Connecting the gap between GWAS and functional targets for lupus susceptibility

摘要 系统性红斑狼疮(SLE或狼疮)是一种目前无法治愈的自身免疫性疾病，其特征是 免疫细胞(如B淋巴细胞)的异常反应和许多致病因素的产生 自身抗体，最终导致多器官损伤(如肾脏、皮肤)。而系统性红斑狼疮的病因尚不完全 理解，实质性的遗传贡献是公认的。几项全基因组关联研究 已经确定了100多个SLE易感基因座(p&lt；5x10-8)，主要是单核苷酸多态 (SNPs)。这些SNP中的大多数不会直接改变蛋白质产物，我们和其他人之前的工作已经 表明许多这样的SNPs在顺式调控元件(CRE)(即增强剂和消音剂)中富含 并有可能调节基因的表达。然而，找出Cre和Cres中的因果、易感SNP 破译其确切的机制是在这一领域取得进展的主要障碍。因此，这 知识差距严重阻碍了GWAS研究成果转化为临床应用。因此，在这里 迫切需要不偏不倚、全面和高吞吐量的方法来解决机械性 数百个潜在的调节性SNPs(RSNPs)与SLE易感性之间存在联系。我们假设SLE- 易感rSNPs异常诱导Cre活性，影响未受刺激的靶基因表达 和/或刺激的B细胞。为了系统地描述rSNPs及其对靶基因的影响，我们建议 建立一个高通量的实验管道，以确定和验证GWAS基因座下的rSNPs。在……里面 目的1、应用高通量技术--自转录活性调控区测序 (STARR-SEQ)从功能上量化数百个含有SNP的区域的监管活动 同时。使用Raji细胞(B淋巴细胞)在未刺激和刺激条件下，我们将应用 STARR-SEQ评估79个不同的Gwas基因座中的756个rSNPs对SLE的易感性。在目标2a中，我们 将应用下一代(NG)捕获-C来检测SNP对顺式分子与内源、 Raji细胞中的同源靶基因，不需要很强的靶基因和功能的先验假设 后果。我们将评估目标1和目标2a中相同的756个SNPs。我们预计这是双管齐下的。 串联策略将弥合GWAS来源的rSNPs与其目标基因的机械性链接之间的差距。 为了验证建议方法的有效性和准确性，Aim 2b将使用基于CRISPR的 (EPI)对选定的rSNP进行基因编辑，以验证等位基因特定的功能对内源性目标的影响 S基因在同基因细胞中的表达。总体而言，提出的发现和验证SLE的无偏见方法 “因果”SNPs是高风险/高回报的，可能导致对SLE病因学和 干预策略。RSNPs、Cres及其靶基因的发现将显著推动我们的 关于系统性红斑狼疮遗传学的知识，并为未来深入机制研究的贡献提供方向 RSNPs和靶基因在狼疮易感性中的作用，它们可以定义来自GWAs的未来治疗靶点。

ABSTRACT Systematic lupus erythematosus (SLE or lupus) is a currently incurable autoimmune disease, characterized by abnormal immune cell (e.g., B-lymphocytes) response and the production of numerous pathogenic autoantibodies, culminating in multi-organ damage (e.g., kidneys, skin). While the etiology of SLE is incompletely understood, a substantial genetic contribution is well established. Several genome-wide association studies (GWAS) have identified over 100 SLE predisposing loci (p<5x10-8), mostly single nucleotide polymorphisms (SNPs). Most of these SNPs do not directly alter protein products, and previous work from us and others have shown that many such SNPs are enriched within cis regulatory elements (cRE) (i.e., enhancers and silencers) and likely to modulate gene expression. However, pinpointing the causal, predisposing SNPs within cREs and deciphering their precise mechanisms represent major obstacles to progress in the field. Consequently, this knowledge gap has severely hindered the translation of GWAS findings into clinical applications. Hence, there is a profound need for unbiased, comprehensive, and high-throughput approaches to address the mechanistic link between hundreds of potential regulatory SNPs (rSNPs) and SLE susceptibility. We hypothesize that SLE- predisposing rSNPs aberrantly induce cRE activities that influence the expression of target genes in unstimulated and/or stimulated B-cells. To systematically delineate rSNPs and their impact on target genes, we propose to establish a high-throughput experimental pipeline to determine and validate rSNPs underlying GWAS loci. In Aim 1, we will apply the high-throughput technique “Self-Transcribing Active Regulatory Region-sequencing” (STARR-seq) to functionally quantify the regulatory activities of hundreds of SNP-containing regions simultaneously. Using Raji cells (B-lymphocyte) in both unstimulated and stimulated conditions, we will apply STARR-seq to evaluate 756 selected rSNPs within 79 distinct GWAS loci for SLE susceptibility. In Aim 2a, we will apply next-generation (NG)-Capture-C to detect SNP-specific effects on cis interactions with endogenous, cognate target genes in Raji cells, needing no strong a priori hypothesis of target genes and functional consequences. We will evaluate the same set of 756 SNPs in Aim 1 and Aim 2a. We anticipate this two-prong tandem strategy will bridge the gap between GWAS-derived rSNPs and mechanistic links to their target genes. To validate the effectiveness and accuracy of the proposed methods, Aim 2b will use CRISPR-based (epi)genetic editing of a selected rSNP to validate the allele-specific functional effects on the endogenous target gene(s) in isogenic cells. Collectively, the proposed unbiased approaches for discovering and validating SLE “causal” SNPs is high risk/high reward and may lead to breakthroughs in the understanding of SLE etiology and intervention strategies. Discovery of rSNPs, cREs, and their target genes will significantly advance our knowledge of SLE genetics, and yield directions for future in-depth mechanistic research on the contribution of rSNPs and target genes in lupus susceptibility, which could define future therapeutic targets derived from GWAS.