LADA prevalence estimation and insulin dependency during follow-up

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1 DIABETES/METABOLISM RESEARCH AND REVIEWS Diabetes Metab Res Rev 2011; 27: Published online in Wiley Online Library (wileyonlinelibrary.com).1278 RESEARCH ARTICLE LADA prevalence estimation and insulin dependency during follow-up Yongsoo Park 1 Sangmo Hong 1 Leejin Park 1 Jungtaek Woo 2 Sehyun Baik 3 Munsuk Nam 4 Kwanwoo Lee 5 Youngseol Kim 2 and on behalf of The KNDP collaboratory Group 1 Department of Internal Medicine and Bioengineering, Hanyang University College of Medicine and Engineering, 2 Department of Endocrinology and Metabolism, Kyung Hee University School of Medicine, 3 Department of Endocrinology, Korea University, 4 Department of Endocrinology, Inha University College of Medicine, Inchon, Korea 5 Department of Endocrinology and Metabolism, Ajou University, Suwon, Korea Correspondence to: Yongsoo Park, Department of Internal Medicine and Bioengineering, Hanyang University, IT/BT Building 11th Floor, Haengdang-dong, Seongdong-gu, Seoul , Korea parkys@hanyang.ac.kr Revised: 5 June 2011 Accepted: 8 June 2011 Abstract Background Latent autoimmune diabetes in adults (LADA) is a form of autoimmune-mediated diabetes in adults, usually defined by GAD autoantibody positivity. Few epidemiological surveys on LADA in Asians did not come to a conclusive answer regarding prevalence and incidence, because of different criteria used in patient ascertainment. Methods We estimated LADA prevalence in a recent type 2 diabetes cohort by the positivity of circulating autoantibodies to pancreatic islet cell antigens (GAD, IA-2 and zinc transporter 8 (ZnT8)) applying a comparable Caucasian criteria. We then observed the development of insulin dependency prospectively for 36 months. Results Applying the European NIRAD LADA group criteria, we found a high prevalence of LADA (4.4%) in Korean patients with phenotypic type 2 diabetes. This high prevalence of LADA in Korea is noteworthy since the previous population-based LADA prevalence survey reported low prevalence (1.7%). When we consider the low-titre GAD antibodies and the low prevalence of multiple autoantibodies, however, increased LADA prevalence does not necessarily mean increase in future insulin dependency. After 36 months of follow-up, only 3 of 39 patients who were initially classified as LADA have become insulin-dependent. Those three were all positive for multiple autoantibodies (GAD, IA-2 and zinc transporter 8 antibody). Other features of insulin secretion or insulin resistance did not determine future insulin necessity. Conclusions Although the LADA prevalence estimated by anti-gad positivity appeared to increase, the true insulin dependency evidenced by multiple antibody positivity did not increase in Korea. Copyright 2011 John Wiley & Sons, Ltd. Keywords LADA; prevalence; incidence; insulin dependency; multiple autoantibody positivity Introduction Diabetes mellitus is a group of metabolic diseases characterized by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. Most diabetic patients fall into two broad categories of diabetes that are type 1 diabetes (T1D) and type 2 diabetes (T2D). The most significant difference between the two categories is that T1D is due to autoimmunity and the patients afflicted with T1D need insulin therapy for survival but T2D patients do not. Copyright 2011 John Wiley & Sons, Ltd.

2 976 Y. Park et al. A subgroup of diabetic patients shows both the features of T1D and T2D. These patients show some evidence of autoimmunity but clinically resembled T2D at diagnosis. Then, they slowly change into insulin-requiring state to survive. Tuomi [1] and Zimmet [2] launched the eponym Latent Autoimmune Diabetes in Adults (LADA) for such patients. LADA is a form of autoimmune-mediated diabetes in adults, usually defined by GAD autoantibody positivity. Although some reviews have widely discussed LADA, this conditions remain poorly understood at both clinical and research level. With the advent of new biochemical autoantibodies, the Immunology of Diabetes Society extended and reproposed the diagnostic criteria for LADA as following: (1) the presence of T2D and 35 years of age; (2) lack of requirement for insulin at least 6 months after diagnosis of T2D; and (3) presence at least one of three circulating autoantibodies to pancreatic islet cell antigens (GAD, IA-2 and insulin) [3]. However, even the new LADA diagnostic criteria could not tell about the possibility of progressive β-cell failure. Because there have been scarce data estimating future insulin dependency in patients with LADA, we investigated whether autoantibodies or biochemical markers might foretell insulin dependency in patients with LADA. We recruited LADA patients from a cross-sectional survey of recent-onset T2D, who were initially diagnosed according to the Immunology of Diabetes Society criteria and have observed the development of insulin dependency prospectively for 36 months. Materials and methods This study was conducted in patients who participated in the Korean National Diabetes Program (KNDP). Briefly, the KNDP is a prospective observational study of T2D patients which has been carried out from March 2005 to understand the characteristics and natural history of Korean T2D and to develop clinical guidelines. At the time of starting this study (31 December 2007), a total of 2622 patients were enrolled. Diagnosis and classification of diabetes were based on the guidelines of the Expert Committee Report of the American Diabetes Association [4]. Out of 2622 patients, 1296 patients met the inclusion criteria for screening for the study. Inclusion criteria were as follows: (1) male or female patients years of age, (2) patients with diagnosis of diabetes according to the World Health Organization classification for more than 2 months and less than 5 years, (3) diabetes controlled by diet or by oral anti-diabetic agents for at least ( ) 2 months after diagnosis or diabetes transiently controlled by insulin therapy for less ( ) than 2 months. Exclusion criteria for screening were: (1) patients with secondary diabetes mellitus due to other conditions or under immunosuppression; (2) pregnant women; (3) heavy ketouria (++) on urine test sticks. Of eligible patients, 884 patients had serum samples. Therefore, Figure 1. Schematic presentation of the study cohort. KNDP, Korea National Diabetes Program individual screening for islet-specific antibody were taken in these 884 patients (Figure 1). Written consent was obtained from each patient before enrollment in the study. The study was approved by the Ethical Committee of Hanyang University Hospital and by the steering committee of the KNDP. As a baseline exam for clinical characteristics, patient s age, sex and body mass index were asked and studied. Body mass index was calculated as weight/height 2 (kg/m 2 ). Serum levels of total cholesterol, triglycerides, creatinine and haemoglobin A1c were also measured. In addition, creatinine clearance and 24-h urine excretion for microalbumin were measured from 24-h urine collection. GAD and IA-2 autoantibodies were measured by a radiobinding assay based on 35 S-labelled recombinant GAD65 or IA-2, as described previously [5], with all samples measured in duplicate. The cutoff point of GAD index was 0.032, and IA-2 index was which were determined according to the 99% upper limit of 128 healthy controls. The sensitivity and specificity of the GAD antibody assay were 71 and 93%, respectively, and those of the IA-2 antibody assay were 71 and 97%, respectively (fourth Diabetes Autoantibody Standardization Program, DASP 2009). Zinc transporter 8 antibody (ZnT8) was also detected by radioligand assay based on 35 S-methioninelabelled human antigens. The ZnT8 cdna constructs used in this study were the cytoplasmic carboxyterminal domains (aa ) of human ZnT8 carrying 325Arg (designated as CR) or a hybrid construct of the CR and 325Trp (CW; designated as CW-CR) with a CLFCEDPCDPSTPPGSSGGGKDFSILLME hinge junction generated by polymerase chain reaction. The cutoff point of ZnT8 index was 0.039, which was determined according to the 99% upper limit of 128 healthy controls. The sensitivity and specificity of the ZnT8 antibody assay were 82 and 81%, respectively.

3 LADA Prevalence Estimation and Insulin Dependency 977 diagnosed by the autoantibody criteria developed insulin dependency. All patients who required insulin during follow-up were positive for all three antibodies initially. Baseline mean titres of GAD antibody in patients who required insulin were significantly higher than those who did not (p < 0.05, Table 2). Figure 2. The progression to insulin dependency during 36 months of follow-up. T2DM, type 2 diabetes mellitus; LADA, latent autoimmune diabetes in adults Statistical analyses All clinical data were summarized as mean ± SD. Continuous clinical data between two groups were compared using student s t-test. Discrete variables were presented as total number (%). Differences in the frequency distribution of the variables were evaluated by Chi-Square or Fisher s exact test, as appropriate. We used SPSS for Windows software for statistical analysis (version 18.0; SPSS, Chicago, IL, USA). p < 0.05 was considered statistically significant. Results Prevalence of LADA Out of 1296 patients who met the inclusion criteria for the study, individual screenings for islet-specific antibodies were taken in 884 patients who had serum samples. Clinical characteristics of those who had serum samples did not differ from those who had not. Among 884 patients, 39 patients had at least one islet-specific antibody [GAD antibody (+): 39 (4.4%), IA-2 antibody (+): 3 (0.3%), ZnT8 antibody (+): 3 (0.3%)] (Figure 2). The prevalence of LADA in Korean population was 4.4% (39 patients out of 884 patients who were diagnosed as T2D). Applying the European NIRAD LADA group criteria [6], we found a high LADA prevalence (4.4%), but very few patients were positive for multiple autoantibodies. Baseline mean titres of GAD antibody positivity in patients with LADA were significantly higher than those who remained in T2D category (n = 845) (0.046 ± versus ± 0.007, p < 0.01). In the comparisons between newly diagnosed LADA patients (n = 39) and those remained as T2D (n = 845), fasting blood glucose, haemoglobin A1c and hscrp were lower in patients with LADA (126.1 ± 29.6 mg/dl, 6.9 ± 1.0% and 0.7 ± 0.73 mg/l, respectively) than T2D patients (142.9 ± 49.7 mg/dl, 7.5 ± 1.9% and 4.1 ± 27.9 mg/l, respectively) (p < 0.05, Table 1). Follow-up of insulin dependency During 36 months of follow-up, only 3 of 39 patients (7.7%) who belonged to the LADA category initially as Discussion Few epidemiological surveys on LADA in Asians did not come to a conclusive answer regarding prevalence and incidence, because of different criteria used in patient ascertainment [7 9]. Applying the European NIRAD LADA group criteria, we found a high prevalence of LADA (4.4%) in Korean patients with phenotypic T2D. This high prevalence of LADA in Korea is noteworthy since the previous population-based T2D prevalence survey reported low prevalence [8]. Our present study population may not represent the overall Korean population, because our cohort is not a population-based, but a hospital-based cohort, composed of 2622 diabetic patients recruited from 13 different university hospitals of Korea. A total of 884 patients with T2D were recruited from the KNDP, a prospective natural history study of T2D. Our present study demonstrated a prevalence of LADA either defined by GAD or by more than one of triple islet-specific antibodies as high as 4.4%, similar to that in Caucasians [10,11]. It is high compared with the previously reported low prevalence (1.7%) which came from GAD autoantibody positivity among new onset T2D patients using a population-based T2D prevalence survey [8]. Since the HLA genetic make-up in the general Koreans are usually composed of T1D protective haplotypes, the prevalence and incidence of T1D and similarly those of LADA were estimated to be low. In other Korean hospital-based studies which also used only GAD antibody to diagnose LADA, however, the increased LADA prevalence in the Koreans was estimated recently (the prevalence estimated ranging from 3.8 to 5.1%) [12], which was similar to our present result applying multiple autoantibodies. In other Asian countries such as Japan and China, LADA prevalence was also reported to be increased recently and similar to our prevalence in this study [9,13]. However, there have been few studies reporting the titres of autoantibodies. Nor did they use triple islet-specific antibodies to diagnose LADA. Although the majority of LADA patients progresses towards insulin dependency within a few years after the diagnosis, this form of diabetes is heterogeneous. The presence of high GAD antibody titres identifies a subgroup of LADA patients with clinical characteristics similar to typical T1D and at very high risk of progression towards insulin dependency [14]. Recently, Maioli et al. suggested that multiple autoantibodies rather than high GAD antibody titre predict insulin dependency in LADA [15]. On the other hand, low GAD antibody titres, or the exclusive presence of GAD antibody directed to middle epitopes of the autoantigen characterizes LADA

4 978 Y. Park et al. Table 1. The comparisons between the latent autoimmune diabetes in adults patients and type 2 diabetes patients Latent autoimmune diabetes Latent autoimmune diabetes in adults (+) in adults ( ) p-value Number Sex (male/female) (19/20) (543/392) ns Age (years) 51.9 ± ± 8.7 ns Duration (years) 1.83 ± ± 1.77 ns Body mass index (kg/m 2 ) 25.3 ± ± 3.1 ns Waist hip ratio 1.12 ± ± 0.83 ns Systolic blood pressure (mmhg) ± ± 14.0 ns Diastolic blood pressure (mmhg) 77.9 ± ± 11.9 ns Fasting blood sugar (mg/dl) ± ± Haemoglobin A1c (%) 6.9 ± ± Total cholesterol (mg/dl) ± ± 40.6 ns Family history of diabetes (%) ns Oral hypoglycaemia agent medication (%) ns Cardiovascular risk factors (%) ns Aspartate transaminase 25.9 ± ± 22.9 ns Alanine transaminase 27.5 ± ± 28.8 ns Gamma-glutamyl transpeptidase 45.4 ± ± ns High-sensitivity C-reactive protein 0.7 ± ± Insulin (µg/dl) 9.3 ± ± 13.4 ns Homeostatic model assessment of insulin resistance 2.95 ± ± 5.37 ns ns, not significant Table 2. Comparison of the mean titres of islet-specific antibodies between those who progressed into insulin dependency and those who did not during follow-up GAD Zinc transporter 8 antibody IA-2 Insulin dependency (n = 3) ± ± ± Insulin independency (n = 36) ± ± ± p-value patients with clinical characteristics more similar to T2D patients. When we consider the low-titre GAD antibodies and low prevalence of multiple autoantibodies in our LADA patients, the relative increase in the proportion of GAD antibody positivity does not necessarily mean a real increase in future insulin treatment. In our study, only three patients developed insulin dependency during 36 months of follow-up. They were all triple antibodies positive (GAD, IA-2 and ZnT8). Their titres of GAD antibody were significantly higher initially. Those who had high-titre GAD antibodies but single antibody (mostly GAD antibody) positive patients did not progress into real insulin dependency. Only the positivity of multiple islet-specific antibodies was suspected to be the marker that would tell about the possible progression to insulin dependency. In a multitude of studies of first-degree relatives of T1D patients, individuals with multiple antibodies showed higher risk of progression to diabetes than those with single antibodies. As far as we know, this is the first study finding an association of multiple islet-specific antibodies with future insulin dependency in Asian populations. Although LADA prevalence estimated by anti-gad positivity appeared to increase, the true insulin dependency evidenced by multiple antibody positivity did not increase in Korea. In conclusion, a more refined new version of diagnostic criteria of LADA is needed which should include variables suggesting future insulin dependency, such as positivity of multiple islet-specific autoantibodies. In our study, it turned out that obesity and other features of insulin resistance do not exclude the presence of LADA. The prevalence of both T1D and T2D among children and adolescents has been steadily increasing over the last few decades. However, as the general paediatric population becomes more obese, reliance on phenotypic characteristics for distinguishing between these types of diabetes is becoming increasingly untenable. Yet, the recognition of differences in treatment strategies, associated disorders, and both short- and long-term diabetes and cardiovascular outcomes supports the importance of diagnostic efforts to make a distinction between diabetes types. At the time of diagnosis, it is generally not possible to be certain of diabetes type, especially LADA and therefore, initial presumptive treatment decisions must be reconfirmed by the individual s course, taking the presence or absence of multiple pancreatic autoantibody positivity into account. Our study has several limitations hampering generalization. First, the KNDP cohort a hospital-based study recruited from various university hospitals in Korea may not represent the true Korean diabetic population. There could be a selection bias in the study population, although we recruited patients from very different part of our

5 LADA Prevalence Estimation and Insulin Dependency 979 country. Second, those three true insulin-dependent LADA patients found during follow-up were too small to evaluate any more statistical analyses. Conflict of interest None declared. Acknowledgements This research was supported by the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A102065). References 1. Tuomi T, Groop LC, Zimmet PZ, et al. Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non-insulindependent onset of disease. Diabetes 1993; 42(2): Zimmet PZ, Tuomi T, Mackay IR, et al. Latent autoimmune diabetes mellitus in adults (LADA): the role of antibodies to glutamic acid decarboxylase in diagnosis and prediction of insulin dependency. Diabet Med 1994; 11(3): Diagnosis and classification of diabetes mellitus. Diabetes Care 2007; 30(Suppl. 1): S42 S Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 2003; 26(Suppl. 1): S5 S Petersen JS, Hejnaes KR, Moody A, et al. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 1994; 43(3): Zampetti S, Spoletini M, Petrone A, et al. Association of TCF7L2 gene variants with low GAD autoantibody titre in LADA subjects (NIRAD Study 5). Diabet Med 2010; 27(6): Tsuruoka A, Matsuba I, Toyota T, et al. Antibodies to GAD in Japanese diabetic patients: a multicenter study. Diabetes Res Clin Pract 1995; 28(3): Park Y, Lee H, Koh CS, et al. The low prevalence of immunogenetic markers in Korean adult-onset IDDM patients. Diabetes Care 1996; 19(3): Qi X, Sun J, Wang J, et al. Prevalence and correlates of latent autoimmune diabetes in adults in Tianjin, China: a population-based cross-sectional study. Diabetes Care 2011; 34(1): Turner R, Stratton I, Horton V, et al. UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group. Lancet 1997; 350(9087): Ruige JB, Batstra MR, Aanstoot HJ, et al. Low prevalence of antibodies to GAD65 in a 50- to 74-year-old general Dutch population. The Hoorn Study. Diabetes Care 1997; 20(7): Yu SH, Park S, Kim HS, et al.theprevalence of GAD antibodies in Korean women with gestational diabetes mellitus and their clinical characteristics during and after pregnancy. Diabetes Metab Res Rev 2009; 25(4): Murao S, Kondo S, Ohashi J, et al. Antithyroid peroxidase antibody, IA-2 antibody, and fasting C-peptide levels predict beta cell failure in patients with latent autoimmune diabetes in adults (LADA) a 5-year follow-up of the Ehime study. Diabetes Res Clin Pract 2008; 80(1): Rosario PW, Reis JS, Fagundes TA, et al. Latent autoimmune diabetes in adults (LADA): usefulness of anti-gad antibody titers and benefit of early insulinization. Arq Bras Endocrinol Metabol 2007; 51(1): Maioli M, Pes GM, Delitala G, et al. Number of autoantibodies and HLA genotype, more than high titers of glutamic acid decarboxylase autoantibodies, predict insulin dependence in latent autoimmune diabetes of adults. Eur J Endocrinol 2010; 163: