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 chromatin landscape on gene expression using RNA-seq in the same cells as profiled in Aim 1; and (3) experimentally validate regions of open chromatin affected by T1D-associated non-coding credible variants that alter gene expression in existing T1DGC EBV-transformed B cells (from the same participants providing PBMCs in Aims 1 and 2) and in T cells using CRISPRa to activate regions of closed chromatin and CRISPRi to inhibit/repress regions of open chromatin. This innovative and cost-effective proposal will leverage existing samples, prior genotyping and phenotyping of T1DGC resources, and an established team of experts in cutting-edge technologies and analysis. The findings obtained from this research will provide mechanisms of the functional basis of T1D- associated variants that will inform therapeutic considerations.
The proposed aims will advance our understanding of the functional impact and mechanisms of action of type 1 diabetes (T1D)-associated credible SNPs that are enriched for non-coding variants residing predominantly in DNA regulatory regions. Our research will focus on gene regulation and the impact of these credible SNPs on T1D risk using our unique resource of samples and data from T1D cases and controls of Caucasian and African-American ancestry who participated in the Type 1 Diabetes Genetics Consortium. The findings obtained from this research will provide mechanisms of the functional basis of T1D-associated variants.
| Rich, Stephen S (2017) The Promise and Practice of Genetics on Diabetes Care: The Fog Rises to Reveal a Field of Genetic Complexity in HNF1B. Diabetes Care 40:1433-1435 |
