ORIGINAL ARTICLE
Polymorphisms of the
b
2
-Adrenergic Receptor
Correlated to Nocturnal Asthma and the
Response of Terbutaline Nebulizer
Ming-Yung Lee
a, Shin-Nan Cheng
a, Shyi-Jou Chen
a, Hui-Ling Huang
b,c,
Chih-Chien Wang
a, Hueng-Chuen Fan
a,*
aDepartment of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan b
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
cInstitute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan
Received Mar 11, 2010; received in revised form May 6, 2010; accepted May 12, 2010
Key Words b2-adrenergic receptor; nocturnal asthma; nonnocturnal asthma; polymorphism; terbutaline
Background: Inhaledb2-adrenergic receptor (b2-AR) agonists are the mainstay of treatment of
acute asthma. Polymorphisms of theb2-AR, especially codons 16, 27, and 164, may affect the
functions of the receptor. This study was conducted to investigate whether different poly-morphisms of theb2-AR are related to the treatment responses of an inhaledb2-AR agonist
in children with nocturnal and nonnocturnal asthma in Taiwan.
Methods: The nocturnal asthma group consisted of 27 children (mean age of 10.3 2.4 years), and the nonnocturnal asthma group consisted of 24 patients (mean age of 9.9 3.0 years). Allele-specific polymerase chain reaction was performed to determine 16, 27, and 164 loci alleles ofb2-AR genetic polymorphisms, and peak expiratory flow (PEF) was measured before
and 1 hour after inhalation of 0.2 mg/kg/dose of terbutaline to determine the treatment response in these patients.
Results: The polymorphisms ofb2-AR 27 but not 16 or 164 were significantly associated with
the response to terbutaline nebulizer (p< 0.05). The polymorphism of b2-AR 16 was associated
with nocturnal asthma (pZ 0.027). The Gly16 allele was more prevalent in the nocturnal asthma group (9/27; 33.3%) than in the nonnocturnal asthma group (3/24; 12.5%). Arg16 allele was less prevalent in the nocturnal asthma (3/27; 11.1%) than in the nonnocturnal asthma group (10/24; 41.7%). There was also a linkage disequilibrium found betweenb2-AR 16 (Arg/
Arg) andb2-AR 27 (Gln/Gln).
* Corresponding author. Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Neihu, Taipei 114, Taiwan.
E-mail address:fanhuengchuen@yahoo.com.tw(H.-C. Fan).
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : h t t p : / / w w w . p e d i a t r - n e o n a t o l . c o m
1875-9572/$36 Copyrightª 2011, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.pedneo.2010.12.011
Conclusion: These findings suggest that polymorphisms ofb2-AR 16 are related to nocturnal
asthma and polymorphisms ofb2-AR 27 are associated with the variable responses to the
inhaled terbutaline in children with nocturnal and nonnocturnal asthma.
Copyrightª 2011, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.
1. Introduction
The caliber of the human airway is not a constant. It may increase during the day and decrease at night.1Evidence shows that this circadian fluctuation in the caliber of both upper and lower airways is amplified in disease states, such as asthma.1 This is one of reasons why most in-hospital sudden deaths and episodes of ventilator arrest from asthma occur at night. Nocturnal asthma, a unique subset of patients with asthma, is of particular interest because patients with this disease show that their caliber of the airways decreases and causes peak dyspnea and wheezing between 2 and 6 AM.2 Moreover, the disturbance of sleep
because of nocturnal asthma may impair performance during the day. Therefore, it will be helpful if patients with nocturnal asthma can be early identified and receive proper treatment.
Although inhaled b2-adrenergic agonists are the
main-stay of treatment of acute asthma, it is still not clear why patients with asthma show various responses to the same inhaledb2 agonists. After the b2-adrenergic receptor (b2
-AR) gene was cloned in 1987,3 the role of theb2-AR gene
in determining disease response and disease severity is now reasonably well accepted.4 The gene encoding this
G-protein-coupled b2-AR is located on the chromosome
5q31-33 and is highly polymorphic. To date, nine distinct polymorphisms in the b2-AR gene have been reported.5
Each of these polymorphisms represents a single base pair substitution. Four of these polymorphisms result in amino acid substitutions at amino acids 16, 27, 34, and 164, whereas the other five are silent mutations located at amino acids 84, 175, 351, 366, and 413.6A question here is whether these polymorphisms might explain altered phar-macologic responses to b2-AR agonist treatment. Indeed,
studies have suggested that these polymorphisms may be associated with asthma of different severity.7,8 Three of these polymorphisms have been found to alter the receptor function by site-directed mutagenesis and recombinant expression studies,8e10including substitutions of glycine for arginine at amino acid position 16 (Arg16/ Gly16), gluta-mic acid for glutamine at position 27 (Gln27/ Glu27), and isoleucine for threonine at position 164 (Thr164/ Ile164). Some studies suggest that the Gly16 allele showed enhanced downregulation of b2-AR, whereas the Glu27
allele was relatively resistant to downregulation ofb2-AR
during exposure to b2-AR agonists. 8,9
The Ile164 variant markedly altered ligand binding and coupling proper-ties.9,10 All these in vitro findings are consistent with the concept that a defective b2-AR may be a primary causal
abnormality in asthma.
Using asthmatic children, we want to know the differ-ence betweenb2-AR polymorphisms at amino acid positions
16, 27, and 164 in Taiwanese children with nocturnal and
nonnocturnal asthma. Moreover, to investigate the responses of patients with nocturnal and nonnocturnal asthma in the treatment with an inhaled adrenergic agonist, we used terbutaline, a selectiveb2-AR stimulator,
which increases the diameter of the airway via relaxation of bronchial smooth muscle within few minutes.11 The
polymorphisms and changes of the peak expiratory flow rate (PEFR) before and after inhaled terbutaline treatment were correlated with when the children required the use of bronchodilator in acute asthma.
2. Materials and Methods
2.1. Study participants
Children with a diagnosis of asthma for at least 1 year attending our emergency department were enrolled for the study. Asthma was defined using the criteria of the American Thoracic Society.12 Exclusion criteria included use of oral or inhaled steroids, cromolyn, antibiotics, or any investigational drug within 2 weeks, moderate to severe asthma exacerbations or upper respiratory tract infection within 2 weeks, and presence of other lung or cardiac diseases as the cause of patient symptoms before this study. Home peak expiratory flow monitoring was per-formed. PEFRs were measured by using a peak flow meter (Mini-Wright; Armstrong Industries, Northbrook, IL, USA). Patients were separated into those with nocturnal asthma or those with nonnocturnal asthma. Nocturnal asthma was defined as a documented fall in PEFR of>20% on at least four of seven nights of testing at home13or on the history of early morning awakening, dyspnea, wheezing, and cough occurs between 2 and 6 AM on 3 consecutive days. The
patients with nonnocturnal asthma were those with asthma attacks beyond the range of 2e6AM. Informed consent was
obtained from the parents in each case. Peak flow meter was performed before and 1 hour after inhalation of 0.2 mg/kg/dose of terbutaline (Brincanyl; Astra-Zeneca, London, UK) in a 3-mL isotonic sodium chloride solution administered with a handheld disposable updraft nebulizer (whisper Jet nebulizer; Intec, Marquest Medical products, Englewood, CO, USA).14 PEFR0 was the PEFR before the
treatment with terbutaline nebulizer when the patients were sent to our pediatric emergency room for asthma attack. PEFR1 was defined as the PEFR 1 hour after
inha-lation of terbutaline.ΔPEFR was the percentage change of PEFR0to PEFR1.
2.2. Genotyping ofb2-AR polymorphism
Genomic DNA from peripheral whole blood was prepared by standard phenol/chloroform extraction procedures.15
Polymorphisms of theb2-AR coding block were delineated
using an allele-specific polymerase chain reaction (PCR) approach.8 Allele-specific PCR was performed to assess polymorphisms at nucleic acids 46, 79, and 491, which result in changes in the encoded amino acids at positions 16, 27, and 164 of the receptor protein. The genotypes are abbreviated as Arg16, Gly16, Gln27, Glu27, Thr164, and Ile164. Genomic DNA was isolated from 2 mL of peripheral blood. PCRs were carried out in a volume of 50mL using 100 ng of genomic DNA. To delineate the two poly-morphisms at nucleic acid 46, the primer pairs used were 50-CTTCTTGCTGGCACCCAATA-30 (sense) and 50-CCAATT TAGGAGGATGTAAACTTC-30 (antisense) or the same anti-sense primer and 50-CTTCTTGCTGGCACCCAATC-30(sense). The generated PCR product size using these primers was 913 base pairs (bp). The primer pair for delineating the two polymorphisms at nucleic acid 79 was 50-GGACCACGACGT CACGCAGC-30(sense) and 50-ACAATCCACACCATCAAGAAT-30 (antisense) or the same antisense primer and 50-GGAC CACGACGTCACGCAGG-3 (sense). Use of these primers resulted in a product with a molecular size of 442 bp. For the detection of polymorphisms at nucleic acid 491, the primer pair used was 50-TGGATTGTGTCAGGCCTTAT-30 (sense) and 50-CACAGCAGTTTATTTTCTTT-30 (antisense) or the same antisense primer and 50-TGGATTGTGTCAGGCCT TAC-30 (sense). The PCR product size from these primers was 662 bp. In general, a 0.1mg (2 mL) of DNA template was added to 50mL of reaction mixtures containing 0.5 mL of Takara Taq DNA polymerase (Takara Shuzo Co.; Kyoto, Japan), 1mL of each primer, 1 mL of dinucleoside 50 -triphosphate, 5mL of 10 PCR reaction buffer, and 39.5 mL of deionized water. The reaction consisted of an initial denaturation at 94C for 5 minutes; followed by denatur-ation at 94C for 2 minutes, 55C for 1 minute, and 72C for 1 minute for 35 cycles; and a final extension of 10 minutes at 72C (DNA Thermal Cycler; Perkin-Elmer Co., Norwalk, CT, USA). The results of electrophoresis of the PCR products could effectively differentiate the polymorphisms in these three alleles. The allele-specific PCR technique was verified by direct dideoxy sequencing (PE Applied Biosystems, Foster City, CA, USA) of PCR products that were generated using sequencing primers that were different from those used in the PCR.
2.3. Statistical analysis
Demographic data, including age, sex, and initial PEFR, were recorded and analyzed. The patients’ ages and pulmonary function test data were expressed as mean standard devi-ation. The association of theb2-AR polymorphisms genotype
between nocturnal asthma and nonnocturnal asthma patients was examined by thec2 and one-way analysis of variance
tests (SPSS 16.0, SPSS Inc., Chicago, IL, USA). The p values <0.05 were considered statistically significant.
3. Results
3.1. Demographic data
There were a total of 51 participants in our study, 16 male and 35 female. Mean age of the participants was 10.1 2.6 years. The nocturnal asthma group consisted of 27 patients (mean age of 10.3 2.4 years; 8 male and 19 female), and the nonnocturnal asthma group consisted of 24 patients (mean age of 9.9 3.0 years; 8 male and 16 female). No patients regularly used inhaled bronchodilator. Mean PEFR0,
PEFR1, and ΔPEFR in the nocturnal asthma group were
204.8 42.5%, 232.2 46.2%, 14.0 10.9%, respectively, whereas those in nonnocturnal asthma group were 185.6 51.9%, 222.5 58.6%, 9.6 11.9%, respectively (Table 1). There were no significant differences in the demographic data and initial PEFR between the nocturnal and the nonnocturnal asthma groups.
3.2. Response to treatment with terbutaline nebulizer according to the polymorphisms ofb2-AR
The allele frequencies found in this study population were 0.49 for Gly16, 0.51 for Arg16, 0.41 for Gln27, 0.59 for Glu27, 0.5 for Thr164, and 0.5 for Ile164. When b2-AR
polymorphisms at amino acid positions 16, 27, and 164 were correlated with meanΔPEFR, the polymorphisms of b2-AR 27
were significantly associated with meanΔPEFR (homozygous Gln/Gln vs. heterozygous Gln/Glu, pZ 0.012; homozygous Gln/Gln vs. homozygous Glu/Glu, pZ 0.001). There was no correlation found between the polymorphisms ofb2-AR 16
or 164 and theΔPEFR after terbutaline treatment (Table 2).
3.3. Correlation of genotypes with nocturnal and nonnocturnal asthma
The distribution of Arg-Gly16, Gln-Glu27, and Thr-Ile164 of b2-AR polymorphisms with nocturnal asthma or
non-nocturnal asthma patients is shown in Table 3. The poly-morphisms at position 27 (Gln or Glu) and position 164 (Thr or Ile) were not different between both groups. A linkage disequilibrium was found in participants carrying Arg16, who also carried Gln27 (84.6%) (Table 4).
4. Discussion
Asthma is a polygenic disease, but so far no clear genotype-phenotype relationships have emerged. Although evidence
Table 1 Demographic data of nocturnal and nonnocturnal asthma groups
Classification of asthma Sex (M/F) Age (yr) PEFR0(L/min) PEFR1(L/min) D PEFR (%)
Nonnocturnal asthma 8/16 10.3 2.4 204.8 42.5 232.2 46.2 14.0 10.9 Nocturnal asthma 8/19 9.9 3.0 185.6 51.9 222.5 58.6 9.6 11.9
Values given as mean standard deviation.
supports that polymorphisms of the human b2-AR are a
critical determinant of functions of this receptor in vitro,10
there are still inconsistencies between in vitro findings and asthma phenotypes. Therefore, it is of interest to investi-gate the associations between polymorphisms ofb2-AR and
nocturnal asthma and clarify whether the variant b2-AR
receptors may affect asthmatic patients’ response to inhaled b2-AR agonists. Because the analysis of
poly-morphisms of b2-AR from peripheral leukocyte can be
surrogate forb2-AR expressed on bronchial smooth muscle
and other cell types relevant to asthma,15DNA of
periph-eral blood from 51 asthmatic children was analyzed by using an allele-specific PCR approach.8 Our results show
that the allele frequencies found in this study were 0.49 for Gly16, which was compatible with the data of Asian chil-dren with asthma (0.4 for Gly16).16The allele frequencies were 0.51 for Arg16, 0.41 for Gln27, 0.59 for Glu27, 0.5 for Thr164, and 0.5 for Ile164. The difference between our data and those of other studies6,8,16,17may be because of variability in allele frequencies ofb2-AR in different ethnic
populations, which has yet to be elucidated.
Airway hyperresponsiveness to a variety of stimuli is one of the essential components of the manifestations of
asthma. Interestingly, individuals with Gln27 may have augmented airway hyperresponsiveness to endogenous catecholamines, resulting in increased airway sensitivity to proinflammatory stimuli and leading to some extent of long-term airway inflammation.7Children in Argentina with homozygous Gln27 developed significantly greater bron-chodilator desensitization tob2-AR agonist than those with
homozygous Glu27.18 In addition, Gln27 has been also reported to be associated with an elevated immunoglobulin E level6and may be more susceptible to inflammation and bronchoconstriction. Another study shows that the poly-morphism ofb2-AR 27 has a linkage disequilibrium in a locus
related to the control of immunoglobulin E levels nearby on chromosome 5q,19 which contains a number of cytokine genes important in the generation of Type 2 T helper cell responses, including those for interleukin (IL)-4, IL-5, IL-9, and IL-13.20 In contrast, Glu27 has been reported to be related to a lower degree of airway reactivity.21
Site-directed mutagenesis experiments have shown that although agonist binding and coupling to adenylyl cyclase is intact in the Glu27b2-AR polymorphism, it was associated
with impaired agonist downregulation.6,10In our study, the
polymorphism ofb2-AR 27, although not related to individuals
Table 2 Response to treatment with terbutaline nebulizer according to polymorphisms ofb2-AR
Genotype ΔPEFR (%) p
b2-AR 16 Homozygous Arg/Arg 12.2 12.1
Heterozygous Arg/Gly 13.1 9.4 NS Homozygous Gly/Gly 8.0 14.9 b2-AR 27 Homozygous Gln/Gln 8.2 9.4 Heterozygous Gln/Glu 10.7 9.0 <0.05 Homozygous Glu/Glu 23.4 14.4 b2-AR 164 Homozygous Thr/Thr 3.8 11.6 Heterozygous Thr/Ile 12.3 11.3 NS Homozygous Ile/Ile 12.1 14.2
ΔPEFR is the percentage change of PEFR before and after 1 hour treatment with terbutaline nebulizer. Values given as mean standard deviation.
NSZ not significant; PEFR Z peak expiratory flow rate.
Table 3 Association between the frequency of genotypes of theb2-AR polymorphism and asthma groups
Genotypes Frequency p
Nocturnal asthma, % (nZ 27)
Nonnocturnal asthma, % (nZ 24)
b2-AR 16 Homozygous Arg/Arg 11.1 41.7
Heterozygous Arg/Gly 55.6 45.8 0.027 Homozygous Gly/Gly 33.3 12.5 b2-AR 27 Homozygous Gln/Gln 48.1 54.2 Heterozygous Gln/Glu 40.8 10.8 NS Homozygous Glu/Glu 11.1 25 b2-AR 164 Homozygous Thr/Thr 11.1 4.2 Heterozygous Thr/Ile 81.5 87.5 NS Homozygous Ile/Ile 7.4 8.3
Values given as percentages.
with or without nocturnal asthma, showed a significant association with the meanΔPEFR (Table 3). Individuals with homozygous Glu27 had a higher increase of ΔPEFR than those with homozygous Gln27. Therefore, patients with b2-AR Glu27 may have a more augmented response to
ter-butaline inhalation than those with b2-AR Gln27 in this
study. We suggest that Glu27 may play a role in assistingb2
-AR to respond to an inhaled terbutaline in patients with asthma.
Recent clinical reports provide evidence that b2-AR
polymorphisms at position 16 have an important role. A cross-sectional survey revealed that possessing with homozygous Arg16 may predispose children and young adults to asthma exacerbation.22Another group found that
PEFR was reduced in patients with homozygous Arg16.23,24 A Chinese group reported that they detected 72% homozy-gous Gly16 in patients with nocturnal asthma.25In addition, a case report also supports the concept that homozygous Arg16 was connected to asthma in one patient with asthma in an uncontrolled situation.26 However, Turki et al8 reported that Gly16 allele appeared more frequently among pediatric patients with nocturnal asthma than among patients with nonnocturnal asthma, and Gly16 ofb2
-AR was significantly associated with nocturnal falls in PEFR in individuals with asthma. Reihsaus et al5concluded that
the Gly16 was associated with the more severe form of asthma, and patients with Gly polymorphism may correlate with a lack of benefit fromb2-AR agonist therapies, thereby
necessitating the use of corticosteroids and/or immuno-therapy. Cell study shows that Gly16 is associated with increased agonist-promoted downregulation of the b2-AR
compared with Arg16.9,10 These results imply that when
compared with patients without this genotype, asthmatic individuals with homozygous Gly16 may be more likely to show less improvement after treatment with short acting b2-AR agonist, even leading to respiratory failure and death
after inhalation of bronchodilators. This enhanced agonist-mediatedb2AR downregulation may also contribute to the
pathogenesis of nocturnal asthma, especially in patients with Gly16 polymorphism.6 Our data show that the Gly16 allele was more prevalent in the individuals with nocturnal asthma than in those with nonnocturnal asthma. Further-more, a strong linkage disequilibrium between the two polymorphisms in patients, with the Arg16 allele having 84.6% of individuals carrying the Gln27 allele. In fact, as in previous reports, above 90% of all chromosomes carrying Arg16 also carried Gln27.23,27We think that the amino acid substitution at codons 16 and 27 of theb2-AR sequence may
result in conformational changes, which may possibly affect the function of b2-AR and the incidence of nocturnal
asthma, and that more studies, such as knock-in variant codons ofb2-AR in vivo may answer these questions.
Amino acid 164 is the third most frequent b2-AR
poly-morphism. This location may be important because it is within the fourth transmembrane domain and adjacent to serine 165, which has been proposed to interact with the b-carbon hydroxyl group of adrenergic ligand.28Site-directed
mutagenesis studies show that the Ile164 protein displays significant dysfunction, with a 50% decrease in maximal stimulation of adenylyl cyclase compared with the wild-type receptor. In addition, the Ile164 form displayed decreased affinities forb-agonists. Therefore, it has been suggested that the Ile164 form may be associated with diminished responsiveness to b-agonists.28 Our data show
that there was no statistically significant association between 164 b2-AR polymorphisms and nocturnal asthma
and the change of PEFR. We think that 164 b2-AR
poly-morphisms may play a minor role in the nocturnal asthma in our study.
In conclusion, the distribution ofb2-AR polymorphisms is
variable among different ethnic groups. Based on our findings, these polymorphisms may be related to the phenotypic modulation of nocturnal asthma and in the determination of the treatment responses of b2-AR
agonists. We suggest that the specific polymorphisms ofb2
-AR may distribute in the particular phenotype of asthma in Taiwanese children. The different genotypes ofb2-AR may
affect the responses to extrinsic or intrinsicb2-AR agonists.
A large-scale prospective study assessing the effects ofb2
-AR haplotypes on the response to chronic use of short- and long-acting b2-AR agonists is required in the design of
clinical trials involving a new generationb2-AR agonists.
Acknowledgments
The authors would like to express their deepest gratitude to the Tri-Service General Hospital for the grant TSGH-C98-108, which fully supported this work.
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Polymorphism 16 Polymorphism 27
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