MLH1 c.790+1G>A, p.Splice_Site
NM_000249.4:c.790+1G>A
COSMIC ID: COSM29723
Pathogenic
The canonical +1 splice site variant c.790+1G>A in MLH1 is predicted to abolish normal splicing, leading to LOF via NMD (PVS1 Very Strong) and is absent from population databases (PM2 Supporting). No additional criteria apply under VCEP guidelines. These combined strengths support a classification of Likely Pathogenic.
ACMG/AMP Criteria Applied
PVS1
PM2
Genetic Information
Gene & Transcript Details
Gene
MLH1
Transcript
NM_000249.4
MANE Select
Total Exons
19
Strand
Forward (+)
Reference Sequence
NC_000003.11
Alternative Transcripts
| ID | Status | Details |
|---|---|---|
| NM_000249.3 | RefSeq Select | 19 exons | Forward |
| NM_000249.2 | Alternative | 19 exons | Forward |
Variant Details
HGVS Notation
NM_000249.4:c.790+1G>A
Protein Change
Splice
Location
Exon 9
(Exon 9 of 19)
5'Exon Structure (19 total)3'
Functional Consequence
Loss of Function
Related Variants
Alternate Identifiers
COSM29723
Variant interpretation based on transcript NM_000249.4
Genome Browser
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HGVS InputNM_000249:c.790+1G>A
Active Tracks
ConservationRefSeqClinVargnomAD
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Clinical Data
Global Frequency
0.0 in 100,000
Extremely Rare
Global: 0.0%
0%
0.05%
0.1%
1%
5%
10%+
Allele Information
Total: 249880Alt: 0Homozygotes: 0
ACMG Criteria Applied
PM2
This variant is present in gnomAD (MAF= 0%, 0/249880 alleles, homozygotes = 0) but does not appear at a higher frequency in any of the selected populations. The variant is rare (MAF < 0.1%), supporting PM2 criterion application.
Classification
7 publications
Likely Pathogenic
Based on 18 submitter reviews in ClinVar
Submitter Breakdown
17 Path
1 LP
Pathogenic
Likely Path.
VUS
Likely Benign
Benign
Publications (7)
Variant summary: The MLH1 c.790+1G>A variant involves the alteration of a conserved intronic nucleotide. One in silico tool predicts a damaging outcome for this variant. 5/5 splice prediction tools predict that this variant ablishes the 5' splicing donor site, which was confirmed by an in vitro study (Auclair_2006). This variant has been found in numerous LS patients and is absent in 115338 control chromosomes. In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as pathogenic.
This variant is considered pathogenic. This variant occurs within a consensus splice junction and is predicted to result in abnormal mRNA splicing of either an out-of-frame exon or an in-frame exon necessary for protein stability and/or normal function. Functional studies indicate this variant impacts protein function [PMID: 8574961, 11781295].
The c.790+1G>A intronic pathogenic mutation results from a G to A substitution one nucleotide after coding exon 9 of the MLH1 gene. This mutation has been seen in multiple individuals with personal and family histories of colon cancer (Cunningham JM et al. Am. J. Hum. Genet. 2001 Oct;69:780-90; Rosty C et al. BMJ Open. 2016 Feb;6(2):e010293; Haraldsdottir S et al. Fam. Cancer 2016 Apr;15(2):253-60; Rossi BM et al. BMC Cancer 2017 Sep;17(1):623). In addition, a functional study utilizing RNA from an individual carrying this mutation found that it leads to skipping of coding exons 9 and 10 (Auclair J et al. Hum. Mutat. 2006 Feb;27:145-54; Ambry internal data). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as a disease-causing mutation.
The MLH1 c.790+1G>A variant (also known as IVS9+1G>A) disrupts a canonical splice site and has been shown to cause an in-frame skipping of exons 9-10 (PMID: 16395668 (2006)), at a level greater the alternatively spliced isoforms observed in normal cells (PMIDs: 9490293 (1998), 7728749 (1995)). The frequency of this variant in the general population, 0.0000066 (1/152090 chromosomes, http://gnomad.broadinstitute.org), is consistent with pathogenicity. The variant has been reported in multiple individuals with colorectal cancer or Lynch syndrome (PMIDs: 26895986 (2016), 25133505 (2014), 25107687 (2014), 24344984 (2013), 20937110 (2010), 20305446 (2010), 17054581 (2006), 16395668 (2006), 15955785 (2005), 15849733 (2005), 11524701 (2001)). A functional study also indicates the variant causes a significant loss of DNA mismatch repair activity (PMID: 11781295 (2002)). Based on the available information, this variant is classified as pathogenic.
Clinical Statement
This variant has been reported in ClinVar as Pathogenic (17 clinical laboratories) and as Likely pathogenic (1 clinical laboratories) and as Pathogenic by International Society for Gastrointestinal Hereditary Tumours (InSiGHT) expert panel.
Expert Panel Reviews
Pathogenic
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)
Functional Impact
Functional Domain
Hotspot Status
Not a hotspot
Domain Summary
This variant is not located in a mutational hotspot or critical domain (0 mutations).
Related Variants in This Domain
Computational Analysis
Pathogenicity Predictions
Predictor Consensus
Unknown
PP3 Applied
No
VCEP Guidelines
Applied ACMG/AMP Criteria (VCEP Specific) VCEP Guidelines
PVS1
PVS1 (Very Strong)
According to VCEP guidelines, the rule for PVS1 Very Strong is: "Variants at IVS±1 or IVS±2 where exon skipping or use of a cryptic splice site disrupts reading frame and is predicted to undergo NMD." The evidence for this variant shows a canonical +1 splice site change (c.790+1G>A) in MLH1, predicted to abolish normal splicing and cause loss of function via NMD. Therefore, this criterion is applied at Very Strong strength because it meets the VCEP definition for a canonical splice disrupting variant predicted to lead to NMD.
PS1
PS1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PS1 Strong is: "Variants affecting the same non-canonical splice nucleotide as a confirmed pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI." The evidence for this variant shows it is at the canonical +1 splice site, not a non-canonical nucleotide, and there is no prior non-canonical splice nucleotide variant at this exact position. Therefore, this criterion is not applied.
PS2
PS2 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PS2 is de novo occurrence points: Very Strong ≥4, Strong 2–3, Moderate 1, Supporting 0.5. There is no information regarding de novo status. Therefore, this criterion is not applied.
PS3
PS3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PS3 Strong is: "Calibrated functional assays with functional odds for Pathogenicity >18.7." No functional assays have been performed for this splice variant. Therefore, this criterion is not applied.
PS4
PS4 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PS4 is: "Prevalence of variant in affected individuals statistically increased over controls." No case-control or segregation data are available. Therefore, this criterion is not applied.
PM1
PM1 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM1 is: "Located in a mutational hot spot and/or critical and well‐established functional domain without benign variation." This variant affects splicing, not a known functional domain hotspot. Therefore, this criterion is not applied.
PM2
PM2 (Supporting) Strength Modified
According to VCEP guidelines, the rule for PM2 Supporting is: "Absent/extremely rare (<1 in 50,000 alleles) in gnomAD v4." The evidence for this variant shows MAF=0% in gnomAD v4. Therefore, this criterion is applied at Supporting strength because the variant is absent in population databases.
PM3
PM3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PM3 applies to recessive disorders with trans observations. No trans occurrences with a pathogenic MLH1 variant have been reported. Therefore, this criterion is not applied.
PM4
PM4 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM4 is: "Protein length changes due to in-frame indels or stop-loss variants." This is a splice site variant leading to LOF covered by PVS1. Therefore, this criterion is not applied.
PM5
PM5 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PM5 Moderate is: "Missense change at an amino acid residue where a different missense change was classified as Pathogenic." This variant is a splice site variant, not a missense change. Therefore, this criterion is not applied.
PM6
PM6 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PM6 is: "Unconfirmed de novo occurrence." No de novo evidence is available. Therefore, this criterion is not applied.
PP1
PP1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PP1 Strong is: "Co-segregation with disease with combined Bayes LR >18.7." No segregation data are available. Therefore, this criterion is not applied.
PP2
PP2 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PP2 is: "Missense variant in a gene with low rate of benign missense variation." This is a splice variant, not missense. Therefore, this criterion is not applied.
PP3
PP3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PP3 Supporting is: "Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score ≥0.2." This is a canonical +1 splice variant for which PVS1 applies and VCEP specifies not to combine PP3 with PVS1. Therefore, this criterion is not applied.
PP4
PP4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for PP4 Strong is: "Phenotype or family history specific for MLH1 pathogenic variants (e.g., MSI-H tumors)." No tumor phenotype data are provided. Therefore, this criterion is not applied.
PP5
PP5 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for PP5 is: "Reputable source reports variant as pathogenic without accessible evidence." Although ClinVar entries exist, VCEP does not recommend PP5. Therefore, this criterion is not applied.
BA1
BA1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BA1 Stand Alone is: "gnomAD v4 Grpmax filtering allele frequency ≥0.001." The variant has MAF=0% in gnomAD. Therefore, this criterion is not applied.
BS1
BS1 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS1 Strong is: "gnomAD v4 Grpmax filtering allele frequency ≥0.0001 and <0.001." The variant has MAF=0% in gnomAD. Therefore, this criterion is not applied.
BS2
BS2 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS2 Strong is: "Co-occurrence in trans with a known pathogenic MLH1 variant in non-CMMRD context." No such co-occurrence data exist. Therefore, this criterion is not applied.
BS3
BS3 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS3 Strong is: "Calibrated assays showing no functional impact." No functional assays demonstrate normal splicing or function. Therefore, this criterion is not applied.
BS4
BS4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BS4 Strong is: "Lack of segregation with disease (Bayes LR <0.05)." No segregation data are available. Therefore, this criterion is not applied.
BP1
BP1 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP1 is: "Missense variant in a gene where truncating variants are the predominant mechanism." This is a splice variant causing truncation, not missense. Therefore, this criterion is not applied.
BP2
BP2 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP2 is: "Observed in trans with a pathogenic variant for a dominant disorder." No such observation is reported. Therefore, this criterion is not applied.
BP3
BP3 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP3 is: "In-frame indels in repetitive regions without known function." This variant is a splice site change, not an in-frame indel. Therefore, this criterion is not applied.
BP4
BP4 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP4 Supporting is: "SpliceAI predicts no splicing impact with delta score ≤0.1." SpliceAI predicts high impact (0.87). Therefore, this criterion is not applied.
BP5
BP5 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP5 Strong is: "Tumor characteristics inconsistent with MLH1 loss." No such tumor data exist. Therefore, this criterion is not applied.
BP6
BP6 (Not Applied) Strength Modified
According to standard ACMG guidelines, the rule for BP6 is: "Reputable source reports variant as benign without accessible evidence." No such benign reports exist. Therefore, this criterion is not applied.
BP7
BP7 (Not Applied) Strength Modified
According to VCEP guidelines, the rule for BP7 Supporting is: "Synonymous or intronic variant at or beyond –21/+7." This is a canonical +1 variant. Therefore, this criterion is not applied.