Published ahead of print on January 25, 2007, doi:10.1164/rccm.200609-1286OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200609-1286OC
Association Analysis of CD40 Polymorphisms with Asthma and the Level of Serum Total IgE1 Genome Research Center for Allergy and Respiratory Diseases, Soonchunhyang University Hospital, Jung Dong, Wonmi Ku, Bucheon, Gyeonggi Do, Republic of Korea; 2 Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea; and 3 Division of Molecular and Life Science, The College of Science and Technology, Hanyang University, Sangrok Ku, Ansan, Gyeonggi Do, Republic of Korea Correspondence and requests for reprints should be addressed to Choon-Sik Park, M.D., Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, 1174, Jung-Dong, Wonmi-Ku, Bucheon, Kyeonggi-Do, 420-020, Korea. E-mail: mdcspark{at}unitel.co.kr
Rationale: The CD40 protein plays important roles in cell-mediated and humoral immune responses, especially in immunoglobulin class-switching to IgE. Objectives: We tested the association of CD40 polymorphisms with the risk of asthma and the level of serum IgE and investigated the functional effect of associated polymorphisms on the expression of CD40. Methods: We identified 17 CD40 single-nucleotide polymorphisms (SNPs) in the Korean population by direct sequencing, and we genotyped 7 of these in 487 subjects with asthma and in 161 normal subjects. Cell-surface expression of CD40 for B-cell lines of various SNP genotypes was measured using flow cytometry. The effects of SNPs in the promoter and 5'-untranslated regions (UTRs) of CD40 were assessed using pGL3 luciferase and enhanced green fluorescent protein (EGFP) reporter systems, respectively.
Measurements and Results: None of the SNPs was associated with asthma risk, but total serum IgE levels were associated with the –580G>A and –1C>T polymorphisms in subjects with asthma (p = 0.007 and 0.005, respectively). The total amount of IgE was highest in the –580A or –1C homozygotes. More CD40 was expressed in B cells with the –1C allele than in those with the –1T allele (p < 0.001). EGFP expression from the CD40 5'-UTR–EGFP construct was higher for the –1C allele than the –1T allele. The –580G>A SNP did not affect promoter activity, even after IFN- Conclusions: CD40 gene polymorphisms exert a genetic effect on IgE production in patients with asthma through translational regulation of CD40 expression on B cells.
Key Words: CD40 • single nucleotide polymorphisms • asthma • IgE
Asthma and its phenotypes are complex traits resulting from interactions between multiple disease susceptibility factors and the surrounding environment, which has a major influence on the onset and severity of the disease (1). The immunopathogenesis of asthma is recognized as a T-helper cell type 2 (Th2) disease with a specific cytokine response profile that includes IL-4, IL-5, and IL-13. In addition, atopy, which is documented by the presence of IgE specific for inhalant allergens or by high IgE production, is closely associated with asthma development (2). IgE production is initiated by Th2 cells that release IL-4 and IL-13 in response to antigen presentation (3) in a cooperative mode with activation of CD40 (MIM #109535) (4). CD40 interacts with its ligand (CD40L; CD154, MIM #300386), which is found primarily on T cells, playing a role in humoral and cell-mediated immune responses in a number of cellular interactions that give rise to inflammatory responses (5). Because high IgE synthesis is a hallmark of atopy, the CD40–CD40L interaction may be important in the development of asthma-associated inflammation. CD40 is expressed not only on B cells but also on bronchial epithelial cells (6), airway macrophages (7) and eosinophils (8), lung myofibroblasts (9), and airway smooth muscle cells (10, 11). These CD40-expressing cells, together with CD40L+ leukocytes, such as activated T cells, eosinophils, mast cells, and basophils (12), modulate asthmatic airway inflammation via production of cytokines, chemokines, and inflammatory mediators (12), and by prolonging survival of inflammatory cells (8). CD40 expression is increased in the bronchial epithelial cells (13) and eosinophils (8), smooth muscle cells (11), and macrophages (7) in the airways of patients with asthma. The CD40 gene is located on chromosome 20q12–13.2, spans 11 kb, and has nine exons of between 29 and 412 bp in length. This chromosomal region is linked with asthma and with combined atopic dermatitis and asthma (14). Based on the biological properties involved in asthma and IgE regulation, we evaluated CD40 for its potential use in a genetic study of asthma and atopy. To discover additional polymorphisms in genes whose variants have been implicated in asthma, we performed extensive screening of CD40 by direct sequencing. We also performed a statistical analysis of the genetic effects of the detected CD40 polymorphisms on asthma. In this article, we present several genetic polymorphisms found in CD40 and the results of an association study with asthma in a Korean asthma cohort (15, 16).
Subjects All patients displayed clinical symptoms consistent with asthma on physical examination (17). Normal subjects were recruited from spouses of the patients and from the general population. Subjects with a history of thyroid disease were excluded. Additional detail on the definition of each population is provided in an online supplement. The subjects and study protocols were approved by the Institutional Review Board of Soonchunhyang University Hospitals. The clinical parameters are summarized in Table 1.
Sequence and Genotype Analysis of the Human CD40 Gene We analyzed DNA samples from 24 Koreans for the presence of single-nucleotide polymorphisms (SNPs) in CD40 by sequencing using an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA). SNPs were genotyped using the single-base extension method as described previously (18). We also genotyped DNA samples from 50 whites and 50 African Americans.
Epstein-Barr Virus Transformation of B Cells
Measurement of CD40 Expression on CD19+ B Cells Using Flow Cytometry
Preparation of CD40 Promoter and 5'-Untranslated Region Reporter Gene Constructs
Transient Transfection and Assays for Luciferase Activity and Intensities of EGFP Fluorescence A549 cells and 293T cells were transiently transfected with the CD40 promoter–pGL3-Basic construct and with the CD40 5'-UTR–pEGFP construct, respectively, using LipofectAMINE F2000 (Invitrogen Life Technologies, Carlsbad, CA). pSV–  -galactosidase vector (Promega) was cotransfected as internal standard. After 24 hours of transfection, the cells were cultured with or without IFN- (10 ng/ml) for an additional 24 hours (19).
Statistical Analysis
SNP Genotyping and Association with Asthma Risk and Serum IgE Levels Using 20 primer sets, we directly sequenced DNA samples from 24 unrelated Korean subjects. Seventeen genetic variants (one simple tandem repeat and 16 SNPs) were identified within the exons and flanking regions of CD40 in this population: two variants in the promoter region, one in the 5'-UTR, and 14 in introns (Figure 1A). Pairwise comparisons among SNPs revealed two sets of absolute LDs (|D'| = 1 and r2 = 1): +104G>A:+10231A>G and +6445A>G:+6526C>T:+9909G>A. Several complete LDs (|D'| = 1 and r2  1) were also found (Figure 1C). Seven SNPs (–580>A, –1C>T, +965G>T, +1117C>T, +6254T>C, +7876A>G, and +9909G>A) were selected for larger scale genotyping based on their LDs, frequencies, and haplotype tagging status.
Haplotypes in the CD40 genes of 642 Korean, 50 African-American, and 50 white subjects were constructed using PHASE software. These three ethnic groups exhibited significant differences in allele and haplotype frequencies. Only three common haplotypes (ht1–ht3) (frequency > 0.05) were used for further haplotype association analysis. Frequencies of each of the seven polymorphisms and the ht1, ht2, and ht3 haplotypes were compared between subjects with asthma and normal control subjects using logistic regression and controlling for age, sex, and smoking status as covariates. None of the polymorphisms or haplotypes manifested a significant association with risk of asthma (Table 3).
In subsequent analysis, –580G>A, –1C>T, +965G>T, +1117C>T, +6254T>C, and ht3 were associated (p = 0.049–0.005) with total serum IgE levels in subjects with asthma (Table 4). When multiple comparison was applied, the association remained significant only for the –580G>A and –1C>T polymorphisms (p = 0.049 and 0.035 in recessive and dominant modes, respectively). The genetic effect of each of these polymorphisms was allelic dose dependent. The total IgE levels of homozygotes for the rare allele of –580G>A (A/A, 338.1 IU/ml) were higher than the levels for the other genotypes (G/A, 181.8 IU/ml and G/G, 168.0 IU/ml; G/*, 174.7 IU/ml; p = 0.007), and the total IgE levels of C homozygotes for 1C>T (C/C, 234.3 IU/ml) were higher than the levels for the other genotypes (C/T, 152.1 IU/ml and T/T, 175.5 IU/ml; */T, 156.6 IU/ml; p = 0.005) (Table 4). These results regarding age, sex, and smoking status as covariates were similar with the covariate-unadjusted results (as shown in Table E1 of the online supplement). No significant association between these two SNPs and serum IgE levels was found in normal control subjects (Table E2).
Effect of SNPs –1C>T and –580G>A on CD40 Protein Expression in B-Cell Lines To investigate the effect of the –1C>T and –580G>A polymorphisms on the production of CD40 protein in B cells, we used flow cytometry to measure surface CD40 levels on B-cell lines from subjects with various alleles of these SNPs. The frequency and level of expression were analyzed (Figure 2). No significant difference was found between the frequencies of CD40 expression in the common and rare allele groups of –580G>A and –1C>T (data not shown).
The amount of CD40 protein expressed was analyzed as the mean fluorescence intensity of the total cells (Figure 2A). The intensity was significantly higher in B-cell lines from subjects with the –1C allele than in those from subjects with the –1T allele (relative mean value of FL1 = 10.66 ± 0.65 vs. 5.93 ± 0.78; p = 0.003) (Figure 2B). In contrast, subjects with the –580G or the –580A allele expressed about the same amount of CD40 (relative mean value of FL1 = 10.75 ± 0.88 vs. 8.58 ± 0.90, p = 0.127) (Figure 2C). A comparison of –580G>A and –1C>T allele-based haplotypes indicated that CD40 expression was affected by the genotype of the latter SNP but not the former (Figure 2D). CD40 expression in B cells with the –1T allele was significantly lower than that in B cells with the –1C allele, regardless of their –580G>A genotype (p = 0.02).
Effect of the –580G and –580A Alleles on the Activity of the CD40 Promoter
Effect of the –1C and –1T Alleles on the Activity of the 5'-UTR of CD40 Given that the –1C>T polymorphism falls in the 5'-UTR region of CD40, we assessed its effect on protein production using an enhanced green fluorescent protein (EGFP) reporter gene. Using flow cytometry, we measured the EGFP fluorescence intensity of 293 T-cell lines transfected with –1C- and –1T-type 5'-UTR–EGFP constructs (Figure 4A). EGFP protein expression was significantly higher for the –1C allele than for the –1T allele of the CD40 5'-UTR–EGFP construct (relative mean value of FL1 = 56.52 ± 3.12 vs. 3.30 ± 0.21; p = 0.004) (Figure 4). These results suggest that the –1C>T genotype affects the efficiency of CD40 translation, leading to differential expression of CD40 protein.
The interaction between CD40 and CD40L is important in a number of cellular processes that give rise to inflammatory responses. In asthma pathogenesis, the role of the CD40–CD40L interaction is complex. By regulating the balance between Th1 and Th2 cells, presumably via induction of IL-12 (7, 23), CD40 may play a protective role in asthma pathogenesis after specific memory T cells are established. In contrast, the constitutional airway cells, including bronchial epithelial cells (6), lung myofibroblasts (9), airway smooth muscle cells (10, 11), and eosinophils (8), modulate asthmatic airway inflammation by interacting with CD40L+ leukocytes, such as activated T cells, eosinophils, mast cells, and basophils (12), via CD40 (6). These biological observations prompted us to evaluate the effect of nucleotide polymorphisms in the CD40 gene on asthma risk and IgE production. We did not find any association between CD40 SNPs and asthma risk, suggesting that CD40 sequence variances have no effect on the development of asthma. Our data do demonstrate, however, that two CD40 SNPs, –1C>T and –580G>A, are associated with total IgE levels in individuals with asthma; to our knowledge, this is a novel finding. To identify the functional effects of these SNPs on CD40 transcription and translation, we first measured CD40 protein expression. Because CD40 is expressed by B cells, we used EB virus to create immortalized B-cell lines from the subjects with asthma whose genotypes we had determined. The flow cytometry results demonstrated that CD40 protein was expressed on these B-cell lines (see Figure 2). In addition, the subjects who were homozygous for the common allele of –1C>T expressed more CD40 protein than did those who were homozygous for the rare allele. In contrast, the subjects who were homozygous for the common or rare alleles of –580G>A expressed approximately the same amount of CD40. Thus, CD40 production in the B-cell lines was dependent on the translation rate, which was genetically affected by the –1C>T polymorphism. In contrast, –580G>A polymorphisms did not affect the CD40 expression. The lack of effect of –580G>A on CD40 expression was demonstrated by our functional analysis of CD40 promoter activity, in which the –580G>A genotype did not affect expression of the luciferase reporter gene (see Figure 3). The –1C>T polymorphism in the 5'-UTR of CD40 has previously been reported as having a genetic effect on the development of Graves disease in white (24) and Korean populations (25). Thus, in our study, we excluded subjects who had a history of thyroid diseases. In addition, Jacobson and coworkers recently demonstrated that the –1C>T polymorphism, which is in the Kozak sequence of CD40, affects CD40 expression in B-cell lines derived from human subjects with Graves' disease and in transfected Rat-2 fibroblasts (26). To our knowledge, no reports of the effect of the –1C>T polymorphism on IgE regulation have previously been published. Jacobson and coworkers suggested that the –1C genotype of the CD40 Kozak sequence might predispose the carrier to Graves disease by increasing the efficiency of translation, but not transcription, of CD40 mRNA (26). The Kozak sequence is a consensus sequence (GCCACCATGG), compiled from the study of some 699 vertebrate genes, which flanks starting methionine codons (AUG) and is of fundamental importance to the initiation of translation (27). Even subtle variations in this sequence can have significant effects on translation (28). Kozak sequence variations have been shown to be important in several diseases associated with genetic dysfunction, including those affected by genes for annexin V (29), factor XII (30), BRCA1 (31), and androgen receptor (32). Hence, CD40 seems to act only as a potentiation factor in concert with polymorphisms of other genes, such as IL-13 and thymus- and activation-regulated chemokine (TARC) (33, 34), IL-4 (35), and IL-4R and IL-18 (36), as well as with other factors such as environmental allergens and endotoxins (37, 38). However, in the time since CD40 and IL-4 were shown to act synergistically in the induction of IgE synthesis in highly purified human B cells (4), many human and animal studies have provided evidence that CD40 plays a role in the regulation of IgE (39). Recently, monoclonal antibodies against CD40–CD40L were proposed as optimal candidates for the treatment of allergic airway disease (5). Further study is needed to elucidate the gene–gene and gene–environment interactions involved in the development of asthma and in IgE regulation. In summary, we identified 17 SNPs in CD40 and selected seven common polymorphic sites for genotyping in an asthma cohort. The –1C allele of –1C>T and the –580A allele of –580G>A were not associated with increased risk of asthma but were associated with higher total IgE in subjects with asthma. In view of these experimental results, we conclude that the –1C>T polymorphism in the CD40 5'-UTR has a great influence on the CD40 protein level. Furthermore, this polymorphism may be useful as a marker to identify subjects prone to high IgE production.
The authors thank Myung-Ran Lee, Seon-Ja Bang, Shin-Ock Lee, and Eun-Young Kim for establishing and maintaining EBV-infected B-cell lines from individuals.
Supported by grants of the Korean Health 21 R&D project. Ministry of Health & Welfare, Republic of Korea (01-PJ10-PG6-01GN14-0003) and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-037-C00032).
* These investigators contributed equally to this article. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200609-1286OC on January 25, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 11, 2006; accepted in final form January 23, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
Other haplotypes were observed with frequencies < 0.05: GCGTCGG, GTTCCGG, GCGCCGG, GCTCTAA, ACGTTAG, ACTCTAA, GCGCCAG, ACGTCGA, ATTCCGG, ATTCTAA, GTGCTAG. (C) LD coefficients (|D'| and r2) among CD40 SNPs.
