GAD-65 and IA-2 Autoantibodies in Relation to HLA Class II DR and DQ. Alleles and Haplotypes in Type 1 Diabetes Mellitus

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1 CVI Accepts, published online ahead of print on 13 April 2011 Clin. Vaccine Immunol. doi: /cvi Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved GAD-65 and IA-2 Autoantibodies in Relation to HLA Class II DR and DQ Alleles and Haplotypes in Type 1 Diabetes Mellitus Mouna Stayoussef 1, Jihen Benmansour 1, Fayza A. Al-Jenaidi 2,3, Hichem B. Said 4, Chiheb B. Rayana 1, Touhami Mahjoub 1, Wassim Y. Almawi 3 * 1 Research unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, 2 Department of Pediatrics, Salmaniya Medical Complex, Manama, 3 College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain, 4 Department of Endocrinology, CHU Farhat Hached, Sousse, Tunisia, Running Title. GAD-65 and IA-2 Antibodies in Type 1 Diabetes Corresponding Author: Wassim Y. Almawi, Ph.D., Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, P. O. Box 22979, Manama, Bahrain. Tel , Fax , . wassim@agu.edu.bh.

2 ABSTRACT Background. The frequency of autoantibodies against glutamic acid decarboxylase-65 (GAD65), and ICA512/IA2 (IA2) are a function of the specific human leukocyte antigen (HLA) in type 1 diabetes mellitus (T1D). We investigated the association of HLA class II (DR, DQ) alleles and haplotypes with the presence of GAD and IA-2 autoantibodies in T1D. Methods. Autoantibodies were tested in 88 Tunisian T1D patients and 112 age-and gendermatched normoglycemic control subjects by enzyme immunoassay. Results. Among T1D patients, mean anti-gad antibody titers were higher in DRB1* allele (P <0.001), together with DRB1*030101/DQB1*0201 (P <0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes, while lower anti-gad titers were associated with DRB1* (P = 0.001) and DRB1* (P < 0.001) alleles, and DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1* (P = 0.001) haplotypes. Mean anti-ia2 antibody titers were higher in DRB1* allele (P = 0.007) and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes, but were lower in DRB1* allele (P = 0.010) and DRB1* (P < 0.001), and DRB1*110101/DQB1* (P = 0.025) haplotype. Multinomial regression analysis confirmed positive association of DRB1*030101, and negative association of DRB1* and DQB1*030101, along with DRB1*070101/DQB1*0201 and DRB1*110101/DQB1* haplotypes with anti-gad levels. By contrast, only DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-ia2 titers. Conclusion. Increased GAD65- and IA-2-Abs positivity is differentially associated with select HLA class II alleles and haplotypes, confirming the heterogeneous nature of T1D Key Words. Autoantibodies; Haplotype; HLA DRB1 and DQB1; Type 1 Diabetes

3 INTRODUCTION Type 1 diabetes mellitus (T1D) is an endocrine disease characterized by autoimmune 52 destruction of pancreatic ß islet cells [2,27]. Several autoantigens were implicated in triggering this process, which include insulin, the 65-kDa isoform of glutamic acid decarboxylase (GAD) [2,19,26], an enzyme involved in the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid in pancreatic ß islet cells, and islet cell antigen-2 (IA-2), a tyrosine phosphatase expressed in β islet cells [12]. Up to 90% of newly diagnosed T1D are positive for anti-ia-2 and/or anti-gad antibodies, compared to the very low prevalence of these autoantibodies seen in non-diabetic control populations ( 1%) [27,28]. Most peptides derived from GAD and IA-2 autoantigens can bind to both T1D-predisposing and -protective HLA molecules, although some exceptions were observed [8]. Functionally, T cell proliferative responses from T1D patients identified GAD65 as a likely candidate in the development of anti-β islet cell immunity [16]. We and others have confirmed the association of select HLA class II alleles and haplotypes with increased risk of T1D [1,21,29], and have identified both susceptibile (DRB1*030101, DRB1* DQB1*0201, and DRB1* DQB1*0302) and protective (DRB1*070101, DRB1*110101, DRB1* DQB1*0201, and DRB1* DQB1*030101) alleles and haplotypes at the class II DR and DQ loci. Homozygosity for DRB1* DQB1*0201 represented the highest-risk genotype, while DRB1* DQB1*0201 homozygosity was associated with significantly decreased risk. Several studies addressed the likely relationship between HLA risk loci and T1D-associated autoantibodies in both children and adults with newly diagnosed T1D. Presentation of β islet cell autoantigens by high- and low-risk alleles needs to be studied, in order to elucidate the mechanism underlying the effect of HLA class II polymorphism on disease risk [2,26].

4 It was suggested that high-risk HLA antigens function in binding and later presenting autoantigens (including GAD65) to autoreactive T cells [6,16,26]. This was highlighted by the HLA-DR2-and HLA-DR4-restricted T cell lines from new onset T1D patients which binds to naturally processed GAD65 [12,23]; no similar T cell clones could be generated from healthy controls. While lymphocytes could be generated to synthetic GAD65 peptides from T1D patients [3], their reactivity to naturally processed GAD65 could not be demonstrated. In this study, we investigate the association between anti-gad and anti-ia-2 antibody titers and HLA class II (DR, DQ) alleles and haplotypes. SUBJECTS AND METHODS Subjects. Study subjects comprised 88 unrelated T1D patients (44 males and 44 females; age 16.4 ± 7.7 years). The diagnosis of T1D was according to clinical features and laboratory data. All T1D patients were ketosis-prone, lacked endogenous insulin secretion, and were dependent on insulin for controlling hyperglycemia. T1D patients were not obese, were free of any concomitant complication, and did not receiving additional treatment at the time of blood collection. Patients with other forms of diabetes (LADA, MODY, type 2 diabetes) were excluded. Control subjects consisted of 112 university students and healthy children (65 males and 47 females; age 28.2 ± 5.8 years), who had normal glucose tolerance and no family history of T1D or other autoimmune diseases. All patients and control subjects were Tunisian Arabs, were from central Tunisia, and were asked to sign a consent form according to the study protocol, and all institutional ethics requirements were met HLA-DRB1 and DQB1 Genotyping. HLA-DRB1 and DQB1 gene alleles were analyzed using the PCR-sequence-specific priming (SSP) technique, using the Micro SSP Generic HLA Class II (DRB/DQB) DNA Typing kit (Lot #05A), according to manufacturer s specifications (One Lambda, Thousand Oaks, CA). PCR products were analyzed on ethidium

5 bromide-stained agarose gel. HLA alleles nomenclature was as previously reported [15]. In total, 16 DRB1 and 7 DQB1 alleles were tested. Autoantibody Screening. IA-2, GAD-65 autoantibodies were measured at the time of initial diagnosis for T1D patients. Serum samples were obtained from all participants, and were 103 stored as small aliquots at or below -40 C. Anti-IA-2 and anti-gad-65 antibodies were measured on two separate occasions by ELISA (Kronus Inc., Boise, ID). Cutoff values for antibody positivity were based on the 99th percentile of antibody levels obtained in nondiabetic controls. Results were expressed as antibody titers (AU/ml), or as percent antibody positive of total. Statistical Analysis. Statistical analysis was performed on SPSS v (SPSS Inc., Chicago, IL). Allele frequencies were determined by the gene counting method, using HLAStat 2000 software, and haplotypes frequencies were determined by the maximum likelihood method. P values were corrected for the number of different alleles or haplotypes tested (Pc) using the Bonferroni inequality method (Pc = 1 (1 P) n ). Data were expressed as P values, odds ratios (OR) and 95% confidence intervals (CI) between patients and controls. Spearman correlation coefficient was used to determine correlation between level of autoantibodies and HLA alleles and haplotypes. Antibody titers were expressed as mean ± SD; differences between cases and controls were made using two-tailed Student t-test. Logistic regression analysis was performed in order to determine OR and 95%CI associated with T1D risk, taking the controls as the reference group. Statistical significance was set at P < RESULTS HLA allele and haplotype frequencies. Significant DRB1 allelic differences were seen between T1D patients and controls. These which comprised DRB1* (Pc = 0.006),

6 which was higher, and DRB1* (Pc = 0.003) and DRB1* (Pc = 0.027) which were lower among patients (Table 1). Similarly, significant DQB1 allelic differences were seen at the DQB1 locus, which comprised DQB1*0302 (Pc = 0.012) which was higher, and DQB1* (Pc = 0.007) and DQB1* (Pc = 0.041) which were lower among patients than control subjects (Table 1). In addition, the frequencies of DRB1* DQB1*0201 (Pc < 0.001), and DRB1* DQB1*0302 (Pc = 0.010) were higher, while those of DRB1* DQB1*0201 (Pc = 0.015) and DRB1* DQB1* (Pc = 0.036) were lower in T1D patients than in control subjects, thereby conferring T1D susceptibility and protection to these haplotypes, respectively (Table 1). Correlation Studies. We examined the functional attributes of HLA alleles and haplotypes on autoantibody levels among T1D patients. Table 2 summarises correlation between level of autoantibodies and HLA alleles and haplotypes among T1D patients positive for a specific allele and haplotype; patients negative for that allele or haplotype serving as controls. Anti- GAD levels were positively correlated with DRB1* allele (r 2 = 0.378; P <0.001), and DRB1*030101/DQB1*0201 (r 2 = 0.572; P <0.001) and DRB1*040101/DQB1*0302 (r 2 = 0.284; P = 0.001) haplotypes. Anti-GAD titers were negatively associated with the protective DRB1* (r 2 = ; P = 0.001), DRB1* (r 2 = ; P <0.001), and DQB1* alleles (r 2 = ; P = 0.002), along with DRB1*070101/DQB1*0201 (r 2 = ; P = 0.001) and DRB1*110101/DQB1* (r 2 = ; P = 0.001) haplotypes. By comparison, anti-ia2 levels were positively correlated only with DRB1*040101/DQB1*0302 (r 2 = 0.312; P = 0.013) haplotype, but were negatively correlated with DRB1* allele (r 2 = ; P = 0.009), and DRB1*110101/DQB1* (r 2 = ; P = 0.006) haplotype. 147

7 Anti-GAD and Anti-IA-2 Antibody Titers. Results from Table 3 showed that the mean anti-gad antibody titers were higher in T1D patients positive for DRB1* allele (P <0.001), and DRB1*030101/DQB1*0201 (P <0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes. Mean anti-gad antibody titers were lower in carriers of DRB1* (P = 0.001) and DRB1* (P < 0.001) alleles, and in DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1* (P = 0.001) haplotypes. By comparison, mean anti- IA2 antibody titers were higher in T1D patients positive for DRB1* (P = 0.007), and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes, but were lower in carriers of DRB1* (P = 0.010) and DRB1* (P < 0.001) alleles, and DRB1*110101/DQB1* (P = 0.025) haplotype. Regression analysis. The selective association of DRB1 and DQB1 alleles and haplotypes with altered anti-gad and anti-ia2 elevated antibody titers were confirmed by regression analysis, after controlling for potential covariates. Multinomial regression analysis confirmed the positive association of DRB1* and DRB1*030101/DQB1*0201, and the negative association of DRB1* and DQB1* alleles, and DRB1*070101/DQB1*0201 and DRB1*110101/DQB1* haplotypes with altered anti-gad levels. By contrast, only DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-ia2 titers (Table 4). DISCUSSION T1DM is an organ-specific autoimmune disease resulting T cell-mediated destruction 170 of pancreatic β islet cells. T1D is also distinguished by the presence of a number of autoantigens [3,6,12,27]. GAD and IA-2 are two of the major and most well characterized, autoantigens. Several studies demonstrated that HLA DQ and DR alleles influence T1D

8 susceptibility [1,20,21,29], and T1D-predisposing or -protective HLA-DQ and -DR alleles were identified [1,29]. Apart from regulation of autoreactive T-cell repertoire by susceptible and protective HLA molecules, presentation of autoantigenic peptides by specific HLA molecules, or induction of regulatory T cells (Treg) explain, at least in part, the modulatory capacity of HLA variants on overall T1D risk [8]. This study is the first report on the association of HLA class II alleles and haplotypes and antibody titers with T1D in Tunisia. We previously demonstrated that the contribution of HLA haplotypes to T1DM genetic susceptibility among Tunisians depends on specific HLA class II haplotypes [29]. The results showed that anti-gad antibody levels were higher in DRB1* allele carriers, in agreement with recent findings in Saudi population [14], but in partial agreement with an earlier study on Taiwanese subjects, in which anti-gad positivity was associated with DR3 and with DR4 [4]. These differences are likely attributed to racial differences in the contribution of HLA class II alleles and haplotypes to T1D pathogenesis [2,8,11]. Our study demonstrated that the prevalence, and increased titre of anti- GAD antibodies are associated with DRB1* and DRB1*030101/DQB1*0201, while anti-ia-2 positivity was associated with DRB1*040101/DQB1*0302 haplotype. Studies on Swedish reported the same association [7]. This association between IA-2 and DRB*04 allele was reported in previous studies [11,13,22,24]. Multinomial regression analysis reveled in our population a positive association of the haplotype DRB1*040101/DQB1*0302 with altered anti-ia2 titers. These results are concordant with previous studies on Canadian [24], Japanese [20], German [10], Finnish [27,28], Italian [13] and Belgian [5] populations that showed that the prevalence and level of IA-2 autoantibody are increased in patients with HLA DR4/DQ8. However, IA-2 was negatively associated with DRB1*110101/DQB1* in titers and in prevalence. Graham et al. showed the same association among Swedes [7].

9 One of the characteristics of MHC genetic associations with T1D is that primary susceptibility is determined by HLA-DQB1*0302, with penetrance of clinical disease significantly influenced by the specific subtype of DR4 present on the same haplotype [30]. It is tempting to speculate that this is based on the selection of specific dominating epitopes favored during disease progression [12,21,30]. Other genetic modifiers of susceptibility, which are also attributed to HLA molecules, the dominant protection conferred by HLA-DQ alleles, may also have a primary mechanistic influence by biasing or adding to the specific epitope recognition cascade [12,17,30]. In this way, T1D can be viewed as a disease of epitope selection and immunological focusing, whereby autoimmunity may progress towards tissue destruction or protection [17,30]. While specific HLA alleles and haplotypes clearly impact the susceptibility to T1D, it is likely that anti-gad positivity and T1D-susceptible loci HLA types translate into increased T cell reactivity. HLA class II molecules expressed on antigen-presenting cells have critical roles for the activation of helper T cells (mainly CD4 + T cells) and may influence antibody status in the periphery. This was highlighted by the findings of Itoh in which HLA DR9/X-positive T1D patients with had significantly higher numbers of GAD-reactive IFN-γproducing T cells, compared to other loci [9]. HLA polymorphism affects autoimmune responses according to the binding capacity of antigenic peptides, and the repertoire of T cell receptors of reactive T cells [25], and different HLA-DR/DQ molecules might have different binding affinities to disease-associated peptides [18]. These may explain our results of unique and different HLA association with autoantibodies from the Caucasoid populations. In conclusion, our study of T1DM among Tunisians reveals that both GAD and IA-2 antibodies and the HLA-DR and DQ alleles are critical in determining the risk for the disease.

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15 TABLE HLA-DRB1* and DQB1* Allele Distribution Allele / Haplotype Patients (n=88) Controls (n=112) P 2 Pc 3 DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DQB1* DQB1* DQB1* DQB1* DRB1* DQB1* <.001 <.001 DRB1* DQB1* DRB1* DQB1* DRB1* DQB1* Allele/haplotype frequency 2. Determined by Fisher's exact test; boldface indicate significant differences. 3. Pc = corrected p for the number of allele tested, calculated using the Bonferonni method.

16 TABLE 2 Correlation Studies 347 Anti-GAD Anti-IA r 2 * P r 2 P Alleles 1 DRB1* DRB1* DRB1* DRB1* DQB1* DRB1* Haplotypes DRB1*030101/DQB1* DRB1*040101/DQB1* DRB1*070101/DQB1* DRB1*110101/DQB1* Spearman correlation coefficient 1. Only alleles and haplotypes significantly associated with altered risk of T1DM in Tunisians (Reference 7).

17 17 TABLE 3 Antibody Titers in HLA Class II Alleles and Haplotypes Anti-GAD Anti-IA2 Positive 1 Negative 1 Z P Positive 1 Negative 1 Z P DRB1* ± ± < ± ± DRB1* ± ± ± ± DRB1* ± ± ± ± DRB1* ± ± < ± ± DQB1* ± ± ± ± DRB1* ± ± ± ± DRB1*030101/DQB1* ± ± < ± ± DRB1*040101/DQB1* ± ± ± ± DRB1*070101/DQB1* ± ± ± ± DRB1*110101/DQB1* ± ± ± ± Positive = patients carrying the specific allele/haplotype ; Negative = other patients. 2. Mean ± SD antibody titers (AU/ml); differences were made using two-tailed Student t-test

18 TABLE 4 Matched Odds Ratios Associated with Antibody Titers in Class II Alleles and Haplotypes 1 Anti-GAD Anti-IA2 P aor (95% CI) 2 P aor (95% CI) DRB1* < ( ) ( ) DRB1* ( ) ( ) DRB1* ( ) ( ) DRB1* < ( ) ( ) DQB1* ( ) ( ) DRB1* ( ) ( ) DRB1*030101/DQB1*0201 < ( ) ( ) DRB1*040101/DQB1* ( ) < ( ) DRB1*070101/DQB1* ( ) ( ) DRB1*110101/DQB1* < ( ) ( ) 1. Only alleles and haplotypes significantly associated with altered risk of T1DM in Tunisians (Reference 7). 2. Reference group being patients negative for the specific allele or haplotype; aor = adjusted odds ratios, controlled for gender, age of onset, and fasting glucose. 18

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