ADRB2 GENE POLYMORPHISMS AND SALBUTAMOL RESPONSIVENESS IN SERBIAN CHILDREN WITH ASTHMA
Jovicic N, Babic T, Dragicevic S, Nestorovic B, Nikolic A
*Corresponding Author: Dr. Nevena Jovicic, Department of Pulmonology and Allergology, University Children’s Hospital, Tirsova 10, 11000 Belgrade, Serbia. Tel: +38-164-115-6721. Fax: +38-111-268-5378. E-mail: jovicic.nevena@gmail.com
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MATERIAL AND METHODS

Subjects. The cross section study was conducted at the University Children’s Hospital in Belgrade during the period from October 2016 to May 2017 and included 54 children of Serbian ethnicity with asthma (6-18 years old). The diagnosis of asthma and disease severity were set in accordance with the Global Initiative for Asthma (GINA) 2016 guidelines. Severity was assessed retrospectively from the level of treatment required to control symptoms and exacerbations. Subjects were classified into three subgroups: mild, moderate and severe asthma. Children with asthma who had some other illnesses that may affect lung function were excluded from the study, as well as children with any chronic disease other than asthma such as bron-chopulmonary dysplasia, tracheobronchial malacia and/or congenital heart disease. The allergic classification was defined by co-occurrence of positive prick skin tests for inhalation allergens, increased serum IgE levels, more than 4.0% eosinophils in peripheral blood in the absence of parasites (three negative stool analyzes for parasites 3 months prior to testing) and co-occurrence of atopic dermatitis as a cumulative nature and which was not present during the study period or in the previous 12 months. None of the subjects had acute exacerbation of asthma. The study was approved by the Ethics Committee of the Faculty of Medicine University of Belgrade (decision No: 29/III-30, 28/3/2016). Detailed anamnesis, physical examination and lung function tests were performed in all subjects. Pulmonary function tests were performed using a spirometric unit (Ganshorn-Schiller SpiroJet 140091; Ganshorn Medizin Electronic GmbH, Niederlauer, Germany). Spirometry was performed according to the standards of the American Thoracic Society [13]. Spirometric measurements included forced expiratory volume in the first second (FEV1), forced vital capacity (FVC) and peak expiratory flow (PEF). The results of pulmonary function tests were expressed as a percentage of predicted values. Children with asthma received an instruction to stop the systemic bron-chodilator or corticosteroid therapy for 72 hours prior to testing, as well as the use of short-acting β2-agonists 12 hours prior to testing. Response to a short-acting bronchodilator was assessed by applying a single dose of salbutamol (0.15 mg/kg) using the Omron Nebuliser (NE-C28P-E; Omron Healthcare Group, Kyoto, Japan), and by performing the lung function test before and 15 min. after administration of nebulized salbutamol. The response to salbutamol was measured by recording a change in the percentage of FEV1 obtained before and after salbutamol administration [13]. Genotyping of ADRB2 +46G>A and +79C>G Polymorphisms/ Variants. Genomic DNA was extracted from peripheral blood using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA). The presence of +46A>G and +79C>G polymorphisms/variants was determined by direct sequencing of polymerase chain reaction (PCR) products obtained with the following primers: 5’-CTG AAT GAG GCT TCC AGG CGT-3’ and 5’-ACA ATC CAC ACC ATC AGA AT-3’. The PCR was conducted in a 50 μL reaction mixture containing: 1 × KAPA Taq Buffer A (KAPA Biosystems, Waltham, MA, USA), 0.3 mM MgCl2, 0.2 mM each dNTP, 10 pmol of each primer, 2U of KAPA Taq DNA Polymerase (KAPA Biosystems) and approximately 300 ng of DNA. The amplifications were performed as follows: initial denaturation for 5 min. at 94 °C; 35 cycles consisting of 30 seconds at 94 °C, 30 seconds at 60 °C and 30 seconds at 72 °C; final extension for 10 min. at 72 °C. The obtained PCR fragments (584 bp long) were purified with PureLink PCR Purification Kit (Thermo Fisher Scientific) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and the same primers as for the amplification. Products of sequencing reactions were analyzed by capillary electrophoresis on an ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and sequencing analysis software (Applied Biosystems). Statistical Analyses. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as percentages and means ± standard deviation (SD) for continuous variables and percentages for categorical variables. To test the normality of the parameters, one sample Kolmogorov-Smirnov test was used. Differences between groups for categorical data were tested by χ2 analysis, while for continuous data Independent Samples Mann-Whitney U test and the Kruskal-Wallis test were used. Hardy-Weinberg equilibrium was analyzed by the Arlequin software. A p value of less than 0.05 was considered statistically significant.



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