Functional consequences of intergenic autoimmune disease risk variants

摘要 系统性红斑狼疮(SLE)是一种多系统自身免疫性疾病，发病率高， 死亡率，以及大部分无效的治疗方法。系统性红斑狼疮遗传复杂，具有较强的家族聚集性和高 遗传力。大多数SLE的风险等位基因都不是蛋白质编码序列的变化，这表明显著的 疾病风险的一部分是由非编码基因组中的调控变异介导的。在最近发现的一家 位于染色体12q12上的基因间SLE风险基因座，我们的计算机分析表明，风险变量是eQTL， 影响多种生物途径的基因转录，这些途径有助于系统性红斑狼疮免疫病理，包括 I型干扰素和抗病毒基因。这个基因间的系统性红斑狼疮风险基因座富含调控元素和 与几个长的非编码RNA(LncRNA)基因相邻。在这项提案中，我们概述了一个全面的 一系列机制和功能实验以了解12q12风险基因在人类免疫中的作用 细胞。我们假设12q12基因座的顺式调控元件中的几个变体会改变表达 ，从而对免疫系统基因产生顺式和反式作用，从而导致 系统性红斑狼疮风险。另一种假设是，12q12基因座可能是附近蛋白质的远程增强剂- 编码和/或非编码基因。这些假设并不是相互排斥的，我们的实验设计将 检测每一种可能性。在目标1中，我们将确定基因间12q12基因座对基因的整体影响 初级免疫细胞和选定的细胞系的调节。综合分析将用于推断lncRNA 对mRNA网络的影响。来自这些分析的候选lncRNAs将被siRNAs敲除以 确认它们的功能。在目标2中，我们将确定基因间隔区12q12区域的因果顺式调控元件。 我们将使用补充方法来确定可能的监管要素，假定的因果要素将是 在细胞系中被敲除，以确认功能并恢复到无风险的细胞表型。我们希望能找到 基因间12q12基因座影响邻近的lncRNAs和/或邻近的蛋白质编码基因的表达 以细胞类型特定的方式非编码基因，导致多种生物的同时调制 参与系统性红斑狼疮发病的途径。这一知识可能会为SLE提供新的治疗靶点 会影响多条致病途径。这代表了SLE的一种新的疾病机制和 提供了基因间风险变量如何在复杂的疾病发病机制中发挥作用的说明。

ABSTRACT Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by high morbidity and mortality, and largely ineffective treatments. SLE is genetically complex with strong familial aggregation and high heritability. Most of the risk alleles for SLE are not protein-coding sequence changes, suggesting that a significant portion of disease risk is mediated by regulatory variants in the noncoding genome. At a recently identified intergenic SLE risk locus on chromosome 12q12, our in silico analyses implicate the risk variants as eQTLs that impact transcription of genes in multiple biological pathways that contribute to SLE immunopathology, including type I interferon (IFN) and anti-viral genes. This intergenic SLE risk locus is enriched in regulatory elements and resides adjacent to several long non-coding RNA (lncRNA) genes. In this proposal, we outline a comprehensive set of mechanistic and functional experiments to understand the role of the 12q12 risk locus in human immune cells. We hypothesize that several variants within cis-regulatory elements at the 12q12 locus alter the expression of adjacent lncRNAs, which in turn exert cis and trans effects on immune system genes resulting in increased SLE risk. An alternate hypothesis is that the 12q12 locus could be a long-range enhancer for nearby protein- coding and/or non-coding genes. These hypotheses are not mutually exclusive, and our experimental design will detect each possibility. In Aim 1, we will determine the overall impact of the intergenic 12q12 locus on gene regulation in primary immune cells and selected cell lines. Integrative analyses will be used to infer lncRNA influences on mRNA networks. Candidate lncRNAs from these analyses will be knocked down with siRNAs to confirm their function. In Aim 2, we will identify causal cis-regulatory elements in the intergenic 12q12 region. We will use complementary methods to identify likely regulatory elements, and putative causal elements will be knocked out in cell lines to confirm function and reversion to a non-risk cellular phenotype. We expect to find that the intergenic 12q12 locus influences the expression of adjacent lncRNAs and/or neighboring protein-coding or non-coding genes in a cell type-specific manner, resulting in simultaneous modulation of multiple biological pathways involved in SLE pathogenesis. This knowledge could suggest novel therapeutic targets for SLE that would influence multiple pathogenic pathways. This represents a novel mechanism of disease in SLE and provides an illustration of how intergenic risk variants can function in complex disease pathogenesis.