Heritability of alternative splicing in the human genome
ABSTRACT: 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 genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. 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. Keywords: Comparative genomic hybridiation within a 3 generation pedigree Overall design: We used 14 individuals from the 3 generation CEPH/UTAH 1444 pedigree for our analysis of looking at heritability of differentially spliced exons within the family. 3 biological growths of individuals NA12739, NA12740, NA12750, and NA12751 were used and a single biological growth for the remaining 10 individuals.
INSTRUMENT(S): [HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [transcript (gene) version]
Project description: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 genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. 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. Keywords: Comparative genomic hybridiation within a 3 generation pedigree We used 14 individuals from the 3 generation CEPH/UTAH 1444 pedigree for our analysis of looking at heritability of differentially spliced exons within the family. 3 biological growths of individuals NA12739, NA12740, NA12750, and NA12751 were used and a single biological growth for the remaining 10 individuals.
Project description: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 5' 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.
Project description: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, alternate splice site usage, intron retention), differential 5’ UTR (initiation of transcription) usage, and differential 3’ UTR (alternative polyadenylation) usage. These results demonstrate that the regulatory effects of genetic variation in a normal human population are drastically more complex than previously observed. This additional layer of molecular diversity may account for natural phenotypic variation and disease susceptibility. Keywords: Comparative genomic hybridiation within the CEU HapMap population Overall design: We used individuals from the HapMap CEU population for our analysis of examining genetic variation in transcript isoforms. 3 biological growths each from 57 individuals (parents of CEU trios) were used.
Project description:Splice isoform structure and abundance can be affected by either noncoding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these nonconstitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays and custom designed q-RT-PCR or TaqMan assays. The majority of these SNPs (15 of 22) as well as an independent set of 24 variants were then subjected to RNAseq analysis using the ValidSpliceMut web beacon (http://validsplicemut.cytognomix.com), which is based on data from the Cancer Genome Atlas and International Cancer Genome Consortium. SNPs from different genes analyzed with gene expression microarray and q-RT-PCR exhibited significant changes in affected splice site use. Thirteen SNPs directly affected exon inclusion and 10 altered cryptic site use. Homozygous SNP genotypes resulting in stronger splice sites exhibited higher levels of processed mRNA than alleles associated with weaker sites. Four SNPs exhibited variable expression among individuals with the same genotypes, masking statistically significant expression differences between alleles. Genome-wide information theory and expression analyses (RNAseq) in tumor exomes and genomes confirmed splicing effects for 7 of the HapMap SNP and 14 SNPs identified from tumor genomes. q-RT-PCR resolved rare splice isoforms with read abundance too low for statistical significance in ValidSpliceMut. Nevertheless, the web-beacon provides evidence of unanticipated splicing outcomes, for example, intron retention due to compromised recognition of constitutive splice sites. Thus, ValidSpliceMut and q-RT-PCR represent complementary resources for identification of allele-specific, alternative splicing.
Project description: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.
Project description:There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5' splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5' splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5' splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only ?14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5' splice site polymorphisms, most notably a strong 3' splice site and the presence of intronic motifs downstream of the 5' splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5' splice site SNPs. In TRIM62, the upstream 3' splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5' splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5' splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5' splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells.
Project description:Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5' and 3' splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events.
Project description:Folate is vital for cell growth and development through its important role in one-carbon metabolism - an essential process in the synthesis of amino acids and nucleic acids. Folate pathway genes have been considered as therapeutic targets of drugs for the treatment of cancer and other diseases. Racial and ethnic disparities of folate metabolism and outcome of antifolate therapies have been reported. In this study, we evaluate the genetic regulation for expression and alternative splicing of folate related genes in HapMap lymphoblastoid cell lines (LCLs) of individuals of European and African descent.Gene and exon level expression and alternative splicing of folate pathway genes were compared in LCLs derived from the Centre d'Etude du Polymorphisme Humain (CEPH) from Utah (CEU) and the Yoruba from Ibadan (YRI) using a permutation-based test. A genome-wide association study was performed to search for SNPs associated with folate pathway gene expressions and alternative splicing in the combined population samples.A total of 52 folate pathway genes were evaluated in the analysis of which 46 were expressed in the LCLs. There were 12 genes (26%) with differential gene-level expression and 23 genes (50%) with differential alternative splicing for exons or UTRs between the CEU and the YRI (permutation p <or= 0.05). The expression level of FPGS and the splicing indices of eight genes (ATP13A2, ASCC3L1, IFIH1, SMARCA5, SMARCA2, SETX, DDX52 and RUVBL2) were found to be associated with SNP genotypes in the combined populations (p < 3.2 x 10(-8), Bonferroni corrected p < 0.05).Our study suggests that LCLs are an in vitro system suitable to evaluate the expression levels of folate pathway genes. The differential transcript-level expressions and the differentially alternative splicing events of exons or UTRs and associated SNP markers in 2 populations will enhance our understanding of the folate pathway and, thus, facilitate research in the areas of nutrition and folate metabolism.
Project description:The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5' splice sites, and alternative 3' splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3' splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ?80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ?50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation.
Project description:Gene duplication and alternative splicing are important sources of proteomic diversity. Despite research indicating that gene duplication and alternative splicing are negatively correlated, the evolutionary relationship between the two remains unclear. One manner in which alternative splicing and gene duplication may be related is through the process of subfunctionalization, in which an alternatively spliced gene upon duplication divides distinct splice isoforms among the newly generated daughter genes, in this way reducing the number of alternatively spliced transcripts duplicate genes produce. Previously, it has been shown that splice form subfunctionalization will result in duplicate pairs with divergent exon structure when distinct isoforms become fixed in each paralog. However, the effects of exon structure divergence between paralogs have never before been studied on a genome-wide scale. Here, using genomic data from human, mouse, and zebrafish, we demonstrate that gene duplication followed by exon structure divergence between paralogs results in a significant reduction in levels of alternative splicing. In addition, by comparing the exon structure of zebrafish duplicates to the co-orthologous human gene, we have demonstrated that a considerable fraction of exon divergent duplicates maintain the structural signature of splice form subfunctionalization. Furthermore, we find that paralogs with divergent exon structure demonstrate reduced breadth of expression in a variety of tissues when compared to paralogs with identical exon structures and singletons. Taken together, our results are consistent with subfunctionalization partitioning alternatively spliced isoforms among duplicate genes and as such highlight the relationship between gene duplication and alternative splicing.