Abstract
Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.
Highlights
The sequencing of the human genome [1,2] and subsequent work describing sequence variation amongst human populations [3] has provided the necessary resources for large-scale studies of the effects of genetic variation on human gene expression
What is not known is whether allele-specific splicing events are an important mechanism by which common genetic variation affects gene expression
Sequence analysis of the relevant splice sites and of the regions surrounding single nucleotide polymorphisms correlated with the splicing events failed to identify any predictive bioinformatic signals
Summary
The sequencing of the human genome [1,2] and subsequent work describing sequence variation amongst human populations [3] has provided the necessary resources for large-scale studies of the effects of genetic variation on human gene expression. Identifying functionally important variation has the potential for increasing understanding of gene regulation and for providing efficient markers to study the effects of variation in gene expression on human disease risk [4]. Novel allele-specific transcript quantification approaches to candidate genes [7,8] have been employed, along with broader approaches to investigate the absolute levels of expression of thousands of genes [9,10]. Using these methods, several cis-acting SNPs that correlate with gene expression have been identified. Fine mapping these effects and determining the mechanisms underlying the associations has been more difficult [11]
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