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

项目摘要 多发性硬化症（MS）是一种慢性炎症性脑和脊髓疾病， 病原性T细胞对髓鞘抗原的反应，导致中枢神经系统的脱髓鞘 （CNS）。髓磷脂反应性T细胞也是诱导实验性自身免疫性脑脊髓炎的关键 (EAE)MS的小鼠模型。MS和EAE的研究涉及疾病发病机制中的CD 4 T辅助（Th）细胞， 包括促炎性Th 17和Th 1细胞。在这方面，这些T细胞的疾病促进能力 亚群来源于它们靶向和浸润CNS的能力以及它们的促炎效应物 功能遗传和环境因素之间的相互作用与MS的发病机制有关。 然而，尽管大量的全基因组关联数据（GWAS）数据揭示了一个强的遗传学特征， 由于对MS的贡献，鉴定许多疾病的真正的疾病促进基因靶标一直是一个挑战。 风险相关的变异，以及它们导致失调的细胞区室。初步 在这项工作中，我们应用全球基因组学方法将MS相关的非编码风险变体映射到它们的 通过物理捕获增强子-启动子相互作用的远程靶基因。这些与MS有关的基因 基于来源于MS的致病性Th 17细胞中失调的表达特征， 患者使用这种方法，我们已经确定了推定的调控MS变体和基因靶点， MS中的CD 4 T细胞功能是本申请的基础。我们假设识别非编码区 改变控制促炎性T细胞的顺式元件的调节功能的变体将提供新的 对MS发展的潜在病因的机械见解。在这里，我们提出了一个 综合策略，优先考虑和验证MS发病机制中的致病遗传变异。目标1的目标是 以确定非编码风险变异对CD 4 T细胞基因调控的贡献。特别是要 采用高通量调控报告基因测定和机器学习方法的组合， 在原代人CD 4 T细胞中鉴定具有调节功能的SNP。在目标2中，我们将确定 这些调节变体有助于MS中T细胞功能和致病性改变的机制。 互补的CRISPR/Cas9介导的遗传和表观遗传扰动将定义增强子背景 的风险等位基因，并描绘真正的调控基因的调控风险变异的目标。从基因型 表型，我们将定义真正的因果变异，有助于疾病病因的背景下，完整的 使用遗传干预的免疫系统，在小鼠中模拟保守的风险变体。在一起，拟议的 实验将促进我们对Th 17的遗传和细胞机制的理解 细胞致病性，发现疾病病因学中功能未知的新位点。

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.