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，其特征在于 异常免疫细胞（例如，B淋巴细胞）反应和许多致病性 自身抗体，最终导致多器官损伤（例如，肾脏、皮肤）。虽然SLE的病因不完全 在理解的基础上，充分确定了实质性的遗传贡献。几项全基因组关联研究 （GWAS）已经确定了超过100个SLE易感基因座（p<5x 10 -8），主要是单核苷酸多态性 （SNP）。这些SNPs中的大多数并不直接改变蛋白质产物，我们和其他人以前的工作已经证明了这一点。 显示许多这样的SNP在顺式调节元件（cRE）内富集（即，增强剂和消音剂） 并可能调节基因表达。然而，精确定位克雷斯内的因果、易感SNP， 破译其确切机制是该领域取得进展的主要障碍。因此，这 知识鸿沟严重阻碍了GWAS研究结果转化为临床应用。因此， 是一个深刻的需要公正的，全面的，和高通量的方法来解决机制， 数以百计的潜在的调节SNPs（rSNPs）和SLE易感性之间的联系。我们假设SLE- 诱发rSNPs异常诱导cRE活性，影响未受刺激的细胞中靶基因的表达。 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基因座的rSNP。在 目的一、应用高通量的自转录活性调控区测序技术 （STARR-seq）来功能性地量化数百个含有SNP的区域的调节活性。 同步使用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GWAS基因座内756个选定rSNP的SLE易感性。在目标2a中，我们 将应用下一代（NG）-Capture-C来检测SNP对顺式与内源性相互作用的特异性影响， Raji细胞中的同源靶基因，不需要强的靶基因和功能的先验假设， 后果我们将评估Aim 1和Aim 2a中相同的756个SNP。我们预计这种双管齐下的 串联策略将弥合GWAS衍生的rSNP和与其靶基因的机制链接之间的差距。 为了验证所提出的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，以验证对内源性靶标的等位基因特异性功能效应 在同基因细胞中的基因。总的来说，提出的无偏的方法，发现和验证系统性红斑狼疮， “因果”SNP是高风险/高回报的，可能导致对SLE病因学的理解取得突破， 干预策略。rSNPs、克雷斯及其靶基因的发现将大大推进我们的研究。 SLE遗传学的知识，以及未来深入研究SLE的贡献机制的方向。 rSNPs和靶基因在狼疮易感性，这可能会定义未来的治疗目标来自GWAS。

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.