b-cell Autoantibodies and Their Function in Taiwanese Children With Type 1 Diabetes Mellitus

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1 ORIGINAL ARTICLE b-cell Autoantibodies and Their Function in Taiwanese Children With Type 1 Diabetes Mellitus Yi-Ching Tung, 1 Mei-Huei Chen, 2 Cheng-Ting Lee, 1 Wen-Yu Tsai 1 * Background/Purpose: To understand the importance of autoimmunity in the development of type 1 diabetes in Taiwanese children, we evaluated the presence of β-cell autoantibodies and their correlation with residual β-cell function. Methods: From 1989 to 2006, 157 Taiwanese children with newly diagnosed type 1 diabetes were enrolled in this study. We determined the presence of β-cell autoantibodies, such as glutamic acid decarboxylase autoantibodies (GADAs), insulinoma antigen 2 autoantibodies (IA-2As), and insulin autoantibodies (IAAs). A 6-minute glucagon test was also performed at diagnosis. Results: At diagnosis, 73% of children tested positive for GADAs, 76% for IA-2As and 21% for IAAs. Ninety-two percent of them had at least one of the β-cell autoantibodies detected. Positivity for IAAs was more frequent in patients younger than 5 years than in those older than 5 years (45% vs. 13%). Using multiple regression analysis, the presence of GADAs or IAAs, or age of onset of these patients was an independent factor for residual β-cell function. Younger patients and those with GADAs had less residual β-cell function at disease onset, whereas those with IAAs had more insulin reserve. Conclusion: Autoimmunity plays an important role in the pathogenesis of type 1 diabetes in Taiwanese children, and the presence of IAAs tends to be more common in younger children. [J Formos Med Assoc 2009;108(11): ] Key Words: autoantibodies, C-peptide, glucagon test, type 1 diabetes mellitus Type 1 diabetes has been the leading form of diabetes among children and adolescents. As a result of sedentary lifestyle and high-fat, highcarbohydrate dietary habits, the prevalence and the secular trends of incidence of type 2 diabetes in early adolescence have increased in the past two decades, including in Taiwan. 1,2 Besides, the incidence for type 1 diabetes has been shown to be steady over these years. 3 Therefore, correct differential diagnosis of pediatric diabetes is important for the management plan. In Caucasians, it has long been reported that type 1 diabetes mellitus is an autoimmune disease associated with the destruction of pancreatic β cells. 4 Several autoantibodies against pancreatic β-cell autoantigens, including insulin, 65-kDa glutamic acid decarboxylase (GAD65) and insulinoma antigen 2 (IA-2), a tyrosine-phosphataselike protein, have been detected in patients with type 1 diabetes and their siblings. 5 7 The presence of β-cell autoantibodies in serum is used as a marker for autoimmunity, and is specific for the diagnosis of type 1 diabetes in children. 8 Studies in Caucasians have shown that about 90% of patients with type 1 diabetes have at least one of these β-cell autoantibodies detected Elsevier & Formosan Medical Association Department of Pediatrics, National Taiwan University Hospital and College of Medicine, National Taiwan University, and 2 Department of Pediatrics, Yung Ho Branch of Cardinal Tien Hospital, Taipei, Taiwan. Received: July 10, 2008 Revised: December 3, 2008 Accepted: June 29, 2009 *Correspondence to: Dr Wen-Yu Tsai, Department of Pediatrics, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan. wenyutsai@ntu.edu.tw 856 J Formos Med Assoc 2009 Vol 108 No 11

2 β-cell autoantibodies in Taiwanese type 1 diabetes The prevalence of type 1 diabetes is lower in Asians than in Caucasians, and the difference in the frequency of susceptible and protective genes can explain partially such an ethnic difference. 11,12 The positive rates of β-cell autoantibodies in patients with recent-onset type 1 diabetes in Japan and Korea are reported to be similar to those in Caucasians. 13,14 However, the positive rate in Chinese patients has been reported as lower than that in Caucasians In the present study, we aimed to elucidate the prevalence of β-cell autoantibodies in newly diagnosed type 1 diabetes in Taiwanese children, and its diagnostic value. Moreover, we evaluated the correlation between the presence of these β-cell autoantibodies and residual β-cell function. Patients and Methods Patients From 1989 to 2006, we enrolled 157 consecutive Taiwanese patients, aged < 18 years, with newly diagnosed type 1 diabetes, who were followed-up at the Pediatric Endocrine Clinic of the National Taiwan University Hospital. All of the patients were evaluated within 3 weeks of diagnosis, which was based on the report of the Expert Committee held in All of the patients currently are receiving insulin therapy. Protocol This study was approved by the Ethics Committee of National Taiwan University Hospital. Informed consent was obtained from the patients or their parents. Metabolic status was evaluated by plasma glycated hemoglobin levels, and metabolic decompensation was assessed by the presence of ketoacidosis, which was defined as the presence of blood or urine ketone bodies, with blood bicarbonate level < 15 mmol/l. Signs of puberty were evaluated at physical examination. The onset of puberty was defined as breast development more than Tanner stage II in girls and testicular size > 4 ml in boys. 21 After the diagnosis was made, blood was drawn for the detection of β-cell autoantibodies. The serum samples were obtained and stored at 80 C until assayed. All of the patients had a 6-minute glucagon test to assess their β-cell function at diagnosis. After the fasting serum C-peptide sample was obtained, 30 μg/kg glucagon (maximum 1 mg) was given intravenously, and the second serum sample was obtained 6 minutes later, to determine the post-glucagon C-peptide level. 22 GAD65 autoantibodies (GADAs), IA-2 autoantibodies (IA-2As) and insulin autoantibodies (IAAs) were assayed using commercially available radioimmunoassay kits (CIS Bio International, Gif-Sur-Yvette Cedex, France). Human recombinant full-length GAD65 protein and intracellular fragment of the IA-2 protein, including amino acids were labeled with I 125. IAA detection was based on the percentage binding of I labelled insulin. Serum levels > 1 U/mL were considered positive for GADAs and IA-2As, whereas serum level > 10% was considered positive for IAAs. The intra-assay coefficients of variance of these three assays were all < 5%, and inter-assay coefficients of variance were all < 14%. These assays had disease sensitivities of 86%, 73% and 37% and specificities of 95%, 99% and 96% for GADAs, IA-2As and IAAs, respectively, in the Diabetes Autoantibody Standardization Program workshop. 23 Serum C-peptide levels were measured by radioimmunoassay (Daiichi Radioisotope Laboratories, Tokyo, Japan). Statistical analysis The mean value and variance of the factors were compared between the subgroups. The χ 2 test was used for categorical variables and the Mann Whitney U test or Kruskal Wallis test was used for numerical data. The correlations among the variables were analyzed by the Pearson method or logistic regression for categorical variables. Multiple linear regression was used to analyze the parameters associated with the dependent variable of residual β-residual cell function at onset, which was shown as post-glucagon C-peptide level. Parameters such as age at onset, sex, glycated J Formos Med Assoc 2009 Vol 108 No

3 Y.C. Tung, et al Table 1. Clinical characteristics of patients with type 1 diabetes Age (yr) 0 5 (n = 40) 5 10 (n = 66) (n = 51) Total (n = 157) p Sex (male:female) 18:22 23:43 19:32 60: HbA1c* < ( ) ( ) ( ) ( ) Ketoacidosis 35 (90) 42 (65) 26 (52) 103 (67) C-peptide (nmol/l)* Fasting ( ) ( ) ( ) ( ) Post-glucagon < ( ) ( ) ( ) ( ) GADA positivity 31 (78) 45 (68) 38 (75) 114 (73) IA-2A positivity 30 (75) 50 (76) 40 (78) 120 (76) IAA positivity 18 (45) 8 (12) 7 (14) 33 (21) < Multiple antibodies 29 (72) 39 (59) 32 (63) 100 (64) No detectable antibodies 1 (3) 8 (12) 4 (8) 13 (8) *Data presented as mean (range); one missing data in each age group; data presented as n (%); more than two b-cell autoantibodies were detected; Kruskal Wallis test; Pearson s c 2 test. HbA1c = hemoglobin A1c; GADA = glutamic acid decarboxylase autoantibody; IA-2A = insulinoma antigen-2 autoantibody; IAA = insulin autoantibody. hemoglobin, and presence of ketoacidosis, GADA, IA-2A or IAA were independent variables of the analysis. A p value of < 0.05 was considered statistically significant. GADA 19 (12%) 67 (43%) 1A 2A 25 (16%) Results 5 (3%) 23 (15%) 5 (3%) The clinical data of patients with type 1 diabetes (60 boys and 97 girls) are shown in Table 1. The median age was 8.0 years (range: years) and 32% had entered puberty at diagnosis. Their median body mass index (BMI) was 14.6 kg/m 2 (range: kg/m 2 ) with a standard deviation score of 1.0 (range: 3.1 to +3.2). Sixty-seven percent had ketoacidosis as the initial presentation. There were 114 patients (73%) positive for GADAs, with a median value of 11.2 U/mL (range: U/mL) (Table 1). One hundred and twenty patients (76%) were positive for IA-2As, with a median value of 11.8 U/mL (range: U/mL), whereas IAAs were positive in 33 patients at diagnosis (21%), with a median value of 12.6% (range: 10 53%). Ninety-two percent had at least one β-cell autoantibody detected and 64% had more than two of the autoantibodies (Figure). IAA 0 (0%) 13 (8%) Figure. The frequency of β-cell autoantibodies in Taiwanese children with newly diagnosed type 1 diabetes. The figures denote the numbers of patients positive for autoantibodies and those in parentheses denote the percentage of these patients. There were no differences in sex or metabolic parameters between patients with and those without β-cell autoantibodies. No age difference was detected between GADA-positive and -negative patients. There was also no difference in age between IA-2A-positive and -negative patients. However, the IAA-positive patients tended to be younger than the IAA-negative patients (5.9 ± J Formos Med Assoc 2009 Vol 108 No 11

4 β-cell autoantibodies in Taiwanese type 1 diabetes Table 2. Multiple linear regression for residual β-cell function Post-glucagon C-peptide level (nmol/l) Parameters Model 1: include all potential variables Model 2: include potential variables significantly associated with Pearson s correlations β±se p β±se p Intercept ± < ± < Age (yr) ± < ± < Sex (male) ± HbA1c (%) ± < ± < GADA positivity ± ± < IA-2A positivity ± IAA positivity ± ± Ketoacidosis ± Adjusted R b = standardized coefficient; SE = standard error; HbA1c = hemoglobin A1c; GADA = glutamic acid decarboxylase autoantibody; IA-2 = insulinoma antigen-2 autoantibody; IAA = insulin autoantibody. vs. 8.7 ± 3.7 years, p < 0.001). Forty-five percent of patients who were < 5 years old had IAAs, whereas IAAs were only detected in 13% of patients > 5 years old. The IAA titers in patients aged < 5 years were also significantly higher than those in older subjects (19.9% vs. 11.5%). With regard to metabolic status, the presence of ketoacidosis at onset was correlated negatively with age of onset (r = 0.314, p < 0.001) and glycated hemoglobin level was correlated positively with age (r = 0.311, p < 0.001), as shown in Table 1. After multiple regression analysis, residual β-cell function was correlated positively with age of onset (β=0.458, p < 0.001) and presence of IAA (β=0.219, p = 0.003). It was correlated negatively with the level of glycated hemoglobin (β= 0.385, p < 0.001) and presence of GADA (β= 0.255, p < 0.001), as shown in Table 2. Discussion Based on the low prevalence of β-cell autoantibodies in Chinese patients with type 1 diabetes, it has been proposed that immune-mediated mechanisms are not as important as in Caucasians However, our study showed that 92% of Taiwanese children with type 1 diabetes had at least one of the β-cell autoantibodies detected and their prevalence was similar to that of other populations. 8 10,13,14 Varied inclusion criteria, such as patient age and disease duration, might explain partly the difference. Our study demonstrates that autoimmunity still plays an important role in the pathophysiology of type 1 diabetes in Taiwanese children. In our study population, 73% had GADAs and 76% had IA-2As. The age of onset had no effect on the positive rate of GADAs and IA-2As. However, the frequency of IAA positivity was significantly higher in patients with an onset age < 5 years than that in older patients, as previously reported Therefore, IAA had greater diagnostic value in young children with type 1 diabetes than in older children. The metabolic state at diagnosis was influenced by the age of onset. Younger patients were more prone to ketoacidosis as an initial presentation than were older patients. The residual β-cell function was correlated well with age of onset, which indicated that younger patients had less insulin reserve, which is consistent with other studies. 24,28 However, even after the exclusion of the confounding effects of age and the presence of other autoantibodies, the residual β-cell function remained correlated positively with the presence J Formos Med Assoc 2009 Vol 108 No

5 Y.C. Tung, et al of IAAs. This indicated that patients with IAAs at onset had more insulin reserve than those without IAAs. In cohort studies in Finland and Norway, IAA has emerged as the first detectable β-cell autoantibody in high-risk pre-diabetic children. 29,30 However, the studies also have shown that IAA disappears more rapidly than the other autoantibodies. 29 Therefore, the fact that patients positive for IAAs had more insulin reserve at disease onset suggested that their pre-clinical period was shorter than those without IAAs. In addition to the factors of age and IAA, the presence of GADA was another independent factor that was correlated negatively with residual β-cell function, which is consistent with other studies. 31,32 On the other hand, Decochez et al have reported that the presence or absence of β-cell autoantibodies does not affect β-cell function at disease onset, but those with these autoantibodies have a more rapid destruction of β-cell function. 33 This is confirmed by our results. In conclusion, our study demonstrates that most type 1 diabetes in Taiwanese children is related to the immune-mediated destruction of pancreatic islet cells. Among the detectable β-cell autoantibodies, IAA is more common in younger children, aged < 5 years, with type 1 diabetes. Acknowledgments Part of this study was supported by a grant (941-02) from the Yung Ho Branch of Cardinal Tien Hospital, Taipei, Taiwan. References 1. Wei JN, Sung FC, Lin CC, et al. National surveillance for type 2 diabetes mellitus in Taiwanese children. JAMA 2003;290: Tseng CH, Tseng CP, Chong CK, et al. Increasing incidence of diagnosed type 2 diabetes in Taiwan: analysis of data from a national cohort. Diabetologia 2006;49: Tseng CH. Incidence of type 1 diabetes in children aged 0 14 years during in Taiwan. Acta Pediatr 2008;97: Esienbarth GS. Type 1 diabetes: a chronic autoimmune disease. N Engl J Med 1986;314: Baekkeskov S, Aanstoot HJ, Christgau S, et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990;347: Lan MS, Wasserfall C, Maclaren NK, et al. IA-2, a transmembrane protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 1996;93: Palmer JP. Insulin autoantibodies: their role in the pathogenesis of IDDM. Diabetes Metab Rev 1987;3: Hagopian WA, Sanjeevi CB, Kockum I, et al. Glutamate decarboxylase-, insulin- and islet cell-antibodies and HLA typing to detect diabetes in a general population-based study of Swedish children. J Clin Invest 1995;95: Bingley PJ, Bonifacio E, Williams AJ, et al. Prediction of IDDM in the general population: strategies based on combinations of autoantibody markers. Diabetes 1997;46: Gorus FK, Goubert P, Semakula C, et al. IA-2- autoantibodies complement GAD65-autoantibodies in new-onset IDDM patients and help predict impending diabetes in their siblings. The Belgian Diabetes Registry. Diabetologia 1997;40: Diabetes Epidemiology Research International Group. Geographic pattern of childhood insulin-dependent diabetes mellitus. Diabetes 1988;37: Park Y. Why is type 1 diabetes uncommon in Asia. Ann N Y Acad Sci 1006;1079: Park Y, Lee H, Takino H, et al. Evaluation of the efficacy of the combination of multiple autoantibodies to islet-specific antigens in Korean type 1 diabetic patients. Acta Diabetol 2001;38: Sera Y, Kawasaki E, Abiru N, et al. Autoantibodies to multiple islet autoantigens in patients with abrupt onset type 1 diabetes and diabetes diagnosed with urinary glucose screening. J Autoimmun 1999;13: Ko GTC, Chan JCN, Yeung VTF, et al. Autoantibodies to glutamic acid decarboxylase in young Chinese diabetic patients. Ann Clin Biochem 1998;35: Ng WY, Lee YS, Todd AL, et al. Tyrosine phosphatase-like protein (IA-2) and glutamic acid decarboxylase (GAD65) autoantibodies: a study of Chinese patients with diabetes mellitus. Autoimmunity 2002;35: Chang YH, Shiau MY, Tsai ST, et al. Autoantibodies against IA-2, GAD, and topoisomerase II in type 1 diabetic patients. Biochem Biophys Res Commun 2004;320: Chen BH, Chung SB, Chiang W, et al. GAD65 antibody prevalence and association with thyroid antibodies, HLA- DR in Chinese children with type a diabetes mellitus. Diabetes Res Clin Pract 2001;54: Wang JP, Zhou ZG, Lin J, et al. Islet autoantibodies are associated with HLA-DQ genotypes in Han Chinese patients with type 1 diabetes and their relatives. Tissue Antigens 2007;70: J Formos Med Assoc 2009 Vol 108 No 11

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