Fine-scale variation and genetic determinants of alternative splicing across individuals.
Fine-scale variation and genetic determinants of alternative splicing across individuals.
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DOI：
10.1371/journal.pgen.1000766
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发表时间：
2009-12
期刊：
影响因子：
4.5
通讯作者：
Majewski J
中科院分区：
文献类型：
--
作者：
Coulombe-Huntington J;Lam KC;Dias C;Majewski J
最近，由于新技术的吞吐量不断增加，我们已经开始探索人类转录组中替代前mRNA剪接(AS)的全部范围。这揭示了个体之间同型异构体水平表达差异的巨大复杂性。我们使用之前发表的来自淋巴母细胞系的剪接敏感微阵列数据对已知和预测的外显子的剪接效率进行了深入的分析。通过将公开可用的AS注释与设计用于搜索AS的新算法相结合，我们表明许多真实的AS事件可以在阵列中通常未被利用的、推测的大多数区域内被检测到，并且在远低于标准多重测试阈值的显著水平上被检测到，这表明顺式调控的个体之间的差异剪接的程度可能远远大于先前报道的程度。具体地说，许多基因在剪接位点的使用上表现出细微但受基因控制的显著差异。PCR验证表明，58个候选基因区域中有42个(72%)经历了可检测到的AS，这是迄今为止对同型eQTL进行规模最大的验证。靶向测序显示，在大多数确诊病例中，可能存在致病的SNP。在所有17个SNP影响剪接位点区域的事件中，在硅胶剪接位点强度建模中，正确地预测了微阵列和PCR结果的方向。在其他13个案例中，我们确定了可能导致破坏预测剪接增强子的SNP。使用FST和REHH分析，我们发现了两个假定的致病SNPs最近经历了正选择的重要证据。我们用体内微基因检测验证了5个SNPs的效果。这项研究表明，个体之间的剪接差异，包括异构体比率的数量差异，在人类群体中是常见的，并且可以利用电子预测来识别致病的SNPs。有几个病例影响了与疾病相关的基因，其中一些差异可能涉及表型多样性和对复杂疾病的易感性。选择性剪接(AS)通过外显子的选择性使用，可以从同一基因组位点产生许多不同的mRNA转录本，从而可能导致许多不同蛋白质的产生。我们知道个体之间存在剪接差异，而且这些变化往往与基因变异有关。到目前为止，这些关联中很少有导致致病多态的准确定位。在这项工作中，通过对之前发表的人类细胞系剪接敏感微阵列数据的深入分析，我们识别并验证了大量与附近基因变异高度相关的剪接变化。然后，我们对候选外显子周围的基因组DNA进行了测序，并将其用于计算机建模工具，以确定大多数候选基因的致病单核苷酸多态。通过构建一个报告质粒，我们进一步证明了所选的5个SNPs在体内复制了预期的效果。我们的结果表明，基因控制的个体之间的剪接差异可能比以前提出的更常见，也可能非常微妙；而且大多数是由影响剪接位点区域或外显子剪接增强子(ESES)序列的SNP引起的。
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. Alternative splicing (AS), through the alternative use of exons, can produce many different mRNA transcripts from the same genomic locus, thus possibly resulting in the production of many different proteins. We know that splicing differences between individuals exist and that these changes are often associated with genetic variants. Thus far, very few of these associations have led to the precise localization of the causative polymorphisms. In this work, using in-depth analysis of previously published splicing sensitive micro-array data from human cell lines, we identified and validated a large number of splicing changes which are highly correlated with nearby genetic variations. We then sequenced the genomic DNA around candidate exons and used in silico modeling tools to identify causative SNPs for most of our candidates. Using a plasmid reporter construct, we further demonstrated that five selected SNPs reproduce the expected effect in vivo. Our results indicate that genetically controlled splicing differences between individuals may be more common than previously suggested and can be very subtle; and most are caused by SNPs affecting either the splice-site region or exonic splicing enhancers (ESEs) sequences.
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影响因子：
14.9
作者：
Cartegni, L;Wang, JH;Krainer, AR
通讯作者：
Krainer, AR
DOI：
10.1038/nrg2544
发表时间：
2009-05
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
Ioannidis JP;Thomas G;Daly MJ
通讯作者：
Daly MJ
影响因子：
3.9
作者：
ElSharawy, Abdou;Hundrieser, Bernd;Hampe, Jochen
通讯作者：
Hampe, Jochen
影响因子：
14.9
作者：
Nickerson, DA;Tobe, VO;Taylor, SL
通讯作者：
Taylor, SL
影响因子：
4.5
作者：
Hull, Jeremy;Campino, Susana;Kwiatkowski, Dominic
通讯作者：
Kwiatkowski, Dominic