NM_000249.3:c.1655C>T (p.Thr552Ile) is a missense variant in exon 14 of MLH1 that is extremely rare in population databases, observed in 2 of 1,612,688 alleles in gnomAD v4.1 (grpmax filtering AF = 3.65e-06), meeting PM2_Supporting under the InSiGHT MLH1 VCEP v2.0.0.1 In silico predictors support a benign computational profile: the MLH1-specific HCI prior probability for pathogenicity is 0.0197, meeting BP4_Supporting (threshold <0.11). SpliceAI predicts no splicing impact (max delta = 0.01). REVEL (0.268) and BayesDel (0.361) are in the indeterminate range.2 This variant has been reported in ClinVar as Uncertain significance by three clinical laboratories (ClinVar Variation ID: 1467499) with no expert panel classification. No publications were identified that specifically report this variant.3 No functional data, segregation data, tumor phenotype data, or de novo observations are available for this variant. Multiple criteria (PS3, PP1, PP4, PS2, BS3, BS4, BS2, BP5) could not be assessed due to absence of evidence. Under the InSiGHT MLH1 VCEP v2.0.0 combining rules, one pathogenic supporting criterion (PM2_Supporting) and one benign supporting criterion (BP4_Supporting) with no other criteria met results in classification as Uncertain Significance — conflicting or insufficient evidence to classify.4
MLH1
Final classification
Uncertain Significance - Conflicting Evidence
MLH1 c.1655C>T · p.Thr552Ile
MLH1
NM_000249.3:c.1655C>T (p.Thr552Ile) is a missense variant in exon 14 of MLH1 that is extremely rare in population databases, observed in 2 of 1,612,688 alleles in gnomAD v4.1 (grpmax filtering AF = 3.65e-06), meeting PM2_Supporting under the InSiGHT MLH1 VCEP v2.0.0.
ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0.0 v2.0.0 criteria-combination framework: matched Rule31 (Benign.Supporting >=1 + Pathogenic.Supporting >=1) with applied criteria: PM2 supporting, BP4 supporting benign; maps to Uncertain Significance - Conflicting Evidence.
Classification rationale
PM2
BP4
Uncertain Significance - Conflicting Evidence
MLH1 c.1655C>T
PM2 + BP4
→
Uncertain Significance - Conflicting Evidence
Gene diagram
· NM_000249.3 · variants mapped to exon structure
MLH1
NM_000249.3
Fetching transcript structure from UCSC…
Applied criteria · 2 applied · 13 assessed
Applied · 2
Strength
Supporting
Moderate
Strong
Very strong
✓
PM2
supporting
Pathogenic
This variant is extremely rare in population databases. In gnomAD v4.1, it is observed in 2 of 1,612,688 alleles (grpmax filtering AF = 3.65e-06), meeting the InSiGHT VCEP threshold for PM2_Supporting (AF < 0.00002, i.e., <1 in 50,000 alleles). It is absent from gnomAD v2.1 and gnomAD-Canada v1.0.
gnomAD v4.1: 2/1612688 alleles
✓
BP4
supporting
Benign
The HCI prior probability for pathogenicity is 0.0197, which meets the InSiGHT VCEP BP4_Supporting threshold for missense variants (HCI prior <0.11). SpliceAI predicts no splicing impact (max delta = 0.01). Multiple in silico predictors (REVEL 0.268, BayesDel 0.361) are consistent with a variant of uncertain significance and do not suggest a damaging effect.
HCI prior: 0.0197meets BP4_Supporting threshold (<0.11).SpliceAI max delta: 0.01 (no predicted splicing impact).
Assessed · not applied
Pathogenic
PS1
No other nucleotide change encoding the same amino acid substitution (Thr552Ile) has been classified as Pathogenic by the InSiGHT VCEP.
PS2
No de novo observations have been reported for this variant.
PS3
No variant-specific functional data are available.
PM5
This is a missense change at residue 552 (p.Thr552Ile).
PP1
No co-segregation data are available for this variant.
PP3
The HCI prior probability for pathogenicity is 0.0197, which does not meet the VCEP thresholds for PP3 (Moderate: >0.81; Supporting: >0.68 and ≤0.81).
PP4
No tumor IHC or MSI data are available for this variant.
Benign
BA1
The gnomAD v4.1 grpmax filtering allele frequency is 3.65e-06 (0.000365%), far below the InSiGHT VCEP BA1 threshold of ≥0.001 (≥0.1%).
BS1
The gnomAD v4.1 grpmax filtering allele frequency is 3.65e-06 (0.000365%), below the InSiGHT VCEP BS1 threshold of ≥0.0001 (≥0.01%).
BS2
No data on co-occurrence in trans with a known pathogenic MLH1 variant are available.
BS3
No variant-specific functional data demonstrating a benign effect are available.
BS4
No co-segregation data are available to assess lack of segregation with disease.
BP5
No data on tumors with MSS or normal MMR protein expression in carriers of this variant are available.
N/A · 13
PVS1 · PS4 · PM1 · PM3 · PM4 · PM6 · PP2 · PP5 · BP1 · BP2 · BP3 · BP6 · BP7
Research & evidence
Population frequency
gnomAD v4.1
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 1.24017e-06; MAF= 0.00012%, 2/1612688 alleles, homozygotes = 0) and has highest observed frequency in the South Asian population (AF= 2.19655e-05; MAF= 0.00220%, 2/91052 alleles, homozygotes = 0); grpmax FAF= 3.65e-06.
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
0.00012%
· 2 / 1,612,688
0 hom · FAF 0.00037%
0 hom · FAF 0.00037%
South Asian 2 / 91,052 |
0.0022% |
+ 9 not observed (Remaining individuals, Admixed American, European (Finnish), Amish, East Asian, Middle Eastern, Ashkenazi Jewish, African/African American, European (non-Finnish))
gnomAD v2.1
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
gnomAD Canada 🇨🇦
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
ClinVar
This variant has been reported in ClinVar as Uncertain significance (3 clinical laboratories). (ClinVarID = 1467499)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01). REVEL score = 0.268. BayesDel score = 0.361201. HCI prior probability for pathogenicity = 0.0197. MAPP score = 4.37. Custom PP2 score = 0.457.
Functional
Unknown Oncogenic Effect
OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. MLH1, a DNA mismatch repair protein, is recurrently altered by deletion and mutation in various cancer types.
COSMIC
Cancer hotspots
Somatic evidence
Not in COSMIC / hotspots
COSMIC
This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).
Hotspots
This variant does not lie in a statistically significant hotspot.
Sources & reference links
9Sources
Triaged references · 6 PMIDs 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
11598466 ↗
Practice parameters for the identification and testing of patients at risk for dominantly inherited colorectal cancer--supporting documentation.
CLINVAR
15604628 ↗
Genetic cancer risk assessment and counseling: recommendations of the national society of genetic counselors.
CLINVAR
22167527 ↗
Identification of individuals at risk for Lynch syndrome using targeted evaluations and genetic testing: National Society of Genetic Counselors and the Collaborative Group of the Americas on Inherited Colorectal Cancer joint practice guideline.
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