Allergy 2005: 60: 1256–1261 Copyright Blackwell Munksgaard 2005 ALLERGY DOI: 10.1111/j.1398-9995.2005.00879.x Original article Association study of mast cell chymase polymorphisms with atopy

Background: Atopic disorders are the result of complex interactions between S. Weidinger1,5, L. Rmmler1, genetic and environmental factors. Associations analyses between the promoter N. Klopp2, S. Wagenpfeil3, polymorphism rs1800875 in the mast cell chymase (CMA1) and atopy- H. J. Baurecht3, G. Fischer 2, R. Holle2, related phenotypes have yielded inconsistent results. A. Gauger1, T. Schfer4, T. Jakob5, Methods: We sequenced the CMA1 locus in 24 unrelated healthy individuals M. Ollert1, H. Behrendt6, with serum IgE levels <50% percentile and 24 individuals with atopic eczema H. E. Wichmann2, J. Ring1,6, T. Illig2 and serum IgE levels >90% percentile. Seven CMA1 single nucleotide poly- 1Department of Dermatology and Allergy morphisms (SNPs) were evaluated for evidence of associations with atopic Biederstein, Technical University Munich, Munich; phenotypes within a large population of German adults (n ¼ 1875). Subjects 2Institute of Epidemiology, GSF-National Research were phenotyped by standardized questionnaires and interviews, skin prick Center for Environment and Health, Neuherberg; 3Institute for Medical Statistics and Epidemiology, testing and serum IgE measurements. Genotyping was performed using MAL- 4 DI-TOF MS (Matrix-Assisted Laser Desorption Ionization–Time of Flight mass Technical University Munich, Munich; Institute of Social Medicine, Medical University Lbeck, Lbeck; spectrometry). 5Department of Dermatology and Allergy TUM and Results: Promoter polymorphism rs1800875 was significantly associated with Division of Experimental Dermatology and Allergy atopic eczema. No associations between any other single SNP and atopic phe- GSF/TUM, Munich, Germany; 6Division of notypes could be detected. Haplotype reconstruction revealed four of 128 Environmental Dermatology and Allergy GSF/TUM, possible haplotypes reaching estimated frequencies of 3% or more. Two of these GSF National Research Center for Environment and haplotypes showed a borderline-significant association with atopic eczema, Health & ZAUM Center for Allergy and Environment, which did not remain significant after correction for multiple testing. Technical University Munich, Munich, Germany Conclusions: Results confirm previous observations of a significant association between the CMA1 promoter polymorphism rs1800875 and atopic eczema, but Key words: association; atopic eczema; chymase; not with serum IgE levels, and support the hypothesis that CMA1 serves as promoter; single nucleotide polymorphisms. candidate gene for atopic eczema. Dr med. Stephan E. Weidinger Department of Dermatology and Allergy Technical University Munich Biedersteiner St 29 80802 Munich Germany

Accepted for publication 20 March 2005

Atopy is defined as personal or familial tendency to Mast cells express a complete and functional high- produce IgE antibodies in response to low doses of affinity IgE receptor, whose aggregation via IgE mole- enviromental allergens, and to develop atopic diseases cules leads to MC activation, granule exocytosis, and (asthma, rhinoconjunctivitis, atopic eczema) (1). Over the release of preformed mediators such as histamine, tryp- last decades, the prevalence of atopic diseases has shown tase and chymase, as well as newly synthesized products a steady increase in Western countries, now having such as prostaglandin D2 and leukotrien C4 [reviewed plateaued (2). Current pathophysiologic concepts suggest in(7)]. Although the pivotal role of MCs in allergic interactions between susceptibility , the host’s envi- inflammation is well known, the role of chymase, which is ronment and immunological factors (3). Mast cells (MCs) present in large quantities in the secretory granules, is not represent key effector cells of IgE-dependent immediate fully understood yet. There is accumulating evidence that reactions, and also contribute significantly to certain chymase contributes to fibrosis and tissue remodelling by features of IgE-associated late-phase reactions and chro- directly activating procollagenase, initiating collagen nic allergic inflammation [reviewed in (4)]. In addition, fibril formation, releasing transforming growth factor MCs have been shown to exert beneficial functions such beta 1 from the and by converting as tissue repair [reviewed in (5)] and as participants in I to angiotensin II. In addition, human MC innate and acquired immune resonses (6). chymase has been shown to promote vascular and

1256 Association study of mast cell chymase polymorphisms with atopy epithelial permeability and to induce the in-vivo-recruit- of Augsburg, South Germany. The sampling frames and study de- ment of neutrophils and other leukocytes [reviewed in signs have been described previously (19, 20). Briefly, from KORA (8)]. C subjects were selected so as to provide 50% with and 50% without demonstration of specific IgE to common environmental Atopy and atopic disorders are likely to result from allergens in the Radio-Allergo-Sorbent-Test (RAST), and, further- complex interactions between several genetic and envi- more, so that within these groups 50% suffered from respiratory ronmental factors. Genome-wide searches have detected a atopy. From KORA S4, subjects which had not been part of KORA number of regions linked to atopic phenotypes, and a C were selected so as to provide 50% with atopic eczema and 50% variety of gene variants have been associated with atopy- without. Finally, we ended up with n ¼ 1991 persons aged 25– related traits [reviewed in (9)]. However, most reported 74 years in our study population for case–control analysis. DNA associations are not robust and lack reproducibility (10). samples were available from 1875 subjects. All study methods were approved by the Ethics committee of the Among the genomic regions linked to atopy is the long ÔBayerische Landesa¨ rtzekammerÕ Munich. arm of 14 (14q11), where CMA1 encoding A written and informed consent was obtained from all partici- chymase is located (11, 12). There have been several pants prior to the beginning of the study. reports about a significant association between a CMA1 promoter polymorphism (rs1800875) and atopic eczema in Japanese adults and school-children (13–15). These Phenotyping results could not be confirmed in another Japanese study All subjects had to complete a standardized questionnaire, which with 100 patients and 69 patient-parents-trios (16) and an next to demographic data included the basis allergy questions of the Italian study with 70 patients (17). Recently, a family- European Community Respiratory Health Survey (ECRHS) on based association study in Caucasians revealed a signifi- respiratory health (21). In addition, all probands were interviewed cant association of this polymorphism with total IgE in a standardized manner to report on symptoms of atopic diseases, basic family data and parental history of atopic diseases. levels in patients with self-reported atopic eczema (18). To All subjects received a conventional lancet skin prick test and and further clarify a possible association between the CMA1 total and specific IgE measurements (enzyme-immuno assay; CAP- gene and atopy related phenotypes we systematically FEIA, Pharmacia, Uppsala, Sweden) using a sample of common sequenced all five exons, parts of the neighbouring in- allergens (grass, birch, rye, mugwort pollen, Alternaria, Cladospo- trons, and the promoter region of the gene in 48 unrelated rium, cat, dog, and Dermatophagoides pteronyssinus). Saline and persons. Seven densely spaced SNPs, which cover the histamine were used as controls (ALK-Scherax, Hamburg, Ger- many). Subjects were classified as having allergic rhinoconjunctivitis whole gene (Fig. 1) were selected for further analyses and or asthma when they reported a doctor’s diagnosis of hay fever or genotyped in a large population-based cohort of 1875 asthma, respectively. Specific sensitization was defined to be present adults. if at least one of the specific IgE antibodies was positive (CAP class ‡1, corresponding to ‡0.35 KU/l) or if a positive skin prick test reaction (wheal diameter ‡3 mm after subtraction of the negative Methods control) against at least one of the allergens was observed. Individuals from KORA S4 received a skin examination by Study population experienced senior dermatologists who had been additionally trained before the start of the study according to the criteria of Subjects were recruited from the KORA C (Cooperative Health Hanifin & Rajka (22) and the UK diagnostic criteria for atopic Research in the Augsburg Region) and KORA S4 survey, which eczema (23). were large population-based cross-sectional studies on Caucasian adults carried out 1994–1995 and 1999–2001 in the city and region Genetic analyses Human CMA1 gene (3,2kb) Sequencing. To detect up to now unknown DNA variants in the human CMA1 gene we sequenced all exons, including parts of the neighbouring introns, and the predicted promoter region of the gene in 24 unrelated healthy individuals with serum IgE levels <50% percentile and 24 individulas with atopic eczema and serum IgE rs5248 rs1956923 rs5250 rs5246 rs5247 rs5244 rs1800875 levels >90%. PCR was performed in a volume of 20 ll in 96-wells each containing 20 pmol of each primer, 20 ng genomic DNA, 0.4 U of HotstarTaq polymerase (Qiagen, Hilden, Germany), 20 lmol of dNTPs and the appropriate PCR-Puffer (Qiagen). Thermocycling started with an initial denaturation step of 95C for 15 min followed by 35 cycles of 95C for 30 s, 60C for 45 s, and Exon 1 2 3 4 5 72C for 60 s. A final extension step at 72C for 10 min was implemeted. PCR-products were purified utilizing MultiScreen- PCR Filter Plate Kit (Millipore, Eschborn, Germany) according to Figure 1. Structure of CMA1 gene. Gene structure of CMA1 the manufacturer’s protocol. Subsequently cycle sequencing of the showing the location of all SNPs genotyped in this study indi- purified PCR products was performed according to a standardized cated by arrows (round-tailed arrows indicate exonic SNPs). protocol for BigDye Terminator v3.1 (Applied Biosystems, Foster The CMA1 locus spans 4.6 kb (CMA1 gene 2.8 kb additional City, CA). Sequence detection was carried out on the ABI prism promotor 1.8 kb). 3730 Genetic Analyser (Applied Biosystems).

1257 Weidinger et al.

Genotyping Table 1. Phenotype characteristics of adults included in analyses (n ¼ 1875)

Genomic DNA was extracted from leukocytes with a commercial Total study population (n ¼ 1875) DNA isolation kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s recommendation. Genotyping Male gender 866 (46.2%) analyses were carried out by using the MassARRAY system (Se- Mean (€SD) age 49.0 (€13.6) quenom, San Diego, CA, USA) as described previously (19). Asthma 167 (8.9%) % with specific sensitization 76.3 Allergic rhinoconjunctivitis 488 (26.0%) Statistics % with specific sensitization 81.8 Atopic eczema 242 (12.9%) Descriptive statistics for quantitative and qualitative values are % with specific sensitization 50.8 given by mean ± standard deviation (SD) and relative frequencies Total serum IgE >100 kU/l 604 (32.2%) or absolute numbers, respectively. For descriptive statistics SPSS Mean (€SD) ln(total serum IgE) 3.9 (€1.4) version 11.5 was used. Association of quantitative traits with SNP genotype was tested with t-tests for two independent samples or Satterthwaite tests using the SAS statistical software package with atopy-related phenotypes. One of the analysed SNPs version 9.1. Deviation from Hardy–Weinberg equilibrium was was rs1800875, which was already tested in different tested with PROC ALLELE in SAS/Genetics for any of the SNP’s previous studies with conflicting results. Considering under consideration. Linkage disequilibrium was assessed with procedure ldmax in the software package gold. Haplotype fre- multiple testing and the two-sided significance level of quencies were estimated from genotype data using the EM algo- 5% none of the genotyped polymorphisms showed a rithm (24, 25). To evaluate associations with quantitative and significant deviation from Hardy–Weinberg equilibrium. qualitative traits, haplotype trend regression models were applied Using a normalized correlation coefficient the degree of including estimated probabilities of haplotypes in a linear and linkage disequilibrium was assessed between single poly- logistic regression approach as independent variables (25), morphisms and expressed in a gold plot as indicated in respectively. Further independent variables were study population Fig. 2. (KORA C or KORA S4), age, family size, and sex. Because smoking is an essential influence factor on IgE levels and atopic In the single SNP analyses homozygosity (GG) at diseases (26), smoking status was included as independent variable rs1800875 was significantly associated with atopic eczema in multiple regressions. with an OR of 1.91 (P ¼ 0.002, 95% CI ¼ 1.25–2.86). The same covariates were included in logistic regressions testing None of the other SNPs genotyped in this study showed associations of qualitative traits with SNP genotypes. Results are any significant association results with atopic eczema. reported after applying backward elimination and stepwise vari- For haplotype analysis 1730 samples were available in able selection. All haplotype analyses were implemented using the which genotyping was successful in each of the seven SAS/genetics module (27). Odds ratios (ORs) are given with two- sided P-values in parentheses. The patterns of linkage disequi- CMA1 polymorphisms (Fig. 1). The EM algorithm librium between markers were visualized using the gold 1.0 showed four of 128 possible haplotypes exceeding a package (http://www.well.ox.ac.uk/asthma/gold). frequency of 3%. 95% confidence intervals for frequen- cies are small because of the large study populations indicating sufficiently high accuracy (Table 3). For hapl- otype GCCGAGC we observed a tendency for associ- Results ation with atopic eczema (P ¼ 0.05, OR ¼ 2.64, 95% Since regression analyses revealed no significant popula- CI ¼ 1.00–7.00), whereas haplotype ACTGAAC was tion effect, both studies (KORA C, KORAS4) were slightly protective against the development of atopic analyzed together. 8.9% (n ¼ 167) of the individuals with eczema (P ¼ 0.05, OR ¼ 0.617, 95% CI ¼ 0.38–1.00). available DNA samples (n ¼ 1875) were diagnosed with However, these associations did not remain significant asthma, 12.9% (n ¼ 242) were suffering from atopic after correction for multiple testing. No associations for eczema, and 26.0% (n ¼ 488) from allergic rhino-con- SNPs or haplotypes were seen for the following traits: junctivitis. IgE serum levels of more than 100 kU/l were asthma, allergic rhinoconjunctivitis, allergic sensitization, detected in 32.2% (n ¼ 604). Between 1809 and 1868 total serum IgE levels, total serum IgE levels in individ- samples (average call rate 97.6%) were genotyped uals with asthma or atopic eczema. When allergic successfully for the respective CMA1 polymorphisms sensitization was defined as CAP class ‡2 against at least (Fig. 1). one of the allergens tested, no associations were Phenotyping as well as genotyping details and allele observed. frequencies are given in Tables 1 and 2. Sequence analysis of all exons of the gene with adjacent intronic sequences as well as of the promoter region in 48 Discussion probands revealed no up to now unknown DNA variants. Seven SNPs with very dense average spacing of 0.7 kB Chromosome 14q contains several important candidate were chosen. Thus we could systematically cover the genes, which could contribute to the genetic predisposi- whole gene region and evaluate evidence for association tion to atopy. Chromosome 14q.11 has been linked with

1258 Association study of mast cell chymase polymorphisms with atopy

Table 2. CMA1 polymorphisms, dbSNP accession number, localization, call rates, allele frequencies, and genotyping primers

SNP Id+ Location Allele Allele frequency (%) Call rate (%) Direction PCR primer Extension primer

rs1800875 5¢ flanking G 47.4 96.4 fwd ACGTTGGATGCCTATTCCATTTCCTCACCC CCAAGACTTAAGTTTTGCT A 52.6 rev ACGTTGGATGGCATCAAGATTCAGATCCAC rs1956923 5¢ flanking C 74.4 97.3 fwd ACGTTGGATGACTGAACGTTCAGTGCAAAG CTCCACTCCATTCCACTACAC T 25.6 rev ACGTTGGATGAAATGGGGTTGTTGCTCAAG rs5244 5¢ flanking C 19.4 97.4 fwd ACGTTGGATGCTCTCACTCTGAATTGGCTG GGTGATTCTAGGGGAACTTC T 80.6 rev ACGTTGGATGTCAGGGCTCAAGTTCCTTTC rs5246 Exon 2 C 0.6 96.5 fwd ACGTTGGATGCGTCAGCACAAAGTTCCGTC ACCACCACAAAATTTTGAGGGACC G 99.4 rev ACGTTGGATGCATGGCCTACCTGGAAATTG rs5247 Exon 2 G 02.0 99.2 fwd ACGTTGGATGACTCTGGTTGTTCATCTCCC CTGTTGTCTCACCTTCCTGCACAA A 98.0 rev ACGTTGGATGAGACGGAACTTTGTGCTGAC rs5248 Intron 2 A 92.0 98.9 fwd ACGTTGGATGCATCACTGTCGCTCCTTTTC CCTTCCTTCTTCCTCACA G 08.0 rev ACGTTGGATGTATGTTATGGGCTCCAAGGG rs5250 3¢ flanking C 88.4 97.7 fwd ACGTTGGATGGAGAGTTCTGTGACCTGTAG TGGTCCCTCAGCCACAA T 11.6 rev ACGTTGGATGTGCCACTCATTCTGGTCTAC

+SNP number according to dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP)

rs5250 comparability of Asian and Caucasian populations. 1.00 Iwanaga et al. (18) recently confirmed the association rs5248 with IgE levels in Caucasian patients with asthma and self-reported atopic eczema, but atopic eczema was not rs5247 the primary trait examined within this study, and the number of atopic individuals without asthma was low, so rs5246 that no conclusions concerning the effect on sensitization alone were possible. rs5244 We aimed to clarify this ambiguity concerning trans- mission of susceptibility to atopy and atopy-related traits rs1956923 by CMA1 polymorphisms. Sequencing of the predicted 0.00 promoter region, all exons and adjacent intronic rs1800875 sequences in 48 persons enabled us to detect all common D' DNA variants. in functional regions of the gene. rs5246 rs1800875 rs5247 rs5250 rs1956923 rs5248 We subsequently assessed seven SNPs in the CMA1 rs5244 locus (4.8 kb), which were very densely spaced (average Figure 2. gold linkage disequilibrium map of the CMA1 SNPs 650 bp), including the above mentioned promoter poly- applying a normalized correlation coefficient. Pairwise estima- morphism rs1800875, on atopic phenotypes in a large tions of Lewontin’s measure of disequilibrium DÕ are shown population-based cohort of German adults (n ¼ 1875). from unrelated subjects on a scale of 1 (complete linkage dis- The traits examined included atopic eczema, asthma, equilibrium: red) to 0 (blue). Marker positions are shown as a allergic rhinoconjunctivitis, specific sensitization and total schematic rather than as actual distances apart. serum IgE levels. In the single marker analyses, polymorphism specific allergic reactions, asthma and total IgE (11, 12, rs1800875 showed significant association with atopic 28), suggesting the presence of a gene (or genes) in the eczema confirming previous findings in small cohorts region that predispose(s) to allergic diseases. Previous (13–15). However, no association was seen with high studies focussing on one BstXI polymorphism total IgE levels in subjects with eczema as reported (rs1800875) within the promoter region have indicated previously (18), or with the other atopic traits. Thus, association with atopy. Initially, in a Japanese cohort of the present study underlines that despite a considerable adults, an association with atopic eczema was observed overlap between atopic traits, they might also be (13). In two subsequent studies this observation was influenced by unshared genetic determinants, and that confirmed, especially for atopic eczema with low total eczema might be largely influenced by a different set of serum IgE levels (14, 15), whereas another study failed to genetic loci (29). replicate the association (16). In contrast, associations Since it has been suggested that haplotype analyses with raised IgE levels have been reported in patients may be of higher informative value to draw associations suffering from both atopic eczema and asthma (15). between phenotypes and genetic variation than SNPs However, the mentioned studies exhibit a variety of (30), we also performed haplotype analyses using the potential limitations, as for example rather low case EM algorithm. A borderline-significant tendency numbers resulting in low statistical power, and limited towards association with atopic eczema was seen for

1259 Weidinger et al.

Table 3. Frequency of CMA1 SNP genotypes and haplotypes in atopic eczema and two CMA1 haplotypes, which did not remain significant controls. Haplotypes with frequencies <3% were not considered after correction for multiple testing. Genotype, haplotype frequencies In conclusion, our data confirm and expand previous observations that the polymorphism rs1800875 in the Location and Atopic Control Total study promoter region of CMA1 is significantly associated with SNP ID genotypes eczema numbers population atopic eczema. Based on our analyses it seems unlikely rs1800875 5¢ flanking that the associations of CMA1 with atopic eczema and AA 26.8 28.8 28.5 related traits are caused by any other allele in the gene AG 44.8 48.6 48.0 region. GG 28.4 22.6 23.5 It may be speculated whether this DNA variant alters rs1956923 5¢ flanking the expression of chymase. Recently, it has been shown CC 54.8 55.4 55.3 that MC chymase is increased in chronic atopic eczema CT 36.1 38.7 38.4 TT 9.1 5.9 6.3 skin lesions (31), and a potential role of chymase in the rs5244 5¢ flanking promotion of the skin barrier defect and cutaneous CC 5.1 3.9 4.1 neovascularization has been suggested (32). Preliminary CT 33.5 30.6 31.0 studies in animal models have indicated a therapeutic TT 61.4 65.5 64.9 potential of chymase inhibitors in atopic eczema (33, 34). rs5246 Exon 3 Functional studies and analyses considering interaction CC 0 0 0 with other loci are needed to clarify the consequences of CG 0.8 1.3 1.2 GG 99.2 98.7 98.8 polymorphism rs1800875 in the CMA1 gene and might rs5247 Exon 3 help to evaluate whether chymase may qualify as target AA 94.0 95.7 95.5 for therapeutical interventions in atopic eczema. AG 6.0 4.3 4.5 GG 0 0 0 rs5248 Intron 4 AA 83.4 85.2 85.0 Acknowledgments AG 16.6 14.1 14.4 GG 0 0.7 0.6 The KORA research platform (KORA: Cooperative Research in rs5250 3¢ flanking the Region of Augsburg) was initiated and financed by the GSF– CC 74.8 77.8 77.4 National Research Centre for Environment and Health, which is CT 22.8 21.1 21.4 funded by the German Federal Ministry of Education, Science, TT 2.4 1.1 1.2 Research and Technology and by the State of Bavaria. A-C-T-G-A-A-C 46.0 50.5 49.9 This work was supported by a research grant within the G-C-C-G-A-A-T 13.5 11.5 11.8 German National Genomic Research Network (NUW-S31T05) G-C-C-G-A-G-C 8.0 7.5 7.6 and by a research grant from the University Hospital ÔÔRechts der G-T-T-G-A-A-C 26.5 24.6 24.9 IsarÕÕ, Technical University Munich (KKF-07/04).

References

1. Johansson SG, Bieber T, Dahl R, 6. Marshall JS, Jawdat DM. Mast cells in 11. Moffatt MF, Hill MR, Cornelis F, Friedmann PS, Lanier BQ, Lockey RF innate immunity. J Allergy Clin Immu- Schou C, Faux JA, Young RP et al, et al. Revised nomenclature for allergy nol 2004;114:21–27. Genetic linkage of T-cell receptor alpha/ for global use: Report of the Nomen- 7. Siraganian RP. Mast cell signal trans- delta complex to specific IgE responses. clature Review Committee of the World duction from the high-affinity IgE Lancet 1994;343:1597–1600. Allergy Organization, October 2003. receptor. Curr Opin Immunol 12. The Collaborative Study on the Genetics J Allergy Clin Immunol 2004;113:832– 2003;15:639–646. of Asthma (CSGA). A genome-wide 836. 8. Miller HR, Pemberton AD. Tissue-spe- search for asthma susceptibility loci in 2. Hertzen L, Haahtela T. Signs of revers- cific expression of mast cell granule ser- ethnically diverse populations. Nat ing trends in prevalence of asthma. ine proteinases and their role in Genet 1997;15:389–392. Allergy 2005;60:283–292. inflammation in the lung and gut. 13. Mao XQ, Shirakawa T, Yoshikawa T, 3. Ring J, Kramer U, Darsow U, Behrendt Immunology 2002;105:375–390. Yoshikawa K, Kawai M, Sasaki S et al. H. Why are allergies increasing? Curr 9. Steinke JW, Borish L, Rosenwasser LJ. Association between genetic variants of Opin Immunol 2001;13:701–708. Genetics of hypersensitivity. J Allergy mast-cell chymase and eczema. Lancet 4. Williams CM, Galli SJ. The diverse Clin Immunol 2003;111:S495-S501. 1996;348:581–583. potential effector and immunoregulatory 10. Hirschhorn JN, Lohmueller K, Byrne E, 14. Mao XQ, Shirakawa T, Enomoto T, roles of mast cells in allergic disease. J Hirschhorn K. A comprehensive review Shimazu S, Dake Y, Kitano H et al. Allergy Clin Immunol 2000;105:847–859. of genetic association studies. Genet Association between variants of mast cell 5. Boyce JA. Mast cells: beyond IgE. Med 2002;4:45–61. chymase gene and serum IgE levels in J Allergy Clin Immunol 2003;111:24–32. eczema. Hum Hered 1998;48:38–41.

1260 Association study of mast cell chymase polymorphisms with atopy

15. Tanaka K, Sugiura H, Uehara M, Sato H, 20. Illig T, Bongardt F, Scho¨ pfer A, Holle R, 28. Deichmann KA, Hildebrandt F, Kuehr J, Hashimoto-Tamaoki T, Furuyama J. Mu¨ ller S, Rathmann W et al. The endo- Forster J. Genetic linkage analysis of Association between mast cell chymase toxin receptor TLR 4 polymorphism is predicted asthma genes and atopy. genotype and atopic eczema: comparison not associated with diabetes or compo- Allergy 1995;50:164. between patients with atopic eczema nents of the metabolic syndrome. Diabe- 29. Cookson W. Genetics and genomics of alone and those with atopic eczema and tes 2003;52:2861–2864. asthma and allergic diseases. Immunol atopic respiratory disease. Clin Exp 21. Burney PG, Luczynska C, Chinn S, Rev 2002;190:195–206. Allergy 1999;29:800–803. Jarvis D. The European Community 30. Stephens JC, Schneider JA, Tanguay DA, 16. Kawashima T, Noguchi E, Arinami T, Respiratory Health Survey. Eur Respir J Choi J, Acharya T, Stanley SE et al. Kobayashi K, Otsuka F, Hamaguchi H. 1994;7:954–960. Haplotype variation and linkage disequi- No evidence for an association between a 22. Hanifin JM, Rajka G. Diagnostic fea- librium in 313 human genes. Science variant of the mast cell chymase gene tures of atoopic dermatitis. Acta Der- 2000;293:489–493. and atopic dermatitis based on case- matovenereol 1980;92:44–47. 31. Badertscher K, Bronnimann M, Karlen S, control and haplotype-relative-risk ana- 23. Williams H, Burney PG, Hay RJ, Archer Braathen LR, Yawalkar N. Mast cell lyses. Hum Hered 1998;48:271–274. CB, Shipley MJ, Hunter J et al. The UK chymase is increased in chronic atopic 17. Pascale E, Tarani L, Meglio P, Businco L, working party’s diagnostic criteria for dermatitis but not in psoriasis. Arch Battiloro E, Cimino-Reale G et al. atopic eczema. I. Derivation of a mini- Dermatol Res 2005;296:503–536. Absence of association between a variant mum set of discriminators for atopic ec- 32. Groneberg DA, Bester C, Grutzkau A, of the mast cell chymase gene and atopic zema. Br J Dermatol 1994;131:383–396. Serowka F, Fischer A, Henz BM et al. dermatitis in an Italian population. Hum 24. Excoffier L, Slatkin M. Maximum-like- Mast cells and vasculature in atopic Hered 2001;51:17717–17719. lihood estimation of molecular haplo- dermatitis–potential stimulus of neoan- 18. Iwanaga T, McEuen A, Walls AF, type frequencies in a diploid population. giogenesis. Allergy 2005;60:90–97. Clough JB, Keith TP, Rorke S et al. Mol Biol Evol 1995;12:921–927. 33. Imada T, Komorita N, Kobayashi F, Polymorphism of the mast cell chymase 25. Slatkin M, Excoffier L. Testing for Naito K, Yoshikawa T, Miyazaki M gene (CMA1) promoter region: lack of linkage disequilibrium in genotypic data et al. Therapeutic potential of a specific association with asthma but association using the Expectation-Maximization chymase inhibitor in atopic dermatitis. with serum total immunoglobulin E lev- algorithm. Heredity 1996;76:377–383. Jpn J Pharmacol 2002;90:214–217. els in adult atopic dermatitis. Clin Exp 26. Jarvis D, Chinn S, Luczynska C, Burney 34. Watanabe N, Tomimori Y, Saito K, Allergy 2004;34:1037–1042. P. The association of smoking with sen- Miura K, Wada A, Tsudzuki M et al. 19. Weidinger S, Klopp N, Wagenpfeil S, sitivity to common allergens: results Chymase inhibitor improves dermatitis Ru¨ mmler L, Schedel M, Kabesch M from the European Community Respir- in NC/Nga mice. Int Arch Allergy et al. Association of a STAT 6 haplotype atory Health Survey. J Allergy Clin Immunol 2002;128:229–234. with elevated serum IgE levels in a pop- Immunol 1999;104:934–940. ulation-based cohort of caucasian adults. 27. SAS II. SAS/Genetics(r) User’s Guide. J Med Gen 2004;41:658–663. NC, USA: Cary, 2002.

1261