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Beta 3 Adrenergic Receptor Trp64Arg Polymorphism and Manifestation of Coronary Artery Disease in Arabs. Abu-Amero, Khaled K. Al-Boudari, Olayan M.
Beta 3 Adrenergic Receptor Trp64Arg Polymorphism and Manifestation of Coronary Artery Disease in Arabs Abu-Amero, Khaled K. Al-Boudari, Olayan M. Mohamed, Gamal H. Dzimiri, Nduna. Human Biology, Volume 77, Number 6, December 2005, pp. 795-802 (Article)

Published by Wayne State University Press DOI: 10.1353/hub.2006.0010

For additional information about this article http://muse.jhu.edu/journals/hub/summary/v077/77.6abu-amero.html

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Beta 3 Adrenergic Receptor Trp64Arg Polymorphism and Manifestation of Coronary Artery Disease in Arabs KHALED K. ABU-AMERO,1 OLAYAN M. AL-BOUDARI,1 GAMAL H. MOHAMED,2 AND NDUNA DZIMIRI1,3

Abstract The substitution of tryptophan (Trp) by arginine (Arg) at position 64 in the ␤3-adrenoceptor (␤3-AR) gene has been associated with obesity, diabetes mellitus, and coronary artery disease (CAD). We have investigated whether the Trp64Arg polymorphism is associated with the manifestation of CAD or one of its important risk factors, such as obesity, diabetes mellitus, elevated cholesterol and triglyceride levels, or hypertension in the Arab population. All participating subjects were genotyped for this polymorphism using the polymerase chain reaction followed by enzymatic digestion and sequencing. In the angiographed normal control subjects (n ⳱ 495), 90.3% were homozygous Trp/ Trp, 9.5% were heterozygous Trp/ Arg, and 0.2% were homozygous for the Arg/Arg genotype, compared to 87%, 12.3%, and 0.7%, respectively, among angiographically confirmed CAD patients (n ⳱ 981). There was no statistical difference in the distribution of genotypes or allele frequencies between the CAD and control groups. We carried out a stepwise logistic regression analysis to study the possible combined effect of the genotypes and other risk factors on CAD. All variables were retained in the model, with p values of 0.014, 0.006, 0.005, ⬍0.001, 0.045, 0.002, ⬍0.001, and 0.016 for genotype, diabetes mellitus, sex, family history of CAD, obesity, myocardial infarction, smoking, and age, respectively. In conclusion, the Trp64Arg polymorphism of the ␤3-AR gene does not represent an independent risk factor for CAD in Arabs. However, in the presence of other CAD risk factors, this polymorphism may be used as a predictor of CAD.

The ␤3-adrenoceptor (␤3-AR) forms an essential component of the cardiovascular sympathetico-adrenoceptor signaling systems. Human ␤3-AR is expressed 1 Department of Genetics, King Faisal Specialist Hospital and Research Center (MBC 03), P. O. Box 3354, Riyadh 11211, Saudi Arabia. 2 Department of Biostatics, Epidemiology, and Scientific Computing, King Faisal Specialist Hospital and Research Center (MBC 03), P. O. Box 3354, Riyadh 11211, Saudi Arabia. 3 Biological and Medical Research Department, King Faisal Specialist Hospital and Research Center (MBC 03), P. O. Box 3354, Riyadh 11211, Saudi Arabia.

Human Biology, December 2005, v. 77, no. 6, pp. 795–802. Copyright 䉷 2005 Wayne State University Press, Detroit, Michigan 48201-1309 KEY WORDS: ␤3 ADRENERGIC RECEPTOR, TRP64ARG POLYMORPHISM, CORONARY ARTERY DISEASE, ARABS.

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796 / abu-amero et al. specifically in adipose tissues, where its activation is stimulated during thermogenesis, and in white adipose tissues, where it is activated during lipolysis. The receptor mediates catecholamine-induced lipolysis and thermogenesis of adipose tissue—processes that are important for the regulation of energy expenditure and therefore body weight. ␤3-AR is thought to play a role in the regulation of energy balance by increasing these processes. Recently, a Trp64Arg polymorphism of the ␤3-AR gene was reported to contribute to the following conditions: the development of obesity in Alaskan Eskimos (Biery et al. 1997), Caucasian German women (Benecke et al. 2000), and Mexican American (Mitchell et al. 1998) and Japanese populations (KimMotoyama et al. 1997); obesity-related metabolic complications in some ethnic groups (Sakane et al. 1997); non-insulin-dependent diabetes mellitus in Japanese (Oizumi et al. 2001) and African Americans (Lowe et al. 2001); earlier onset of type 2 diabetes mellitus and features of the insulin resistance syndrome (de Silva et al. 2001); and essential hypertension in the Sardinian population (Tonolo et al. 1999). The association of the Trp64Arg polymorphism with obesity has been related to sex and leisure-time physical activity (Marti et al. 2002). The contribution to the development of obesity in Alaskan Eskimos and Caucasian German women and the fact that ␤3-AR plays a critical role in lipid metabolism has led to the notion that alterations in its function may be involved in metabolic syndrome. In addition, the Trp64Arg polymorphism is associated with hypertension, higher serum triacylglycerol levels, and, to a degree, glucose metabolism in the Sardinian population (Melis et al. 2002), leading to the conclusion that this polymorphism confers increased sensitivity to the pressure effect of noradrenaline. In obese Chinese women the Trp64Arg polymorphism has been associated with increased plasma leptin levels (Lin et al. 1999). In addition, Corbalan et al. (2002) observed that Trp64Arg carriers, age 20–35 years old, had a substantially higher risk of developing obesity, independent of their sex or leisure-time physical activity. Patients with the Trp64Arg polymorphism showed a significantly younger onset age of diabetes than those carrying the wild type (Inukai et al. 2001). However, many studies have failed to detect any effect of this variant. Hence the importance of the Trp64Arg variant in human obesity remains controversial. Furthermore, although the studies cited so far indicate an association of the Trp64Arg polymorphism with cardiac-related and circulatory disorders, other investigators have found no association with obesity (Hinney et al. 1997; Buettner et al. 1998), body mass index (Hinney et al. 1997), insulin resistance (Rissanen et al. 1997), different cardiovascular risk factors (de Silva et al. 2001; Santos et al. 2002), or diabetes mellitus (Rissanen et al. 1997; Buettner et al. 1998; Vendrell et al. 1998) in various populations. The role of the Trp64Arg variant in the manifestation of coronary artery disease (CAD) is even less clear. Some available data in the literature indicated an association of this variant with CAD among Japanese (Higashi et al. 1997), whereas others failed to establish such a

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Beta 3 Adrenergic Receptor Polymorphism in Arabs / 797 link (Stangl et al. 2000, 2001). It appears therefore that the Trp64Arg polymorphism may invariably be associated with CAD and some of its risk factors in different ethnic groups. In this study we investigated the potential role of the Trp64Arg polymorphism as a predictor of CAD or its risk factors in the Middle Eastern Arab population of the Arabian peninsula.

Materials and Methods Study Population. The study group was composed of 981 individuals (555 males and 426 females with a mean age of 60.1 Ⳳ 12.3 years) of Saudi Arabian descent with angiographically documented CAD (CAD group). The inclusion criteria for CAD included, among other things, the presence of angiographically determined narrowing of the coronary vessels by at least 70%. In addition, 495 individuals (351 males and 144 females, mean age 58.2 Ⳳ 10.3 years) with no CAD, as established angiographically, were recruited as a normal control group. This control group included individuals undergoing surgery for heart valve diseases and individuals who reported chest pain but were established to be free of CAD. This study was performed in accordance with the regulations laid down by the Hospital Ethics Committee, and all participants signed an informed consent. Sample Collection and DNA Extraction. Five milliliters of peripheral blood were collected in tubes containing EDTA from all individuals who participated in this study, after obtaining their consent. DNA extraction was performed using the Purgene DNA isolation kit from Gentra Systems (Minneapolis, Minnesota); samples were stored in aliquots at ⳮ20⬚C until analysis. Determination of the Trp64Arg Polymorphism Genotype. A 367-bp fragment of the ␤3-AR gene encompassing the polymorphism site was amplified by PCR using the following primers: 5⬘-TTC CTT CTT TCC CTA CCG CCC-3⬘ (sense) and 5⬘-GCA GCC AGT GGC GCC CAA CGG-3⬘ (antisense) under the standard conditions described previously (Clement et al. 1995). Ten microliters of the amplified product was digested with BstNI restriction enzyme (Stratagene, La Jolla, California), and all fragments were resolved on a 4% MetaPhor agarose gel (FMC Bioproducts, Rockland, Maine) in TE buffer containing 0.5 ␮g/ml ethidium bromide. To differentiate between the homo- and heterozygous mutants, we sequenced non-wild-type PCR samples using the reverse primer and the BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, California). Samples were then run on the ABI Prism 3100 sequencer (Applied Biosystems). Statistical Analysis. Genotype frequencies in various groups were compared using a chi-square test. Multivariable logistic regression was used to study the

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798 / abu-amero et al. Table 1. Genotype and Allele Distribution of the Trp64Arg Polymorphism of the ␤3-AR Gene Among Angiographically Confirmed CAD Patients and Angiographed Normal Control Subjects Genotype or Allele Genotype Trp/ Trp Trp/Arg Arg/Arg Allele Trp Arg

CAD Group (n ⳱ 981) 853 (87%) 121 (12.3%) 7 (0.7%) 1,827 (93%) 135 (7%)

Control Group (n ⳱ 495)

Odds Ratio

95% Confidence Interval

447 (90.3%) 47 (9.5%) 1 (0.2%)

– 1.35 3.67

Reference [0.93–1.96] [0.46–79.5]

– 0.09 0.27

941 (95%) 49 (5%)

– 1.42

reference [1.0–2.01]

– 0.05

P Value

effect of the Trp64Arg genotype on CAD status, incorporating other variables (coronary risk factors) into the model. All analyses were performed using SPSS, version 10 (SPSS Inc., Chicago), statistical analysis software. A two-tailed p value less than 0.05 was considered statistically significant.

Results Digestion of the PCR product (367 bp) with BstNI will result in 6 fragments of length 97, 94, 66, 61, 34, and 15 bp in the wild-type genotype (Trp/ Trp), whereas the Trp64Arg polymorphism genotype (Trp/Arg) eliminates one of the BstNI sites, yielding an additional 158-bp product. The homozygous genotype (Arg/Arg) will have 158-, 94-, 66-, 34-, and 15-bp fragments. Because of the difficulty in resolving the 97-bp and 94-bp fragments or the 66-bp and 61-bp fragments on the agarose gel, we sequenced all non-wild-type samples to distinguish between the heterozygous (Trp/Arg) and the homozygous (Arg/Arg) genotypes. Among the angiographically confirmed CAD patients (CAD group; n ⳱ 981), 853 (87%) carried the Trp/Trp genotype, 121 (12.3%) were heterozygous Trp/Arg, and 7 (0.7%) were homozygous for the Arg/Arg genotype (Table 1). A comparison of the CAD group to the control group, using the Trp/ Trp genotype as the (wild-type) reference, showed that the Trp/Arg and Arg/Arg genotypes were not associated with CAD ( p values of 0.09 and 0.27, respectively). Similarly, the frequency of the Arg allele was not significantly different between the control and CAD groups ( p ⳱ 0.056). All other CAD risk factors were associated with CAD, except for genotype ( p ⳱ 0.062), obesity ( p ⳱ 0.089), and hypertension ( p ⳱ 0.554) (see Table 2). Next, we subjected the variables in Table 2 with p values less than 0.1 to a stepwise logistic regression to study the possible combined effect of genotype and other CAD risk factors on disease manifestation. All variables were retained

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Beta 3 Adrenergic Receptor Polymorphism in Arabs / 799 Table 2. Crude Odds Ratio for All CAD Risk Factors Among CAD and Control Groups

Risk Factor

Status

Genotype

Trp/ Trp Trp/Arg Arg/Arg ⬍40 ⱖ40 No Yes No Yes No Yes Female Male Trp/ Trp Trp/Arg Ⳮ Arg/Arg No Yes No Yes No Yes No Yes No Yes

Age (years) Elevated total cholesterol level (⬎5.2 mmol/L) Diabetes mellitus Family history of CAD Sex Genotypea Hypertension Obesity (BMI ⱖ 30) Smoking Elevated triglyceride level (⬎1.8 mmol/L) Myocardial infarction

CAD Group

Control Group

Odds Ratio (95% Confidence Interval)

853 121 7 220 759 186 468 550 384 682 250 426 555 853 128 77 733 888 89 763 122 138 444 888 93

447 47 1 25 446 49 384 148 265 245 166 144 351 447 48 45 381 435 59 283 114 47 358 490 5

Reference 1.35 [0.94–1.93] 3.66 [0.45–29.8] Reference 0.193 [0.13–0.30] Reference 0.321 [0.23–0.45] Reference 0.39 [0.31–0.50] Reference 0.54 [0.42–0.69] Reference 0.53 [0.42–0.66] Reference 1.39 [0.98–1.98] Reference 1.12 [0.76–1.66] Reference 0.74 [0.52–1.05] Reference 0.39 [0.29–0.53] Reference 0.42 [0.29–0.60] Reference 10.2 [4.14–25.4]

p Value 0.100 0.225 ⬍ 0.001 ⬍ 0.001 ⬍0.001 ⬍ 0.001 ⬍ 0.001 0.062 0.554 0.089 ⬍ 0.001 ⬍ 0.001 ⬍ 0.001

a. Because of the small number of individuals with the Arg/Arg genotype and for statistical power, we combined individuals with the Trp/Arg and Arg/Arg genotypes into one group.

in the model, with p values of 0.014, 0.006, 0.005, ⬍0.001, 0.045, 0.002, ⬍0.001, and 0.016 for genotype, diabetes mellitus, sex, family history of CAD, obesity, myocardial infarction, smoking, and age, respectively. It was difficult for us to test for Hardy-Weinberg equilibrium because random mating is not satisfied, as a result of the high consanguinity rate (⬎65%) in the Saudi population (Panter 1991).

Discussion We established that Trp/ Trp is the most abundant genotype, followed by the Trp/Arg genotype; the Arg/Arg genotype was the least common genotype among the Middle Eastern Arab population. In the present study the heterozygous (Trp/Arg) rate obtained in this population (9.5%) was slightly lower than

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800 / abu-amero et al. that observed in the German Caucasian (10.8%) (Buettner et al. 1998), Italian (11.2%) (Strazzullo et al. 2001), Dutch (12.2%) (Janssen et al. 1998), and Japanese (28.8%) (Oizumi et al. 2001) populations. The frequency of the Arg allele among our angiographed control subjects (5%) was lower than that among Bolivians (18%) (Karasaki and Kashiwazaki 2004), Pima Indians (32%) (Karasaki and Kashiwazaki 2004), Alaskan Eskimos (38%) (Biery et al. 1997), Japanese (18%) (Kawamura et al. 1999), southern Chinese (13%) (Thomas et al. 2000), and African Americans (10%) (Lowe et al. 2001) but similar to the rate observed in the Caucasian population (5%) (Oeveren van-Dybicz et al. 2001), thus indicating that variability in the Arg allele frequencies depends on region and ethnicity. Early detection of genetically susceptible individuals to CAD can lead to early intervention. However, data are lacking regarding the efficacy of this approach in preventing clinical symptoms. Despite this lack of evidence, knowledge of genetic CAD susceptibility has value in providing risk information and guiding decision making. Further research that investigates outcomes regarding genetic risk assessment for CAD is necessary. To date, studies attempting to establish the relationship between prevalence of the Trp64Arg polymorphism in the ␤3-AR gene and CAD have yielded conflicting results (Higashi et al. 1997; Stangl et al. 2000, 2001). Thus one of the aims of our study was to evaluate the potential relevance of this polymorphism to CAD or its associated risk factors in the Arab population. It is clear from our results that the prevalences of different genotypes in both the angiographed normal control subjects and the angiographically confirmed CAD patients were comparable, with only slight variations, which were not statistically significant (see Table 1). Our univariable analysis showed that all risk factors, except genotype, obesity, and hypertension, were associated with CAD. As for genotype, our results were similar to previous studies that also failed to establish a link between CAD and the Trp64Arg polymorphism (Stangl et al. 2000, 2001). In order to study the possible combined effect of genotype and other risk factors on CAD, we subjected variables from Table 2 with p values less than 0.1 to a stepwise logistic regression. After analysis, the following variables were retained in the model: combined genotype (Trp/Arg and Arg/Arg), diabetes mellitus, sex, family history of CAD, obesity, myocardial infarction, smoking, and age. This analysis indicates that genotype (Trp/Arg and Arg/Arg) is associated with the disease only in the presence of other variables. In summary, the Trp64Arg polymorphism of the ␤3-AR gene cannot be used as an independent risk factor for CAD in Arabs. However, in the presence of other CAD risk factors, this polymorphism can be used as a predictor of CAD. Further studies on different populations are required to ascertain the role of this gene variant in the development of CAD.

Received 17 November 2004; revision received 9 October 2005.

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