Variant classification summary

NM_000249.4:c.222T>C

MLH1 · NP_000240.1:p.(Asp74=) · NM_000249.4

GRCh37: chr3:37042460 T>C · GRCh38: chr3:37000969 T>C

Gene: MLH1 Transcript: NM_000249.4

Final call

Likely Benign

BS3 supporting BP4 supporting BP7 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Asp74=)

gnomAD AF

2.3189728329198876e-05 (v4.1)

ClinVar

Likely benign

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.222T>C is a synonymous variant (p.Asp74=) in exon 3 of MLH1.

2

SpliceAI predicts no splicing impact (max delta score 0.03), satisfying BP4_Supporting under InSiGHT MLH1 VCEP v2.0.0 rules for synonymous variants.

3

The variant is located within the -21 exonic splice region (c.222 in exon 3, acceptor c.208), satisfying BP7_Supporting.

4

An RNA splicing assay performed by a clinical diagnostic laboratory indicates this variant does not significantly alter splicing, consistent with a benign functional effect (BS3_Supporting).

5

The variant is present in gnomAD v4.1 at a low frequency (37/1,595,534 alleles; AF=2.32e-05) that exceeds the VCEP PM2_Supporting threshold of <0.00002, therefore PM2 is not met. The frequency does not reach BS1 or BA1 thresholds.

6

The variant has been reported in ClinVar as Likely benign by six clinical laboratories (ClinVar ID 237333).

7

No evidence of pathogenicity was identified: PVS1, PS1, and PM5 are not applicable (synonymous); PS2/PM6, PP1, PP4, BS2, BS4, and BP5 lack data; PS3, PP3, BA1, and BS1 are not met.

Final determination: Rule19 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 Likely Benign.

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. NM_000249.4:c.222T>C is a synonymous variant (p.Asp74=) and is neither a null variant (nonsense, frameshift, canonical splice) nor predicted to result in a loss-of-function mechanism. The variant falls outside the PVS1 decision tree for MMR genes.	pvs1_gene_context pvs1_variant_assessment vcep_pvs1_decisiontree_mmr
PS1	N/A	PS1 is not applicable. This variant is synonymous (p.Asp74=) and does not encode an amino acid change; there is no amino acid substitution to compare against a previously established pathogenic missense change.	cspec
PS2	Not assessed	No de novo occurrences of NM_000249.4:c.222T>C were identified in any database or publication. Absent de novo evidence precludes application of PS2.
PS3	Not met	PS3 is not met. No well-established functional studies demonstrate a damaging effect for this synonymous variant. An RNA splicing assay reported by a clinical laboratory (Invitae, cited in ClinVar) indicates this variant does not significantly alter splicing, which contradicts a damaging functional effect. The variant is not listed in the MMR calibrated functional assay dataset.	vcep_functional_assay_svi_documentation_mmr clinvar
PS4	N/A	PS4 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
PS5	N/A	PS5 is not defined in the InSiGHT MLH1 VCEP v2.0.0 criteria. The corresponding PP5 criterion is explicitly Not Applicable per the VCEP. In VCEP mode, PS5 is not used.	cspec
PM1	N/A	PM1 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
PM2	Not met	PM2_Supporting is not met. Under the VCEP rule, PM2_Supporting requires an allele frequency <0.00002 (fewer than 1 in 50,000 alleles) in gnomAD v4. The variant is present in gnomAD v4.1 at an allele frequency of 2.32e-05 (37/1,595,534 alleles, grpmax FAF=2.35e-05), which exceeds the PM2_Supporting threshold.	gnomad_v4 cspec
PM5	N/A	PM5 is not applicable. The variant is synonymous (p.Asp74=) and does not produce a missense change. PM5 requires a missense alteration at an amino acid residue where a different missense change has been classified as Pathogenic or Likely Pathogenic. No candidate comparators were identified.	pm5_candidates cspec
PM6	N/A	PM6 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
PP1	Not assessed	No co-segregation data are available for this variant. No published family studies or segregation analyses mentioning NM_000249.4:c.222T>C were identified.
PP2	N/A	PP2 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
PP3	Not met	PP3 is not met. Under VCEP rules, for synonymous variants, PP3_Supporting requires a SpliceAI delta score >=0.2 for non-canonical splicing nucleotides. The SpliceAI max delta score for this variant is 0.03, well below the threshold. No HCI prior score is available for synonymous variants.	spliceai cspec revel
PP4	Not assessed	No tumor MSI/IHC data specific to this variant are available. PP4 under the VCEP requires MSI-H tumor data with loss of MMR protein expression consistent with the variant location. Absent such data, PP4 cannot be assessed.
PP5	N/A	PP5 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
BA1	Not met	BA1 is not met. The VCEP rule requires a gnomAD v4 grpmax filtering allele frequency >=0.001 (0.1%). The variant has a grpmax FAF of 2.35e-05 (0.00235%), far below the BA1 threshold.	gnomad_v4 cspec
BS1	Not met	BS1 is not met. The VCEP rule requires a gnomAD v4 grpmax filtering allele frequency >=0.0001 and <0.001 (0.01-0.1%). The variant has a grpmax FAF of 2.35e-05 (0.00235%), below the BS1 lower bound of 0.0001.	gnomad_v4 cspec
BS2	Not assessed	No data on co-occurrence in trans with a known pathogenic MLH1 variant are available. BS2 requires confirmed phase with a pathogenic variant in a patient meeting specific clinical criteria.
BS3	Met	BS3_Supporting is met. An RNA splicing assay performed by a clinical diagnostic laboratory (Invitae, reported via ClinVar) indicates that NM_000249.4:c.222T>C does not significantly alter splicing. This is direct functional evidence of no splicing aberration for a synonymous variant where aberrant splicing would be the only plausible pathogenic mechanism. SpliceAI independently predicts no splicing impact (max delta 0.03). Under the VCEP BS3_Supporting rule, variant-specific proficient function in mRNA-based lab assays qualifies.	cspec spliceai clinvar
BS4	Not assessed	No segregation or lack-of-segregation data are available for this variant. BS4 requires a Bayes likelihood ratio from pedigree analysis demonstrating lack of co-segregation with disease.
BP1	N/A	BP1 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
BP2	N/A	BP2 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
BP4	Met	BP4_Supporting is met. Under the VCEP rule for synonymous variants, BP4_Supporting applies when SpliceAI predicts no splicing impact with a delta score <=0.1. SpliceAI max delta for NM_000249.4:c.222T>C is 0.03, meeting this threshold. Additionally, REVEL score is 0.035 (strongly benign-leaning).	spliceai cspec revel
BP5	Not assessed	No tumor phenotype data (MSS status, MMR protein expression, BRAF/MLH1 methylation) are available for this variant. BP5 requires specific tumor characteristics inconsistent with LS.
BP6	N/A	BP6 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel VCEP for MLH1 v2.0.0.	cspec
BP7	Met	BP7_Supporting is met. NM_000249.4:c.222T>C is a synonymous variant located at nucleotide position 15 within exon 3 (c.208-c.306). This falls within the -21 position from the acceptor splice site (positions c.208-c.228), satisfying the VCEP BP7 rule for synonymous variants at or beyond -21/+7 (5'/3' exonic). SpliceAI also predicts no splicing impact (delta 0.03). BP7 and BP4 may both be applied.	spliceai cspec

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