Systematic mRNA analysis for the effect ofMLH1 andMSH2 missense and silent mutations on aberrant splicing

Abstract

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Integrated analysis of unclassified variants in mismatch repair genes

“…However, a combination of analyses seems to be more reliable than a single-software test, even if an analysis performed using different web tools is complicated by prediction discordance, particularly for ESE/exon splicing silencer creation or elimination, due to different algorithms used to evaluate nucleotide variations. A study conducted on ESEfinder and then confirmed on RescueESE8, 40,41 demonstrated that the prediction does not always correlate with an in vitro effect. Protein-prediction software tools are more reliable and there is greater agreement in their output data.…”

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome

“…The single case (DSALLG15_R1) with MSH6 mutation (p. T915A) and no increase in the mutational burden carried an amino acid alteration predicted as benign by PolyPhen2 (31). Interestingly, the two cases with MLH1 mutation presented an identical alteration, a splicing mutation (X264_splice; rs267607789) observed in patients with Lynch syndrome (32). In addition, the second relapse of patient DSALLG11 displayed an exceptionally high number of SNVs (1225), possibly because of a synergistic effect of mutations both in MSH6 and SETD2 (33).…”

Prediction of single‐nucleotide substitutions that result in exon skipping: identification of a splicing silencer inBRCA1exon 6

“…Splicing defects resulting from exonic substitutions have been found in a growing number of disease genes, such as CFTR [Pagani et al, 2005;Raponi et al, 2007], MLH1 [Auclair et al, 2006;Stella et al, 2001;Tournier et al, 2008], ATM [Teraoka et al, 1999], NF1 [Ars et al, 2000], SMN2 [Lorson and Androphy, 2000] or BRCA1 . The latter gene is one of the two most important breast cancer susceptibility genes [King et al, 2003], encoding a 1863 amino-acid protein involved in DNA damage repair and transcription regulation [Gowen et al, 1998].…”