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，其特点是发病率高， 死亡率和大部分无效的治疗。SLE是一种遗传复杂的疾病，具有很强的家族聚集性， 遗传性SLE的大多数危险等位基因不是蛋白质编码序列的改变，这表明SLE的显著危险性是由基因突变引起的。 部分疾病风险是由非编码基因组中的调节变体介导的。在最近确定的 染色体12 q12上的基因间SLE风险位点，我们的计算机模拟分析表明风险变异是eQTL， 影响多种生物学途径中的基因转录，这些途径有助于SLE免疫病理学，包括 I型干扰素（IFN）和抗病毒基因。该基因间SLE风险基因座富含调节元件， 位于几个长的非编码RNA（lncRNA）基因附近。在这份提案中，我们概述了一个全面的 一组机制和功能实验，以了解12 q12风险基因座在人类免疫中的作用 细胞我们假设12 q12基因座顺式调控元件内的几个变体改变了 相邻的lncRNA，这反过来又对免疫系统基因产生顺式和反式效应，导致增加 SLE风险。另一种假设是，12 q12位点可能是附近蛋白质的远程增强子- 编码和/或非编码基因。这些假设并不相互排斥，我们的实验设计将 发现每一种可能性。在目的1中，我们将确定基因间12 q12位点对基因的总体影响， 在原代免疫细胞和选择的细胞系中的调节。整合分析将用于推断lncRNA 影响mRNA网络。来自这些分析的候选lncRNA将用siRNA敲低， 确认其功能。在目标2中，我们将识别基因间12 q12区域中的因果顺式调节元件。 我们将使用互补的方法来确定可能的调控因素，并假定的因果因素将是 在细胞系中敲除，以确认功能和回复到无风险的细胞表型。我们希望能发现 基因间12 q12基因座影响相邻lncRNA和/或相邻蛋白质编码或 以细胞类型特异性的方式调节非编码基因，从而同时调节多种生物学特性。 参与SLE发病机制的途径。这些知识可能为SLE提供新的治疗靶点， 会影响多种致病途径这代表了SLE疾病的一种新机制， 提供了基因间风险变异如何在复杂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.