Integrated high-throughput functional assays to identify ulcerative colitis causal risk variants

摘要 溃疡性结肠炎(UC)影响着全球100多万人。成交时相对风险增加13.8倍 受影响个人的亲属。全基因组关联研究(GWAS)，包括由 CO-I Cho，已经确定了133个UC-或共有的克罗恩病(CD)/UC相关基因。由于单倍型 结构，GWAS通常提名大量的单核苷酸多态(SNPs)在 连锁不平衡(LD)，使得在LD中很难区分因果和中性侧翼SNPs。 此外，这些SNP大多位于非编码区，这使得功能解释变得更加重要 由于对非编码监管要素的了解不完整，这一点很困难。在这里，我们测试假设 UC的遗传风险是通过结肠固有机制介导的。本研究的总体目标是理性地 选择在正常结肠上皮细胞中活跃的UC相关非编码和编码变异体。我们 通过计算确定了1,407个与GWASUC相关的非编码变体，映射到 在结肠中活跃的增强子/启动子区域，以及248个与GWAUC相关的错义变体。在目标1中， 我们将通过实验检测结肠类器官中的1,407个候选非编码SNP 集成新型大规模并行的创新高通量突变转录读出流水线 染色体整合自转录活性调控区测序分析(ISARR-SEQ) 高通量定量双荧光素酶试验用于命名致因性UC非编码风险变异。在目标2中， 我们将通过我们的整合蛋白对248个候选错义SNPs进行实验检测 结合6个高通量突变功能的互作组扰动筛选(INPOINT)管道 量化编码变异对蛋白质稳定性和特定蛋白质相互作用的影响的分析。 基于我们的实验结果，我们将进行综合网络分析，以提名10个因果变量 使用CRISPR/Cas9基因组进行体内功能评估的候选对象(7个非编码变体和3个编码变体) 在AIM 3中以冒号器官进行编辑。成功完成这些目标将提供对 溃疡性结肠炎的遗传机制和建立广泛适用于病因识别的策略 其他疾病复杂性状的潜在变异。

SUMMARY Ulcerative colitis (UC) affects over 1 million people worldwide. Relative risk is increased by >13.8 fold for close relatives of affected individuals. Genome-wide association studies (GWAS), including a meta-analysis lead by Co-I Cho, have identified 133 UC- or shared Crohn’s Disease (CD)/UC-associated loci. Due to haplotype structure, GWAS usually nominate clusters of large numbers of single nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD), making it difficult to distinguish causal vs neutral flanking SNPs in LD. Furthermore, most of these SNPs are in non-coding regions, making functional interpretation even more difficult due to incomplete knowledge of non-coding regulatory elements. Here, we test the hypothesis that genetic risk to UC is mediated through colon-intrinsic mechanisms. The overall goal of this study is to rationally select the causal UC-associated non-coding and coding variants active in normal colon epithelial cells. We have computationally identified 1,407 GWAS UC-associated non-coding variants mapping to enhancer/promoter regions active in the colon, and 248 GWAS UC-associated missense variants. In Aim 1, we will experimentally examine each of the 1,407 candidate non-coding SNPs in colon organoids through an innovative high-throughput mutagenesis transcriptional readout pipeline integrating a novel massively parallel chromosome-integrated self-transcribing active regulatory region sequencing assay (iSTARR-seq) with the high-throughput quantitative dual luciferase assay to nominate causal UC non-coding risk variants. In Aim 2, we will experimentally examine each of the 248 candidate missense SNPs through our INtegrated PrOtein INteractome perTurbation screening (InPOINT) pipeline combining six high-throughput mutagenesis functional assays to quantify the impact of coding variants on protein stability and specific protein-protein interactions. Based on our experimental results, we will perform integrated network analysis to nominate 10 causal variant candidates (7 non-coding and 3 coding variants) for functional evaluation in vivo using CRISPR/Cas9 genome editing in colon organoids in Aim 3. Successful completion of these aims will provide important insights into the genetic mechanisms driving Ulcerative Colitis and establish a strategy broadly applicable for identifying causal variants underlying complex traits for other diseases.