Abstract
The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes. The analysis identified a splice variant of LAMA3 (Laminin α 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative RT-PCR. In a series of 59 prospectively collected head and neck tumours, expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants.
Highlights
Alternative splicing is the process by which cells can selectively include different sections of pre-mRNA during RNA processing
Affymetrix Exon microarrays, which feature multiple probesets targeting different locations throughout each gene, allow the changes in transcription that result from alternative splicing to be investigated in a single genome-wide assay
We developed a novel algorithm to exploit both these aspects of exon arrays and applied it to tumour hypoxia in clinical samples
Summary
Alternative splicing is the process by which cells can selectively include different sections of pre-mRNA during RNA processing. These feature probesets targeting at intervals throughout each transcript, rather than at the 39 end interrogated by most other arrays This enables the assembly, in silico, of expression levels across genes providing a more complete representation of transcription for each gene, and allowing the identification of loci where there are changes in the splicing pattern across experimental samples. Another useful consequence of the increased resolution of the arrays is that since most transcripts are targeted by multiple probesets, their signals can be combined in order to increase statistical power [6]. This increased performance is not without additional challenges, since detailed analysis of the arrays requires annotation describing the known relationships between genes, transcripts and exons, and the ability to combine this with appropriate statistics [7,8,9]
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