Variant classification summary

NM_000249.4:c.1989G>A

MLH1 · NP_000240.1:p.(Glu663=) · NM_000249.4

GRCh37: chr3:37090100 G>A · GRCh38: chr3:37048609 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

Pathogenic

PVS1 very strong PM2 supporting PP5 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Glu663=)

gnomAD AF

ClinVar

Pathogenic

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

PVS1_Very_Strong: c.1989G>A at the last nucleotide of exon 17 results in exon 17 skipping confirmed by patient mRNA analysis in two independent laboratories (Terdiman 2001, Tang 2009). Exon 17 skipping (93 bp, out-of-frame) introduces a premature termination codon predicted to undergo NMD.

2

PM2_Supporting: c.1989G>A is absent from gnomAD v2.1, v4.1, and gnomAD-Canada, meeting the InSiGHT VCEP threshold of < 0.00002 (< 1 in 50,000 alleles).

3

Final classification: Pathogenic. Per InSiGHT VCEP MLH1 v2.0 combining rules (Rule 1): 1 PVS1_Very_Strong yields a Pathogenic classification. This is concordant with the ClinVar InSiGHT Expert Panel classification of Pathogenic (ClinVar ID 89979).

Final determination: Rule4 in the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0 v2.0 criteria-combination framework is satisfied by the adjudicated criteria, so the variant is classified as Pathogenic.

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	Met	c.1989G>A at the last nucleotide of exon 17 disrupts the splice donor consensus. Patient mRNA analysis in two independent laboratories (Terdiman 2001, Tang 2009) confirms exon 17 skipping, which is out-of-frame (93 bp) and predicted to introduce a premature termination codon subject to NMD. This matches the InSiGHT VCEP PVS1_Very_Strong rule for variants where mRNA from patient constitutional samples demonstrates splicing aberration leading to premature stop codon, confirmed by an independent laboratory.	PMID:11208710 PMID:19419416 vcep_pvs1_decisiontree_mmr
PS1	N/A	PS1 requires a predicted missense substitution encoding the same amino acid change with a different nucleotide change. c.1989G>A is a synonymous variant (p.Glu663=) with no amino acid change.
PS2	Not assessed	No de novo data available in the evidence package. VCEP PS2 requires de novo point accumulation based on confirmed parentage.
PS3	Not assessed	The VCEP-calibrated functional assay spreadsheet (Functional-assay-SVI-documentation-MMR.xlsx) does not list c.1989G>A. No calibrated functional odds ratio data is available for this variant. The functional consequence of this variant is a splicing defect (exon 17 skipping), which is captured under PVS1 rather than PS3.	vcep_functional_assay_svi_documentation_mmr
PS4	N/A	PS4 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
PS5	Not assessed	No evidence provided to evaluate PS5. No reported de novo variant observations for this specific variant.
PM1	N/A	PM1 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
PM2	Met	c.1989G>A is absent from gnomAD v2.1 (exomes), gnomAD v4.1 (exomes/genomes), and gnomAD-Canada v1.0. This satisfies the InSiGHT VCEP PM2_Supporting threshold of allele frequency < 0.00002 (absent/extremely rare in gnomAD v4).	gnomad_v2 gnomad_v4 gnomad_canada cspec
PM5	N/A	VCEP PM5 requires a missense change at an amino acid residue where a different missense change was classified as Pathogenic. c.1989G>A is a synonymous variant (p.Glu663=), not a missense change.
PM6	N/A	PM6 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
PP1	Not assessed	No co-segregation data provided. VCEP PP1 requires Bayes likelihood ratio from pedigree analysis.
PP2	N/A	PP2 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
PP3	Not met	VCEP PP3 for MLH1 requires either a missense variant with HCI prior >0.68 (not applicable; this is a synonymous variant not in the HCI table) or a predicted splice defect with SpliceAI delta >= 0.2 (SpliceAI max delta = 0.00, no splice impact predicted). Neither condition is satisfied.	spliceai vcep_hci_priors_mlh1 cspec
PP4	Not assessed	No tumor data (MSI status, IHC for MMR protein expression) provided in the evidence package. VCEP PP4 requires MSI-H tumors with MMR protein expression loss consistent with the variant location.
PP5	Met	Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Pathogenic.	cspec clinvar
BA1	Not met	VCEP BA1 requires gnomAD v4 grpmax filtering allele frequency >= 0.001 (0.1%). c.1989G>A is absent from gnomAD. The BA1 threshold is not satisfied.	gnomad_v4 cspec
BS1	Not met	VCEP BS1 requires gnomAD v4 grpmax filtering allele frequency >= 0.0001 and < 0.001. c.1989G>A is absent from gnomAD. The BS1 threshold is not satisfied.	gnomad_v4 cspec
BS2	Not assessed	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant. VCEP BS2 requires confirmed trans co-occurrence in a CRC patient after age 45 without CMMRD features.
BS3	Not met	VCEP BS3 requires calibrated functional assays with odds for pathogenicity <= 0.05, or variant-specific proficient function in protein/mRNA assays. The experimental data available shows the opposite: patient mRNA assays in two independent laboratories confirm exon 17 skipping (a loss-of-function splicing defect). No evidence of normal or proficient function exists for this variant.	PMID:11208710 PMID:19419416 cspec
BS4	Not assessed	No segregation data available. VCEP BS4 requires Bayes likelihood ratio for lack of co-segregation.
BP1	N/A	BP1 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
BP2	N/A	BP2 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
BP3	N/A	In-frame indels in repetitive regions without known function; not applicable to a single-nucleotide substitution.
BP4	Not met	VCEP BP4 for synonymous variants requires SpliceAI delta score <= 0.1 predicting no splicing impact. SpliceAI delta = 0.00 satisfies this threshold in silico. However, direct experimental evidence from patient mRNA (Terdiman 2001, Tang 2009) demonstrates that c.1989G>A does cause exon 17 skipping. In silico prediction is overridden by experimental data showing a splicing defect.	spliceai PMID:11208710 PMID:19419416 cspec
BP5	Not assessed	No tumor data (MSS status, MMR protein expression by IHC) provided. VCEP BP5 requires MSS tumors or inconsistent MMR protein loss in the tumor.
BP6	N/A	BP6 is not applicable per InSiGHT VCEP MLH1 v2.0 specifications.	cspec
BP7	Not met	VCEP BP7 applies to synonymous or intronic variants at or beyond -21/+7 from the splice junction. c.1989G>A is at the last nucleotide of exon 17, which is position -1 of the splice donor consensus. This position is within the splice consensus region, not at or beyond -21/+7. Furthermore, patient mRNA confirms this variant does affect splicing (exon 17 skipping), precluding BP7 application.	PMID:11208710 PMID:19419416 cspec

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