Deciphering the role of noncoding variation in the pathogenesis of multiple sclerosis

项目摘要 多发性硬化症(MS)是大脑和脊髓的一种慢性炎症性疾病，主要由 致病T细胞对髓鞘抗原的反应，导致中枢神经系统脱髓鞘 (CNS)。髓鞘反应性T细胞在实验性自身免疫性脑脊髓炎的诱导中也起关键作用 多发性硬化症(EAE)小鼠模型的研究表明，CD4T辅助细胞(Th)参与了疾病的发病机制。 包括促炎症的Th17和Th1细胞。在这方面，这些T细胞的促病能力 亚群来源于它们靶向和渗透中枢神经系统及其促炎效应因子的能力 功能。遗传和环境因素之间的相互作用在MS的发病机制中有牵连。 然而，尽管大量的基因组范围关联数据(GWAS)数据揭示了强大的基因 对于多发性硬化症的贡献，确定许多人真正的促病基因靶点(S)一直是一个挑战 与风险相关的变种，以及它们导致调节失调的细胞室。在预赛中 工作中，我们应用了全球基因组学方法将MS相关的非编码风险变量映射到其 通过物理捕捉增强子-启动子相互作用获得长距离靶基因。这些涉及多发性硬化症的基因 根据MS来源的致病Th17细胞中的异常表达特征，进一步优先考虑 病人。使用这种方法，我们已经确定了推测的调节MS变异体和控制 CD4T细胞在MS中的功能是这一应用的基础。我们假设识别非编码 改变控制促炎T细胞的顺式元件调节功能的变体将提供新的 对多发性硬化症发生的潜在病因的机械论见解在这里，我们提出一个 综合策略，优先考虑和验证MS发病机制中的因果遗传变异。目标1的目标是 确定非编码风险变异体在CD4T细胞基因调控中的作用。特别是，我们将 采用高通量监管记者分析和机器学习方法相结合的方式 在原代人类CD4T细胞中鉴定具有调节功能的SNPs。在目标2中，我们将确定 这些调节变异体导致多发性硬化患者T细胞功能和致病性改变的机制 互补CRISPR/Cas9介导的遗传和表观遗传扰动将定义增强子上下文 风险等位基因，并描绘出真正的调控风险变异体的调控基因靶点。从基因型转变为 对于表型，我们将在完整的背景下定义有助于疾病病因学的真正因果变异 在小鼠中使用遗传干预来模拟保守的风险变异的免疫系统。总而言之，建议的 实验将促进我们对控制Th17的遗传和细胞机制的理解 MS的细胞致病性，并发现在疾病病因学中功能未知的新基因座。

Project Summary Multiple sclerosis (MS) is a chronic, inflammatory condition of the brain and spinal cord mediated largely by pathogenic T cell responses to myelin antigens, resulting in demyelination of the central nervous system (CNS). Myelin-reactive T cells are also crucial for induction of the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Studies of MS and EAE implicate CD4 T helper (Th) cells in disease pathogenesis, including pro-inflammatory Th17 and Th1 cells. In this regard, the disease-promoting ability of these T cell subsets stems from both their capacity to target and infiltrate the CNS and their pro-inflammatory effector function. An interplay between genetic and environmental factors is implicated in the pathogenesis of MS. However, despite a wealth of genome wide association data (GWAS) data revealing a strong genetic contribution to MS, it has been challenging to identify the bona fide disease-promoting gene target(s) of many risk-associated variants, and the cellular compartment in which they contribute to dysregulation. In preliminary work, we have applied global genomics approaches to map MS-associated noncoding risk variants to their long-range target gene by physically capturing enhancer-promoter interactions. These MS-implicated genes were further prioritized based on dysregulated expression signatures in pathogenic Th17 cells derived from MS patients. Using this approach, we have identified putative regulatory MS variants and gene targets that control CD4 T cell function in MS that are the basis of this application. We hypothesize that identifying noncoding variants that alter the regulatory function of cis elements controlling proinflammatory T cells will provide novel mechanistic insights into the underlying etiology governing the development of MS. Here, we propose an integrative strategy to prioritize and validate causal genetic variants in MS pathogenesis. The goal of Aim 1 is to determine the contribution of noncoding risk variants to CD4 T cell gene regulation. In particular, we will employ a combination of high-throughput regulatory reporter assays and machine learning approaches to identify SNPs with regulatory function in primary human CD4 T cells. In Aim 2, we will determine the mechanism by which these regulatory variants contribute to altered T cell function and pathogenicity in MS. Complementary CRISPR/Cas9-mediated genetic and epigenetic perturbations will define the enhancer context of risk alleles and delineate bona fide regulatory gene targets of regulatory risk variants. Moving from genotype to phenotype, we will define true causal variants that contribute to disease etiology in the context of an intact immune system using genetic interventions that model conserved risk variants in mice. Together, the proposed experiments will advance our understanding of the both the genetic and cellular mechanisms governing Th17 cell pathogenicity in MS and discover new loci with unknown functions in disease etiology.