rs267607760
Variant summary
Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PM2PP3PP5_Very_Strong
The NM_000249.4(MLH1):c.545+3A>G variant causes a splice region, intron change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★).
Frequency
Consequence
NM_000249.4 splice_region, intron
Scores
Clinical Significance
Conservation
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ACMG classification
Verdict is Pathogenic. Variant got 11 ACMG points.
Transcripts
RefSeq
Ensembl
Frequencies
GnomAD3 genomes
GnomAD4 exome Cov.: 29
GnomAD4 genome
ClinVar
Submissions by phenotype
not provided Pathogenic:4
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This variant is denoted MLH1 c.545+3A>G or IVS6+3A>G and consists of an A>G nucleotide substitution at the +3 position of intron 6 of the MLH1 gene. In-silico analyses, including splice predictors and evolutionary conservation, support a deleterious effect. RNA studies and protein truncation testing confirm this prediction, demonstrating abnormal splicing leading to a frameshift that results in a truncated protein (Pensotti 1997, Thiffault 2004). This variant was observed in multiple individuals with colon cancer, including two who had absent MLH1 on immunohistochemistry, and is considered an Italian pathogenic founder variant (Pensotti 1997, Hendriks 2003, Liu 2004, Thiffault 2004, Mangold 2005, Valentin 2011). This variant has been shown to segregate with disease in two affected siblings in one family and with three affected relatives in another family (Thiffault 2004). MLH1 c.545+3A>G was not observed in large population cohorts (Lek 2016). The adenine (A) nucleotide that is altered is conserved across species. Based on the current evidence, we consider this variant to be pathogenic. -
The variant has been reported in individuals and families with Lynch syndrome in the published literature (PMIDs: 28874130 (2017), 26437257 (2015), 21681552 (2011), 15849733 (2005)). In addition, published studies indicate that this variant causes a damaging effect on normal MLH1 splicing (PMIDs: 15253764 (2004), 9218993 (1997)). This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). Based on the available information, this variant is classified as pathogenic. -
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Lynch syndrome Pathogenic:3
Variant summary: The MLH1 c.545+3A>G variant involves the alteration of a conserved nucleotide located at the intron/exon boundary. One in silico tool predicts a damaging outcome for this variant. 4/4 splice prediction tools predict loss/weakening effect on the canonical splicing donor site. This variant was absent in 121426 control chromosomes but has been reported in multiple affected individuals/families, and some families showed evidence of co-segregation of the variant with disease. In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. RT-PCR showed the variant lead to an aberrant splicing product (Pensotti_GCC_1997). Taken together, this variant was classified as pathogenic. -
Variant causes splicing aberration, >2 MSI-H, co-segregation with disease & absent in 1000 genomes -
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Hereditary cancer-predisposing syndrome Pathogenic:3
This variant causes an A to G nucleotide substitution at the +3 position of intron 6 of the MLH1 gene. A functional RNA study has shown that this variant causes aberrant splicing and a significant truncation of exon 6 due to the use of a cryptic donor site leaving all but the first 3 nucleotides of exon 6 (PMID: 9218993). This variant has been reported in multiple individuals affected with hereditary nonpolyposis colorectal cancer (PMID: 9218993, 12547705, 15253764, 15309712) and shown to segregate with disease in family studies (PMID: 9218993, 15253764). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -
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The c.545+3A>G intronic pathogenic mutation results from an A to G substitution 3 nucleotides after coding exon 6 in the MLH1 gene. This mutation has been identified in probands whose Lynch syndrome-associated tumor demonstrated high microsatellite instability and/or loss of MLH1 or MLH1/PMS2 expression by immunohistochemistry (Hendriks Y et al. Am. J. Pathol., 2003 Feb;162:469-77; Thiffault I et al. Clin. Genet., 2004 Aug;66:137-43; Carneiro da Silva F et al. PLoS ONE, 2015 Oct;10:e0139753; Ambry internal data). This alteration has been shown to cause a deleterious impact on splicing by RT-PCR and cDNA analyses (Pensotti V et al. Genes Chromosomes Cancer, 1997 Jul;19:135-42; Morak M et al. Eur J Hum Genet, 2019 Dec;27:1808-1820). In addition, RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -
Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2
The splice site c.545+3A>G variant has been reported in multiple individuals affected with hereditary nonpolyposis colorectal cancer (Rossi BM. et al., 2017, Pensotti V. et. al., 1997). A functional RNA study has shown that this variant causes aberrant splicing and a significant truncation of exon 6 due to the use of a cryptic donor site leaving all but the first 3 nucleotides of exon 6 (Pensotti V. et. al., 1997). This variant has been reported to the ClinVar database as Pathogenic with a status of reviewed by expert panel. The c.545+3A>G variant is novel (not in any individuals) in gnomAD Exomes and 1000 Genomes. Loss of function variants have been previously reported to be disease causing. For these reasons, this variant has been classified as Pathogenic. -
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Lynch-like syndrome Pathogenic:1
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Hereditary nonpolyposis colorectal neoplasms Pathogenic:1
This sequence change falls in intron 6 of the MLH1 gene. It does not directly change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or altered protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individuals with Lynch syndrome (PMID: 9218993, 12547705, 15253764, 15309712, 15849733, 26437257, 28874130). It has also been observed to segregate with disease in related individuals. Invitae Evidence Modeling of clinical and family history, age, sex, and reported ancestry of multiple individuals with this MLH1 variant has been performed. This variant is expected to be pathogenic with a positive predictive value of at least 99%. This is a validated machine learning model that incorporates the clinical features of 1,370,736 individuals referred to our laboratory for MLH1 testing. ClinVar contains an entry for this variant (Variation ID: 90260). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in activation of a cryptic splice site, and produces a non-functional protein and/or introduces a premature termination codon (PMID: 9218993). For these reasons, this variant has been classified as Pathogenic. -
Lynch syndrome 1 Other:1
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Computational scores
Source:
Splicing
Find out detailed SpliceAI scores and Pangolin per-transcript scores at