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

综合高通量功能测定，以确定溃疡性结肠炎的因果风险变异

• 批准号: 9918930
• 负责人: ROBERT J. KLEIN
• 金额: $ 65.72万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918930
• 关键词: Affect; Automobile Driving; Binding; Biological Assay; Biological Models; CRISPR/Cas technology; Chromosomes; Code; Colon; Colorectal; Complex; Crohn' s disease; Data; Diagnosis; Disease; Enhancers; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Family; Gene Expression; Gene Frequency; Genes; Genetic; Genetic Diseases; Genetic Risk; Genetic Transcription; Genotype; Goals; Haplotypes; Human; Individual; Inflammatory Bowel Diseases; Inherited; Knowledge; Lead; Letters; Libraries; Linkage Disequilibrium; Luciferases; Mediating; Meta-Analysis; Methodology; Missense Mutation; Mutagenesis; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Natural Immunity; Nucleic Acid Regulatory Sequences; Organoids; Parents; Pathway Analysis; Patients; Promoter Regions; Proteins; Recording of previous events; Regulator Genes; Regulatory Element; Relative Risks; Resources; Risk; Risk Factors; Single Nucleotide Polymorphism; Structure; Susceptibility Gene; Testing; Transcript; Ulcerative Colitis; Untranslated RNA; Variant; Work; base; causal variant; epigenomics; follow-up; genetic variant; genome editing; genome wide association study; improved; in vivo evaluation; innovation; insight; interest; novel; protein protein interaction; risk variant; screening; therapy development; tool; trait; transcription factor; transcriptome sequencing; transcriptomics

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.

总结 溃疡性结肠炎（UC）影响全球超过100万人。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等人已经鉴定了133个UC-或共有的克罗恩病（CD）/UC相关基因座。由于单倍型 结构，GWAS通常提名集群的大量单核苷酸多态性（SNP）， 连锁不平衡（LD），使得难以区分LD中的因果与中性侧翼SNP。 此外，这些SNPs中的大多数位于非编码区，使得功能解释更加复杂。 由于对非编码调控元件的不完全了解，在这里，我们测试的假设， UC的遗传风险通过结肠内在机制介导。本研究的总体目标是合理地 选择在正常结肠上皮细胞中有活性的致病UC相关非编码和编码变体。我们 通过计算确定了1，407个GWAS UC相关的非编码变体， 增强子/启动子区域在结肠中的活性，和248 GWAS UC相关的错义变体。在目标1中， 我们将通过一个实验来检查结肠类器官中1,407个候选非编码SNP中的每一个。 创新性高通量诱变转录读出流水线，其整合了新的大规模并行 染色体整合自转录活性调控区测序测定（iSTARR-seq）， 高通量定量双荧光素酶测定法，以确定病因UC非编码风险变体。在目标2中， 我们将通过我们的整合蛋白， Interactome微扰筛选（InPOINT）流水线结合六种高通量诱变功能 定量编码变体对蛋白质稳定性和特异性蛋白质-蛋白质相互作用的影响的测定。 根据我们的实验结果，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物（7种非编码变体和3种编码变体） 在Aim 3中编辑结肠类器官。成功地完成这些目标将为我们提供重要的见解， 遗传机制驱动溃疡性结肠炎，并建立广泛适用于确定病因的策略 其他疾病的复杂特征的潜在变异。