Variant classification summary

NM_000249.3:c.1655C>T

MLH1 · NP_000240.1:p.(Thr552Ile) · NM_000249.3

GRCh37: chr3:37081773 C>T · GRCh38: chr3:37040282 C>T

Gene: MLH1 Transcript: NM_000249.3

Final call

PM2 supporting BP4 supporting benign

Variant details

Gene

MLH1

Transcript

NM_000249.3

Protein

NP_000240.1:p.(Thr552Ile)

gnomAD AF

1.2401654876826764e-06 (v4.1)

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

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.

2

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.

3

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.

4

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.

5

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.

Final determination: Rule31 in the 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 is satisfied by the adjudicated criteria, so the variant is classified as Uncertain Significance - Conflicting Evidence.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	PVS1 is not applicable to missense substitutions under the InSiGHT MLH1 VCEP v2.0.0 framework. The VCEP reserves PVS1 for null variants (nonsense, frameshift, canonical splice ±1,2, initiation codon, or confirmed splicing aberrations). NM_000249.3:c.1655C>T is a missense change (p.Thr552Ile) and does not fall into any PVS1 bucket.	pvs1_gene_context pvs1_variant_assessment
PS1	Not met	No other nucleotide change encoding the same amino acid substitution (Thr552Ile) has been classified as Pathogenic by the InSiGHT VCEP. The VCEP pilot variant spreadsheet does not include any codon 552 variant, and no VCEP-classified comparator exists.	hci_prior vcep_vcep_pilot_variants_mmr
PS2	Not assessed	No de novo observations have been reported for this variant. No publications or ClinVar submissions document a confirmed de novo occurrence.
PS3	Not assessed	No variant-specific functional data are available. The variant was not found in the VCEP Functional-assay-SVI-documentation-MMR spreadsheet. OncoKB reports Unknown Oncogenic Effect with no variant-specific functional evidence. No publications report functional assay data for c.1655C>T.	oncokb vcep_functional_assay_svi_documentation_mmr
PS4	N/A	PS4 is not applicable under the InSiGHT MLH1 VCEP v2.0.0. The VCEP states: 'Due to the availability of tumor IHC data for variant classification (see PP4), PS4 has not been utilized for MMR variant classification using proband counting.'	cspec
PS5	N/A	PS5 is not defined in the InSiGHT MLH1 VCEP v2.0.0 criteria set. This criterion does not exist in the current framework.	cspec
PM1	N/A	PM1 is not applicable under the InSiGHT MLH1 VCEP v2.0.0. The VCEP states: 'There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains.'	cspec
PM2	Met	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 gnomad_v2 gnomad_canada
PM5	Not met	This is a missense change at residue 552 (p.Thr552Ile). However, no different missense change at the same residue has been classified as Pathogenic or Likely Pathogenic by the InSiGHT VCEP. The two other codon 552 missense variants (c.1655C>A/p.T552N and c.1655C>G/p.T552S) are not present in the VCEP pilot variant spreadsheet, and no VCEP-classified comparator exists.	hci_prior vcep_vcep_pilot_variants_mmr pm5_candidates
PM6	N/A	PM6 is not applicable under the InSiGHT MLH1 VCEP v2.0.0. The VCEP directs users to PS2 instead for de novo assessment.	cspec
PP1	Not assessed	No co-segregation data are available for this variant. No publications or ClinVar submissions report segregation analysis in affected families.
PP2	N/A	PP2 is not applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
PP3	Not met	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). SpliceAI predicts no splicing impact (max delta = 0.01), below the 0.2 threshold for predicted splice defects. REVEL (0.268) and BayesDel (0.361) are in the indeterminate range and do not independently establish PP3 under VCEP rules.	hci_prior spliceai revel bayesdel
PP4	Not assessed	No tumor IHC or MSI data are available for this variant. No publications or ClinVar submissions report MSI-H status or MMR protein expression in tumors from carriers of this variant.
PP5	N/A	PP5 is not applicable under the InSiGHT MLH1 VCEP v2.0.0. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. No expert panel has classified this variant as pathogenic.	cspec
BA1	Not met	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%). BA1 is not met.	gnomad_v4
BS1	Not met	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%). BS1 is not met.	gnomad_v4
BS2	Not assessed	No data on co-occurrence in trans with a known pathogenic MLH1 variant are available. Assessment of BS2 would require confirmed phase testing in an individual with colorectal cancer after age 45 without CMMRD features.
BS3	Not assessed	No variant-specific functional data demonstrating a benign effect are available. The variant was not found in the VCEP functional assay documentation, and no publications report functional evidence of normal MMR activity for c.1655C>T.	vcep_functional_assay_svi_documentation_mmr
BS4	Not assessed	No co-segregation data are available to assess lack of segregation with disease. No publications or ClinVar submissions report segregation analysis for this variant.
BP1	N/A	BP1 is not applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP2	N/A	BP2 is not applicable under the InSiGHT MLH1 VCEP v2.0.0.	cspec
BP4	Met	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 spliceai revel bayesdel
BP5	Not assessed	No data on tumors with MSS or normal MMR protein expression in carriers of this variant are available. No BRAF V600E or MLH1 methylation data have been reported in association with this variant.
BP6	N/A	BP6 is not applicable under the InSiGHT MLH1 VCEP v2.0.0. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP7	N/A	BP7 applies only to synonymous (silent) or intronic variants at or beyond -21/+7 splice positions. NM_000249.3:c.1655C>T is a missense substitution (p.Thr552Ile) and does not meet the variant type requirement for BP7.	cspec
BP3	N/A	BP3 applies only to in-frame insertions/deletions in repetitive regions; not applicable to a single-nucleotide missense substitution.
PM3	N/A	Skipped by user directive. PM3 requires trans observation with a known pathogenic variant.
PM4	N/A	PM4 applies to protein-length-changing variants (in-frame deletions/insertions, stop-loss); not applicable to a missense substitution.

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