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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

基本信息

批准号：
9752313
负责人：
ROBERT J. KLEIN
金额：
$ 66.07万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/9752313
关键词：
Affect Automobile Driving Binding Biological Assay Biological Models CRISPR/Cas technology Chromosomes Code Colon Colorectal Complex Crohn&apos s disease Data Diagnosis Disease Enhancers Epigenetic Process Epithelial Epithelial Cells 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

项目摘要

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