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

使用高通量方法在 MS 上识别/表征功能变异

• 批准号: 9670361
• 负责人: Gang Li
• 金额: $ 16.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-20 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9670361
• 关键词: Using; Throughput; Approach; Identify; Characterize

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.

抽象的 多发性硬化症（MS）是一种自身免疫性疾病 致病理解，无法治愈。基因组广泛的研究 （GWAS）在MS上确定了> 200 ms相关的局部。这些地方代表 数千种遗传变异，通常在单个核苷酸多态性中 （SNP），在链接二动（LD）中。但是，GWAS不知道哪一个 它们是每个基因座中的因果/功能性SNP（FSNP）。这个技术缺点 在GWAS和特定机构之间留下“差距”，该机制转化为 生物学洞察力和治疗干预的机会有限。到 克服了这一限制，我们开发了两种新型技术：功能 单核苷酸多态性seq（FSNP-SEQ）和侧翼限制 酶介导的DNA下拉（FREP）。 FSNP-Seq是一种高通量方法 通过实验确定哪些SNP可能会结合调节蛋白和 因此代表FSNP。 FREP使用FSNP序列作为“诱饵”来识别 相关的调节蛋白以半吞吐量的方式。使用这些 技术，我们已经在MS相关的CD40基因座上确定了三个FSNP EMSA已确认了等位基因特异性的荧光素酶报告器组件，并且 CRISPR/CAS9，我们还确定了调节CD40的四种调节蛋白 通过这些FSNP表达。根据这些初步数据，我们建议 两个目的是将我们的新方法应用于MS的整个GWAS数据。首先，我们会的 利用我们对CD40基因座的新见解来定义潜在的目标CD40 调节蛋白复合物。其次，我们将采用FSNP-Seq进行高级 LD中4573个SNP中FSNP的吞吐量鉴定，有196个风险基因座 多发性硬化症。我们将使用FREP系统地识别控制的调节蛋白 通过关注MS FSNP的MS相关基因的表达 MS相关的抗原呈现基因，例如CD86，CD80和MHCI R21应用程序。这些研究将共同​​帮助我们产生MS- 相关的信号转导和等位基因特异性转录网络（MS- 停止网络），目的是确定MS治疗的新靶标。