The Identification of Genetic and Epigenetic Changes that Contribute to Type 1 Diabetes

Abstract

Type 1 diabetes (T1D) results from an immune cell mediated destruction of insulin-producing pancreatic β cells. Currently there is no cure for T1D. The exact cause for T1D is unknown but growing evidence points to the contribution of both genetic and environmental factors, leading to a breakdown in immunological tolerance normally maintained by Regulatory T (Treg) cells. The exact environmental contributions to T1D progression are not well characterised but emerging studies suggest that they may alter the immune system via epigenetic modification. Recent data strongly link the breakdown in tolerance in T1D and other autoimmune diseases to alterations in the transcriptional program in CD4+ T cells, however, the molecular mechanisms are not well understood. This work proposes that in T1D causal genetic risk SNPs alter the gene expression patterns in CD4+ Treg and or T helper cells by either disrupting or creating new TF (transcription factor) binding sites in regulatory elements (enhancers) located in genetic susceptibility regions and this may combine with environmentally induced epigenetic change and alter chromatin accessibility. Current methods to identify the functional consequences and mechanisms of these changes are complex, time consuming and expensive as generally they can only examine one TF/binding site at a time, involve TF binding site prediction, which has a high degree of false positives/negatives and require large quantities of starting material making them challenging for application on limited clinical samples. To overcome these limitations, and to functionally annotate genetic risk of T1D, this study employs genome wide approaches including ATAC-seq and RNA-seq to compare the DNA accessibility and transcriptomes in CD4+ Treg and Th (Helper T)/Tconv (Conventional T) cells isolated from individuals with established T1D and sibling-matched healthy controls. By incorporating case-control ATAC-seq and TF footprints this study prioritises 111 and 96 T1D-associated SNPs in Treg and Tconv cells, respectively, that may play a role in mediating the disease susceptibility and subsequently contributing to the loss of tolerance in T1D. Using a bioinformatic pipeline to integrate case-control ATAC-seq differentially accessible peaks and RNA-seq differentially expressed genes with Hi-C 3D connectivity maps this study identifies 42 and 21 dysregulated gene targets in Treg and Tconv cells, respectively. Those targets include TIGIT, MAF and IL2 and the enhancers regulating those loci showed differential accessibility and are enriched for T1D SNPs and differential TF footprint signals. One theory to explain such observation is T1D SNPs and epigenetic alterations may alter or disrupt TF occupancy at these loci contributing to dysregulated target gene regulation. This study identifies changes in chromatin structure in T1D samples relative to healthy controls, enabling the identification of changes driven by both genetic and epigenetic variation that correlates with an altered transcriptional program in T1D. T1D associated SNPs at these regions can then be correlated with alterations in TF binding and putative epigenetically modified T1D regions can be validated in follow-up functional assays to demonstrate causality. This study captures chromatin and transcriptional changes between T1D and healthy individuals but it does not have the capability to distinguish if the changes are the driver or the consequence of the disease because the case cohort contains only established T1D from a single time point. In order to infer causality those changes would need to be tracked and validated over a timeline of disease progression in a longitudinal cohort. Nonetheless, this work provides a novel 3D genomic approach to functionally annotating the genetic risk and epigenetic changes that directly or indirectly result in altered gene expression, and promising preliminary data warranting further investigation on the causal functional role of the dysregulated gene targets in T1D.