Caroline Brorsson

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.

Identification of a SIRT1 mutation in a family with type 1 diabetes

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and beta-cell apoptosis

Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico “phenome–interactome analysis” on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β–induced NF-κB activity and protection against IL-1β–mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

CTSH regulates β-cell function and disease progression in newly diagnosed type 1 diabetes patients

Significance In type 1 diabetes (T1D), the insulin-producing pancreatic β-cells are destroyed by the immune system. Both genetic and environmental factors contribute to T1D risk. Candidate genes for T1D identified by genome-wide association studies have been proposed to act at both the immune system and the β-cell levels. This study shows that the risk variant rs3825932 in the candidate gene cathepsin H (CTSH) predicts β-cell function in both model systems and human T1D. Collectively, our data indicate that higher CTSH expression in β-cells may protect against immune-mediated damage and preserve β-cell function, thereby representing a possible therapeutic target. Our study reinforces the concept that candidate genes for T1D may affect disease progression by modulating survival and function of the β-cells. Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat β-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh−/− mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower β-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of β-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic β-cells, the target cells of the autoimmune assault.

Correlations between islet autoantibody specificity and the SLC30A8 genotype with HLA-DQB1 and metabolic control in new onset type 1 diabetes

We hypothesised that the correlation between autoantibody specificity for the ZnT8 Arg325Trp isoforms and the type 2 diabetes-associated rs13266634 may affect β-cell function at type 1 diabetes (T1D) onset. To study this, we tested 482 newly diagnosed diabetic probands and 478 healthy siblings from the Danish population-based T1D registry for autoantibodies to ZnT8 (ZnT8A) in addition to GAD65 and IA-2. The prevalence and titres of autoantibodies were correlated with genotypes for rs13266634 and HLA-DQB1, age at diagnosis (AAD) and insulin dose-adjusted HbA1c (IDAA1c), as a proxy for residual β-cell function. We replicated the correlation between rs13266634 genotypes and specificity for the ZnT8-Argenine (ZnT8R) and ZnT8-Tryptophan (ZnT8W) isoforms previously reported. ZnT8A overlapped substantially with autoantibodies to glutamate decarboxylase 65 (GADA) and IA-2 (IA-2A) and correlated significantly with IA-2A prevalence (p < 2e-16). No effect on IDAA1c was demonstrated for ZnT8A or rs13266634. We found a correlation between ZnT8R positivity and HLA-DQB1*0302 genotypes (p = 0.016), which has not been shown previously. Furthermore, significantly lower ZnT8R and GADA prevalence and titres was found among probands with AAD < 5 years (prevalence: p = 0.004 and p = 0.0001; titres: p = 0.002 and p = 0.001, respectively). The same trend was observed for IA-2A and ZnT8W; however, the difference was non-significant. Our study confirms ZnT8 as a major target for autoantibodies at disease onset in our Danish T1D cohort of children and adolescents, and we have further characterised the relationship between autoantibody specificity for the ZnT8 Arg325Trp epitopes and rs13266634 in relation to established autoantibodies, AAD, measures of β-cell function and HLA-DQB1 genotypes in T1D.

Effects of GWAS-Associated Genetic Variants on lncRNAs within IBD and T1D Candidate Loci

Long non-coding RNAs are a new class of non-coding RNAs that are at the crosshairs in many human diseases such as cancers, cardiovascular disorders, inflammatory and autoimmune disease like Inflammatory Bowel Disease (IBD) and Type 1 Diabetes (T1D). Nearly 90% of the phenotype-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) lie outside of the protein coding regions, and map to the non-coding intervals. However, the relationship between phenotype-associated loci and the non-coding regions including the long non-coding RNAs (lncRNAs) is poorly understood. Here, we systemically identified all annotated IBD and T1D loci-associated lncRNAs, and mapped nominally significant GWAS/ImmunoChip SNPs for IBD and T1D within these lncRNAs. Additionally, we identified tissue-specific cis-eQTLs, and strong linkage disequilibrium (LD) signals associated with these SNPs. We explored sequence and structure based attributes of these lncRNAs, and also predicted the structural effects of mapped SNPs within them. We also identified lncRNAs in IBD and T1D that are under recent positive selection. Our analysis identified putative lncRNA secondary structure-disruptive SNPs within and in close proximity (+/−5 kb flanking regions) of IBD and T1D loci-associated candidate genes, suggesting that these RNA conformation-altering polymorphisms might be associated with diseased-phenotype. Disruption of lncRNA secondary structure due to presence of GWAS SNPs provides valuable information that could be potentially useful for future structure-function studies on lncRNAs.

Residual β-Cell Function 3–6 Years After Onset of Type 1 Diabetes Reduces Risk of Severe Hypoglycemia in Children and Adolescents

OBJECTIVE To determine the prevalence of residual β-cell function (RBF) in children after 3–6 years of type 1 diabetes, and to examine the association between RBF and incidence of severe hypoglycemia, glycemic control, and insulin requirements. RESEARCH DESIGN AND METHODS A total of 342 children (173 boys) 4.8–18.9 years of age with type 1 diabetes for 3–6 years were included. RBF was assessed by testing meal-stimulated C-peptide concentrations. Information regarding severe hypoglycemia within the past year, current HbA1c, and daily insulin requirements was retrieved from the medical records and through patient interviews. RESULTS Ninety-two children (27%) had RBF >0.04 nmol/L. Patients with RBF <0.04 nmol/L were significantly more likely to have severe hypoglycemia than patients with RBF >0.04 nmol/L (odds ratio, 2.59; 95% CI, 1.10–7.08; P < 0.03). HbA1c was significantly higher in patients with RBF <0.04 nmol/L compared with patients with RBF >0.04 nmol/L (mean, 8.49 ± 0.08% [69.3 ± 0.9 mmol/mol] vs. 7.92 ± 0.13% [63.1 ± 1.4 mmol/mol]; P < 0.01), and insulin requirements were significantly lower in patients with RBF >0.2 nmol/L (mean ± SE: 1.07 ± 0.02 vs. 0.93 ± 0.07 units/kg/day; P < 0.04). CONCLUSIONS We demonstrated considerable phenotypic diversity in RBF among children after 3–6 years of type 1 diabetes. Children with RBF are at lower risk for severe hypoglycemia, have better diabetes regulation, and have lower insulin requirements compared with children without RBF. There appears to be a lower limit for stimulated RBF of ∼0.04 nmol/L that confers a beneficial effect on hypoglycemia and metabolic control.

Functional SOCS1 polymorphisms are associated with variation in obesity in whites

Aims/hypothesis:  The suppressor of cytokine signalling 1 (SOCS1) is a natural inhibitor of cytokine and insulin signalling pathways and may also play a role in obesity. In addition, SOCS1 is considered a candidate gene in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective was to perform mutation analysis of SOCS1 and to test the identified variations for association to T2D‐related quantitative traits, T2D or T1D.

The Type 1 Diabetes - HLA Susceptibility Interactome - Identification of HLA Genotype-Specific Disease Genes for Type 1 Diabetes

Background The individual contribution of genes in the HLA region to the risk of developing type 1 diabetes (T1D) is confounded by the high linkage disequilibrium (LD) in this region. Using a novel approach we have combined genetic association data with information on functional protein-protein interactions to elucidate risk independent of LD and to place the genetic association into a functional context. Methodology/Principal Findings Genetic association data from 2300 single nucleotide polymorphisms (SNPs) in the HLA region was analysed in 2200 T1D family trios divided into six risk groups based on HLA-DRB1 genotypes. The best SNP signal in each gene was mapped to proteins in a human protein interaction network and their significance of clustering in functional network modules was evaluated. The significant network modules identified through this approach differed between the six HLA risk groups, which could be divided into two groups based on carrying the DRB1*0301 or the DRB1*0401 allele. Proteins identified in networks specific for DRB1*0301 carriers were involved in stress response and inflammation whereas in DRB1*0401 carriers the proteins were involved in antigen processing and presentation. Conclusions/Significance In this study we were able to hypothesise functional differences between individuals with T1D carrying specific DRB1 alleles. The results point at candidate proteins involved in distinct cellular processes that could not only help the understanding of the pathogenesis of T1D, but also the distinction between individuals at different genetic risk for developing T1D.

Shared Genetic Basis for Type 1 Diabetes, Islet Autoantibodies, and Autoantibodies Associated With Other Immune-Mediated Diseases in Families With Type 1 Diabetes.

Type 1 diabetes (T1D) is a polygenic autoimmune disease that is often present with autoantibodies directed against pancreatic islet proteins. Many genetic susceptibility loci are shared with other autoimmune or immune-mediated diseases that also cosegregate in families with T1D. The aim of this study was to investigate whether susceptibility loci identified in genome-wide association studies (GWAS) of T1D were also associated with autoantibody positivity in individuals with diabetes. Fifty single nucleotide polymorphisms (SNPs) were genotyped in 6,556 multiethnic cases collected by the Type 1 Diabetes Genetics Consortium (T1DGC). These were tested for association with three islet autoantibodies—against autoantibodies to GAD (GADA), IA-2 (IA-2A), and zinc transporter 8 (ZnT8A)—and autoantibodies against thyroid peroxidase (TPOA) in autoimmune thyroid disease, gastric parietal cells (PCA) in autoimmune gastritis, transglutaminase (TGA) in celiac disease, and 21-hydroxylase (21-OHA) in autoimmune hypoadrenalism. In addition to the MHC region, we identify SNPs in five susceptibility loci (IFIH1, PTPN22, SH2B3, BACH2, and CTLA4) as significantly associated with more than one autoantibody at a false discovery rate less than 5%. IFIH1/2q24 demonstrated the most unrestricted association, as significant association was demonstrated for PCA, TPOA, GADA, 21-OHA, and IA-2A. In addition, 11 loci were significantly associated with a single autoantibody.