ClinVar Genomic variation as it relates to human health

Interrupts canonical donor splice site

This sequence change affects a donor splice site in intron 8 of the MSH2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or disrupted protein product. This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with Lynch syndrome (PMID: 15849733, 16736289). ClinVar contains an entry for this variant (Variation ID: 90641). Studies have shown that disruption of this splice site results in skipping of exon 8 and introduces a premature termination codon (PMID: 16395668, 19669161; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic.

Variant summary: MSH2 c.1386+1G>A 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. Several computational tools predict a significant impact on normal splicing: four predict the variant abolishes a 5' splicing donor site. Publications reported experimental evidence, demonstrating that the variant resulted in the skipping of exon 8 (Auclair_2006, Betz_2010). The variant was absent in 248984 control chromosomes (gnomAD). c.1386+1G>A has been reported in the literature in multiple individuals affected with Hereditary Nonpolyposis Colorectal Cancer (e.g. Auclair_2006, Mangold_2005, Mueller-Koch_2005, Betz_2010, Baert-Desurmont_2018, Jiang_2019). These data indicate that the variant is likely to be associated with disease. Three other submitters, including an expert panel (InSiGHT), have provided clinical-significance assessments for this variant in ClinVar after 2014, and classified the variant as pathogenic (2x) / likely pathogenic (1x). Based on the evidence outlined above, the variant was classified as pathogenic.

This variant is considered likely 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.

The c.1386+1G>A intronic pathogenic mutation results from a G to A substitution one nucleotide after coding exon 8 of the MSH2 gene. This mutation has been previously identified in individuals with personal and/or family histories suggestive of Lynch syndrome (Mangold E et al. Int. J. Cancer 2005 Sep;116:692-702; Mueller-Koch Y et al. Gut 2005 Dec;54:1733-40; Jiang W et al. Int J Cancer, 2019 05;144:2161-2168). This mutation has been identified as somatic in conjunction with a somatic pathogenic MSH2 variant in a Lynch syndrome-associated tumor that demonstrated loss of MSH2/MSH6 expression by immunohistochemistry (IHC; Watkins JC et al. Int. J. Gynecol. Pathol. 2017 Mar;36:115-127). This variant has been identified in probands whose Lynch syndrome-associated tumor demonstrated high microsatellite instability and/or loss of MSH2 or MSH2/MSH6 expression by IHC (Ambry internal data). Additionally, functional analyses have demonstrated that this alteration leads to the skipping of coding exon 8 (Auclair J et al. Hum. Mutat. 2006 Feb;27:145-54; Betz B et al. J. Cancer Res. Clin. Oncol. 2010 Jan;136:123-34; 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. 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 c.1386+1G>A variant was identified in 3 of 3562 proband chromosomes (frequency: 0.001) from individuals or families with Lynch Syndrome (Auclair, 2006; Mangold, 2005). The variant was also identified in dbSNP (ID: rs267607957) “With pathogenic allele”, HGMD, “MMR Gene Unclassified Variants Database”, “InSiGHT Colon Cancer Database” (as likely pathogenic), and the ClinVar database. The c.1386+1G>A variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. Studies by Auclair and Betz have shown this variant to cause the loss of exon 8 and Mueller-Koch links this loss to a loss of expression of the MSH2 protein in colon tumours. In addition, in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing in 4 of 5 programs. Loss of function variants of the MSH2 gene are an established mechanism of disease in Lynch syndrome and this is the type of variant expected to cause the disorder. In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic.