ClinVar Genomic variation as it relates to human health

Multifactorial likelihood analysis posterior probability >0.99

This variant disrupts a canonical splice-donor site and interferes with normal MLH1 mRNA splicing. It has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in individuals with or at high risk of colorectal cancer (PMIDs: 27601186 (2016), 23544471 (2013), 17312306 (2007), 15342696 (2004), 12624141 (2003)). Based on the available information, this variant is classified as pathogenic.

The c.677+1G>T intronic pathogenic mutation results from a G to T substitution one nucleotide after coding exon 8 of the MLH1 gene. This mutation, also referred to as IVS8+1G>T in published literature, has been reported in a family that met modified Amsterdam criteria (Parc Y et al. J Med Genet. 2003 Mar;40(3):208-13). This mutation was also detected in several individuals whose tumors showed high microsatellite instability and negative BRAF V600E analyses (Domingo E et al. J Med Genet. 2004 Sep;41(9):664-8; Lagerstedt Robinson K et al. J Natl Cancer Inst. 2007 Feb 21;99(4):291-9). This mutation has been identified in at least one proband who met Amsterdam I/II criteria for Lynch syndrome and tumor demonstrated high microsatellite instability with loss of MLH1 expression by immunohistochemistry (IHC; Ambry internal data). Furthermore, this mutation has been identified in probands whose Lynch syndrome-associated tumor demonstrated loss of MLH1/PMS2 expression by IHC (Ambry internal data). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). 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 and may result in the creation or strengthening of a novel splice donor site; however, direct evidence is insufficient at this time (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.

This pathogenic variant is denoted MLH1 c.677+1G>T or IVS8+1G>T and consists of a G>T nucleotide substitution at the +1 position of intron 8 of the MLH1 gene. The variant destroys a canonical splice donor site and is predicted to cause abnormal gene splicing, leading to either an abnormal message that is subject to nonsense-mediated mRNA decay or to an abnormal protein product. This variant has been reported in individuals with familial and/or early-onset colorectal cancer, including one individual whose colorectal tumor demonstrated microsatellite instability (Parc 2003, Domingo 2004, Lagerstedt Robinson 2007). The International Society for Gastrointestinal Hereditary Tumours Incorporated (InSiGHT) classifies this variant as pathogenic based on their multifactorial likelihood analysis (Thompson 2014). Based on the current evidence, we consider MLH1 c.677+1G>T to be pathogenic.

For these reasons, this variant has been classified as Pathogenic. Studies have shown that disruption of this splice site results in skipping of exon 8 and introduces a premature termination codon (Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 24362816). ClinVar contains an entry for this variant (Variation ID: 90312). This variant is also known as IVS8+1G>T. Disruption of this splice site has been observed in individuals with a MLH1-related disease (PMID: 12624141, 15342696, 17312306, 27601186; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change affects a donor splice site in intron 8 of the MLH1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or disrupted protein product.

Variant summary: MLH1 c.677+1G>T is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. 4 computational tools predict a significant impact on normal splicing and abolishes a 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant was absent in 250982 control chromosomes (gnomAD). The variant, c.677+1G>T, has been reported in the literature in individuals affected with Lynch Syndrome and early-onset colorectal cancer (examples: Parc_2003, Domingo_2004, Tanskanen_2013 and FerrerAvargues_2021). These data indicate that the variant is likely to be associated with disease. Two ClinVar submissions from clinical diagnostic laboratories (evaluation after 2014) cite the variant as pathogenic. Based on the evidence outlined above, the variant was 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: 12655562].

The MLH1 c.677+1G>A variant was identified in 4 of 1350 proband chromosomes (frequency: 0.003) from individuals or families with Lynch Syndrome (Parc 2003, Domingo 2004, Lagerstedt Robinson 2007, Schneider 2018). The variant was identified in dbSNP (rs267607778) as “with pathogenic allele”, ClinVar (classified as pathogenic by GeneDx and InSiGHT and likely pathogenic by Invitae) and UMD-LSDB (observed 6x). The variant was not identified in the following control databases: the Exome Aggregation Consortium (August 8th 2016), or the Genome Aggregation Database (Feb 27, 2017). The c.677+1G>A variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. In addition, in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a loss of the 5’ splice site. In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.