Diagnostic Value Of Zinc Transporter 8 Autoantibody In Type 1 Diabetes In China

Part 1 Establishment of the Radioligand Assay for Autoantibodies to Zinc Transporter 8Objective:The aim of this part is to establish a radioligand binding assay (RLA) for measuring autoantibodies to zinc transporter 8 (ZnT8A). And to explore its preliminary diagnostic role in type 1 diabetes in China.Methods:35S labeling recombinant human ZnT8 antigen was obtained by in vitro transcription and translation, and then mixed with serum samples in self-made rotating incubator overnight. The ZnT8 immunocomplex was precipitated with protein A-agarose, and then washed with TBST buffer and counted in CPM using liquid scintillation and luminescence counters. The results were expressed by ZnT8A index. The sensitivity and specificity of the assay were evaluated in the 6th Diabetes Autoantibody Standardization Program (DASP 2009) sponsored by the Immunology of Diabetes Society (IDS) and Centers for Disease Control and Prevention (CDC). And ZnT8A was screened in Chinese diabetes patients(type 1, n=429; type 2, n=555) and healthy controls(n=405).Results:①The intra-assay coefficient of variation(CV) and inter-assay CV were 3.9%-9.8% and 4.3%-13.8% respectively; The results from DASP 2009 showed that the sensitivity was 66%(33 positive out of 50 type 1 diabetes) and its specificity reached 100% (no positive out of 100 healthy controls).②ROC analysis showed that the area under curve(AUC) was 0.879±0.034 and 95% confidence interval was 0.813-0.945.③The positive cut-off point of ZnT8A index was 0.011 which determined by the 99% percentile in 405 healthy controls.④The positive frequency of ZnT8A was 24.1% (130 of 539)in type 1 and 1.3% (6 of 555, x2=182.3, P<0.0001) in type 2 diabetes patients, but only 0.99% (4 of 405, x2=98.6, P<0.0001) in healthy controls.Conclusions:①The RLA for ZnT8A is a highly sensitive, specific and reproducible method.②There was a high ZnT8A prevalence in type 1 diabetes, which was very useful for classification and diagnosis in type 1 diabetes. Part 2 Investigation on distribution of autoantibodies to zinc transporter 8 in type 1 diabetesObjective:The aim of this part was to investigate the distribution of autoantibodies to zinc transporter 8 (ZnT8A) in Chinese type 1 diabetes patients.Methods:ZnT8A were measured in sera of 539 type 1 diabetic (T1DM) patients,555 type 2 diabetic (T2DM) patients and 405 healthy controls using radioligand binding assays based on human ZnT8. T1DM patients were recruited from Xiangya 2nd Hospital of Central South University and Hunan Province T1DM Cooperative Study between Oct 1999 and Dec 2009. The average age at diagnosis was 24.0 years (range 1.0-70.0 years) and median duration of diabetes 2 months (range 0-348 months) with 293 males and 246 females. The measurements of GADA and IA-2A were carried out by radioligand binding assays.Results:①The prevalence of ZnT8A in T1DM patients of 24.1% (130/539)was markedly higher than that in T2DM 1.1%(6/555) and controls 1.0%(4/405) (both P<0.001).②ZnT8A were detected in 101 (31.2%) patients, among them 324 of 539 (53.4%) with positive other islet autoantibodies (GADA, IA-2A) and 29 of 177(15.8%) patients who were negative for other autoantibodies (P<0.001).③Matching onset age and disease duration to others, ZnT8A in newly diagnosed Chinese TIDM patients were less prevalent than reported in Caucasian 61.5%(259/421) and German 60.0%(122/202)(both P<0.001) populations, though not different from that in Japanese 27.8%(75/270) (P > 0.05).④The prevalence of ZnT8A (24.1%) is obviously lower than that of GADA(53.4%) (P=0.000).But there were no significant difference between that of ZnT8A and IA-2A (25.8%) (P>0.05).⑤The prevalence of ZnT8A,GADA,IA-2A simultaneously positive is 9.5%(51/539).The overlap among the three antibodies were highest between GADA and IA-2A 19.1%(103/539), followed by GADA and ZnT8A 16.0%(86/539), then IA-2A and ZnT8A 12.3%(66/539).⑥Combined ZnT8A,GADA, and IA-2A testing can increase the sensitivity of diagnosis of T1D for 65.5%(353/539). On the base of testing GADA and IA-2A,the positive rate of autoimmune will from 60.1% increase to 65.5% when ZnT8A is further tested.⑦ZnT8A were more often found in patients who already had autoantibodies against multiple islet autoantigens other than ZnT8: 101 of 324 (31.2%) patients previously classified as single autoantibody-positive were ZnT8A-positive, as compared to 51 of 103 (49.5%) with two autoantibodies (P<0.01).Conclusions:①ZnT8A constitute an additional valuable marker of T1D diabetes, especially in GADA/IA-2A negative T1D cases.②There have different prevalence about ZnT8A in various races. ZnT8A in newly diagnosed Chinese and Japanese T1DM patients were less prevalent than reported in Caucasian and German populations.③There is a certain overlap between ZnT8A and GADA/IA-2A.④ZnT8A can improve the diagnostic sensitivity of autoimmune-related T1D when combined with GADA and/or IA2-A.⑤ZnT8A are more often found in patients with a more widespread antibody-response to multiple islet autoantigens. Part 3 Study on clinical features of ZnT8A in type 1 diabetesObjective:To explore clinical features of ZnT8A in type 1 diabetes.Methods:Radioligand binding assays were used to measure the sera of ZnT8A in a group of 539 T1D diabetic patients. The characteristics of these patients are the same as the second part. GADA and IA-2A were determined by radioligand binding assays.Results:①The prevalence of ZnT8A in the patients with BMI< 21.0 kg/m2, BMI 21.0-25.0 kg/m2 is 24.3%,25.0% respectively, obviously higher than that of BMI>25.0 kg/m2 (9.3%) (both P<0.05).②There was no significant difference in the prevalence of ZnT8A between FCP<150pmol/L(24.4%) and FCP>250pmol/L(31.8%)(P> 0.05).③ZnT8A+individuals had younger age of onset, lower body index, lower blood pressure, higher 2-hour postprandial C peptide(all P< 0.05),and with higher level of GADA and IA-2A titer, higher prevalence of GADA+and IA-2A+(both P<0.05)than ZnT8A-subjects.④ZnT8+ alone group had longer median duration of disease, a higher requirement for insulin (both P<0.01), yet presented with lower proportion of metabolic syndrome, lower systolic pressure (both P< 0.05) than antibodies negative group. ZnT8+alone group had higher body index, higher waist-to-hip ratio and with lower HbAlc, higher fasting C peptide (all P<0.05) as compared to GADA+alone group. ZnT8+alone group had higher body index as compared to IA-2A+alone group.⑤ZnT8A,GADA and IA-2A all positive subjects had younger age of onset, lower body index, lower waist-to-hip ratio, lower triglyceride and with lower proportion of metabolic syndrome an compared to all antibodies negative subjects (all P<0.05).⑥ZnT8A index was correlated weakly with IA-2A index (r=0.214, P=0.000), but not with GADA (r=0.068, P=0.127).Conclusions:①ZnT8A were more prevalent in the leaner cases in TIDM subjects. ZnT8A did not associate with beta-cell function.②It was difficult to define clinical features of classic T1DM or both GADA and IA-2A negative T1DM with ZnT8A. ZnT8A could identify another T1DM phenotype which was between those of classic T1DM and antibodies negative T1DM.