Computational approaches for detecting disease-associated alternative splicing events.

Alternative splicing (AS) is a key transcriptional regulation pathway. Recent studies have shown that AS events are associated with the occurrence of complex diseases. Various computational approaches have been developed for the detection of disease-associated AS events. In this review, we first describe the metrics used for quantitative characterization of AS events. Second, we review and discuss the three types of methods for detecting disease-associated splicing events, which are differential splicing analysis, aberrant splicing detection and splicing-related network analysis. Third, to further exploit the genetic mechanism of disease-associated AS events, we describe the methods for detecting genetic variants that potentially regulate splicing. For each type of methods, we conducted experimental comparison to illustrate their performance. Finally, we discuss the limitations of these methods and point out potential ways to address them. We anticipate that this review provides a systematic understanding of computational approaches for the analysis of disease-associated splicing.

A 584 bp deletion in CTRB2 inhibits chymotrypsin B2 activity and secretion and confers risk of pancreatic cancer.

Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where germline variants associate with risk of pancreatic ductal adenocarcinoma (PDAC) in populations of European ancestry. Here, we fine-mapped one such locus on chr16q23.1 (rs72802365, p = 2.51 × 10-17, OR = 1.36, 95% CI = 1.31-1.40) and identified colocalization (PP = 0.87) with aberrant exon 5-7 CTRB2 splicing in pancreatic tissues (pGTEx = 1.40 × 10-69, ßGTEx = 1.99; pLTG = 1.02 × 10-30, ßLTG = 1.99). Imputation of a 584 bp structural variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (p = 2.83 × 10-16, OR = 1.36, 95% CI = 1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsinogen B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). We propose that intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein leads to ER stress and pancreatic inflammation, which may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles.

Structural variants and short tandem repeats impact gene expression and splicing in bovine testis tissue.

Structural variants (SVs) and short tandem repeats (STRs) are significant sources of genetic variation. However, the impacts of these variants on gene regulation have not been investigated in cattle. Here, we genotyped and characterized 19,408 SVs and 374,821 STRs in 183 bovine genomes and investigated their impact on molecular phenotypes derived from testis transcriptomes. We found that 71% STRs were multiallelic. The vast majority (95%) of STRs and SVs were in intergenic and intronic regions. Only 37% SVs and 40% STRs were in high linkage disequilibrium (LD) (R2 > 0.8) with surrounding SNPs/insertions and deletions (Indels), indicating that SNP-based association testing and genomic prediction are blind to a nonnegligible portion of genetic variation. We showed that both SVs and STRs were more than 2-fold enriched among expression and splicing QTL (e/sQTL) relative to SNPs/Indels and were often associated with differential expression and splicing of multiple genes. Deletions and duplications had larger impacts on splicing and expression than any other type of SV. Exonic duplications predominantly increased gene expression either through alternative splicing or other mechanisms, whereas expression- and splicing-associated STRs primarily resided in intronic regions and exhibited bimodal effects on the molecular phenotypes investigated. Most e/sQTL resided within 100 kb of the affected genes or splicing junctions. We pinpoint candidate causal STRs and SVs associated with the expression of SLC13A4 and TTC7B and alternative splicing of a lncRNA and CAPP1. We provide a catalog of STRs and SVs for taurine cattle and show that these variants contribute substantially to gene expression and splicing variation.

The Genetic Regulation of Alternative Splicing in Populus deltoides.

Alternative splicing (AS) is a mechanism of regulation of the proteome via enabling the production of multiple mRNAs from a single gene. To date, the dynamics of AS and its effects on the protein sequences of individuals in a large and genetically unrelated population of trees have not been investigated. Here we describe the diversity of AS events within a previously genotyped population of 268 individuals of Populus deltoides and their putative downstream functional effects. Using a robust bioinformatics pipeline, the AS events and resulting transcript isoforms were discovered and quantified for each individual in the population. Analysis of the AS revealed that, as expected, most AS isoforms are conserved. However, we also identified a substantial collection of new, unannotated splice junctions and transcript isoforms. Heritability estimates for the expression of transcript isoforms showed that approximately half of the isoforms are heritable. The genetic regulators of these AS isoforms and splice junction usage were then identified using a genome-wide association analysis. The expression of AS isoforms was predominately cis regulated while splice junction usage was generally regulated in trans. Additionally, we identified 696 genes encoding alternatively spliced isoforms that changed putative protein domains relative to the longest protein coding isoform of the gene, and 859 genes exhibiting this same phenomenon relative to the most highly expressed isoform. Finally, we found that 748 genes gained or lost micro-RNA binding sites relative to the longest protein coding isoform of a given gene, while 940 gained or lost micro-RNA binding sites relative to the most highly expressed isoform. These results indicate that a significant fraction of AS events are genetically regulated and that this isoform usage can result in protein domain architecture changes.

Identification and Quantification of Splicing Quantitative Trait Loci.

Most complex traits, including diseases, have a large genetic component. Identifying the genetic variants and genes underlying phenotypic variation remains one of the most important objectives of current biomedical research. Unlike Mendelian or familial diseases, which are usually caused by mutations in the coding regions of individual genes, complex diseases are thought to result from the cumulative effects of a large number of variants, of which, the vast majority are noncoding. Therefore, to discern the genetic underpinnings of a complex trait, we must first understand the impact of noncoding variation, which presumably affects gene regulation. In this chapter, we outline the recent progress made and methods used to discover putative regulatory regions associated with complex traits. We will specifically focus on mapping splicing quantitative trait loci (sQTL) using Yoruba samples from GEUVADIS as a motivating example.

Environmental influences on RNA processing: Biochemical, molecular and genetic regulators of cellular response.

RNA processing has emerged as a key mechanistic step in the regulation of the cellular response to environmental perturbation. Recent work has uncovered extensive remodeling of transcriptome composition upon environmental perturbation and linked the impacts of this molecular plasticity to health and disease outcomes. These isoform changes and their underlying mechanisms are varied-involving alternative sites of transcription initiation, alternative splicing, and alternative cleavage at the 3' end of the mRNA. The mechanisms and consequences of differential RNA processing have been characterized across a range of common environmental insults, including chemical stimuli, immune stimuli, heat stress, and cancer pathogenesis. In each case, there are perturbation-specific contributions of local (cis) regulatory elements or global (trans) factors and downstream consequences. Overall, it is clear that choices in isoform usage involve a balance between the usage of specific genetic elements (i.e., splice sites, polyadenylation sites) and the timing at which certain decisions are made (i.e., transcription elongation rate). Fine-tuned cellular responses to environmental perturbation are often dependent on the genetic makeup of the cell. Genetic analyses of interindividual variation in splicing have identified genetic effects on splicing that contribute to variation in complex traits. Finally, the increase in the number of tissue types and environmental conditions analyzed for RNA processing is paralleled by the need to develop appropriate analytical tools. The combination of large datasets, novel methods and conditions explored promises to provide a much greater understanding of the role of RNA processing response in human phenotypic variation. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients.

Our comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, we identified ≈930 exon-exon junctions ("neojunctions") in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, we confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders ("putative neoantigens").

A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize.

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis-sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans-sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.