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 M (kg/m BMI aaaemean are Data eaesx6(75 4.)1 5.)4 4.)2 4.)6(45 3(00 0(00 1(36 7(50.0) 17 (63.6) 21 (50.0) 10 (50.0) 33 (54.5) 6 (40.8) 20 (49.5) 47 (51.9) 14 (43.8) 7 (37.5) (0 6 0 (years) Duration sex Female atn -etd n/L 0.4 (ng/mL) C-peptide Fasting ubro ujcs1 62 54 16 03 34 33 20 66 11 49 95 27 16 42 16 (years) Age subjects of Number HbA 12% of adults ,50 years of age, and very rarely for adults 1 Table .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 (%) — lnclcaatrsiso subjects of characteristics Clinical 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 LADA. excluding *T1DM indicated. otherwise unless (range) median or (%), ahigh-density,ﬂuorescence-based protein microarray containing duplicate spots of 9,480 human proteins derived

from the Ultimate ORF NGT Clone T1DM Collection Graves (Life NGT Technolo- LADA T2DM T1DM* NGT T2DM T1DM 6 6 6 6 gies) to immunologically characterize a broad spectrum – )1 (1 15 1) . 1.8 0.4 668 16 . 8.1 2.4 of T1DM. Among 9,480 proteins, we selected two candi- 24.4 3.0 date autoantibodies—anti–eukaryote translation elongation factor 1 a 1 (EEF1A1) cohort Third autoantibody (EEF1A1-AAb) cohort Second cohort First and anti–ubiquitin-conjugating enzyme 2L3 (UBE2L3) au- 6 6 6 6 — – 21) . 1.8 0.7 toantibody (UBE2L3-AAb) based on multivariate partial 5.2 1.5 368 . 23.7 3.5 least squares–discriminant analysis (PLS-DA) and immunofluorescence staining and validated them in two independent cohorts. –– 6 6 6 6 . 0.3 0.7 RESEARCH DESIGN AND METHODS 9.4 0.3 232 . 21.3 3.3 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 – 4 (0 6 14) . 2.5 0.3 . 8.1 2.9 150 11 control subjects with normal glucose tolerance (NGT). 25.7 3.2 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- (0 3 21) . 0.5 0.9 . 9.1 1.7 548 15 cagon loading (10), 2) an initiation of insulin treatment 20.5 3.5 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) . 1.6 0.5 . 5.2 2.0 control serum samples were obtained from T2DM 66 11 . 24.1 3.0 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 autoantibody 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 . 5.4 2.2 410 11 years, and median duration of diabetes was 3 (range 0– 17.7 2.5 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 children 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. among T1DM, T2DM, and NGT samples (21). To deter- Mean age of healthy children was 10 6 1 years (Table 1). mine a cutoff for significant VIPs, we estimated an em- For patients with T1DM, serum GADA concentration pirical distribution of VIP for the null hypothesis (i.e., an was measured by a radioimmunoassay (RSR Ltd.). GADA autoantibody has no contribution to the separation) by values .1.45 International Units/mL were considered applying PLS-DA followed by Gaussian kernel density es- positive. timation method (22) to randomly permutated experi- This study was conducted in accordance with the ments. We then determined a VIP cutoff of 1.59 as the provisions of the Declaration of Helsinki for participation 90th percentile of the empirical VIP distribution. Thus, of subjects in human research. The institutional review the autoantibodies with VIP .1.59 were selected as those board of the Clinical Research Institute at Seoul National significant contributing to the separation among T1DM, University Hospital approved the study protocol, and T2DM, and NGT samples. written informed consent was obtained from each subject. Immunofluorescence Staining and Protein Microarray Experiments Immunohistochemistry Protein microarray platform version 5.0 (ProtoArray; Primary anti-EEF1A1 (Merck Millipore), anti-UBE2L3 Invitrogen) contains duplicate spots of 9,480 N-terminal (Santa Cruz), and anti-chromosome 7 open reading frame glutathione S-transferase–tagged human proteins printed 53 (c7orf53) (Santa Cruz) antibodies were loaded onto on a nitrocellulose slide. In this array, the antigens have slides of pancreas tissue of a subject who underwent their native cellular enzymatic activities and conforma- partial pancreatectomy due to serous adenoma. Alexa tions. Assays were performed according to the instruc- Fluor 647– (Merck Millipore), 594– (Invitrogen), or 488– tions of the manufacturer (13) as follows: Serum diluted (Invitrogen) conjugated secondary antibodies were used 1:500 in PBS with Tween was incubated on microarray to visualize insulin, glucagon, and candidate autoantigens, slides for 90 min, and Alexa Fluor 647–conjugated anti- respectively. The cell nuclei were counterstained with human IgG was used to detect the reaction. The array DAPI (Invitrogen). An Olympus FluoView FV1000 confo- slides were scanned with a GenePix 4000B fluorescent cal laser scanning microscope was used to acquire data. To scanner (Molecular Devices). investigate the expression of candidate autoantigens in other tissues, including liver, kidney, stomach, intestine, Analysis of ProtoArray Data muscle, brain, and thyroid gland, immunohistochemistry Signal intensities were acquired using ProtoArray Pros- using tissue array (SuperBioChips) was performed with pector, a software tool developed for ProtoArray data by EnVision System-HRP–labeled polymer (Dako) according fl Invitrogen (14). We converted uorescent intensities for to the manufacturer’s instructions. the spot proteins (or autoantigens) to log2 intensities and then normalized across the total 59 data sets by using the ELISA and Immunoprecipitation quantile normalization method (15). Protein microarray The differential expression of autoantibodies identified by data used in this study are available from the Gene Ex- ProtoArray in T1DM patients was confirmed by ELISA pression Omnibus database (http://www.ncbi.nlm.nih. using corresponding recombinant proteins. Plates coated gov/gds) under submission number GSE 50866. To deter- with EEF1A1 or UBE2L3 (Origene) were incubated with mine the presence of autoantibodies for each probe, we diluted serum samples (1:1,000–1:10,000) in PBS. All calculated a z score defined as follows: (spot intensity – experiments were performed in triplicate for each patient the averaged intensity of all probes) / SD of all probes. and control sample. The recombinant proteins and their The autoantibody with z score .1.64 (95th percentile of corresponding antibody, anti-EEF1A1, and anti-UBE2L3 all probe intensities) was considered present. To identify were used as the positive control for the assay. Autoanti- autoantibodies showing a significant difference between body bound to the protein was detected with goat anti- two groups (T1DM vs. NGT, T1DM vs. T2DM, or T1DM human IgG-HRP (Jackson ImmunoResearch). The cutoff vs. T2DM + NGT), the M-statistic test in ProtoArray Pros- point for the presence or absence of each autoantibody pector was applied to the normalized intensities as was determined according to the mean absorbance + 3 3 reported previously (16–20). For each probe, M test 1) SD of NGT, which corresponded to ;99% of normal con- produced the number of samples (M-statistic counts) trol. Because titers of autoantibodies may differ between with the intensity greater than or equal to the M-statistic the solid and liquid phases as a result of the difference in threshold in two groups and 2) computed the P value (the exposure of binding epitopes, the relative levels of significance of the difference between two groups) from EEF1A1-AAb and UBE2L3-AAb were analyzed using an the sample sizes and the M-statistic counts in the two immunoprecipitation–Western blot method. T1DM pa- groups. As an initial set of autoantibody candidates, the tient samples categorized as high, medium, and low proteins with P , 0.05 and z score .1.64 in at least one were selected from the second cohort based on ELISA of T1DM samples were selected. absorbance values at 450 nm. Autoantibodies present in diabetes.diabetesjournals.org Koo and Associates 3025 these groups of T1DM and NGT samples were immuno- T1DM samples (Fig. 1A). Of these, we focused on the precipitated using EEF1A1-flag or UBE2L3-flag. Autoanti- 69 autoantibodies (Fig. 1B and Supplementary Table 2) body:antigen immune complex captured with antiflag identified from T1DM versus NGT + T2DM, which over- M2-antibody–conjugated magnetic beads was eluted from lapped with those in either T1DM versus NGT or T1DM the beads following the manufacturer’s protocol (Sigma) versus T2DM. and analyzed by Western blot using anti-human IgG- Selection of Autoantibodies for Validation in a Large- HRP. Antigens coeluted from the beads were detected Scale Cohort using antibodies for EEF1A1 and UBE2L3. An irrelevant To identify a set of biomarkers predictive of T1DM, it is human IgG antibody (ForteBio) was used as a control for important to evaluate the relative contribution of the 69 the immunoprecipitation assay. proteins to the separation between T1DM and T2DM/ Statistical Analysis NGT samples. To this end, we applied multivariate PLS- Statistical analysis for the microarray studies was as just DA (21) to the abundance of the 69 autoantibodies to described. Results of ELISA experiments were evaluated evaluate their collective contribution to the separation by means of independent t test. The x2 test was used to (Fig. 2A). The PLS-DA result showed that a clear separa- evaluate differences in the proportion of antibody-positive tion between T1DM and T2DM/NGT samples can be subjects per group. The number of test sets for ELISA was achieved by the differential expression of the 69 proteins determined from the results of ELISA in the second cohort. (Fig. 2B and Supplementary Table 3). The relative col- That is, to reliably identify a significant group difference by lective contribution of individual autoantibodies to the the two-sample t test with a type I error rate of 0.05 and statistical power of 0.8, 32 subjects in disease and control groups were needed for the test sets. RESULTS Serum Autoantibody Profiling We profiled autoantibodies in the sera from 16 recent- onset T1DM, 16 T2DM, and 27 NGT subjects by using the ProtoArray human protein microarray version 5 containing 9,480 different proteins spotted in duplicate. These 9,480 proteins correspond to 6,867 of the 19,050 annotated proteins (36.0%) (Swiss-Prot 2013_05), which include 1,632 of 4,673 genes (34.9%) encoding proteins localized in plasma membrane or extracellular regions based on their gene ontology cellular component annota- tions and 809 of 1,929 proteins (41.9%) previously detected in human serum and plasma samples according to the human plasma proteome project (23). The autoantibodies against 3,060 among the 9,480 proteins were found to be present in at least one of the samples. To assess the data quality, we examined whether the signal intensity of a known autoantigen, GAD65, obtained from ProtoArray reflects a serum concentration of GADA that was independently measured by a conventional radioimmunoassay and confirmed a significant correlation between them (Spearman r = 0.863, P , 0.001). On the other hand, none of IA-2A, ICA, or ZnT8 is spotted in the ProtoArray platform (Supplementary Table 1). Figure 1—Identification of T1DM-associated autoantibodies using fi Identi cation of Autoantibodies Predominantly serum autoantibody profiling. A: T1DM-associated autoantibody Expressed in T1DM candidates whose abundance increased in T1DM. The Venn dia- To identify autoantibodies predominantly expressed in gram shows the numbers of these candidates identified from the three comparisons: 1) T1DM vs. NGT, 2) T1DM vs. T2DM, and 3) T1DM samples, the relative abundance of 3,060 autoanti- B fi 1 T1DM vs. NGT + T2DM. : Increased abundance of the 69 autoantibodies detected in the pro ling were compared among ) bodies in T1DM revealed by hierarchical clustering analysis. The T1DM versus NGT, 2) T1DM versus T2DM, and 3) T1DM columns and rows in the heat map represent samples and autoanti- versus NGT + T2DM by using M test. From these com- bodies, respectively. In each row, the intensities were divided by the parisons, we identified 103, 27, and 79 autoantibodies, SD after subtracting the mean intensity from them (i.e., autoscaling). Yellow and blue represent the increase and decrease in abundance, respectively, whose abundance increased in T1DM with respectively. The color bar represents the gradient of the auto- M test P , 0.05 and z . 1.64 in at least one of the scaled log2 intensities. 3026 Novel Autoantibodies in Type 1 Diabetes Diabetes Volume 63, September 2014

Figure 2—Selection of three autoantibodies associated with T1DM. A: The overall scheme for selection of two novel autoantibodies tested in large-scale cohorts. See RESEARCH DESIGN AND METHODS for detailed procedures. B: Discrimination of T1DM from T2DM and NGT using PLS- DA. The two-dimensional PLS-DA score plot shows a separation among T1DM, T2DM, and NGT samples. The line indicates a decision function between T1DM and T2DM + NGT samples. C: Selection of a second list of autoantibodies with signiﬁcant collective contribution to the separation-based VIP values. The cutoff of 1.59 (line) was determined as described in RESEARCH DESIGN AND METHODS. Among the 50 autoantibodies, four (GADA, EEF1A1-AAb, c7orf53-AAb, and UBE2L3-AAb) were selected (see RESEARCH DESIGN AND METHODS). D: A heat map showing the increased abundance of GADA, EEF1A1-AAb, c7orf53-AAb, and UBE2L3-AAb in T1DM patients compared with T2DM patients and NGT subjects. See Fig. 1B legend for the descriptions of the heat map. separation achieved by PLS-DA was estimated as their VIP also evaluated the contribution of individual samples to values (21) (Supplementary Table 4). A large VIP value the separation captured by the PLS-DA model by using indicates a high contribution to the separation. Among the sample contribution analysis. This analysis showed no the 69 autoantibodies, we selected 50 with VIP values signiﬁcant correlation between disease duration and the .1.59 (Fig. 2C)(seeRESEARCH DESIGN AND METHODS). We distances to the PLS-DA model (r = –0.465 and P = 0.070) diabetes.diabetesjournals.org Koo and Associates 3027

Figure 3—Localization of candidate autoantigens in pancreatic b-cells. Triple immunofluorescence staining of the pancreas using specific antibodies against EEF1A1 (A), UBE2L3 (F), and c7orf53 (K) (green) along with anti-insulin (B, G, and L) (white) and anti-glucagon (C, H, and M) (red) antibodies. The cell nuclei were counterstained with DAPI (D, I, and N) (blue). EEF1A1 and UBE2L3 were mainly enriched in the b-cells in the islets (E and J), whereas c7orf53 was expressed exclusively in a-cells (O). Original magnification 3400, scale bar = 50 mm.

(Supplementary Fig. 1). We further selected the following (Fig. 4A). Of note, EEF1A1-AAb showed a significantly three autoantibodies that were detected in none of NGT higher abundance in T1DM compared with Graves disease samples (i.e., z , 1.64 in all NGT samples) (Fig. 2D): 1) (P = 0.003). On the other hand, UBE2L3-AAb was not EEF1A1-AAb, 2) UBE2L3-AAb, and 3) c7orf53-AAb. For significantly different between T1DM and Graves disease. the selection of the autoantibodies targeting b-cells, we The prevalence of EEF1A1-AAb and UBE2L3-AAb was performed immunofluorescence staining of the pancreas 29.5% and 35.8% in T1DM, respectively, which was sig- using antibodies with specificity for EEF1A1, UBE2L3, nificantly higher than T2DM and NGT (P , 0.001 in and c7orf53. EEF1A1 and UBE2L3 were predominantly both). These antibodies were detected in only 2.0% of expressed in b-cells, whereas c7orf53 was exclusively T2DM and 1.5% of NGT subjects (Table 2). To quantita- found in a-cells (Fig. 3). On the basis of these results, tively assess the accuracy for prediction of T1DM, we EEF1A1-AAb and UBE2L3-AAb were selected as the can- generated receiver operating characteristic curves for the didates for the T1DM-associated autoantibodies for the autoantibodies and computed the areas under the curve subsequent studies. Both EEF1A1 and UBE2L3 were (AUCs). The AUCs (0.73 for EEF1A1-AAb, 0.78 for expressed in skin, stomach, colon, kidney, lung, and ad- UBE2L3-AAb) revealed high accuracy of the autoantibodies renal gland tissues (Supplementary Fig. 2), which was in predicting T1DM (Fig. 4B). The comparison of EEF1A1- consistent with previous reports showing extrapancreatic AAb and UBE2L3-AAb in the third cohort of children expression of EEF1A1 and UBE2L3 (24–27). between T1DM and NGT showed a significantly higher abundance of these autoantibodies in T1DM than in NGT Validation of Novel Autoantibodies subjects (P =0.007and0.020,respectively)(Fig.4C), which We measured the amount of EEF1A1-AAb and UBE2L3- is consistent with the results in the adult T1DM cohort. AAb in serum samples by ELISA in a large cohort, which Western blot analysis of the pull-down autoantibodies comprised 95 T1DM, 49 T2DM, 11 LADA, and 66 NGT showed that the relative abundance of autoantibodies in subjects as well as 20 patients with Graves disease. the T1DM samples measured by the immunoprecipitation EEF1A1-AAb and UBE2L3-AAb were significantly higher method reasonably agrees with the titers of autoanti- in their abundance in T1DM samples than in NGT (P , bodies measured by ELISA (Supplementary Fig. 3), and 0.001 in both) and T2DM (P , 0.001 in both) samples they were well correlated with each other (r = 0.779, 3028 Novel Autoantibodies in Type 1 Diabetes Diabetes Volume 63, September 2014

Figure 4—Testing the validity of the selected autoantibodies in independent cohorts. A: Relative titers of EEF1A1-AAb and UBE2L3-AAb measured by ELISA absorbance at 450 nm in T1DM, T2DM, and NGT subjects in the second cohorts. The signal above (mean + 3 3 SD) of healthy controls (dotted line) was considered positive. The solid line for each group represents the mean value of ELISA measurements. B: Receiver operating characteristic analysis representing the accuracy (AUCs) of the novel autoantibodies in predicting T1DM. C: Relative titers of EEF1A1-AAb and UBE2L3-AAb measured by ELISA absorbance at 450 nm in T1DM, T2DM, and NGT subjects in the third cohorts. The solid line of each group represents the mean value of ELISA measurements. AAb, autoantibody. *P < 0.05, **P < 0.01.

P = 0.039, for EEF1A1-AAb and r = 0.940, P = 0.002, for ZnT8A in adult T1DM (28). The most prevalent autoan- UBE2L3-AAb) (Supplementary Fig. 4). tibody, GADA in T1DM (8), was detected in 76.3% of T1DM patients (71 of 93). To further examine the clinical Clinical Implications of Novel Autoantibodies importance of this ﬁnding, the prevalence of the novel Clinical characteristics of the novel autoantibodies were autoantibodies in the T1DM patients without GADA evaluated in the second cohort. The prevalence of was analyzed. Of the 22 GADA-negative T1DM patients, EEF1A1-AAb and UBE2L3-AAb in T1DM was 29.5% and an equal distribution (31.8%) of autoantibody between 35.8%, respectively, which was comparable to that of EEF1A1-AAb and UBE2L3-AAb was observed, and a total

Table 2—Prevalence of anti-EEF1A1 and anti-UBE2L3 antibodies from ELISA Prevalence of positivity* P values† Graves T1DM T2DM LADA disease NGT T1DM vs. T1DM vs. T1DM vs. T1DM vs. (n = 95) (n = 49) (n = 11) (n = 20) (n = 66) NGT T2DM LADA Graves disease EEF1A1 28 (29.5) 1 (2.0) 0 2 (10.0) 1 (1.5) ,0.001 ,0.001 0.035 0.094 UBE2L3 34 (35.8) 1 (2.0) 0 4 (20.0) 1 (1.5) ,0.001 ,0.001 0.015 0.201 Data are n (%). *The signal above (mean + 3 3 SD) healthy controls was considered as positive. †P values from x2 test. diabetes.diabetesjournals.org Koo and Associates 3029 of 40.9% (9 of 22) had either EEF1A1-AAb or UBE2L3- of subjects with fulminant T1DM (3 of 11), whereas AAb (Fig. 5A), which was higher than that of ZnT8A in an GADA was not detected in fulminant T1DM patients Asian population (29). Additionally, it is worth noting (Fig. 5F and Table 3). There was no difference in the that the proportion of patients with evidence of autoim- prevalence of other autoimmune diseases or history of munity in T1DM was increased from 76.3% to 86.0% diabetic ketoacidosis between T1DM patients with or when two newly identified novel autoantibodies were without EEF1A1-AAb. However, EEF1A1-AAb notably added to GADA. was not detected among the patients with LADA (Table We also observed the trend of negative correlation 2). Furthermore, the characteristics found in patients between the age of onset and the prevalence of EEF1A1- with EEF1A1-AAb were similar to those with UBE2L3- AAb. The age of disease onset was significantly younger in AAb (Table 3 and Fig. 5B, D, and G). UBE2L3-AAb was T1DM patients with EEF1A1-AAb than in those without detected in 18.2% of patients with fulminant T1DM, EEF1A1-AAb (P , 0.001) (Table 3). Categorical analysis whereas UBE2L3-AAb was not detected in patients with showed that the prevalence of EEF1A1-AAb in T1DM in- LADA. creased when the age of disease onset was young, which was not observed for GADA (P , 0.001 for trend) (Fig. DISCUSSION 5B), which was maintained even in the stratified analysis Novel autoantibodies indicative of the autoimmune de- according to duration of diabetes (Fig. 5C). Duration of struction in the serum of T1DM patients can be im- diabetes did not affect the prevalence of EEF1A1-AAb portant in developing new diagnostic and therapeutic (Table 3), and EEF1A1-AAb titer was not associated means for T1DM patients. In this study, we extensively with disease duration (r = 0.153, P = 0.139), whereas profiled autoantibodies by using a protein-antigen micro- ZnT8A has been shown to decline rapidly in the first years array in the sera of 16 T1DM, 16 T2DM, and 27 NGT after disease onset and is less persistent than IA-2A or subjects. Among 9,480 different proteins in the array, GADA in the longer term (30). Of note, the prevalence of we initially selected three novel autoantibody candi- EEF1A1-AAb increased as the C-peptide level decreased dates (EEF1A1-AAb, c7orf53-AAb, and UBE2L3-AAb) by (P = 0.03 for trend), which was not observed for GADA M test coupled with PLS-DA. Considering potential auto- (Fig. 5E). In addition, EEF1A1-AAb was detected in 27.3% antigens to be expressed in pancreatic b-cells, EEF1A1-AAb

Figure 5—Clinical implications of the selected autoantibodies. A: The Venn diagram shows the prevalence of EEF1A1-AAb and UBE2L3- AAb in T1DM with and without GADA. Nine of 22 GADA-negative T1DM patients had either EEF1A1-AAb or UBE2L3-AAb. B: The prevalence of EEF1A1-AAb in T1DM was 45.6% (26 of 57), 8.3% (2 of 24), and 0% (0 of 14) in patients aged <30, 30–39, and $40 years at onset, respectively (P < 0.001 for trend). The negative correlation trend between the age of onset and the prevalence of EEF1A1-AAb (C) and UBE2L3-AAb (D) autoantibodies was maintained even in the analysis stratiﬁed according to duration of diabetes (0, 1–2, 3–5, and >5 years). E: Prevalence of EEF1A1-AAb and UBE2L3-AAb according to fasting C-peptide level. Relative titers of EEF1A1-AAb (F) and UBE2L3-AAb (G) measured by ELISA absorbance at 450 nm among typical patients with T1DM, fulminant T1DM, and LADA in the second cohort. Dotted lines represent the signal above (mean + 3 3 SD) of healthy controls, and the solid lines are the mean value of ELISA measurements in each group. AAb, autoantibody. 3030 Novel Autoantibodies in Type 1 Diabetes Diabetes Volume 63, September 2014

Table 3—Clinical characteristics of T1DM patients with anti-EEF1A1 antibody, anti-UBE2L3 antibody, and anti-GADA Anti-GADA Anti-EEF1A1 antibody Anti-UBE2L3 antibody Positive Negative Positive Negative Positive Negative (n = 71) (n = 22) (n = 28) (n = 67) (n = 34) (n = 61) Female sex 36 (50.7) 9 (40.9) 12 (42.9) 35 (52.2) 12 (35.3) 35 (57.4) Age (years) 29 (17–69) 32 (19–58) 25 (17–33)† 34 (17–69) 23 (17–37)† 34 (17–69) Age of onset (years) 25 (13–66) 29 (10–58) 21 (13–33)† 30 (10–66) 19 (14–36)† 32 (10–66) Disease duration (years) 3.5 6 3.0 2.6 6 3.2 3.3 6 2.7 3.2 6 3.7 2.9 6 2.7 3.4 6 3.7 Insulin requirement (International Units/day/kg) 0.68 6 0.30 0.75 6 0.21 0.73 6 0.28 0.69 6 0.28 0.73 6 0.27 0.69 6 0.29 History of DKA 20 (28.2)‡ 16 (72.7) 11 (35.3) 27 (40.3) 14 (41.2) 24 (39.3) Fulminant T1DM 0‡ 11 (50.0) 3 (10.7) 8 (11.9) 2 (5.9) 9 (14.8) Other autoimmune disease 15 (21.4) 3 (14.3) 5 (18.5) 14 (21.2) 7 (21.1) 12 (20.0) BMI (kg/m2) 21.1 6 3.1 21.6 6 3.3 20.9 6 3.2 21.4 6 3.2 20.5 6 3.1 21.4 6 3.3 Fasting C-peptide (ng/mL) 0.33 6 0.32 0.22 6 0.24 0.22 6 0.23 0.33 6 0.33 0.25 6 0.26 0.33 6 0.33

HbA1c (%) 9.7 6 3.1 8.5 6 2.0 9.7 6 3.6 9.4 6 2.9 9.8 6 3.3 9.3 6 3.0 Prevalence of GADA ——21 (75.0) 50 (76.9) 27 (79.4) 44 (74.6) Prevalence of anti-UBE2L3 27 (38.0) 7 (31.8) 26 (92.9)‡ 8 (11.9) —— Prevalence of anti-EEF1A1 21 (29.6) 7 (31.8) ——26 (76.5)‡ 2 (3.3) Data are n (%), median (range), or mean 6 SD. Anti-EEF1A1 and anti-UBE2L3 antibodies were measured by ELISA, and anti-GADA was measured by radioimmunoassay. DKA, diabetic ketoacidosis. †P , 0.05 from independent t test comparing the subjects without respective autoantibody. ‡P , 0.05 from x2 test comparing the subjects without respective autoantibody. and UBE2L3-AAb were selected as autoantibody candidates they were detected in 27% of patients with fulminant based on immunofluorescence staining for further valida- T1DM without GADA but were not detected in LADA tion studies. The increase in abundance of the selected patients. Thus, the two autoantibodies can provide novel novel autoantibodies in T1DM was confirmed by ELISA diagnostic information and new insights into the classifi- in a second cohort and a third test set of children. cation of and therapy for T1DM as well as into T1DM The prevalence of EEF1A1-AAb and UBE2L3-AAb was pathogenesis. ;30% for T1DM patients and 1.5% for NGT subjects in EEF1A1 and UBE2L3 are involved in protein trans- the present study, which is comparable to that of ZnT8A lation (31) and degradation (24), respectively. We previ- in adult T1DM patients (9). The two identified autoanti- ously reported aminoacyl-transfer RNA synthetase as bodies EEF1A1-AAb and UBE2L3-AAb have several clini- a novel marker of T1DM (32), which is also involved in cal advantages compared with ZnT8A. First, the disease protein translation. EEF1A1 is associated with Felty syn- duration did not affect the prevalence of these autoanti- drome, an autoimmune disease (33), and UBE2L3 is as- bodies. By contrast, ZnT8A was shown to decline rapidly sociated with the risk of such autoimmune diseases as in the first years after disease onset and to be less persis- rheumatoid arthritis (34), Crohn disease (35), and sys- tent than IA-2A or GADA in the longer term (30). This temic lupus erythematosus (36). These data indicate may imply that both EEF1A1-AAb and UBE2L3-AAb are that protein homeostasis represented by these autoanti- efficient diagnostic markers of T1DM for patients with bodies can reflect the autoimmunity in T1DM. Of note, a long duration of diabetes. Second, the prevalence of the two autoantibodies correlated well in their levels (Sup- EEF1A1-AAb in GADA-negative T1DM was 31.8%, which plementary Fig. 5). Additionally, the immunofluorescence is higher than that of ZnT8A in an Asian population (29). staining of the corresponding two autoantigens in the The prevalence further increased to 40.9% when UBE2L3- pancreas using antibodies specific for EEF1A1 and AAb was added. Considering that the prevalence of these UBE2L3 showed that both autoantigens were expressed autoantibodies increased for young T1DM patients at higher levels in islets of Langerhans in the pancreas, (45.6% in T1DM patients with an age of onset ,30 more specifically in b-cells, than in the acinar cells and years), the use of both autoantibodies should be more a-cells. The tissue array data showed that they are efficient for the young patients without GADA. Third, expressed in many tissues, suggesting that autoantibodies T1DM patients with and without GADA showed no sig- targeting EEF1A1 and UBE2L3 may come from epitope nificant difference in the prevalence of EEF1A1-AAb and spreading, which is a typical phenomenon occurring dur- UBE2L3-AAb (38.0 and 28.2% in GADA-positive T1DM ing the preclinical and clinical disease process in T1DM and 31.8 and 27.3% in GADA-negative T1DM for (37). Nonetheless, all these data suggest that the protein EEF1A1-AAb and UBE2L3-AAb, respectively). In addition, homeostasis represented by these proteins can be used to diabetes.diabetesjournals.org Koo and Associates 3031 show the autoimmune destruction of pancreatic islet b-cells. a 2014 Research Grant from Kangwon National University (to K.M.K.), the Pro- Detailed functional studies can be designed to elucidate the teogenomic Research and Priority Research Centers Program (to D.H. and E.C.Y.), mechanisms underlying the links of the protein homeostasis the World Class University program (to E.C.Y. and K.S.P.), Institute for Basic to the T1DM-related autoimmunity in the pancreas. Science grant CA1308 (to D.H.), and the 21C Frontier Functional Proteomics Project (to D.H., E.C.Y., and K.S.P.). Furthermore, our approach involving autoantibody fl fi Duality of Interest. No potential con icts of interest relevant to this article pro ling provided a comprehensive list of autoantibody were reported. candidates that extends extensively the current list of Author Contributions. B.K.K. contributed resources and to the research fi autoantibodies identi ed by previous approaches, such design, experiments, and writing of the manuscript. S.C. contributed to the data as protein expression profiling of pancreas (4) and mea- analysis and writing of the manuscript. K.M.K. contributed resources and to the surements of molecular targets associated with insulin research design, experiments, data analysis, and writing of the manuscript. M.J.K. secretion in b-cells (3,38) or pathologically proven auto- contributed to the experiments, data analysis, and writing of the manuscript. E.G.K. antigens (2,39). Other than the two autoantibodies tested contributed to the experiments and data analysis. S.H.K., H.S.J., Y.M.C., S.H.C., in study, an additional 66 potential novel autoantibodies, Y.J.P., C.H.S., and H.C.J. contributed to the experiments. C.S.S. contributed to including fms-like tyrosine kinase receptor 3 ligand the data analysis. D.H., E.C.Y., and K.S.P. contributed resources and to the (FLT3LG), were previously shown to be associated with research design, data analysis, and writing of the manuscript. D.H., E.C.Y., and K.M.K. are the guarantors of this work and, as such, had full access to all diabetes (40) and identified in the current study (Fig. 1B the data in the study and take responsibility for the integrity of the data and the and Supplementary Table 2). FLT3LG treatment was accuracy of the data analysis. reported to delay diabetes onset in the NOD mouse model Prior Presentation. Parts of this study were presented in abstract form at of autoimmune diabetes (41). Protein kinase C (PRKCA), the 74th Scientific Sessions of the American Diabetes Association, San Francisco, also shown in our list, is associated with multiple sclerosis CA, 13–17 June 2014. (42), which is known to be initiated by a misdirected References immune response against myelin autoantigens (43). In addition, our list includes the molecules associated with 1. Baekkeskov S, Nielsen JH, Marner B, Bilde T, Ludvigsson J, Lernmark A. Autoantibodies in newly diagnosed diabetic children immunoprecipitate human T1DM-related pathophysiology in pancreatic b-cells (Sup- pancreatic islet cell proteins. Nature 1982;298:167–169 plementary Fig. 6), such as insulin secretion [PRCKA (44), 2. Bottazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies in di- ACVR2B (45), GLUL (46), and PSMD9 (47)] and glucose abetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974;2:1279– metabolism [GPI (48)], indicating their potential roles in 1283 the autoimmune destruction of the pancreatic b-cells 3. Rabin DU, Pleasic SM, Shapiro JA, et al. Islet cell antigen 512 is a diabetes- in T1DM. Thus, the list of autoantibody candidates we specific islet autoantigen related to protein tyrosine phosphatases. J Immunol identified in this study can be a comprehensive basis for 1994;152:3183–3188 understanding the links of autoantibodies to T1DM di- 4. Wenzlau JM, Juhl K, Yu L, et al. The cation efflux transporter ZnT8 agnosis and pathogenesis. (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl Acad Sci The clinical implications of the two novel autoantibodies U S A 2007;104:17040–17045 can be further tested with a larger number of GADA- 5. Imagawa A, Hanafusa T, Uchigata Y, et al. Fulminant type 1 diabetes: negative and fulminant T1DM patients. In addition, a nationwide survey in Japan. Diabetes Care 2003;26:2345–2352 longitudinal studies can be designed to further demon- 6. Stenström G, Gottsäter A, Bakhtadze E, Berger B, Sundkvist G. Latent autoimmune diabetes in adults: definition, prevalence, beta-cell function, and strate the nature of dynamic changes of these autoanti- treatment. Diabetes 2005;54(Suppl. 2):S68–S72 bodies during the course of disease progression, and new 7. Imagawa A, Hanafusa T. Fulminant type 1 diabetes—an important subtype subtypes of T1DM patients might be further characterized in East Asia. Diabetes Metab Res Rev 2011;27:959–964 based on the dimension of protein homeostasis repre- 8. Vermeulen I, Weets I, Asanghanwa M, et al.; Belgian Diabetes Registry. sented by these autoantibodies when larger numbers of Contribution of antibodies against IA-2b and zinc transporter 8 to classification adult T1DM patients are used. Nevertheless, our approach of diabetes diagnosed under 40 years of age. Diabetes Care 2011;34:1760– successfully demonstrated the possibility of the use of the 1765 two autoantibodies as diagnostic markers and/or thera- 9. Lampasona V, Petrone A, Tiberti C, et al.; Non Insulin Requiring Autoim- peutic targets. Finally, our autoantibody profiling approach mune Diabetes (NIRAD) Study Group. Zinc transporter 8 antibodies complement can be applied to other autoimmune diseases for which GAD and IA-2 antibodies in the identification and characterization of adult-onset autoantibody markers are unknown, and the newly iden- autoimmune diabetes: Non Insulin Requiring Autoimmune Diabetes (NIRAD) 4. tified autoantibodies can contribute to the understanding Diabetes Care 2010;33:104–108 fi of the mechanisms underlying autoimmune destruction. 10. Hother-Nielsen O, Faber O, Sørensen NS, Beck-Nielsen H. Classi cation of newly diagnosed diabetic patients as insulin-requiring or non-insulin-requiring In summary, our approach successfully identified two novel based on clinical and biochemical variables. Diabetes Care 1988;11:531–537 autoantibodies, EEF1A1-AAb and UBE2L3-AAb, that can fi 11. Imagawa A, Hanafusa T. Fulminant type 1 diabetes mellitus. Endocr J 2006; add a new dimension to the diagnosis, classi cation, therapy, 53:577–584 and pathogenesis of T1DM. 12. Burman KD, Baker JR Jr. Immune mechanisms in Graves’ disease. Endocr Rev 1985;6:183–232 13. Invitrogen. ProtoArray Human Protein Microarray v5.0 for Immune Response Funding. This work was supported by the Seoul National University Hospital BioMarker Profiling (IRBP): IRBP Experienced Users Guide. Grand Island, NY, Life Research Fund (to B.K.K.), the National Research Foundation of Korea (to K.M.K.), Technologies, 2011 3032 Novel Autoantibodies in Type 1 Diabetes Diabetes Volume 63, September 2014

14. Vermeulen N, de Béeck KO, Vermeire S, et al. Identification of a novel 32. Park SG, Park HS, Jeong IK, et al. Autoantibodies against aminoacyl-tRNA autoantigen in inflammatory bowel disease by protein microarray. Inflamm Bowel synthetase: novel diagnostic marker for type 1 diabetes mellitus. Biomarkers Dis 2011;17:1291–1300 2010;15:358–366 15. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normali- 33. Ditzel HJ, Masaki Y, Nielsen H, Farnaes L, Burton DR. Cloning and ex- zation methods for high density oligonucleotide array data based on variance and pression of a novel human antibody-antigen pair associated with Felty’s syn- bias. Bioinformatics 2003;19:185–193 drome. Proc Natl Acad Sci U S A 2000;97:9234–9239 16. Le Roux S, Devys A, Girard C, Harb J, Hourmant M. Biomarkers for the 34. Orozco G, Eyre S, Hinks A, et al.; UKRAG consortium. Study of the common diagnosis of the stable kidney transplant and chronic transplant injury using the genetic background for rheumatoid arthritis and systemic lupus erythematosus. ProtoArrayÒ technology. Transplant Proc 2010;42:3475–3481 Ann Rheum Dis 2011;70:463–468 17. Hu S, Vissink A, Arellano M, et al. Identification of autoantibody biomarkers 35. Fransen K, Visschedijk MC, van Sommeren S, et al. Analysis of SNPs with for primary Sjögren’s syndrome using protein microarrays. Proteomics 2011;11: an effect on gene expression identifies UBE2L3 and BCL3 as potential new risk 1499–1507 genes for Crohn’s disease. Hum Mol Genet 2010;19:3482–3488 18. Babel I, Barderas R, Díaz-Uriarte R, Martínez-Torrecuadrada JL, Sánchez- 36. Harley JB, Alarcón-Riquelme ME, Criswell LA, et al.; International Consor- Carbayo M, Casal JI. Identification of tumor-associated autoantigens for the di- tium for Systemic Lupus Erythematosus Genetics (SLEGEN). Genome-wide as- agnosis of colorectal cancer in serum using high density protein microarrays. Mol sociation scan in women with systemic lupus erythematosus identifies Cell Proteomics 2009;8:2382–2395 susceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nat Genet 2008; 19. Pallier A, Hillion S, Danger R, et al. Patients with drug-free long-term graft 40:204–210 function display increased numbers of peripheral B cells with a memory and 37. Bonifacio E, Lampasona V, Bernasconi L, Ziegler AG. Maturation of the inhibitory phenotype. Kidney Int 2010;78:503–513 humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 20. Sboner A, Karpikov A, Chen G, et al. Robust-linear-model normalization to diabetes. Diabetes 2000;49:202–208 reduce technical variability in functional protein microarrays [published correction 38. Kawasaki E, Hutton JC, Eisenbarth GS. Molecular cloning and character- appears in J Proteome Res 2010;9:636]. J Proteome Res 2009;8:5451–5464 ization of the human transmembrane protein tyrosine phosphatase homologue, 21. Hyung SW, Lee MY, Yu JH, et al. A serum protein profile predictive of the phogrin, an autoantigen of type 1 diabetes. Biochem Biophys Res Commun 1996; resistance to neoadjuvant chemotherapy in advanced breast cancers. Mol Cell 227:440–447 Proteomics 2011;10:M111.011023 39. Maclaren NK, Huang SW, Fogh J. Antibody to cultured human insulinoma 22. Bowman AW, Azzalini A. Applied Smoothing Techniques for Data Analysis: cells in insulin-dependent diabetes. Lancet 1975;1:997–1000 The Kernel Approach With S-Plus Illustrations (Oxford Statistical Sciences Series). 40. Vasu C, Dogan RN, Holterman MJ, Prabhakar BS. Selective induction of New York, Clarendon Press, Oxford University Press, 1997 dendritic cells using granulocyte macrophage-colony stimulating factor, but 23. Farrah T, Deutsch EW, Omenn GS, et al. A high-confidence human plasma not fms-like tyrosine kinase receptor 3-ligand, activates thyroglobulin-specific proteome reference set with estimated concentrations in PeptideAtlas. Mol Cell CD4+/CD25+ T cells and suppresses experimental autoimmune thyroiditis. Proteomics 2011;10:M110.006353 J Immunol 2003;170:5511–5522 24. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and 41. Van Belle TL, Juntti T, Liao J, von Herrath MG. Pre-existing autoimmunity cell life. EMBO J 1998;17:7151–7160 determines type 1 diabetes outcome after Flt3-ligand treatment. J Autoimmun 25. Knudsen SM, Frydenberg J, Clark BF, Leffers H. Tissue-dependent variation 2010;34:445–452 in the expression of elongation factor-1 alpha isoforms: isolation and charac- 42. Barton A, Woolmore JA, Ward D, et al. Association of protein kinase C alpha terisation of a cDNA encoding a novel variant of human elongation-factor 1 alpha. (PRKCA) gene with multiple sclerosis in a UK population. Brain 2004;127:1717–1722 Eur J Biochem 1993;215:549–554 43. Wang H, Munger KL, Reindl M, et al. Myelin oligodendrocyte glycoprotein 26. Chambers DM, Peters J, Abbott CM. The lethal mutation of the mouse antibodies and multiple sclerosis in healthy young adults. Neurology 2008;71: wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of 1142–1146 translation elongation factor 1alpha, encoded by the Eef1a2 gene. Proc Natl Acad 44. Leitges M, Plomann M, Standaert ML, et al. Knockout of PKC alpha en- Sci U S A 1998;95:4463–4468 hances insulin signaling through PI3K [published correction appears in Mol En- 27. Lee S, Francoeur AM, Liu S, Wang E. Tissue-specific expression in mam- docrinol 2002;16:1203]. Mol Endocrinol 2002;16:847–858 malian brain, heart, and muscle of S1, a member of the elongation factor-1 alpha 45. Yamaoka T, Idehara C, Yano M, et al. Hypoplasia of pancreatic islets in gene family. J Biol Chem 1992;267:24064–24068 transgenic mice expressing activin receptor mutants. J Clin Invest 1998;102: 28. Kawasaki E, Uga M, Nakamura K, et al. Association between anti-ZnT8 294–301 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients 46. Tholey G, Sena AH, Ledig M. Specific insulin-mediated regulation of glu- with type 1 diabetes. Diabetologia 2008;51:2299–2302 tamine synthetase in cultured chick astroglial cells. J Neurochem 1986;47:1490– 29. Yang L, Luo S, Huang G, et al. The diagnostic value of zinc transporter 1492 8 autoantibody (ZnT8A) for type 1 diabetes in Chinese. Diabetes Metab Res Rev 47. Mahtani MM, Widén E, Lehto M, et al. Mapping of a gene for type 2 diabetes 2010;26:579–584 associated with an insulin secretion defect by a genome scan in Finnish families. 30. Wenzlau JM, Walter M, Gardner TJ, et al. Kinetics of the post-onset decline Nat Genet 1996;14:90–94 in zinc transporter 8 autoantibodies in type 1 diabetic human subjects. J Clin 48. Farese RV, Standaert ML, Yamada K, et al. Insulin-induced activation of Endocrinol Metab 2010;95:4712–4719 glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin me- 31. Merrick WC. The Protein Biosynthesis, Elongation Cycle. New York, Cold diator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki Spring Harbor Laboratory Press, 2009 rats. Proc Natl Acad Sci U S A 1994;91:11040–11044