TBX21 gene variants increase childhood asthma risk in combination with HLX1 variants

TBX21 gene variants increase childhood asthma risk in combination with HLX1 variants Kathrin Suttner, MSc, a Philip Rosenstiel, MD, b Martin Depner, MSc, c Michaela Schedel, PhD, a Leonardo A. Pinto, MD, c Andreas Ruether, PhD, b Jerzy Adamski, PhD, d Norman Klopp, PhD, e Thomas Illig, PhD, e Christian Vogelberg, MD, f Stefan Schreiber, MD, b Erika von Mutius, MD, c and Michael Kabesch, MD a Hannover, Kiel, Munich, Neuherberg, and Dresden, Germany Background: The T cell specific T-box transcription factor (TBX21) plays a crucial role in the regulation of the immune system because this factor induces the differentiation of T H 1 and blocks T H 2 commitment together with the homeobox transcription factor HLX1. Objective: The role of genetic variants in TBX21 alone and in combination with HLX1 polymorphisms was investigated in the development of T H 2-associated atopy and asthma. Methods: The TBX21 gene was resequenced in 37 adult volunteers. Polymorphisms identified were genotyped in a crosssectional (N 5 3099) and nested asthma case-control population (N 5 1872) using mainly matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Effects of promoter polymorphisms on TBX21 gene expression were studied by reporter gene assays. Furthermore, the impact of combinations of TBX21 and HLX1 polymorphisms on the development of asthma was assessed by using a risk score model. Statistical analyses were performed by using SAS/Genetics. Results: Forty-three polymorphisms were identified in the TBX21 gene. Considering a minor allele frequency of at least 10%, single nucleotide polymorphisms were assigned to 7 linkage disequilibrium blocks. Three tagging single nucleotide polymorphisms increased childhood asthma risk significantly (odds ratio [OR], 2.60, 95% CI, 1.34-5.03, P 5.003; OR, 1.39, 95% CI, 1.02-1.90, P 5.039; and OR, 1.97, 95% CI, 1.18-3.30, P 5.009). TBX21 promoter polymorphisms contained in 2 From a the Clinic for Paediatric Pneumology and Neonatology, Hannover Medical School; b the Institute of Clinical Molecular Biology, University Hospital Schleswig- Holstein, Campus Kiel; c the University Children s Hospital, Ludwig-Maximilians- Universität, Munich; d the Institute of Experimental Genetics and e the Institute of Epidemiology, Helmholtz Zentrum Munich, Neuherberg; and f the University Children s Hospital Dresden. Supported by the German Research Foundation as part of trans-regional collaborative research program TR22, Allergic Immune Responses of the Lung, grants A15 and Z3, and National Genome Research Network research grants NGFN 01GS 0429 to M.K and NUW-S23T16 to P.R. Genotyping was performed in the Genome Analysis Center of the Helmholtz Zentrum Munich. Disclosure of potential conflict of interest: P. Rosenstiel is a consultant for UCB S.A. Inc and receives grant support from Applied Biosystems. S. Schreiber receives grant support from the German Research Council, the German Ministry of Science, and the European Union Commission. M. Kabesch receives grant support from the European Union, Bundesministerium f ur Gesundheit, and Deutsche Forschungsgemeinschaft. The rest of the authors have declared that they have no conflict of interest. Received for publication April 6, 2008; revised February 2, 2009; accepted for publication February 4, 2009. Available online April 13, 2009. Reprint requests: Michael Kabesch, MD, Center for Paediatrics, Clinic for Paediatric Pneumology and Neonatology, Medical School Hannover, Carl-Neuberg-Str 1, D-30625 Hannover, Germany. E-mail: Kabesch.Michael@mh-hannover.de. 0091-6749/$36.00 Ó 2009 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2009.02.025 blocks significantly influenced TBX21 promoter activity. In a risk score model, the combination of TBX21 and HLX1 polymorphisms increased the asthma risk by more than 3-fold. Conclusions: These data suggest that TBX21 polymorphisms contribute to the development of asthma, potentially by altering TBX21 promoter activity. A risk score model indicates that TBX21 and HLX1 polymorphisms may have synergistic effects on asthma risk. (J Allergy Clin Immunol 2009;123:1062-8.) Key words: TBX21, HLX1, asthma, association study, genetic analysis, functional promoter analysis, risk score model Asthma is the most common chronic disease in childhood, affecting as many as 30% of children worldwide. 1 Asthma risk is determined by genetic susceptibility and environmental factors. 2 A lack of T H 1 responses and a deviation toward a T H 2 reaction is observed in many patients with asthma, suggesting a profound involvement of deregulation and imbalance of the immune system in the onset of atopic disorders such as asthma. Transcription factors play an important role in determining T-cell development. Although GATA-3 is crucial for T H 2development, 3 the T cell specific T-box transcription factor (TBX21) is essential for directing T cells toward T H 1 by inducing IFN-g. 4 TBX21 is significantly less expressed in the lung tissue of patients with asthma than controls without asthma. 5 TBX21-deficient mice (TBX21 1/ and TBX21 / ) developed characteristic asthma symptoms such as enhanced bronchial hyperresponsiveness (BHR) and airway remodeling. 5 Further studies showed that airway remodeling and eosinophilic airway inflammation after allergen exposure were significantly reduced in TBX21- overexpressing mice compared with GATA-3 overexpressing animals. 6 However, to obtain maximal IFN-g expression, an interaction between TBX21 and the homeobox transcription factor HLX1 (H. 20-like homeobox) is required. 7 In addition, TBX21 and HLX1 have the ability to redirect T H 2 cells into T H 1 cells. 7 Although these functional observations indicate an important role of TBX21 in asthma and other atopic diseases, a mixed picture evolves from genetic association studies investigating the effect of TBX21-related single nucleotide polymorphisms (SNPs) on atopic diseases. Whereas some studies report no association with asthma, 8,9 others describe effects of different TBX21 SNPs on asthma phenotypes. 10-12 Because the role of TBX21 variants in asthma and atopy is not yet clear, we resequenced the TBX21 gene to extend and verify the genetic information available for TBX21. In a next step, comprehensive association and interaction studies were performed, and functional analyses on selected TBX21 SNPs in the promoter region of the gene were conducted. 1062

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1063 Abbreviations used 39UTR: 39 flanking region BHR: Bronchial hyperresponsiveness HLX1: H 2.0-like homeobox LD: Linkage disequilibrium MAF: Minor allele frequency OR: Odds ratio SNP: Single nucleotide polymorphism TBX21: T cell specific T-box transcription factor previously described. 20 PCR assays and associated extension reactions were designed by using the SpectroDESIGNER software (Sequenom Inc). All amplification and extension reaction conditions have been previously described, 21 and specific primers are given in this article s Tables E2 and E3 in the Online Repository at www.jacionline.org. Primer extension products were loaded onto a 384-elements chip with a nanoliter pipetting system (SpectroCHIP, SpectroJet; Sequenom Inc) and analyzed by a MassARRAY mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). The resulting mass spectra were analyzed for peak identification by using the SpectroTYPER RT 2.0 software (Sequenom Inc). For genotyping quality control, Hardy-Weinberg calculations were performed to ensure that each marker was within the expected allelic population equilibrium. METHODS Population Between 1995 and 1996, cross-sectional studies were carried out in the German cities of Munich, Leipzig, and Dresden to assess the prevalence of asthma and allergies in white German schoolchildren age 9 to 11 years. 13,14 Informed written consent was obtained from all parents, and all study methods were approved by the local ethics committees. All children with asthma and/or BHR were selected (n 5 624) and matched with a stratified random selection of healthy, nonatopic children without asthma or BHR (n 5 1248; age 9-11) at a 1:2 ratio from the same population as previously described. 15 To investigate the influence of TBX21 tagging SNPs on other atopic phenotypes, polymorphisms were also genotyped in the original cross-sectional population from Munich and Dresden (N 5 3099; Munich, n 5 1159; Dresden, n 5 1940). Consequently, overlaps between the case-control and the cross-sectional study populations exist (see this article s Fig E1 in the Online Repository at www.jacionline.org). 16 Parental questionnaires for self-completion were sent through the schools to the families. Children whose parents reported a physician s diagnosis of asthma or recurrent spastic or asthmatic bronchitis were classified as having asthma. Atopic asthma was defined as the concomitant co-occurrence of asthma with a positive skin prick test result, whereas nonatopic asthma was defined as asthma without a positive skin test result. Current environmental smoke exposure was defined as any current environmental tobacco smoke exposure at the age of 9 to 11 years. Resequencing and mutation screening To cover 15,199 bp in and around the TBX21 gene (exons, introns, 2297 bp upstream and 990 bp downstream of the gene), 36 overlapping fragments were designed. Gene fragments of interest were amplified by PCR with specific primers (see this article s Table E1 in the Online Repository at www.jacionline.org). Primers were designed using the NetPrimer software and obtained from Metabion GmbH (Planegg-Martinsried, Germany). PCR was carried out on standard cyclers (Eppendorf GmbH, Eppendorf, Germany), in a total volume of 50 ml with 60 ng genomic DNA, 0.5 mmol/l of each amplification primer, 0.2 mmol/l of each deoxynucleotide triphosphate, and 0.8 U of Taq DNA Polymerase. PCR fragments were sequenced in at least 37 unrelated randomly selected adult volunteers by using ABI 310 and ABI 3730 sequencers (Applied Biosystems, Lincoln, Calif). Genotyping Genomic DNA was extracted from whole blood by a standard salting out method. 17 To minimize the use of genomic DNA, a modified primer extension preamplification 18 or alternatively the GenomiPhi procedure (Amersham Biosciences, Freiburg, Germany) was applied for random DNA preamplification. DNA samples were genotyped by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom Inc, San Diego, Calif), 19 except for G-999A, for which a restriction-endonuclease based assay was used, and A12406C, for which a solid-phase oligonucleotide ligation assay was performed (Variom Biotechnology AG, Berlin, Germany). SNPs A4704T, T7729C, and A8385T were genotyped by using the TaqMan MGB bialleleic discrimination system (Applied Biosystems) as Luciferase assay Jurkat cells were seeded in a 96-well plate at a density of 8 3 10 4 per well. The next day, cells were transfected with 35 ng of the plasmids expressing a luciferase gene under the control of the TBX21 promoter containing either the risk or the nonrisk allele at position T-1993C (block 2) or the combination of risk alleles at positions T-1514C and G-999A (both block 1) together with 15 ng prl-tk Renilla reporter plasmid (Promega, Madison, Wis) for normalization of transfection efficiency and cell viability. Plasmids always contained wild-type/nonrisk alleles at those polymorphic positions that were not tested. Xtreme Gene Q was used as transfection reagent according to the manufacturer s protocol (Roche, Mannheim, Germany). Eight hours after transfection, medium was exchanged by medium containing 50 ng/ml ionomycin or pure medium. After 18 hours of incubation, cells were washed in PBS and lysed in 13 passive lysis buffer (Promega). A dual luciferase assay was performed by using a dual luciferase reporter assay system (Promega) and a Genios Pro luminometer (Tecan, Crailsheim, Germany). Bioinformatics and statistical analysis Linkage disequilibrium (LD) patterns were assessed by Haploview, 22 and a threshold of r 2 0.8 was used to build LD blocks (also denoted as bins ). Deviations from Hardy-Weinberg equilibrium were tested by using the x 2 test, with expected frequencies derived from allele frequencies. x 2 Tests and logistic regression models were used to test for associations between dichotomous traits and single SNPs. We tested a recessive model and used a conservative Bonferroni correction for all tagging SNPs in every phenotype to adjust for multiple testing. A risk score was established for TBX21 and previously described HLX1 variants 23 showing associations with asthma in the single SNP analyses to account for possible gene-by-gene interaction effects. Risk alleles were determined and given a risk value of either 1 (presence of risk in dominant or recessive model) or 0 (absence of genetic risk variant). Risk values were calculated and combinations were compared to the reference population with a risk score of 0. Risk score effects were assessed by using logistic regression in the cross-sectional study population. All calculations were carried out by using the SAS/Genetics software package (version 9.13) (SAS Institute Inc, Cary, NC). RESULTS Mutation screening and polymorphism identification in the TBX21 gene Resequencing of at least 37 adult volunteers led to the identification of 43 polymorphisms in the TBX21 gene with minor allele frequencies (MAFs) of at least 3% (see this article s Table E4 in the Online Repository at www.jacionline.org). Thirteen polymorphisms (C533G, G2011A, T2473A, G2761A, C3075T, G3078A, T4716A, C5287T, A6618G, T6902C, G8760A, T10386C, delt12564) were previously not described in public SNP databases (dbsnp) and were submitted to dbsnp. Three polymorphisms were found in the promoter region, 1 in the 59 untranslated region, 31 in the intronic regions, and 5 in the 39

1064 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 FIG 1. Structure of the TBX21 gene, location of polymorphisms, and linkage disequilibrium (r 2 ) between TBX21 polymorphisms (MAF 10%) genotyped in the case-control population (N 5 1872). TBX21 tagging SNPs are underlined. Color code for the LD plot is given by Haploview: white (r 2 5 0), shades of gray (0 < r 2 < 1), and black (r 2 5 1). flanking region (39UTR). Three SNPs were located in the coding region, 1 of them (C98G) resulting in an amino acid change in the TBX21 protein. Association studies with selected TBX21 SNPs According to power calculations (see this article s Fig E2 in the Online Repository at www.jacionline.org), SNPs with a MAF of at least 10% were considered for LD-based SNP selection for genotyping. LD analysis was performed in the resequenced screening population (see this article s Fig E3 in the Online Repository at www.jacionline.org) and reassessed after genotyping in the study population MDL (Munich, Dresden, Leipzig) (n 5 1872; Fig 1). TBX21 polymorphisms could be assigned to 5 LD blocks that were genotyped by using SNPs tagging LD blocks (bins). Two common SNPs could not be assigned to LD blocks. One of these was genotyped successfully, but SNP G8766C genotyping failed repeatedly with different methods. Thus, 6 SNPs (Table E4) capturing the genetic information of 25 common SNPs (MAF 10%) at the TBX21 locus were used for further association studies (T-1514C [tagging for block 1: G-999A, G728T, G2844A, A4708T, T6902C, C11271T], C9902T [for block 2: T-1993C, A389G, C9886G, T10154C, T12080C, A12406C], C1667A [for block 3: G2613A, T4716A, G8760A], A4704T [for block 4: G1167T, A2404C, G6546A], A8385T [for block 5: T1303C], and SNP T7729C [single SNP]). The tagging SNPs T-1514C (block 1), A4704T (block 4), and C9902T (block 2) of the TBX21 gene showed significant associations with asthma in the case-control study population (Table I) in a recessive model. In all cases, the polymorphic alleles significantly increased the risk for the development of asthma (tagging SNP T-1514C: odds ratio [OR], 2.60, 95% CI, 1.34-5.03, P 5.0034; tagging SNP A4704T: OR, 1.39, 95% CI, 1.02-1.90, P 5.0389; tagging SNP C9902T: OR, 1.97, 95% CI, 1.18-3.30, P 5.0088). Interestingly, SNP G2844A contained in block 1 showed even stronger associations than the tagging SNP T-1514C itself (G2844A: OR, 3.09, 95% CI, 1.56-6.12, P 5.0007). The 2 tagging SNPs (T-1514C and C9902T) stayed significant after correction for multiple testing (C9902T in the sex/environmental tobacco smoke (ETS)-adjusted analyses only; data not shown). Haplotype analyses did not reveal any extra effects (data not shown). To investigate also other atopic phenotypes such as atopy, hay fever, atopic dermatitis, wheeze, and BHR, a cross-sectional study population of 3099 children from Dresden (n 5 1940) and Munich (n 5 1159) was genotyped and analyzed. However, no significant associations with other atopic phenotypes were observed. TBX21 SNP selection for functional studies The results of the association studies suggested that 3 LD blocks identified in the TBX21 gene may contribute to the development of asthma. Of the SNPs contained in the 3 blocks, 3 SNPs (2 from block 1 and 1 from block 2) are located within the TBX21 promoter region, 2 SNPs are located in the coding region of TBX21 but do not lead to amino acid changes, and the rest of the SNPs are located in intronic and 39UTR regions of the gene. Thus, we focused further investigations on the 3 promoter SNPs. The promoter SNP T-1993C contained in block 2 is in relatively low LD (r 2 5 0.81) with the block 2 tagging SNP C9902T. Thus, we genotyped T-1993C in the study

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1065 TABLE I. Associations in terms of ORs and (95% CIs) for recessive effects* in the case-control population (N 5 1872, unadjusted analysesy) T-1514C C1667A A4704T T7729C A8385T C9902T T-1993Ck n 5 1477 n 5 1453 n 5 1329 n 5 1534 n 5 1566 n 5 1468 n 5 1429 Risk, n 5 37 Risk, n 5 18 Risk, n 5 234 Risk, n 5 10 Risk, n 5 16 Risk, n 5 66 Risk, n 5 91 Asthma N 5 369 2.60 (1.34-5.03) 1.29 (0.46-3.65) 1.39 (1.02-1.90) 0.37 (0.05-2.90) 2.03 (0.73-5.63) 1.97 (1.18-3.30) 1.73 (1.10-2.72) with asthma N 5 1248 P 5.0034à P 5.6262 P 5.0389 P 5.3215 P 5.1640 P 5.0088 P 5.0175 supercontrols n 5 1299 n 5 1273 n 5 1168 n 5 1343 n 5 1374 n 5 1292 n 5 1258 Risk, n 5 29 Risk, n 5 16 Risk, n 5 202 Risk, n 5 10 Risk, n 5 14 Risk, n 5 54 Risk, n 5 78 Atopic N 5 171 2.77 (1.21-6.36) 1.69 (0.48-6.00) 1.59 (1.05-2.40) 0.79(0.10-6.26) 2.93(0.91-9.45) 2.11 (1.09-4.10) 2.12 (1.21-3.73) asthma N 5 1248 supercontrols P 5.0123 P 5.4120 P 5.0261 P 5.8211 P 5.0594 P 5.0241 P 5.0076à Nonatopic asthma n 5 1294 n 5 1275 n 5 1170 n 5 1341 n 5 1374 n 5 1288 n 5 1260 Risk, n 5 29 Risk, n 5 15 Risk, n 5 200 Risk, n 5 9 Risk, n 5 12 Risk, n 5 54 Risk, n 5 74 N 5 171 2.86 (1.25-6.58) 1.11 (0.25-4.97) 1.47 (0.97-2.24) 1.47 (0.32-6.75) 2.17 (1.12-4.21) 1.60 (0.86-2.99) N 5 1248 supercontrols P 5.0097 P 5.8898 P 5.0686 P 5.2630 P 5.6226 P 5.0194 P 5.1352 T-1514C: with asthma 5 351, atopic with asthma 5 165, nonatopic with asthma 5 160, without asthma 5 1163. C1667A: with asthma 5 338, atopic with asthma 5 156, nonatopic with asthma 5 157, without asthma 5 1133. A4704T: with asthma 5 359, atopic with asthma 5 166, nonatopic with asthma 5 166, without asthma 5 1204. T7729C: with asthma 5 358, atopic with asthma 5 167, nonatopic with asthma 5 164, without asthma 5 1186. A8368T: with asthma 5 363, atopic with asthma 5 169, nonatopic with asthma 5 167, without asthma 5 1219. C9902T: with asthma 5 354, atopic with asthma 5 166, nonatopic with asthma 5 162, without asthma 5 1180. T-1993C: with asthma 5 347, atopic with asthma 5 163, nonatopic with asthma 5 161, without asthma 5 117. Atopic and nonatopic with asthma do not add to 369 because of missing data for atopy. *Nonrisk 5 wild-type 1 heterozygous genotype. Case definition is based on doctor s diagnosis of asthma. BHR only is not considered here. àsignificant after correction for multiple testing. Exact numbers for the phenotypes as a result of missing values are as follows: kassociations of SNP T-1993C, contained in block 2 tagged by SNP C9902T, are also shown because this SNP is used for further functional studies. population and compared association results between both SNPs to assess the contribution of T-1993C to the block 2 association signal. Although the OR for asthma for -1993C (OR, 1.73; 95% CI, 1.10-2.72; P 5.0175) is lower than for the tagging SNP 9902T (OR, 1.97; 95% CI, 1.18-3.30; P 5.0088), -1993C seems to have a stronger impact on the development of atopic asthma (OR, 2.12; 95% CI, 1.21-3.73; P 5.0076) than the tagging SNP (OR, 2.11; 95% CI, 1.09-4.10; P 5.0241; Table I), indicating that T-1993C may substantially contribute to the association effect observed for block 2 SNPs. Because previous studies also indicated that genetic variants in the promoter/59 untranslated region of a gene most likely influence gene expression, we investigated the functional properties of promoter SNPs (T-1514C and G-999A from block 1 and T-1993C from block 2). Promoter polymorphism dependent TBX21 gene expression analysis Luciferase reporter constructs were generated harboring a 2.1- kb TBX21 promoter fragment carrying either the nonrisk alleles or risk alleles at positions T-1514C and G-999A to model the block 1 haplotype. In addition, a construct carrying the risk allele at position T-1993C was engineered. The constructs were transfected into the human Jurkat T-cell line, and gene expression levels were measured without and after mitogen stimulation (ionomycin) by luciferase reporter assays. The combination of both minor alleles of the block 1 promoter polymorphisms (T-1514C and G-999A) led to an increase in gene expression in unstimulated (P 5.0008) and stimulated Jurkat T cells (P 5.0013), whereas the minor allele of T-1993C decreased TBX21 gene expression (in unstimulated cells, P 5.00001; and in stimulated cells, P 5.00023; Fig 2, A and B). Risk score analysis Because TBX21 is able to induce optimal quantities of IFN-g only in combination with a second transcription factor, the homeobox factor HLX1, we investigated how combinations of polymorphisms in the TBX21 and HLX1 genes (previously analyzed in the same cross-sectional study population 23 ) influence the development of asthma. Two SNPs within the HLX1 gene previously shown to be associated with asthma (C-1407T: OR, 1.44, 95% CI, 1.11-1.86, P 5.0061; and T346C: OR, 0.73, 95% CI, 0.56-0.95, P 5.0172) 23 and the 3 associated TBX21 tagging SNPs (T-1514C, A4704T, and C9902T) were entered in the model (Table II) and assigned risk values ranging from a minimum of 0 points to a maximum of 5 points.

1066 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 FIG 2. TBX21 polymorphisms influence TBX21 promoter activity in Jurkat T cells. Jurkat T cells were transiently transfected with 2.1-kb TBX21 promoter reporter constructs carrying the nonrisk allele, combinations of the risk alleles -1514C and -999A (A), or 1 risk allele at position -1993C (B). Cells were left unstimulated or stimulated with ionomycin (50 ng/ml) and harvested after 18 hours (n 5 6). The relative luciferase activity is presented in relative light units (RLUs). These analyses suggest that children with the maximal risk score of 5 have a more than 3-fold risk to develop asthma (OR, 3.21; 95% CI, 1.04-9.88; P 5.0420) compared with the reference population with a risk score of 0 (Table III). The combination of the 3 risk alleles in TBX21 leads to an increased asthma risk compared with the reference group (OR, 1.82; 95% CI, 1.01-3.27; P 5.0451). DISCUSSION The transcription factor TBX21 is responsible for the activation and maintenance of T H 1 development by inducing IFN-g production and suppressing T H 2-cell commitment. Because of this prominent role in determining T H cell function, TBX21 represents a promising functional candidate gene for asthma and atopy. Indeed, several studies have shown that TBX21 may play a role in the pathogenesis of asthma. Therefore, we screened the complete length of the TBX21 gene for genetic variants and studied the influence of common TBX21 polymorphisms on asthma and atopy in large and well phenotyped German populations, using a case-control and a cross-sectional design. By resequencing at least 74 chromosomes, we identified 43 polymorphisms. Thirteen of them were previously not described and were submitted to dbsnp. Using the information of LD pattern and considering all SNPs with a minor allele frequency of 10%, the 26 remaining TBX21 SNPs could be divided into 7 LD blocks tagged by the polymorphisms T-1514C, C1667A, A4704T, T7729C, A8385T, G8766C, and C9902T. Association studies revealed that 3 SNPs (T-1514C, A4704T, C9902T) tagging for 3 LD blocks increased asthma risk significantly in the asthma case-control population. When the influence of TBX21 SNPs on other atopic phenotypes was investigated in a cross-sectional population from Munich and Dresden, the associations remained limited to asthma but did not affect other atopic phenotypes. Three promoter SNPs associated with asthma were studied for their functional properties, and indeed, these SNPs were capable of changing TBX21 promoter activity significantly in a standardized in vitro model. When TBX21 SNPs were assessed in a risk score model in combination with SNPs in HLX1, a cotranscription factor for TBX21, synergistic effects of these polymorphisms were identified. Until now, a number of studies have investigated the impact of polymorphisms in and around the TBX21 gene on atopic phenotypes. 8-12 Interestingly, most of the positive studies report associations with asthma but not with other atopic phenotypes. This was confirmed by our own observations. Also, no consensus existed on which SNPs needed to be studied to capture the genetic information of the TBX21 gene locus. Therefore, we resequenced all areas of the TBX21 gene locus that could putatively affect gene function and expression, including the promoter region, all exons, all introns, and the 39 untranslated region of TBX21. On the basis of extensive genotyping information of these SNPs in a rather large cross-sectional population, LD could be assessed and tagging SNPs for the TBX21 locus could be determined unambiguously, replacing previous random genotyping strategies. Thus, our results can easily be compared with studies that had covered these tagging SNPs, 10 but comparisons may be more difficult with other previous work. Recently, Munthe-Kaas et al 10 described a haplotype consisting of 12 TBX21 SNPs to increase the OR significantly for childhood atopic asthma, which was exceptionally well phenotyped in that population. Covering that haplotype with tagging SNPs, our results confirm these associations. Previous association reports linking SNP T-1993C to asthma 11 are also in line with our results, even though in those studies, aspirin-induced asthma and asthma with polynosis were studied, whereas we investigated childhood asthma. It is more difficult to compare our results with those of Raby et al 12 because the SNPs associated in this study with asthma and BHR were all located outside the region we and others studied. Association studies performed in Korea 8 and Finland 9 detected no significant associations between TBX21 polymorphisms and any asthma phenotypes, even though both groups genotyped at least 1 of the associated tagging SNPs described in this work. Although the Finnish study population may have been too small to pick up the association effects, the study from Korea reports very different TBX21 allele frequencies compared with white populations. Furthermore, some studies had investigated rare mutations in the TBX21 gene (C98G) 24 that are not covered by the common tagging SNP approach. However, when the effect of this rare mutation leading to an amino acid change was investigated (data not shown), no effect on childhood asthma was observed. Taken together, these data indicate that TBX21 SNPs are capable of modifying the risk to develop asthma, at least in white populations. Our results and those of previous reports 11 hint at a role of promoter SNPs in these effects, putatively influencing gene expression of TBX21. To investigate whether promoter SNPs may modify TBX21 gene expression, luciferase reporter assays were performed in Jurkat T cells. Interestingly, the TBX21 reporter constructs carrying the risk promoter alleles for block 1 increased gene expression, and the promoter construct

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1067 TABLE II. Creation of risk scores concerning asthma based on variants from TBX21 (T-1514C, A4704T, and C9902T) and HLX1 (C-1407T and T346C) Allele combinations Risks No risks TBX21* T-1514C AA Aa aa aa 5 1 Non aa 5 0 A4704T AA Aa aa aa 5 1 Non aa 5 0 C9902T AA Aa aa aa 5 1 Non aa 5 0 HLX1 C-1407T AA Aa aa Aa or aa 5 1 AA 5 0 T346C AA Aa aa AA 5 1 Non AA 5 0 Maximum score 5 5 Minimum score 5 0 AA, Major allele; Aa, heterozygous; aa, minor allele. Definition of risk alleles (bold) or combinations based on single SNP analyses: *A recessive model was used for single SNP analyses. A dominant model was used for single SNP analyses. TABLE III. ORs and 95% CIs for risk scores based on TBX21 and HLX1 risk allele combinations Number OR OR lower CI OR upper CI P value of Wald test in logistic regression Only TBX21 Risk score, 0 2508 1 Risk score, 1 vs 0 304 0.95 0.62 1.46.8180 Risk score, 2 vs 0 69 1.18 0.53 2.62.6795 Risk score, 3 vs 0 96 1.82 1.01 3.27.0451 TBX21 and HLX1 Risk score, 0 781 1 Risk score, 1 vs 0 1134 1.27 0.90 1.81.1756 Risk score, 2 vs 0 704 1.77 1.23 2.54.0022 Risk score, 3 vs 0 129 1.44 0.74 2.77.2804 Risk score, 4 vs 0 65 1.98 0.90 4.38.0901 Risk score, 5 vs 0 22 3.21 1.04 9.88.0420 carrying the risk allele of block 2 decreased TBX21 promoter activity. Although our experiments were performed in Jurkat T cells, increased transcriptional activity of the TBX21 gene was observed in HEK293 and HeLa cells. These findings indicate that SNPs in the TBX21 promoter are capable of affecting TBX21 gene expression significantly. However, the direction of this effect may differ by cell line and even cell status. Similar observations were previously described for IL13 promoter polymorphisms enhancing IL13 gene expression only in primary CD4 1 T H 2 cells, not in nonpolarized CD4 1 T cells. 25 Thus, these preliminary experiments can confirm the functional relevance of TBX21 promoter polymorphisms but cannot provide a model of how these effects may occur in vivo. Evidence suggests a specific role of TBX21 in pulmonary immunology and asthma, not just in directing T-cell development in general. Thus, it may be necessary in a next step to study the functional effects of these genetic variants in pulmonary cells. Because these experiments are not feasible in human beings, the generation of TBX21 transgenic mice has been initiated. In addition, TBX21 interacts closely with the homeobox transcription factor HLX1. Recent studies demonstrated that HLX1 polymorphisms also influence the development of asthma significantly. 23 Because of the biologic relation between TBX21 and HLX1, we used a risk score model to investigate how combinations of TBX21 and HLX1 polymorphisms influence asthma risk. The analyses of the risk score model demonstrated that carriers of combinations of different TBX21 and HLX1 SNPs have a 3-fold higher risk for the onset of asthma compared with a nonrisk group. It needs to be acknowledged that the risk score model is a statistical model that does not allow deducing biological causality or interaction between SNPs. Indeed, no significant interaction terms (data not shown) were observed for the TBX21 and HLX1 SNPs in question, which may very well be a result of the low numbers of these SNP combinations in our cohort. Only experimental studies investigating protein interaction as well as SNP function in both genes concomitantly will be able to elucidate the mechanisms underlying our observations. Independent of yet understanding these mechanisms, risk score models may prove useful in identifying individuals with elevated asthma risk in future studies. Wilfried Peters, Ilona Dahmen, and Sonja Zeilinger are acknowledged for expert technical assistance performing sequencing, genotyping, and cell culture. In addition, we thank Thomas Meindl from the Department of Human Genetics of the Ludwig-Maximilians-Universität, Munich for his kind support in sequencing the TBX21 gene. This work is part of the PhD thesis of Kathrin Suttner. Clinical implications: Knowledge of TBX21 polymorphism effects may help identify and better define one of the pathways leading to the clinical manifestation of childhood asthma. REFERENCES 1. Worldwide variations in the prevalence of asthma symptoms: the International Study of Asthma and Allergies in Childhood (ISAAC). Eur Respir J 1998;12:315-35.

1068 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 2. Cookson W. The alliance of genes and environment in asthma and allergy. Nature 1999;402:B5-11. 3. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 1997;89:587-96. 4. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000;100:655-69. 5. Finotto S, Neurath MF, Glickman JN, Qin S, Lehr HA, Green FH, et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 2002;295:336-8. 6. Kiwamoto T, Ishii Y, Morishima Y, Yoh K, Maeda A, Ishizaki K, et al. Transcription factors T-bet and GATA-3 regulate development of airway remodeling. Am J Respir Crit Care Med 2006;174:142-51. 7. Mullen AC, Hutchins AS, High FA, Lee HW, Sykes KJ, Chodosh LA, et al. Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction. Nat Immunol 2002;3:652-8. 8. Chung HT, Kim LH, Park BL, Lee JH, Park HS, Choi BW, et al. Association analysis of novel TBX21 variants with asthma phenotypes. Hum Mutat 2003;22: 257. 9. Ylikoski E, Kinos R, Sirkkanen N, Pykalainen M, Savolainen J, Laitinen LA, et al. Association study of 15 novel single-nucleotide polymorphisms of the T-bet locus among Finnish asthma families. Clin Exp Allergy 2004;34:1049-55. 10. Munthe-Kaas MC, Carlsen KH, Haland G, Devulapalli CS, Gervin K, Egeland T, et al. T cell-specific T-box transcription factor haplotype is associated with allergic asthma in children. J Allergy Clin Immunol 2008;121:51-6. 11. Akahoshi M, Obara K, Hirota T, Matsuda A, Hasegawa K, Takahashi N, et al. Functional promoter polymorphism in the TBX21 gene associated with aspirininduced asthma. Hum Genet 2005;117:16-26. 12. Raby BA, Hwang ES, Van Steen K, Tantisira K, Peng S, Litonjua A, et al. T-bet polymorphisms are associated with asthma and airway hyperresponsiveness. Am J Respir Crit Care Med 2006;173:64-70. 13. Weiland SK, von Mutius E, Hirsch T, Duhme H, Fritzsch C, Werner B, et al. Prevalence of respiratory and atopic disorders among children in the East and West of Germany five years after unification. Eur Respir J 1999;14:862-70. 14. von Mutius WS, Fritzsch C, Duhme H, Keil U. Increasing prevalence of hay fever and atopy among children in Leipzig, East Germany. Lancet 1998;351:862-6. 15. Kormann MS, Carr D, Klopp N, Illig T, Leupold W, Fritzsch C, et al. G-proteincoupled receptor polymorphisms are associated with asthma in a large German population. Am J Respir Crit Care Med 2005;171:1358-62. 16. Kormann MS, Depner M, Hartl D, Klopp N, Illig T, Adamski J, et al. Toll-like receptor heterodimer variants protect from childhood asthma. J Allergy Clin Immunol 2008;122:86-92. 17. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. 18. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A 1992;89:5847-51. 19. Ding C, Cantor CR. A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc Natl Acad Sci U S A 2003;100:3059-64. 20. Haas SL, Ruether A, Singer MV, Schreiber S, Bocker U. Functional P2X7 receptor polymorphisms (His155Tyr, Arg307Gln, Glu496Ala) in patients with Crohn s disease. Scand J Immunol 2007;65:166-70. 21. Schedel M, Carr D, Klopp N, Woitsch B, Illig T, Stachel D, et al. A signal transducer and activator of transcription 6 haplotype influences the regulation of serum IgE levels. J Allergy Clin Immunol 2004;114:1100-5. 22. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-5. 23. Suttner K, Ruoss I, Rosenstiel P, Depner M, Pinto LA, Schedel M, et al. HLX1 gene variants influence the development of childhood asthma. J Allergy Clin Immunol 2009;123:82-8. 24. Tantisira KG, Hwang ES, Raby BA, Silverman ES, Lake SL, Richter BG, et al. TBX21: a functional variant predicts improvement in asthma with the use of inhaled corticosteroids. Proc Natl Acad Sci U S A 2004;101:18099-104. 25. Cameron L, Webster RB, Strempel JM, Kiesler P, Kabesch M, Ramachandran H, et al. Th2 cell-selective enhancement of human IL13 transcription by IL13-1112C>T, a polymorphism associated with allergic inflammation. J Immunol 2006;177:8633-42.

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1068.e1 REFERENCES E1. Kormann MS, Depner M, Hartl D, Klopp N, Illig T, Adamski J, et al. Toll-like receptor heterodimer variants protect from childhood asthma. J Allergy Clin Immunol 2008;122:86-92. E2. Menashe I, Rosenberg PS, Chen BE. PGA: power calculator for case-control genetic association analyses. BMC Genet 2008;9:36.

1068.e2 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 FIG E1. Overview of cross-sectional and case-control selection based on children from the International Study of Asthma and Allergies in Childhood II (Munich, Dresden) and Leipzig. E1

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1068.e3 FIG E2. Power calculator for case-control genetic association analyses. E2 Parameters were set according to the used case-control study population MDL.

1068.e4 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 FIG E3. Structure of the TBX21 gene, location of polymorphisms, and LD (r 2 ) between TBX21 polymorphisms (MAF 10%) identified in the screening population (n 5 26). Full information on all mutations and SNPs in TBX21 was available for 26 of 37 individuals. Thus, r 2 may differ from that in Table E4. Color code for the LD plot is given by Haploview: white (r 2 5 0), shades of gray (0 < r 2 < 1), and black (r 2 5 1).

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1068.e5 TABLE E1. Description of the primers used for PCR amplification and sequencing Fragment Length Sense 59-39 Antisense 59-39 Additional sequencing primer 1 552 GGG GAT GAA TCA CTT GAC C CCA GCT CTA CAT TTC TGT CCC 2 558 CCC CTA AGG GTG AAG CC CTC CAT TTT CCT TTT ATG TTA AC 3 552 CGG ATA GTT TTC ATC ATA AAA GG CTG CAT CTT GTA GCT CTA GCC 4 548 GTG CGC TTT AAG GAA CATTTC C CCC ACT CCG CCA CCT CG 5 549 CTA GTA TTA GCC ACG AGA GGG CTC CAA GGA AGC GGC TCG 6 544 CGT CGC GGG GGC GGC AGC GCA ACA GCC TGG GCA CAG ACG 7 548 CTG GTT CTT GTG AGT GGG AGG GGA CAG AAC CCT GGT GAT GTA GG 8 484 CGT CTC GTC TGT TTT TCT GGC TCG CTC CCC CCG TAC ACA CAC ACC AC 9 578 CCC CTG CGC CCA CCT CC GGT TTC TGT TTC TTT CCT TGC GCA G 10 568 GGC TTC ATG GCT CAG GGT TC GGT TGG GGG GAG CAG AGA G 11 605 CTG GGA TGA ACC CAG GAA AGT TG GGC AGG AGG AGG CAG AGG C 12 610 GTG GGG CCT CTT AAC CTT CC CTT TTC AGG TTT GCT AAT GGT AAT G 13 607 CTT CGC TTC TTC TGT CTG CAA C GGC TGT AGG CTG TAG GTG GG 14 542 CTT CCT GCT TTT GTG GGC TG CAC ACA CCC ACA GAG ACA CAG G 15 581 GTG TGG CAG TGT GTG TGT GTT G GCT GGG CTT GGT GGC TTC CCT GTG TCT CTG TGG GTG TGT G (sense 59-39) GTA ATG ACT GTG AGG ATA GCA AGG ATA G (antisense 59-39) 16 552 GAC TTG AAC CTC CCA CAT AGA TAA C CAA GAC AAA TGA AAC CAG TTA AGC 17 526 CTC CCG CCT CAG CCT CCC GAC TAT TGG TGT GGG CGT AAA TTG G TGA GCC ATA ATC GCA CCA CTG (antisense 59-39) 18 541 GAG GTT GGT AAG ACG AGG AGT TC GCA AAT TAA AAT AGC AAG GAG ATA GAT AG 19 547 GAT CCA CCT GCC TCG GC CCG AGT CAC CCG AGC G 20 558 CCT GTG TCT CCA TTT CCC TCT ACT CGG CTC ACT GCA ACC TCT G 21 334 GAG AAT AAT GGC GTG AAC CTT G GGT GGG AGA GCA GAG GTG AG CAG GCG CAG TGT TGG GTG (sense 59-39) GAC AGA GTC TCG CTC TGT CCC (antisense 59-39) 22 398 TGA ATA TGA AGA AAT GGG GAC CAC GCC GAG GAG GGT GGA TTT C 23 281 GAG GAA AGA TGG ACA GGA GTT AGA C TCA CTC TAA CCT CCA CCT TCC AG CCA ACG TGA AAC TCC ATC TCT AC (sense 59-39) 24 382 CAC CGC AAC CTT CGC C CTC AAG TGG TCT ACC CGC C 25 547 CCA ACG TGA AAC TCC ATC TCT AC CTA ACA CAA GCA GGA AGA GCA G 26 520 GAG AGG AGG GGG AAG TGT G CTC TAT TGA ACA CGG GGC TC ACT CTG CTC TTC CTG CTT GTG TTA G (sense 59-39) 27 558 CAC AAC AGC GGA ATC ATA CAG C GCA TGG AGG AGG TAC TAA ATC ACA G 28 412 CCC TGT GGT GTA AAT ACT CCT GC GGC GAA ACC CCG TCT CTA C CTT GCT CTT GTC ATC CAG GC (sense 59-39) CAT CAC TGC CCA GTT ATT TTA CG (antisense 59-39) 29 511 GGG TTC AAG CCA TTC TCC TGC CAT CCT GTA GTG GCT GGT GGG 30 551 CCT TCC CTG CCT GGT CCT CC GCA ATG GCA ACC CAT GAT TTG G 31 562 GGA CGG GGG TCA TAT TCA GG CCC TCT TCT ACC TCC AGA TGT C 32 555 AAA GTG CCC TTG CCC TAA AG GGG AAA TAG AGT CAC CTG AGT CC 33 552 GGC CCA CTG TCT TCC TTG G GCC TCA TAG CTG TGG TCC 34 549 CTA CCC CGA CCT TCC TGG C CGG TGT CCT CCA ACC TAA TAA CAC 35 558 CTA TTT TCC CAA CTG AGC AG CCA CTG TGT TTG AGC AGG 36 539 CGT ATG TTA TAA CCA TCA GCC CAA GGG ATT CTT CTC TGT CC

1068.e6 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 TABLE E2. Description of the primers used for genotyping of the TBX21 gene in the study population MDL MDL (n 5 1872) PCR primers Extension primer ACG TTG GAT GTA CCA GAA ACA CAG GAC TGG T-1993C 1st ACG TTG GAT GAG GCA GAA ACT TCC CTG TTC TCC CCA ACA CCT TAC CC ACG TTG GAT GTT CAG TGA ACA CCC TCT GAG T-1514C 1st ACG TTG GAT GTT CCA TGA CAC CTT GTG GAG TCT GTC TCT GTC TTT TGC ACA C G-999A 1st Restriction-endonuclease based assay ACG TTG GAT GCA AGC CCC ACG TTT GGT ATC C1667A 1st ACG TTG GAT GTG GAA GGG CTG TTG TCA TTG TGG TAT CCA CAC CTC TC ACG TTG GAT GCT AAT ATA GTT GCA GAC AG G2844A 1st ACG TTG GAT GTA ACA GAC TTC CAT CCC CAG TAG TTG CAG ACA GAA GAA G Fwd GCA TGC TTA ACT GGT TTC ATT TGT CT A4704T* Rev GGT GAC AGA GCA AGA CCA TGT Assay-on-demand T7729C* HCV: 2545053 Fwd GAG CAA CTG ACC CTC TGA AAG AA A8385T* Rev CCA CAG GGA TCA GCA AAC ACT AC G8766C ACG TTG GAT GTG GAC CAC AAC AGG TGG TTG A389G 1st ACG TTG GAT GGT GAG GAC TAC GCG CTA CC TTT CCC CGA CAC CTC CAG ACG TTG GAT GGA GAA ACA GCC AGA GTT TAG C9902T 1st ACG TTG GAT GGG AAG GAA GGT TCG TTT TTC ACA GCC AGA GTT TAG GAA GGA A ACG TTG GAT GTG CCG AGT TTC TCT AGG TTG T10154C 1st ACG TTG GAT GAA GTG CTG GGA TTA TGG GTG CCC TTG CTG GCT GGG TG ACG TTG GAT GTG ACT GGT TCT GCT TGT GAC C11271T 1st ACG TTG GAT GGA TGC TGG TGT CAA CAG ATG TGC TTG TGA CCC GTT TTC ACG TTG GAT GTC TCA GGT TTC ATC GTG GGC T12080C 1st ACG TTG GAT GGG CCC TTC TCT GTT TAG TAG TCA TCG TGG GCC AGG AAG C A12406C Kit specific primer set SPOLA Fwd, Forward; Rev, reverse; SPOLA, solid-phase oligonucleotide ligation assay. *Genotyping was performed by TaqMan. Genotyping of SNP G8766C failed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and TaqMan.

J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 5 SUTTNER ET AL 1068.e7 TABLE E3. Description of the primers used for genotyping of the TBX21 gene in the study population MD (Munich, Dresden) MD (n 5 3009) PCR primers Extension primer ACG TTG GAT GCT CTG AGA CCT CAC TCC TTA T-1514C 1st ACG TTG GAT GAT AAA GCA GCA TGT GTA GTG GGC TCT GTC TTT TGC ACA C Fwd GCA TGC TTA ACT GGT TTC ATT TGT CT A4704T* Rev GGT GAC AGA GCA AGA CCA TGT ACG TTG GAT GCA TCT CAT CTT CCT CCC AAG C9902T 1st ACG TTG GAT GGG TTC GTT TTT CTT CTG TCC GGG CCA GAG TTT AGG AAG GA Fwd, Forward; Rev, reverse. *Genotyping was performed by TaqMan.

1068.e8 SUTTNER ET AL J ALLERGY CLIN IMMUNOL MAY 2009 TABLE E4. Description of all identified TBX21 polymorphisms and rs numbers, their respective position within the gene, allele frequencies, LD and tagging SNPs, success rate of genotyping call in percentage (Callrate R %), and the P value for a deviation from Hardy-Weinberg equilibrium (HWE) SNP rs No. Position in relation to ATG* Base change Position in the gene structure MAFy LD (r 2 ) with tagging SNPy Tagging SNP (block) Callrate R% HWE** 1 rs4794067-1993 T/C Promoter 0.26 0.81 (2) 93.86.0863 2 rs17250932-1514 T/C Promoter 0.16 1.0 T-1514C (1) 93.54.1242 3 rs11650451-999 G/A Promoter 0.15 0.93 (1) 98.02.7517 4 rs17244544-79 C/T 59-UTR 0.07à k 5 rs2240017 98{ C/G Exon 1 0.04à k 6 rs2074190 389 A/G Exon 1 0.26 0.80 (2) 93.80.0710 7 rs41444548# 533 C/G Intron 1 0.07à k 8 rs41519545 728 G/T Intron 1 0.13à 1.0à (6) 9 rs57781320 1167 G/T Intron 1 0.44à 1.0à (4) 10 rs10514934 1303 T/C Intron 1 0.18à 1.0à (5) 11 rs8081095 1667 C/A Intron 1 0.10 1.0 C1667A (3) 90.76.3514 12 ss107793991# 2011 G/A Intron 1 0.04à k 13 rs11079787 2404 A/C Intron 1 0.44à 0.85à (4) 14 ss107793992# 2473 T/A Intron 1 0.04à k 15 rs16946264 2613 G/A Intron 1 0.14à 1.0à (3) 16 ss107793993# 2761 G/A Intron 1 0.09à k 17 rs41321047 2844 G/A Intron 1 0.16 0.99 G2844A (6) 1 T-1514C (1)àà 91.35.0555 18 ss107793995# 3075 C/T Intron 1 0.09à k 19 ss107793996# 3078 G/A Intron 1 0.09à k 20 rs8078974 4704 A/T Intron 1 0.39 1.0 A4704T (4) 96.53.7296 21 rs11653146 4708 A/T Intron 1 0.12à 1.0à (6) 22 ss107793997# 4716 T/A Intron 1 0.14à 1.0à (3) 23 ss107793998# 5287 C/T Intron 1 0.08à k 24 rs11652969 6546 G/A Intron 1 0.44à 1.0à (4) 25 ss107795097# 6618 A/G Intron 1 0.05à k 26 ss107794000# 6902 T/C Intron 1 0.16à 0.82à (6) 27 rs2158079 7729 T/C Intron 1 0.11 1.0 T7729C 95.25.0895 28 rs56308324 8385 A/T Intron 1 0.13 1.0 A8385T (5) 97.54.0175 29 ss107794002# 8760 G/A Intron 1 0.13à 1.0à (3) 30 rs58067360 8766 G/C Intron 1 0.28à 1.0 G8766C 31 rs11657388 9886 C/G Intron 3 0.25à 1.0à (2) 32 rs11079788 9902 C/T Intron 3 0.22 1.0 C9902T (2) 94.60.0704 33 rs12451801 10154 T/C Intron 3 0.22 0.99 (2) 94.55.0499 34 rs41407050# 10386 T/C Intron 3 0.08 3 5 35 rs16946878 10689 T/C Intron 3 0.09 3 5 36 rs17250953 11023 C/G Intron 4 0.08 3 5 37 rs11650354 11271 C/T Intron 5 0.16 0.98 (1) 94.93 0.1109 38 rs12721470 11758 G/A Exon 6 0.08 3 5 39 rs11657479 12080 T/C 3 -UTR 0.23 0.94 (2) 93.54 0.0616 40 rs17244587 12214 G/A 3 -UTR 0.07 3 5 41 rs7502875 12406 A/C 3 -UTR 0.22 0.95 (2) 96.85 0.0452 42 rs41447544 7 12564 DEL/T 3 -UTR 0.07 3 5 43 rs17250967 12641 T/C 3 -UTR 0.03 3 5 *Based on the TBX21 sequence obtained from the SNPper database (http://snpper.chip.org). Data determined on population-based genotyping (N 5 1872). àdata determined in screening population only (n 37). Data determined in a random selection of children from the cross-sectional study population from Dresden (n 5 711). kpolymorphism was excluded from further analysis because of its MAF <10%. {SNP leads to an amino acid change. #SNP previously not described in dbsnp (http://www.ncbi.nlm.nih.gov/projects/snp/). All these variations have been submitted to the National Center for Biotechnology Information (NCBI) database (handle: asthmagene; submitter: Michael Kabesch), and have now been included in dbsnp, or the process is still ongoing (ssnumbers). **HWE determined by controls. The genotyping of this SNP failed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and TaqMan. ààbecause G2844A was in high LD with SNP T-1514C in the case-control population (N 5 1872), block 6 was joined to block 1 (Fig 1).