Variant classification summary

NM_000249.3:c.1738G>A

MLH1 · NP_000240.1:p.(Ala580Thr) · NM_000249.3

GRCh37: chr3:37089016 G>A · GRCh38: chr3:37047525 G>A

Gene: MLH1 Transcript: NM_000249.3

Final call

VUS

PM2 supporting PP3 supporting

Variant details

Gene

MLH1

Transcript

NM_000249.3

Protein

NP_000240.1:p.(Ala580Thr)

gnomAD AF

1.2390345442830946e-06 (v4.1)

ClinVar

Uncertain significance

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.3:c.1738G>A (p.Ala580Thr) is a missense variant in MLH1 exon 16. It is extremely rare in population databases (gnomAD v4.1 grpmax FAF = 2.8e-07), meeting PM2_Supporting per the InSiGHT VCEP v2.0 specification.

2

In silico analysis yields an HCI prior probability of 0.8146, which falls in the PP3_Supporting range (>0.68 and ≤0.81) per the InSiGHT VCEP v2.0 specification. SpliceAI predicts no splicing impact (max delta score = 0.00). REVEL score is 0.703.

3

No variant-specific functional data, co-segregation data, tumor phenotype data, or de novo observations were identified in the reviewed literature or ClinVar submissions. ClinVar reports this variant as Uncertain Significance (6 submissions; review status: criteria provided, single submitter).

4

Under the InSiGHT VCEP v2.0 framework, the only criteria met are PM2_Supporting and PP3_Supporting. Multiple criteria are designated as Not Applicable per the VCEP specification (PS4, PM1, PM6, PP2, PP5, BP1, BP2, BP6, PM4) or are not applicable to a missense substitution (PVS1, BP3, BP7). No benign criteria are met. With only two supporting pathogenic criteria and no moderate, strong, or very strong criteria met, the variant remains as Uncertain Significance per the VCEP combination rules.

Final determination: No criteria-combination rule matched the adjudicated criteria 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 framework, so the variant remains a Variant of Uncertain Significance pending human review.

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. NM_000249.3:c.1738G>A is a missense substitution (p.Ala580Thr) and does not fall into any PVS1 null-variant category (nonsense, frameshift, canonical splice, initiation codon, or large deletion). The VCEP PVS1 rules are restricted to null variants and do not apply to missense changes.	pvs1_variant_assessment pvs1_gene_context cspec
PS1	Not met	No evidence that a different underlying nucleotide change producing the same amino acid substitution (p.Ala580Thr) has been classified as Pathogenic or Likely Pathogenic by this VCEP. No such variant was identified in the VCEP pilot variants database or in the reviewed literature.	cspec
PS2	Not met	No de novo occurrence data were identified for NM_000249.3:c.1738G>A in the reviewed literature or ClinVar submissions. The VCEP PS2 rule requires de novo points from confirmed parentage testing.	clinvar cspec
PS3	Not met	No variant-specific functional data were identified for p.Ala580Thr (NM_000249.3:c.1738G>A) in the reviewed literature, the VCEP calibrated functional assay documentation, or OncoKB. The variant is not listed in the VCEP pilot variants spreadsheet with any PS3 or BS3 functional code assignment.	oncokb vcep_functional_assay_svi_documentation_mmr cspec
PS4	N/A	PS4 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
PS5	N/A	PS5 is not defined in the ClinGen InSiGHT VCEP v2.0 specification for MLH1; this criterion is not available under the governing VCEP framework.	cspec
PM1	N/A	PM1 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
PM2	Met	The variant is extremely rare in gnomAD v4.1 with a grpmax filtering allele frequency of 2.8e-07 (2/1,614,160 alleles, 0 homozygotes), which is well below the VCEP PM2_Supporting threshold of <0.00002 (<1 in 50,000 alleles). Absent from gnomAD v2.1.	gnomad_v4 gnomad_v2 cspec
PM5	Not met	No different missense change at amino acid residue 580 was identified that has been classified as Pathogenic or Likely Pathogenic by this VCEP. The VCEP pilot variants spreadsheet does not list any codon 580 comparator. PM5 also requires PP3 to be supporting; although PP3_Supporting is met (HCI prior 0.8146), the absence of a qualifying comparator variant precludes PM5 application.	cspec pm5_candidates
PM6	N/A	PM6 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
PP1	Not met	No co-segregation data were identified for this variant in the reviewed literature or ClinVar submissions. The VCEP PP1 rule requires Bayes Likelihood Ratio calculation from pedigrees.	clinvar cspec
PP2	N/A	PP2 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
PP3	Met	The HCI prior probability for pathogenicity for p.Ala580Thr is 0.8146, which falls in the PP3_Supporting range (>0.68 and ≤0.81) per the InSiGHT VCEP v2.0 specification. SpliceAI predicts no splicing impact (max delta score 0.00), so the splice-based PP3 rule does not apply. REVEL score is 0.703 and BayesDel score is 0.291.	hci_prior spliceai revel bayesdel cspec
PP4	Not met	No tumor data (MSI status, MMR protein expression by IHC) were identified for patients carrying NM_000249.3:c.1738G>A in the reviewed literature or ClinVar submissions. The VCEP PP4 rule requires one or more CRC/endometrial MSI-H tumors with loss of MMR protein expression consistent with the variant location.	clinvar cspec
PP5	N/A	PP5 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
BA1	Not met	The gnomAD v4.1 grpmax filtering allele frequency is 2.8e-07, which is far below the VCEP BA1 threshold of ≥0.001 (0.1%). The variant is extremely rare and does not meet the stand-alone benign allele frequency criterion.	gnomad_v4 cspec
BS1	Not met	The gnomAD v4.1 grpmax filtering allele frequency is 2.8e-07, which is below the VCEP BS1 threshold of ≥0.0001 (0.01%). The variant is too rare to satisfy BS1.	gnomad_v4 cspec
BS2	Not met	No observation of co-occurrence in trans with a known pathogenic MLH1 variant was identified in the reviewed literature or ClinVar submissions. The VCEP BS2 rule requires documented trans co-occurrence with a known pathogenic variant in a CRC patient after age 45 without CMMRD features.	clinvar cspec
BS3	Not met	No functional data demonstrating a benign effect for p.Ala580Thr were identified in the reviewed literature, the VCEP calibrated functional assay documentation, or OncoKB. The variant is not listed in the VCEP pilot variants spreadsheet with any BS3 code assignment.	oncokb vcep_functional_assay_svi_documentation_mmr cspec
BS4	Not met	No lack of co-segregation data were identified for this variant. The VCEP BS4 rule requires Bayes Likelihood Ratio calculation from pedigrees showing absence of segregation with disease.	cspec
BP1	N/A	BP1 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
BP2	N/A	BP2 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec
BP3	N/A	BP3 does not apply to substitution variants. NM_000249.3:c.1738G>A is a single nucleotide substitution, not an in-frame indel.
BP4	Not met	The HCI prior probability for pathogenicity for p.Ala580Thr is 0.8146, which is well above the VCEP BP4_Supporting threshold of <0.11. REVEL score is 0.703 and BayesDel score is 0.291, which do not suggest a benign in silico profile. SpliceAI predicts no splice impact (delta 0.00), but the BP4 splice rule applies only to intronic and synonymous variants, not missense.	hci_prior revel bayesdel spliceai cspec
BP5	Not met	No tumor data demonstrating MSS status or retained MMR protein expression were identified for patients carrying this variant. The VCEP BP5 rule requires multiple tumors with MSS and/or no loss of MMR protein expression, or BRAF V600E/MLH1 methylation with MSI-H.	cspec
BP6	N/A	BP6 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0. 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 positions -21/+7. NM_000249.3:c.1738G>A is a missense variant (p.Ala580Thr) in exon 16 and does not qualify for BP7.	cspec
PM3	N/A	PM3 was designated for skip in the case instructions; not assessed.
PM4	N/A	PM4 is designated as Not Applicable by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications for MLH1 Version 2.0.	cspec

Disclaimer: The content and results provided by LYFE Sciences are for research and educational purposes only and must not be used as a substitute for professional medical judgment, diagnosis, or treatment. Always consult a qualified healthcare professional before making any clinical decisions.