Variant classification summary

NM_000249.4:c.1094G>A

MLH1 · NP_000240.1:p.(Ser365Asn) · NM_000249.4

GRCh37: chr3:37067183 G>A · GRCh38: chr3:37025692 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

PM2 supporting BP4 supporting benign

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Ser365Asn)

gnomAD AF

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.1094G>A (p.Ser365Asn) is a missense variant in exon 12 of MLH1, absent from gnomAD v4.1 and v2.1 (PM2_Supporting per VCEP v2.0.0).

2

In silico predictors strongly favor a benign effect: HCI prior probability is 0.0025 (BP4_Supporting per VCEP, threshold <0.11), REVEL score is 0.321, and BayesDel score is -0.220172.

3

SpliceAI predicts no splicing impact (max delta score 0.01), confirming the variant does not create a cryptic splice site.

4

The InSiGHT Locus-Specific Database (Thompson et al. 2013, PMID:22949387) classifies this variant as Class 5 (Pathogenic) based on a multifactorial likelihood model incorporating yeast-based MMR functional defect data and co-segregation in multiple families, but the quantitative calibrated functional odds and segregation likelihood ratios required for VCEP PS3 and PP1 strength assignment were not available in the case materials.

5

ClinVar reports this variant as Uncertain Significance (4 clinical laboratories, criteria provided single submitter, ClinVar ID 1790197).

6

No tumor pathology data (MSI status, MMR IHC, BRAF V600E, MLH1 methylation) was available to assess PP4 or BP5.

7

Several criteria designated as Not Applicable by the VCEP were not assessed: PVS1 (missense, not null), PS4, PM1, PM6, PP2, PP5, BP1, BP2, BP6, BP7.

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 variants under the InSiGHT/ClinGen MLH1 VCEP v2.0.0. NM_000249.4:c.1094G>A is a missense substitution (p.Ser365Asn) in exon 12. SpliceAI predicts no splicing impact (max delta score 0.01), and the variant does not fall into any PVS1-eligible bucket (nonsense, frameshift, canonical ±1/2 splice, initiation codon, or confirmed splicing aberration).	spliceai
PS1	Not assessed	PS1 requires a different nucleotide change encoding the same amino acid (Ser365Asn) previously established as Pathogenic by this VCEP. No such comparator variant was identified in the VCEP pilot variants spreadsheet or in the available evidence. The InSiGHT Locus-Specific Database classification of this variant itself is Class 5 (Pathogenic), but that does not constitute a comparator for PS1.
PS2	Not assessed	No de novo occurrence reports were identified for NM_000249.4:c.1094G>A. The exploratory literature search found no evidence of de novo status in Lynch syndrome or constitutional mismatch repair deficiency (CMMRD). De novo events in adult-onset cancer syndromes are rarely documented.
PS3	Not assessed	The InSiGHT Locus-Specific Database (Thompson et al. 2013, PMID:22949387) classifies c.1094G>A (p.Ser365Asn) as Class 5 (Pathogenic) based on a multifactorial likelihood model incorporating functional data (yeast-based MMR defect assay). However, the calibrated functional odds ratio required by the VCEP PS3 strength table (Strong: >18.7; Moderate: >4.3 and ≤18.7; Supporting: >2.08 and ≤4.3) was not available. The VCEP Functional-assay-SVI-documentation-MMR.xlsx does not contain variant-specific entries for c.1094G>A, and the calibrated assays listed therein (Drost 2018/2020, Jia/Scott 2021/2022, Rath 2022) do not include this variant. Full text of PMID:22949387 was not available in the case materials for direct verification of the functional data.	hci_prior
PS4	N/A	PS4 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
PS5	Not assessed	PS5 (reputable source reports variant as pathogenic) is not defined in the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specification. The equivalent criterion PP5 is explicitly listed as Not Applicable by the VCEP. Under generic ACMG/AMP 2015, PS5 would require a reputable source recently reporting the variant as pathogenic with evidence not available to the laboratory for independent evaluation. The InSiGHT Locus-Specific Database (PMID:22949387) classifies this variant as Class 5 (Pathogenic), but this classification predates the VCEP framework and relies on the same underlying evidence being adjudicated here.
PM1	N/A	PM1 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
PM2	Met	NM_000249.4:c.1094G>A is absent from gnomAD v4.1 (and v2.1), satisfying the VCEP PM2_Supporting rule: absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in the gnomAD v4 dataset.	gnomad_v4 gnomad_v2 cspec
PM5	Not assessed	PM5 requires a different missense change at the same amino acid residue (Ser365) previously classified as Pathogenic or Likely Pathogenic by this VCEP. The pm5_candidates search identified no same-residue comparator variants in ClinVar meeting VCEP Pathogenic/Likely Pathogenic criteria. Without an established pathogenic comparator at codon 365, PM5 cannot be applied.
PM6	N/A	PM6 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
PP1	Not assessed	The InSiGHT Locus-Specific Database (PMID:22949387) reports co-segregation of c.1094G>A with Lynch syndrome-spectrum cancers in multiple families, contributing to its Class 5 (Pathogenic) classification. However, the specific Bayes Likelihood Ratio required for VCEP PP1 strength assignment (Strong: >18.7 in ≥2 families; Moderate: >4.3 and ≤18.7; Supporting: >2.08 and ≤4.3) was not available in the case materials. The number of meioses, family structures, and LOD scores are recorded in the InSiGHT database but were not retrieved.	hci_prior
PP2	N/A	PP2 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
PP3	Not met	Per the MLH1 VCEP v2.0.0, PP3 for missense variants is assessed by HCI prior probability: >0.81 = PP3_Moderate, >0.68 and ≤0.81 = PP3_Supporting. The HCI prior for c.1094G>A (p.Ser365N) is 0.0025, well below both thresholds. SpliceAI delta score is 0.01, also below the non-canonical splice threshold of 0.2. Neither criterion for PP3 is met.	hci_prior spliceai cspec
PP4	Not assessed	The VCEP PP4 criterion requires MSI-H colorectal or endometrial tumors with loss of MMR protein expression consistent with the variant location, with MLH1 promoter methylation excluded. No tumor pathology data (MSI status, IHC for MMR proteins, BRAF V600E, or MLH1 methylation) was available for this variant in the case materials.
PP5	N/A	PP5 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BA1	Not met	Per the MLH1 VCEP v2.0.0, BA1 requires gnomAD v4 Grpmax filtering allele frequency ≥0.001 (0.1%) with exclusion as a founder pathogenic variant. The variant is absent from gnomAD v4.1 (0 alleles), far below the BA1 threshold.	gnomad_v4 cspec
BS1	Not met	Per the MLH1 VCEP v2.0.0, BS1 requires gnomAD v4 Grpmax filtering allele frequency ≥0.0001 and <0.001 (0.01-0.1%) with exclusion as a founder pathogenic variant. The variant is absent from gnomAD v4.1, so BS1 is not met.	gnomad_v4 cspec
BS2	Not assessed	BS2 requires co-occurrence in trans with a known pathogenic MLH1 variant in a patient with colorectal cancer after age 45 (or other LS cancer above median age of onset) without clinical manifestations of CMMRD. No such co-occurrence data was identified for this variant.
BS3	Not met	BS3 requires calibrated functional assays showing functional odds for pathogenicity ≤0.05 (Strong) or >0.05 and ≤0.48 (Supporting), or proficient function per the MMR functional assay flowchart. The available functional evidence (InSiGHT database, PMID:22949387) indicates a damaging effect in a yeast-based MMR assay, which is inconsistent with normal/proficient function. The VCEP Functional-assay-SVI-documentation-MMR.xlsx lists calibrated assays but does not contain variant-specific data for c.1094G>A. No evidence of normal MMR function for this variant was identified.	hci_prior vcep_functional_assay_svi_documentation_mmr
BS4	Not met	BS4 requires lack of co-segregation with disease in pedigrees (combined Bayes Likelihood Ratio <0.05 for Strong, >0.05 and ≤0.48 for Supporting). The InSiGHT Locus-Specific Database reports co-segregation of this variant with Lynch syndrome-spectrum cancers, which is the opposite of what BS4 requires. No evidence of non-segregation was identified.	hci_prior
BP1	N/A	BP1 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP2	N/A	BP2 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP4	Met	Per the MLH1 VCEP v2.0.0, BP4_Supporting is met when the HCI prior probability of pathogenicity is <0.11 for missense variants. The HCI prior for c.1094G>A (p.Ser365N) is 0.0025, which is well below the threshold. Additionally, REVEL score is 0.321 and BayesDel score is -0.220172, both consistent with a benign in silico prediction profile.	hci_prior revel bayesdel cspec
BP5	Not assessed	BP5 requires specific tumor data: ≥4 MSS tumors with no loss of MMR expression (Strong), or 2-3 such tumors (Supporting), or BRAF V600E/MLH1 methylation in MSI-H tumors. No tumor pathology data was available for this variant in the case materials.
BP6	N/A	BP6 is listed as Not Applicable under the InSiGHT/ClinGen MLH1 VCEP v2.0.0 specifications. 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 positions -21/+7 (5'/3' exonic). NM_000249.4:c.1094G>A is a missense variant (p.Ser365Asn) and is therefore not eligible for BP7.	cspec

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