1 Clinical Epidemiology Unit, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
2 Centre for Clinical Epidemiology and Biostatistics, University of Newcastle, Newcastle, New South Wales, Australia
3 Centre for Biostatistics and Genetic Epidemiology, Department of Health Sciences, University of Leicester, Leicester, United Kingdom
4 Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute, John Hunter Hospital, Newcastle, New South Wales, Australia
5 Department of Respiratory Medicine, Waikato Hospital, Hamilton, New Zealand
6 Queensland Institute of Medical Research, Brisbane, Queensland, Australia
7 Division of Therapeutics and Molecular Medicine, University Hospital, Nottingham, United Kingdom
8 Department of Environmental and Occupational Health Sciences, School of Public Health and Community Medicine, University of Washington, Seattle, WA
9 Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, Special Administrative Region, China
10 Department of Child Health, Medical School, University of Aberdeen, Aberdeen, United Kingdom
11 deCODE Genetics, Inc., Reykjavik, Iceland
12 Department of Genetics, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Correspondence to Dr. John Attia, Centre for Clinical Epidemiology and Biostatistics, University of Newcastle, Newcastle, NSW 2300, Australia (e-mail: John.Attia{at}newcastle.edu.au).
Received for publication January 17, 2005. Accepted for publication March 14, 2005.
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ABSTRACT |
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asthma; epidemiology; genetics; haplotypes; linkage disequilibrium; meta-analysis; polymorphism, genetic; receptors, adrenergic
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INTRODUCTION |
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The ß2-adrenoceptor gene is a key gene to study in asthma. ß2-Adrenoceptors are present on many airway cells, including smooth muscle cells which are hyperreactive in asthma, and ß2-adrenoceptor agonists form a major treatment class in asthma. Functional polymorphisms of this gene may influence both disease susceptibility and treatment response in asthma.
A number of studies have investigated polymorphisms in the ß2-adrenoceptor gene in relation to asthma. Two common polymorphisms are Arg/Gly16 and Gln/Glu27; in the former polymorphism, glycine is substituted for arginine at codon 16 (Arg16Gly) and, in the latter, glutamic acid is substituted for glutamine at codon 27 (Gln27
Glu) (2
, 3
). In vitro studies indicate that the Gly16 allele enhances agonist-induced down regulation of the receptor, whereas the Glu27 allele enhances resistance to down regulation (4
, 5
). It is plausible that these differences in receptor regulation influence the reactivity of airway smooth muscle in response to airway inflammation and thereby alter the risk of asthma. However, epidemiologic studies have yielded conflicting results, with the direction of the effects not always congruent with the in vitro results. Several narrative reviews of these two polymorphisms and asthma (4
6
) have been conducted; however, neither a magnitude nor a mode of gene effect was provided in these reviews. Furthermore, new studies that examine this association have been reported since those reviews, and there have been new developments in the methodology of meta-analysis of genetic studies (1
, 7
, 8
). We therefore performed a systematic review of the association between Arg/Gly16 and Gln/Glu27 and asthma with the following objectives: first, to estimate allele frequencies; second, to ascertain if there is an effect of these polymorphisms on asthma susceptibility, and if so to estimate the magnitude of that effect and the possible mode of inheritance (1
, 7
, 8
); third, to determine linkage disequilibrium between these two polymorphisms; and fourth, to infer haplotypes of these polymorphisms and link them with asthma susceptibility.
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MATERIALS AND METHODS |
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Inclusion criteria
For allele frequency, any human studies that estimated the prevalence of ß2-adrenoceptor polymorphisms at codon 16 (Arg/Gly16) and/or codon 27 (Gln/Glu27) and reported on ethnically homogeneous populations were included, regardless of size. For assessing association, human studies, regardless of sample size, were included if they met the following criteria:
Data extraction
Data were extracted independently and in duplicate by two reviewers (A. T. and M. M.) who used a standardized data extraction form. Any disagreement was adjudicated by a third author (J. A.). Covariables, such as mean age, gender, and ethnicity, were also extracted for each study.
Quality score assessment
The quality of studies was also independently assessed by the same two reviewers who used quality assessment scores that were modified from our previous meta-analysis of molecular association studies (7) (appendix table 1). These scores were based on both traditional epidemiologic considerations and genetic issues (1
). Total scores ranged from 0 (worst) to 13 (best).
Statistical analysis
Data analyses were performed as follows. First, the frequency of Arg16 and Gln27 alleles in various ethnic groups was estimated by the inverse variance method, as described in the Appendix.
Second, estimation of the gene effect on asthma was performed by a logistic regression approach described previously (8). In brief, the steps were as follows. Hardy-Weinberg equilibrium was assessed for each study by use of the
2 test or Fisher's exact test, where appropriate, and only in control groups. A Q test for heterogeneity was performed separately for three odds ratios (ORs), that is, Gly/Gly versus Arg/Arg (OR1), Arg/Gly versus Arg/Arg (OR2), and Gly/Gly versus Arg/Gly (OR3) for the Arg/Gly16 polymorphism and Glu/Glu versus Gln/Gln (OR1), Gln/Glu versus Gln/Gln (OR2), and Glu/Glu versus Gln/Glu (OR3) for the Gln/Glu27 polymorphism. If there was heterogeneity on at least one of these odds ratios, the cause of heterogeneity was explored by fitting a covariable (e.g., ethnicity, age, gender, or quality score) in a meta-regression model (9
11
). If there was no heterogeneity, logistic regression analysis with the fixed-effect model was used to determine the gene effect; otherwise, the random-effect model was used to pool. A likelihood ratio test was used to gauge whether the overall gene effect was significant. If the main effect of the genotype was statistically significant, further comparisons of OR1, OR2, and OR3 were explored. These pairwise differences were used to indicate the most appropriate genetic model as follows.
Third, the gene effect was estimated by use of a newer, "parsimonious" approach detailed elsewhere (C. Minelli et al., University of Leicester, unpublished manuscript). In brief, this approach summarizes the genetic model in terms of a parameter lambda (), which is the ratio between log(OR1) (Glu/Glu vs. Gln/Gln) and log(OR2) (Gln/Glu vs. Gln/Gln). This parameter, which represents the heterozygote effect as a proportion of the homozygote variant effect, captures information about the genetic mode of action as follows: a recessive model if
= 0, a dominant model if
= 1, a codominant model if
= 0.5, and homozygous or overdominant if
is greater than 1 or less than 0. The two log odds ratios are modeled as either fixed or random effects, as described in the second statistical analysis enumerated above.
Once the best genetic model is identified, this model is used to collapse the three genotypes into two groups (except in the case of a codominant model) and to pool the results again. Sensitivity analyses were performed by including or excluding studies not in Hardy-Weinberg equilibrium.
Fourth, with haplotype analysis, the haplotype frequencies of Arg/Gly16 and Gln/Glu27 polymorphisms were inferred using the expectation-maximization algorithm (12). The odds ratio was then estimated by use of the profile likelihood. The linkage disequilibrium coefficient was then estimated (13
). The likelihood ratio test was used to test whether the linkage disequilibrium was significant.
All analyses were performed using Stata software, version 8.0 (14), apart from the parsimonious approach, for which WinBugs 1.4 (15
) with vague prior distributions was used. A p value of less than 0.05 was considered statistically significant, except for tests of heterogeneity where a level of 0.10 was used.
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RESULTS |
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Allele frequencies
Arg allele
To estimate the pooled frequency, we used data only from control groups where a case-control design was used or from the entire group where a cohort design was used. Twenty-six studies (2, 3
, 17
, 18
, 20
22
, 24
, 26
43
) reported Arg allele frequencies (table 1), with 13 studies of Caucasian adults, three of Caucasian children, four of Black adults, six of Oriental adults, two of Oriental children, and one of Semite (Jews/Arabs) adults. Of these, six were not in Hardy-Weinberg equilibrium, leaving 12 studies of Caucasians, three of Blacks, and seven of Orientals for pooling.
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Gln allele
Twenty-six studies (3, 16
, 17
, 19
, 20
, 22
25
, 27
43
) reported the frequency of the Gln/Glu27 polymorphism, 12 studies of Caucasian adults, three of Caucasian children, three of Black adults, seven of Oriental adults, two of Oriental children, one of Jewish adults, and one of Polynesian adults (table 2). Three studies, all of Caucasians, did not observe Hardy-Weinberg equilibrium and were not included in pooling.
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Assessing association between gene polymorphisms and asthma
Across both Embase and Medline databases, 435 studies were identified in total, of which 113 were duplicates, leaving 322 study abstracts that were reviewed. From these, 30 studies seemed to be relevant, and therefore the full papers were retrieved. Sixteen studies were judged to have met the inclusion criteria, of which eight provided complete data in the paper. Requests for additional data on the other eight studies were made, of which four were granted. Two additional studies (36, 43
) were identified by a known expert (D. D.), and the authors provided additional data. The characteristics of the adult and pediatric study populations, for example, mean age, gender, ethnicity, type of subjects, and allele frequency, are given in table 3.
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Childhood asthma.
Five studies (3741
) determined the association between the Arg/Gly16 polymorphism and asthma in children (table 4), and all observed Hardy-Weinberg equilibrium. The total sample size was 334 with asthma and 842 controls.
No heterogeneity was detected for OR1 (Gly/Gly vs. Arg/Arg), OR2 (Arg/Gly vs. Arg/Arg), or OR3 (Gly/Gly vs. Arg/Gly) (for OR1: p = 0.74; for OR2:
p = 0.85; for OR3:
p = 0.30). Logistic regression with the fixed-effect model was used to assess the overall gene effect, and this was close to the formal significance level (LR2 = 5.15, p = 0.08). The estimated OR1, OR2, and OR3 were 0.75 (95 percent CI: 0.50, 1.12), 1.08 (95 percent CI: 0.76, 1.55), and 0.70 (95 percent CI: 0.51, 0.96) (table 5). These estimates suggest a recessive protective effect of the Gly allele, and therefore Arg/Arg and Arg/Gly were combined and compared with Gly/Gly. The estimated odds ratio was 0.71 (95 percent CI: 0.53, 0.96); that is, children with the Gly/Gly genotype had about 29 percent lower risk of having asthma than did children with the Arg/Arg and Arg/Gly genotypes. Using the parsimonious approach gave similar results: OR1 and OR2 of 0.88 (95 percent CI: 0.52, 1.20) and 1.04 (95 percent CI: 0.76, 1.54), respectively. The estimated
was 0.16 (95 percent CI: 3.85, 4.39), close to what would be expected for a recessive model, that is, 0, although the confidence interval was wide.
Gln/Glu27 polymorphism
Adult asthma.
Eight studies (3, 32
36
, 42
, 43
) assessed the association between the Gln/Glu27 polymorphism and asthma in adult patients (table 6). The sample size was 1,162 for asthma and 1,745 for control groups. All studies except one (34
) observed Hardy-Weinberg equilibrium, and seven studies were therefore pooled to assess gene effect.
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The estimated OR1, OR2, and by the parsimonious approach were 0.97 (95 percent CI: 0.75, 1.27), 0.88 (95 percent CI: 0.63, 1.18), and 0.61 (95 percent CI: 4.66, 5.54), respectively. Sensitivity analysis was performed by adding the one study (34
) not observing Hardy-Weinberg equilibrium, and the gene effect was robust: The estimated OR1, OR2, and OR3 were 0.88 (95 percent CI: 0.68, 1.13), 0.71 (95 percent CI: 0.60, 0.84), and 1.22 (95 percent CI: 0.95, 1.59), respectively This seems to indicate a homozygous or overdominant mode of effect, with heterozygotes being at lower risk of asthma than either homozygote. Pooling according to this model yielded an odds ratio of 0.73 (95 percent CI: 0.62, 0.87); that is, the chance of having asthma was about 27 percent less with Gln/Glu compared with Gln/Gln + Glu/Glu. Although this is a nonintuitive model, there is precedent for other genes acting in this manner (see Discussion); alternatively, this may be a spurious result due to the distribution of data and the possibility of interaction between the two polymorphic sites. We address this possibility further in the next section using haplotype analysis.
Childhood asthma.
There were five studies (3741
) addressing the association between the Gln/Glu27 polymorphism and asthma in children (table 6). All studies observed Hardy-Weinberg equilibrium except one (38
).
The four studies observing Hardy-Weinberg equilibrium were pooled (37, 39
41
). Since the studies by Lin et al. (40
) and Leung et al. (41
) had cells with no counts, we added 1 for each cell for these two studies. There was no evidence of heterogeneity for OR1 (Glu/Glu vs. Gln/Gln), OR2 (Gln/Glu vs. Gln/Gln), or OR3 (Glu/Glu vs. Gln/Glu) (for OR1:
p = 0.93; for OR2:
p = 0.53; for OR3:
p = 0.68). Logistic regression with the fixed-effect model was then used to pool; the estimated OR1 and OR3 of 0.62 (95 percent CI: 0.36, 1.07) and 0.59 (95 percent CI: 0.35, 0.99), respectively, were similar, whereas the estimated OR2 of 1.05 (95 percent CI: 0.75, 1.48) was close to one (table 5). Although the overall gene effect was not significant (p = 0.12), there is the suggestion of a recessive protective effect. The Gln/Gln and Gln/Glu genotypes were therefore combined and compared with Glu/Glu. We found that the estimated odds ratio was 0.60 (95 percent CI: 0.37, 1.00); that is, children who had the Glu/Glu genotype were about 40 percent less likely to have asthma than were children who had genotype Gln/Glu or Gln/Gln. Sensitivity analysis was performed by including the study not in Hardy-Weinberg equilibrium; this did not change the indication of a recessive protective effect (OR = 0.61, 95 percent CI: 0.38, 0.98). The parsimonious model was compatible with this effect, with an OR1 of 0.90 (95 percent CI: 0.49, 1.22), an OR2 of 1.02 (95 percent CI: 0.76, 1.40), and an estimated
of 0.04 (95 percent CI: 3.63, 4.30). Hence, these results suggested a recessive protective effect of Glu, although neither model was statistically significant.
Haplotype analysis of Arg/Gly16 and Gln/Glu27 polymorphisms
Three studies of adults provided data for haplotype analysis (3, 33
, 36
). The study by Weir et al. (30
) reported inferred haplotype data among subjects who had only homozygous wild or mutant genotypes at one locus, so this study was not included in the present analysis. The expectation-maximization algorithm was applied to infer haplotypes for the three studies, and linkage disequilibrium was assessed. The estimated linkage disequilibrium coefficient was 0.48 (p < 0.001).
The haplotype frequency in asthmatics and controls is described in table 7. The three most common haplotypes were Arg/Gln (37.5 percent), Gly/Glu (31.7 percent), and Gly/Gln (28.2 percent). The estimated odds ratios were 0.39 (95 percent CI: 0.29, 0.58), 0.99 (95 percent CI: 0.74, 1.49), and 0.83 (95 percent CI: 0.62, 1. 24) for haplotypes Arg/Glu, Gly/Gln, and Gly/Glu compared with Arg/Gln. These numbers seem to indicate that, when Gln is present at position 27, the risk of asthma is the same regardless of what allele is present at position 16. However, with Glu at position 27, the risk of asthma is lower, and this decreased risk is modified by the allele at position 16, being lower with Arg16 than with Gly16.
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DISCUSSION |
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Second, the protective effect of Glu27 may be due to the haplotype. It is probable that this is not an effect of this SNP in isolation but, instead, reflects a common haplotype that includes this allele. Drysdale et al. (44) investigated 13 SNPs in the human ß2-adrenergic receptor gene promoter and coding regions in relation to responsiveness to ß2 agonists. They found that, although there was no association when SNPs were analyzed individually, there was a clear relation between one of the common haplotypes (haplotype 2 in their paper, which included Glu27) and good response to ß2 agonists in vivo, as well as increased messenger RNA levels and gene expression in vitro. Haplotypes that included Gln27 (e.g., haplotype 4 in their paper) had overall poorer response to ß2 agonists and lower expression levels. Presumably, good response to exogenous agonists also reflects good response to endogenous agonists and, hence, a protective effect against asthma.
Third, the genetic model suggested by the data appears to be an overdominant protective effect of Glu27. This model is also called heterozygote advantage or positive heterosis, and although it may appear counterintuitive, a recent review indicates that this mode of action is perhaps more common than previously thought and cites numerous examples (45). Indeed, the IL12B promoter polymorphism has been associated with severity of asthma in children, and this also seems to observe a pattern of heterozygote advantage (46
). The mechanism of such a model is still speculative but may include 1) advantages in having variation in a multimeric protein, such as better Vmax (47
); 2) an allele with a selective advantage that is detrimental when homozygous (e.g., sickle cell and falciparum malaria); and 3) a greater range of expression of gene products and plasticity with heterozygotes than homozygotes (45
). Alternatively, this may be a spurious result due to other untyped loci in the haplotypes analyzed.
Fourth, there may be interaction or synergism between different SNPs. The haplotype analysis raises the possibility that the position 16 polymorphism may be an effect modifier: The protective effect of Glu27 was accentuated with Arg16 compared with Gly16, although there was no independent effect of the position 16 polymorphism on its own. This would indicate that it may be difficult to predict a haplotype effect from its constituent SNPs.
Fifth, the linkage disequilibrium between position 16 and 27 polymorphisms is not high. This may be surprising given that they are only 30 nucleotides apart and there are no intervening introns. However, this is congruent with other studies indicating that recombination frequency is not strictly proportional to chromosomal distance, and it is sensitive to ancestral effects; for example, Drysdale et al. found that "some pairs of close sites have reduced levels of linkage disequilibrium relative to more spaced pairs of sites" (44, p. 10485).
The pooled allele frequencies at both the Arg16 and Gln27 sites confirm the presence of significant variation between racial groups and are similar to values generally recognized, for example, in ALFRED (Allele Frequency Database) (48). Although crude, these results do support a role of these polymorphisms in asthma susceptibility, given the varying incidence of asthma in these racial groups. Interestingly, the variation was more marked at the Gln27 locus than at Arg16, and it was the former that was more strongly implicated in asthma susceptibility in our results.
These findings must be taken with caution at the present time for a number of reasons. First, these estimates are obtained by pooling despite heterogeneity.
Second, the asthma phenotype was often not fully specified, and details of asthma diagnoses were often scanty. Future studies should clearly identify whether asthma cases were diagnosed from symptoms or on population screening, and they should include results of atopic testing, spirometry, or methacholine challenge. Without sufficient information in individual studies, the condition labeled as asthma in this meta-analysis is likely to be heterogeneous and may be contributing to the inconsistency of results.
Third, the haplotype results are very different from those found in the longitudinal Normative Aging Study cohort (49), where the Gly16/Gln27 haplotype had a protective effect compared with Arg16/Glu27 (a different reference genotype), whereas in our study there was an increased risk. This discrepancy, however, may be due to the fact that, in the latter, the outcome was airway hyperresponsiveness (which does not always correspond to asthma) and that the population was general, community-dwelling males screened with a methacholine challenge test, not diagnosed asthmatics.
Fourth, these findings do not take into account smoking status, since data were available from only two studies (3, 42
). There are some indications that the genotype effects may be more apparent among nonsmokers (49
).
Fifth, the findings in childhood and adult asthma are inconsistent. This may be due to chance, or, alternatively, there may be a genuinely different mode of action in adults compared with children, in that asthma is a clinically different disease in these two populations. Asthma in late childhood, which was the age range studied in these papers, is predominantly atopic in nature, more likely to be eosinophilic, more likely to be symptom diagnosed and episodic, and less likely to be associated with persistent airway hyperresponsiveness (4952
). Since the Glu27 polymorphism is associated with less airway hyperresponsiveness (53
), this may explain differences between the associations in adults and children. Alternatively, given the incomplete understanding of asthma pathogenesis, there may be pleiotropic effects of the ß2-receptor at different stages or etiologies of disease. Indeed, one of us has observed such an age-specific association for another gene candidate in a population of children followed from childhood into early adult life (54
).
In summary, these results are suggestive of a protective effect of the Glu27 allele, probably as part of a haplotype, and they raise the possibility of interactions with the position 16 alleles and possibly other SNPs. This warrants further investigation in larger studies. The clinical implications of these findings are not clear. These polymorphisms may be involved in both conferring the risk to develop asthma and influencing the response to ß2-agonists; this has been the subject of a recent randomized crossover trial (55) and is the topic of an ongoing meta-analysis (56
).
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APPENDIX |
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Heterogeneity of prevalences across studies was checked as follows:
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The Q statistic follows a 2 distribution with number of studies (k) 1 df. If heterogeneity was present, between-study variation was then estimated as follows:
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The 95 percent confidence interval was estimated as follows:
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* FEV1, forced expiratory volume in 1 second; PEFR, peak expiratory flow rate.
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ACKNOWLEDGMENTS |
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NOTES |
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References |
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