Impact of 27gln\glu polymorphisms of β2-adrenergic receptor gene on pulmonary function in asthmatic children treated with nebulized salbutamol

Haidar A N. Abood

doi:10.4103/bbrj.bbrj_67_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 5 | Issue : 4 | Page : 435-439

Impact of 27gln\glu polymorphisms of β2-adrenergic receptor gene on pulmonary function in asthmatic children treated with nebulized salbutamol

Haidar A N. Abood
Department of Pharmacology, College of Medicine, University of Kerbala, Karbala, Iraq

Date of Submission	30-Apr-2021
Date of Acceptance	03-May-2021
Date of Web Publication	14-Dec-2021

Correspondence Address:
Haidar A N. Abood
Department of Pharmacology, College of Medicine, University of Kerbala, Karbala
Iraq

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_67_21

Abstract

Background: The β₂-adrenergic receptor gene is a typical candidate for the study of genetic polymorphism. Many patients with asthma do not respond to β2-agonist; in addition, a wide interindividual variability in pharmacological response exists, likely because of the interaction between clinical, environmental, and genetic factors. Methods: A total of 80 children (55 boys and 25 girls) with mild-moderate acute asthma attacks, ages range between 5 and 18 years, attended the Asthma Clinic at Karbala Teaching Hospital for Children were included in this study. Spirometry (Spirolab III) was used for lung function assessment before and after treatment with nebulized salbutamol, and allele-specific polymerase chain reaction was performed to determine single nucleotide polymorphisms (SNP) of β2-adrenergic receptor gene at nucleic acid 79 (27 amino acid position). Results: The effect of treatment with nebulized salbutamol on lung function in asthmatic children having different genotypes of 27Gln\Glu SNP demonstrated that patients having GG and CG genotypes showed significant improvement (P < 0.05) in forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and FEV1% predicted after treatment, while patients having CC genotype showed significant improvement (P < 0.05) in FEV1, FVC, and FEV1% predicted after treatment with nebulized salbutamol without significant improvement in FEV1/FVC ratio. Patients having CG genotype of 27Gln\Glu SNP showed highest change (increase) in FEV1 (185.38 ± 174.93 ml) and in FEV1% predicted (14.96 ± 15.45%) after treatment with nebulized salbutamol in comparison with other genotype groups although this change was not statistically significant between groups. Conclusion: Asthmatic children having CG and GG genotypes of β2-adrenergic receptor gene (27Gln\Glu) showed better improvement in lung function after treatment with nebulized salbutamol in comparison with those having CC (27Gln\Glu) genotype during mild-to-moderate acute asthma attacks.

Keywords: Acute asthma attacks, salbutamol, spirometry, β2-adrenergic receptor gene

How to cite this article:
Abood HA. Impact of 27gln\glu polymorphisms of β2-adrenergic receptor gene on pulmonary function in asthmatic children treated with nebulized salbutamol. Biomed Biotechnol Res J 2021;5:435-9

How to cite this URL:
Abood HA. Impact of 27gln\glu polymorphisms of β2-adrenergic receptor gene on pulmonary function in asthmatic children treated with nebulized salbutamol. Biomed Biotechnol Res J [serial online] 2021 [cited 2023 Mar 28];5:435-9. Available from: https://www.bmbtrj.org/text.asp?2021/5/4/435/332463

Introduction

Asthma is the most common chronic disease of childhood and has many environmental and genetic factors, resulting in airway hyperresponsiveness, obstruction, mucus hyperproduction, and airway wall morbidity.^[1] The disease influences both adults and children, with high morbid and relative little mortality as compared with other chronic disease.^[2],[3] Spirometry is the first-line test to diagnose asthma in children. The introduction of this test across all health-care settings has the ability to reduce misdiagnosis, developed asthma management and decrease the health-care spending for asthma.^[4] Forced expiration calculations may help in diagnosis and monitoring asthma and in assessing efficacy of treatment. Pulmonary function test is mainly helpful in children with asthma who are small perceivers of airflow obstruction, or when physical signs of asthma are not severe.^[5] Salbutamol is the first-line therapy of acute attack of asthma, it has a rapid onset of action (3–5 min), bronchodilation is maximum within 15–30 min and continuous for 3–4 h, so these agents considered as the drugs of choice for the acute asthma symptoms and for suppressing exercise-induced bronchoconstriction in children of all ages.^[6] The reversibility to bronchodilator drugs is usually calculated in a standardized way by first measuring lung function, usually forced expiratory volume in 1 s (FEV1), then inhaling a bronchodilator drug and then measuring FEV1 after a proper time, enabling the bronchodilator drug to have an effect on a bronchial smooth muscle.^[7] In many methods, the β₂-adrenergic receptor (ADRB2) gene is a typical candidate for the study of genetic polymorphism, the biological relevance, and clinical consequences of polymorphisms. The receptor is expressed on most of cells, and agonists are used in therapy of a number of diseases, such as asthma.^[8] The 27Gln\Glu single nucleotide polymorphisms (SNP) is encoded by a common nonsynonymous polymorphism (rs1042714) in the ADRB2 gene; at amino acid position 27, Gln or Glu can be found. The estimated frequency of the Glu isoform is 24.6% among Caucasian, 18.7% among African Americans, and 9% among Chinese.^[9] Other investigations, however, recorded no association between this SNP and different drug response^{[10],[11],[12]} whereas some groups showed contradictory outcomes.^[13],[14] Earlier studies have suggested that the Glu27 isoform does not downregulate the expression of the β2-adrenergic receptor.^[15],[16] Individuals who were homozygous for Glu27 had higher maximal response than those who were homozygous for Gln27, suggesting that the Gln to Glu change is associated with increased agonist-mediated responsiveness, and the Glu27 allele was relatively resistant to downregulation of ADRB2 during exposure to ADRB2 agonists.^[17] This study aimed to investigate the effect of β2-adrenergic receptor polymorphisms 27Gln\Glu C 79 > G (rs1042714) on the therapeutic response (assessed by pulmonary function test) to salbutamol in asthmatic children during acute attacks.

Methods

The protocol of this study was approved by the ethical committee of clinical research; College of Pharmacy, University of Kerbala No. 2651 on 17-November-2019. Approval was also taken from Karbala Health Directorate, Administration of Karbala Teaching Hospital for Children. In addition, consent was taken from parents of each patient after explaining the nature and purpose of the study.

Patients

This study included a total of 80 asthmatic children with mild-moderate acute attack of asthma (55 boys and 25 girls) attending the Asthma Clinic at Karbala Teaching Hospital for Children, Karbala, Iraq, in the period from September 2019 to the end of February 2020 with age of asthmatic children ranged between 5 and 18 years. All children had the American Thoracic Society criteria for asthma^[18] and have no other chronic illness.

Patients assessment and follow-up

Asthmatic children with acute attacks were assessed clinically and those with sign and symptoms of severe attacks were excluded from the study in addition to those who received any bronchodilator drug in the past 12 h from hospital attendance. Assessment of lung function was done by Spirometer with disposable turbine system (Spirolab III) using forced vital capacity (FVC) mode.^[19] Standard dose of nebulized salbutamol was used in this study (2.5 mg (0.5 ml of Ventolin® respiratory solution) mixed with 2 ml normal saline) and administered through jet nebulizer using face mask. The reversibility to the bronchodilator drug, salbutamol, was calculated in a standardized way by first measuring lung function parameters at baseline using FEV1, FVC, FEV1/FVC ratio, and FEV1% predicted then nebulizing the bronchodilator drug and then repeated the pulmonary function parameter after 15 min from treatment.

Genotyping

Blood samples were obtained from eligible children to extract genomic DNA using G-DEXTM IIb (intron, Korea). The polymorphism of the ADRB2 gene was detected using allele-specific polymerase chain reaction (PCR) to assess 27Gln\Glu SNP of β₂-adrenergic receptor gene at nucleic acid 79 (27 amino acid position) and the primers used were 5'-GGACCACGACGTCACGCAGC-3' (sense) and 5'-ACAATCCACACCATCAAGAAT-3' (antisense) or the same antisense primer and 5'-GGACCACGACGTCACGCAGG-3' (sense).^[20],[21] For polymorphisms, the 25-μL PCR reaction mixtures included 10 μg of genomic DNA, 0.5 μmoL of each primer (Promega), and 1X PCR mix (Taq PCR Master Mix Kit, QIAGEN), containing 200 μmol/L of each dNTP, 5 μL of 10× reaction buffer, 1.25 U of Taq Gold Polymerase, and 4 mmol/L of MgCl2. Amplification was carried out using a PTC-100 thermal cycler (Perkin-Elmer). Temperature cycling was 98°C for 30 s, 66°C to 68°C for 45 s, and 72°C for 45 s for 30 cycles. Twenty μL of the PCR mixture was then electrophoresed on 1% agarose gels and visualized with ethidium bromide staining and ultraviolet illumination. The sizes of the PCR products were 442 bps for ADRB2 27.

Statistical analysis

Data were introduced into the Statistical Package for the Social Sciences (SPSS) software version 20 (Graph Pad Software, San Diego, California, USA) to do statistical analysis. The data were expressed as mean ± standard deviation and as percent (%). Differences of data express by percent (%) were analyzed by Chi-square test in genotype groups and by Fisher's exact when cell count was <5. Differences within genotypes groups were examined by analysis of variance test. The paired t-test was used to calculate the P value at baseline and after treatment in genotype groups. All statistical procedures and tests were applied under a level of significance P < 0.05 to be considered as significant difference or correlation.

Results

The distribution of genotyping groups of asthmatic patients is shown in [Table 1] and [Figure 1]. [Table 2] shows the demographic profile of asthmatics children, and there was no significant association (P > 0.05) regarding age, weight, height, BMI, and gender between different genotypes of 27Gln\Glu SNP in asthmatic children. There was significant improvement (P < 0.05) in all recorded pulmonary function parameters (FEV1, FVC, FEV1\FVC ratio, and FEV1% predicted) for asthmatic children after treatment with nebulized salbutamol as summarized in [Table 3]. The effect of treatment with salbutamol on lung function in asthmatic children with different genotypes of 27Gln\Glu SNP is shown in [Table 4]. Patients having GG and CG genotypes showed significant increase (improvement) (P < 0.05) in FEV1, FVC, FEV1/FVC, and FEV1% predicted after treatment with nebulized salbutamol in compared with those patients having CC genotype. Patients having CC genotype were having significantly increased (improvement) (P < 0.05) in FEV1, FVC, and FEV1% predicted after treatment with nebulized salbutamol. There was no significant increase (improvement) (P > 0.05) in the ratio FEV1/FVC of this genotype. [Table 5] and [Figure 2] and [Figure 3] demonstrate that patients having CG genotype of Gln27Glu SNP showed highest change (increase) in FEV1 (185.38 ± 174.93 ml) and in FEV1% predicted (14.96% ±15.45%) after treatment with nebulized salbutamol in compared with other genotypes groups although this change is not statistically significant (P > 0.05).

Table 1: Distribution of different genotype of 27Gln\Glu SNP of ADRB2 gene in asthmatic children

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Table 2: Association of demographic characteristic between different genotypes of 27Gln\Glu SNP of ADRB2 gene

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Table 3: Effect of treatment with salbutamol on pulmonary function parameters in asthmatic children

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Table 4: Effect of different genotypes of 27Gln\Glu SNP of ADRB2 gene on pulmonary function parameters at baseline and after administration the nebulized salbutamol in asthmatic children

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Table 5: Effect of different genotypes of 27Gln\Glu SNP of ADRB2 gene on change in forced expiratory volume in 1 s from baseline after administration the nebulized salbutamol in asthmatic children

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Figure 1: Distribution of different genotype of 27Gln\Glu single nucleotide polymorphisms of β2-adrenergic receptor gene in asthmatic children

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Figure 2: Change (increase) in forced expiratory volume 1% predicted after treatment in different genotypes of 27Gln\Glu single nucleotide polymorphisms

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Figure 3: Change (increase) in forced expiratory volume 1 s after treatment in asthmatic children with different genotypes of 27Gln\Glu single nucleotide polymorphisms

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Discussion

Asthmatic children show a large heterogeneity in response to bronchodilator short-acting β₂-agonist. Rapidly evolving genotyping technologies have led to the identification of various genetic variants associated with treatment outcomes.^[22] This was the first interventional prospective study done on Iraqi asthmatic children to demonstrate the rationale behind conducting asthma pharmacogenetic studies, provide an overview of asthma pharmacogenetic phenotypes, and detail the most important results of asthma pharmacogenetic studies in Iraqi population. Regarding the distribution of ADRB2 Gene 27Gln\Glu SNP, [Table 1] and [Figure 1] show that the distribution of 27Gln\Glu genotypes in asthmatic children was 38.7% of homozygous Gln\Gln, 28.8% of homozygous Glu\Glu, and 32.5% of heterozygous Gln\Glu. These results were supported by results reported by Martinez et al. in Tucson, Arizona.^[23]

The results of this study on polymorphism of 27Gln\Glu SNP of ADRB2 demonstrated that patients having GG and CG genotypes showed significant improvement (P < 0.05) in FEV1, FVC, FEV1/FVC ratio, and FEV1% predicted after treatment with nebulized salbutamol in comparison with those having CC genotype who had significant improvement (P < 0.05) in FEV1, FVC, and FEV1% predicted after treatment with nebulized salbutamol, without significant increase improvement in FEV1/FVC ratio as shown in [Table 4]. These findings revealed that patients having heterozygous CG (Gln\Glu) are associated with higher response (improvement in lung function) to nebulized salbutamol in compared with patients having other genotypes. In line with previous studies, several studies support this finding, Ashgan Abdallah et al 2018 studied on β₂-adrenergic receptor gene polymorphism effect on childhood asthma severity and response to treatment in Egypt who investigated that asthmatic patients having CG (Gln\Glu) give a better response to drug therapy than patients having CC (Gln\Gln) and GG (Glu\Glu) genotypes.^[24] Other study reported by Verónica et al. 2008 which concluded that the homozygosity for 27Gln\Glu polymorphism was associated with a desensitization of the receptor with a decline in the bronchodilator response to albuterol after chronic use in Argentinean population.^[25]

Conclusion

Asthmatic children having CG (Gln\Glu) genotype of 27Gln\Glu SNP of β₂-adrenergic receptor gene showed better improvement in lung function after treatment with nebulized salbutamol during mild-to-moderate acute attacks in comparison with those having CC (Gln\Gln) and those having heterozygous GG (Glu\Glu).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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