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.

Public Health Relevance

Ulcerative colitis (UC), which affects over 1 million people worldwide, is caused in part by genetic changes inherited from one's parents. Here, we propose to take advantage of novel, massively parallel experimental approaches we have developed to screen through genetic changes statistically associated with UC to identify those changes most likely to directly cause the disease and then test these candidates in more complex biological models. Our study will not only provide important genetic insights into UC for diagnosis and treatment development, but also establish a strategy broadly applicable to other complex genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK115398-01A1
Application #
9600802
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Karp, Robert W
Project Start
2018-08-01
Project End
2023-04-30
Budget Start
2018-08-01
Budget End
2019-04-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Cornell University
Department
Miscellaneous
Type
Organized Research Units
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850