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INTRODUCTION
Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) cause symptoms in 20-30% of patients with chronic urticaria (CU).1,2 In the Korean population, 35.7% of CU patients are affected, as confirmed by oral provocation testing.3 Several genes are likely to be involved in the etiology and pathogenesis of aspirin-intolerant urticaria (AIU), including both the acute and chronic forms as well as intermediate phenotypes. The candidate genes include HLA-DRB1 *1302-DQB1*0609,4 ALOX5,5 FCER1A,6 HNMT,7 and FCER1G.8
The cytokine interleukin (IL)-13 is produced primarily by activated Th2 cells9 and is thought to play a role in the mechanisms of several atopic conditions. IL-13 promotes B cell differentiation and is capable of inducing isotype-switching in B cells, resulting in the production of IgG4 and IgE.10 Human lung mast cells express IL-13Rα1, and activation of these cells by the cytokine for 5 days increased FcεRI expression and proliferation.11 IL-13 maps to chromosome 5q31, which has been implicated in the development of asthma and atopy.12 It is a critical effector in the induction and maintenance of IgE production and IgE-mediated allergic airway responses.13
Analyses of IL-13 gene polymorphisms have produced various results depending on the specific study population. For example, genetic association studies in German14 and Chinese populations have suggested a role for the IL13 variant Arg110Gln in the increased IgE production and atopic sensitization that characterizes allergic rhinitis, whereas this allele was found to be associated with asthma in British and Japanese populations.15 Previous studies have reported that AICU patients had a significantly higher rate of atopy than ATCU patients and normal controls, suggesting that atopy is a risk factor for AICU.3,8 A study on FcER1α promoter polymorphisms found that the prolonged exposure of AICU patients to IgE induces the persistent accumulation and activation of mast cells and basophils via increased surface expression of high-affinity IgE receptors.6
To identify genetic factors in the development of AICU, we analyzed the genotypes and haplotypes of three polymorphisms of IL-13 to determine whether genetic variants of IL-13 play an important role in AICU.
MATERIALS AND METHODS
Study subjects and phenotyping
Two groups of CU patients, AICU (n=135) and ATCU (n=146), who were identified based on the results of n oral provocation test with up to 500 mg of aspirin (Rhonal; KunWha Pharmaceutical Co., Seoul, Korea), were enrolled in the study, as described previously.4 AICU patients who had aspirin-intolerant asthma (AIA) were excluded. Normal healthy controls (NC; n=430) with no personal or family history of allergic diseases, or aspirin or drug hypersensitivity were recruited from the general Korean population. Informed consent was obtained from all participants, and the institutional review board of Ajou University Hospital approved this study. Skin-prick tests were performed with 55 common aeroallergens (Bencard Co., West Sussex, UK), and atopy was defined as one or more positive reactions to common inhalant allergens. Total IgE concentrations were measured using a UniCAP system (Phadia, Uppsala, Sweden), according to the manufacturer's instructions. Anti-thyroglobulin levels in serum were measured using anti-Tg (B.R.A.H.M.S Aktiengesellschaft, Hennigsdorf, Germany) and serum antimicrosomal antibodies with a thyroid peroxidase IgG ELISA (Zeus Scientific, Zierikzee, Netherlands).
SNP identification and genotyping
Peripheral blood samples from 40 healthy Korean volunteers were used to identify single nucleotide polymorphisms (SNPs). Genomic DNA was prepared from the blood samples using a Puregene DNA purification kit (Gentra, Minneapolis, MN, USA), according to the manufacturer's protocol.
The IL-13 gene was examined for SNPs using an ABI Prism 3100 DNA analyzer (Applied Biosystems, Foster City, CA, USA). Three polymorphisms of IL-13, -1510 A>C, -1055C>T, and Arg110Gln (110G>A), were genotyped using a primer-extension method and a SNAPshot ddNTP primer-extension kit (Applied Biosystems). The primers used were: for IL-13 -1510A>C, forward (F) 5'-TGTGGGAGATGCCGTGGG-3', reverse (R) 5'-TCTGACTCCCAGAAGTCTGC-3', and extension 5'-TACAGATTAGGAAACAGGCCCGTAG-3'; for IL-13-1055C>T, (F) 5'-AGAGAGGGTGGGAATGAC-3', (R) 5'-CCAGTCTCTGCAGGATCA-3', and extension 5'-TGTCGCCTTTTCCTGCTCTTCCCTC-3'; and for IL-13 110G>A, (F) 5'-AGTTTGTAAAGGACCTGCTCTTAC-3', (R) 5'-TCAGGTCCTGTCTCTGCAA-3', and extension 5'-TTAAAGAAACTTTTTCGCGAGGGAC-3'.
Statistical analysis
Chi-squared tests were used to detect significant departures in genotype frequency from the Hardy-Weinberg equilibrium at each SNP. Haplotypes of IL-13 were analyzed using Haploview v2.0, based on the expectation maximization algorithm. Differences in the mean values of phenotypic characteristics among AICU patients were compared using the chi-squared test, or Fisher's exact test and independent t-test. Continuous variables that did not have a normal distribution, such as the levels of serum total IgE, were log-transformed. Differences in genotype frequency between groups were examined with the chi-squared test, and three logical regression models (co-dominant, dominant, recessive) were used after accounting for age and gender covariates. The level of statistical significance was set at P<0.05. SPSS 12.0 for Windows software (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses.
RESULTS
In our study, genetic associations among three genetic polymorphisms of IL-13, -1510A>C, -1055C>T, and Arg110Gln (110G>A), and the three study groups (AICU, ATCU, and NC) were examined. No significant association was detected between the IL-13 polymorphisms and susceptibility to AICU (P>0.05).
Significant differences in AICU phenotypes, based on the presence of atopy and log serum total IgE levels, were found between the AICU, ATCU, and NC groups (P<0.001, respectively) (Table 1). However, there were no significant differences between the two CU groups with respect to allele and genotype frequencies (P>0.05) (Table 2). Furthermore, the three polymorphisms of IL-13 were not in linkage disequilibrium.
Using Haploview 2.0 and the expectation maximization algorithm, five common haplotypes were constructed: ht1 (ACG), ht2 (CTA), ht3 (ACA), ht4 (CCG), and ht5 (CTG). Haplotype analysis further confirmed that there were no significant differences among the three study groups (P>0.05) (data not shown).
DISCUSSION
This study evaluated three AICU-susceptibility loci in the IL-13 gene: two SNPs in the promoter region (-1510 A>C and -1055C>T) and one nonsynonymous SNP (Arg110Gln, 110G>A). In a case control study, we evaluated the three polymorphisms for evidence of association with AICU and related phenotypes. Based on the results, no evidence was found to support a significant association between these three SNPs and the diagnosis of AICU.
The cytokines IL-13 and IL-4 share biological properties such as isotype switching to IgE and expression of endothelial cell adhesion molecules.16 Accordingly, it was reasonable to propose that in chronic urticaria, IL-13 increases mast cell FcεR1 expression and mediator release,17 leading to the development of hives and the enhancement of cutaneous inflammation. In a previous report, patients with more frequent itch were found to have higher cytokine levels, suggesting that IL-13 may serve as a serological marker of disease activity.18
Owing to the lack of previously published data on IL-13 SNPs in patients with AICU, our results cannot be compared directly with others. However, several studies showed a linkage between IL-13 and a high serum total IgE level,19,20 including an association of IL-13 Arg110Gln with serum IgE levels in patients with asthma21,15 and atopic dermatitis.22 IL-13 variants have been associated with airway responsiveness23 and with eosinophil count.24 Recently, IL-13 Arg110Gln was reported to be significantly associated with reduced lung function in Korean children and adolescents.25 Nevertheless, we found no significant association of either of the promoter SNPs or the coding SNP with AICU traits, including serum total IgE and atopy. Our findings indicate that a genetic variation in the form of any of the three SNPs at the IL-13 locus is not likely to be involved in the development of AICU.
This result is consistent with an earlier report showing that there was no relationship between the coding SNP and allergic rhinitis induced by artemisia pollen and/or DerpI in a Chinese population. Marginal significance was found for atopy-related phenotypes associated with the Arg110Gln variant and in vitro specific IgE responses to common inhalant allergens.26 In our previous study, two SNPs, at FcεR1β E237G and FcεR1γ, were associated with atopy in AICU patients, but not in ATCU patients, and were proposed to explain the increased release of histamine from basophils and subsequent development of the AICU phenotype.8 Furthermore, other studies have noted a significant association of the FcεR1β-109T>C and FcεR1β E237G polymorphisms with serum IgE levels in asthmatic patients,27,28 and the FcεR1α -344C>T polymorphism in AICU.6 However, in our study, IL-13 genetic polymorphisms were not related to either atopy or total IgE levels in AICU patients, implying that neither the two SNPs in the functional promoter nor the SNP in the coding sequence contribute significantly to AICU susceptibility. This is the first detailed investigation showing the absence of a genetic association between IL-13 and AICU and its phenotypes, i.e., serum total IgE and atopy. There is no evidence that these three SNPs are associated with AICU; instead, SNPs of IL-13 may confer a greater susceptibility to asthma or other inflammatory diseases.
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