Missense mutations of MLH1 and MSH2 genes detected in patients with gastrointestinal cancer are associated with exonic splicing enhancers and silencers

Abstract

The MLH1 and MSH2 genes in DNA mismatch repair are important in the pathogenesis of gastrointestinal cancer. Recent studies of normal and alternative splicing suggest that the deleterious effects of missense mutations may in fact be splicing-related when they are located in exonic splicing enhancers (ESEs) or exonic splicing silencers (ESSs). In this study, we used ESE-finder and FAS-ESS software to analyze the potential ESE/ESS motifs of the 114 missense mutations detected in the two genes in East Asian gastrointestinal cancer patients. In addition, we used the SIFT tool to functionally analyze these mutations. The amount of the ESE losses (68) was 51.1% higher than the ESE gains (45) of all the mutations. However, the amount of the ESS gains (27) was 107.7% higher than the ESS losses (13). In total, 56 (49.1%) mutations possessed a potential exonic splicing regulator (ESR) error. Eighty-one mutations (71.1%) were predicted to be deleterious with a lower tolerance index as detected by the Sorting Intolerant from Tolerant (SIFT) tool. Among these, 38 (33.3%) mutations were predicted to be functionally deleterious and possess one potential ESR error, while 18 (15.8%) mutations were predicted to be functionally deleterious and exhibit two potential ESR errors. These may be more likely to affect exon splicing. Our results indicated that there is a strong correlation between missense mutations in MLH1 and MSH2 genes detected in East Asian gastrointestinal cancer patients and ESR motifs. In order to correctly understand the molecular nature of mutations, splicing patterns should be compared between wild-type and mutant samples.

Keywords: MLH1; MSH2; exonic splicing enhancer; exonic splicing silencer; gastrointestinal cancer; missense mutation.

Figure 1

High-score splicing regulator (SR) protein motif analysis in MSH2 exon 6 (A) and a single point mutation variant c.1012G>A (B). High-score motifs are shown in dark gray for SF2/ASF (IgM-BRCA1), black for SC35, light gray for SRp40 and white for SRp55, and only the scores above the threshold for each SR protein are shown. The height of each bar indicates the score value, and its width and placement on the x-axis represent the length of the motif (6–8 nt) and its position along the sequence. The arrow indicated that the c.1012G>A transversion in MSH2 exon 6 affected a SF2/ASF motif, reducing the score from 2.620 to 0.840; a SC35 motif, reducing the score from 2.669 to 0.917; and a SRp40 motif, reducing the score from 4.353 to 1.971.

Figure 2

Fluorescence-activated screen for exonic splicing silencers (FAS-ESS) analysis of MLH1 exon 4 (A) and a single point mutation variant c.332C>T (B). The arrow indicates that the c.332C>T transversion in MLH1 exon 4 created a new ESS motif.