Bridging the gap between GWAS and mechanism in JIA

项目摘要 幼年特发性关节炎（JIA）是一个开始于儿童时期的炎性关节炎家族。 在最常见的表型，血清阴性少关节加 多关节型JIA（有时为简单起见称为“polygoJIA”），已经确定了27个非HLA基因座相关 疾病风险。最具破坏性的变异体系统性JIA的GWAS定义了额外的位点。中的每 这些“命中”标志着JIA发病机制中的生物学途径。 不幸的是，从GWAS数据中精确定位这些机制已经被证明是困难的。GWAS击中马克大 DNA片段，称为单倍型。这些单倍型中的大多数不含SNP（单核苷酸多态性）。 多态性）或其他影响编码的变体，这表明大多数致病性多态性是 监管。事实证明，寻找调节性SNP和结合它们的蛋白质是非常困难的， 因此，GWAS迄今为止对JIA生物学的了解有限。 为了解决这个问题，我们的实验室最近开发了两种新的实验方法。SNP-seq 使用酶限制性内切酶询问候选调控变体， 测序以鉴定结合转录因子和其他调节蛋白的那些。侧翼 限制性增强下拉（FREP）是一种有效的方法来下拉相关的蛋白质， 通过质谱鉴定，从而将疾病相关变体与特定细胞 途径。这种串联策略使我们能够弥合GWAS和机制之间的差距。 这一提议的假设是，对遗传关联的实验性解剖将确定 新的机制，从而新的潜在的治疗JIA的目标。在目标I中，我们将追求SNP-seq + FREP以确定驱动polygoJIA风险的新型DNA-蛋白质关联。我们筛选了1,223个基因 多态性与27个polygoJIA风险位点的紧密连锁不平衡，并确定了多个强 候选功能变体，包括几种使用技术进行实验验证的变体，包括 电泳迁移率变化、荧光素酶和CRISPR。我们将确认这些高概率的变体， 然后将工作扩展到其他SNP-seq匹配。在Aim II中，从独立的SNP-seq筛选开始， TRAF 1/C5位点，我们将采用FREP和其他上游和下游验证方法， 定义这个基因座如何驱动polygoJIA的风险。我们将使用SNP-seq + FREP来询问最近- GWAS在系统性JIA中的发现，以确定这种独特的高度炎症性疾病的机制。 总之，这些研究将使我们能够确定驱动JIA发病机制的DNA-蛋白质关联。

Project Summary Juvenile idiopathic arthritis (JIA) is a diverse family of inflammatory arthritides that begin in childhood. Genome-wide association studies in the most common phenotype, seronegative oligoarticular plus polyarticular JIA (sometimes for simplicity termed “polygoJIA”), have identified 27 non-HLA loci associated with disease risk. GWAS of the most destructive variant, systemic JIA, has defined additional loci. Each of these “hits” marks a biological pathway in the pathogenesis of JIA. Unfortunately, pinpointing these mechanisms from GWAS data has proven difficult. GWAS hits mark large segments of DNA, termed haplotypes. Most of these haplotypes contain no SNPs (single nucleotide polymorphisms) or other variants that affect coding, suggesting that most causative polymorphisms are regulatory. Finding regulatory SNPs and the proteins that bind them has proven to be exceptionally difficult, and as a result GWAS have so far provided limited insight into JIA biology. To address this roadblock, our lab has recently developed two novel experimental methods. SNP-seq interrogates candidate regulatory variants using enzymatic restriction followed by next-generation sequencing to identify those that bind transcription factors and other regulatory proteins. Flanking Restriction Enhanced Pulldown (FREP) is an efficient method to pull down the associated protein(s) for identification by mass spectrometry, thereby linking disease-associated variants to particular cellular pathways. This tandem strategy allows us to bridge the gap between GWAS and mechanism. The hypothesis underlying this proposal is that experimental dissection of genetic associations will identify new mechanisms and thus new potential therapeutic targets in JIA. In Aim I, we will pursue SNP-seq + FREP to identify novel DNA-protein associations that drive polygoJIA risk. We screened 1,223 genetic polymorphisms in close linkage disequilibrium with 27 polygoJIA risk loci and identified multiple strong candidate functional variants, including several validated experimentally using techniques including electrophoretic mobility shift, luciferase and CRISPR. We will confirm these high-probability variants and then extend the work to other SNP-seq hits. In Aim II, beginning with an independent SNP-seq screen of the TRAF1/C5 locus, we will employ FREP and other upstream and downstream validation approaches to define how this locus drives risk of polygoJIA. We will employ SNP-seq + FREP to interrogate recently- available GWAS findings in systemic JIA to define mechanisms in this unique, highly-inflammatory disease. Together, these studies will allow us to identify DNA-protein associations that drive the pathogenesis of JIA.