Differentially expressed alternatively spliced genes in malignant pleural mesothelioma identified using massively parallel transcriptome sequencing

Abstract

Background: Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.

Methods: We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.

Results: We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).

Conclusion: Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

Figure 1

Identification of alternatively spliced transcripts using second generation sequencing data. The 454 sequencing reads that span exon junctions provide information about alternatively spliced variants in the human transcriptome. For example, two typical alternatively spliced variants ('a' and 'b') of a single gene are shown in the top half of the figure. The lower half of the figure shows how different 454 reads may align to cDNA sequences generated from the mRNA providing evidence of alternatively splicing events.

Figure 2

Differential expression of specific exon junctions in MPM and normal lung samples. Average expression levels of exon junctions in ACTG2 (A) and CDK4 (B) differ statistically significantly between MPM and normal lung samples. Individual expression levels of exon junctions in ACTG2 (C) and CDK4 (D) can be used to classify MPM and normal tissue samples using the median value as a cut-off. Error bars, SEM. Arrows indicate misclassified samples.