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.

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.

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.

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.

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.

Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes.

Type 1 diabetes (T1D) is a common autoimmune disorder that arises from the action of multiple genetic and environmental risk factors. We report the findings of a genome-wide association study of T1D, combined in a meta-analysis with two previously published studies. The total sample set included 7,514 cases and 9,045 reference samples. Forty-one distinct genomic locations provided evidence for association with T1D in the meta-analysis (P < 10(-6)). After excluding previously reported associations, we further tested 27 regions in an independent set of 4,267 cases, 4,463 controls and 2,319 affected sib-pair (ASP) families. Of these, 18 regions were replicated (P < 0.01; overall P < 5 × 10(-8)) and 4 additional regions provided nominal evidence of replication (P < 0.05). The many new candidate genes suggested by these results include IL10, IL19, IL20, GLIS3, CD69 and IL27.

Genome-wide scan for linkage to type 1 diabetes in 2,496 multiplex families from the Type 1 Diabetes Genetics Consortium.

OBJECTIVE: Type 1 diabetes arises from the actions of multiple genetic and environmental risk factors. Considerable success at identifying common genetic variants that contribute to type 1 diabetes risk has come from genetic association (primarily case-control) studies. However, such studies have limited power to detect genes containing multiple rare variants that contribute significantly to disease risk. RESEARCH DESIGN AND METHODS: The Type 1 Diabetes Genetics Consortium (T1DGC) has assembled a collection of 2,496 multiplex type 1 diabetic families from nine geographical regions containing 2,658 affected sib-pairs (ASPs). We describe the results of a genome-wide scan for linkage to type 1 diabetes in the T1DGC family collection. RESULTS: Significant evidence of linkage to type 1 diabetes was confirmed at the HLA region on chromosome 6p21.3 (logarithm of odds [LOD] = 213.2). There was further evidence of linkage to type 1 diabetes on 6q that could not be accounted for by the major linkage signal at the HLA class II loci on chromosome 6p21. Suggestive evidence of linkage (LOD > or =2.2) was observed near CTLA4 on chromosome 2q32.3 (LOD = 3.28) and near INS (LOD = 3.16) on chromosome 11p15.5. Some evidence for linkage was also detected at two regions on chromosome 19 (LOD = 2.84 and 2.54). CONCLUSIONS: Five non-HLA chromosome regions showed some evidence of linkage to type 1 diabetes. A number of previously proposed type 1 diabetes susceptibility loci, based on smaller ASP numbers, showed limited or no evidence of linkage to disease. Low-frequency susceptibility variants or clusters of loci with common alleles could contribute to the linkage signals observed.

A human type 1 diabetes susceptibility locus maps to chromosome 21q22.3.

OBJECTIVE: The Type 1 Diabetes Genetics Consortium (T1DGC) has assembled and genotyped a large collection of multiplex families for the purpose of mapping genomic regions linked to type 1 diabetes. In the current study, we tested for evidence of loci associated with type 1 diabetes utilizing genome-wide linkage scan data and family-based association methods. RESEARCH DESIGN AND METHODS: A total of 2,496 multiplex families with type 1 diabetes were genotyped with a panel of 6,090 single nucleotide polymorphisms (SNPs). Evidence of association to disease was evaluated by the pedigree disequilibrium test. Significant results were followed up by genotyping and analyses in two independent sets of samples: 2,214 parent-affected child trio families and a panel of 7,721 case and 9,679 control subjects. RESULTS- Three of the SNPs most strongly associated with type 1 diabetes localized to previously identified type 1 diabetes risk loci: INS, IFIH1, and KIAA0350. A fourth strongly associated SNP, rs876498 (P = 1.0 x 10(-4)), occurred in the sixth intron of the UBASH3A locus at chromosome 21q22.3. Support for this disease association was obtained in two additional independent sample sets: families with type 1 diabetes (odds ratio [OR] 1.06 [95% CI 1.00-1.11]; P = 0.023) and case and control subjects (1.14 [1.09-1.19]; P = 7.5 x 10(-8)). CONCLUSIONS: The T1DGC 6K SNP scan and follow-up studies reported here confirm previously reported type 1 diabetes associations at INS, IFIH1, and KIAA0350 and identify an additional disease association on chromosome 21q22.3 in the UBASH3A locus (OR 1.10 [95% CI 1.07-1.13]; P = 4.4 x 10(-12)). This gene and its flanking regions are now validated targets for further resequencing, genotyping, and functional studies in type 1 diabetes.

Treatment of Danish adolescent diabetic patients with CSII - a matched study to MDI.

OBJECTIVE: To compare two intensified insulin therapy regimens - continuous subcutaneous insulin infusion (CSII) against multiple daily insulin injection (MDI) - in Danish adolescents examined in a prospective, matched controlled study design. RESEARCH DESIGN AND METHODS: Thirty type 1 diabetic adolescents at CSII and 26 matched MDI controls were included in this open intention-to-treat study. Actrapid was used in both groups. Before study entry, all participants followed a brush-up course in order to minimize study effect. At each visit, the following parameters were recorded: hemoglobin A1c (HbA1c), insulin dose, weight, number of hypoglycemic and diabetic ketoacidosis (DKA) events, and the time resources used. At entry and exit of the study, diet registration and validated quality-of-life (QoL) questionnaires were filled by the participants. RESULTS: A non-significant decline in HbA1c was seen in both groups (p = 0.468); HbA1c decreased from 9.5 to 8.9% and from 9.7 to 9.5% in the CSII and MDI group, respectively. The insulin dose and the number of severe hypoglycemic events per patient were lower (non-significant) in the CSII group. Both groups showed increased body mass index - highest in the CSII group - and mild to moderate DKA episodes were only seen among CSII users. No differences could be demonstrated within the QoL or diet registrations. CONCLUSIONS: CSII treatment is beneficial as an intensified insulin therapy for selected type 1 diabetic patients and both MDI and CSII can be offered by the professional diabetes team to better tailor therapy. In future, there is a strong need to identify the characteristics of responders to CSII treatment in order to increase the efficacy and safety of CSII treatment.

Posttranslational Protein Modifications in Type 1 Diabetes - Genetic Studies with PCMT1, the Repair Enzyme Protein Isoaspartate Methyltransferase (PIMT) Encoding Gene.

BACKGROUND: Posttranslational protein modifications have been implicated in the development of autoimmunity. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) repairs modified proteins and is encoded by PCMT1, located in a region linked to type 1 diabetes (T1D), namely IDDM5. AIM: To evaluate the association between genetic variations in the PCMT1 gene and T1D. METHODS: Firstly, PCMT1 was sequenced in 26 patients with T1D (linked to IDDM5) and 10 control subjects. The variations found in PCMT1 were then tested (alone and interacting with a functional polymorphism in SUMO4 and with HLA) for association with T1D in 253 families (using transmission disequilibrium test). In a third step, the association of the functional variation in PCMT1 (rs4816) with T1D was analyzed in 778 T1D patients and 749 controls (using chi-square test). In vitro promoter activity was assessed by transfecting INS-1E cells with PCMT1 promoter constructs and a reporter gene, with or without cytokine stimulation. RESULTS: Four polymorphisms in complete linkage disequilibrium were identified in PCMT1 (5' to the gene (rs11155676), exon 5 (rs4816) and exon 8 (rs7818 and rs4552)). In the whole cohort of 253 families, the allele associated with increased PIMT enzyme activity (rs4816, allele A) was less frequently transmitted to the affected than to the non-affected offspring (46% vs. 53%, p = 0.099). This finding was even more evident in the subset of families where the proband had high-risk SUMO4 (p = 0.069) or low-risk HLA (p = 0.086). Surprisingly, in the case-control study with 778 cases and 749 controls, an inverse trend was found (40.36% of patients and 36.98% of controls had the allele, p = 0.055). PCMT1 promoter activity increased with cytokine stimulation, but no differences were detected between the constructs adjacent to rs11155676. CONCLUSION: PCMT1 was virtually associated with T1D in groups defined by other risk genes (SUMO4 and HLA). A general association in a not further defined sample of T1D patients was not evident. Verification in a larger population is needed.

Integrative analysis for finding genes and networks involved in diabetes and other complex diseases.

We have developed an integrative analysis method combining genetic interactions, identified using type 1 diabetes genome scan data, and a high-confidence human protein interaction network. Resulting networks were ranked by the significance of the enrichment of proteins from interacting regions. We identified a number of new protein network modules and novel candidate genes/proteins for type 1 diabetes. We propose this type of integrative analysis as a general method for the elucidation of genes and networks involved in diabetes and other complex diseases.

Type 1 diabetes risk analysis on dried blood spot samples from population-based newborns: design and feasibility of an unselected case-control study.

Development of type 1 diabetes mellitus (T1D) may be triggered pre- or perinatally by multiple factors. Identifying new predisposing T1D markers or combinations of markers in a large, well-characterised case-control collection may be important for future T1D prevention. The present work describes the design and feasibility of a large and unselected case-control study, which will define and evaluate prediction criteria for T1D at the time of birth. Danish registries (Biological Specimen Bank for Neonatal Screening, and the National Discharge Registry) made it possible to identify and collect dried blood spots (DBS) from newborns who later developed T1D (cases) born 1981-2002. DBS samples from 2086 cases and two matching control subjects per case were analysed for genetic and immune factors that are associated with T1D: (a) candidate genes (HLA, INS and CTLA4), (b) cytokines and inflammatory markers, (c) islet auto-antibodies (GAD65A, IA-2A). The objective of the study was to define reliable prediction tools for T1D using samples available at the time of birth. In a unique approach, the study linked a large unselected and population-based sample resource to well-ascertained clinical databases and advanced technology. It combined genetic, immunological and demographic data to develop prediction algorithms. It also provided a resource for future studies in which new genetic markers can be included as they are identified.

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.

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).

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.

Immune-mediated beta-cell destruction in vitro and in vivo-A pivotal role for galectin-3.

Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets and insulinoma cells confirmed IL-1beta regulated gal-3 expression of several gal-3 isoforms and a complex in vivo expression profile during diabetes development in rats. Over-expression of gal-3 protected beta-cells against IL-1beta toxicity, with a complete blockage of JNK phosphorylation, essential for IL-1-mediated apoptosis. Mutation scanning of regulatory and coding regions of the gal-3 gene (LGALS3) identified six polymorphisms. A haplotype comprising three cSNPs showed significantly increased transmission to unaffected offspring in 257 T1D families and replicated in an independent set of 170 T1D families. In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies.