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(Chest. 2004;126:1832-1839.)
© 2004 American College of Chest Physicians

Polymorphisms of IL4, IL13, and ADRB2 Genes in COPD*

Ahmed E. Hegab, MSc; Tohru Sakamoto, MD; Wataru Saitoh, BS; Hosam H. Massoud, MD; Hosny M. Massoud, MD; Khalid M. Hassanein, MD and Kiyohisa Sekizawa, MD

* From the Department of Pulmonary Medicine (Drs. Hegab, Sakamoto, Saitoh, and Sekizawa), Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan; and the Department of Chest Diseases and Tuberculosis (Drs. Massoud, Massoud, and Hassanein), Faculty of Medicine, Cairo University, Cairo, Egypt.

Correspondence to: Kiyohisa Sekizawa, MD, Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan; e-mail: kiyo-se{at}md.tsukuba.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Interleukin (IL)-4, IL-13, and ß2-adrenoceptor (ADRB2) are involved in airway hyperresponsiveness (AHR), and their coding genes are located on chromosome 5q31-q33. AHR is one of the risk factors for COPD. Investigating polymorphisms within these genes may help to pinpoint the genetic susceptibility to COPD.

Subjects and measurements: A case-control association study was conducted on two different ethnic groups: Japanese subjects (88 patients with COPD and 61 control subjects) and Egyptian subjects (106 patients with COPD and 72 control subjects). The following polymorphisms were genotyped: – 589 C/T, – 33 C/T, and variable number of tandem repeat (VNTR) in IL4, – 1111 C/T and + 2044 G/A in IL13, and + 46 A/G and + 79 C/G in ADRB2. Pairwise haplotype frequencies as well as genotype and allele frequencies were analyzed.

Results: The distribution of the genotype frequencies of ADRB2 + 79 C/G was significantly different between the COPD and the control groups in the Egyptians (p = 0.002). The distributions of the haplotypes in the Japanese (IL4 – 589 C/T: IL4 VNTR; IL4 – 33 C/T: IL4 VNTR) [corrected p values < 0.001 and 0.022, respectively], and those in the Egyptians (IL4 – 589 C/T: ADRB2 + 79 C/G; IL4 VNTR: ADRB2 + 79 C/G) [corrected p values, 0.033 and 0.001, respectively] showed significant differences between the COPD and the control groups.

Conclusions: The ADRB2 + 79 C/G polymorphism and the haplotypes shown in this study may be involved in the pathogenesis of COPD.

Key Words: ß2-adrenoceptor • COPD • interleukin-4 • interleukin-13 • polymorphism


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cigarette smoking is by far the most important risk factor for COPD. However, not all smokers acquire clinically significant COPD. It is suggested that multiple genetic factors and genotype-by-environment interactions are involved in the development of complex-trait diseases such as COPD.1 An individual genetic factor has only a small contribution for the susceptibility to the diseases. To date, > 20 polymorphisms of candidate genes have been shown to be related to COPD.2 Some of these, however, could not be replicated. Therefore, it is important to confirm the associations in different ethnic groups.

There is a strong interaction between airway hyperresponsiveness (AHR) and smoking.3 AHR is a risk factor for COPD mortality.4 Genetic susceptibility to AHR has been shown to be coinherited with genetic markers on chromosome 5q31-q33.5 Coding genes for interleukin (IL)-4, IL-13, and ß2 adrenoceptor (ADRB2) are located on chromosome 5q31-q33, and it has been demonstrated that they are associated with AHR.67 In addition, ß2-adrenergic agonists are the main bronchodilators used for the treatment of COPD. Also, IL-4 and IL-13 are known for their capacity to induce mucin production and secretion.89 Zheng et al10 demonstrated that overexpression of IL-13 in the adult murine lung causes emphysema.

Some studies have analyzed an association of polymorphisms of the IL4, IL13, and ADRB2 genes with COPD. It has been shown that the IL13 – 1111 C/T polymorphism (– 1055 relative to the transcription start site) is associated with COPD in the white population from the Netherlands.11 In the same article,11 however, they could not detect any significant association of the IL13 + 2044 G/A and two IL4 polymorphisms, – 589 C/T (– 524 relative to the transcription start site) and – 33 C/T (+ 33 relative to the transcription start site), with the development of COPD. As for ADRB2 polymorphisms, Ho et al12 reported that the + 46 A/G polymorphism (amino acid change Arg16Gly) is related to the susceptibility to COPD, and that the + 79 C allele (amino acid Gln27) may be associated with the severity of the disease in Chinese. By contrast, Joos et al13 reported that the ADRB2 +79C/G heterozygosity (amino acid Gln27Glu) may be protective against the fast decline of lung function in smokers in white subjects collected from multiple North American medical centers. It has been described that there are ethnic differences in the prevalence, presentation, and mortality of COPD.1415

Based on these contradictory results among the studies in different populations and on the possible effect of the ethnic differences on the case-control studies, we conducted a retrospective case-control study to assess the association of these polymorphisms of the IL4, IL13, and the ADRB2 genes with COPD in two diverse ethnic groups, Japanese and Egyptians. These two populations are widely separated geographically and genetically. Therefore, an additional purpose of this study was to investigate the effect of the ethnic difference on the association of the polymorphisms with COPD.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
The investigation was a case-control association study in two different ethnic groups, Japanese and Egyptians. All the subjects were long-term heavy smokers with a Brinkman index (the number of cigarettes per day times the number of years) of > 450. The subjects had no blood relationship to each other. Part of the subjects were the same as those in a previous study.16 COPD was diagnosed based on history, physical examination, and spirometric data, and FEV1/FVC ratio < 70%, according to the Global Initiative for Chronic Obstructive Lung Disease criteria.17 Control subjects included were smokers with normal pulmonary function. For the COPD group, we excluded those with FEV1 > 70% predicted. Chest radiographs and histories were evaluated in all the subjects to exclude other causes of airflow limitation such as pulmonary tuberculosis, bronchial asthma, bronchiectasis, and heart failure. To eliminate possible effect of population stratification on genotype and allele frequencies, ethnic and geographic matching was considered. All the subjects and their parents had to be born in the region of recruitment and have grandparents born inside the country.

The Japanese subjects, 88 patients with COPD and 61 control subjects, were recruited from the Department of Pulmonary Medicine, University of Tsukuba. The Egyptian subjects, 106 patients with COPD and 72 control subjects, were recruited from the Department of Chest Diseases and Tuberculosis at Cairo University Hospital and affiliated hospitals. The study was approved by the ethics committees of the hospitals involved, and written informed consent was obtained from all the subjects.

Genotyping
The nucleotide positions in this study are given relative to the translation start site. The reference sequences used were GeneBank accession numbers AF395008 for the IL4 gene, U31120 for the IL13 gene, and Y00106 for the ADRB2 gene. Genomic DNA was extracted from whole blood using a DNA blood kit (QIAGEN; Hilden, Germany).

The IL4 polymorphisms, – 589 C/T and – 33 C/T, were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism analysis. A pair of primers was designed to include both single nucleotide polymorphisms (SNPs) in a single PCR product of 646 base-pairs (bp). Forward and reverse primers were 5'-ACTAGGCCTCACCTGATACG-3' and 5'-AGGTGTCGATTTGCAGTGAC-3', respectively. BsmFI digested only the – 589 C allele to produce 45-bp and 601-bp fragments, whereas the uncut – 589 T allele gave a 646-bp fragment. The digest products were resolved by electrophoresis on a 3% agarose gel and stained with ethidium bromide. For the – 33 C/T SNP, BsmAI digestion yielded two fragments of 24 bp and 622 bp in the presence of the – 33 T mutant allele, while the uncut – 33 C gave a 646-bp fragment. The digest products were resolved by electrophoresis on a 10% acrylamide gel. The IL4 variable number of tandem repeat (VNTR) has been described as a 70-bp repeat in intron 3.18 The primers used were 5'-TAGGCTGAAAGGGGGAAAGC-3' and 5'-CTGTTCACCTCAACTGCTCC-3'. The amplified products were size separated by 3% agarose gel electrophoresis. The size of the products was directly diagnostic of number of repeats in the intervening sequence. Alleles were named as follows: allele 1 = three repeats (254 bp), and allele 2 = two repeats (184 bp).

The IL13 polymorphisms, – 1111 C/T and + 2044 G/A, and the ADRB2 polymorphisms, + 46 A/G and + 79 C/G, were genotyped using TaqMan allelic discrimination technique (Applied Biosystems; Foster City, CA). For each SNP, a pair of primers flanking the SNP and a pair of oligonucleotide probes, one homologous to the mutant type labeled with VIC and another homologous to the wild type labeled with 6-carboxyfluorescein (FAM), were designed and synthesized by Applied Biosystems (Table 1 ). PCR cycling conditions (ABI PRISM 7000; Applied Biosystems) were as follows: 50°C for 2 min; 95°C for 10 min, followed by 40 cycles of 95°C for 15 s; and 60°C for 1 min. The different alleles were discriminated according to the fluorescence intensity of FAM and VIC.


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  		Table 1. Primers and Probes Used for TaqMan Allelic Discrimination*

 
Statistical Analysis
Differences in clinical data between the patients with COPD and the control subjects in each ethnic group were checked by unpaired Student t test. Significance was accepted at p < 0.01. Hardy-Weinberg equilibrium was assessed using a goodness-of-fit {chi}2 test for biallelic markers. A Fisher exact test (two-by-three and two-by-two tables) was used to analyze distribution of genotype and allele frequency. Statistical significance was defined as p < 0.05. Odds ratios and 95% confidence intervals were calculated to quantitatively assess the degree of association observed. The polymorphisms in the present study were common, the frequencies of which had been expected to be > 20% in either of the two populations. Under the condition of the minor allele frequency of 20% and the significance level of 0.05, the sample size in this study offers > 80% power to detect a significant association at the relative risk of 2.5 calculated by the power calculator available at http://calculators.stat.ucla.edu/. We did not adopt the Bonferroni correction for multiple comparisons in the analysis of the genotype and the allele frequencies for the following reasons19: (1) the individual alleles studied were not independent each other, (2) the polymorphisms were known to be functional, and (3) the genes under consideration were susceptibility genes for COPD. Maximum-likelihood haplotype frequencies were estimated using the expectation-maximization algorithm with SNPAlyze program (Dynacom; Mobara, Japan). The expectation-maximization algorithm is an iterative method, whereby initial arbitrary haplotype frequencies are used to estimate genotype frequencies (the E-step). These expected genotypes are used in turn to estimate haplotype frequencies at the next iteration (the M-step) and so on until the haplotype frequencies reach equilibrium. Haplotype distributions for the COPD and control groups were assessed by a Fisher exact test. The Bonferroni correction was applied for multiple tests. Pairwise linkage disequilibrium (LD) was estimated as D = x11 – p1q1, where x11 is the frequency of haplotype A1B1, and p1 and q1 are the frequencies of alleles A1 and B1 at loci A and B, respectively. The Lewontin coefficient,20 D’, is given by D’ = D/Dmax, where Dmax = min (p1q2, p2q1) when D < 0 or Dmax = min (p1q1, p2q2), when D is > 0. p2 and q2 are the frequencies of the other alleles A2 and B2 at loci A and B, respectively. A {chi}2 test was also performed to test for significant departure from the null hypothesis that pairwise LD was due to random association. Significant LD was accepted at D’ value of > 0.3321 or p value of < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The characteristics of all participants in this study are summarized in Table 2 . Since we excluded those with FEV1 values > 70% predicted for the COPD group, all the patients in both ethnic groups had moderate-to-severe COPD according to the Global Initiative for Chronic Obstructive Lung Disease classification of severity: mild = FEV1 ≥ 80% of predicted; moderate = FEV1 ≥ 30% to < 80% of predicted; and severe = FEV1 < 30% of predicted.17 Age and Brinkman index were not significantly different between the patient group and the control group.


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  		Table 2. Clinical Features of Study Populations*

 
Table 3 shows the genotype frequencies of the IL4, IL13, and ADRB2 gene polymorphisms. Allele 1 of IL4 VNTR consists of three 70-bp repeats, while allele 2 contains two repeats. We found a much rarer allele of IL4 VNTR with a single repeat (allele 3). DNA sequencing confirmed that this allele was a single-repeat variant. We registered it in the dbSNP database of the National Center for Biotechnology Information under accession numbers ss12568631 and ss12568633. Since the frequency of this allele with a single repeat was < 1%, it was counted among the minor allele in the statistical analysis. For all the biallelic polymorphisms, distributions of homozygotes and heterozygotes conformed to expectations based on Hardy-Weinberg analysis. The allelic frequencies of all the three IL4 polymorphisms and the ADRB2 +46 A/G SNP were opposite between the Japanese and the Egyptian population. Of the seven polymorphisms studied, only the ADRB2 +79 C/G in Egyptians showed a significant difference between the patient group and the control group (p = 0.002). Furthermore, the mutant allele + 79 G frequency in the Egyptian population was significantly higher in the COPD group than in the control group (31% in the COPD group vs 15% in the control group, p = 0.0006; odds ratio, 2.59; 95% confidence interval, 1.50 to 4.48).


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  		Table 3. Genotype Frequencies of IL4, IL13, and ADRB2 Gene Polymorphisms*

 
Pairwise LD was measured among the seven polymorphisms in the Japanese and the Egyptian populations (Tables 4, 5 , respectively). The polymorphisms within each gene were in strong LD in both ethnic groups. Furthermore, in the Japanese population, significant LD was detected across the two genes between the following pairs of the polymorphisms: IL4 – 589 C/T: IL13 – 1111 C/T; IL4 – 33 C/T: IL13 – 1111 C/T; IL4 – 33 C/T: ADRB2 + 46 A/G; IL4 VNTR: IL13 – 1111 C/T; IL4 VNTR: IL13 + 2044 G/A; IL13 – 1111 C/T: ADRB2 + 79 C/G; and IL13 + 2044 G/A: ADRB2 + 79 C/G. By contrast, in the Egyptian population, the IL4 VNTR was significantly in LD with the IL13 – 1111 C/T and the IL13 + 2044 G/A.


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  		Table 4. Pairwise LD (D’ Above Diagonal) and Statistical Significance (p Value Below Diagonal) in the Japanese Population

 

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  		Table 5. Pairwise LD (D’ Above Diagonal) and Statistical Significance (p Value Below Diagonal) in the Egyptian Population

 
We compared pairwise haplotype frequencies of the seven SNPs between the patients and the control subjects in both ethnic groups (Table 6 ). In the Japanese population, significant differences were observed in the haplotypes IL4 – 589 C/T: IL4 VNTR and IL4 – 33 C/T: IL4 VNTR (corrected p values, 0.0009 and 0.022, respectively). For the first pair, 8% of the Japanese control subjects displayed IL4 – 589 T: IL4 VNTR allele 1, while none of the patients with COPD had it. Sixteen percent of the Japanese controls showed IL4 – 589 C: IL4 VNTR allele 2, while it only accounted for 7% in the COPD group. As for the second pair, 5% and 16% of the Japanese control subjects displayed IL4 – 33 T: IL4 VNTR allele 1 and IL4 – 33 C: IL4 VNTR allele 2, while 0% and 8% of the Japanese patients with COPD showed the former and the latter haplotypes, respectively. In the Egyptian population, significant differences were observed in haplotypes IL4 – 589 C/T: ADRB2 + 79 C/G and IL4 VNTR: ADRB2 + 79 C/G (corrected p values, 0.033 and 0.001, respectively). Thirteen percent of the Egyptian patients with COPD displayed IL4 – 589 T: ADBR2 + 79 G, while 4% of the control subjects had this haplotype. Ten percent of the Egyptian patients with COPD showed IL4 VNTR allele 2: ADBR2 + 79 G, while 1% of the control subjects had the same haplotype.


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  		Table 6. Haplotype Frequencies Having Significantly Different Distributions Between the COPD Group and the Control Group*

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have investigated whether polymorphisms of the IL4, IL13, and ADRB2 genes are associated with the development of COPD in two different populations. Pairwise haplotype frequencies as well as genotype and allele frequencies were analyzed between the COPD and the control groups. The allelic frequencies of the three IL4 polymorphisms and the ADRB2 + 46 A/G SNP were opposite between the Japanese and the Egyptian populations. The frequencies of these polymorphisms in the Japanese population in the present study are consistent with those in Japanese samples reported previously,2223 which are opposite to those in white population,1113 suggesting the ethnic variability in the allelic frequencies of these genes.

The present study demonstrated a significant association between the ADRB2 + 79 C/G SNP (Gln27Glu) and the development of COPD in the Egyptian population. The frequency of the ADRB2 +79 G allele (Glu 27) in the COPD group was significantly higher than that in the control group. To our knowledge, there has been only one article analyzing the relationship between the ADRB2 polymorphisms and COPD.12 Our results were inconsistent with theirs,12 who showed no significant difference in the frequency of the ADRB2 +79 C/G between patients with COPD and healthy control subjects in the Chinese population, and even demonstrated that the patients with severe COPD were more likely to have the + 79 C allele (Gln27). In vitro studies2425 showed that the ADRB2 +79 G allele (Glu27) appears to protect against agonist-promoted downregulation of receptor number. In regard to bronchial asthma, asthmatic patients with the + 79 C allele (Gln27) have been shown to have increased airway reactivity for methacholine,26 and this allele may be associated with childhood asthma.27 However, another study28 has demonstrated no association of the ADRB2 +79 C/G with bronchial asthma. Since the ADRB2 +79 C (Gln 27) may be associated with serum IgE concentration,29 it is likely that this allele is related to childhood asthma only and has no association with nonatopic asthma in adult populations, in whom IgE is not important for the development of the disease. Since COPD has even less close relationship with IgE than bronchial asthma, it is reasonable that + 79 G (Glu 27) but not + 79 C (Gln 27) of the ADRB2 gene is associated with the development of COPD in this study. Although the airflow limitation of COPD is not fully reversible, ß2-adrenergic agonists have significant clinical effects on COPD. Therefore, in addition to bronchodilation, the efficacy of the ADRB2 activation in COPD might be due to inhibition of airway smooth-muscle cell proliferation,30 stimulation of mucociliary transport,31 reduction of mucosal damage,32 and inhibition of neutrophil accumulation.33 The ADRB2 + 79 C/G SNP might be related to these functions. It is also possible that other yet to be identified functional polymorphisms of ADRB2 gene or its nearby inflammatory mediator genes are in LD with + 79 C/G, and are relevant to the pathophysiology of COPD. In regard to the study of Joos et al13 on ADRB2 polymorphisms, their ascertainment method is based on rate of decline in lung function, which is not exactly equivalent to COPD. Additionally, their population is highly liable for population stratification error because it is a multicenter study.

In this study, no significant differences were detected in the genotype frequencies of IL4 and IL13 polymorphisms between the COPD and the control groups. IL4 and IL13 are T-helper type 2 cytokines and have been studied mainly for the pathogenesis of bronchial asthma. However, it has been demonstrated that these cytokines promote goblet-cell hyperplasia and induce mucus production,89 and that cells expressing either of the cytokines increase in the bronchial submucosa of smokers with chronic bronchitis.34 van der Pouw Kraan et al11 analyzed the association between COPD and the polymorphisms of IL4 and IL13. They have reported that only the IL13 – 1111 C/T is associated with the development of COPD, but they did not investigate haplotype analysis among the different polymorphisms. In addition, the VNTR in the IL4 gene was not included, which has been shown to be associated with COPD by haplotype analysis in this study. The difference between their studies and ours may be also due to the genetic heterogeneity among different ethnic groups. Since the susceptibility to COPD is recognized to be affected by multiple genetic factors and genotype-by-environment interactions,1 each of which has a small influence on the development of the disease, it is possible that some polymorphisms shown to be responsible for COPD in one ethnic group cannot be confirmed in others.

Haplotype analysis, testing associations using several polymorphisms, has greater statistical power than analyzing individual polymorphisms independently. Three genes examined in this research are located in order on chromosome 5q31-q33: IL13, IL4, and ADRB2. The IL4 gene is very close to the IL13 gene, approximately 20 kilobase apart. Although the distance between the ADRB2 and the IL13 genes is approximately 1,600 kb, significant LD was detected between the ADRB2 + 79 SNP and the IL13 SNPs in the Japanese population. Since the genetic factors responsible for complex-trait diseases such as COPD involve an interaction between multiple alleles located on different genes and even on different chromosomes, pairwise haplotype analyses were performed using all the combinations of the seven polymorphisms in this study, and the p values were corrected by the Bonferroni method. Although no significant difference was detected in the Japanese population by the analysis based on individual polymorphisms, the frequencies of two haplotypes (IL4 – 589 C/T: IL4 VNTR and IL4 – 33 C/T: IL4 VNTR), were significantly different between the COPD and the control groups. Also in the Egyptian population, the ADRB2 + 79 C/G in combination with IL4 VNTR showed more significant differences than the single SNP analysis of the ADRB2 + 79 C/G. Previous studies2435 have shown that these polymorphisms are functional when studied independently. However, no study, to date, has evaluated their functional effects when combined in the haplotypes.

Again, the differences of the haplotype frequencies between the Japanese and the Egyptians may be due to genetic diversity between the different ethnic groups. It is possible that different haplotypes can cause the same COPD phenotype. Another explanation for the differences is that the component of patients with COPD may be different between the Japanese and the Egyptians. COPD is a heterogeneous disorder characterized by dysfunction of airways and destruction of the lung parenchyma, both of which lead to the progressive airflow limitation. Therefore, it may happen that patients with COPD in different ethnic groups have distinct type of haplotype causing a specific aspect of the COPD spectrum: chronic bronchitis and/or emphysema.

We assessed the seven common polymorphisms in the IL4, IL13, and ADRB2 genes on chromosome 5q31-q33 for evidence of association with COPD. All the minor allele frequencies, except for ADRB2 + 79 G in the Japanese population, which was 6%, were > 17%. There are many less common polymorphisms in these genes, and any of them may be associated with the COPD susceptibility. However, their low minor allele frequencies need a much larger number of subjects in order to have sufficient power to detect such association. In order to clarify the relationship between the polymorphisms in this study and the development of COPD, further case-control studies in other ethnic groups are required. Chromosome 5q31-q33 contains many other genes related to inflammation, such as IL3, IL5, IL9, GM-CSF, corticosteroid receptor, and ß-chain of IL12. Cocarriage of polymorphisms of these genes with the polymorphisms in the present study may be associated with the susceptibility COPD.


   Footnotes
 
Abbreviations: ADRB2 = ß2-adrenoceptor; AHR = airway hyperresponsiveness; bp = base-pair; FAM = 6-carboxyfluorescein; IL = interleukin; LD = linkage disequilibrium; PCR = polymerase chain reaction; SNP = single nucleotide polymorphisms; VNTR = variable number of tandem repeat

This study was performed at the University of Tsukuba, Ibaraki, Japan; and the Department of Chest Diseases and Tuberculosis, Faculty of Medicine, Cairo University, Cairo, Egypt.

Received for publication April 14, 2004. Accepted for publication July 21, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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