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* From the Channing Laboratory (Drs. Litonjua, Silverman, Tantisira, and Weiss, and Ms. Sylvia), Department of Medicine, Brigham and Women’s Hospital, Boston; and the Normative Aging Study (Dr. Sparrow), Veterans Affairs Medical Center, Boston, MA.
Correspondence to: Augusto A. Litonjua, MD, FCCP, Channing Laboratory, 181 Longwood Ave, Boston, MA 02115; e-mail: augusto.litonjua{at}channing.harvard.edu
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Abstract |
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Design: A case-control study in 543 white men (152 case patients and 391 control subjects), who were nested in an ongoing longitudinal cohort.
Setting: Subjects were selected from the Normative Aging Study, an ongoing longitudinal cohort of healthy aging.
Participants: Case patients were defined as those having a positive response to methacholine challenge testing. Control subjects were selected among those who did not have a diagnosis of asthma and who had no response to methacholine.
Results: There was a trend for an association of the Arg16 SNP genotype with airways hyperresponsiveness (odds ratio, 1.25; 95% confidence interval, 0.96 to 1.64 [in an additive model]). In stratified analyses, the effect of the Arg16 variant was seen mainly among nonsmokers. Smokers had increased risks for airway hyperresponsiveness regardless of genotype at either SNP. Using a program to estimate haplotype frequencies, three common haplotypes were identified. Adjusting for age, baseline FEV1, serum IgE level, and smoking status, the Gly16/Gln27 haplotype was negatively associated with airways hyperresponsiveness in the full complement of case patients and control subjects (score statistic, – 2.43; p = 0.02). The effect of the ß2-AR haplotypes was much stronger among lifelong nonsmokers, among whom the Gly16/Gln27 haplotype remained negatively associated with airways hyperresponsiveness (score statistic, – 3.114; p = 0.002), whereas the Arg16/Gln27 haplotype was positively associated with airways hyperresponsiveness (score statistic, 3.142; p = 0.002). No effects were seen among ever-smokers.
Conclusions: In this cohort of middle-aged to older white men, ß2-AR polymorphisms were associated with airways hyperresponsiveness, particularly among lifelong nonsmokers. Our results illustrate an instance in which greater power is obtained by performing haplotype analyses as opposed to single SNP analysis.
Key Words: ß2-adrenergic receptor gene • genetic • haplotype • polymorphisms • smoking
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Introduction |
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It has been shown4 that a significant interaction exists between ß2-AR polymorphisms at position 16 and smoking status in the risk for asthma, in a Chinese population. In addition, using haplotypes may be more informative than single nucleotide polymorphisms (SNPs) in association studies,5 because there may be interactions between multiple SNPs within a haplotype. Moreover, haplotypes may characterize the linkage disequilibrium pattern of a region more precisely, so that associations with other interesting variants can be identified. However, to date, there has been no reported study utilizing both haplotype information of the ß2-AR gene and environmental exposure information (eg, smoking) in the risk for asthma and related phenotypes in population-based association studies. This may reflect the difficulty and expense of obtaining haplotype information in these types of studies.
Because airway hyperresponsiveness is an important intermediate phenotype in asthma, we sought to determine whether ß2-AR polymorphisms at positions 16 and 27, individually and as haplotypes, were associated with this intermediate phenotype in a cohort of middle-aged to older white men, and whether this association would be modified by exposure to cigarette smoke.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since entry, volunteers have reported for periodic examinations, each consisting of a uniform medical history and physical examination, along with blood tests and spirometry. Beginning in 1984, as part of the Epidemiology of Airways Responsiveness project, 1,580 subjects also have been studied with a detailed respiratory symptom and smoking questionnaire, pulmonary function tests, and methacholine challenge tests, in addition to their regular NAS examination. This study has been approved by the Human Studies Subcommittee of the Research and Development Committee, Veterans Affairs Medical Center, Boston, MA, and by the Institutional Review Board of the Brigham and Women’s Hospital.
Definition of Case Patients and Control Subjects
Case patients and control subjects were selected from NAS subjects who were taking part in the Epidemiology of Airways Responsiveness study and who reported for their regularly scheduled examinations from 1984 through 1993. During this time period, 1,535 subjects participated, of whom 1,169 (76.2%) had agreed to perform at least one methacholine challenge test. Of the 2,313 methacholine challenge tests performed on these 1,169 subjects, 1,703 test results (73.6%) were negative. Subjects were selected as a case patient if they had a positive result for the methacholine challenge test (defined below) at any point during this time period. Control subjects were selected from among the subjects who had undergone at least two examinations over the study period, who never had received a doctor’s diagnosis of asthma (asthmaMD), and who never had a positive methacholine challenge test result. The asthmaMD was obtained from responses to the respiratory questionnaire. We identified a total of 543 subjects (152 case patients and 391 control subjects) who underwent methacholine challenge testing and had stored blood that was available for genotyping. All subjects in this study were white.
Symptom and Smoking Information
Information on symptoms and smoking habits was obtained from standard questionnaires, which were based on the American Thoracic Society DLD-78 questionnaire.7 Any wheeze was defined as a positive response to any of the following questions: (1) "Do you wheeze when you have a cold?" (2) "Do you wheeze occasionally apart from colds?" or (3) "Do you wheeze most days or nights?" Wheeze with shortness of breath was defined as a positive response to the question, "Have you ever had an attack of wheezing that has made you feel short of breath?" Current smokers were defined as those men who had smoked at least one cigarette per day during the previous year and still had been smoking at least 1 month before their examination. Former smokers were defined as those men who previously had smoked at least one cigarette per day for at least 1 year, but who had stopped smoking > 1 month before their examination. Never-smokers were defined as those men who had never smoked cigarettes. The number of pack-years smoked was calculated from the information obtained in the questionnaires.
Skin Testing
Skin testing was performed as previously described.8 Briefly, after cleaning the volar surface of each forearm with alcohol and allowing it to dry, four allergens plus a glycerin control were applied to the skin 5 cm apart. The allergens were ragweed (1:20), mixed trees (1:20), mixed grasses (1:20), and house dust (1:10), and the control was a 50% solution of glycerin. Wheal reactions were read after 20 min by a specially trained physician. The size of the reaction was determined by measuring the largest diameter of the wheal and that perpendicular to it, then halving the sum. When wheal reactions occurred to the glycerin control, they were subtracted from the size of the allergen reactions. A positive skin test was defined as a wheal diameter of 
2 mm in reaction to any of the four allergens after subtraction of any wheal reaction to the control.
Serum IgE Measurements
Phlebotomy was performed during the subjects’ scheduled examinations. The total serum IgE concentration was determined by paper radioimmunosorbent test (Pharmacia Diagnostics; Piscataway, NJ), and the mean of two determinations was used for analysis.
Methacholine Challenge Testing
Methacholine challenge testing was performed as previously reported.89 Subjects underwent a methacholine challenge protocol that had been adapted from that of Chatham and colleagues.10 Saline and methacholine solutions were aerosolized using a nebulizer (model 646; DeVilbiss; Somerset, PA) attached to an air compressor (DeVilbiss). All inhalations were 6-s inspiratory maneuvers from residual volume to total lung capacity, followed by 2 s of breathholding. Incremental doses of methacholine were inhaled at 5-min intervals according to the following schedule: five inhalations of 0 mg/mL (phenol-buffered saline alone); one inhalation of 1 mg/mL; one inhalation of 5 mg/mL; four inhalations of 5 mg/mL; one inhalation of 25 mg/mL; and four inhalations of 25 mg/mL. Previous determination of nebulizer output by weight in a subset of the whole cohort8 indicated that the methacholine inhalation schedule corresponded to cumulative doses of methacholine of 0, 0.330, 1.98, 8.58, 16.8, and 49.8 µmol/L. Spirometry was performed 30, 90, and 180 s after each inhalation level. If the first two spirograms at each level were consistent (ie, FEV1 within 5%), then the higher measurement of these two was chosen for analysis. Otherwise, the higher FEV1 measurement from the most consistent pair of acceptable spirograms was used. The test was terminated when a 20% decline in FEV1 from the post-saline solution inhalation value occurred, or at the end of the dose schedule, if such a decline did not occur. This abbreviated method was compared with the standard methacholine challenge protocol in a subset of the cohort, and the results from both methods were highly correlated (r = 0.95; p < 0.001).11
For this analysis, subjects were defined as having a positive response to methacholine challenge when a 20% decline in FEV1 from the post-saline solution inhalation value occurred at or before four inhalations of 25 mg/mL methacholine (equivalent cumulative dose of methacholine, 49.8 µmol/L). A negative response was defined as the absence of a 20% decline in FEV1 from the post-saline solution inhalation value at the end of the dose schedule.
Genotyping
The Arg16 Gly SNP genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism.12 The genomic DNA was extracted from frozen buffy coat of whole blood, using DNA blood kits (Qiamp; Qiagen; Valencia, CA). DNA was amplified with the following two primers: 5'- GCC TTC TTG CTG GGC ACC CAT-3'; and antisense primer 5'-CAT ACG CTC GAA CTT GGC CAT C-3'. The italicized bases were modified from the reported sequence to create NcoI restriction sites. The 5'- primer creates a NcoI site in the PCR product generated from the Gly16 allele, but not from the Arg16 allele. The 3'-primer contains a complete restriction site, and thus NcoI digests the PCR product from both alleles, which serves as a control for assessing whether digestion was complete. The PCR reaction volume was 25 µL, containing 100 µg genomic DNA. The PCR conditions included a 10-min incubation at 95°C (Amplitaq Hot Start; PerkinElmer; Wellesley, MA) followed by 35 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR products are digested by 2 U NcoI (New England Biolabs; Beverly, MA) at 37°C for 4 h. The digested products then underwent electrophoresis on a 3.5% one-half agarose (MetaPhor; Cambrex; Rockville, ME) gel at 120 V for 1.5 h. The Gly16 variant creates an NcoI site. Thus, the 168-base pair (bp) PCR product digested with NcoI shows one fragment of 128 bp in Gly16 samples with two small fragments of 18 bp and 22 bp. In samples with the Arg16 variant, the 5' NcoI site is not created, and therefore, a 144-bp fragment and a small 22-bp fragment are created.
The Gln27 Glu SNP was amplified using the same PCR primers and conditions indicated above. The PCR products were digested with 1 U BbvI restriction enzyme (New England Biolabs) at 37°C for 2 h. The digested products then underwent electrophoresis on a 3.5% 3:1 agarose (NuSieve; Cambrex) gel. BbvI digestion of the Gln27 variant produced 105-bp and 63-bp fragments. The Glu27 variant remained uncut. Genotyping at both positions was confirmed in a random sample of 100 subjects at another laboratory, who were blinded to previous genotyping results.
Statistical Analysis
SNPs at both positions were first analyzed separately, and genotype categories were created. For position 16, the genotype was categorized as 0, 1, or 2, depending on the number of Arg16 alleles that were present. Similarly, for position 27 categories were created for the number of Gln27 alleles that were present. Univariate relationships were explored using t tests and analysis of variance for continuous variables, and 
2 analyses for categoric variables. Both univariate and multivariate logistic regression models were used to further explore the relationships between the independent and dependent variables (LOGISTIC Procedure, SAS software; SAS Institute; Cary, NC).13
Haplotype analyses were conducted using software designed for that purpose (haplo.score14 [which is a suite of S-PLUS (Insightful; Seattle, WA)15 routines that computes score statistics to test associations between haplotypes and a wide variety of traits]). In this method, score tests derived from generalized linear models were used for global tests of association, as well as for haplotype-specific tests. Linkage-phase ambiguity (which is inherent in methods that infer haplotypes from unphased marker data) was addressed by computing the conditional distribution of haplotypes, given the observed marker data for all individuals in the study. These conditional distributions served as weights in the construction of the score tests. Analyses were run with and without adjustment for nongenetic factors. The minimum haplotype frequency was set at 0.05. We conducted the haplotype analyses on the whole sample and stratified them by smoking status.
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Results |
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2 values: position 16, 0.22; position 27, 2.91; p > 0.05 for both).
ß2-AR Polymorphisms, Smoking, and Airways Hyperresponsiveness
There were no significant associations between polymorphisms of the ß2-AR at either position and airways hyperresponsiveness. However, there was a trend for increasing risk for airways hyperresponsiveness with an increasing number of Arg16 alleles (odds ratio [OR], 1.25; 95% confidence interval [CI], 0.96 to 1.64]), and with a dominant model for Arg16 (OR, 1.4; 95% CI, 0.95 to 2.07). When the joint effects of ß2-AR polymorphisms and smoking status were considered, the risk associated with the Arg16 allele was seen only among nonsmoking men (Table 2
). Smokers had increased risks for airways hyperresponsiveness regardless of the genotype at position 16. There were no associations among polymorphisms at position 27, smoking, and airways hyperresponsiveness.
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8.58 µmol), or when we stratified the analyses by categories of pack-years (0, < 20, and 20 pack-years).
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Discussion |
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Since the original cloning of the ß2-AR,16 in vitro studies have been conducted to determine the functional significance of the common polymorphisms in positions 16 and 27 (reviewed in Liggett17 and in Raby and Weiss1). However, since these studies investigated receptor down-regulation and expression, it is difficult to interpret our findings in terms of these previous in vitro results. More relevant are the studies that have investigated these polymorphisms as asthma susceptibility loci or as disease-modifying loci in human populations.118 Overall, there is conflicting evidence regarding the role of ß2-AR polymorphisms in the risk for asthma and airways hyperresponsiveness. Among patients with mild-to-moderate asthma, Hall et al19 found that subjects with the Glu27 polymorphism had significantly less airways reactivity to methacholine. They did not find any association with the codon 16 SNP. Our study differs in that we studied airways hyperresponsiveness in a cohort of men sampled from a general population rather than in an asthmatic population. Ramsay et al20 assessed airway responsiveness to histamine in 332 subjects from 76 families who were not selected for asthma. They also found that subjects with the Glu27 allele were less responsive than those with the Gln27 allele. While codon 16 alleles were not associated with airway responsiveness in their study, patients who were homozygous for the Arg16 polymorphism were more likely to wheeze during a cold compared with those homozygous for the Gly16 polymorphism. In a large, multigenerational pedigree, SNPs in the ß2-AR gene were not linked to asthma, asthma symptoms, or airways hyperresponsiveness.21
Our finding that polymorphisms in position 16 of the ß2-AR gene are associated with adult-onset airway hyperresponsiveness is consistent with pharmacogenetic studies of asthma, in which the response to the regular use of ß2-agonists is also associated with the same polymorphisms. Israel et al22 showed that regular ß2-agonist use was associated with a deterioration in pulmonary function measures over a period of 20 weeks in subjects who were homozygous for the Arg16 polymorphism. Polymorphisms in position 27 did not alter the response to regular ß2-agonist use in these asthmatic patients. Taylor et al23 also found that subjects who were homozygous for the Arg16 polymorphism had a greater frequency of major exacerbations during long-term treatment with salbutamol compared with treatment with placebo, whereas no specific adverse events occurred in patients with the other genotypes.
More recently, it was shown that the associations between ß2-AR polymorphisms and asthma may be modified by exposure to cigarette smoke. The study by Wang et al,4 which was conducted on a Chinese population, showed that although there were no overall associations between ß2-AR polymorphisms and asthma, subjects who were homozygous for the Arg16 allele and who smoked had an almost eightfold increased risk for asthma compared with subjects who did not have the Arg16 allele and who did not smoke. We were unable to replicate the findings of Wang et al in our current analysis and, in fact, only found an effect of the Arg16 allele in lifelong never-smokers. Several differences between that study and ours need to be highlighted to shed light on the apparent contradictory results. First and most important, the cohorts of subjects in whom we conducted our studies were different. While our allele frequencies are similar to those reported in other samples of white subjects (in whom the Arg16 and Glu27 polymorphisms occur less frequently),2312 these allele frequencies are very different from those in the Chinese population.4 In that study, allele frequencies were as follows: Gly16 = 0.436 and Arg16 = 0.564; Glu27 = 0.085 and Gln27 = 0.915. Next, our cohort was composed entirely of men while the Chinese cohort was composed of men and women, and there is evidence that associations of ß2-AR polymorphisms with airways hyperresponsiveness may differ by gender.24 Third, phenotype definitions of case patients differed in the two studies. While the study by Wang et al4 identified asthma case patients by a combination of asthmaMD, airway hyperresponsiveness, and allergy, our case patients were chosen based solely on the presence of airway hyperresponsiveness, regardless of symptoms, asthma status, or allergy status. We emphasize that airway hyperresponsiveness is not equivalent to asthma. While only 30% of the case patients in our study had wheezing symptoms, we have shown in this cohort that methacholine airways responsiveness is associated with the subsequent development of wheezing symptoms.25 Others have also shown2627 that airways hyperresponsiveness predicts the subsequent development of asthma.
Two previous studies have investigated the associations between ß2-AR haplotypes and airways hyperresponsiveness. D’Amato et al28 conducted an analysis in a cohort of 248 young Italian men and found that the haplotype with Gly16 and Gln27 polymorphisms was associated with persistent airways hyperresponsiveness over a period of 8 months. Haplotype frequencies in their study were as follows: Gly16/Gln27 = 0.343; Gly16/Glu27 = 0.310; and Arg16/Gln27 = 0.347. Alternatively, Ulbrecht et al24 found that the Gly16/Gln27/Thr164 haplotype was underrepresented in the case group, indicating a "protective" effect against airways hyperresponsiveness, in a cohort of German individuals. The haplotype frequencies in the study by Ulbrecht et al24 (Gly16/Gln27/Thr164 = 0.185; Gly16/Glu27/Thr164 = 0.449; and Arg16/Gln27/Thr164 = 0.365) differed from those of the study by D’Amato et al.28 While we estimated haplotype frequencies in our study, in contrast to the direct molecular methods that the two previous association studies utilized, our estimated haplotype frequencies were very similar to those of Ulbrecht et al.24 Although we did not genotype codon 164, we also find that the haplotype containing Gly16/Gln27 was protective against airways hyperresponsiveness. We additionally showed that this effect was mainly seen among nonsmokers, whereas the study by Ulbrecht et al24 did not stratify by smoking status. Furthermore, the haplotype containing Arg16/Gln27 was associated with airways hyperresponsiveness among nonsmokers, which is consistent with our SNP analysis. None of the haplotypes was associated with airways hyperresponsiveness among smokers. We stress that while our results appear to be consistent with the results of the study by Ulbrecht et al,24 which used a study design that was similar to ours, studies of ß2-AR genetics from other researchers have had different results (some of which have been reviewed here). Our study used a case-control design in a cohort of older white men with adult-onset airways hyperresponsiveness, and, thus, our results may not be applicable to other populations (eg, children with asthma) using different study designs (eg, family-based designs). Finally, we do not have SNP information in other regions in and around the gene, which have been associated with other asthma phenotypes.
Given the nonsignificant findings for the analysis on the whole cohort, we performed power calculations utilizing the genotype frequencies at position 16 for a dominant model. Our study had 80% power to detect a minimum OR of 1.8, and this explains the trend for significance in our analysis for the dominant model for Arg16 (OR, 1.4). However, because the effect among nonsmokers was much bigger, we were able to find a significant effect in the stratified analyses despite smaller numbers. Although there remains the possibility that these positive results may have been due to chance alone, the fact that the results from the individual SNP analyses and the haplotype analyses were consistent suggests that this may not be the case. Furthermore, the haplotype analysis that was performed on the whole complement of case patients and control subjects found a negative association between the Gly16/Gln27 haplotype and airways hyperresponsiveness, hinting at the fact that in this case, haplotype analysis was a more powerful approach. While we did not have a replication data set, our results are consistent with the findings of Ulbrecht et al,24 as we have discussed above. We also note that case patients were slightly older than the control subjects. This is not surprising since we did not perform any matching between case patients and control subjects. However, since genotype does not change during the lifetime of the individual, it is not likely that age is a confounder in the association between genotype and methacholine airways responsiveness. Finally, all analyses were adjusted for age.
We did not find a statistical interaction between cigarette smoking and ß2-AR polymorphisms in the risk for airways hyperresponsiveness, despite the different effects seen in the smoking-stratified analyses, and this may have been due to inadequate power to detect a true interaction effect. We did, however, find a very strong effect of smoking on airways hyperresponsiveness. Being exposed to cigarette smoke, either from the environment or from personal smoking, is a known risk factor for airways hyperresponsiveness among children and adults.2930 A possible explanation for finding an effect of ß2-AR polymorphisms only among lifelong nonsmokers and not among ever-smokers (former or current smokers) is that subjects who carry the Arg16 allele may be less likely to smoke because they already have airways hyperresponsiveness. However, we did not find differences in the proportion of alleles between former smokers and current smokers (data not shown), thus, it is not likely that this is the explanation for this finding. A more likely explanation is that smoking in our cohort has a strong effect on airways hyperresponsiveness and that this overwhelms any effect of ß2-AR gene variants.
In genetic case-control studies, one of the possible explanations for a positive association is population stratification. All of the subjects in this current analysis were white. Although we did not formally assess for the presence of population stratification, there is emerging evidence that in white populations in the United States, the problem with stratification is likely to not be severe enough to be a cause of the positive association.3132
In conclusion, we have reported an association between the ß2-AR gene and airway hyperresponsiveness in this cohort of white men, and these results were strongest among lifelong nonsmokers. Smoking had a very strong association with airway hyperresponsiveness and may have overwhelmed any effect of the ß2-AR gene in smokers. Our study suggests that the use of estimated haplotype information may be more powerful than SNP analysis for association studies, at least for the case of the ß2-AR gene, and for instances in which only a few SNPs are genotyped. Finally, our study illustrates the need to investigate the joint effects of environmental exposures and genes in the genetics of complex disorders. Because of the small numbers in the stratified analyses, our results need to be replicated in other cohorts with larger numbers of nonsmokers.
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Footnotes |
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This study was supported by grants K08-HL03870 and HL34645 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, and by grant RG-024-N from the American Lung Association. The Normative Aging Study is supported by the Cooperative Studies Program/Epidemiology Research and Information Center of the US Department of Veterans Affairs and is a component of the Massachusetts Veterans Epidemiology Research and Information Center.
Received for publication July 18, 2003. Accepted for publication March 23, 2004.
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