INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Asthma, characterized by reversible airflow obstruction and enhanced airway responsiveness to a variety of environmental stimuli, is a phenotypically heterogeneous disorder with variable disease expression. Although environmental factors are clearly important determinants of asthma, numerous studies have revealed that asthma also has strong genetic components. Current analyses indicate that asthma does not follow simple monogenic patterns of inheritance (1). Multiple regions of the human genome that are likely to contain susceptibility genes for asthma and associated phenotypes have been reported (4, 5).

Szentivanyi proposed in the late 1960s that the pathogenesis of asthma was an imbalance between bronchodilation and bronchoconstriction leading to airway obstruction (6). One of the effective mechanisms for bronchodilation is stimulation at the ₂-adrenergic receptor (₂AR) (7, 8). Thus, on the basis of physiologic considerations, the ₂AR gene is considered a candidate gene for asthma (9, 10) whose potential in this regard was enhanced when it was mapped to chromosome 5q (11) in a region genetically linked to the diagnosis of asthma (12, 13). Although this gene is in a region linked to asthma, previous studies have failed to establish a relationship between the diagnosis of asthma and known sequence variants (at codons 16 and 27) in this gene (14, 15).

Previous studies have revealed that cigarette smoking can induce airway inflammation and increase airway responsiveness (16), and it is associated with an asthma phenotype (19, 20). Environmental tobacco smoke exposure has been linked to the onset of asthma in early childhood (21), increased emergency room visits for children with asthma (22), and increased respiratory symptoms among the general population (23). Although active and passive cigarette smoking are well-recognized risk factors for bronchial asthma, only a portion of individuals manifest asthma. Some have speculated that individuals may vary in genetic susceptibility to cigarette smoke (24, 25), but gene-smoking interactions have not been examined in asthma or chronic obstructive lung disease.

We conducted a nested case-control study to assess the association of asthma with ₂AR gene polymorphisms and cigarette smoking, and their potential interactions in Anqing, China. The prevalence of asthma in this region was approximately 1.6 to 1.7% in men, and 1.2 to 1.8% in women according to a previous investigation (26).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subject Selection

The subjects of this report were identified through a community-based screening survey of local residents of Anqing, China (27). Anqing encompasses three urban areas and eight rural counties, with a total area of 15,000 km² and a total population of 5.8 million in 1990. Anqing was selected for genetic studies because local residents are relatively homogeneous with respect to ethnic origin, environment, occupation, and diet, and because the villages have existed for several thousand years with a stable resident population. Since 1993, 2,054 asthma index families, with at least two physician-diagnosed asthmatics comprising a total of 10,014 individuals, have been identified. All subjects were surveyed by trained interviewers using a modified American Thoracic Society Division of Lung Disease questionnaire to specifically address the issue of asthma genetics; this approach has been validated by the U.S. Asthma Genetics Collaborative Study (5). Standardized pulmonary function tests were conducted with equipment which met ATS criteria (Schiller; Boar, Switzerland), and the maneuvers were performed in a standardized manner (subject seated with nose clip) (28). The standardized airway challenge tests (methacholine) were performed on any subject whose baseline FEV₁ was more than 60% of predicted value (29). In other words, methacholine challenge test was performed in cases as well as control subjects. Of 10,014 subjects, 7,141 have completed the methacholine challenge test; of the 7,191 subjects, 2,450 had positive responses (provocative dose causing a 20% reduction in FEV₁ [PD₂₀]  9.74 mg); of these 2,450 subjects, 1,171 had PD₂₀  1.68 mg. Therefore, of the 2,450 subjects with positive methacholine responses, 1,279 were excluded because of the cutoff. No lower limit of PD₂₀ was applied; the reason to have a lower limit for predicted FEV₁ (%) was that methacholine challenge test should not be performed for those subjects with measured FEV₁ (%) below 60% of their predicted FEV₁.

All the cases and control subjects were selected from these 10,014 individuals (2,054 nuclear families). Of the 10,014 individuals, we selected cases based on the following criteria: (1) physician-diagnosed asthma or persistent wheeze; (2) bronchodilator response  12%; (3) their actual measurements of FEV₁ were lower than their predicted values of FEV₁, based on their age, sex, and other covariates (the predicted model was established using a randomly selected sample from the same geographic region); (4) PD₂₀  1.68 mg; (5) one case (must be of lowest age among the offspring) per nuclear family to ensure that all individuals were unrelated to each other; and (6) DNA samples must have been successfully extracted. Among the entire population of 10,014, only 128 individuals met these criteria and were included in this report.

We selected control subjects based on the following criteria: (1) no physician-diagnosed asthma or persistent wheeze; (2) no chronic phlegm; (3) no shortness of breath; (4) their actual measurements of FEV₁ were greater than their predicted values of FEV₁, and their actual measurements of FEV₁ should be the highest possible FEV₁ values in the age group that is within the ± 5 yr of the case group; (5) bronchodilator response < 5%; (6) methacholine challenge test negative; (7) one control per nuclear family to ensure that all individuals were unrelated to each other; and (8) DNA samples must have been successfully extracted. Among the entire population of 10,014, 136 individuals who met these criteria were selected and were included in this report. Actually, all control subjects did not respond to methacholine doses as high as 9.74 mg (Table 1 provides a summary of these criteria).

Ethics

Each subject is a mainland Chinese family member who has completed a questionnaire, given blood, and performed pulmonary function tests as part of a protocol approved by Brigham and Women's Hospital Human Subjects Committee and local Human Subject Committees. All subjects have signed a consent form, and for children, consent forms were signed by a parent or guardian.

₂AR Gene Polymorphism Genotyping

Genomic DNA was isolated from peripheral blood using a salt-precipitation method (30). The polymorphisms of the ₂AR were assigned by an allele-specific approach; a modification of polymerase chain reaction (PCR) that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. The primer pairs for detecting two polymorphisms at nucleic acid 46 (amino acid 16, ₂AR-16) were 5'-CTT CTT GCT GGC ACC CAA AA-3' for Arg-16 and 5'-CTT CTT GCT GGC ACC CAA AG-3' for Gly-16. The underlined base pair (bp) of each primer indicates the mismatched nucleic acid of the polymorphic site. By using the same reverse primer 5'-TGA TGA AGT AGT TGG TGA CC-3', the size of PCR product is 191 bp. The primer pairs 5'-GGA CCA CGA CGT CAC GCA AC-3' and 5'-GGA CCA CGA CGT CAC GCA AG-3' were used to delineate the polymorphisms at nucleic acid 79 (amino acid 27, ₂AR-27), Gln-27 and Glu-27, respectively, and the PCR product is 158 bp. An additional PCR primer pair, 5'-GAA CTG CCA CTT CAG CTG TCT-3' and 5'-CAG CTG CAT TTG GAA GTG CTC-3', was included in each PCR to amplify a 320-bp DNA fragment of gene CYP1A1, which served as the internal positive control of PCR. PCR was carried out in a volume of 10 µl containing approximately 50 ng of genomic DNA, 1.5 mM MgCl₂, 200 µM of each deoxynucleotide triphosphate, 200 nM of each primer, 0.45 unit of Taq DNA polymerase (Qiagen Inc., Valencia, CA), and 1× reaction buffer. An initial denaturation at 94° C for 3 min was followed by 40 cycles of 94° C for 60 s, 57° C for 60 s, and 68° C for 60 s with a final extension for 7 min at 68° C. The PCR products were electrophoresed on 2% SeaKem LE (FMC Bioproducts, Rockland, ME) agarose gels and visualized with ethidium bromide staining and ultraviolet illumination. All the genotype assignments were based on two consistent experimental results and were made without knowledge of any clinical variables. The genotype error of our study should be extremely low, if any, given that all genotypes were confirmed by two independent assays, 15% of randomly selected samples were regenotyped by a different laboratory, and the results were all consistent between the two different laboratories.

Statistical Analysis

Our analyses focused on the relationship between ₂AR gene polymorphisms and asthma and on the gene-smoking interaction. We first examined the frequency distribution of ₂AR polymorphic genotypes by case-control status by chi-square tests. The linkage disequilibrium (LD) was calculated by the EH program (URL: http://linkage.rockefeller. edu/soft/). We used logistic regression to estimate the odds ratio (OR) and 95% confidence interval (CI) of asthma in relation to ₂AR polymorphic genotypes, with adjustment for age, sex, level of FEV₁, cigarette smoking status (never, and ever coded as 0/1), and occupational exposure to dust. We also computed ORs for six groups based on genotype and smoking status using nonsmoking-wild-type genotypes as the reference group. In addition, we estimated the dose-response relationship between asthma and cigarette smoking by the ₂AR-16 genotypes. The reported p values are two-sided. Finally, we used the likelihood ratio test (LRT) to evaluate gene-smoking interactions.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Table 2 presents the demographic, cigarette smoking, lung function, and genotype data by cases and control subjects. Cases were slightly younger, comprised a higher proportion of male subjects than control individuals, and were more likely to be current smokers or ever-smokers. As expected, cases had reduced FEV₁, and increased bronchodilator response.

The overall allele frequencies of the two ₂AR polymorphisms were as follows: Arg-16, 56.4%; Gly-16, 43.6%; and Gln-27, 91.5%; Glu-27, 8.5%. The genotypic distributions were in Hardy-Weinberg equilibrium. Cases tended to have a different distribution of the polymorphic genotypes at codon 16 than control subjects, i.e., cases had a higher percentage of the Arg-16/Arg-16 genotype and a lower percentage of the Gly-16/Gly-16 genotype than control subjects. We performed a chi-square test to compare the ₂AR-16 genotype distribution of the asthmatics (i.e., cases) versus the control subjects. The results showed that it was marginally significant [²(df, 2) = 5.359, p = 0.069]. We also performed a similar test regarding the ₂AR-27 polymorphism, and the cases and control individuals turned out to be similar at this locus [²(df, 2) = 0.090, p = 0.956]. Because the frequency of homozygosity at the Glu-27 locus was rare (< 1%), we combined the Glu-27/Glu-27 and Glu-27/Gln-27 genotypes in the subsequent analyses.

We calculated the LD (between ₂AR-16 and ₂AR-27) (31), and the LD coefficient D' (defined as D*/D_max) in the control group. The LD between ₂AR-16 and ₂AR-27 in the control sample (n = 136) turned out to be highly significant ²(df, 1) = 19.19, p = 1.8 × 10⁵; the D' was 95%.

Given an individual is Gln-27, according to the estimation based on our data, the predictive power that this person has a position 16 variant is 44.1%. This result is not surprising, because most people carry the Gln-27 allele regardless of their genotypes at position 16. On the other hand, given an individual is Glu-27, the predictive power that this person has a position 16 variant is 94.6%, which is similar to that reported in Asian populations (32).

There was no significant association between asthma and ₂AR-27 polymorphism in this population (OR = 1.38; 95% CI: 0.69 to 2.73; reference group Gln-27 homozygotes). However, we found a significant linear association between the number of Arg-16 alleles harbored by each subject and asthma (OR = 1.47, 95% CI: 1.05-2.06; reference group is Gly-16 homozygotes). We also examined the distribution of ₂AR gene polymorphisms at codon 16 in cases and control subjects by smoking status (Table 3). Among ever-smokers, cases were more than twice as likely as control individuals to carry the Arg-16/Arg-16 genotype. Hence, we compared the ORs in six groups based on polymorphic genotypes (Gly-16/Gly-16, Arg-16/Gly-16, and Arg-16/Arg-16) and smoking status (never and ever) (Table 3). In the nonsmoking group, the Arg-16 alleles were not significantly related to an increased risk of asthma. In the ever-smoking group, the risk of asthma significantly increased among subjects carrying two Arg-16 alleles (OR = 7.81; 95% CI: 2.07 to 29.5). After removing 12 ex-smokers from the analysis, the observed association was still statistically significant (crude OR = 5.6, 95% CI: 1.73 to 18.17; adjusted OR = 5.43, 95% CI: 1.37 to 21.54). Further analysis was performed by grouping the dose of cigarette smoking in tertiles of pack-years. The interaction between homozygous variant Arg-16 genotype and ever cigarette smoking on the increased risk of asthma appeared dose-dependent (Figure 1). However, there was no increased risk seen in subjects with only one Arg-16 allele. We found no associations when data were stratified by genotype at the ₂AR-27 locus, and when the cases with the rare Glu-27 genotype were removed the ORs for ever-smokers and asthma for the Arg-16 genotype remained unchanged.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Dose-response curves for cigarette smoking on OR of asthma with the 2 adrenoreceptor gene polymorphisms, with adjustment for age, sex, level of FEV1, cigarette smoking status (never, and ever coded as 0/1), and occupation. Dose 1: < 8 pack-years; dose 2: 8-16 pack-years; and dose 3:  16 pack-years. The asterisks indicate a statistical significance (*p < 0.05; **p < 0.01).

To control for the confounding of smoking status, we have restricted our logistic regression analysis to ever-smokers. Using the Gly-16/Gly-16 (of the ₂AR-16 polymorphism) ever-smokers as the reference group, the adjusted OR is 4.97 (95% CI: 1.02 to 24.17), which reached statistical significance (Table 4).

In order to see the effect of ₂AR-16 per se, we restricted our sample to those subjects who were homozygous wild-type at position 27 (i.e., we restricted our analysis to those individuals with Gln-27/Gln-27 genotypes only). The results are shown in Table 5. We can see that the results are consistent with those obtained without excluding the codon 27 variant.

Finally, to assess the presence of gene-environment interactions between the ₂AR-16 polymorphism of the ₂-adrenergic receptor gene and smoking, we compared the risk of asthma for subjects in each category of joint exposure with that of subjects who were homozygous wild-type for the ₂AR-16 and who were nonsmokers. Estimates for the individual and joint associations were calculated from logistic regression models using indicator variables created for each category, omitting the hypothesized low-low risk category (i.e., the baseline category). For categorical variables with more than two categories, the interaction was evaluated using the LRT, comparing the model with indicator variables for the cross-classified variables with a reduced model containing indicator variables for the main effects only (33). By treating smoking as a binary variable (Yes/No), the LRT gave us a ² (df, 2) of 5.66, which corresponds to a p value of 0.059. Because smoking appeared to be the most relevant environmental variable, we did not examine the gene-environment interaction of other variables.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We examined the relationships among sequence variants of the ₂AR gene, cigarette smoking, and asthma using 128 asthmatics and 136 control subjects identified from 10,014 studied subjects. Although, as expected, there was a marginally significant association between the number of Arg-16 alleles carried by each subject and the occurrence of bronchial asthma when smoking was not considered, our results suggested a marginally significant interaction between cigarette smoking and ₂AR-16 genotype after adjusting for important confounding factors by the LRT (p = 0.06). Specifically, we found that compared with never-smoking Gly-16 homozygotes, those ever-smokers who are Arg-16 homozygotes had a significantly increased risk of asthma (OR = 7.81; 95% CI: 2.07 to 29.5). As shown in Figure 1, the Arg-16/Gly-16 and Gly-16/Gly-16 genotypes had similar patterns of dose-response curves of adjusted ORs, both of which were quite divergent from the Arg-16/ Arg-16 curve. Thus, Arg-16 appeared to be a recessive allele.

A number of studies have suggested that individuals homozygous for Gly-16 are more prone to bronchodilator desensitization (34), nocturnal asthma (35), and more severe disease as indicated by steroid treatment (31). Martinez and coworkers reported that children homozygous for the Arg-16 allele were more likely to have a positive response to bronchodilators than individuals homozygous for the Gly-16 allele; patients who were heterozygous at this locus have an intermediate response (15). Taken together, these findings are consistent with the concept that patients homozygous for the Gly-16 form of the ₂AR have already downregulated their receptors. As a consequence, environmental exposures, such as ₂-agonist use or endogenous adrenergic stimuli, such as cigarette smoking, may have less of an effect on individuals carrying the Gly-16/ Gly-16 ₂AR variant. On the other hand, patients who are homozygous for the Arg-16 form of the receptors have a much greater capacity for receptor downregulation in response to environmental exposures.

Previous studies have shown that cigarette smoking is a risk factor for the development of asthma and increased airway responsiveness in both children and adults (24, 25, 36). Increased airway mucosal permeability and nonspecific airway responsiveness have also been observed in cigarette smokers with normal lung function (37, 38). The effects of cigarette smoking on ₂AR were studied by Laustiola and coworkers using 10 monozygotic male twin-pairs discordant for smoking for an average discordant time for smoking of 23 yr (39). Compared with their nonsmoking cotwins, smoking twins had a lower density of ₂AR on the surface of lymphocytes (6.7 ± 1.2 and 11.1 ± 1.8 fmol/10⁶ cells, respectively, p < 0.05), decreased ligand binding to the ₂AR (31.7 ± 5.5 and 26.7 ± 5.4, respectively) resulting in decreased formation of cyclic adenosine monophosphate (cAMP) (16.2 ± 3.3 versus 29.2 ± 6.5 pmol/10⁶ cells, p < 0.05), and an impaired catecholamine response. These investigators concluded that long-term cigarette smoking resulted in downregulation of ₂AR on lymphocytes, and implied a similar downregulation of ₂AR in other cell systems. In a follow-up study, Laustiola and coworkers demonstrated that the downregulation of ₂AR by chronic smoking was reversible after an 8-wk period of smoking cessation (40). These findings suggested a mechanism for a gene-environment interaction between the ₂AR gene and cigarette smoking.

In the present study, we found the risk of asthma is remarkably increased (OR = 7.81; 95% CI: 2.07 to 29.5) if one both smokes and is homozygous for the Arg-16 allele, and there was a clear dose-response relationship with the amount smoked. Our results thus provide a novel and important demonstration of a gene-environment interaction. Because different polymorphic forms of the ₂AR at position 16 have different effects on the downregulation of the receptor promoted by ₂-adrenergic agonists and because long-term cigarette smoking results in downregulation of the ₂AR, it is interesting to speculate that the smoking-induced downregulation of the ₂AR might be different in subjects carrying different polymorphic forms of this receptor. The different outcomes of this interaction may contribute to expression of the asthmatic phenotype or its various component parts.

The allele frequencies for polymorphisms of the ₂AR at codons 16 and 27 are different from those seen in Western industrialized populations (14, 15). Nevertheless, the Arg-16 allele frequency estimated in this study is not different from that reported by Weir and coworkers (41) (Arg-16 = 60.0%) or Drysdale and coworkers (32) (Arg-16 = 57.5%) in Asian populations. Also the Gln-27 allele frequency appears similar to the frequency reported for Asians by Drysdale and coworkers (32) (Gln-27 = 10.0%), both of which are lower than those reported by Weir and coworkers (41). Our population is also ethnically and geographically homogeneous, and these two factors make population stratification between cases and control subjects an unlikely explanation for our results. Theoretically, LD is an explanation for our results, yet polymorphisms of the ₂AR known to be in LD with position 16, such as the sequence variant at position 27, showed no independent effects on the asthma phenotype either directly or as an interaction with cigarette smoking in this study. Although there were differences between cases and control subjects in terms of sex, level of FEV₁, and cigarette smoking, these factors were controlled for in the analysis and, hence, could not have influenced our results. Furthermore, when the analysis is limited to current smokers, our results are unchanged (crude OR = 5.6, 95% CI: 1.73 to 18.17; adjusted OR = 5.43, 95% CI: 1.37 to 21.54).

Our use of the composite diagnostic criteria assures the diagnosis of asthma, but does not allow us to dissect which of the phenotypic characteristics upon which the diagnosis was made are most strongly responsible for the observed gene by environmental interaction. It is interesting to speculate that the sequence variants identified as linked to asthma in patients with a history of cigarette smoking may allow identification of asthmatics most susceptible for the development of chronic obstructive pulmonary disease, rather than for asthma per se, but this speculation awaits further study.

Asthma is considered a complex disease associated with many genes and interactions between genes and gene-environment factors. Our results are consistent with this theoretical construct and suggest the importance of both factors in disease causation.