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

Our plain-English summary of Differentially expressed alternatively spliced genes in malignant pleural mesothelioma identified using massively parallel transcriptome sequencing, by L. Dong, R.V. Jensen, A. De Rienzo, et al (BMC Medical Genetics, 2009).

The study

• Authors: L. Dong, R.V. Jensen, A. De Rienzo, G.J. Gordon, Y. Xu, D.J. Sugarbaker, R. Bueno
• Journal: BMC Medical Genetics
• Published: 2009;10:149
• Cited by: 19 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory pilot study examined alternative splicing (genes producing different versions of their messages) in malignant pleural mesothelioma (MPM) compared with normal lung tissue. It did not study asbestos or any other exposure. The authors re-analyzed 13,274,187 gene-sequence readings from an earlier study of 4 MPM tumors and 1 normal lung sample, matching them to 151,486 known splice junctions. Of these, 70,953 junctions were covered by at least one reading. They then retested 10 junctions with a second laboratory method (qRT-PCR): the top 3 ranked higher in normal lung and the top 7 ranked higher in MPM. The predicted difference was confirmed for 8 of the 10. Two junctions, in the ACTG2 and CDK4 genes, were tested in another 18 MPM and 18 normal lung samples, and both differed with statistical significance. Using the middle value of all 36 samples as a cut-off, ACTG2 correctly sorted 89% of samples and CDK4 sorted 78%. The authors concluded these splice variants could be useful as diagnostic markers and possible treatment targets for MPM. They named the small sample size as a limitation.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory gene-expression study (re-analysis of existing transcriptome sequencing data, with validation by quantitative real-time PCR); the authors describe it as a pilot study
• Who was studied: Discovery set: 4 malignant pleural mesothelioma tumors and 1 normal lung sample, a subset of samples sequenced in an earlier study. Validation set: 36 additional discarded tissue specimens (18 MPM, 18 normal lung) from the Tumor Bank at Brigham and Women's Hospital, Boston. Collection dates are not given.
• Exposure: Not studied. The article does not examine asbestos or any fiber exposure; it compares gene splicing patterns between MPM tumors and normal lung tissue.
• Main results:
  • Of 151,486 known exon junctions, 70,953 were covered by at least one sequence read in at least one of the 5 discovery samples
  • 76 exon junctions were identified as highly expressed in the MPM samples and 32 as highly expressed in the normal lung sample
  • For qRT-PCR testing in the 5 discovery samples, the authors chose 10 exon junctions: the top 3 ranked as higher in normal lung and the top 7 ranked as higher in MPM. They state they arbitrarily weighted the list towards junctions over-expressed in MPM.
  • Of these 10 junctions, the predicted differential expression was confirmed for 8. The junctions in EMP2 and DNAJB11 could not be confirmed.
  • In the additional 18 MPM and 18 normal lung samples, average expression of the ACTG2 and CDK4 exon junctions differed statistically significantly between tumor and normal samples in the predicted direction (P = 0.00054 and P = 0.00039, respectively). ACTG2 was predicted to be higher in normal lung and CDK4 higher in tumor.
  • Using the median of all 36 samples as the cut-off, the ACTG2 junction correctly classified 89% of samples (16/18 tumor, 16/18 normal; P = 5.2 × 10 to the minus 6)
  • The CDK4 junction correctly classified 78% of samples (14/18 tumor, 14/18 normal; P = 2.2 × 10 to the minus 3)
  • All 36 validation samples were correctly classified by at least one of the two exon junctions
• The authors' conclusion: The authors concluded that whole-transcriptome shotgun sequencing with their bioinformatics pipeline can identify differentially expressed exon junctions, and that the alternative transcripts found could be useful as diagnostic markers and potential therapeutic targets for MPM
• Limitations the authors state:
  • The small discovery sample (only 4 MPM and 1 normal lung) meant the authors could not test for statistical differences in the sequencing data
  • They could not compare results across different sequencing platforms
  • Not all exon junctions predicted to be differentially expressed proved to be so to the same extent by qRT-PCR. The authors attribute this to the limited number of specimens.
  • The signal-to-noise ratio may have been over-amplified so as not to miss candidates
  • Sequencing artifacts, insufficient sequencing depth, SNPs near the exon junctions and an incomplete database of exon junctions are further possible limitations

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Lung Adenocarcinoma, Mesothelioma.

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