Departments of 1Pharmacology & Clinical Pharmacy and 2Biochemistry, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, Oman; 3Department of Laboratory Medicine & Pathology, Royal Hospital, Muscat, Oman; 4Department of Small Animal Health, Directorate General of Veterinary Services, Royal Court Affairs, Muscat, Oman; 5Department of Basic Medical Sciences, Dubai Healthcare City, Dubai, United Arab Emirates *Corresponding Author e-mail: mme51@squ.edu.om تكرار التعدد اجليين ملورثات Arg16Gly, Gln27Glu and Thr164Ile يف مستقبالت بيتا 2 األدرينريجية عند العمانيني خالد البلو�سي، فهد الزدجايل، �سو�سن ال�سنانية، املعت�سم الزدجايل، ريا�ض بيومي abstract: Objectives: This study aimed to assess the distribution of missense mutations in the adrenoceptor β2 (ADRB2) gene in an Omani cohort. Methods: This study was carried out between May 2014 and March 2015 at the Sultan Qaboos University, Muscat, Oman. Blood samples were taken from 316 unrelated Omani subjects. Genotyping for rs1042713 (c.46A>G, p.Arg16Gly), rs1042714 (c.79C>G, p.Gln27Glu) and rs1800888 (c.491C>T, p.Thr164Ile) polymorphisms was performed by real-time polymerase chain reaction using single nucleotide polymorphism (SNP) genotyping assays. The allelic frequencies of these polymorphisms were estimated on the basis of the observed numbers of specific alleles from the genotype data for male and female subjects. The genotype frequencies for each polymorphism were tested for deviation from the Hardy-Weinberg equilibrium. Results: Gly16 and Glu27 were the most frequent variants found among the cohort (63% and 75%, respectively). The Ile164 variant was not detected in the study population. There was a significant linkage disequilibrium between the rs1042713 and rs1042714 SNPs (r2 = 0.209; P ≤0.001). The most observed haplotypes were Gly16-Gln27 and Arg16-Gln27 (0.37 and 0.38, respectively). The frequency of Gly16-Glu27 was 0.25, comprising all Glu27 carriers. Conclusion: The allelic distribution of variants in this Omani cohort was similar to distributions reported among Caucasian populations. Keywords: beta-2 Adrenergic Receptor; Genetic Polymorphisms; Single Nucleotide Polymorphisms; Allele Freq- uencies; Genotype; Oman. امللخ�ص: الهدف: هدف هذا البحث اإىل درا�سة توزيع الطفرات املغلطة يف مورثات م�ستقبالت بيتا 2 الأدرينريجية )ADRB2( يف جمموعة من العمانيني. الطريقة: اأجريت الدرا�سة بني مايو 2014م ومار�ض 2015م بجامعة ال�سلطان قابو�ض مب�سقط يف عمان. متجمع عينات دم من p.Gln27Glu( ،rs1042713 )c.46AG, p.Arg16Gly), rs104- 2714 (c.79C>G, p.Gln27Glu), rs1800888 (c.491C>T, p.Thr164Ile) and rs1141370 (c.100G>A, p.Val34Met). The first three of the nonsynonymous SNPs de- monstrated in vivo functional effects on the receptor activity.7 The rs1800888 and rs1141370 SNPs were infrequent and found only in the heterozygous state. Arg16Gly and Glyn27Glu were the most common nonsynonymous polymorphisms reported in the ADRB2 gene.7 The Arg16Gly polymorphism is the most common and functionally relevant SNP at the amino terminus of the receptor; it occurs with allelic frequencies of between 67–72% in different populations.8 The frequency of the Gln27Glu polymorphism is approximately 29% in Caucasian populations.7 The Thr164Ile polymorphism, which is located in the fourth transmembrane domain of β2AR, exhibits an allelic frequency of 2–5% in Caucasian populations; however, it has only been reported in the heterozygous state so far.7 Allelic frequencies of ADRB2 polymorphisms, especially the SNPs in the coding region, have been studied in different ethnic groups. Studies on African American, European American, Saudi, Southwest Asian, Kenyan and Chinese populations all show inter-ethnic variation in the frequency of Gln27Glu and Arg16Gly polymorphisms.9,10 Since β2AR is an important target for many asthma drugs, these variations in the frequency of ADRB2 genotypes may influence disease susceptibility and drug responses in different populations.11 Jamil et al. reported that the prevalence of asthma in the USA was lower among Arabs in comparison to non-Middle Eastern Caucasians, independent of environmental factors.12 This further suggests the role of ethnic-specific gene-environment interactions in the predisposition to asthma. Therefore, the current study aimed to determine the frequencies of alleles and haplotypes of major missense mutations in the ADRB2 gene in an Omani cohort. Methods This study was carried out between May 2014 and March 2015 at the Sultan Qaboos University (SQU), Muscat, Oman. A total of 316 unrelated Omani subjects were recruited either as volunteers from the community or from patients visiting the Family Medicine & Community Clinic at SQU Hospital for regular medical check-ups. The inclusion criteria for the subjects were Omani nationality and an age of ≥35 years old. A minimum sample size of 261 was calculated based on 5.5% precision, 5% type I error and the reported proportion of Gln27Glu in a Caucasian population (0.29).8 Blood was collected from all participants for the purposes of DNA extraction. Genomic DNA was isolated from 200 µL of whole blood using a DNA kit (QIAGEN GmbH, Hilden, Germany) according to the protocols provided by the manufacturer. Genotyping for the rs1042713, rs1042714 and rs1800888 SNPs was performed by real-time polymerase chain reaction using SNP genotyping assays (TaqMan®, Applied Biosystems, Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer’s instructions. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), Version 20.0 (IBM Corp., Chicago, Illinois, USA). Data were compiled by genotype and the derived allelic frequencies were estimated based on the observed numbers of the specific alleles from the genotype data for male and female subjects. The genotype frequencies for each polymorphism were tested for deviation from the Hardy-Weinberg equilibrium (HWE) using a Chi-squared goodness-of-fit analysis with one degree of freedom. Haplotypes were constructed from the combination of both polymorphisms. Frequencies were inferred using PHASE 2.1.1 software (Matthew Stephens Laboratory, Department of Human Genetics, University of Chicago, Chicago, Illinois, USA).13,14 Ethical permission to conduct this study was obtained from the Medical Research & Ethics Committee of the College of Medicine & Health Sciences at SQU (MREC #284-A). All participants gave informed consent before inclusion in the study. Frequencies of the Arg16Gly, Gln27Glu and Thr164Ile Adrenoceptor β 2 Polymorphisms among Omanis e488 | SQU Medical Journal, November 2015, Volume 15, Issue 4 Results Among the Omani cohort, there were 129 male and 187 female subjects. The overall mean age was 46.0 ± 8.0 years, with a mean age of 45.0 ± 9.0 years for males and 46.0 ± 7.9 years for females (P = 0.291). Three variants of the ADRB2 gene were genotyped: rs1042713 (p.Arg16Gly), rs1042714 (p.Gln27Glu) and rs1800888 (p.Thr164Ile). No significant differences were observed in the genotype frequencies between genders [Table 1]. The rare rs1800888 SNP was not detected in the cohort; all individuals were homozygous for the wild-type allele. Gly16 and Gln27 were the most frequent variants in the study population (63% and 75%, respectively), with average frequencies of 0.63 and 0.75, respectively [Table 2]. The P values for observed and expected genotype frequencies of the rs1042713 and rs1042714 SNPS were 0.171 and 0.708, respectively, with no deviation from the HWE distribution. Allelic frequencies for the rs1042713, rs1800888 and rs1042714 SNPs were 0.63/0.37, 1.00/0.00 and 0.75/0.25, respectively [Table 3]. There was a significant linkage disequilibrium between the rs1042713 and rs1042714 SNPs (R2 = 0.209; P ≤0.001). Random segregation of alleles showed that the Arg16 allele did not occur together with Glu27, as shown in the distribution of the AG haplotype. The most observed haplotypes in the cohort were Gly16- Gln27 (GC) and Arg16-Gln27 (AC) with frequencies of 0.38 and 0.37, respectively. The frequency of Gly16- Glu27 (GG) was also common (0.25), comprising all Glu27 carriers [Table 4]. Table 1: Genotype distributions of adrenoceptor β2 genetic polymorphisms by gender among an Omani cohort (N = 316) SNP n (%) P value Male (n = 129) Female (n = 187) rs1042713 G>A 0.937 GG 54 (41.9) 77 (41.2) AG 56 (43.4) 80 (42.8) AA 19 (14.7) 30 (16.0) rs1800888 C>T - CC 129 (100.0) 187 (100.0) CT 0 (0.0) 0 (0.0) TT 0 (0.0) 0 (0.0) rs1042714 C>G 0.715 CC 70 (54.3) 109 (58.3) CG 49 (38.0) 67 (35.8) GG 10 (7.8) 11 (5.9) SNP = single nucleotide polymorphism. Table 2: Allelic frequencies and percentages of adrenoceptor β-2 genetic polymorphisms among an Omani cohort (N = 316) SNP Allelic frequency Allelic percentage rs1042713 GA 0.63/0.37 63/37 rs1800888 CT 1.00/0.00 100/0 rs1042714 CG 0.75/0.25 75/25 Table 3: Observed and expected genotype distributions and allelic frequencies of the adrenoceptor β-2 gene among an Omani cohort (N = 316) SNP n (%) 95% CI HWE P value Observed Expected rs1042713 G>A 1.88 0.171 GG 131 (41.5) 125.3 (39.7) 34.4–45.1 AG 136 (43.0) 147.4 (46.6) 41.2–52.2 AA 49 (15.5) 43.3 (13.7) 10.3–18.0 rs1800888 C>T - - CC 316 (100.0) 316 (100.0) - CT 0 (0.0) 0 (0.0) - TT 0 (0.0) 0 (0.0) - rs1042714 C>G 0.14 0.708 CC 179 (56.6) 177.8 (56.3) 50.8–61.7 CG 116 (36.7) 118.5 (37.5) 32.2–42.8 GG 21 (6.6) 19.8 (6.3) 3.6–8.9 SNP = single nucleotide polymorphism; CI = confidence interval; HWE = Hardy-Weinberg equilibrium. Table 4: Haplotype frequencies and linkage disequilibrium pattern of adrenoceptor β-2 genetic polymorphisms (rs1042713/rs1042714) among an Omani cohort (N = 316) Haplotype n Frequency Linkage R2 χ2 P value GG 158 0.25 0.209 127.54 ≤0.001 GC 240 0.38 AG 0 0.00 AC 233 0.37 Khalid Al-Balushi, Fahad Zadjali, Sawsan Al-Sinani, Al-Muatasim Al-Zadjali and Riad Bayoumi Clinical and Basic Research | e489 Discussion In the current study, Gly16 and Gln27 were the most common alleles among the Omani cohort. There were no differences in the genotype frequencies between genders, suggesting random selection; this was further supported by the lack of deviation from the HWE distribution. Previous research in different populations has suggested inter-ethnic differences in ADRB2 gene polymorphisms. The allelic frequencies of two SNPs (rs1042713 and rs1042714) in the current Omani cohort were compared with those of various ethnic groups reported in the literature [Table 5].9,10,15–21 Gly16 and Gln27 polymorphisms occur with high allelic frequency in Caucasian populations;7,8 the frequencies of Gly16 and Gln27 polymorphisms in the current Omani cohort were similar. However, regardless of population, the frequency of the Gln27 variant showed higher inter-ethnic variation than the Gly16 variant (standard deviation: 0.14 versus 0.07). The haplotype distributions in the population of the current study were similar to those reported in a previous study of the Turkish population.15 There is strong linkage disequilibrium between SNPs in the ADRB2 gene since limited sets of haplotypes are observed in different ethnic groups. Drysdale et al. reported three common haplotypes in Caucasians and four common haplotypes in African Americans based on 13 ADRB2 SNPs.22 Examinations of the results of genetic association studies between ADRB2 and diseases like asthma should take into account differences in genotype frequencies between populations and the existence of the strong linkage disequilibrium between ADRB2 SNPs. Variations in the ADRB2 gene affect patient responses to drug actions; therefore, these variations could be of importance for the clinical management of diseases in which β2AR plays a role, such as asthma, obesity and hypertension. Inter-ethnic differences in this receptor have been proposed to explain differences in responses to drugs such as terbutaline, isoproterenol and albuterol.23 Conclusion The present study provides further evidence of inter- ethnic differences in ADRB2 gene polymorphisms. The allelic distribution of variants in this Omani population was similar to distributions reported in Caucasian populations. a c k n o w l e d g e m e n t This study was funded by The Research Council, Oman (#RD/MED/PHAR/12/01). c o n f l i c t o f i n t e r e s t The authors report no conflicts of interest. Table 5: Comparative analysis of allelic frequencies of Gly16 (rs1042713) and Gln27 (rs1042714) polymorphisms in various ethnic groups reported in the literature Author and year of study Population Frequency of polymorphism Total sample size Gly16 Gln27 Maxwell et al.9 2005 Saudi 0.53 0.83 100 Maxwell et al.9 2005 Ghanaian 0.47 0.90 100 Maxwell et al.9 2005 Kenyan 0.43 0.91 100 Maxwell et al.9 2005 Sudanese 0.57 0.84 52 Maxwell et al.9 2005 Filipino 0.46 0.91 78 Maxwell et al.9 2005 Chinese 0.41 0.93 99 Xie et al.10 1999 African American 0.51 0.79 123 Maxwell et al.9 2005 Southwest Asian 0.46 0.84 99 Maxwell et al.9 2005 Scottish 0.59 0.54 100 Kato et al.17 2001 Japanese 0.51 0.93 1,681 Ehrenborg et al.18 2000 Swedish 0.59 0.62 180 Hall et al.19 2006 British 0.64 0.55 8,018 Hamdy et al.16 2002 Egyptians 0.43 0.76 240 Aynacioglu et al.15 1999 Turkish 0.60 0.68 104 Ramasy et al.20 1999 Australian 0.54 0.60 332 Xie et al.10 1999 American Caucasian 0.62 0.58 212 Candy et al.21 2000 South African 0.52 0.83 123 Present study Omani 0.63 0.75 316 Mean of all populations ± SD - 0.53 ± 0.07 0.77 ± 0.14 - SD = standard deviation. Frequencies of the Arg16Gly, Gln27Glu and Thr164Ile Adrenoceptor β 2 Polymorphisms among Omanis e490 | SQU Medical Journal, November 2015, Volume 15, Issue 4 References 1. Kobilka BK, Dixon RA, Frielle T, Dohlman HG, Bolanowski MA, Sigal IS, et al. cDNA for the human beta 2-adrenergic receptor: A protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A 1987; 84:46–50. 2. Turki J, Pak J, Green SA, Martin RJ, Liggett SB. Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma: Evidence that Gly16 correlates with the nocturnal phenotype. J Clin Invest 1995; 95:1635–41. doi: 10.1172/JCI117838. 3. Yako YY, Echouffo-Tcheugui JB, Balti EV, Matsha TE, Sobngwi E, Erasmus RT, et al. Genetic association studies of obesity in Africa: A systematic review. Obes Rev 2015; 16:259–72. doi: 10.1111/obr.12260. 4. Ortega VE. Pharmacogenetics of beta2 adrenergic receptor agonists in asthma management. Clin Genet 2014; 86:12–20. doi: 10.1111/cge.12377. 5. Johnson AD, Newton-Cheh C, Chasman DI, Ehret GB, Johnson T, Rose L, et al. Association of hypertension drug target genes with blood pressure and hypertension in 86,588 individuals. Hypertension 2011; 57:903–10. doi: 10.1161/ HYPERTENSIONAHA.110.158667. 6. Green SA, Turki J, Bejarano P, Hall IP, Liggett SB. Influence of beta 2-adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am J Respir Cell Mol Biol 1995; 13:25–33. doi: 10.1165/ajrcmb.13.1.7598936. 7. Reihsaus E, Innis M, MacIntyre N, Liggett SB. Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. Am J Respir Cell Mol Biol 1993; 8:334–9. doi: 10.1165/ajrcmb/8.3.334. 8. Fenech A, Hall IP. Pharmacogenetics of asthma. Br J Clin Phar- macol 2002; 53:3–15. doi: 10.1046/j.0306-5251.2001.01509.x. 9. Maxwell TJ, Ameyaw MM, Pritchard S, Thornton N, Folayan G, Githang’a J, et al. Beta-2 adrenergic receptor genotypes and haplotypes in different ethnic groups. Int J Mol Med 2005; 16:573–80. 10. Xie HG, Stein CM, Kim RB, Xiao ZS, He N, Zhou HH, et al. Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals. Pharmacogenetics 1999; 9:511–16. 11. Liggett SB. The pharmacogenetics of beta2-adrenergic receptors: Relevance to asthma. J Allergy Clin Immunol 2000; 105:S487–92. doi: 10.1016/S0091-6749(00)90048-4. 12. Jamil H, Raymond D, Fakhouri M, Templin T, Khoury R, Fakhouri H,et al. Self-reported asthma in Chaldeans, Arabs, and African Americans: Factors associated with asthma. J Immigr Minor Health 2011; 13:568–75. doi: 10.1007/s10903- 010-9390-0. 13. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001; 68:978–89. doi: 10.1086/319501. 14. Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet 2003; 73:1162–9. doi: 10.1086/379378. 15. Aynacioglu AS, Cascorbi I, Güngör K, Ozkur M, Bekir N, Roots I, et al. Population frequency, mutation linkage and analytical methodology for the Arg16Gly, Gln27Glu and Thr164Ile polymorphisms in the beta2-adrenergic receptor among Turks. Br J Clin Pharmacol 1999; 48:761–4. doi: 10.1046/j.1365-2125.1999.00082.x. 16. Hamdy SI, Hiratsuka M, Narahara K, El-Enany M, Moursi N, Ahmed MS, et al. Allele and genotype frequencies of polymorphic DCP1, CETP, ADRB2, and HTR2A in the Egyptian population. Eur J Clin Pharmacol 2002; 58:29–36. doi: 10.1007/s00228-002-0423-z. 17. Kato N, Sugiyama T, Morita H, Kurihara H, Sato T, Yamori Y, et al. Association analysis of beta(2)-adrenergic receptor polymorphisms with hypertension in Japanese. Hypertension 2001; 37:286–92. doi: 10.1161/01.HYP.37.2.286. 18. Ehrenborg E, Skogsberg J, Ruotolo G, Large V, Eriksson P, Arner P, et al. The Q/E27 polymorphism in the beta2- adrenoceptor gene is associated with increased body weight and dyslipoproteinaemia involving triglyceride-rich lipoproteins. J Intern Med 2000; 247:651–6. doi: 10.1046/j.1365- 2796.2000.00669.x. 19. Hall IP, Blakey JD, Al Balushi KA, Wheatley A, Sayers I, Pembrey ME, et al. Beta2-adrenoceptor polymorphisms and asthma from childhood to middle age in the British 1958 birth cohort: A genetic association study. Lancet 2006; 368:771–9. doi: 10.1016/S0140-6736(06)69287-8. 20. Ramsay CE, Hayden CM, Tiller KJ, Burton PR, Goldblatt J, Lesouef PN. Polymorphisms in the beta2-adrenoreceptor gene are associated with decreased airway responsiveness. Clin Exp Allergy 1999; 29:1195–203. doi: 10.1046/j.1365- 2222.1999.00570.x. 21. Candy G, Samani N, Norton G, Woodiwiss A, Radevski I, Wheatley A, et al. Association analysis of beta2 adrenoceptor polymorphisms with hypertension in a black African population. J Hypertens 2000; 18:167–72. 22. Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS, Nandabalan K, et al. Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000; 97:10483–8. doi: 10.1073/pnas.97.19.10483. 23. Litonjua AA, Gong L, Duan QL, Shin J, Moore MJ, Weiss ST, et al. Very important pharmacogene summary ADRB2. Pharmacogenet Genomics 2010; 20:64–9. doi: 10.1097/ FPC.0b013e328333dae6. http://dx.doi.org/10.1172/JCI117838 http://dx.doi.org/10.1111/obr.12260 http://dx.doi.org/10.1111/cge.12377 http://dx.doi.org/10.1161/HYPERTENSIONAHA.110.158667 http://dx.doi.org/10.1161/HYPERTENSIONAHA.110.158667 http://dx.doi.org/10.1165/ajrcmb.13.1.7598936 http://dx.doi.org/10.1165/ajrcmb/8.3.334 http://dx.doi.org/10.1046/j.0306-5251.2001.01509.x http://dx.doi.org/10.1016/S0091-6749%2800%2990048-4 http://dx.doi.org/10.1007/s10903-010-9390-0 http://dx.doi.org/10.1007/s10903-010-9390-0 http://dx.doi.org/10.1086/319501 http://dx.doi.org/10.1086/379378 http://dx.doi.org/10.1046/j.1365-2125.1999.00082.x http://dx.doi.org/10.1007/s00228-002-0423-z http://dx.doi.org/10.1161/01.HYP.37.2.286 http://dx.doi.org/10.1046/j.1365-2796.2000.00669.x http://dx.doi.org/10.1046/j.1365-2796.2000.00669.x http://dx.doi.org/10.1016/S0140-6736%2806%2969287-8 http://dx.doi.org/10.1046/j.1365-2222.1999.00570.x http://dx.doi.org/10.1046/j.1365-2222.1999.00570.x http://dx.doi.org/10.1073/pnas.97.19.10483 http://dx.doi.org/10.1097/FPC.0b013e328333dae6 http://dx.doi.org/10.1097/FPC.0b013e328333dae6