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Egypt J Pediatr Allergy Immunol 2012;10(2):81-86. Original article

Effects of β2-adrenergic receptor polymorphisms on asthma severity and
response to salbutamol in Egyptian children
Background: Several polymorphisms of the β2-adrenergic receptor (ADRB2) Khalid Salah,
gene have been identified. There is mounting evidence that these polymorphisms Saed Morsy,
are associated with significant variability in response to bronchodilator therapy and thus in severity and duration of asthmatic symptoms. Amal Atta *
Objectives: to assess
the frequency of ADRB2 polymorphisms at codon 16 in Egyptian asthmatic
children and to study the association of these polymorphisms with asthma
severity and response to inhaled salbutamol. Methods: This case-control study Departments of
was conducted at pulmonology unit, Zagazig University children's hospital Pediatrics and
during the period from December 2010 to December 2011. One hundred Microbiology*, Faculty
children (50 asthmatics and 50 controls) were enrolled into the study. For all of Medicine, Zagazig study population, detailed history taking, systematic physical examination, chest University, Egypt. x-ray, pulmonary function testing and ADRB2 genotyping were performed. Results: There was a significant increase in frequencies of Arg/Gly and Gly/Gly
genotypes among asthmatic children in comparison with healthy controls (OR =
7.9; CI: 0.94-67.4, P<0.05 and OR= 4.5; CI: 0.91-22.7, P<0.05 respectively).
On the other hand, there was a lower frequency of Arg/Arg genotype in
asthmatic children than that in healthy controls (OR = 0.14; CI: 0.04-0.55,
P<0.05). Moreover, statistical analysis revealed association of Arg/Arg
genotype with mild asthma (OR = 5.77; 95% CI: 1.55-21.5, P<0.05) and
association of Gly/Gly genotype with moderate/severe asthma (OR=0.057; 95%
CI: 0.006-0.516, P<0.05). However, no difference in distribution of Arg/Gly
genotypes among mild and moderate/severe asthmatics (OR = 0.79; 95% CI:
0.156-3.99, P>0.05). Regarding bronchodilator responsiveness, Gly/Gly and Arg/Gly genotypes were associated with reduced response, while Arg/Arg Correspondence:
genotype was associated with favorable response to nebulized salbutamol. Dr. Saed Morsy, Conclusion: Polymorphisms of ADRB2 at codon 16 may be a determinant of
Department of asthma severity and response to salbutamol in Egyptian asthmatic children. Pediatrics, Faculty of Further studies are needed to demonstrate effects of other polymorphisms of ADRB2 gene on these outcomes. Medicine, Zagazig University, Zagazig, El- Keywords: Polymorphism; β2-adrenergic receptor (ADRB2) gene; asthma; Sharkiah, Egypt.
Egyptian children; bronchodilator therapy.
E-mail: saedkid2002@ yahoo.com. Asthma aggregates within families and is a complex multifactorial disease with the Asthma is a disease characterized by hyper- involvement of environmental and genetic responsiveness of the airways to various stimuli, components5. Understanding of the genetic basis of which results in airway obstruction that is reversible asthma may contribute toward identifying better either spontaneously or as a result of treatment1. targets for asthma drugs6. Asthma prevalence has increased very considerably Inhaled β2-adrenergic agonist medications are in recent decades such that it is now one of the the foundation of therapy for acute asthma commonest chronic disorders in the world2. Asthma exacerbation7. The β2-adrenergic receptor is estimated to affect 300 million people world- (ADRB2) protein is expressed on bronchial smooth wide, with an expected increase to 400 million muscle cells and mediates physiologic responses worldwide by 20253. In a recent Egyptian study, the including bronchodilation, vasodilatation and overall prevalence of wheezing in the last year was lipolysis8. ADRB2 gene located on chromosome 14.7% and of physician-diagnosed asthma was 5q31-32 is one of the candidate genes most consistently identified as being associated with asthma-related phenotypes 9. Salah et al. Several polymorphisms of ADRB2 have been years. Subjects were included as controls only if described in particular at codons 16 they reported no history of asthma or allergies, no Arginine/Glycine and 27 Glutamine /Glutamic acid history or report of having experienced symptoms which alter the receptor function in vitro10,11. In of coughing, wheezing and shortness of breath in adults, genetic variations in this receptor have been the past 2 years and no other history of lung linked to asthma severity12, bronchial hyper- diseases, chronic illnesses, or medications. responsiveness13 and lung function tests14. For all study groups, detailed history taking, The relationship between ADRB2 genotypes systematic physical examination, chest x-ray, and response to inhaled β2-adrenergic agonists is pulmonary function testing and ADRB2 genotyping controversial. Some studies15-21 have found that the were performed. Informed consent was obtained Arg/Arg genotype is associated with reduced from the children's guardians. The study protocol response to these medications, whereas others22-25 was approved by the Pediatric Department Ethics have found that the Gly/Gly genotype is associated Committee of Zagazig University. with reduced response. ADRB2 genotyping:
Knowledge of the genotype which is Three milliliters of venous blood were collected associated with favorable response, could aseptically from every subject in a tube containing significantly affect treatment selection for asthma in EDTA. This blood was incubated at -20°C children. The current study will focus on the temperature. After samples were enough, the blood polymorphisms of ADRB2 gene at codon 16 as a was prepared for DNA extraction and amplification. previous study22 found no association between DNA extraction was done using Axyprep Blood polymorphisms at position 27 and response to Genomic DNA Miniprep Kit (Axygen Biosciences, inhaled β2-adrenergic agonists in asthmatic USA). DNA amplification was carried out by using children. polymerase chain reaction (PCR) Master-Mix Gold So, the objectives of this study were to assess beads (Bioron, Germany). The nucleotide the frequencies of ADRB2 polymorphisms at codon sequences of the forward and reverse prime used for 16 in Egyptian asthmatic children and the effects of these polymorphisms on asthma severity and 5'-GCCTTCTTGCTGGCACCCCAT-3' and
bronchodilator response to salbutamol.
Then, for detection of the ADRB2 polymorphisms, restriction fragment assay was done. This case-control study was conducted at Pulmonary function testing:
pulmonology unit, Zagazig University children's
Pulmonary function tests were performed using hospital during the period from December 2010 to Lilly Pneumatocho- graph (D-97204 Hochberg, December 2011. One hundred children were Germany). Spirometry was performed according to enrolled in the study. They were divided into two the American Thoracic Society standards27. groups; Group I and Group II. Spirometeric measurements included forced Group I (asthmatic group) included 50 expiratory volume in one second (FEV1), forced children, 26 males and 24 females. Their ages vital capacity (FVC) and peak expiratory flow ranged from 5-12 years with a mean value of 6.8 years. The diagnosis of asthma in these patients was Pulmonary function test results were expressed based on National Asthma Education and as a percentage of the predicted normal value. Prevention Program Guidelines26. Cases were Asthmatic children were instructed to stop any classified and subgrouped as follow: mild systemic bronchodilator or corticosteroid therapy intermittent and mild persistent asthmatics were 72 hours before tests and short acting β2-adrenergic included into the group of mild asthma and agonists were stopped 12 hours before the moderate persistent and severe persistent asthmatics were included as the group of moderate/severe To assess response to a bronchodilator in asthma. Recruited asthma patients were not in asthmatic group, one dose of salbutamol (0.15 exacerbation state and did not have other mg/kg) was administered using ultrasonic nebulizer concomitant diseases that might affect lung (eMed A100, Italy) and measurement of lung function. Children with any chronic disease (other function was performed before and 15 minutes after than asthma) were excluded. salbutamol nebulization. Response to salbutamol Group II (control group) included 50 healthy was reported as a percent change in FEV1 between children, 26 males and 24 females. Their ages baseline and after salbutamol administration. We ranged from 5-12 years with a mean value of 7.2 chose to explore changes in FEV1 after salbutamol ADRB2 polymorphisms in asthmatic children mobilizations as a measure of response to a distribution between asthmatic and healthy children bronchodilator, because this was the most objective, (P<0.05). immediate, and most common endpoint studied by Distribution of ADRB2 gnenotypes at codon 16 among asthmatic and non-asthmatic children is illustrated in table 2. There was a significant Statistical analysis:
increase of carriers of Arg/Gly and Gly/Gly Data were analyzed using Statistical Package for genotypes among asthmatic children in comparison Social Sciences (SPSS), release 16. The to controls (OR = 7.9; CI: 0.94 - 67.4, P<0.05 and quantitative variables were expressed as means and OR= 4.5; CI: 0.91-22.7, P<0.05 respectively). On standard deviations. For comparison between two the other hand, there was a lower frequency of group means, t-test was used. For comparison Arg/Arg genotype in asthmatic children than in between three group means, one way ANOVA controls (OR = 0.14; CI: 0.04- 0.55, P<0.05). (analysis of variance) was used. Qualitative Relation between polymorph-isms of ADRB2 variables were expressed by frequency and at codon 16 and asthma severity is shown in table 3. percentage and compared using chi-square test. Statistical analysis revealed an association of Also, odds ratio (OR) and 95% confidence interval Arg/Arg genotype with mild asthma (OR= 5.77; (CI) were calculated. For all tests a probability (P) 95% CI: 1.55-21.5, P<0.05). On other hand, less than 0.05 was considered significant and less Gly/Gly genotype was less frequent in mild asthma than 0.001 was considered highly significant. (OR= 0.057; 95% CI: 0.006-0.516, P<0.05). However, no difference in distribution of Arg/Gly genotype was noticed among mild and The characteristics of the two study groups are moderate/severe asthmatics (OR=0.79; 95% CI: shown in table 1. The two groups were similar in 0.156-3.99, P> 0.05). age and gender. A highly significant difference was Regarding bronchodilator responsiveness, observed in spirometric parameters between Gly/Gly and Arg/Gly genotypes were associated asthmatic and healthy children (P<0.001). Also, with reduced response (FEV1 change =11.4% and significant difference was observed in genotype 9.1%, respectively), while Arg/Arg genotype was associated with favorable response (FEV1change = 17.3%) (Table 4). Table 1. Demographic and clinical data of the study groups.
Variable
Asthmatic group
Control group
Age( year) , Mean ±SD
Male gender, n (%)
Genotype, n (%)
Spirometric parameters, Mean±SD
Asthma severity, n (%)
Mild intermittent Moderate persistent Severe persistent Table 2. Distribution of ADRB2 genotypes at codon 16 in asthmatic and healthy children.
Asthmatic group
Control group
Genotype
Salah et al. Table 3. Relation between polymorphisms of β2AR at codon 16 and asthma severity.
Moderate/
Mild asthma
Genotype
severe asthma
Table 4. Relation between polymorphisms of β2AR at codon 16 and response to salbutamol
Percentage of change
<0.05a <0.05b >0.05c of FEV1 (%), Mean±SD
FEV1: forced expiratory volume in one second , a Arg/Arg group vs. Arg/Gly group , b Arg/Arg group vs. Gly/Gly group, c Arg/Gly group vs. Gly/Gly group DISCUSSION
moderate/severe asthma. On the other hand, Genetic assessment revealed that asthmatic children Gly/Gly genotype was associated with had frequencies of Arg/Arg of 70%, Arg/Gly of moderate/severe asthma when compared with mild 14%, and Gly/Gly of 16%, while in the healthy asthmatics. This indicates that the polymorphism at children the frequency of Arg/Arg was 94%, codon 16 of ADRB2 is a possible determinant of Arg/Gly was 2%, and Gly/Gly was 4%. These asthma severity. results revealed a significant difference of A meta-analysis including a total of 28 studies distribution of ADRB2 genotypes at codon 16 performed by Contopoulos-Ioannidis et al.32, among asthmatics and healthy children. concluded that, Gly/Gly had a much higher risk for Globally, there are marked interethnic nocturnal asthma and asthma severity than the differences in the frequency of ADRB2 Arg/Arg. polymorphisms. In Caucasian–American and In contrast, Salama et al.30 found an association African–American healthy individuals, the Arg/Gly between Arg/Gly genotype with severe asthma genotype was the most predominant (38.3% and when compared to mild/moderate asthma. Also, 50.4% respectively), while Arg/Arg genotype was Gly/Gly genotype was present with lower present in lower frequencies (26.6% and 23.6%, frequencies in severe asthma when compared with respectively)28. In another study conducted on 128 mild/moderate asthma. Chinese asthmatics by Wang et al., the frequency of In vitro functional studies33,34 indicated that Arg/Arg was 40.6%, Arg/Gly was 42.2%, and down-regulation of ADRB2 receptors occurs in Gly/Gly was 17.2%, while in the healthy people the individuals expressing Gly16 allele in response to frequency of Arg/Arg was 27.9%, Arg/Gly was circulating catecholamines or exogenously 47.1%, and Gly/Gly was 25.0% 29. administered β2-agonists. The Arg16 allele, which In Egyptian population, Salama et al.30 found demonstrates resistance to down-regulation, might that the frequencies of ADRB2 genotypes at therefore be expressed at greater levels than the position 16 among healthy children, was 52.6% for Gly16 allele within airways. Accordingly, Arg/Arg, 5.3% for Arg/Gly and 42.1% for Gly/Gly. individuals carrying the Gly/Gly genotype might be In asthmatic children, these genotype frequencies more sensitive to stimuli resulting in were different; 17.5% for Arg/Arg, 45% for bronchoconstriction, and therefore have more Arg/Gly and 37.5% for Gly/Gly. Although these reactive airways than individuals carrying the frequencies were different from our results, yet the final conclusion was the same: higher frequencies In our study, the Gly allele (Arg/Gly and of Arg/Gly and Gly/Gly genotypes in asthmatic Gly/Gly genotypes) was associated with resistance children when compared with controls. In another to the bronchodilator effect of inhaled short-acting study, the frequencies of ADRB2 genotypes at β 2-agonist (salbutamol) when compared with position 16 among Egyptian healthy individuals, Arg/Arg genotype. The association between was 32.6% for Arg/Arg, 49% for Arg/Gly and ADRB2 genotypes and response to inhaled β2 18.5% for Gly/Gly31. These differences reflect the agonists is controversial, and discordant findings need for wide-based, population studies. have been reported. Finkelstein et al.22 conducted a There was an association of Arg/Arg genotype meta-analysis to examine the association between with mild asthma when compared with ADRB2 polymorphisms and the response to inhaled ADRB2 polymorphisms in asthmatic children β2 -adrenergic agonists in children with asthma. 3. Masoli M, Fabian D, Holt S, Beasley R. The They included 3 studies9,35,36 and found a significant global burden of asthma: executive summary of the association between favorable therapeutic response GINA Dissemination Committee report. Allergy to inhaled β2-adrenergic agonists and the Arg/Arg 2004; 59:469-78. genotype. Several studies came up with the same 4. Georgy V, Fahim HI, El Gaafary M, Walters S. conclusion23-25. Prevalence and socioeconomic associations of asthma However, when examining the influence of and allergic rhinitis in northern Africa. Eur Respir J 2006; 28:756-62 ADRB2 polymorphisms on the response to long- term and repeated dosages of inhaled β2 -adrenergic 5. Bijanzadeh M, Mahesh PA, Ramachandra NB. An understanding of the genetic basis of asthma. agonist therapy, the Arg/Arg genotype has been Indian J Med Res 2011; 134:149-61. more closely associated with reduced response15-20. Moreover, in children with severe asthma Hall IP. The future of asthma. BMJ 1997; 314(7073): 45–49. exacerbations, Carroll et al.21 found that children whose genotypes were Gly/Gly had a more rapid Baren JM, Zorc JJ. Contemporary approach to the emergency department management of pediatric response to inhaled β2 -adrenergic agonists. asthma. Emerg Med Clin North Am 2002; 20:115– Five reasons may underlie the somewhat discordant results reported by different authors. 8. Insel PA. Seminars in medicine of the Beth Israel First, studies had included different patient Hospital, Boston. Adrenergic receptors-evolving populations, regarding age, disease severity, and concepts and clinical implications. N Engl J Med ethnic background. Second, authors had 1996; 334:580–5. administered different β2-agonists to study patients. 9. Silverman EK, Kwiatkowski DJ, Sylvia JS, Third, authors had used different outcome measures Lazarus R, Drazen JM, Lange C, et al. Family- and endpoints to assess drug-responsiveness. based association analysis of beta2-adrenergic Fourth, some authors had suggested that the receptor polymorphisms in the childhood asthma conflicting results among studies could be management program. J Allergy Clin Immunol 2003; explained by specific combinations of polymorphisms that are commonly inherited 10. Giubergia V, Zelazko M, Roy A, Gravina LP, together (ADRB2 haplotypes), rather than by a Gonzalez Pena H, Chertkoff L. Beta 2-adrenergic single allele polymorphism37. Finally, race is both polymorphisms and total serum IgE levels in children with asthma from Argentina. Ann Allergy Asthma a biologic and a social construct and constitutes not Immunol 2009;102(4):308–13. only genetic differences in individuals, but also the behaviors, beliefs, and experiences that vary among Qiu YY, Zhang XL, Yin KS . 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