PM2_Supporting is met: the variant is extremely rare in gnomAD v4.1 (AF=1.86e-6, grpmax FAF=2.8e-7, well below the VCEP threshold of <0.00002).1 PP1_Strong is met: the variant cosegregates with Lynch syndrome in 11 individuals from an extended Scottish kindred and 5 Polish cancer families (PMID:23403630), with a combined Bayes Likelihood Ratio exceeding 18.7.2 PP4_Strong is met: among 43 reported carriers, 88% (15/17) of tumors tested were MSI-H and 100% (15/15) showed loss of MLH1 protein by IHC, exceeding the VCEP threshold of ≥3 independent MSI-H tumors in ≥2 families (PMID:23403630).3 BP4_Supporting is met: the HCI prior probability of pathogenicity is 0.0273 (<0.11), suggesting a benign computational prediction. However, this is contradicted by strong clinical evidence.4 The InSiGHT VCEP MLH1 v2.0.0 combination rules yield a classification of Pathogenic: ≥2 Pathogenic Strong criteria (PP1_Strong + PP4_Strong) triggers Rule 5 (Pathogenic). The single Benign Supporting criterion (BP4_Supporting) does not trigger a conflicting-evidence rule against Pathogenic Strong criteria.5
MLH1
Final classification
Pathogenic
MLH1 c.2041G>A · p.Ala681Thr
MLH1
PM2_Supporting is met: the variant is extremely rare in gnomAD v4.1 (AF=1.86e-6, grpmax FAF=2.8e-7, well below the VCEP threshold of <0.00002).
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 Rule5 (Pathogenic.Strong >=2) with applied criteria: PM2 supporting, PP1 strong, PP4 strong, BP4 supporting benign; maps to Pathogenic.
Classification rationale
PM2PP1PP4
BP4
Pathogenic
MLH1 c.2041G>A
PM2 + PP1 + PP4 + BP4
→
Pathogenic
4
hci_prior
5
cspec ↗final_classification_framework
Gene diagram
· NM_000249.4 · variants mapped to exon structure
MLH1
NM_000249.4
Fetching transcript structure from UCSC…
Applied criteria · 4 met · select any tile
Met
Not met
Not assessed
N/A
Strength
very strong
supporting
Pathogenic evidence
PVS
PS
PM
PP
Benign evidence
BA
BS
BP
—
—
—
Rationale
Select a criterion.
Sources
Evidence used
Gaps remaining
Rule
—
Research & evidence
Population frequency · supports pathogenic
gnomAD v4.1
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 1.85862e-06; MAF= 0.00019%, 3/1614098 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 1.66644e-05; MAF= 0.00167%, 1/60008 alleles, homozygotes = 0); grpmax FAF= 2.8e-07.
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
0.00019%
· 3 / 1,614,098
0 hom · FAF 2.8e-05%
0 hom · FAF 2.8e-05%
Admixed American 1 / 60,008 |
0.0017% |
European (non-Finnish) 2 / 1,179,974 |
0.00017% |
+ 8 not observed (Remaining individuals, European (Finnish), Amish, East Asian, Middle Eastern, South Asian, Ashkenazi Jewish, African/African American)
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 is present in ClinVar (Variation ID: 17099); submission details unavailable.
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01). REVEL score = 0.831. BayesDel score = 0.470252. HCI prior probability for pathogenicity = 0.0273. MAPP score = 8.22. Custom PP2 score = 0.198.
Functional
Inconclusive
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Inconclusive; curated oncogenicity label: Inconclusive.
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.
Literature · how each cited paper was used
1papers cited
Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 4 further PMIDs triaged but not cited — see Sources & References.
Expression defect size among unclassified MLH1 variants determines pathogenicity
Searched
c.2041G>Ap.Ala681ThrA681T
Found
MLH1 p.Ala681T was one of 38 missense variants functionally characterized. The variant was MMR proficient but showed a protein expression defect (~52% of wild-type) due to reduced stability (half-life decreased to 43% of wild-type). Clinical evaluation of 43 carriers showed average age at diagnosis of 42.6 years, 55% Amsterdam-positive, 88% tumor MSI, 100% loss of MLH1 IHC, and strong cosegregation in 11 individuals from a Scottish kindred and 5 Polish families. The variant was classified as pathogenic based on expression defect falling below the established pathogenicity threshold between neutral V716M and pathogenic A681T.
Variant
✓ Names this variant — characterised directly
Applied to
→PP1 supports · met
→PP4 supports · met
Why
Variant-specific functional and clinical evidence confirmed; expression defect and clinical data support PP1_Strong and PP4_Strong. Functional assay does not meet VCEP PS3 calibrated thresholds (MMR proficient, expression 52% not <10%).
Three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression.
Location Abstract; Results (Table 1, Figure 4); Discussion · Context Transient transfection in HEK293T cells; in vitro MMR assay; qPCR for transcript quantification; pulse-chase protein stability assay; differential scanning fluorimetry for thermal stability · full text
Sources & reference links
9Sources
Triaged references · 4 PMIDs not cited in assessment