Advances in Genetics Endocrine Research. Site: The study was conducted at a university referral center.

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1 JCEM ONLINE Advances in Genetics Endocrine Research HLA Class II Haplotypes Differentiate Between the Adult Autoimmune Polyglandular Syndrome Types II and III B. K. Flesch, N. Matheis, T. Alt, C. Weinstock, J. Bux, and G. J. Kahaly Laboratory of Immunogenetics/HLA (B.K.F., C.W.), German Red Cross Blood Service West, Bad Kreuznach 55543, Germany; Molecular Thyroid Research Laboratory (N.M., G.J.K.), Johannes Gutenberg University Medical Center, Mainz 55101, Germany; Bioinformatics Unit (T.A.) and Center for Transfusion Medicine (J.B.), German Red Cross Blood Service West, Hagen 58097, Germany Background: Genetics of the adult autoimmune polyglandular syndrome (APS) is poorly understood. Aim: The aim of this study was to gain further insight into the genetics of the adult APS types. Site: The study was conducted at a university referral center. Methods: The human leukocyte antigen (HLA) class II alleles, haplotypes, and genotypes were determined in a large cohort of patients with APS, autoimmune thyroid disease (AITD), and type 1 diabetes and in healthy controls by the consistent application of high-resolution typing at a four-digit level. Results: Comparison of the allele and haplotype frequencies significantly discriminated patients with APS vs AITD and controls. The HLA class II alleles DRB1*03:01 *04:01, DQA1*03:01, *05:01, DQB1*02:01, and *03:02 were observed more frequently (P.001) in APS than in AITD and controls, whereas the alleles DRB1*15:01, DQB1*03:01, and *06:02 were underrepresented in APS vs AITD (P c.001) and controls (P c.01), respectively. The DRB1*03:01-DQA1*05:01-DQB1*02:01 (DR3-DQ2) and DRB1*04:01-DQA1*03:01:DQB1*03:02 (DRB1*04:01-DQ8) haplotypes were overrepresented in APS (P c.001). Combination of both haplotypes to a genotype was highly prevalent in APS vs AITD and controls (P c.001). Dividing the APS collective into those with Addison s disease (APS type II) and those without Addison s disease but including type 1 diabetes and AITD (APS type III) demonstrated DR3-DQ2/DRB1*04:01-DQ8 as a susceptibility genotype in APS III (P c.001), whereas the DR3-DQ2/DRB1*04:04-DQ8 genotype correlated with APS II (P c.001). The haplotypes DRB1*11:01-DQA1*05:05-DQB1*03:01 and DRB1*15:01-DQA1*01:02-DQB1*06:02 are protective in APS III but not in type II (P c.01). Conclusions: HLA class II haplotypes differentiate between the adult APS types II and III. Susceptible haplotypes favor the development of polyglandular autoimmunity in patients with AITD. (J Clin Endocrinol Metab 99: E177 E182, 2014) Autoimmune polyglandular syndromes (APS) are defined as the coexistence of two endocrine autoimmune diseases (1). Distinction between APS types is determined by disease presentation, age of syndrome onset, and mode of inheritance (2). The juvenile APS type I is a monogenic, autosomal recessive disease linked to an autoimmune regulator mutation with a peak incidence during early childhood, whereas the more common APS types II and III present during adulthood. The defining endocrine component of APS II is Addison s disease (AD), ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by The Endocrine Society Received July 15, Accepted October 25, First Published Online November 1, 2013 Abbreviations: AD, Addison s disease; AITD, autoimmune thyroid disease; APS, autoimmune polyglandular syndromes; GD, Graves disease; HLA, human leukocyte antigen; HT, Hashimoto s thyroiditis; OR, odds ratio; P c, Bonferroni-Holm-method; SSO, sequence-specific oligonucleotides; T1D, type 1 diabetes. doi: /jc J Clin Endocrinol Metab, January 2014, 99(1):E177 E182 jcem.endojournals.org E177

2 E178 Flesch et al Genetics of APS J Clin Endocrinol Metab, January 2014, 99(1):E177 E182 which is absent in APS III. The APS III phenotype is characterized by the presence of autoimmune thyroid disease (AITD), either Hashimoto s thyroiditis (HT) or Graves disease (GD), coexisting with type 1 diabetes (T1D) (3). APS comprising T1D and/or AD in combination with a further glandular autoimmune disease is associated with the HLA-DR3-DQ2 and HLA-DR4-DQ8 haplotypes. Haplotypes DR3-DQB1*02:01 increase patient vulnerability to both T1D and AITD, whereas DR4-DQB1*03:02 is associated with T1D only (4). However, susceptibility for the HLA DR3 and DR4 alleles does not fully describe the genetic association between AITD and T1D (5). The few previous studies on genetics of APS have seldom used the high resolution technique for each of the human leukocyte antigen (HLA) class II loci. In addition, the APS group was not well characterized defining combined glandular and nonglandular disease as APS. Furthermore, the patient number included was frequently low. Therefore, to gain further insight into the genetics of the adult APS types, we aimed to compare the HLA class II alleles, haplotypes, and genotypes in a large and welldefined collective of patients with APS, AITD, T1D, and healthy controls exclusively using reliable high-resolution HLA typing methods at the four-digit level. Materials and Methods A total of 449 consecutive unrelated subjects of a university referral center were included. The study was approved by the local ethical committee and was performed according to the Helsinki Declaration. Written informed consent was obtained from each investigated individual. Endocrine, serological, and immunological investigation was performed to confirm suspected diagnoses of autoimmune glandular disease, and data acquisition followed standardized diagnostic criteria. The study collective encompasses patients with adult APS (n 124, mean age y, 64% females), AITD (n 222, including 111 GD patients, y, 89% females and 111 patients with HT, y, 82% females), T1D (n 103, y, 54% females) as well as 121 nonrelated healthy euthyroid controls (40 15 y, 54% females) from the same geographic region. The APS cohort consisted of 25 APS II patients with AD and an additional autoimmune glandular disease, predominantly HT (n 20); 93 APS III patients without AD but with T1D and AITD; and six APS IV patients. Mean ages of the APS II and III groups were and years, respectively. In 66 of 449 patients, family members were available for typing to verify the linkage-based calculation of HLA haplotypes. Twentyone of the family members were healthy unrelated spouses of an index patient, thus being included in the 121 controls. HLA typing by PCR-sequence-specific oligonucleotides (SSO) DNA was prepared from EDTA-anticoagulated blood either by an automated method (BioRobot EZ1; QIAGEN) or by a manual DNA extraction kit (QIAmp DNA blood minikit; QIA- GEN) following the manufacturer s instructions. HLA-DRB1-, DQA1, and -DQB1 alleles were typed by PCR followed by hybridization with SSO as previously described (6). If PCR-SSO analysis reported a high-resolution result at the four-digit level without manual correction, the result was accepted. Otherwise, the sample was typed by high-resolution PCR with sequence specific primers (Olerup) and analysis of the amplification products with the Helmberg-Score version 5.0.T software (Olerup). Assignment of haplotypes The HLA-DRB1-DQA1-DQB1 haplotypes of the 66 index patients, 21 healthy spouses, and their family members in parallel were deduced according to the known linkage of HLA alleles and according to segregation. Determination of the haplotypes of the remaining samples exclusively followed the known linkage. Statistical analyses Differences in the allele, haplotype, and genotype frequencies were evaluated by the 2 test. P values were calculated using Fisher s exact probability test. Due to multiple comparisons, the P values were corrected according to the Bonferroni-Holmmethod (P c ). Values of P c.05 were considered statistically significant. Odds ratios (ORs) and 95% confidence intervals were determined for each comparison. All data analyses were performed with Microsoft Excel version Results The HLA class II alleles DRB1*03:01 *04:01, DQA1*03: 01, *05:01, DQB1*02:01, and *03:02 were observed more frequently (P c.001) in APS than in AITD and controls, whereas the alleles DRB1*15:01, DQB1*03:01, and *06:02 were underrepresented in APS vs AITD (P c.001) and vs controls (P c.01). In HT, the DQA1*05:05 allele was more frequent (P c.05) than in GD; however, HT and GD did not discriminate in any other allele or haplotype (P c.05). Therefore, GD and HT patients are considered as one collective. DQA1*01:02 (P c.001) and *05:05 were underrepresentedinaps(p c.01)comparedwithaitdbut not vs controls. HLA class II haplotypes Of the 75 different four-digit haplotypes determined in all subjects, 19 haplotypes leading to the DRB1*04- DQA1*03-DQB1*03 type at a two-digit resolution were demonstrated, 10 of them being present in APS. However, exclusively the DRB1*04:01-DQA1*03:01- DQB1*03:02 haplotype was overrepresented in APS vs AITD and controls, indicating that only high-resolution typing at the four-digit level resolves significant differences. Additionally, DRB1*03:01-DQA1*05: 01-DQB1*02:01 was highly overrepresented in contrast

3 doi: /jc jcem.endojournals.org E179 Figure 1. Relative frequency of HLA-DRB1-DQA1-DQB1 haplotypes in patients with autoimmune monoglandular and polyglandular diseases and in euthyroid healthy controls. The patients had clinically and biochemically confirmed APS, T1D, and AITD, either GD or HT. GD was defined as hyperthyroidism with the presence of TSH receptor autoantibodies and a typical ultrasound pattern with enhanced vascularization of the thyroid gland. HT was defined as a tenfold increased serum level of thyroid peroxidase autoantibodies, a hypoechoic ultrasound pattern with enhanced vascularization of a thyroid gland, and a primary hypothyroidism or a normal thyroid function. T1D was defined as complete insulin deficiency with the presence of -cell autoantibodies against the islet cell antigens, tyrosine phosphatase IA-2, insulin, and/or glutamic acid decarboxylase-65. AD was diagnosed as very low serum cortisol levels, elevated baseline serum ACTH levels, abnormal ACTH stimulation test, and the presence of cytochrome P hydroxylase autoantibodies. Primary ovary failure was diagnosed as low serum peripheral sexual hormone levels, elevated serum gonadotropic hormone levels, pathological LHRH stimulation test, and positive 17-hydroxylase autoantibodies. Primary hypoparathyroidism was diagnosed as low PTH levels with low serum calcium and elevated serum phosphate levels. Only haplotypes with significant differences after correction for the number of comparisons are shown. The OR and 95% confidence interval for the different haplotypes are listed only for comparisons with P c.05. These included the following: DRB1*03:01-DQA1*05:01-DQB1*02:013, OR 2.9 ( ) for APS vs AITD, OR 4.6 ( ) for APS vs controls, OR 3.2 ( ; P c.05) for APS II vs AITD, OR 5.2 ( ) for APS II vs controls, OR 2.9 ( ) for APS III vs AITD, OR 4.6 ( ) for APS III vs controls; DRB1*04:01-DQA1*03:01-DQB1*03:023, OR 7.4 ( ) for APS vs AITD, OR 4.7 ( ) for APS vs controls, OR 9.7 ( ) for APS III vs AITD, OR 6.1 ( ) for APS III vs controls; DRB1*04:04-DQA1*03:01-DQB1*03:023, OR 6.9 ( ) for APS II vs AITD, OR 11.3 ( ) for APS II vs controls; DRB1*11:01-DQA1*05:05-DQB1*03:013, OR 0.07 ( ) for APS III vs AITD, OR 0.1 ( ) for APS III vs controls; DRB1*15:01-DQA1*01:02-DQB1*06.023, OR 0.2 ( ) for APS vs AITD, OR 0.2 ( ; P c.05) for APS vs controls, OR 0.04 ( ) for APS III vs AITD, OR 0.05 ( ; P c.01) for APS III vs controls. to AITD and controls (Figure 1), whereas the DRB1*15: 01-DQA1*01:02-DQB1*06:02 haplotype was rare in APS. HLA class II haplotype distribution in T1D was similar to that observed in APS. Comparison of APS II vs III Differences (P c.05) were observed between APS II and T1D but not between APS III and T1D. The DRB1*04:01- DQA1*03:01-DQB1*03:02 haplotype frequency was equally high in APS III and in T1D as a singular disease, whereas in APS II the frequency of this haplotype was low and comparable with controls/aitd. In contrast, the frequency of the DRB1*04:04-DQA1*03:01-DQB1*03:02 haplotype in APS II was 3-fold higher than in APS III and T1D and the 10-fold of controls. In comparison, DRB1*03: 01-DQA1*05:01-DQB1*02:01 was equally overrepresented in APS II and III. DRB1*11:01-DQA1*05:05- DQB1*03:01 was rare in APS III and in T1D but frequent in APS II. Finally, the DRB1*15:01- DQA1*01:02-DQB1*06:02 haplotype was significantly underrepresented only in APS III and T1D. HLA class II genotypes The genotypes DRB1*03:01-DQA1*05:01- DQB1*02:01/DRB1*04:01-DQA1*03:01- DQB1*03:02 (DR3-DQ2/DRB1*04:01-DQ8) and DRB1*03:01-DQA1*05:01-DQB1*02:01/DRB1*04: 04-DQA1*03:01-DQB1*03:02 (DR3-DQ2/DRB1*04: 04-DQ8) predominated in APS and T1D compared with AITD and controls (P.0008). Differences increased when separately analyzing APS II and III (Figure 2). Although the DR3-DQ2/DRB1*04:01-DQ8 genotype was overrepresented in APS III and T1D, the DR3-DQ2/ DRB1*04:04-DQ8 genotype was more frequent in APS II compared with controls. The combination of DRB1*03: 01-DQA1*05:01-DQB1*02:01 and DRB1*11:01- DQA1*05:05-DQB1*03:01 was observed more frequently in APS II than in controls (P.0018; P c.05) or T1D patients (P.0004; P c.05).

4 E180 Flesch et al Genetics of APS J Clin Endocrinol Metab, January 2014, 99(1):E177 E182 Figure 2. HLA-DRB1-DQA1-DQB1 genotypes in patients with APS, APS type II, APS type III, T1D, AITD, and in euthyroid healthy controls Only genotypes with significant differences (without correction for the number of comparisons) between the groups are shown. P c values are indicated only for differences that were highly significant after correction for the number of comparisons (P c.001). DR3-DQ2/DRB1*04:01-DQ83 APS vs AITD: OR 19.9 ( ); APS vs controls: OR 21.7 ( ); APS III vs AITD: OR 26.4 ( ); APS III vs controls: OR 28.8 ( ). Discussion The power of this study is the large panel of Caucasian APS patients in comparison with equally large collectives of patients with monoglandular autoimmune disease. HLA alleles were consistently determined at a four-digit level, and the assignment of single alleles to HLA class II-haplotypes was verified in a smaller collective by family-based segregation. The HLA DQA1 alleles were also determined by PCR and were not deduced from the known DQA1- DQB1 linkage because in GD patients a stepwise logistic regression analysis had demonstrated the higher priority of DQA1 loci in contrast to DQB1 for the disease association (7). Previous studies pertaining to the prevalence of the DQA1*03:01 and *05:01 alleles in German APS patients (8) and the DQB1*02:01 and *03:02 alleles in Tunisian APS patients (9) used lower-resolution techniques and included less patients with APS. Also, the APS entity was poorly defined in a report (8) in which patients with glandular and nonglandular diseases were mixed. HLA-DRB1*03:01-DQA1*05:01-DQB1*02:01 and HLA-DRB1*04:01-DQA1*03:01-DQB1*03:02 were identified as susceptibility haplotypes in APS due to their significant overrepresentation compared with AITD or controls. This is in line with a reported prevalence of the HLA-DRB1*03, *04, and DQB1*02 alleles in APS applying low-resolution typing (6, 10) and with familybased studies (11, 12), which reported a prevalence of the DR3-DQB1*02:01 and DR4-DQB1*03:02 haplotypes in patients with both T1D and AITD but refused a prevalence of the DR4-DQB1*03:02 haplotype in AITD alone. In our study, we detected at a high-resolution level 19 different haplotypes leading to the DRB1*04- DQA1*03-DQB1*03 haplotype, five of these including DQB1*03:02 in combination with DQA1*03:01. Only the DRB1*04:01-DQA1*03:01-DQB1*03:02 haplotype was prevalent at a significant level in the complete APS and APS III collective. Low-resolution typing would not have resolved this differentiation. The HLA- DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype is protective in APS confirming earlier findings (6, 10). The DRB1*04-DQ8 haplotype differentiates between APS II and III with DRB1*04:01 in combination with DQA1*03:01*-DQB1*03:02 (DRB1*04:01-DQ8) asa susceptibility haplotype in APS III and DRB1*04:04 combined with DQA1*03:01-DQB1*03:02 (DRB1*04:04- DQ8) overrepresented in APS II. Previously, using a lowresolution typing, we could not identify these differences (6), but two studies demonstrated the prevalence of the DRB1*04:04-DQ8 haplotype in AD (13, 14). According to the National Center for Biotechnology Information dbmhc Sequence Alignment Viewer (www. ncbi.nlm.nih.gov) the DRB1*04:01 and DRB1*04:04 al-

5 doi: /jc jcem.endojournals.org E181 leles discriminate within exon 2 in the codons 71 and 86. DRB1*04:01 codes for Lys71 and Gly86, whereas DRB1*04:04 codes for Arg71 and Val86. In autoimmune glandular disease, the critical amino acid positions within the peptide binding pocket of the HLA-DR molecules, belonging to a region of shared epitopes are at positions 71 and 74. The positively charged combination of Lys71 and Arg74 is a susceptibility type for APS III (15). Within HLA-DR-antigens, Lys 71 is exclusively encoded by all of the DRB1*03 alleles and the DRB1*04:01 allele but not by the DRB1*04:04 allele. This difference in the peptide binding pocket is critical for antigen presentation and the pathomechanism of APS. Additionally, the HLA class II haplotype distribution within APS and T1D was similar in this study, confirming earlier data and reflecting the known prevalence of DRB1*03 and DRB1*04 in T1D (6). Finally, our data are not comparable with those reported in Japanese patients (16, 17) due to the different distribution and frequency of HLA alleles, haplotypes, and genotypes in Asian vs Caucasian populations. The combination of both APS susceptibility haplotypes to the DR3-DQ2/DRB1*04:01-DQ8 or DR3-DQ2/ DRB1*04:04-DQ8 genotypes defined a high-risk genotype for APS compared with AITD or controls, whereas the effect was less pronounced for homozygosity for one of these haplotypes. The DRB1*04:01 or DRB1*04:04 encompassing genotypes are associated with either the presence of T1D in APS III or AD in APS II, irrespective on the presence of AITD. This corresponds to the finding that HLA-DRB1*04:04 is prevalent in AD because 43% of AD patients had the high-risk genotype DR3-DQB1*02: 01-B8/DRB1*04:04-DQB1*03:02 (18) in contrast to 1.5% of the general population. In conclusion, HLA haplotypes in APS are associated either with APS including AD with a prevalence of the DRB1*04:04-DQA1*03:01-DQB1*03:02 haplotype or with APS including T1D with an overrepresentation of the DRB1*04:01-DQA1*03:01-DQB1*03:02 haplotype and an underrepresentation of the DRB1*11:01- DQA1*05:05-DQB1*03:01 and DRB1*15:01-DQA1*01: 02-DQB1*06:02 haplotypes. Subject to confirmation in a largercollectiveofapspatients, haplotypeandgenotypetesting in patients with monoglandular autoimmune disease may be prognostically and thus clinically useful. Acknowledgments We thank S. Barkia, M. C. Hager, S. Göbel, and A. Busch (Molecular Thyroid Research Lab, Gutenberg University Medical Center) for data collection. Single aspects of this manuscript include parts of their doctoral thesis. We gratefully acknowledge the technical expertise of M. Steitz, A. Janson, and I. Kuhn. This work was supported by. Authors tasks included the following: B.K.F. discussed the study concept, analyzed and interpreted the data, and wrote the manuscript. N.M. provided blood samples and clinical data, discussed the study concept, and revised the manuscript. T.A. performed the statistical analyses. C.W. provided part of the data and revised the manuscript. J.B. supervised the research and critically revised the manuscript. G.J.K. initiated the project, was responsible for the patient management, provided the study concept, discussed the obtained data with the first three authors, supervised the research, and wrote and critically revised the manuscript. Address all correspondence and requests for reprints to: Professor George J. Kahaly, Department of Medicine I, Johannes Gutenberg University Medical Center, Langenbeckstrasse 1, Mainz 55131, Germany. kahaly@ukmainz.de. addresses of the other authors are as follows: b.flesch@bsdwest.de; nina.matheis@unimedizin-mainz.de; t.alt@ bsdwest.de; c.weinstock@blutspende.de; juergen.bux@web.de; and j.bux@web.de. Present address for C.W.: Institute of Clinical Transfusion Medicine and Immunogenetics, Ulm, Germany; German Red Cross Blood Service Baden-Württemberg Hessen, Germany; and Institute of Transfusion Medicine, University of Ulm, Ulm, Germany. This work was supported by an unrestricted grant from the German Red Cross Blood Service West (Germany) and by financial support from the Johannes Gutenberg University Medical School. Disclosure Summary: The authors have nothing to disclose. References 1. Eisenbarth GS, Gottlieb PA. Autoimmune polyendocrine syndromes. N Engl J Med. 2004;350: Dittmar M, Kahaly GJ. 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6 E182 Flesch et al Genetics of APS J Clin Endocrinol Metab, January 2014, 99(1):E177 E182 dular failure is associated with HLA-DRB1*03. Eur J Endocrinol. 2008;159: Golden B, Levin L, Ban Y, Concepcion E, Greenberg DA, Tomer Y. Genetic analysis of families with autoimmune diabetes and thyroiditis: evidence for common and unique genes. J Clin Endocrinol Metab. 2005;90: Huber A, Menconi F, Corathers S, Jacobson EM, Tomer Y. Joint genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms. Endocr Rev. 2008;29: Myhre AG, Undlien DE, Lovas K, et al. Autoimmune adrenocortical failure in Norway autoantibodies and human leukocyte antigen class II associations related to clinical features. J Clin Endocrinol Metab. 2002;87: Yu L, Brewer KW, Gates S, et al. DRB1*04 and DQ alleles: expression of 21-hydroxylase autoantibodies and risk of progression to Addison s disease. J Clin Endocrinol Metab. 1999;84: Menconi F, Osman R, Monti MC, Greenberg DA, Concepcion ES, Tomer Y. Shared molecular amino acid signature in the HLA-DR peptide binding pocket predisposes to both autoimmune diabetes and thyroiditis. Proc Natl Acad Sci USA. 2010;107: Hashimoto K, Maruyama H, Nishiyama M, et al. Susceptibility alleles and haplotypes of human leukocyte antigen DRB1, DQA1, and DQB1 in autoimmune polyglandular syndrome type III in Japanese population. Horm Res. 2005;64: Horie I, Kawasaki E, Ando T, et al. Clinical and genetic characteristics of autoimmune polyglandular syndrome type 3 variant in the Japanese population. J Clin Endocrinol Metab. 2012;97: Baker PR, Baschal EE, Fain PR, Triolo TM, et al. Haplotype analysis discriminates genetic risk for DR3-associated endocrine autoimmunity and helps define extreme risk for Addison s disease. J Clin Endocrinol Metab. 2010;95: Need Part IVMOC Points? Learn more about the Endocrine Society s Practice Improvement Modules (PIMs).