Variant classification summary

NM_000249.4:c.677G>A

MLH1 · NP_000240.1:p.(Arg226Gln) · NM_000249.4

GRCh37: chr3:37053590 G>A · GRCh38: chr3:37012099 G>A

Gene: MLH1 Transcript: NM_000249.4

Final call

Pathogenic

PVS1 very strong PM2 supporting PP3 supporting PP4 supporting PP5 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Arg226Gln)

gnomAD AF

6.233924267794426e-07 (v4.1)

ClinVar

Pathogenic

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

PVS1_VeryStrong: c.677G>A (last nucleotide of exon 8) produces no full-length transcript; patient mRNA analysis demonstrates complete exon 8 skipping (r.589_677del), frameshift, and NMD in two independent studies.

2

PM2_Supporting: Extremely rare in population databases — gnomAD v4.1 AF = 6.23e-7 (1/1,604,126 alleles), absent from v2.1 and gnomAD-Canada, meeting VCEP threshold <0.00002.

3

PP3_Supporting: SpliceAI predicts splicing impact (max delta = 0.44), meeting VCEP threshold (delta ≥0.2) for non-canonical splice position.

4

PP4_Supporting: One CRC tumor from a carrier demonstrates MSI-H (5/5 markers) with loss of MLH1 protein expression by IHC.

5

Evidence for pathogenicity includes confirmed RNA-level splicing aberration causing frameshift, extreme population rarity, in silico splice prediction, and tumor phenotype consistent with MMR deficiency.

Final determination: Rule4 in the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0.0 v2.0.0 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.677G>A (last nucleotide of exon 8) results in complete exon 8 skipping (r.589_677del) as demonstrated by patient mRNA analysis in two independent studies, leading to a frameshift and premature termination codon subject to nonsense-mediated decay. The variant allele produces no full-length or reference transcript. Confirmed in an affected sibling carrying the same variant.	PMID:15300854 PMID:16341550 cspec
PS1	N/A	No alternative nucleotide substitution encodes p.Arg226Gln. The known variant c.677G>T at the same position encodes p.Arg226Leu, a different amino acid change, and therefore does not satisfy the PS1 requirement for same amino acid change via a different nucleotide.
PS2	Not assessed	No confirmed de novo occurrence of c.677G>A has been identified in ClinVar submissions, the published literature reviewed, or the InSiGHT LOVD database. PS2 requires confirmed de novo observations with maternity/paternity confirmation.
PS3	Not assessed	Variant was not found in the VCEP-calibrated functional assay documentation (Functional-assay-SVI-documentation-MMR.xlsx). Putative functional evidence from PMID:12362047 (yeast-based MMR assay) could not be verified as the full-text was not available for review. PS3 requires calibrated functional odds per the VCEP framework.
PS4	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark PS4 as Not Applicable for this gene.	cspec
PS5	N/A	The VCEP framework deprecates PP5 (reputable source reporting) as Not Applicable. PS5 is analogous and not specified in the CSPEC rule set. Sufficient primary evidence is available for independent evaluation, making reliance on external assertions unnecessary.	cspec
PM1	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark PM1 as Not Applicable for MLH1.	cspec
PM2	Met	c.677G>A is present in gnomAD v4.1 at an allele frequency of 6.23e-7 (1/1,604,126 alleles, 0 homozygotes), which is below the VCEP PM2_Supporting threshold of <0.00002 (<1 in 50,000 alleles). The variant is absent from gnomAD v2.1 and gnomAD-Canada.	gnomad_v4 gnomad_v2 cspec
PM5	N/A	c.677G>A has been demonstrated to cause complete exon 8 skipping via patient mRNA analysis, acting as a splicing defect rather than a missense change at the protein level. VCEP PM5 requires a missense change at the amino acid residue not due to aberrant splicing. As the pathogenic mechanism is splicing aberration, not an expressed missense protein, PM5 is not applicable.	PMID:15300854 PMID:16341550 cspec
PM6	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark PM6 as Not Applicable for MLH1.	cspec
PP1	Not assessed	Pagenstecher et al. (2006) report co-segregation of c.677G>A with disease ('Yes') in one family, but provide insufficient detail (number of meioses, pedigree structure, affected/unaffected counts) to calculate a Bayes Likelihood Ratio per VCEP PP1 rules. VCEP PP1 requires a combined LR >2.08 for Supporting, >4.3 for Moderate, or >18.7 for Strong. Specific segregation quantification is needed.	PMID:16341550
PP2	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark PP2 as Not Applicable for MLH1.	cspec
PP3	Met	SpliceAI predicts a splicing defect for this variant (max delta score = 0.44), meeting the VCEP PP3_Supporting threshold of delta ≥0.2 for non-canonical splicing nucleotides. The HCI prior probability of 0.4702 does not meet the missense PP3 thresholds (>0.68).	spliceai hci_prior cspec
PP4	Met	One CRC tumor from a carrier of c.677G>A demonstrated MSI-H (5/5 microsatellite markers) and loss of MLH1 protein expression by immunohistochemistry (Pagenstecher et al. 2006). This meets VCEP PP4_Supporting criteria: 1 CRC with MSI-H and MMR protein loss consistent with variant location.	PMID:16341550 cspec
PP5	Met	Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Pathogenic.	cspec clinvar
BA1	Not met	gnomAD v4.1 allele frequency is 6.23e-7, well below the VCEP BA1 stand-alone benign threshold of ≥0.001 (0.1%).	gnomad_v4 cspec
BS1	Not met	gnomAD v4.1 allele frequency is 6.23e-7, below the VCEP BS1 strong benign threshold range of ≥0.0001 and <0.001.	gnomad_v4 cspec
BS2	Not assessed	No evidence of co-occurrence in trans with a known pathogenic MLH1 variant in a patient without clinical manifestations of CMMRD was identified in the reviewed literature.
BS3	Not met	All identified functional evidence demonstrates a damaging effect on splicing (complete exon 8 skipping). No well-established functional studies show normal MMR activity or benign protein function for this variant.	PMID:15300854 PMID:16341550
BS4	Not met	Pagenstecher et al. (2006) report co-segregation of c.677G>A with disease, which is inconsistent with BS4 (lack of co-segregation). No published reports demonstrate absence of co-segregation for this variant.	PMID:16341550
BP1	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark BP1 as Not Applicable for MLH1.	cspec
BP2	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark BP2 as Not Applicable for MLH1.	cspec
BP3	N/A	In-frame deletion/insertion not present; variant is a single nucleotide substitution. Skipped per workflow directive.
BP4	Not met	HCI prior probability of pathogenicity is 0.4702, far above the VCEP BP4_Supporting threshold of <0.11. SpliceAI delta score of 0.44 predicts splicing impact (≥0.1), further contraindicating BP4.	hci_prior spliceai cspec
BP5	Not met	The available tumor evidence shows MSI-H with loss of MLH1 protein expression (Pagenstecher et al. 2006), which is inconsistent with BP5 requirements for MSS tumors with intact MMR protein expression.	PMID:16341550
BP6	N/A	VCEP ClinGen InSiGHT MMR specifications (v2.0.0) explicitly mark BP6 as Not Applicable as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP7	Not met	c.677G>A is a missense (non-synonymous) coding variant, not a synonymous or intronic variant. BP7 requires a synonymous (silent) or intronic variant at or beyond -21/+7 exonic positions.	cspec

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