4 / 10,280 |
0.039% |
Admixed American 3 / 17,440 |
0.017% |
Remaining individuals 3 / 24,634 |
0.012% |
East Asian 2 / 18,806 |
0.011% |
European (non-Finnish) 49 / 573,780 |
0.0085% |
European (Finnish) 1 / 16,150 |
0.0062% |
+ 2 not observed (Amish, Middle Eastern)
gnomAD v2.1
0.092%
· 34 / 36,902
0 hom · FAF 0.2%
0 hom · FAF 0.2%
African/African American 16 / 5,300 |
0.3% |
Admixed American 2 / 1,248 |
0.16% |
Ashkenazi Jewish 2 / 1,624 |
0.12% |
South Asian 5 / 4,298 |
0.12% |
European (non-Finnish) 8 / 18,924 |
0.042% |
European (Finnish) 1 / 2,462 |
0.041% |
+ 2 not observed (East Asian, Remaining individuals)
gnomAD Canada 🇨🇦
0.041%
· 7 / 17,144
0 hom
0 hom
⚠ LCR
indel · split
Middle Eastern 1 / 138 |
0.72% |
Ashkenazi Jewish 2 / 746 |
0.27% |
African/African American 1 / 734 |
0.14% |
European (non-Finnish) 3 / 11,068 |
0.027% |
+ 5 not observed (Latino/Admixed American, East Asian, European (Finnish), Remaining individuals, South Asian)
ClinVar
This variant has been reported in ClinVar as Likely benign (2 clinical laboratories). (ClinVarID = 492685)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.03).
Functional
No data
No calibrated functional assay or RNA evidence was identified for this variant.
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.
Sources & reference links
Triaged references · 1 PMID not cited in assessment
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