Do post-translational beta cell protein modifications trigger type 1 diabetes?

Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading to the T cell attack against beta cells is presented. In this model, PTM plays a prominent role in triggering beta cell destruction. We discuss literature of relevance and perform genetic and human islet gene expression analyses. Both direct and circumstantial support for the involvement of PTM in type 1 diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1 diabetes as a classical autoimmune disease is open for debate.

Association analysis in type 1 diabetes of the PRSS16 gene encoding a thymus-specific serine protease.

We have previously mapped a separate type 1 diabetes (T1D) association in the extended MHC class I region, marked by D6S2223, on the DRB1*03-DQA1*0501-DQB1*0201 haplotype. The associated region encompasses a gene encoding a thymus-specific serine protease (PRSS16), presumably involved in positive selection of T cells or in T-cell regulation. Fourteen PRSS16 polymorphisms were genotyped in two steps using a total of six T1D family data sets, as well as case-control materials for both T1D and celiac disease (CD). An association with a 15 base-pair deletion in exon 12 of PRSS16 was found on the DRB1*03-DQA1*0501-DQB1*0201 haplotype for both T1D and CD, but it could not explain the more pronounced disease associations observed at marker D6S2223. We compared the performance of the 14 tested PRSS16 polymorphisms, selected after our previous comprehensive screen, against HapMap selected tag SNPs. Use of a HapMap based SNP selection strategy would result in loss of a large proportion of the genetic variation in PRSS16. Our data suggest that it is unlikely that polymorphisms within the PRSS16 gene are involved in the predisposition to T1D. However, we cannot rule out that regulatory polymorphisms located some distance away from the gene may be involved.

Type 1 diabetes: evidence for susceptibility loci from four genome-wide linkage scans in 1,435 multiplex families.

Type 1 diabetes is a common, multifactorial disease with strong familial clustering (genetic risk ratio [lambda(S)] approximately 15). Approximately 40% of the familial aggregation of type 1 diabetes can be attributed to allelic variation of HLA loci in the major histocompatibility complex on chromosome 6p21 (locus-specific lambda(S) approximately 3). Three other disease susceptibility loci have been clearly demonstrated based on their direct effect on risk, INS (chromosome 11p15, allelic odds ratio [OR] approximately 1.9), CTLA4 (chromosome 2q33, allelic OR approximately 1.2), and PTPN22 (chromosome 1p13, allelic OR approximately 1.7). However, a large proportion of type 1 diabetes clustering remains unexplained. We report here on a combined linkage analysis of four datasets, three previously published genome scans, and one new genome scan of 254 families, which were consolidated through an international consortium for type 1 diabetes genetic studies (www.t1dgc.org) and provided a total sample of 1,435 families with 1,636 affected sibpairs. In addition to the HLA region (nominal P = 2.0 x 10(-52)), nine non-HLA-linked regions showed some evidence of linkage to type 1 diabetes (nominal P < 0.01), including three at (or near) genome-wide significance (P < 0.05): 2q31-q33, 10p14-q11, and 16q22-q24. In addition, after taking into account the linkage at the 6p21 (HLA) region, there was evidence supporting linkage for the 6q21 region (empiric P < 10(-4)). More than 80% of the genome could be excluded as harboring type 1 diabetes susceptibility genes of modest effect (lambda(S) > or = 1.3) that could be detected by linkage. This study represents one of the largest linkage studies ever performed for any common disease. The results demonstrate some consistency emerging for the existence of susceptibility loci on chromosomes 2q31-q33, 6q21, 10p14-q11, and 16q22-q24 but diminished support for some previously reported locations.

Different islet protein expression profiles during spontaneous diabetes development vs. allograft rejection in BB-DP rats.

Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing beta-cells in the islets of Langerhans. When the beta-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets. Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared. In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations. In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.

The Type 1 Diabetes Genetics Consortium.

The Type 1 Diabetes Genetics Consortium (T1DGC) is an international, multicenter program organized to promote research to identify genes and their alleles that determine an individual's risk for type 1 diabetes (T1D). The primary goal of the T1DGC is to establish resources and data that can be used by, and that is fully accessible to, the research community in the study of T1D. All the information on T1DGC can be accessed at the following web address: http://www.t1dgc.org. A resource base of well-characterized families is being assembled that will facilitate the localization and characterization of T1D susceptibility genes. From these families, the T1DGC is establishing banks of DNA, serum, plasma, and cell lines, as well as useful databases. The T1DGC also sponsors training opportunities (bioinformatics) and technology transfer (HLA genotyping).

CBLB variants in type 1 diabetes and their genetic interaction with CTLA4.

Type 1 diabetes (T1D) is a multifactorial disease with genetic and environmental components involved. Recent studies of an animal model of T1D, the Komeda diabetes-prone rat, have demonstrated that the Casitas-B-lineage lymphoma b (cblb) gene is a major susceptibility gene in the development of diabetes and other autoimmune features of this rat. As a result of the inhibitory role of Cbl-b in T cell costimulation, dysregulation of Cbl-b may also contribute to autoimmune diseases in man. Different isoforms of Cbl-b exist; we evaluated expression levels of two known transcript variants. Constitutive expression of both isoforms was demonstrated, as well as an increased expression, after cytokine exposure, of an isoform lacking exon 16, suggesting a possible role of this variant in the pathogenesis of autoimmunity. We screened coding regions of the human CBLB gene for mutations in a panel of individuals affected with several autoimmune diseases. Eight single nucleotide polymorphisms (SNPs) were detected. One SNP in exon 12 of the CBLB gene was significantly demonstrated to be associated to T1D in a large Danish T1D family material of 480 families. Evidence for common genetic factors underlying several autoimmune diseases has come from studies of cytotoxic T lymphocyte antigen 4 (CTLA4), which encodes another negatively regulatory molecule in the immune system. Gene-gene interactions probably play substantial roles in T1D susceptibility. We performed stratification of CBLB exon 12 SNP data, according to an established CTLA4 marker, CT60, and evidence for a genetic interaction between the CTLA4 and CBLB genes, involved in the same biological pathway of T cell receptor signaling, was observed.

Increased risk of childhood type 1 diabetes in children born after 1985.

OBJECTIVE: The incidence rate of childhood type 1 diabetes is thought to be increasing; however, Danish studies have not confirmed this. Using a national diabetes register initiated in 1996 and two previous regional incidence studies, we studied the age-specific incidence of type 1 diabetes over 30 years. Here, we describe the incidence rates of type 1 diabetes in Danish children from 1996 to 2000 and evaluate trends in age-specific incidence rates from 1970 to 2000. RESEARCH DESIGN AND METHODS: A nationwide registration of all newly diagnosed cases of type 1 diabetes among children under the age of 15 years was established in Denmark in 1996. Incidence rates of type 1 diabetes in Denmark were obtained from this register. Age-specific incidence rates were compared with data collected from 1970 to 1976 and from 1980 to 1984, both population-based studies using existing national routine registration of hospitalizations within the survey areas. Population data were obtained from Statistics Denmark. RESULTS: During the study period, 1,421 Danish children developed type 1 diabetes before the age of 15 years. The incidence rates by age-groups were: 12.7, 19.4, and 26.3 for the 0-4, 5-9, and 10-14 years age-groups, respectively, and 19.5 for the 0-14 years age-group per 100,000 in the period 1996-2000. An age-period-cohort analysis showed a modest drift effect (yearly increase) of 1.2% (0.7-1.8) from 1970 to 2000, and a significant birth cohort effect with an increased risk for children born after 1985 was observed. CONCLUSIONS: The incidence rate of type 1 diabetes is rising in children living in Denmark. The steep increase in the youngest age-group was explained by the increased risk for cohorts born at the beginning of the 1980s.

No evidence of type 1 diabetes susceptibility genes in the region centromeric of the HLA complex.

There is strong evidence that DQB1, DQA1, and DRB1 alleles are not the only contributors to the human leukocyte antigen (HLA) linked type 1 diabetes (T1D) predisposition. Although the HLA complex is much studied for disease association, little is known about the neighboring centromeric region. We have previously found suggestive association on DQ2-DR3 haplotypes for marker D6S291, located 3.6-Mb centromeric of HLA-DQB1. This region on human chromosome 6 is syntenic to a part of the region adjacent to the mouse major histocompatibility complex (MHC) on chromosome 17, which has been suggested to harbor a susceptibility gene in mouse (Idd16). To evaluate a possible role of the region centromeric of HLA-DQB1 in human T1D, we have scanned the region with nine microsatellite markers in 267 T1D families from five different populations. Our results indicate that the characteristic strong linkage disequilibrium in the HLA complex does not extend into this region. Furthermore, we did not detect any consistent T1D association for the markers analyzed in the study. In conclusion, our data argue against the presence of any strong genetic susceptibility factors for T1D in the region centromeric of the HLA complex.

Prophylactic insulin treatment of syngeneically transplanted pre-diabetic BB-DP rats.

UNLABELLED: Type 1 Diabetes Mellitus is characterized by selective destruction of the pancreatic beta-cells in the islets of Langerhans and insulitis. Subcutaneous insulin injections given to diabetes prone BioBreeding (BB-DP) rats reduce diabetes incidence. The underlying mechanism(s) are not known in detail. Previously, we showed that transplantation of 200 syngeneic neonatal islets under the kidney capsule is useful for studying molecular events during diabetes development in BB-DP rats. In the present study we tested if prophylactic insulin treatment of syngeneically transplanted BB-DP rats would protect both islets in situ and transplanted islets from destruction. METHODS: BB-DP rats received transplants of 200 syngeneic neonatal islets under the kidney capsule at 30 days of age. They were given a subcutaneous insulin or placebo implant and were compared to control rats. Blood glucose was measured three times weekly. In total, 193 rats were transplanted and rats were sacrificed 7, 23, 50, 90 days post-transplantation or at onset of diabetes. Pancreatic and transplant sections were stained for insulin and mononuclear cell infiltration and insulitis was graded. RESULTS: Eight (19%) rats developed diabetes in the insulin-treated group and 19 (63%) and 19 (65%) rats in the control and placebo, respectively (p = 0.0002 and p = 0.0001). Onset of diabetes in the insulin treated group was delayed compared to control and placebo, (102, 77 and 81 days of age, respectively (p = 0.0001 and p = 0.0001)). Insulin treatment diminished mononuclear cell infiltration in the islets at day 50 after transplantation compared to placebo. Infiltration pattern in islets in situ correlates with infiltration in transplants (r is 0.9076 and p < 0.001). CONCLUSION/INTERPRETATION: These results suggest that insulin-treatment of syngeneically transplanted BB-DP rats considerably decreases the incidence of diabetes and that this model is well suited for studying molecular changes in the islet transplants.

Strain dependent rat iNOS promoter activity--correlation to identified WT1 transcription factor binding site.

The free radical nitric oxide (NO) has been implicated in cytokine mediated destruction of rat beta-cells in islets of Langerhans. Cytokine mediated NO production is associated with increased expression of the inducible nitric oxide synthase (iNOS). We have previously shown a strain dependent difference between Wistar Kyoto (WKY) and Brown Norway (BN) rats of IL-1beta mediated destruction of islets of Langerhans to be related to expression levels of iNOS and NO production. The aim of the present study was to clone and screen the iNOS gene promoter region from WKY and BN rats for polymorphisms and to functionally test such nucleotide differences. Within the total 2077 bp sequenced from both rat strains we identified three polymorphisms in two separate areas: (i) a GT-repeat polymorphism linked to (ii) a C/T polymorphisms, leading to a WT1 binding site approximately 1650bp upstream the BN iNOS promoter and (iii) a G/A SNP in exon 1. Apart from these polymorphisms the homology between all published rat iNOS sequences including the presently described are about 96%. Promoter activity was detected for both genes in a luciferase assay followed cloning of 2012 bp fragments and transient transfection into RIN cells. For both strains IL-1beta induced dose-dependent activity and strain dependent iNOS promoter activity was demonstrated when WT1 was co-expressed. To our knowledge, this is the first demonstration of functional WT1/iNOS promoter interaction. We conclude that the iNOS promoter is strain-dependently regulated which may relate to quantitatively as well as qualitatively strain dependent differences in transcription factor expression, in this study exemplified by WT1.

Is mortalin a candidate gene for T1DM ?

Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1beta mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1beta stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population.

Application of genomics and proteomics in Type 1 diabetes pathogenesis research.

Type 1 diabetes is a polygenic, multifactorial autoimmune disease characterized by selective and irreversible destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. An exogenous supply of insulin is required to sustain life after the onset of Type 1 diabetes. Despite decades of intensive research into its pathogenesis, no single gene or protein has been found to be responsible for Type 1 diabetes. This review will describe the use of large-scale genomics and proteomics in studying the pathogenesis of Type 1 diabetes, and will discuss future directions of research in the field.

Evidence of gene-gene interaction and age-at-diagnosis effects in type 1 diabetes.

The common genetic loci that independently influence the risk of type 1 diabetes have largely been determined. Their interactions with age-at-diagnosis of type 1 diabetes, sex, or the major susceptibility locus, HLA class II, remain mostly unexplored. A large collection of more than 14,866 type 1 diabetes samples (6,750 British diabetic individuals and 8,116 affected family samples of European descent) were genotyped at 38 confirmed type 1 diabetes-associated non-HLA regions and used to test for interaction of association with age-at-diagnosis, sex, and HLA class II genotypes using regression models. The alleles that confer susceptibility to type 1 diabetes at interleukin-2 (IL-2), IL2/4q27 (rs2069763) and renalase, FAD-dependent amine oxidase (RNLS)/10q23.31 (rs10509540), were associated with a lower age-at-diagnosis (P = 4.6 × 10⁻6 and 2.5 × 10⁻5, respectively). For both loci, individuals carrying the susceptible homozygous genotype were, on average, 7.2 months younger at diagnosis than those carrying the protective homozygous genotypes. In addition to protein tyrosine phosphatase nonreceptor type 22 (PTPN22), evidence of statistical interaction between HLA class II genotypes and rs3087243 at cytotoxic T-lymphocyte antigen 4 (CTLA4)/2q33.2 was obtained (P = 7.90 × 10⁻5). No evidence of differential risk by sex was obtained at any loci (P ≥ 0.01). Statistical interaction effects can be detected in type 1 diabetes although they provide a relatively small contribution to our understanding of the familial clustering of the disease.

Danish children born with glutamic acid decarboxylase-65 and islet antigen-2 autoantibodies at birth had an increased risk to develop type 1 diabetes.

OBJECTIVE: A large, population-based case-control cohort was used to test the hypothesis that glutamic acid decarboxylase-65 (GAD65) and islet antigen-2 autoantibodies (IA-2A) at birth predict type 1 diabetes. DESIGN AND METHODS: The design was an individually matched case-control study of all Danish type 1 diabetes patients born between 1981 and 2002 and diagnosed before May 1 2004 (median age at diagnosis was 8.8 years). Dried blood spot samples collected 5 days after birth in the 1981-2002 birth cohorts and stored at -25 °C were identified from 2023 patients and from two matched controls (n = 4042). Birth data and information on parental age and diabetes were obtained from Danish registers. GAD65A and IA-2A were determined in a radiobinding assay. HLA-DQB1 alleles were analyzed by PCR using time-resolved fluorescence. RESULTS: GAD65A and IA-2A were found in 70/2023 (3.5%) patients compared to 21/4042 (0.5%) controls resulting in a hazard ratio (HR) of 7.49 (P < 0.0001). The HR decreased to 4.55 but remained significant (P < 0.0003) after controlling for parental diabetes and HLA-DQB1 alleles. Conditional logistic regression analysis showed a HR of 2.55 (P < 0.0001) for every tenfold increase in the levels of GAD65A and IA-2A. This HR decreased to 1.93 but remained significant (P < 0.001) after controlling for parental diabetes and HLA-DQB1 alleles. CONCLUSION: These data suggest that GAD65A and IA-2A positivity at birth are associated with an increased risk of developing type 1 diabetes in Danish children diagnosed between 1981 and 2004.

Tests for genetic interactions in type 1 diabetes: linkage and stratification analyses of 4,422 affected sib-pairs.

OBJECTIVE: Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1 diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions. RESEARCH DESIGN AND METHODS: To address this issue, the Type 1 Diabetes Genetics Consortium recruited 3,892 families, including 4,422 affected sib-pairs. After genotyping 6,090 markers, linkage analyses of these families were performed, using a novel method and taking into account factors such as genotype at known susceptibility loci. RESULTS: Evidence for linkage was robust at the HLA and INS loci, with logarithm of odds (LOD) scores of 398.6 and 5.5, respectively. There was suggestive support for five other loci. Stratification by other risk factors (including HLA and age at diagnosis) identified one convincing region on chromosome 6q14 showing linkage in male subjects (corrected LOD = 4.49; replication P = 0.0002), a locus on chromosome 19q in HLA identical siblings (replication P = 0.006), and four other suggestive loci. CONCLUSIONS: This is the largest linkage study reported for any disease. Our data indicate there are no major type 1 diabetes subtypes definable by linkage analyses; susceptibility is caused by actions of HLA and an apparently random selection from a large number of modest-effect loci; and apart from HLA and INS, there is no important susceptibility factor discoverable by linkage methods.

Associations between features of glucose exposure and A1C: the A1C-Derived Average Glucose (ADAG) study.

OBJECTIVE: Various methods are used to quantify postprandial glycemia or glucose variability, but few have been compared and none are standardized. Our objective was to examine the relationship among common indexes of postprandial glycemia, overall hyperglycemia, glucose variability, and A1C using detailed glucose measures obtained during everyday life and to study which blood glucose values of the day provide the strongest prediction of A1C. RESEARCH DESIGN AND METHODS: In the A1C-Derived Average Glucose (ADAG) study, glucose levels were monitored in 507 participants (268 type 1 diabetic, 159 type 2 diabetic, and 80 nondiabetic subjects) with continuous glucose monitoring (CGM) and frequent self-monitoring of blood glucose (SMBG) during 16 weeks. We calculated several indexes of glycemia and analyzed their intercorrelations. The association between glucose measurements at different times of the day (pre- and postprandial) and A1C was examined using multiple linear regression. RESULTS: Indexes of glucose variability showed strong intercorrelation. Among postprandial indexes, the area under the glucose curve calculated from CGM 2 h after a meal correlated well with the 90-min SMBG postprandial measurements. Fasting blood glucose (FBG) levels were only moderately correlated with indexes of hyperglycemia and average or postprandial glucose levels. Indexes derived with SMBG strongly correlated with those from CGM. Some SMBG time points had a stronger association with A1C than others. Overall, preprandial glucose values had a stronger association with A1C than postprandial values for both diabetes types, particularly for type 2 diabetes. CONCLUSIONS: Indexes of glucose variability and average and postprandial glycemia intercorrelate strongly within each category. Variability indexes are weakly correlated with the other categories, indicating that these measures convey different information. FBG is not a clear indicator of general glycemia. Preprandial glucose values have a larger impact on A1C levels than postprandial values.

[Glycated haemoglobin may in future be reported as estimated mean blood glucose concentration--secondary publication]. / Glykeret haemoglobin kan i fremtiden rapporteres som estimeret middelblodglukosekoncentration--sekundaerpublikation.

Glycated haemoglobin (HbA 1c ) is widely used to determine levels of chronic glycaemia, to judge the adequacy of diabetes treatment and to adjust therapy. HbA 1c results are expressed as the percentage of HbA that is glycated. Day-to-day management is guided by self-monitoring of capillary glucose concentrations in mmol/l. The ADAG study determined the relationship between HbA 1c and average glucose concentration (AG) and concluded that for most patients with diabetes, HbA 1c can - with reasonable precision - be expressed as an estimated AG in the same units as self-monitoring.