Major histocompatibility complex class II transactivator gene polymorphism: associations with Löfgren s syndrome

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1 Tissue Antigens ISSN Major histocompatibility complex class II transactivator gene polymorphism: associations with Löfgren s syndrome J. Grunewald 1,F.Idali 1,2,I.Kockum 3, M. Seddighzadeh 4,M.Nisell 1,A.Eklund 1 & L. Padyukov 4 1 Department of Medicine, Division of Respiratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 2 Department of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 3 Department of Clinical Neurosciences, Neuroimunology Unit, Center for Molecular Medicine (CMM), Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 4 Department of Medicine, Rheumatology Unit, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Key words genetics; Löfgren s syndrome; major histocompatibility complex class II transactivator; sarcoidosis Correspondence Dr Johan Grunewald Department of Medicine Division of Respiratory Medicine Karolinska Institutet Lung Research Laboratory L4:01 Karolinska University Hospital Solna S Stockholm Sweden Tel: Fax: johan.grunewald@ki.se Received 30 November 2009; revised 20 January 2010; accepted 4 February 2010 doi: /j x Abstract The major histocompatibility complex (MHC) class II transactivator (MHC2TA) is known as a master regulator for expression of MHC class II molecules. In the present study, we investigated the influence on the risk for sarcoidosis of two variants of the MHC2TA gene, selected from previous association studies of inflammatory diseases. Seven hundred and twenty-eight sarcoidosis patients and 873 controls matched by ethnicity were included in the study. Patients were classified as with Löfgren s syndrome (or not) as subphenotypes. Individuals were genotyped for two single nucleotide polymorphisms (SNPs) of the MHC2TA gene, rs A/G and rs C/T, and were human leukocyte antigen (HLA)-DRB1-typed. After correction for multiple testing, our data showed a significant association with Löfgren s syndrome in allelic model for the rs SNP, which was not detected in non-löfgren s patients. A similar trend was noted for the rs SNP. These risk factors were independent of HLA-DRB1*03, which is known to be associated with Löfgren s syndrome. The finding of a new genetic association between Löfgren s syndrome and MHC2TA gene polymorphisms, which seems independent of HLA- DRB1*03 and relates to the expression of MHC class II molecules, strongly supports theideathatlöfgren s syndrome is a separate disease entity. Introduction Sarcoidosis is a granulomatous disease of unknown aetiology, affecting mostly middle aged individuals and involving the lungs in more than 90% of cases (1). There is a genetic influence, as suggested by family studies (2) and by variations in incidence as well as in clinical manifestations between different ethnic groups (1, 3, 4). Detailed genetic analyses have identified distinct DNA regions associated with sarcoidosis (5), and especially the major histocompatibility complex (MHC) region has been pointed out (6, 7). Specific human leukocyte antigen (HLA) alleles have also been shown to associate not only with susceptibility to disease but also to distinct forms of the disease (4). Thus, in Scandinavia, HLA-DRB1*03-positive patients often have a benign disease course, while DRB1*14- or DRB1*15-positive patients more often develop a chronic disease (8). A recent genome-wide association (GWA) study of 499 sarcoidosis patients and 490 controls showed reproducible risk from the ANXA11 gene, but also several associations with MHC class II loci, including BTNL2, DRB1, DRB5 and DRA (5). No significant association was found at the major histocompatibility complex class II transactivator (MHC2TA) genetic locus in this study. However, a limited number of SNPs were used in this region with little linkage disequilibrium according to HapMap data. Because of the multitude of clinical manifestations, it has been discussed whether sarcoidosis consists of several distinct disease entities, each with its own separate pathogenesis (3, 4). One such specific disease entity may be Löfgren s syndrome, characterized by an acute disease onset, bilateral hilar lymphadenopathy (BHL) and in some cases parenchymal infiltrates, erythema nodosum (EN) and/or bilateral ankle arthritis, and usually fever. This syndrome, first described by Dr S Löfgren (9), associates with HLA- DRB1*03/DQB1*02 and a good prognosis (8, 10 15). In particular, HLA-DRB1*03-positive patients with Löfgren s syndrome have an excellent prognosis (16). Another genetic 2010 John Wiley & Sons A/S 1

2 MHC class II transactivator gene polymorphism J. Grunewald et al. association specific for Löfgren s syndrome was recently reported, i.e. between a CCR2 haplotype and Löfgren s syndrome, supporting the hypothesis that Löfgren s syndrome is a disease entity with specific genetic factors, which may be different from other subgroups of sarcoidosis (17). Given the well-established associations between sarcoidosis and the MHC class II region, it was of interest to study any influence of gene polymorphisms of the promoter region of the MHC2TA gene, which has a key function in controlling the expression of MHC class II molecules (18). In this study, we analyzed associations between sarcoidosis and two single nucleotide polymorphisms (SNPs), one of which (rs A G) recently was shown to associate with the inflammatory disorders, rheumatoid arthritis (RA), multiple sclerosis (MS) and also with myocardial infarction (19), while another was found in association with RA in a follow-up study (20). The study was designed as explorative with limited number of genetic markers, because no experimental data was available regarding particular functional variations on this locus. For both two MHC2TA SNPs, we found associations with Löfgren s syndrome, especially for rs , while for non-löfgren s patients no associations were detected. These findings add to the genetic delineation of Löfgren s syndrome and further support the concept that it is a disease entity by its own. Materials and methods Study subjects Altogether, 728 sarcoidosis patients (412 men) with a median age of 38 (min 19; max 77) years were included in this study. Patient samples were obtained when they were investigated at the Division of Respiratory Medicine, Karolinska University Hospital, Stockholm, Sweden, because of clinical symptoms compatible with sarcoidosis. Most patients were examined by one of the authors (AE) according to our routines at the outpatient clinic, and in a few cases data was collected through records. Patients were in general not on treatment. All patients were diagnosed with sarcoidosis through typical clinical manifestations, findings at bronchoscopy with bronchalveolar lavage (BAL) including an elevated CD4/CD8 BAL cell ratio and positive biopsies, using the criteria as outlined by the World Association of Sarcoidosis and other Granulomatous disorders (WASOG) (21). Löfgren s syndrome was defined as having an acute onset of the disease with BHL and in some cases parenchymal infiltrates, EN and/or bilateral ankle arthritis or distinct periarticular inflammation, and usually fever. We included 262 (36%; median age 36; min 22, max 71) individuals with Löfgren s syndrome (143 men; median age 35; min 22, max 63) and 466 (64%; median age 39; min 19, max 77) without Löfgren s syndrome (269 men; median age 38; min 19, max 75) in our study. There were 873 healthy controls matched by ethnicity (243 men), with a median age of 54 (min 17, max 70) years. From known history of smoking, we identified 25.8% ever smokers among patients (29.7% among patients without Löfgren s syndrome and 18.6% among patients with Löfgren s syndrome) and 64% ever smokers among controls. All included subjects gave their informed consent for participation in the study, which was approved by the local Ethics Committee at the Karolinska University Hospital/Institute. In total, 717 patients were analyzed for rs C/T and 717 for rs A/G. Altogether, 258 patiens with Löfgren s syndrome were analyzed for rs C/T and 256 for rs A/G. All the patients with Löfgren s syndrome were HLA-typed and grouped into HLA-DRB1*03 pos (n = 176) or DRB1*03 neg (n = 86). Among 873 controls, 864 were successfully analyzed for the MHC class II transactivator gene polymorphism rs A/G and 862 for rs C/T and all were genotyped for HLA-DRB1. Genotyping procedures Genomic DNA was extracted from whole blood samples using the salting-out method. MHC2TA SNPs were analyzed as previously described (19) by TaqMan allelic discrimination assay (Applera, NY) using a 384-well format, and HLA-DRB1 alleles were determined using the polymerase chain reaction with sequence-specific primers (PCR-SSP) technique (DR low resolution kit, Olerup SSP AB, Saltsjöbaden, Sweden) (22). Genotyping rate was 98.8% for rs and 98.6% for rs In addition, we performed genotyping of 70 DNAs in duplicates, which showed 100% consistency for the replicates. Statistical analyses SNP data was analyzed for association with whole sarcoidosis disease group and with subgroups with and without Löfgren s syndrome in allelic and co-dominant models by the Chisquared test. The Bonferroni correction was used to adjust the P-values because of multiple comparison of data for subgroups. Because our initial hypothesis was based on comparison of the whole case group against controls, this analysis does not require such correction. All analyzed genotypes were in Hardy Weinberg equilibrium according to assessment in Haploview. Results Association of MHC2TA variants with sarcoidosis We performed genotyping of two pre-selected SNPs from the MHC2TA gene, which were found to be highly linked in our study (D = 0.96; r 2 = 0.73). There was a significant increase of the rs G allele frequency in all patients John Wiley & Sons A/S

3 J. Grunewald et al. MHC class II transactivator gene polymorphism Table 1 Allele frequencies of rs in all patients, in patients with Löfgren s syndrome and in patients with non-lögren s syndrome Group A chromosomes G chromosomes χ 2 vs controls P-value P-value, corrected All patients 1062 (74.1) 372 (25.9) NA Löfgren s 371 (72.5) 141 (27.5) Non-Lögren s 691 (74.9) 231 (25.1) NS Controls 1339 (77.5) 389 (22.5) Figures show number of chromosomes and (%). NA, not applicable; NS, not significant. vs controls, with 372 (25.9%) chromosomes in patients with rs G allele compared with 389 (22.5%) chromosomes in controls (χ 2 = 5.045, P = ) (Table 1). Also, there was a trend towards an increased rs GG genotype in the patient group, as 54 (7.5%) patients had the rs GG genotype vs 44 (5.1%) controls (χ 2 = 5.51, P = 0.064) (Table 2). We also found a moderate increase in rs C allele frequency in the patient group, with 438 (30.5%) chromosomes carrying rs C allele in patients compared with 468 (27.1%) in controls (χ 2 = 4.42, P = 0.036) (Table 3). However, there was no difference in the rs genotype distribution between patients and controls (Table 4). Owing to the fact that in this analysis we were testing our initial hypothesis about association and because of the high linkage disequilibrium between SNPs (r 2 = 0.73 in our study), we did not correct these findings for multiple comparisons. Additional haplotype analysis did not influence the probability for differences (data not shown). Table 2 Genotypes for rs in all patients, in patients with Löfgren s syndrome and in patients with non-lögren s syndrome Group AA AG GG χ 2 vs controls P-value All patients 399 (55.6) 264 (36.8) 54 (7.5) Löfgren s 135 (52.7) 101 (39.5) 20 (7.8) Non-Lögren s 264 (57.3) 163 (35.4) 34 (7.4) Controls 519 (60.1) 301 (34.8) 44 (5.1) Figures show number of individuals and (%). Association of MHC2TA variants with subgroups of sarcoidosis Patients were subgrouped into patients with Löfgren s syndrome or without. Among the former, there was a significantly increased frequency of the rs G allele: 141 (27.5%) positive chromosomes in patients compared with 389 (22.5%) in controls (χ 2 = 5.53, P = 0.019, non-corrected; P = with correction) (Table 1). In contrast, in non- Löfgren s patients, there was no difference (Table 1). The rs C allele was found to be moderately increased in patients with Löfgren s syndrome, with 166 (32.2%) positive chromosomes in patients compared with 468 (27.1%) in controls (χ 2 = 4.94, P = 0.026, non-corrected; P = with correction) (Table 3). In contrast, there was no difference in rs C allele frequency between non-löfgren s patients and controls (Table 3). No significant differences were found for frequencies of rs and rs genotypes between patient subgroups and controls (Table 4). Because of the known association between HLA-DRB1*03 and Löfgren s syndrome, we also analyzed the rs and rs SNPs separately in DRB1*03 pos and DRB1*03 neg patients with Löfgren s syndrome, but found no difference in MHC2TA allele frequencies or genotypes (data not shown). Discussion Together with previously reported differences in genetic background between sarcoidosis patients with or without Löfgren s syndrome, our finding of an association between variants of the MHC2TA gene and Löfgren s syndrome provides additional support for a pathogenetic heterogeneity in sarcoidosis, with Löfgren s syndrome as a potential distinct subgroup of the disease. Table 3 Allele frequencies of rs in all patients, in patients with Löfgren s syndrome and in patients with non-lögren s syndrome Group C chromosomes T chromosomes χ 2 vs controls P-value P-value, corrected All patients 438 (30.5) 996 (69.5) NA Löfgren s 166 (32.2) 350 (67.8) Non-Lögren s 272 (29.6) 646 (70.4) NS Controls 468 (27.1) 1256 (72.9) Figures show number of chromosomes and (%). NA, not applicable; NS, not significant John Wiley & Sons A/S 3

4 MHC class II transactivator gene polymorphism J. Grunewald et al. Table 4 Genotypes for rs in all patients, in patients with Löfgren s syndrome and in patients with non-lögren s syndrome Group CC CT TT χ 2 vs controls P-value All patients 70 (9.8) 298 (41.6) 349 (48.7) Löfgren s 24 (9.3) 118 (45.7) 116 (45.0) Non-Lögren s 46 (10.0) 180 (39.2) 233 (50.8) Controls 63 (7.3) 342 (39.7) 457 (53.0) Figures show number of individuals and (%). A large number of studies, including more recent GWA studies, have pointed out the MHC region as well as specific MHC class II (HLA-DRB1* and -DQB1*) alleles to be associated with sarcoidosis (3, 5 8, 23). In addition, there is also a well-described association between distinct HLA-DRB1 alleles and the disease course in sarcoidosis (4). The essential function of MHC class II molecules is to present antigens for T-cells, promoting development of specific signals for the adaptive immunity. Different MHC allelic variants will be able to present distinct antigenic peptides. However, also the cellular surface expression of the MHC class II molecules differs depending on, for example, differentiation and cell activation, which might also influence the immune response. Inflammation, with increased levels of cytokines such as IFN-γ, results in an increased MHC class II molecule expression at the surface level. This is regulated through the MHC II transactivator gene (MHC2TA, aka CIITA), which encodes a protein that orchestrates the binding of other proteins to the promoter regions of MHC class II genes and thereby influences the transcription of these genes (18). The recently described association among RA, MS and myocardial infarction and polymorphisms in the promotor regions of the MHC2TA gene, suggested to influence the degree of MHC class II expression, indicates a new aspect of the MHC complex which may be of importance for developing autoimmunity (19, 20). A reduced MHC class II cell surface expression as a result of specific MHC2TA variants was suggested to be linked to a reduced capacity to induce T-cell tolerance via interaction with T-regulatory cells (19). However, more studies on the role of MHC2TA SNPs in inflammatory diseases are required, as the association with RA and MS has not been uniformly reproduced (24 26). Previous studies on genetic associations in sarcoidosis have included patient populations with rather variable phenotypes, e.g. patients with or without Löfgren s syndrome, or subgroups of distinct ethnic origin. We believe it is crucial in sarcoidosis, which may represent a family of diseases, to thoroughly classify and subgroup the patients clinically according to their phenotype, and we and others have accordingly described distinct HLA-DRB1 allele correlations with patients with Löfgren s syndrome only (8, 10 15). Also, an association with a specific CCR2 gene polymorphism was described in patients with Löfgren s syndrome only (17). In the present study, we analyzed two SNPs of the promoter region of the MHC2TA gene, i.e. rs and rs One of those was suggested to be important for the degree of MHC class II mrna expression and therefore with a potential influence on level of MHC class 2 expression and subsequent antigen presentation, i.e. a key feature in antigendriven inflammation such as is the case believed to be in sarcoidosis (27, 28). The sarcoidosis patients included in our study were recruited from one single centre in Stockholm, Sweden, and represent a large and homogeneous patient population. Furthermore, patients were subgrouped into those with Löfgren s syndrome, with distinct clinical manifestations, and those without this syndrome. The strongest association was found between patients with Löfgren s syndrome and the rs in allelic model, and, in addition, an association with the rs GG genotype was indicated in Löfgren s syndrome patients. Interestingly, these associations were not seen at all in patients with non-löfgren s sarcoidosis. A similar trend was noted for the rs in allelic co-dominant model and in C-dominant model, although these associations were somewhat weaker. We like to acknowledge the fact that different genetic models may be instrumental at the discovery stage in genetic studies and the allelic model helps to gain additional statistical power to the study. However, it is important not to oversee consistency between different genetic models as additional evidence for association, which was the case for our study. Knowing the strong association between HLA-DRB1*03 and Löfgren s syndrome, we also analyzed the MHC2TA SNPs in DRB1*03 pos and DRB*03 neg Löfgren s patients, but found no difference, i.e. the described MHC2TA associations were independent of DRB1*03. The finding of a separate and distinct genetic association for patients with Löfgren s syndrome is in line with the hypothesis that these patients make up a distinct disease unit, further supported by their distinct clinical disease manifestations. Also, patients with Löfgren s syndrome compared with non- Löfgren s patients have a less pronounced local lung Th1 immune response (29). Thus the genetic background, the immune response and the clinical phenotype all are distinct from non-löfgrens patients with sarcoidosis. These findings may also support the notion that Löfgren s syndrome could be an autoimmune disease, because the level of MHC class II expression might interfere with maintenance of T-cell mediated tolerance, as suggested for RA and MS (19), i.e. two typical autoimmune disorders. This is in line with our recently reported identification of antigen peptides derived from HLA molecules of BAL cells of DRB1*03 pos sarcoidosis patients, because these were all derived from selfproteins, some of which are known auto-antigens in other autoimmune diseases (30). Finally, the Löfgren s-associated DRB1*03 allele is part of the 8.1 ancestral haplotype, including several other genes of importance for the immune John Wiley & Sons A/S

5 J. Grunewald et al. MHC class II transactivator gene polymorphism system, and associated with a large number of autoimmune diseases (31). Altogether, these results may suggest that Löfgren s syndrome could be an autoimmune disease of its own entity, but with several similarities with non-löfgren s sarcoidosis, e.g. granuloma formation. It is also worth mentioning that, even though we analyzed a relatively large group of patients and controls from the same geographical area and of the same ethnicity, there was no perfect matching by gender and age between the two groups. Thus, to establish whether our data is influenced by such parameters, replication in independent age- and sex-matched cohorts will be necessary. In conclusion, we present a new genetic association with sarcoidosis, which seems confined to one particular patient subgroup, i.e. sarcoidosis patients with Löfgren s syndrome. This group of patients also have other genetic associations that differ from non-löfgren s patients including HLA DRB1 alleles and CCR2 receptor gene polymorphism (17), besides the special clinical features of this syndrome. Altogether, this indicates that Löfgren s syndrome is in fact a disease entity on its own. Acknowledgments The authors would like to thank Benita Dahlberg, Lotta Müller-Suur, Margita Dahl, Gunnel De Forest, Heléne Blomquist and Eva-Marie Karlsson for assistance and EIRA study group for access to control material. This study was supported by The Swedish Heart-Lung Foundation, The King Oscar II Jubilee Foundation, the Swedish Medical Research Council, Torsten and Ragnar Söderbergs Foundation, Karolinska Institutet and through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute. LP is supported by grant from The Swedish Research Council. References 1. Iannuzzi M, Rybicki B, Teirstein A. 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