HLA-DR-DQ haplotypes and specificity of the initial autoantibody in islet specific autoimmunity.

OBJECTIVE: We aimed to clarify the association of various HLA risk alleles with different types of autoantibodies initiating islet specific autoimmunity. METHODS: Follow-up cohorts from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and children diagnosed with type 1 diabetes (T1D) from the Finnish Pediatric Diabetes Register (FPDR) were analyzed for the presence of autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), IA-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A); and genotyped for HLA DR/DQ alleles. In the DIPP study, autoantibodies were regularly analyzed from birth up to 15 years of age. RESULTS: In the DIPP cohort, 621 children developed one single persistent autoantibody, GADA in 284, IAA in 268, and IA-2A in 40 cases. Highly significant differences in the specificity of the first autoantibody were observed between HLA genotypes. Homozygotes for the DR3-DQ2 haplotype had almost exclusively GADA as the first autoantibody, whereas a more even distribution between GADA and IAA was found in DR3-DQ2/DR4-DQ8 as well as DR3-DQ/x and DR4-DQ8/x genotypes (x referring to neutral haplotypes). In DR4-DQ8 positive genotypes with the DRB1*04:01 allele IAA was more often the first autoantibody than in DRB1*04:04 positive genotypes. Various neutral haplotypes also significantly affected the relative proportions of different initial autoantibodies. These findings were confirmed and expanded in a series of 1591 T1D children under the age of 10 years from FPDR. CONCLUSIONS: These results emphasize the importance of HLA class II polymorphisms in the recognition of autoantigen epitopes in the initiation of various pathways of the autoimmune response.

'25-Hydroxyvitamin D, Autoantigenic and Total Antibody Concentrations: Results from a Danish Case-control Study of Newly Diagnosed Patients with Childhood Type 1 Diabetes and their Healthy Siblings'.

B cells have recently entered the stage as an important accessory player in type 1 diabetes (T1D) etiopathogenesis. Experimental studies suggest regulatory functions of vitamin D on B cells. However, only a few human studies, with considerable methodological limitations, have been conducted within this field. Our objective was to investigate whether higher 25-hydroxyvitamin D (25(OH)D) concentrations were inversely associated with ß-cell autoantigens glutamic acid decarboxylase (isoform 65) (GADA) and insulinoma-associated antigen-2A (IA-2A). Further, we also wanted to examine the relationship between 25(OH)D and total antibody concentrations. We randomly selected 500 patients with newly diagnosed T1D and 500 siblings for 25(OH)D, antibody and genetic analysis from the population-based Danish Registry of Childhood and Adolescent Diabetes. The relative change (RC) in the mean concentration of GADA, IA-2A and antibody isotypes by a 10 nmol/l increase in 25(OH)D concentration was modelled by a robust log-normal regression model. We found no association between 25(OH)D and GADA [adjusted RC per 10 nmol/l increase: 1.00; 95% confidence interval (CI): 0.98-1.02] and IA-2A [adjusted RC per 10 nmol/l increase: 0.92; CI: 0.76-1.12]. Further, 25(OH)D was not associated with the total concentration of antibody isotypes [immunoglobulin (Ig)A, IgE, IgG and IgM]. All null findings were unaltered after adjustment for genetic variation in the vitamin D pathway. Physiological concentrations of 25(OH)D are unlikely to have a clinically important effect on antibody concentrations in a paediatric population of newly diagnosed patients with T1D and their healthy siblings.

Biomarkers related to severe hypoglycaemia and lack of good glycaemic control in ACCORD.

AIMS/HYPOTHESIS: In patients with diabetes, intensive glycaemic control reduces microvascular complications. However, severe hypoglycaemia frequently complicates intensive glycaemic control. Blood biomarkers that predict successful intensification of glycaemic control in patients with type 2 diabetes without the development of severe hypoglycaemia would advance patient care. In patients who received intensive treatment for type 2 diabetes from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, we hypothesised that insulin deficiency and islet autoantibodies would be associated with severe hypoglycaemia and failure to achieve near-normal glycaemia (HbA1c <6.0% [42 mmol/mol]). METHODS: A nested case-control design was used. Cases (n = 326) were defined as participants having severe hypoglycaemia and failure to achieve an HbA1c level of <6.0% (42 mmol/mol) prior to the ACCORD transition or death. Controls (n = 1,075) were those who achieved an HbA1c level of <6.0% (42 mmol/mol) prior to the ACCORD transition or death without severe hypoglycaemia. Each case was matched (for race, age and BMI) by up to four controls. Baseline insulin deficiency (fasting C-peptide ≤0.15 nmol/l) and islet autoantibodies (glutamic acid decarboxylase [GAD], tyrosine phosphatase-related islet antigen 2 [IA2], insulin [IAA] and zinc transporter 8 [ZnT8]) were measured. Conditional logistic regression with and without adjustment for age, BMI and diabetes duration was used on the full cohort and after removal of patients who died and their respective controls. RESULTS: Severe hypoglycaemia accompanied by an inability to achieve an HbA1c level of <6.0% (42 mmol/mol) was associated with insulin deficiency (adjusted OR 23.2 [95% CI 9.0, 59.5], p < 0.0001), the presence of IAA autoantibodies or baseline insulin use (adjusted OR 3.8 [95% CI 2.7, 5.3], p < 0.0001), GAD autoantibodies (OR 3.9 [95% CI 2.5, 6.0], p < 0.0001), IA2 autoantibodies (OR 16.7 [95% CI 3.9, 71.6], p = 0.0001) and ZnT8 autoantibodies (adjusted OR 3.9 [95% CI 1.2, 12.4], p = 0.02). CONCLUSIONS: C-peptide and islet autoantibodies may serve as biomarkers to predict the risk of severe hypoglycaemia during intensification of type 2 diabetes treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000620 (original ACCORD study).

[Obesity and risk factors for metabolic syndrome in young people with type 1 diabetes]. / Obesidad y factores de riesgo del síndrome metabólico en jóvenes con diabetes tipo 1.

Changes in the clinical presentation of diabetes mellitus in childhood and adolescence associated with obesity have resulted in an overlap of the two most common types of diabetes with a greater clinical heterogeneity. In order to characterize the type of diabetes at onset and assess the effect of obesity, 50 children with diabetes were studied. The patients were divided into two groups according to their nutritional status at diagnosis (over-weight/obese vs. normal weight). Insulin reserve was evaluated by measuring basal C-peptide and stimulated C-peptide in response to a mixed meal (MMTT) as well as HLA-DQB1 genotype, antibodies, and family history of risk factors for metabolic disease. Of all 50 patients, 38% was overweight/obese, 84% had a positive family history of metabolic syndrome, 82% had positive antibodies, and 100% were positive for the high-risk HLA-DQB1 genotype. No significant differences were found in fasting C-peptide or glycemic index/C-peptide levels between the two groups. In the overweight/obese group C-peptide response to MMTT showed higher levels at 60 and 120 minutes (p = 0.02 and 0.03) and the area under the curve for C-peptide was also higher (1.77 ng / ml vs. 5.5 ng/ ml, p = 0.0007) than in the normal-weight group. In conclusion, overweight/obese patients with type 1A diabetes had a greater pancreatic reserve, suggesting that nutritional status may accelerate disease onset.

Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of ß-cell autoimmunity.

BACKGROUND: The aim of this study was to create a new research strategy to obtain high-quality pancreatic tissues from subjects with preclinical or clinical type 1 diabetes, which would open up new avenues for studying the mechanisms of the ß-cell damaging process in humans. RESEARCH DESIGN AND METHODS: A nationwide collaboration network (the PanFin network) was established in Finland to start an on-call screening of diabetes-associated autoantibodies from deceased organ donors and subsequent processing of pancreases from autoantibody-positive donors. This protocol was integrated into the national organ transplantation procedure. RESULTS: Only a few modifications were needed to the normal transplantation practices. One additional blood sample was obtained from donors for autoantibody analyses, the transplantation team was informed about the autoantibody result and the pancreas of autoantibody-positive donors was transported to the core laboratory. Altogether, 307 donors were screened and 22 (7.2%) were positive for at least one autoantibody and 3 tested positive for two or more autoantibodies out of the five tested (islet cell antibodies, insulin autoantibodies and autoantibodies to glutamic acid decarboxylase, islet antigen 2 and zinc transporter 8). The quality of collected pancreatic tissue was superior to that from autopsies and allowed the detection of both RNA and proteins. CONCLUSIONS: The study protocol was proven feasible to be carried out on a nationwide scale. It did not interfere with the normal transplantation activities and provided valuable tissue material for research.

The diagnostic value of zinc transporter 8 autoantibody (ZnT8A) for type 1 diabetes in Chinese.

BACKGROUND: Zinc transporter-8 (ZnT8) was recently identified as a novel autoantigen in human type 1 diabetes (T1D). Autoantibody to ZnT8 (ZnT8A) was detected in up to 80% of patients with new-onset T1D and 26% of patients with T1D otherwise classified as negative on the basis of existing markers. As no data of ZnT8A in Chinese have been reported, we aim to evaluate the utility of ZnT8A for diagnosis of autoimmune T1D in Chinese relative to other autoantibody markers. METHODS: Radioligand binding assays were performed on 539 T1D sera using human ZnT8 carboxyterminal 325Arg construct or a dimer incorporating 325Arg and 325Trp alongside antibodies to glutamic acid decarboxylase (GADA) or insulinoma-associated protein 2 (IA-2A). The antigenic specificity was analysed in the context of clinical characteristics of the patients. RESULTS: ZnT8A were present in 24.1% (130 of 539) of patients with T1D versus 1.8% (10 of 555; P < 0.001) in type 2 diabetes. At diagnosis, ZnT8A and IA-2A were less prevalent in Chinese subjects with T1D than in Caucasian populations (both P < 0.001) but similar to Japanese. The diagnostic sensitivity of combined GADA, IA-2A and ZnT8A measurements reached 65.5% with ZnT8A detected in 13.5% (29 of 215) of GADA and/or IA-2A-negative subjects. ZnT8A prevalence was lower in older and fatter patients. ZnT8A+ alone patients were distinguished from Ab- ones (P < 0.05-0.001) on the basis of higher insulin requirement and lower systolic blood pressure level. CONCLUSION: ZnT8A is an independent marker for T1D in Chinese and combined with GADA and IA-2A enhances diagnostic sensitivity. ZnT8A may be associated with different clinical phenotypes than GADA or IA-2A.

3 Screen islet cell autoantibody ELISA: A sensitive and specific ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8.

3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8, has been developed and evaluated. In the assay serum samples were incubated (overnight; 2-8°C) in ELISA plate wells coated with GAD65, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD65, IA-2 and ZnT8 (1h; 2-8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine. Samples tested in the 3 Screen were also analysed in ELISAs and radiobinding assays for the three individual autoantibodies. 129/132 (98%) samples from newly diagnosed T1DM children and 1/100 non-diabetic children controls were positive in 3 Screen. There was good agreement between 3 Screen and the individual autoantibody assays. Dilution of positive samples showed good linearity characteristics. In the 2015 Islet Autoantibody Standardization Program 3 Screen achieved 94% sensitivity, 95.6% specificity and 0.948 area under curve by ROC analysis. 3 Screen provides a simple and sensitive method for combined measurement of three major diabetes associated autoantibodies in a single sample. The assay should be a useful tool for large scale population screening for individuals at risk of developing T1DM.

Baseline heterogeneity in glucose metabolism marks the risk for type 1 diabetes and complicates secondary prevention.

AIMS: Non-diabetic children with multiple islet autoantibodies were recruited to a secondary prevention trial. The objective was to determine the predictive value of baseline (1) HbA1c and metabolic variables derived from intravenous (IvGTT) and oral glucose tolerance tests (OGTT), (2) insulin resistance and (3) number, type and levels of islet autoantibodies, for progression to type 1 diabetes. METHODS: Children [n = 50, median 5.1 (4-17.9) years] with autoantibodies to glutamate decarboxylase (GAD65A) and at least one of insulinoma-associated protein 2 (IA-2A), insulin or ZnT8 transporter (ZnT8RA, ZnT8WA, ZnT8QA) were screened with IvGTT and OGTT and followed for a minimum of 2 years. RESULTS: Baseline first phase insulin response (sum of serum-insulin at 1 and 3 min during IvGTT; FPIR) ≤3 µU/mL [HR 4.42 (CI 1.40-14.0) p = 0.011] and maximal plasma glucose ≥11.1 mmol/L measured at 30, 60 and/or 90 min during OGTT [HR 6.13 (CI 1.79-21.0) p = 0.0039] were predictors for progression to diabetes. The combination of FPIR from IvGTT and maximal plasma glucose during OGTT predicted diabetes in 10/12 children [HR 9.17 (CI 2.0-42.0) p = 0.0043]. High-level IA-2A, but not number of autoantibodies, correlated to dysglycemia during OGTT (p = 0.008) and to progression to type 1 diabetes [HR 4.98 (CI 1.09-22.0) p = 0.039]. CONCLUSIONS: Baseline FPIR, maximal plasma glucose ≥11.1 at 30, 60 or 90 min during OGTT and high-level IA-2A need to be taken into account when randomizing islet autoantibody positive non-diabetic children to secondary prevention.

Prevalence of ZnT8 antibody in relation to phenotype and SLC30A8 polymorphism in adult autoimmune diabetes: results from the HUNT study, Norway.

Zinc transporter 8 (ZnT8), a product of the SLC30A8 gene, is a target-antigen in autoimmune diabetes. Associations between ZnT8 antibody (ZnT8A), phenotype and the genetic variant rs13266634 in the SLC30A8 gene have primarily been studied in patients with young-onset diabetes. We explored such associations in adult-onset autoimmune diabetes identified from the all-population based Nord-Trøndelag health Study (HUNT) ZnT8A (assayed by a fusion probe of C-terminal Arg325 and Trp325), and antibodies against glutamic decarboxylase (GADA) and tyrosine phosphatase-like protein insulinoma antigen-2 (IA-2A) were analysed in 266 subjects classified as having adult-onset autoimmune diabetes ( ≥ 25 years of age at diagnosis). Of these, 161 subjects fulfilled the criteria of latent autoimmune diabetes in adults (LADA), whereas 105 subjects were termed "classical" type 1 diabetes. Ten out of 161 LADA (6.2%) and 23 out of 105 adult-onset "classical" type 1 diabetic patients (22%) were ZnT8A positive. Adult-onset diabetic subjects positive both for GADA and IA-2A (n = 17), had lower waist circumference (p = 0.024) and higher fasting glucose levels (p = 0.023) than those positive both for GADA and ZnT8A (n = 13). Genotyping results of rs13266634 (available in 178 adult-onset diabetic subjects), showed a tendency for association between ZnT8A positivity and the TT- and CC genotypes of SNP rs13266634 (p = 0.101) using the standard cut-off level of 0.06ai, and a significant association at a lower cut-off level of 0.01ai (p = 0.005). We conclude that ZnT8A positivity in a population of adult-onset autoimmune diabetes is a less strong marker of autoimmunity than IA-2A. Further, positivity could be influenced by polymorphism of the SLC30A8 gene.

Seroconversion to islet autoantibodies between early pregnancy and delivery in non-diabetic mothers.

Islet autoantibodies are currently used to classify type 1 diabetes at diagnosis as they reflect the autoimmune pathogenesis of the disease. The presence of maternal autoantibodies reactive with pancreatic islet antigens is thought to increase the risk for type 1 diabetes in the offspring. The objective of this study was to determine seroconversion to islet autoantibodies in non-diabetic mothers during pregnancy. Screening of 33,682 mothers between September 2000 and August 2004 in the Diabetes Prediction in Skåne (DiPiS) study showed that at delivery, 242 non-diabetic mothers had increased titers of islet autoantibodies reactive with glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or insulin (IAA), alone or in combination. Control mothers (n=1419), who were islet autoantibody negative at delivery, were randomly selected and matched by age, parity and pregnancy sampling date. Mothers positive for GADA (92%), IA-2A (84%) or IAA (65%) at delivery had increased titers already evident in early pregnancy. Titers declined for GADA (p<0.0001), IA-2A (p<0.0001) and IAA (p<0.0001). Seroconversion during pregnancy was observed for GADA in 10 (8%), IA-2A in 3 (16%) and IAA in 37 (35%) mothers. It is concluded that non-diabetic mothers with islet autoantibodies at delivery had significantly higher titers during early pregnancy than at delivery. As the statistical power in the seroconverting mothers was insufficient, further studies are needed to determine if the risk for type 1 diabetes in the offspring differs between mothers who already had increased titers of islet autoantibodies during early pregnancy or acquired them during pregnancy.

Islet autoantibody seroconversion in the DPT-1 study: justification for repeat screening throughout childhood.

OBJECTIVE: Although type 1 diabetes autoimmunity frequently begins in childhood, little is known about the relationship between age and autoimmunity development. Our aim was to determine the timing of seroconversion to diabetes-associated autoantibody (DAA) positivity and risk in first- and second-degree relatives of patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Study subjects were identified through the Diabetes Prevention Trial-Type 1 (DPT-1). Children 3-18 years of age (n = 42,447) were screened for DAAs; 1,454 were ICA positive (≥ 10 JDF units), 1,758 were GAD65 positive, and 899 were ICA512 positive at the time of initial screening. Subjects who were initially antibody negative (n = 39,212) were recalled for rescreening, and 11,813 returned for rescreening. RESULTS: DAA seroconversion occurred in 469 (4%) children; 258 seroconverted to ICA, 234 to GAD65, and 99 to ICA512. The median time to seroconversion was 2 years. The 2-year risk for DAAs was highest in early childhood. For each 1-year increase in age in this cohort, the risk of any autoantibody seroconversion (HR 0.95, 95% CI 0.92-0.97) decreased by 5%, and for any two autoantibodies risk decreased by 13% (0.87, 0.82-0.93). CONCLUSIONS: Risk of autoantibody seroconversion among children followed in DPT-1 is age dependent. Younger children have the highest risk for DAAs, with the majority of children seroconverting by 13 years of age (75%). This suggests that annual screenings should be started in early childhood and continued through early adolescence to identify the majority of subjects at risk for type 1 diabetes and eligible for prevention trials.

Prediction of type 1 diabetes among siblings of affected children and in the general population.

AIMS/HYPOTHESIS: To compare the predictive characteristics of autoantibodies to GAD (GADA) and islet antigen 2 (IA-2A) for type 1 diabetes between siblings of affected children and children from the general population. METHODS: Seven-hundred and fifty-five siblings and 3,475 population-derived children were screened for GADA and IA-2A and observed for type 1 diabetes for 15 years. Sensitivity and cumulative disease risks from GADA, IA-2A and double positivity were compared between the cohorts. RESULTS: Fifty-six siblings (7.4%) tested positive for GADA, 39 (5.2%) for IA-2A and 29 (3.8%) for both autoantibodies. Thirty-four population derived participants (1.0%) had GADA, 22 (0.6%) had IA-2A and 7 (0.2%) had double positivity. Fifty-one siblings (6.8%) and 15 participants in the population cohort (0.4%) progressed to type 1 diabetes. The predictive sensitivity of GADA was 68% (95% CI 53-81%) among siblings and 50% (95% CI 23-77%) in the general population, while the corresponding values were 58 (95% CI 43-72%) and 43% (95% CI 18-71%) for IA-2A. Double-autoantibody positivity had a sensitivity of 48% (95% CI 34-63%) among siblings and 36% (95% CI 13-65%) in the population cohort. Cumulative disease risks from GADA, IA-2A and double positivity were, respectively, 61% (95% CI 48-74%), 74% (95% CI 61-88%) and 83% (95% CI 69-97%) among siblings compared with those of 24% (95% CI 9-38%), 32% (95% CI 12-51%) and 86% (95% CI 60-100%) in the general population. CONCLUSIONS/INTERPRETATION: There were no significant differences in the disease-predictive sensitivity of GADA and IA-2A positivity or their combination between siblings and the population cohort, whereas, for each antibody, positivity was associated with a higher cumulative disease risk among siblings. Double-antibody positivity conferred similar cumulative disease risk both among siblings and in the general population.