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Identification of Novel Autoantibodies in Type 1 Diabetic Patients Using A 3022 Diabetes Volume 63, September 2014 Bo Kyung Koo,1,2 Sehyun Chae,3 Kristine M. Kim,4 Min Jueng Kang,5 Eunhee G. Kim,4 Soo Heon Kwak,1 Hye Seung Jung,1 Young Min Cho,1 Sung Hee Choi,1 Young Joo Park,1 Choong Ho Shin,6 Hak C. Jang,1 Chan Soo Shin,1 Daehee Hwang,3,7 Eugene C. Yi,5 and Kyong Soo Park1,5 Identification of Novel Autoantibodies in Type 1 Diabetic Patients Using a High- Density Protein Microarray Diabetes 2014;63:3022–3032 | DOI: 10.2337/db13-1566 Autoantibodies can facilitate diagnostic and therapeutic Type 1 diabetes (T1DM) results from immune-mediated means for type 1 diabetes (T1DM). We profiled autoanti- pancreatic b-cell destruction. Several autoantibodies, such bodies from serum samples of 16 T1DM patients, 16 type 2 as GAD65 antibody (GADA) (1), islet cell antibody (ICA) diabetic (T2DM) patients, and 27 healthy control subjects (2), protein tyrosine phosphatase antibody (IA-2 antibod- with normal glucose tolerance (NGT) by using protein ies [IA-2A]) (3), and zinc transporter antibody (ZnT8A) microarrays containing 9,480 proteins. Two novel autoanti- (4), were identified and used for diagnosis and prediction bodies, anti-EEF1A1 and anti-UBE2L3, were selected from of T1DM. fl microarrays followed by immuno uorescence staining of However, the nature and mechanism of b-cell destruc- pancreas. We then tested the validity of the candidates by tion in humans seem to be variable, which results in a broad 1 ELISA in two independent test cohorts: ) 95 adults with spectrum of T1DM spanning from fulminant T1DM (5) to T1DM, 49 with T2DM, 11 with latent autoimmune diabetes latent autoimmune diabetes in adults (LADA) (6). Fulmi- in adults (LADA), 20 with Graves disease, and 66 with NGT nant T1DM is a rapidly progressing form of T1DM without and 2) 33 children with T1DM and 34 healthy children. Con- evidence of autoimmunity, whereas LADA is autoimmune centrations of these autoantibodies were significantly higher in T1DM patients than in NGT and T2DM subjects diabetes not requiring insulin at diagnosis. In most cases of P < fi LADA, which account for 10% of incident cases of diabetes IMMUNOLOGY AND TRANSPLANTATION ( 0.01), which was also con rmed in the test cohort of b children (P < 0.05). Prevalence of anti-EEF1A1 and anti- in adults, -cell function is severely impaired within 6 years, UBE2L3 antibodies was 29.5% and 35.8% in T1DM, re- leading to insulin dependency (6). Fulminant T1DM is an spectively. Of note, 40.9% of T1DM patients who lack important subtype of T1DM in Asian adults (7), accounting anti-GAD antibodies (GADA) had anti-EEF1A1 and/or anti- for 15%–20% of T1DM with ketosis or ketoacidosis in UBE2L3 antibodies. These were also detected in patients Japan (5). Moreover, T1DM in adults often lacks evidence with fulminant T1DM but not LADA. Our approach identi- of autoimmunity. The prevalence of autoantibodies associ- fied autoantibodies that can provide a new dimension of ated with T1DM declines as the onset age increases, espe- information indicative of T1DM independent of GADA and cially in the case of IA-2A (8) and ZnT8A (4,9). ZnT8A was new insights into diagnosis and classification of T1DM. observed in 80% of individuals in late adolescence (4,9), 1Department of Internal Medicine, Seoul National University College of Medicine, 7Center for Systems Biology of Plant Aging Research, Institute for Basic Science, Seoul, Korea Daegu Gyeongbuk Institute of Science and Technology, Daegu, Korea 2 Department of Internal Medicine, Boramae Medical Center, Seoul, Korea Corresponding author: Kyong Soo Park, [email protected]; Eugene C. Yi, euyi@ 3 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of snu.ac.kr; or Daehee Hwang, [email protected]. Science and Technology, Pohang, Korea Received 14 October 2013 and accepted 11 April 2014. 4Department of Systems Immunology, College of Biomedical Science, and Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Korea This article contains Supplementary Data online at http://diabetes 5Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1. School of Convergence Science and Technology, and College of Medicine or B.K.K., S.C., K.M.K., and M.J.K. contributed equally to this work. College of Pharmacy, Seoul National University, Seoul, Korea © 2014 by the American Diabetes Association. Readers may use this article as 6Department of Pediatrics, Seoul National University College of Medicine, Seoul, long as the work is properly cited, the use is educational and not for profit, and Korea the work is not altered. diabetes.diabetesjournals.org Koo and Associates 3023 BMI (kg/m Data are mean Duration (years)Female sex 0 (0 6 (37.5) 7 (43.8) 14 (51.9) 47 (49.5) 20 (40.8) 6 (54.5) 33 (50.0) 10 (50.0) 21 (63.6) 17 (50.0) Fasting C-peptide (ng/mL) 0.4 Age (years)Number of subjects 16 42 16 27 95 49 11 66 20 33 34 HbA 12% of adults ,50 years of age, and very rarely for adults Table 1 .60 years of age (9). Consequently, there has been a need 1c for novel markers that can reflect b-cell destruction in such (%) 8.9 — Clinical characteristics of subjects a broad spectrum of T1DM, which in turn can be used to 2 ) 21.5 differentiate T1DM from other classes of diabetes. Thus, we systematically explored autoantibodies asso- 6 SD, ciated with T1DM in a large-scale screening study of n autoantibody repertoires in subjects with T1DM by using (%), or median (range) unless otherwise indicated. *T1DM excluding LADA. ahigh-density,fluorescence-based protein microarray con- taining duplicate spots of 9,480 human proteins derived from the Ultimate ORF Clone Collection (Life Technolo- T1DM T2DM NGT T1DM* T2DM LADA NGT Graves T1DM NGT 6 6 6 6 gies) to immunologically characterize a broad spectrum – 1) 15 (1 0.4 1.8 16 68 2.4 8.1 of T1DM. Among 9,480 proteins, we selected two candi- 3.0 24.4 date autoantibodies—anti–eukaryote translation elonga- tion factor 1 a 1 (EEF1A1) autoantibody (EEF1A1-AAb) First cohort Second cohort Third cohort and anti–ubiquitin-conjugating enzyme 2L3 (UBE2L3) au- 6 6 6 6 — – 21) 0.7 1.8 toantibody (UBE2L3-AAb) based on multivariate partial 1.5 5.2 368 3.5 23.7 least squares–discriminant analysis (PLS-DA) and immuno- fluorescence staining and validated them in two indepen- dent cohorts. –– 6 6 6 6 0.7 0.3 RESEARCH DESIGN AND METHODS 0.3 9.4 232 3.3 21.3 Sample Preparation For autoantibody profiling, we carefully selected the first cohort of 16 Korean patients with recent-onset T1DM, 16 3(0 6 6 6 6 patients with type 2 diabetes (T2DM), and 27 healthy – 14) 6 (0 0.3 2.5 2.9 8.1 11 50 control subjects with normal glucose tolerance (NGT). 3.2 25.7 This cohort was used to screen candidate autoantibodies using protein microarrays. Serum samples were drawn from T1DM patients with 1) a fasting C-peptide level 6 6 6 6 , , – 0.3 nmol/L or serum C-peptide 0.6 nmol/L after glu- 21) 3 (0 0.9 0.5 1.7 9.1 15 48 cagon loading (10), 2) an initiation of insulin treatment 3.5 20.5 within 6 months after diagnosis, and 3) a duration of diabetes #12 months. Mean age of patients with T1DM in the first cohort was 42 6 16 years (Table 1). The 6 6 6 6 – 9) 0.5 1.6 2.0 5.2 control serum samples were obtained from T2DM 11 66 3.0 24.1 patients who were treated only with oral antidiabetic drugs for at least 5 years and from subjects with NGT who had no history of diabetes, no first-degree relatives , —— 6 6 6 with diabetes, a fasting plasma glucose concentration of 6.1 6 0.6 0.3 2.9 mmol/L, and an HbA1c value of ,5.8% (40 mmol/mol) 230 (Table 1). The second cohort to test the validity of the autoanti- body candidates comprised 95 T1DM, 49 T2DM, 11 LADA, — — — and 66 NGT subjects, where T1DM patients met the first 6 and second selection criteria of the first cohort. Among 88 them, 11 patients with fulminant T1DM, as defined 16.8 0.4 9.6 according to a previous report (11), were included. Mean 0(0 6 6 6 6 age of patients with T1DM in the second cohort was 32 6 – 7) 0.3 2.2 5.4 410 11 years, and median duration of diabetes was 3 (range 0– 2.5 17.7 14) years (Table 1). The same criteria for T2DM and NGT in the first cohort were used. Patients with LADA defined as previously reported (6) (n = 11) and patients with — — 6 6 Graves disease who had no history of diabetes as the au- 6 0.5 1 toimmune disease control (12) (n = 20) were also included 2.5 in the second cohort. The third cohort, comprising 33 chil- dren with T1DM and 34 healthy children, was used as an 3024 Novel Autoantibodies in Type 1 Diabetes Diabetes Volume 63, September 2014 independent test set. The same criteria for T1DM in the We then applied PLS-DA to compute the variable second cohort were used for the third cohort. Age of importance in projection (VIP) representing the relative patients with T1DM in the third cohort was 8 6 4years, contribution of each autoantibody to the separation and median duration of diabetes was 0 (range 0–7) years.
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