Dataset Information

Overestimation of alternative splicing caused by variable probe characteristics in exon arrays.

ABSTRACT: In higher eukaryotes, alternative splicing is a common mechanism for increasing transcriptome diversity. Affymetrix exon arrays were designed as a tool for monitoring the relative expression levels of hundreds of thousands of known and predicted exons with a view to detecting alternative splicing events. In this article, we have analyzed exon array data from many different human and mouse tissues and have uncovered a systematic relationship between transcript-fold change and alternative splicing as reported by the splicing index. Evidence from dilution experiments and deep sequencing suggest that this effect is of technical rather than biological origin and that it is driven by sequence features of the probes. This effect is substantial and results in a 12-fold overestimation of alternative splicing events in genes that are differentially expressed. By cross-species exon array comparison, we could further show that the systematic bias persists even across species boundaries. Failure to consider this effect in data analysis would result in the reproducible false detection of apparently conserved alternative splicing events. Finally, we have developed a software in R called COSIE (Corrected Splicing Indices for Exon arrays) that for any given set of new exon array experiments corrects for the observed bias and improves the detection of alternative splicing (available at www.fmi.ch/groups/gbioinfo).

SUBMITTER: Gaidatzis D

PROVIDER: S-EPMC2760813 | biostudies-other | 2009 Sep

REPOSITORIES: biostudies-other

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Overestimation of alternative splicing caused by variable probe characteristics in exon arrays.

Gaidatzis Dimos D Jacobeit Kirsten K Oakeley Edward J EJ Stadler Michael B MB

Nucleic acids research 20090615 16

In higher eukaryotes, alternative splicing is a common mechanism for increasing transcriptome diversity. Affymetrix exon arrays were designed as a tool for monitoring the relative expression levels of hundreds of thousands of known and predicted exons with a view to detecting alternative splicing events. In this article, we have analyzed exon array data from many different human and mouse tissues and have uncovered a systematic relationship between transcript-fold change and alternative splicing ...[more]

PMID: 19528075

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Project description:BACKGROUND: The vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. METHODOLOGY/PRINCIPAL FINDINGS: We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. CONCLUSIONS/SIGNIFICANCE: Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatibility gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF(2) and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches.

Project description:BackgroundThe contribution of alternative splicing and isoform expression to cellular response is emerging as an area of considerable interest, and the newly developed exon arrays allow for systematic study of these processes. We use this pilot study to report on the feasibility of exon array implementation looking to replace the 3' in vitro transcription expression arrays in our laboratory.One of the most widely studied models of cellular response is T-cell activation from exogenous stimulation. Microarray studies have contributed to our understanding of key pathways activated during T-cell stimulation. We use this system to examine whole genome transcription and alternate exon usage events that are regulated during lymphocyte proliferation in an attempt to evaluate the exon arrays.ResultsPeripheral blood mononuclear cells form healthy donors were activated using phytohemagglutinin, IL2 and ionomycin and harvested at 5 points over a 7 day period. Flow cytometry measured cell cycle events and the Affymetrix exon array platform was used to identify the gene expression and alternate exon usage changes. Gene expression changes were noted in a total of 2105 transcripts, and alternate exon usage identified in 472 transcript clusters. There was an overlap of 263 transcripts which showed both differential expression and alternate exon usage over time. Gene ontology enrichment analysis showed a broader range of biological changes in biological processes for the differentially expressed genes, which include cell cycle, cell division, cell proliferation, chromosome segregation, cell death, component organization and biogenesis and metabolic process ontologies. The alternate exon usage ontological enrichments are in metabolism and component organization and biogenesis. We focus on alternate exon usage changes in the transcripts of the spliceosome complex. The real-time PCR validation rates were 86% for transcript expression and 71% for alternate exon usage.ConclusionsThis study illustrates that the Exon array technology has the potential to provide information on both transcript expression and isoform usage, with very little increase in expense.

Project description:Background: The vast majority of human genes (.70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. Methodology/Principal Findings: We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. Conclusions/Significance: Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatability gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MGthymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches Keywords: Disease analysis, Tumor type comparison analysis

Dataset Information

Overestimation of alternative splicing caused by variable probe characteristics in exon arrays.

Publications

Overestimation of alternative splicing caused by variable probe characteristics in exon arrays.

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