Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.
Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.
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DOI：
10.1371/journal.pgen.1002078
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发表时间：
2011-05
期刊：
影响因子：
4.5
通讯作者：
Brown CD
中科院分区：
文献类型：
--
作者：
Innocenti F;Cooper GM;Stanaway IB;Gamazon ER;Smith JD;Mirkov S;Ramirez J;Liu W;Lin YS;Moloney C;Aldred SF;Trinklein ND;Schuetz E;Nickerson DA;Thummel KE;Rieder MJ;Rettie AE;Ratain MJ;Cox NJ;Brown CD
表达数量性状基因座(EQTL)的发现有助于揭示复杂性状的遗传贡献。我们使用安捷伦(n = 206)和Illumina(n = 60)表达阵列和Illumina SNP基因分型(550K)分析的两个独立的初级组织集合来确定人类肝脏基因表达变异的遗传决定因素，我们还纳入了一项已发表的研究(n = 266)的数据。我们发现，在一项研究中，∼30%的SNP表达相关性未能在其他任何一个中复制，即使在模拟中产生高重复性的阈值，我们还量化了许多影响重复性的因素。我们的数据表明，药物暴露、临床描述和与组织确定和分析相关的未知因素对基因表达有实质性影响，控制隐藏的混杂变量显著提高复制率。此外，我们发现可重复的eQTL SNPs在基因起始点和结尾处高度富含，随后对14个基因的启动子和3‘UTRs进行了重新测序，并用荧光素酶分析检测了所鉴定的单倍型。对于这三个基因，显著的单倍型特异性体外功能差异与表达水平直接相关，这表明许多真正的eQTL是由可以高通量机械分离的功能变体产生的。最后，根据我们的研究设计，我们能够发现并验证数百个肝脏eQTL。其中许多与复杂的特征直接相关，肝脏特异性分析可能与之相关，我们确定了数十个与疾病相关基因的潜在联系。这些研究包括先前确定的糖尿病、药物反应和血脂水平的eQTL贡献者，他们提出了新的候选因素，如NOD2表达在麻风病风险中的作用以及C2orf43在前列腺癌中的作用。总体而言，本文介绍的工作将对未来准确识别各种复杂性状的遗传贡献并从功能上确定其特征具有重要意义。许多与疾病相关的遗传变异不会改变蛋白质序列，很难准确识别。表达数量性状基因座(EQTL)的发现，或遗传变异和基因表达水平之间的相关性，为解决这一挑战提供了一种手段。然而，在原代细胞中的eQTL研究有几个缺点。特别是，它们的重复性在很大程度上是未知的，产生不可靠关联的变量没有特征化，他们的发现的解决受到连锁不平衡的限制。我们在原人类肝脏中进行了eQTL的三向复制研究。我们在一项独立的研究中证明了∼67%的顺式-eQTL关联是重复的，已知的多态、重叠的表达探针、SNP到基因的距离以及未测量的混杂变量都会影响复制率。我们对14个eQTL进行了精细定位，并确定了3个基因启动子或3‘非编码区的致病多态，表明相当一部分eQTL是由功能分离的近端变异所驱动的。最后，我们发现与复杂性状相关的SNPs和复制的eQTL SNPs之间有数百个重叠。我们的数据为未来的eQTL分析及其影响的复杂性状提供了警示(即许多强eQTL的不可重复性)和乐观(即精确识别功能非编码变体)预测。
The discovery of expression quantitative trait loci (“eQTLs”) can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3′UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits. Many disease-associated genetic variants do not alter protein sequences and are difficult to precisely identify. Discovery of expression quantitative trait loci (eQTL), or correlations between genetic variants and gene expression levels, offers one means of addressing this challenge. However, eQTL studies in primary cells have several shortcomings. In particular, their reproducibility is largely unknown, the variables that generate unreliable associations are uncharacterized, and the resolution of their findings is constrained by linkage disequilibrium. We performed a three-way replication study of eQTLs in primary human livers. We demonstrated that ∼67% of cis-eQTL associations are replicated in an independent study and that known polymorphisms overlapping expression probes, SNP-to-gene distance, and unmeasured confounding variables all influence the replication rate. We fine-mapped 14 eQTLs and identified causative polymorphisms in the promoter or 3′UTR for 3 genes, suggesting that a considerable fraction of eQTLs are driven by proximal variants that are amenable to functional isolation. Finally, we found hundreds of overlaps between SNPs associated with complex traits and replicated eQTL SNPs. Our data provide both cautionary (i.e. non-reproducibility of many strong eQTLs) and optimistic (i.e. precise identification of functional non-coding variants) forecasts for future eQTL analyses and the complex traits that they influence.
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