Type 1 diabetes (T1D) results from progressive loss of pancreatic islet mass through autoimmunity targeted at a diverse, yet limited, series of molecules that are expressed in the pancreatic ␤ cell. Identification of these molecular targets provides insight into the pathogenic process, diagnostic assays, and potential therapeutic agents. Autoantigen candidates were identified from microarray expression profiling of human and rodent pancreas and islet cells and screened with radioimmunoprecipitation assays using newonset T1D and prediabetic sera. A high-ranking candidate, the zinc transporter ZnT8 (Slc30A8), was targeted by autoantibodies in 60 -80% of new-onset T1D compared with <2% of controls and <3% type 2 diabetic and in up to 30% of patients with other autoimmune disorders with a T1D association. ZnT8 antibodies (ZnTA) were found in 26% of T1D subjects classified as autoantibody-negative on the basis of existing markers [glutamate decarboxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), antibodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)]. Individuals followed from birth to T1D showed ZnT8A as early as 2 years of age and increasing levels and prevalence persisting to disease onset. ZnT8A generally emerged later than GADA and IAA in prediabetes, although not in a strict order. The combined measurement of ZnT8A, GADA, IA2A, and IAA raised autoimmunity detection rates to 98% at disease onset, a level that approaches that needed to detect prediabetes in a general pediatric population. The combination of bioinformatics and molecular engineering used here will potentially generate other diabetes autoimmunity markers and is also broadly applicable to other autoimmune disorders.autoantibody ͉ zinc transport ͉ prediabetes

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

Islet cell antibodies (ICAs) are predictive of type I diabetes in first-degree relatives, but this immunohistochemical assay has proven difficult to standardize. As an alternative, we assessed the use of radioassays for antibodies against three molecularly characterized islet autoantigens, including ICA512bdc (amino acid residues 256-979 of the IA-2 molecule, incorporating the intracellular domain). We measured insulin autoantibodies (IAAs), GAD autoantibodies (GAAs), and ICA512bdc autoantibodies (ICA512bdcAAs) by radioassay, in addition to ICAs, in 882 first-degree relatives of patients with type I diabetes, 50 of whom later developed diabetes with a median follow-up of 2.0 years (maximum 11.3 years). The cutoff for each radioassay was determined by testing >200 control subjects. When autoantibody frequencies among the relatives were analyzed according to relationship to the proband, the offspring of diabetic fathers had a higher frequency of ICA5I2bdcAAs (P = 0.008), IAAs (P = 0.0001) and GAAs (P = 0.0001) than the offspring of diabetic mothers. ICA512bdcAAs and IAAs both showed a significant association with HLA-DR4-DQ8 (P = 0.0005). Among relatives developing diabetes, 98% had one or more of IAAs, GAAs, or ICA512bdcAAs, and 80% had two or more of these autoantibodies, compared with none of the control subjects. Using survival analysis to allow for different lengths of follow-up, there was a significant increase in the risk of diabetes with the number of these autoantibodies present, comparing zero, one, two, and three autoantibodies (P < 0.0001, log-rank test), and by Cox regression analysis, this was independent of ICAs and age. For relatives with two or more of these autoantibodies, the risk of diabetes within 3 years was 39% (95% CI, 27-52) and the risk within 5 years was 68% (95% CI, 52-84). Relatives with all three autoantibodies had a risk within 5 years estimated to be 100%. The presence of low first-phase insulin release further increased the risk for relatives with one or two autoantibodies. We conclude that the presence of two or more autoantibodies (out of IAAs, GAAs, and ICA512bdcAAs) is highly predictive of the development of type I diabetes among relatives.

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16 -18 weeks of age (NOD mice IAA ؉ at 8 weeks: 83% (5͞6) diabetic by 18 weeks versus 11% (1͞9) of IAA negative at 8 weeks; P < .01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively ''fixed'' early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.insulin autoantibodies ͉ radioassay ͉ type 1 diabetes T ype 1A diabetes mellitus, as defined by an expert panel of the American Diabetes Association, is characterized by the presence of antiislet autoantibodies (1). There has been tremendous progress in defining islet autoantigens and developing antiislet autoantibody assays (2-4). In the most recent Immunology of Diabetes Workshop, a series of antiislet autoantibody assays were compared (2). Although the GAD65 autoantibody (GAA) and ICA512 (IA-2) autoantibody (ICA512AA) assays showed good concordance between laboratories, the insulin autoantibody (IAA) assays were divergent, with marked differences between laboratories in sensitivity and specificity. In this workshop, many of the IAA assays utilizing Ͻ600 l of sera had sensitivities Ϸ1͞2 of those utilizing a larger volume of serum (2). It is likely that such marked differences in interlaboratory measurement of IAA and the technical difficulty of current assays contributes to differences in the reported importance of IAA for disease prediction.Despite the difficulties of IAA determination, a number of laboratories have routinely measured IAA with large sera volume assays for large series of patients followed to the development of diabetes. Studies from multiple countries have reported that IAA has an important role in diabetes prediction (5-8). Antiinsulin autoantibodies appear to be unique in that their levels are ...

Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements.

Diabetes Autoimmunity Study in the Young (DAISY) has followed 1972 children for islet autoimmunity and diabetes: 837 first-degree relatives of persons with type 1 diabetes and 1135 general population newborns identified through human leukocyte antigen (HLA) screening. During follow-up of 4.06 yr (range, 0.17-9 yr), serial determination of autoantibodies to glutamic acid decarboxylase, protein tyrosine phosphatase IA2, and insulin has generated approximately 20,000 results. Among 162 children with at least one positive autoantibody, in 31% the test was false positive (autoantibodies were negative twice on blinded duplicate aliquots), in 31% it was transiently positive (confirmed on blinded duplicate aliquots but negative on follow-up), and in 36% it was persistently positive. Using proportional hazards modeling, the HLA-DR3/4 DQ8 genotype, another positive autoantibody at the first positive visit, and level of autoantibody were predictive of persistent positivity. Only HLA-DR3/4 DQ8 genotype was predictive of progression to diabetes in proportional hazards modeling. This prospective study reveals that cross-sectional determination of islet autoantibodies in a population with relatively low previous probability of autoimmunity identifies as "positive" a large number of individuals who are either false or transiently positive. Predictive value of autoantibodies increases with blinded duplicate and independent sample retesting and incorporation of the level of autoantibody in the predictive algorithm.

Twin studies have helped to define the importance of genetic and environmental factors in the aetiology of disease [1±9]. By measuring concordance rates (both twins affected) in identical (monozygotic) and nonidentical (dizygotic) twins, estimates of genetic influence can be obtained. In primarily genetic disorders, concordance rates should be higher in monozygotic than in dizygotic twins. Differences between monozygotic twins must be due to factors not coded in the germ line, i. e. non-germ-line genetic (e. g. somatic) or environmental factors. Monozygotic twins also show differences in genes that undergo random rearrangement such as immunoglobulin and T-cell receptor genes.Comprehensive population-based studies of twins have only recently been initiated, so most of the information available on diabetic twins is from clinic- AbstractAims. To determine the risk, hazard rate and factors affecting progression to diabetes in monozygotic twins of patients with Type I (insulin-dependent) diabetes mellitus. Methods. Prospective analysis was done of two cohorts of non-diabetic monozygotic twins of patients with Type I diabetes from Great Britain (n = 134) and the United States (n = 53). Results. The diabetes-free survival analysis was similar between both cohorts (p = 0.6). The combined survival analysis (n = 187, median follow-up = 17.7 years, range = 0.01±57) at 40 years of discordance estimated a 39 % probability of diabetes for the initially discordant twin. Survival analysis with left truncation of data estimated that probability to be 50 %. For twins who became concordant (n = 47), the median discordance time was 4.2 years (range 0.4 to 39), exceeding 15 years in 23.4 %. Twins of probands diagnosed at 24 years of age or younger had a 38 % probability of diabetes by 30 years of discordance, compared with 6 % for twins of probands diagnosed after 24 years of age (p = 0.004). The twins of probands diagnosed before 15 years of age had the highest diabetes hazard rate in the first discordance year, decreasing thereafter. By survival analysis, diabetes risk was higher in twins who were heterozygous for DR3-DQ2 and DR4-DQ8 than in twins with neither DR3-DQ2 nor DR4-DQ8 (p < 0.05). Conclusion/interpretation. Monozygotic twins of patients with Type I diabetes from two different countries had similar rates of progression to diabetes. Whereas most twins did not develop diabetes, 25 % of the twins who progressed did so after more than 14 years of discordance. An age-related heterogeneity was observed, with higher progression to diabetes for twins of patients diagnosed at a younger age. [Diabetologia (2001) 44: 354±362]

OBJECTIVEThere is limited information from large-scale prospective studies regarding the prediction of type 1 diabetes by specific types of pancreatic islet autoantibodies, either alone or in combination. Thus, we studied the extent to which specific autoantibodies are predictive of type 1 diabetes.RESEARCH DESIGN AND METHODSTwo cohorts were derived from the first screening for islet cell autoantibodies (ICAs) in the Diabetes Prevention Trial–Type 1 (DPT-1). Autoantibodies to GAD 65 (GAD65), insulinoma-associated antigen-2 (ICA512), and insulin (micro-IAA [mIAA]) were also measured. Participants were followed for the occurrence of type 1 diabetes. One cohort (Questionnaire) included those who did not enter the DPT-1 trials, but responded to questionnaires (n = 28,507, 2.4% ICA+). The other cohort (Trials) included DPT-1 participants (n = 528, 83.3% ICA+).RESULTSIn both cohorts autoantibody number was highly predictive of type 1 diabetes (P < 0.001). The Questionnaire cohort was used to assess prediction according to the type of autoantibody. As single autoantibodies, ICA (3.9%), GAD65 (4.4%), and ICA512 (4.6%) were similarly predictive of type 1 diabetes in proportional hazards models (P < 0.001 for all). However, no subjects with mIAA as single autoantibodies developed type 1 diabetes. As second autoantibodies, all except mIAA added significantly (P < 0.001) to the prediction of type 1 diabetes. Within the positive range, GAD65 and ICA autoantibody titers were predictive of type 1 diabetes.CONCLUSIONSThe data indicate that the number of autoantibodies is predictive of type 1 diabetes. However, mIAA is less predictive of type 1 diabetes than other autoantibodies. Autoantibody number, type of autoantibody, and autoantibody titer must be carefully considered in planning prevention trials for type 1 diabetes.