Identifying Causal Variants in Juvenile Arthritis Using a Massively Parallel Reporter Assay

摘要 该项目旨在朝着儿科风湿病领域一直以来的目标迈出重要的一步 30多年来致力于：识别导致遗传风险的实际因果变异 幼年特发性关节炎。就像复杂性状的典型情况一样，我们发现，大多数遗传风险 贾跃亭位于基因组的非编码区。这些区域富含H3K4me1/H3K27ac 组蛋白标记，增强剂的表观遗传标记。因此，JIA的遗传风险涉及到 调节基因，而不是改变蛋白质功能的氨基酸取代(S)。而不是 认为JIA主要是一种自身免疫性疾病，我们假设JIA的出现是因为白细胞 基因和表观遗传介导的干扰，削弱了它们调节和协调的能力 整个基因组的转录。这种协调监管的丧失导致了不适当的表达 在缺乏正常外部信号的情况下的炎症介质，通常需要启动或维持 炎症反应。 我们检验这一假设的一个重大障碍是，JIA的实际因果变异是未知的。 目前已知的JIA危险基因座有38个以上，这些基因座包含18,000多个变异。我们最近 48例JIA患者的全基因组测序完成进一步增加了候选患者的数量 基因座和变种。鉴于这些挑战，以及因果变量存在于监管范围内(而不是 蛋白质编码)区域，该领域迫切需要一种高通量的方法来查询数以万计的 变异，确定它们对转录的影响，并识别这些变异在哪些细胞中发挥其作用 效果。 在这项先导性研究中，我们将介绍实验室开发的大规模平行报告分析(MPRA) 我们的合作者，帕迪斯·萨贝蒂博士，作为一种通过它们的能力来识别潜在的因果变异的方法 改变髓系和淋巴系细胞系的基因表达。这个测试是专门设计用来询问 基因组非编码区内的遗传变异。我们将测试表现出强大的变种 与所有已知的JIA相关风险基因座中的指数SNPs的连锁不平衡 研究(n=7,312)，检查它们对基因表达的影响。然后我们将使用荧光素酶分析来 证实了MPRA的结果。在这项为期两年的试点研究结束时，我们预计将有显著的 缩小了JIA中可能的因果变异的列表，并将准备进行所需的研究 阐明特定等位基因与特定转录网络和细胞中的变化之间的因果联系 功能。好了！

Abstract This project aims to take an important step toward a goal that the field of pediatric rheumatology has been working towards for more than 30 years: identifying the actual causal variants that confer genetic risk in juvenile idiopathic arthritis (JIA). As is typical for complex traits, we have found that most of the genetic risk for JIA resides within non-coding regions of the genome. These regions are enriched for H3K4me1/H3K27ac histone marks, epigenetic signatures of enhancers. Thus, genetic risk for JIA involves changes in the regulation of genes, rather than amino acid substitutions that alter the function(s) of proteins. Rather than viewing JIA as primarily an autoimmune disease, we hypothesize that JIA emerges because leukocytes suffer genetically and epigenetically-mediated perturbations that blunt their capacity to regulate and coordinate transcription across the genome. This loss of coordinate regulation leads to inappropriate expression of inflammatory mediators in the absence of the normal external signals typically required to initiate or sustain an inflammatory response. A significant impediment to our testing that hypothesis is that the actual causal variants for JIA are unknown. There are more than 38 known risk loci for JIA, and these loci contain more than 18,000 variants. Our recent completion of whole genome sequencing on 48 patients with JIA has further added to the number of candidate loci and variants. Given these challenges, and the fact that the causal variants lie within regulatory (rather than protein-coding) regions, the field is in urgent need of a high throughput method to query tens of thousands of variants, determine their effects on transcription, and identify the cells in which these variants exert their effects. In this pilot study, we will introduce a massively parallel reporter assay (MPRA), developed in the laboratory of our collaborator, Dr. Pardis Sabeti, as a way of identifying potential causal variants through their capacity to alter gene expression in myeloid and lymphoid cell lines. This assay was specifically designed to query genetic variants within non-coding regions of the genome. We will test variants that demonstrate strong linkage disequilibrium with index SNPs within all known JIA associated risk loci as identified from previous studies (n= 7,312), examining their effects on gene expression. WE will then use luciferase assays to corroborate the results from the MPRA. At the end of this 2-year pilot study, we expect to have significantly narrowed the list of potential causal variants in JIA and will be prepared to undertake the studies required to elucidate a causal link between specific alleles and alterations in specific transcriptional networks and cellular functions. !