DNA Elements Underlying Celiac and Crohn's Susceptibility

乳糜泻和克罗恩病易感性的 DNA 元素

• 批准号: 9664318
• 负责人: Benjamin Tycko
• 金额: $ 16.84万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-09 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9664318
• 关键词: DNA; Elements; Underlying; Celiac; Crohn

Project Summary/Abstract Well powered genome-wide association studies (GWAS) for celiac disease and Crohn's disease have identified numerous non-HLA susceptibility loci. Genetic fine-mapping and expression quantitative trait loci (eQTL) can narrow in on which genes probably account for the GWAS signals, but even after these approaches the locations of the causal nucleotide changes often remain unknown. This gap in understanding is a roadblock to targeted prevention and therapy. As developed in our prior work, mapping haplotype-dependent allele-specific CpG methylation (hap-ASM) and methylation quantitative trait loci (mQTLs) in cells relevant to a given disease, and then overlapping these epigenetic maps with GWAS data, can help to hone in on the DNA regulatory sequences that causally underlie the GWAS signals. Our hypothesis is that this combined genetic-epigenetic mapping strategy, followed by functional assays, will be able to identify regulatory DNA sequences that contribute to celiac disease and Crohn's disease susceptibility and pathogenesis. First, we will carry out Methyl-Seq of CD4+ and CD8+ T cells and peripheral blood monocytes, to map hap-ASM genome-wide. These data will pinpoint hap- ASM differentially methylated regions (DMRs) in the same haplotype blocks as GWAS peaks for celiac and Crohn's. Since hap-ASM often reflects allele-specific transcription factor binding site (TFBS) or insulator occupancies, we will cross-validate our findings using an independent method, Assay for Transposase- Accessible Chromatin Sequencing (ATAC-Seq). In our second aim, we will rank and prioritize the hap-ASM DMRs based on the strength of the allelic asymmetries, ATAC-Seq overlaps, and linkage disequilibrium (LD) with GWAS peak SNPs, and perform high-throughput targeted bisulfite sequencing to fine-map the top-ranked DMRs, both in the blood-derived cells and in mucosal T cells from celiac and Crohn's patients. In our third aim, to definitively test the functional roles of specific TFBS in the DMRs, we will use CRISPR to delete these sequences in Jurkat cells and in normal T cells. We will score effects of the deletions on local methylation patterns and mRNA levels of each of the nearby genes. These data will clarify our fundamental understanding of susceptibility and pathogenesis of celiac disease and Crohn's disease.

项目概要/摘要 针对乳糜泻和克罗恩病的强有力的全基因组关联研究 (GWAS) 已确定 许多非 HLA 易感位点。遗传精细定位和表达数量性状位点（eQTL）可以 缩小了哪些基因可能解释 GWAS 信号，但即使在这些方法之后，位置 引起核苷酸变化的原因通常仍然未知。这种理解上的差距是实现目标的障碍 预防和治疗。正如我们之前的工作中所开发的，映射单倍型依赖的等位基因特异性 CpG 与给定疾病相关的细胞中的甲基化（hap-ASM）和甲基化数量性状位点（mQTL），以及 然后将这些表观遗传图谱与 GWAS 数据重叠，可以帮助磨练 DNA 调控序列 GWAS 信号的因果关系。我们的假设是，这种结合的遗传-表观遗传图谱 策略，随后进行功能测定，将能够识别有助于乳糜泻的调控 DNA 序列 疾病和克罗恩病的易感性和发病机制。首先，我们将进行CD4+和的甲基测序 CD8+ T 细胞和外周血单核细胞，以绘制 hap-ASM 全基因组图谱。这些数据将精确定位 ASM 差异甲基化区域 (DMR) 位于与乳糜泻和 GWAS 峰值相同的单倍型块中 克罗恩病。由于 hap-ASM 通常反映等位基因特异性转录因子结合位点 (TFBS) 或绝缘子 占用率，我们将使用独立的方法交叉验证我们的发现，转座酶测定- 可访问染色质测序 (ATAC-Seq)。在我们的第二个目标中，我们将对 hap-ASM 进行排名和优先排序 基于等位基因不对称强度、ATAC-Seq 重叠和连锁不平衡 (LD) 的 DMR 具有 GWAS 峰值 SNP，并执行高通量靶向亚硫酸氢盐测序以精细定位排名靠前的 DMR，存在于乳糜泻和克罗恩病患者的血液来源细胞和粘膜 T 细胞中。在我们的第三个目标中， 为了明确测试 DMR 中特定 TFBS 的功能作用，我们将使用 CRISPR 删除这些 Jurkat 细胞和正常 T 细胞中的序列。我们将评估缺失对局部甲基化的影响 每个附近基因的模式和 mRNA 水平。这些数据将澄清我们的基本认识 乳糜泻和克罗恩病的易感性和发病机制。