118 Diabetes Care Volume 39, January 2016

Matilda Juusola,1,2 Anna Parkkola,1,2 Positivity for Zinc Transporter 8 Taina Hark¨ onen,¨ 1,2 Heli Siljander,1,2 Jorma Ilonen,3,4 Hans K. Akerblom,˚ 1,2 Autoantibodies at Diagnosis Is Mikael Knip,1,2,5,6 and the Childhood Subsequently Associated With Diabetes in Finland Study Group* Reduced b-Cell Function and Higher Exogenous Insulin RequirementinChildrenand Adolescents With Type 1 Diabetes Diabetes Care 2016;39:118–121 | DOI: 10.2337/dc15-1027

OBJECTIVE This study assessed the relationship between autoantibodies against zinc trans- porter 8 (ZnT8A) and disease characteristics at diagnosis of type 1 diabetes and during the first 2 years.

RESEARCH DESIGN AND METHODS Children, younger than 15 years of age (n = 723) who were newly diagnosed with diabetes, were analyzed for ZnT8A, other diabetes-associated autoantibodies, HLA DR-DQ alleles, and metabolic status, which was monitored by pH, plasma 1Children’s Hospital, University of Helsinki and glucose, and occurrence of ketoacidosis at diagnosis and through follow-up of Helsinki University Hospital, Helsinki, Finland C-peptide concentrations, exogenous insulin dose, and glycosylated hemoglobin 2Research Programs Unit, Diabetes and Obesity, for 2 years after the diagnosis. University of Helsinki, Helsinki, Finland 3Immunogenetics Laboratory, University of RESULTS Turku, Turku, Finland 4Department of Clinical Microbiology, University ZnT8A positivity was detected in 530 children (73%). Positivity for ZnT8A was of Eastern Finland, Kuopio, Finland associated with older age (median 8.9 vs. 8.2 years, P = 0.002) and more frequent 5Folkhalsan¨ Research Center, University of ketoacidosis (24% vs. 15%, P = 0.013). Children carrying the HLA DR3 allele were Helsinki, Helsinki, Finland 6 less often ZnT8A positive (66% vs. 77%, P = 0.002) than others. ZnT8A-positive Tampere Center for Child Health Research, Tampere University Hospital, Tampere, Finland children had lower serum C-peptide concentrations (P = 0.008) and higher insulin P Corresponding author: Mikael Knip, mikael. doses ( = 0.012) over time than their ZnT8A-negative peers. knip@helsinki.fi. NOVEL COMMUNICATIONS IN DIABETES CONCLUSIONS Received 15 May 2015 and accepted 26 Septem- ber 2015. Positivity for ZnT8A at diagnosis seems to reflect a more aggressive disease pro- This article contains Supplementary Data online cess before and after diagnosis. at http://care.diabetesjournals.org/lookup/ suppl/doi:10.2337/dc15-1027/-/DC1. The clinical diagnosis of type 1 diabetes is preceded by an asymptomatic preclinical *A complete list of the investigators of the phase, during which autoantibodies against intracellular antigens of the b-cells Childhood Diabetes in Finland Study Group can be found in the Supplementary Data online. appear in the circulation (1). Zinc transporter 8 (ZnT8) is the most recently discov- © 2016 by the American Diabetes Association. ered diabetes-associated autoantigen (2). The aim of this study was to assess the Readers may use this article as long as the work is relationship between ZnT8A on the one hand and demographic characteristics, properly cited, the use is educational and not for other diabetes-associated autoantibodies, HLA risk markers, the degree of profit, and the work is not altered. care.diabetesjournals.org Juusola and Associates 119

metabolic decompensation at diagnosis, Markers of Metabolic Status antibodies. When ZnT8A was added, and the natural course of the disease Metabolic parameters included pH and this number decreased by 31% (n =9). during the first 2 years after diagnosis plasma glucose at diagnosis, analyzed in The most sensitive (97.9%) combination on the other hand. the local laboratories. Diabetic ketoaci- conferred by three autoantibody assays dosis was defined as blood pH #7.30. was the analysis of ZnT8A, GADA, and RESEARCH DESIGN AND METHODS Random serum C-peptide concentra- IA-2A, leaving only 2.1% of the subjects Subjects tions, glycosylated hemoglobin (GHb), undetected. The population-based, nationwide and exogenous insulin dose were moni- Childhood Diabetes in Finland (DiMe) tored for 2 years after diagnosis. The Relationship Between ZnT8A and study was conducted from 1986 to C-peptide concentrations were mea- HLA Genotypes 1989. All patients younger than 15 sured with a radioimmunoassay (8), Among the 226 children carrying the years who were diagnosed with type 1 using antiserum K6 (Novo Research In- HLA DR3 allele, only 65.9% tested posi- diabetes according to the World Health stitute, Bagsvaerd, Denmark). The intra- tive for ZnT8A, whereas 76.7% of the Organization criteria were invited to assay coefficient of variation was 1.8%, DR3-negative subjects were ZnT8A pos- participate. The study involved 801 par- and the interassay coefficient of varia- itive (P , 0.001). Of the HLA-associated ticipants; serum samples were initially tion was 10%. We have previously genotypes, the highest frequency available from 758 children. The current shown that random serum C-peptide (79.9%) and median ZnT8A titer levels study population comprised 723 chil- levels correlate strongly with other stan- (3.0 RU, P = 0.009) were seen among dren (55.4% male) because no serum dardized C-peptide analyses (9). Stan- those carrying the non-DR3/DR4 was any longer available from 35 chil- dard methods for blood GHb analyses genotype. dren. These 723 patients represent the were used in the various hospitals. To index cases in the DiMe study, described compare the results, data were expressed Metabolic State at Diagnosis At diagnosis, children who were positive in detail elsewhere (3). The ethical com- as SD above mean for subjects without for ZnT8A had lower blood pH (mean mittees of all participating hospitals diabetes in each laboratory (10). 7.33 vs. 7.36, P = 0.002) and were approved the study protocol, and the more likely to have ketoacidosis than parents gave written informed consent Statistical Analysis children who were negative for ZnT8A to their child’s participation. Serum The data were statistically evaluated us- (23.9% vs. 15.0%, P = 0.013). samples were stored at –708C. ing cross-tabulation, the Kruskal-Wallis test, and the Mann-Whitney U test. Autoantibody Assays Mixed between- and within-subjects Natural Course of Type 1 Diabetes Serum ZnT8A levels were analyzed by a ANOVA was used to explore the effect At the time of diagnosis, ZnT8A-positive radiobinding assay as described earlier of ZnT8A positivity on serum C-peptide and -negative children had serum (4,5). Islet cell antibody (ICA) was de- concentrations, GHb levels, and the ex- C-peptide concentrations of the same tected with indirect immunofluores- ogenous insulin dose during the 2-year magnitude and a similar need of exoge- cence, whereas GAD antibody (GADA), follow-up period. Serum C-peptide con- nous insulin. The serum C-peptide con- IA-2A, and insulin autoantibody (IAA) centrations were not normally distributed centrations were, however, higher in were quantified with specific radiobind- and were log transformed. Statistical the ZnT8A-negative patients during the ing assays (6). We used a cutoff limit for analyses were performed with SPSS follow-up period (P = 0.008). When the ICA positivity of 2.5 JDFU. Antibody lev- 21.0 software (IBM Corp., Armonk, serum C-peptide concentrations were fi els for ZnT8A, GADA, IA-2A, and IAA NY). A P value ,0.05 was considered compared at speci c times, these were were expressed in relative units (RU). significant. lower in the ZnT8A-positive children The cutoff limits corresponding to the during the second year (Fig. 1A). The 99th percentile in 374 nondiabetic chil- RESULTS daily insulin dose was significantly dren are 0.61 RU for ZnT8A, 3.48 RU for Frequencies and Levels of ZnT8A higher in ZnT8A-positive children during IAA, 5.36 RU for GADA, and 0.43 RU for Of723childrentestedforZnT8A, the follow-up (Fig. 1B, P = 0.012). There IA-2A. The disease sensitivity and spec- 530 (73.3%) were positive (median was no significant difference in GHb ificity of the ZnT8A assay were 60% and 4.13 RU, range 0.61–733.8 RU). The over time (Fig. 1C). 100%, respectively, according to the ZnT8A-positive children were generally CONCLUSIONS 2010 Diabetes Autoantibody Standardi- older (median age 8.85 vs. 8.23 years, zation Program. P = 0.002). The association between ZnT8A positiv- ity, C-peptide concentrations, and insu- HLA Typing Combinations and Associations lin requirement over the first 2 years HLA typing of the main predisposing Between Autoantibodies suggests that a strong initial humoral DQA1-DQB1 genotypes and DRB1*04 ZnT8A positivity concurred consistently immune response against ZnT8 sup- subtypes was performed with a PCR- with ICA and/or IA-2A positivity but less presses the recovery ability of the resid- based oligonucleotide hybridization often with GADA- or IAA-positivity. ual b-cell function after diagnosis. ZnT8 and time-resolved fluorometry (7). The These proportions varied according to might be a regulator of b-cell function, DR3-DQA1*05-DQB1*02 haplotype has age (Supplementary Table 1). When an- the observed association between a beenshortenedtoDR3andHLA- alyzed for the four classical antibo- polymorphism in the SLC30A8 gene DRB1*04-DQB1*0302 to DR4. HLA typ- diesdICA, IAA, GADA, and IA-2Ad13 and type 2 diabetes supporting such a ing data were available for 682 patients. children (1.8%) had no detectable role (11). Positivity for ZnT8A increases 120 ZnT8A Positivity and Type 1 Diabetes Diabetes Care Volume 39, January 2016

Figure 1—Serum C-peptide concentrations (A), exogenous daily insulin dose (B), and GHb SDS values (C) in 530 ZnT8A-positive children and 193 ZnT8A-negative children over the first 2 years after the diagnosis of type 1 diabetes. The top, middle, and bottom horizontal bars show the 25th, 50th, and 75th percentiles, respectively, and the whiskers describe the highest and lowest value that is #1.5 times the interquartile range. The ZnT8A-positive children had lower C-peptide concentrations over time (P = 0.008 according to the mixed between- and within-subjects ANOVA). The significant P values for specific time points (Mann-Whitney U test) are indicated in the figure. The ZnT8A-positive children had higher daily insulin doses over time (P = 0.012 according to the mixed between- and within-subjects ANOVA). The most conspicuous difference was seen at 2 years. There was no significant difference in GHb standard deviation score (SDS) values over time (P =0.819)accordingtothemixedbetween- and within-subjects ANOVA.

the risk of ketoacidosis at diagnosis, by a recent report on T-cell responses to may accordingly complement each other in reflecting a more aggressive disease. ZnT8 in type 1 diabetes (12). Although our relation to HLA associations. In this study we observed a strong in- study indicates that the frequency of ZnT8A Prospective studies have shown that verse relationship between the HLA DR3 is reduced in DR3-positive individuals, high positivity for two or more diabetes- allele and ZnT8A, suggesting that the GADA levels have been reported to be as- associated autoantibodies is a strong pre- DR3-DQ2 haplotype protects against auto- sociatedwithHLADR3andhighIA-2Atiters dictor of progression to type 1 diabetes, immunity to ZnT8. This view is supported with DR4 (7). These three autoantibodies with ;70% of such individuals presenting care.diabetesjournals.org Juusola and Associates 121

with clinical disease during a 10-year fol- Life Insurance Companies, and the University of diabetes. Diabetes Metab Res Rev 2013;29: low-up (13). The current results suggest Helsinki. 646–654 fl ¨ that the combination of GADA, ZnT8A, Duality of Interest. No potential con icts of 6. Kukko M, Kimpimaki T, Korhonen S, et al. interest relevant to this article were reported. Dynamics of diabetes-associated autoanti- and IA-2A assays might be a feasible and Author Contributions. M.J., A.P., T.H., H.S., bodies in young children with human leukocyte cost-effective approach for the detection and J.I. researched data. M.J., H.S., and M.K. antigen-conferred risk of type 1 diabetes re- of b-cell autoimmunity because only wrote the manuscript. H.K.A.˚ was principal in- cruited from the general population. J Clin En- 2.1% of the subjects in the current study vestigator of the DiMe study. All authors re- docrinol Metab 2005;90:2712–2717 viewed and edited the manuscript. M.K. is the 7. Hermann R, Turpeinen H, Laine AP, et al. HLA would have remained undetected. guarantor of this work and, as such, had full DR-DQ-encoded genetic determinants of Positivity for ZnT8A at diagnosis was access to all data in the study and takes respon- childhood-onset type 1 diabetes in Finland: an anal- associated with reduced b-cell function, sibility for the integrity of the data and the ac- ysis of 622 nuclear families. Tissue Antigens 2003;62: increased insulin requirement, and a curacy of the data analysis. 162–169 higher frequency of diabetic ketoacido- 8. Heding LG. Radioimmunological determina- tion of human C-peptide in serum. Diabetologia sis at diagnosis. Altogether, positivity for References 1975;11:541–548 ZnT8A seems to reflect a more aggres- 1. Knip M, Siljander H. Autoimmune mecha- 9. Karjalainen J, Knip M, Mustonen A, Akerblom˚ sive disease process both before and nisms in type 1 diabetes. Autoimmun Rev HK. Insulin autoantibodies at the clinical mani- after diagnosis. 2008;7:550–557 festation of type 1 (insulin-dependent) diabetes– 2. Wenzlau JM, Juhl K, Yu L, et al. The cation a poor predictor of clinical course and antibody efflux transporter ZnT8 (Slc30A8) is a major response to exogenous insulin. Diabetologia autoantigen in human type 1 diabetes. Proc 1988;31:129–133 Acknowledgments. Sirpa Anttila (Department Natl Acad Sci U S A 2007;104:17040–17045 10. Komulainen J, Lounamaa R, Knip M, Kaprio of Pediatrics, University of Oulu, Oulu, Finland), 3. Tuomilehto J, Lounamaa R, Tuomilehto-Wolf EA, Akerblom HK; Childhood Diabetes in Finland Berta Davydova (Children’s Hospital, University E, et al. Epidemiology of childhood diabetes mel- Study Group. Ketoacidosis at the diagnosis of of Helsinki, Helsinki, Finland), and Riitta Pakkil¨ a¨ litus in Finland–background of a nationwide study type 1 (insulin dependent) diabetes mellitus is (Department of Pediatrics, University of Oulu, of type 1 (insulin-dependent) diabetes mellitus. related to poor residual beta cell function. Arch Oulu, Finland) are acknowledged for their excel- The Childhood Diabetes in Finland (DiMe) Study Dis Child 1996;75:410–415 lent technical assistance. Group. Diabetologia 1992;35:70–76 11. Rutter GA, Chimienti F. SLC30A8 muta- Funding. This study was supported by grants 4. Wenzlau JM, Liu Y, Yu L, et al. A common tions in type 2 diabetes. Diabetologia 2015; from Folkhalsan¨ Research Foundation, Academy nonsynonymous single nucleotide polymor- 58:31–36 of Finland (Centre of Excellence in Molecular phism in the SLC30A8 gene determines ZnT8 12. Dang M, Rockell J, Wagner R, et al. Human Systems Immunology and Physiology Research autoantibody specificity in type 1 diabetes. type 1 diabetes is associated with T cell autoim- 2012–2017, Decision No. 250114), Helsinki Uni- Diabetes 2008;57:2693–2697 munity to zinc transporter 8. J Immunol 2011; versity Hospital Research Funds, and the Liv och 5. Salonen KM, Ryhanen¨ S, Hark¨ onen¨ T, Ilonen 186:6056–6063 Halsa¨ Foundation. The DiMe project has been J, Knip M; Finnish Pediatric Diabetes Register. 13. Ziegler AG, Rewers M, Simell O, et al. Sero- supported by grants from the National Insti- Autoantibodies against zinc transporter 8 are conversion to multiple islet autoantibodies tutes of Health (grant DK-37957), the Sigrid related to age, metabolic state and HLA DR ge- and risk of progression to diabetes in children. Juselius´ Foundation, the Association of Finnish notype in children with newly diagnosed type 1 JAMA 2013;309:2473–2479