Genetics, Vol. 169, 1583-1587, March 2005, Copyright © 2005
doi:10.1534/genetics.104.037812

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Haplotype Analysis of the ß2 Adrenergic Receptor Gene and Risk of Myocardial Infarction in Humans

Robert Y. L. Zee^*,1, Nancy R. Cook^*, Rebecca Reynolds, Suzanne Cheng and Paul M. Ridker^*

^* Center for Cardiovascular Disease Prevention, LeDucq Center for Molecular and Genetic Epidemiology and Harvard-Reynolds Center for Cardiovascular Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215
Bayer HealthCare, East Walpole, Massachusetts 02032
Department of Human Genetics, Roche Molecular Systems, Alameda, California 94501

¹ Corresponding author: Center for Cardiovascular Disease Prevention, Brigham and Women's Hospital, Harvard Medical School, 900 Commonwealth Ave. East, Boston, MA 02215.
E-mail: rzee{at}rics.bwh.harvard.edu

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Polymorphisms in the ß2 adrenergic receptor (ADRB2), in particular G16R, Q27E, and T164I, have been implicated in the pathogenesis of cardiovascular and metabolic phenotypes. However, no prospective, genetic-epidemiological data are available on the risk of cardiovascular disease associated with these variants. Using DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we evaluated the G16R, Q27E, and T164I polymorphisms among 523 individuals who subsequently developed myocardial infarction and among 2092 individuals who remained free of reported cardiovascular events during follow-up. The haplotype frequency distribution was significantly different among cases and controls (, P = 0.0039). Haplotype-based logistic regression, adjusting for age, smoking, and randomized treatment group, indicated that G16-Q27-I164 (odds ratio 0.178, 95% C.I. 0.043–0.737, P = 0.017) and (non-G16-Q27)-T164 (odds ratio 1.235, 95% C.I. 1.031–1.480, P = 0.022) haplotypes were significantly associated with altered risk of myocardial infarction. These findings remained after further adjustment for BMI, history of hypertension, and presence or absence of diabetes. In conclusion, variation in haplotype frequencies for the ß2 adrenergic receptor gene was found to be associated with risk of myocardial infarction.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Patient selection and clinical investigation:
We used a nested case-control design within the Physicians' Health Study (STEERING COMMITTEE OF THE PHYSICIANS' HEALTH STUDY RESEARCH GROUP 1989). In brief, of 22,071 American men aged 40–84 years who were free of prior MI, stroke, transient ischemic attack, and cancer, 14,916 provided baseline blood samples, which were available for genetic analysis. In our overall nested case-control approach, each participant who provided an adequate sample of whole blood at baseline and had a confirmed MI, ischemic stroke, or venous thrombosis during follow-up was matched wherever possible to two or three controls. The controls were study participants who had also provided a baseline blood sample and who remained free of any reported cardiovascular disease at the time the index event occurred in the case patient. Controls were selected at random from among those who met the matching criteria of age (±2 years), smoking habits, and time since study entry. As previously described (STEERING COMMITTEE OF THE PHYSICIANS' HEALTH STUDY RESEARCH GROUP 1989), yearly follow-up self-report questionnaires provide reliable updated information on newly developed diseases and the presence or absence of other cardiovascular risk factors. For all reported incident vascular events occurring after study enrollment, hospital records, death certificates, and autopsy reports were requested and reviewed by an end points committee using standardized diagnostic criteria. The diagnosis of MI was confirmed by evidence of symptoms in the presence of either diagnostic elevations of cardiac enzymes or diagnostic changes on electrocardiograms. In the case of fatal events, the diagnosis of MI was also accepted on the basis of autopsy findings. To increase power for statistical analysis, all controls were pooled in a common reference group. A total of 523 cases of incident MI and 2092 controls were available for the current analysis. The study was approved by the Brigham and Women's Hospital Institutional Review Board for Human Subjects Research.

Genotyping:
Genotype determination for the ADRB2 polymorphisms was accomplished using multiplex PCR and immobilized probe-based assays developed for multi-locus variant detection (Roche Molecular Systems, Alameda, CA) essentially as described elsewhere (CHENG et al. 1999; ZEE et al. 2002). Genotype scoring was carried out by two independent observers. Discordant results (<2%) were resolved by a further joint reading and, where necessary, by a repeat genotyping. All results were scored blinded to case-control status. Because of the rarity of the I164 allele observed in the case group, a second method, polymerase chain reaction-restriction fragment length polymorphism as described by KAY et al. (2003), was used to regenotype the entire case group, and we found 100% concordance with our original genotyping score.

Statistical analysis:
Demographic and clinical data between cases and controls were compared using ² analysis (categorical variables) and t-test (continuous variables). Genotype frequencies between cases and controls were compared by ² analysis. Pairwise linkage disequilibrium (LD) was examined using the software Haploview (http://www.broad.mit.edu/personal/jcbarret/haploview/). Haplotype frequencies were estimated from genotype data using the partition ligation-expectation maximization algorithm (QIN et al. 2002). Haplotype distributions were compared between cases and controls by a likelihood-ratio test (TERWILLIGER and OTT 1994). A likelihood-ratio statistic was computed from the estimated haplotype frequency likelihoods for cases and controls separately vs. combined. In addition, the relationship between the haplotypes and risk of MI was examined by a haplotype-based logistic regression analysis with baseline-haplotype parameterization (WALLENSTEIN et al. 1998), adjusting for age, body mass index (BMI), hypertension, smoking habits, diabetes, and randomized aspirin/ß-carotene treatment assignment. This parameterization approach, using an additivity assumption, is particularly appropriate for case-control studies. A global test statistic, comparing the model with genetic data to the model without genetic data using the likelihood-ratio test, was also performed to check for the overall association of haplotypes with the disease outcome. For each odds ratio, we calculated 95% confidence intervals (C.I.'s). A two-tailed P-value of 0.05 was considered a statistically significant result.

	RESULTS

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Baseline characteristics of the study population are shown in Table 1. As expected in a prospective cohort study, participants who subsequently developed MI had a higher prevalence of conventional athero-sclerotic risk factors at baseline than did the control subjects. The observed genotype distributions were in Hardy-Weinberg equilibrium among the controls. Using a standard marker-by-marker ² analysis, genotype distribution was significantly different between cases and controls only for T164I (P = 0.0022; Table 2). The polymorphisms tested were in strong LD among one another (Table 3). Further analysis using haplotypic information showed a significant difference in haplotype distribution between cases and controls (P = 0.004; Table 4). As shown in Table 4, three of the eight possible haplotypes were not observed in our study sample (G16-E27-I164, R16-Q27-I164, R16-E27-I164), and the R16-E27-T164 haplotype was extremely rare and not present in the control group; these haplotypes were not included in the haplotype-based logistic regression analysis. As also shown in Table 4, virtually all of the I164 carriers were G16-Q27 whereas a minority of the T164 carriers were G16-Q27, a result of an apparent linkage disequilibrium between the G16-Q27 haplotype and the T164 allele. Thus, the haplotype patterns were further collapsed and stratified using T164 vs. I164 and G16-Q27 vs. non-G16-Q27 as presented in Table 5. Further analysis of the combined-genotype distribution after excluding the T164I heterozygotes yielded similar significant results (P < 0.0001; APPENDIX). Thus, G16-Q27-T164 was used as the baseline haplotype for the regression analysis. Results from the crude logistic regression analysis showed significant association between haplotype patterns G16-Q27-I164 and (non-G16-Q27)-T164 with risk of MI (P = 0.017 and P = 0.022, respectively; Table 6). Further adjustment for BMI, hypertension status, and diabetes yielded similar findings (P = 0.022 and P = 0.015, respectively; Table 6). The global test statistic—comparing the model with genetic data to the model without—again showed similar significant results (crude, P < 0.001; adjusted, P < 0.001; Table 6). The haplotype-based logistic regression approach proposed by WALLENSTEIN et al. (1998) to analyze the data in the present study was made possible by the particular LD pattern among the three polymorphisms investigated, which allowed unambiguous assignment of haplotype pairs to each individual (confidence level for the haplotype estimation and inference >99.90%). Virtually identical study results were obtained in an analysis limited to the Caucasian male participants (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

The I164 allele has recently been shown to display a threefold reduction in receptor affinity, a markedly depressed basal and agonist-stimulated adenylyl cyclase activity, an impaired stabilization of mast cells, and a decrease in adipocyte sensitivity as compared to the T164 allele (DRYSDALE et al. 2000; HOFFSTEDT et al. 2001; HERRMANN et al. 2002; JOHNSON and TERRA 2002; KAY et al. 2003). Moreover, the I164 allelic form of the receptor has been associated with increased downregulation as compared to the T164 form (GREEN et al. 1993). Interestingly, a recent report by LIGGETT et al. (1998), examining the role of G16R, Q27E, and T164I polymorphisms in heart failure, has shown an association of the T164I polymorphism with the outcome of patients with congestive heart failure; individuals with heart failure carrying the I164 allele were at significant risk for rapid progression as compared to those carrying the T164 allele. However, this finding was based on only 10 patients with the I164 allelic form and should be interpreted with caution (JOHNSON and TERRA 2002).

By comparison, the G16R and Q27E polymorphisms have not been shown to have an effect on either ligand binding or adenylyl cyclase activity. However, they have been reported to have influence on receptor desensitization (JOHNSON and TERRA 2002). A recent study by D'AMATO et al. (1998) examining the association of G16R and Q27E with bronchial hyperresponsiveness (BHR) using haplotypic information found an increased frequency of the G16-Q27 haplotype among BHR-positive subjects, suggesting that this haplotype may be an independent risk marker for BHR. In experimental studies, the G16 and Q27 alleles have been independently associated with increased downregulation (GREEN et al. 1994, 1995), further suggesting that this haplotype carrying these allelic forms has the greatest potential for receptor downregulation. Interestingly, in our investigation, a discrepancy was observed between the prespecified combined-genotype analysis (APPENDIX) and the haplotype-based analysis (Table 5) on G16R and Q27E with risk of MI. The G16R16-Q27Q27 combined genotype is common among the cases, but has a relatively low frequency among the controls. On the other hand, the G16R16-Q27E27 combined genotype is common among the controls, but is relatively infrequent among the cases (APPENDIX), suggesting that having one R16 is associated with increased risk, while having one R16 and one E27 is associated with reduced risk. The discrepancy might result from the nature of the additivity assumption that underlies the haplotype-based logistic regression approach used and/or suggest additional complexity of heterogeneous interaction between G16R and Q27E.

Taken together, our observed associations of the T164I polymorphism, either alone or in a haplotype context, with reduced risk of MI provide further evidence of roles for ß2 adrenergic receptor gene polymorphisms in athero-thrombotic disease, although the exact mechanism(s) by which the gene variants/haplotypes that were tested influence disease progression remain unclear. We cannot exclude the possibility that the polymorphisms/haplotypes examined are in linkage disequilibrium with an unidentified susceptability gene(s)/polymorphism(s) that is responsible for the observed significant associations and for the additional complexity of heterogenous interaction observed between G16R and Q27E. These results do, however, suggest the importance of including a haplotype-based analysis, as others have recommended (SCHAID 2002; SCHAID et al. 2002), for proper assessment of multi-locus genetic association studies (JOHNSON and TERRA 2002).

The Physician's Health Study cohort is almost exclusively composed of Caucasian middle-aged men, and therefore the present data may not be generalized to other ethnic groups, women, or other populations. Nonetheless, there are considerable strengths of our present study design. It is a large prospective, nested case-control cohort in which the determination of case status is based solely on the subsequent development of disease rather than on arbitrary selection criteria. The background allele frequencies observed in our control group were similar to those previously reported in other populations (D'AMATO et al. 1998; LIGGETT et al. 1998; HOFFSTEDT et al. 2001; JOHNSON and TERRA 2002; KAY et al. 2003).

In conclusion, on the basis of the studied population, we found an association of the ß2 adrenergic receptor gene variants with altered risk of myocardial infarction. Furthermore, if replicated in other studies, our findings provide further evidence of an important role for the ADRB2 gene and its variants in athero-thrombotic disorders.

APPENDIX Prespecified combined-genotype frequency between G16R and Q27E pairs among study participants who subsequently developed myocardial infarction (cases) and those who remained free of vascular disease during follow-up (controls)

Combined genotypea Cases Controls G16G16-Q27Q27 0.029 0.042 G16G16-Q27E27 0.140 0.183 G16G16-E27E27 0.200 0.181 G16R16-Q27Q27 0.311 0.166 G16R16-Q27E27 0.150 0.270 R16R16-Q27Q27 0.171 0.157

^a Only T164T164 homozygotes were considered. ; P = <0.0001.

	ACKNOWLEDGEMENTS

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We are grateful to Daniel Chasman for insightful comments on the manuscript. This work was supported by grants from the National Heart Lung and Blood Institute (HL-58755, HL-63293), the Doris Duke Charitable Foundation, the American Heart Association, and the Donald W. Reynolds Foundation, Las Vegas, Nevada.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 

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