Diabetes Care Volume 40, April 2017 561 fi Maria Acevedo-Calado,1 Eddie A. James,2 Identi cation of Unique Antigenic Michael P. Morran,3 Susan L. Pietropaolo,1 Qin Ouyang,1 David Arribas-Layton,2 Determinants in the Amino Marco Songini,4 Marco Liguori,4 Anna Casu,5 Richard J. Auchus,6 Terminus of IA-2 (ICA512) in Shuai Huang,7 Liping Yu,8 Aaron Michels,8 Roberto Gianani,1,9 and Childhood and Adult Autoimmune Massimo Pietropaolo1 Diabetes: New Biomarker Development Diabetes Care 2017;40:561–568 | DOI: 10.2337/dc16-1527 OBJECTIVE EMERGING TECHNOLOGIES AND THERAPEUTICS The characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes. RESEARCH DESIGN AND METHODS 1Division of Diabetes, Endocrinology and Metab- Clinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: olism, Department of Medicine, Baylor College of 0.01–31 years) were evaluated using a new biomarker detecting autoantibodies Medicine, Houston, TX 2Benaroya Research Institute, University of directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA- Washington, Seattle, WA 2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly di- 3Department of Medicinal Chemistry, College of agnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04–0.49 Pharmacy, University of Toledo, Toledo, OH 4Diabetes Clinic, Department of Internal Medicine, years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 and Laboratory of Microbiology & Immunology, autoantibodies were assayed. Azienda Ospedaliera G. Brotzu, Cagliari, Italy 5Istituto Mediterraneo per i Trapianti e Terapie RESULTS ad Alta Specializzazione, Palermo, Italy 6 IA-2ec autoantibodies were detected in patients with type 1 diabetes and, sur- Division of Metabolism, Endocrinology & Diabe- tes, Department of Internal Medicine, University prisingly, in 5% of patients with type 2 diabetes without serologic responses to of Michigan Medical School, Ann Arbor, MI other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the 7Department of Industrial and Systems Engineer- ability of IA-2ec–derived peptides to elicit CD4+ T-cell responses by stimulating ing, University of Washington, Seattle, WA 8 peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and Barbara Davis Center for Childhood Diabetes, Uni- n versity of Colorado School of Medicine, Aurora, CO HLA-matched healthy subjects ( = 13) with peptides and staining with the pep- 9Rocky Vista University College of Osteopathic tide/DQ8-specific tetramers, observing disease-associated responses to previ- Medicine, Parker, CO ously unreported epitopes within IA-2ec. Corresponding author: Massimo Pietropaolo, [email protected]. CONCLUSIONS Received 14 July 2016 and accepted 11 January We developed a new antibody biomarker identifying novel antigenic determi- 2017. nants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in © 2017 by the American Diabetes Association. patients with type 1 diabetes and in a subgroup of adult autoimmune patients Readers may use this article as long as the work with type 2 diabetes phenotype negative for conventional islet autoantibody is properly cited, the use is educational and not for profit, and the work is not altered. More infor- testing. These observations suggest that islet autoimmunity may be more com- mation is available at http://www.diabetesjournals mon in clinically diagnosed type 2 diabetes than previously observed. .org/content/license. 562 Autoimmune Responses to IA-2ec Diabetes Care Volume 40, April 2017 Autoimmune diabetes is considered to evidence for humoral responses directed was in vitro transcribed/translated in the be the end result of an immune-mediated to IA-2ec in patients with type 1 diabetes presence of [35S]methionine (PerkinElmer) injury of the b-cells within the islets of and, surprisingly, in a subgroup of patients using the TNT-coupled rabbit reticulocyte Langerhans (1,2). Circulating autoanti- with clinically diagnosed type 2 diabetes. system (Promega, Madison, WI) with T7 bodies and T-cell responses to islet auto- Finally, we demonstrated cell-mediated RNA polymerase. IA-2ec autoantibodies antigens have allowed the development immunity directed against posttranslation- were detected by radiobinding assay using of diagnostic biomarkers that aid in the ally modified epitopes of the extracellular 50% protein A–Sepharosetoseparatefree identification of subjects at risk for domain of IA-2 in patients with type 1 di- [35S]methionine from antibody-bound la- type 1 diabetes and of a subset of abetes, suggesting that IA-2ec may play a beled products. The assay was run in trip- type 2 diabetes with evidence for islet role in the pathogenesis of autoimmune licate, and the results were expressed autoimmunity (3–6). The latter condition diabetes. as an index calculated as follows: index = is often termed latent autoimmune dia- (serum sample counts per minute [cpm] 2 betes in adults (LADA) (7,8). RESEARCH DESIGN AND METHODS negative control cpm)/(positive control In autoimmune diabetes, several ele- Subjects cpm 2 negative control cpm). We used ments of the secretory pathway of pan- The study population consists of 150 pa- the protein tyrosine phosphatase IA-2 creatic b-cells, such as insulin and tients with type 1 diabetes (74 male and Q-20 antibody (sc-54749; Santa Cruz Bio- protein islet tyrosine phosphatase-like 76 female; mean age 13.40 6 10.35 technology, Santa Cruz, CA) as positive protein (IA-2), are targeted by autoanti- years) and 258 patients with type 2 di- control and serum from individuals with- body and T-cell responses. In type 1 di- abetes (135 male and 123 female; mean out diabetes as negative controls. The cut- abetes, the neuroendocrine molecule age 52.67 6 9.14 years). The sex distri- off point was established as the 99th IA-2 is one of major targets of immune- bution in the two groups was not statis- percentile of values from serum samples mediated responses (9–12). IA-2 is a trans- tically significantly different (P = 0.6078). obtained from 178 healthy volunteers. The membrane glycoprotein of the tyrosine Patients with type 2 diabetes were signif- interassay coefficient of variation (CV) was phosphatase-like protein family, which is icantly older than patients with type 1 13.4% (n = 10), and the intra-assay CV was localized in the insulin-secretory granules diabetes (P , 0.0001). 5.45% (n = 15). The IA-2ec autoantibody of the pancreatic b-cell. This molecule We assayed sera from 150 patients assay achieved ratings of 4% sensitivity contains three domains: the N-terminal with type 1 diabetes from the Barbara and 99% specificity at the Islet Autoanti- extracellular (or luminal) domain (amino Davis Center for Childhood Diabetes, Uni- body Standardization Program 2012. acids 1–556), the transmembrane do- versity of Colorado School of Medicine; The intracellular domain of IA-2 (IA-2ic) main (amino acids 557–600), and the C 100 patients with clinically diagnosed construct (amino acid residues 605–979) terminus intracellular (or cytoplasmic) type 2 diabetes from the Division of Me- was provided by Dr. E. Bonifacio (DFG- domain (amino acids 601–979) containing tabolism, Endocrinology and Diabetes, Center for Regenerative Therapies Dres- a juxtamembrane domain (amino acids University of Michigan Health System; den, TU Dresden, Dresden, Germany). 601–686) and an inactive protein tyrosine and 158 patients with clinically diagnosed The IA-2ic autoantibody radioimmuno- phosphatase domain (amino acids 687– type 2 diabetes from the Diabetes Clinic, assay has a similar assay format as that 979). IA-2 is a pseudophosphatase that Azienda Ospedaliera G. Brotzu, Cagliari, to detect IA-2ec autoantibodies (14). The plays a number of roles within the pancre- Italy. The study was approved by the interassay CV was 9.9%, and the intra- atic islet such as contributing in b-cell pro- respective Institutional Review Boards. assay CV was 4.8%. IA-2ic autoantibodies liferation, aiding in regulating insulin The percentage of Caucasians was achieved ratings of 72% sensitivity and exocytosis, and acting to tether secretory 87 and 92% in the patients with type 1 99% specificity at the 2007 4th assay granules to the cytoskeleton. During insulin and type 2 diabetes, respectively. Diabe- proficiency evaluation of the Diabetes secretion, the cytoplasmic domain of IA-2/ tes was diagnosed according to standard Autoantibody Standardization Program. ICA512 is cleaved and traffics to the nu- American Diabetes Association criteria cleus, whereby it stimulates the transcrip- (15). Serum samples from 178 healthy IA-2ec Antibody Specificity tion of the insulin gene. Albeit the biological control subjects (72 male and 106 fe- To demonstrate specificity of IA-2ec au- role of IA-2 extracellular domain (IA-2ec) male; mean age 34.62 6 10.15 years; toantibodies, we performed inhibition has not been entirely elucidated, stability and 76% Caucasian) were assayed for of autoantibody binding studies. Unla- of pro-ICA512/IA-2 and its targeting to IA-2ec autoantibodies. beledIA-2ec(aminoacidresidues26– insulin secretory granules require b4- Subjects with type 2 diabetes evalu- 577) and IA-2ic (amino acid residues sheet–mediated dimerization of its ecto- ated in this study were treated with diet, 605–979) were expressed in the reticu- domain in the endoplasmic reticulum (13). oral hypoglycemic agents, or insulin locyte lysate system with amino acid We previously found indirect evidence therapy. All participants were assayed mixture containing unlabeled methio- for autoantibodies binding to IA-2ec. The for GAD65, IA-2ic, IA-2ec, and zinc trans- nine (Fig. 3). For competitive binding presence of these autoantibodies was as- porter 8 (ZnT8) autoantibodies. studies, unlabeled antigens were each sociated with a high risk of progression of incubated separately with test human type 1 diabetes (14).