Systematic identification of functional T1D-associated non-coding SNPs using genetic, transcriptomic and epigenetic methods

Type 1 diabetes (T1D) is an autoimmune disease that arises from the action of both genetic and environmental factors. The Type 1 Diabetes Genetics Consortium (T1DGC) assembled data and biospecimens, including serum, plasma, DNA, PBMCs, and EBV-transformed B cell lines from more than 4,000 affected sibpair (ASP) families. As leaders of the T1DGC, we conducted a genome-wide association scan (GWAS) that identified more than 40 statistically significant T1D risk loci. We followed the GWAS with fine mapping each T1D locus with the ImmunoChip. This resulted in identifying sets of 99% credible SNPs (those most likely to be causal) within each locus. Our interrogation of data from ENCODE and the Epigenomics Roadmap revealed that the set of T1D credible causal SNPs was enriched for overlap with enhancer chromatin states in immunologically relevant tissues (CD4+ and CD8+ T cells, CD19+ B cells, and CD34+ stem cells). In response to these findings, the NIDDK has issued RFA-DK-15-025, “Mechanisms Underlying the Contribution of Type 1 Diabetes Risk-Associated Variants (DP3)”. As the T1D-associated credible SNPs are enriched in regulatory regions, we propose to directly examine these SNPs in T and B cells from our viably frozen T1DGC PBMC collection. We will focus on the accessible chromatin landscape at T1D risk loci, using ATAC-seq and characterize the impact of these SNPs on gene expression using RNA-seq in the same cells from the same individuals. Finally, we will test the function of the variants that impact chromatin accessibility and gene expression by perturbation experiments using CRISPR/Cas9 gene editing, followed by validation by gene expression and other functional assays. Our Specific Aims are to (1) identify T1D-associated SNPs that alter chromatin accessibility using ATAC-seq in T cells (CD4+ and CD8+) and B cells (CD19+) from 200 unrelated T1D cases and 200 unrelated controls (Caucasian and African-American); (2) define the effect of SNPs influencing the chrom