MLH1

Final classification

Pathogenic

MLH1 c.1989G>A · p.Glu663=

MLH1

Gene

MLH1

Transcript

NM_000249.4

HGVS · transcript:coding

NM_000249.4:c.1989G>A

Consequence

N/A

GRCh38

chr3:37048609 G>A

GRCh37

chr3:37090100 G>A

Basis 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: matched Rule4 (1 Pathogenic.Very Strong + Pathogenic.Supporting >=2) with applied criteria: PVS1 very strong, PM2 supporting, PP5 supporting; maps to Pathogenic. ▾

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: matched Rule4 (1 Pathogenic.Very Strong + Pathogenic.Supporting >=2) with applied criteria: PVS1 very strong, PM2 supporting, PP5 supporting; maps to Pathogenic.

Classification rationale

PVS1PM2PP5 Pathogenic

MLH1 c.1989G>A

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.¹ 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).² 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).³

PVS1 + PM2 + PP5 → Pathogenic

1 PMID:11208710 ↗PMID:19419416 ↗

2 gnomad_v2 ↗gnomad_v4 ↗gnomad_canada ↗

3 cspec ↗clinvar ↗

Gene diagram · NM_000249.4 · variants mapped to exon structure

MLH1 NM_000249.4

Fetching transcript structure from UCSC…

Applied criteria · 3 applied · 13 assessed

Applied · 3

Strength Supporting Moderate Strong Very strong

✓

PVS1 very strong Pathogenic

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.

c.1989G>A at last base of exon 17 (splice donor position -1)mRNA assay from two independent laboratories confirms exon 17 skippingExon 17 is 93 bp

PMID:11208710 ↗PMID:19419416 ↗

✓

PM2 supporting Pathogenic

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).

Absent from gnomAD v2.1Absent from gnomAD v4.1Absent from gnomAD-Canada v1.0

gnomad_v2 ↗gnomad_v4 ↗gnomad_canada ↗cspec ↗

✓

PP5 supporting Pathogenic

Expert panel International Society for Gastrointestinal Hereditary Tumours (InSiGHT) classified as Pathogenic.

ClinVar expert panel classification

cspec ↗clinvar ↗

Assessed · not applied

Pathogenic

PS2 No de novo data available in the evidence package.

PS3 The VCEP-calibrated functional assay spreadsheet (Functional-assay-SVI-documentation-MMR.xlsx) does not list c.1989G>A.

PP1 No co-segregation data provided.

PP3 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).

PP4 No tumor data (MSI status, IHC for MMR protein expression) provided in the evidence package.

Benign

BA1 VCEP BA1 requires gnomAD v4 grpmax filtering allele frequency >= 0.001 (0.1%).

BS1 VCEP BS1 requires gnomAD v4 grpmax filtering allele frequency >= 0.0001 and < 0.001.

BS2 No evidence of co-occurrence in trans with a known pathogenic MLH1 variant.

BS3 VCEP BS3 requires calibrated functional assays with odds for pathogenicity <= 0.05, or variant-specific proficient function in protein/mRNA assays.

BS4 No segregation data available.

BP4 VCEP BP4 for synonymous variants requires SpliceAI delta score <= 0.1 predicting no splicing impact.

BP5 No tumor data (MSS status, MMR protein expression by IHC) provided.

BP7 VCEP BP7 applies to synonymous or intronic variants at or beyond -21/+7 from the splice junction.

N/A · 10 PS1 · PS4 · PM1 · PM5 · PM6 · PP2 · BP1 · BP2 · BP3 · BP6

Research & evidence

Population frequency · supports pathogenic

gnomAD v4.1

gnomAD v2.1

v4.1

Absent from gnomAD v4.1.

v2.1

Absent from gnomAD v2.1.

🇨🇦 CA

Absent from gnomAD-Canada v1.0.

Allele frequency by ancestry

three datasets · side by side

gnomAD v4.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v2.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD Canada 🇨🇦

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗

ClinVar

This variant has been reported in ClinVar as Likely pathogenic (4 clinical laboratories) and as Pathogenic by International Society for Gastrointestinal Hereditary Tumours (InSiGHT) (expert panel). (ClinVarID = 89979)

ClinVar ↗

In silico

SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).

SpliceAI ↗

Functional Unknown Oncogenic Effect

OncoKB identified curated literature and non-variant-specific oncogenicity context for review; listed oncogenicity label: Unknown Oncogenic Effect.

OncoKB ↗

COSMIC

Cancer hotspots

Somatic evidence Not in COSMIC / hotspots

COSMIC

This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).

Hotspots

This variant does not lie in a statistically significant hotspot.

COSMIC ↗ Hotspots ↗

Literature · how each cited paper was used

2papers cited

Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 7 further PMIDs triaged but not cited — see Sources & References.

Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.

PMID 11208710 ↗ · Terdiman JP et al. · 2001 Jan CLINVAR · functional

Searched

c.1989G>A1989G>A1989G A

Found

c.1989G>A at the splice donor of MLH1 exon 17 identified in two independent HNPCC/Lynch syndrome families (2AN7, 5EJ9). mRNA analysis from patient lymphocytes confirmed exon 17 skipping in both families.

Variant

✓ Names this variant — characterised directly

Applied to

→PVS1 supports · met

Why

Confirms exon 17 skipping from c.1989G>A in two independent families. Used to support PVS1_Very_Strong.

1989G A, Splice donor, Exon skipping (M)

Location Table listing mutations with mRNA assay results for MLH1 exon 17 · Context RT-PCR mRNA analysis from patient lymphocyte-derived RNA; exon skipping detected by gel electrophoresis · full text

Germ line MLH1 and MSH2 mutations in Taiwanese Lynch syndrome families: characterization of a founder genomic mutation in the MLH1 gene.

PMID 19419416 ↗ · Tang R et al. · 2009 Apr CLINVAR · splicing rna

Searched

c.1989G>A1989G.A1989G>A

Found

c.1989G>A in MLH1 exon 17 reported in a Taiwanese Lynch syndrome family. Aberrant splicing confirmed as the functional consequence.

Variant

✓ Names this variant — characterised directly

Applied to

→PVS1 supports · met

Why

Independent confirmation from a separate laboratory that c.1989G>A causes aberrant splicing. Provides the required independent confirmation for PVS1_Very_Strong application at a non-canonical splice site.

c.1989G.A, 663, Aberrant splicing

Location Table summarizing MLH1 mutations identified in Taiwanese Lynch syndrome families · Context Mutation screening by DHPLC and DNA sequencing in 93 Taiwanese families fulfilling Amsterdam criteria II · full text

Sources & reference links

9Sources

CSpec VCEP

ClinVar

gnomAD v2.1

gnomAD v4.1

gnomAD-Canada

SpliceAI

OncoKB

COSMIC

Cancer hotspots

Triaged references · 7 PMIDs not cited in assessment

10480359 ↗ Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer. CLINVAR

16395668 ↗ Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. CLINVAR

17576681 ↗ Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. CLINVAR

18561205 ↗ A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. CLINVAR

21404117 ↗ Missense variants in hMLH1 identified in patients from the German HNPCC consortium and functional studies. CLINVAR

21615986 ↗ Distinct mutations in MLH1 and MSH2 genes in hereditary non-polyposis colorectal cancer (HNPCC) families from China. CLINVAR

28492532 ↗ Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. CLINVAR