Alternative Isoform Detection Using Exon Arrays

Introduction

Alternative pre-mRNA splicing is a process allowing the production of several distinct gene isoforms from a single genomic locus. The most common type of alternative splicing events in mammals results in cassette exons, where each such exon can be either included or excluded from the mature mRNA. Other events include alternative use of donor or acceptor splice sites, and intron retention. In addition, processes such as alternative promoter usage and alternative polyadenylation, resulting in differences in initiation and termination of the transcript, respectively, further diversify eukaryotic transcriptomes and proteomes. Such processes have been suggested to be at least partly responsible for mammalian complexity, which is otherwise difficult to explain in view of our relatively low number of genomic loci - less than 25,000 genes in humans, versus approximately 20,000 in the nematode worm C. elegans (Claverie, 2001). It is estimated that a high percentage of mammalian genes is alternatively spliced, and this frequency is highest in specialized and complex tissues, such as the brain and the liver. Differences in splicing patterns have been shown to exist across species, and within populations of the same species. In humans, splicing defects are known to result in numerous genetic disorders (Faustino & Cooper, 2003) and may confer susceptibility to complex genetic diseases. Thus, the process of alternative splicing attracts the interest of researchers across the entire biomedical sciences spectrum, ranging from evolutionary biology, through development, to medicine.

In recent years, alternative transcript investigation in a genome-wide context has been carried out using expressed sequence tag libraries (ESTs). Generally, ESTs (short cDNA sequence reads) are mapped to the genomic sequence, and different isoforms can be inferred from incongruence of splicing patterns (Modrek, Resch, Grasso, & Lee, 2001). However, EST library analyses are prone to sequencing errors, biased towards highly expressed genes, and influenced by cancer-derived ESTs, which may not generally be present in healthy tissues.

More recently, microarray platforms have been proposed as a tool for studying gene expression at the isoform level (Black & Graveley, 2006; Lee & Roy, 2004; Zhang et al., 2006). Splicing sensitive microarrays employ a number of exon body oligonucleotide probes, or exon junction probes, or a combination of the two designs, to determine mRNA levels at the resolution of a single exon or splice site. The Affymetrix GeneChip® Human Exon 1.0 ST Array is the first commercially available microarray product designed for genome-wide, exon level expression analysis. The array relies on targeting multiple probes to individual exons and allows simultaneous, exon-level detection of expression intensity for 1.4 million probesets covering over 1 million known and predicted human exons. The Exon Array is a flexible tool, which can be used to perform the function of classical expression arrays and concurrently provide supplementary information on isoform changes. However, because of the complexity of the design, statistical analysis of the data becomes much more intensive, both at the theoretical and computational level. The simplest illustration is the multiple testing problem; whereas in classical expression arrays the number of tests is of the order of the number of genes, in an exon array, the number of tests is over ten-fold higher – and can vary between a few hundred thousand to over 1 million (if computationally predicted exons are included). The statistical approaches need to be able to distinguish between whole gene expression differences and isoform differences, which introduces a new level of complexity. The robustness of measurement is also an issue, since the exon array has on average four probes per probeset, whereas Affymetrix expression arrays relied on more than 10 probes per probeset to estimate expression.