MSH2 Overexpression Due to an Unclassified Variant in 3’-Untranslated Region in a Patient with Colon Cancer

Abstract

Background: The loss or low expression of DNA mismatch repair (MMR) genes can result in genomic instability and tumorigenesis. One such gene, MSH2, is mutated or rearranged in Lynch syndrome (LS), which is characterized by a high risk of tumor development, including colorectal cancer. However, many variants identified in this gene are often defined as variants of uncertain significance (VUS). In this study, we selected a variant in the 3′ untranslated region (UTR) of MSH2 (c*226A > G), identified in three affected members of a LS family and already reported in the literature as a VUS. Methods: The effect of this variant on the activity of the MMR complex was examined using a set of functional assays to evaluate MSH2 expression. Results: We found MSH2 was overexpressed compared to healthy controls, as determined by RTqPCR and Western blot analyses of total RNA and proteins, respectively, extracted from peripheral blood samples. These results were confirmed by luciferase reporter gene assays. Conclusions: We therefore speculated that, in addition to canonical inactivation via a gene mutation, MMR activity may also be modulated by changes in MMR gene expression.

MiR-137 Targets the 3′ Untranslated Region of MSH2: Potential Implications in Lynch Syndrome-Related Colorectal Cancer

“…Total RNA and protein were extracted 48 h after miRNA and anti-miRNA transfection. RNA was isolated with TRIZOL solution according to the manufacturer’s protocols, and was used to determine endogenous mRNA levels of MSH2 and actin (internal control) according to the same protocol reported for RNA analysis of patient carrying the variant in MSH2 3′UTR gene [ 8 ]. The dosage of miR-137 levels was performed using a TaqMan MiRNA RT and Assay kit (Applied Biosystems, Inc.,Waltham, Massachusetts, Stati Uniti), with small nucleolar RNA RNU6B (Applied Biosystems, Inc.) as a normalization gene.…”

“…Total protein was isolated using cell lysis buffer containing 10% glycerol, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA pH 8, and 10 µL of protease inhibitor cocktail (Sigma-Aldrich). Quantification of MSH2 and actin (loading control) was performed as described for the patient protein analysis [ 8 ].…”

“…At the somatic level, this condition determines microsatellite instability (MSI) [ 2 , 3 , 4 , 5 ]. In some instances, MMR deficiency is associated with MLH1 promoter hypermethylation which is responsible for sporadic CRC with MSI [ 6 , 7 ], whereas on other occasions, it correlates with germline pathogenic variants in MMR genes responsible for the Lynch Syndrome (LS) phenotype [ 3 , 4 , 5 , 8 ]. MSH2 and MLH1, but also MSH6 and PMS2 are the most common pathogenetic variants [ 2 , 3 , 4 , 9 , 10 , 11 , 12 ] determining MMR deficiency in tumoral tissue; this is due to altered MMR protein functionality.…”

“…To date, several hypotheses purporting to account for loss of function of the MMR system have been postulated. For example, etiological factors such as microRNAs (miRNAs) strongly influence repair functions within the MMR complex, with significant effects on disease progression [ 8 , 19 , 20 , 21 , 22 ]. MiRNAs constitute a family of small, single-stranded endogenous, non-coding, RNAs of approximately 21–25 nucleotides, the fundamental role of which is to negatively regulate gene expression at post-transcriptional levels.…”

“…Recently, we described a patient case where MSH2 overexpression was likely related to a loss of MSH2 down-regulation due to a nucleotide variant in the 3′UTR [ 8 ]. Preliminary bioinformatics analyses showed this region was a putative target site for miRNAs.…”