We have performed a genome-wide analysis of common genetic variation controlling differential expression of transcript isoforms in the CEU HapMap population using a comprehensive exon tiling microarray covering 17,897 genes. We detected 324 genes with significant associations between flanking SNPs and transcript levels. Of these, 39% reflected changes in whole gene expression and 55% reflected transcript isoform changes such as splicing variants (exon skipping, alternative splice site use, intron retention), differential 5' UTR (initiation of transcription) use, and differential 3' UTR (alternative polyadenylation) use. These results demonstrate that the regulatory effects of genetic variation in a normal human population are far more complex than previously observed. This extra layer of molecular diversity may account for natural phenotypic variation and disease susceptibility.

Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5Ј splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled.[The microarray data from this study have been submitted to GEO under accession no. GES7952.]The human genome is estimated to contain ∼20,000-25,000 genes, and recent studies suggest that ∼50%-75% of multi-exon genes undergo alternative splicing (AS), generating multiple mRNA isoforms and greatly increasing human proteomic diversity (Lander et al. 2001;Modrek et al. 2001). The splicing of mRNA is a highly regulated process involving the interactions of trans-acting splicing factors and cis-acting regulatory motifs. Disruptions of this process through mutations within these factors and regulatory signals may play an important role in phenotypic diversity and genetic disorders ( ) and applied to identification of AS events that are tissue-specific, for the most part. However, one caveat of these studies utilizing customized arrays is a bias toward genes with solid EST and cDNA evidence for known AS events and that are therefore limited in their usefulness as a discovery tool for de novo splicing events. Here, we have chosen to use an alternative array design, the Affymetrix GeneChip Human Exon 1.0 ST Array, which is less biased toward known AS events by targeting multiple probes to individual exons and allowing simultaneous, exon-level detection of expression levels for 1.4 million probe sets covering over one million known and predicted human exons (Fig. 1). Exon-tiling arrays have several advantages over exon-junction arrays: flexibility of probe placement, exact trans...

Recently, thanks to the increasing throughput of new technologies, we have begun to explore the full extent of alternative pre–mRNA splicing (AS) in the human transcriptome. This is unveiling a vast layer of complexity in isoform-level expression differences between individuals. We used previously published splicing sensitive microarray data from lymphoblastoid cell lines to conduct an in-depth analysis on splicing efficiency of known and predicted exons. By combining publicly available AS annotation with a novel algorithm designed to search for AS, we show that many real AS events can be detected within the usually unexploited, speculative majority of the array and at significance levels much below standard multiple-testing thresholds, demonstrating that the extent of cis-regulated differential splicing between individuals is potentially far greater than previously reported. Specifically, many genes show subtle but significant genetically controlled differences in splice-site usage. PCR validation shows that 42 out of 58 (72%) candidate gene regions undergo detectable AS, amounting to the largest scale validation of isoform eQTLs to date. Targeted sequencing revealed a likely causative SNP in most validated cases. In all 17 incidences where a SNP affected a splice-site region, in silico splice-site strength modeling correctly predicted the direction of the micro-array and PCR results. In 13 other cases, we identified likely causative SNPs disrupting predicted splicing enhancers. Using Fst and REHH analysis, we uncovered significant evidence that 2 putative causative SNPs have undergone recent positive selection. We verified the effect of five SNPs using in vivo minigene assays. This study shows that splicing differences between individuals, including quantitative differences in isoform ratios, are frequent in human populations and that causative SNPs can be identified using in silico predictions. Several cases affected disease-relevant genes and it is likely some of these differences are involved in phenotypic diversity and susceptibility to complex diseases.

BackgroundAlternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.ResultsA genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, Fox2, we demonstrate that changes in Fox2 expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.ConclusionsWe propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies