Identification of Novel Type 1 Diabetes Candidate Genes by Integrating Genome-Wide Association Data, Protein-Protein Interactions, and Human Pancreatic Islet Gene Expression

Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated genes (CD83, IFNGR1, IL17RD, TRAF3IP2, IL27RA, PLCG2, MYO1B, and CXCR7) in these networks also harbored single nucleotide polymorphisms nominally associated with type 1 diabetes. Finally, the expression and cytokine regulation of these new candidate genes were confirmed in insulin-secreting INS-1 β-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.


Supplementary
. Input genes from type 1 diabetes-associated regions identified by GWAS. The SNP (dbSNP) demonstrating the highest association in each type 1 diabetes associated region are listed together with its physical position on the chromosome. For each associated region the positional genes contained in the linkage disequilibrium interval are listed together with their physical location and a short gene description. *The interval defined by rs2292239 did in NCBI, build 36, contain 28 genes also contained in the subsequent interval defined by rs1678536. The identification of input genes were based on NCBI, build 36.

IL17D
Interleukin 17 D regulates cytokine production in endothelial cells and inhibits hemopoiesis in vitro. The protein encoded by this gene is a cytokine that shares sequence similarity with IL17. The treatment of endothelial cells with this cytokine has been shown to stimulate the production of other cytokines including IL6, IL8 and CSF2/ GM-CSF. The increased expression of IL8 induced by this cytokine was NF-kappa B-dependent (1)

IL17RE
Interleukin 17 receptor E, can activate the MAPK signaling pathway and seems to function at or upstream of Ras.

IL17RD
Interleukin 17 receptor D. IL-17 produced by Th17 cells is associated with inflammatory disease (2). IL17RD associates and co-localizes with IL-17R, the receptor for IL-17, and mediates IL-17 signaling (3). IL17RD also acts a tumor suppressor regulating nuclear Ras/ERK signaling pathway by inhibiting the nuclear translocation of activated ERK (4) . Il17RD is a feedback inhibitor of fibroblast growth factor mediated Ras-MAPK signaling and ERK activation. The nuclear ERK signaling pathway is regulated by spatially blocking nuclear translocation of activated ERK without inhibiting cytoplasmic phosphorylation of ERK. Mediates JNK activation and may be involved in apoptosis.

IFNGR1
Interferon gamma receptor type 1 is the ligand-binding chain (alpha) of the gamma interferon receptor. Human interferon-gamma receptor is a heterodimer of IFNGR1 and IFNGR2. Viruses induce type I interferon, subdivided into alpha-interferon, produced by leukocytes or lymphoblastoid cells, and beta-interferon, produced by fibroblasts.

TRAF3IP2
Tumor necrosis factor receptor-associated factor 3 (TRAF3) interacting protein 2 or Nuclear factor kappa-B activator 1. a protein involved in regulating responses to cytokines by members of the Rel/NF-kappaB transcription factor family. These factors play a central role in innate immunity in response to pathogens, inflammatory signals and stress. This gene product interacts with TRAF proteins and either I-kappaB kinase or MAP kinase to activate either NF-kappaB or Jun kinase. Could be involved in the activation of both NF-kappa-B via a NF-kappa-B inhibitor kinase (IKK)-dependent mechanism and stressactivated protein kinase (SAPK)/JNK.

CD83
Cluster of differentiation 83 antigen (activated B lymphocytes, immunoglobulin superfamily). Involved in antigen presentation and its expression important for CD4 + T-cell development in the thymus (4; 5)

CCR7
Chemokine (C-C motif) receptor 7, important for immune cell migration and a normal antigen-specific immune response (6; 7). CCR7-deficient mice are prone to development of multi-organ autoimmunity and show an increased susceptibility to streptozotocin-induced diabetes (8). Interestingly, although NOD mice deficient for CCR7 develop multi-organ autoimmunity, they have less islet infiltration and do not develop diabetes (9). Additionally, the expression of CCR7 is increased on CD8+ cells from recent onset T1D patients (10).

CXCL11
Chemokine (C-X-C motif) ligand 11, mediating chemotaxis through interaction with the chemokine receptor CXCR3. Chemotactic for interleukin-activated Tcells but not unstimulated T-cells, neutrophils or monocytes. Induces calcium release in activated T-cells. IFN-gamma is a potent inducer of transcription of this gene. May play an important role in CNS diseases which involve T-cell recruitment. May play a role in skin immune responses.

CXCL10
Chemokine (C-X-C motif) ligand 10, mediates chemotaxis through interaction with the chemokine receptor CXCR3. Binding to CXCR3 results in pleiotropic effects, including stimulation of monocytes, natural killer and T-cell migration, and modulation of adhesion molecule xpression. CXCL10 levels are elevated in serum and in pancreatic islets from T1D patients compared to healthy controls (11)(12)(13). Neutralization of CXCL10 prevents autoimmune diabetes in various experimental models (14)(15)(16) and transgenic mice expressing CXCL10 in the β-cells have insulitis and impaired β-cell function (17).

EBI3
Epstein-Barr virus-induced gene 3 protein, a subunit in 2 distinct heterodimeric cytokines: interleukin-27 (IL27) and IL35. IL27 can trigger signaling in T cells, B cells, and myeloid cells (18). IL35 is an inhibitory cytokine involved in regulatory T-cell function. a secreted glycoprotein belonging to the hematopoietin receptor family, and heterodimerizes with a 28 kDa protein to form interleukin 27 (IL-27). IL-27 regulates T cell and inflammatory responses, in part by activating the Jak/STAT pathway of CD4+ T cells.

CXCL9
Chemokine (C-X-C motif) ligand 9 mediating chemotaxis of activated T-cells through interaction with the chemokine receptor CXCR3. The function has not been specifically defined; however, it is thought to be involved in T cell trafficking. Affects the growth, movement, or activation state of cells that participate in immune and inflammatory response.

IL27RA
The Interleukin 27 receptor consists of the unique IL27RA chain and the common gp130 (IL6ST; 600694) chain, which is used by several other cytokines. IL27RA is widely expressed in the immune system, and IL27 trigger signaling in T cells, B cells, and myeloid cells (18).

PDCD1LG2
Programmed cell death 1 ligand 2 play an essential role in immune tolerance by dampening T-cell activation (19). Involved in the costimulatory signal, essential for T-cell proliferation and IFNG production in a PDCD1-independent manner. Interaction with PDCD1 inhibits T-cell proliferation by blocking cell cycle progression and cytokine production.

FLT3LG
FMS-related tyrosine kinase-3 ligand. Stimulates the proliferation of early hematopoietic cells. Synergizes with other colony stimulating factors and interleukins.

IL15
Interleukin-15 a cytokine that affects T-cell activation and proliferation similarly to IL2. IL15 use components of the IL2 receptor. Neutralization of IL15 attenuate diabetes in the MLD-STZ mouse model (20).

PLCG2
Phospholipase C, gamma 2A has a lipase-independent role in intracellular Ca 2+ entry in immune cells (21). In PLCG2 deficient NK cells, cytotoxicity was abrogated due to defective secretion of cytotoxic granules, which is dependent on the rise of Ca2+44. Plcg2-/-mice display decreased B-cell count and altered platelet, mast cell and NK-cell functions45.

MYO1B
Myosin, a molecular motor interacting with actin filaments and utilize energy from ATP hydrolysis to generate mechanical force, may participate in process critical to neuronal development and function such as cell migration, neurite outgrowth and vesicular transport.

CXCR7
Chemokine (C-X-C motif) receptor 7, proposed as important scavenger for CXCL12 (22) . a family of chemoattractant molecules involved in the directed migration of immune cells.

PANX2
Pannexin 2 modify voltage-activated outward currents mediated by Panx1. Structural component of gap junctions.Highest expression in brain.
Supplementary Figure 1. Protein-protein interaction networks. 395 positional candidate genes from linkage disequilibrium intervals of 44 non-HLA type 1 diabetes-associated regions identified from GWAS were used as input genes for construction of biological protein networks by use of the STRING software. Only networks containing proteins encoded by genes from at least two type 1 diabetes input regions are shown and used for further analysis. In total, seventeen networks were identified, numbered 1 to 17. Networks not enriched in cytokineregulated gene expression are shown. Name of proteins encoded by genes from input regions and their chromosomal position are in red.