Using High Throughput Approach to Identify/Characterize Functional Variants on MS

摘要 多发性硬化症(MS)是一种自身免疫性疾病，对其治疗有限 对病原学有一定了解，但无法治愈。全基因组关联研究 在MS上发现了200个与MS相关的基因座。这些基因座代表 数以千计的遗传变异，通常是单核苷酸多态性 (SNPs)，连锁不平衡(LD)。然而，Gwas并不知道是哪一个 它们是每个基因座上的因果/功能性SNP(FSNP)。这一技术缺陷 在全球气候变化框架和具体机制之间留下了一个“鸿沟”，这将转化为 生物洞察和治疗干预的机会有限。至 克服了这一限制，我们开发了两种新技术：泛函 单核苷酸多态-序列(fSNP-seq)与侧翼限制 酶介导的DNA下拉反应(FREP)。FSNP-SEQ是一种高通量的方法 通过实验确定哪些SNPs可能结合调节蛋白和 因此，代表fSNPs。FREP使用fSNP序列作为“诱饵”来识别 相关的调节蛋白以半高通量的方式。使用这些 技术，我们已经在MS相关的CD40基因座上发现了三个fSNPs 已经通过EMSA，一种等位基因特异的荧光素酶报告试验和 CRISPR/Cas9，我们还鉴定了四种调节CD40的调节蛋白 通过这些fSNP表达。根据这些初步数据，我们建议 两个目标是将我们的新方法应用于关于MS的整个Gwas数据。首先，我们将 使用我们对CD40基因座的新见解来定义潜在的靶向CD40 调节蛋白复合体。第二，我们将使用fSNP-seq来承担高 利用196个易感基因座对4573个基因座中的fSNPs进行吞吐量鉴定 我们将使用FREP系统地识别控制 MS相关基因通过MS fSNPs的表达 MS相关抗原提呈基因CD86、CD80和MHCI与此相关 R21应用。总之，这些研究将帮助我们产生多发性硬化症- 相关信号转导和等位基因特异性转录网络 STAST网络)，目的是确定多发性硬化症治疗的新靶点。

Abstract Multiple sclerosis (MS) is an autoimmune disease for which there is limited pathogenic understanding and no cure. Genome wide association studies (GWAS) on MS have identified >200 MS-associated loci. These loci represent thousands of genetic variants, usually in single nucleotide polymorphisms (SNPs), in linkage disequilibrium (LD). However, GWAS doesn't tell which one of them is the causal/functional SNP (fSNP) in each locus. This technical drawback leaves a “gap” between GWAS and specific mechanism that translates into limited opportunities for biological insight and therapeutic intervention. To overcome this limitation, we have developed two novel techniques: functional Single Nucleotide Polymorphism-seq (fSNP-seq) and Flanking Restriction Enzyme-mediated DNA Pulldown (FREP). fSNP-seq is a high throughput method to identify experimentally which SNPs are likely to bind regulatory proteins and therefore to represent fSNPs. FREP uses an fSNP sequence as “bait” to identify associated regulatory proteins in a semi high throughput way. Using these techniques, we have identified three fSNPs on a MS-associated CD40 locus that have been confirmed by EMSA, an allele-specific luciferase reporter assay and CRISPR/Cas9, and we also identified four regulatory proteins that regulate CD40 expression via these fSNPs. On the basis of these preliminary data, we propose two aims to apply our new methods to the entire GWAS data on MS. First, we will use our new insights into CD40 locus to define a potentially targetable CD40 regulatory protein complex. Second, we will employ fSNP-seq to undertake high- throughput identification of fSNPs among 4573 SNPs in LD with 196 risk loci for MS. We will use FREP to systematically identify regulatory proteins that control the expression of MS-associated genes via the MS fSNPs by focusing on the MS-associated antigen presenting genes such as CD86, CD80 and MHCI for this R21 application. Together, these studies will help us to generate a MS- associated signal transduction and allele-specific transcription network (MS- STAST network), with the goal of identifying novel targets for MS therapy.