Association between beta-2 adrenergic receptor variants and clinical outcomes in children and adolescents with acute asthma

ABSTRACT Objective To investigate whether different genotypes of p.Arg16Gly, p.Gln27Glu, p.Arg19Cys and p.Thr164Ile variants interfere in response to treatment in children and adolescents with moderate to severe acute asthma. Methods This sample comprised patients aged 2 to 17 years with a history of at least two wheezing episodes and current moderate to severe asthma exacerbation. All patients received multiple doses of albuterol and ipratropium bromide delivered via pressurized metered-dose inhaler with holding chamber and systemic corticosteroids. Hospital admission was defined as the primary outcome. Secondary outcomes were changes in forced expiratory volume in the first second after 1 hour of treatment, and for outpatients, length of stay in the emergency room. Variants were genotyped by sequencing. Results A total of 60 patients were evaluated. Hospital admission rates were significantly higher in carriers of the genotype AA relative to those with genotype AG or GG, within the p.Arg16Gly variant (p=0.03, test χ2, alpha=0.05). Secondary outcomes did not differ between genotypes. Conclusion Hospital admission rates were significantly higher among carriers of the genotype AA within the p.Arg16Gly variant. Trial registration: ClinicalTrials.gov: NCT01323010


❚ INTRODUCTION
Asthma is a highly prevalent condition in childhood. Asthma exacerbation is a common cause of emergency visit and, hospital admission, school absenteeism and loss of quality of life in children. (1)(2)(3) Asthma attacks may also have negative impacts on asthma-related lung function. Maintenance treatment aimed to prevent exacerbations and appropriate management of these attacks are vital to minimize lung function compromise. (4) Asthma is a heterogeneous condition. Wide variations regarding age of onset, presence or absence of atopy, severity and reversibility of airway obstruction, among other factors, have been reported. Hence, associations between phenotype and endotype-related factors and different clinical outcomes, including the occurrence and severity of exacerbations, have been increasingly reported. (1)(2)(3) Short-acting bronchodilators are the cornerstone of asthma attack treatment. In mild attacks, a few doses given at home are enough to relieve symptoms. However, moderate to severe attacks require multiple doses at einstein (São Paulo). 2022;20:1-7 short intervals, especially in the first hour of treatment (usually, every 20 minutes,). (1)(2)(3)5,6) The efficacy and safety of these drugs -particularly albuterol-when delivered via pressurized metered-dose inhalers with holding chambers (with mask or mouthpiece) have been demonstrated in several scientific studies. (1)(2)(3)5,6) Therefore, the role of beta-2 adrenergic receptors (the target of bronchodilators) and the respective codifying gene (ADRB2) must be understood. (7,8) The beta-2 adrenergic receptor is a member of a family of seven transmembrane receptors. This receptor contains 413 amino acids, with a molecular weight of approximately 46.500 daltons. Type 1 and type 3 beta adrenergic receptors are found in the cardiac muscle and fat tissue, respectively, whereas type 2 receptors are found primarily in airway smooth muscles and other lung cells, such as mast cells, epithelial cells, endothelial cells and type II pneumocytes. These receptors tend to be more abundant the higher the airway generation, and are present in particularly high numbers in small airways. (9) ADRB2 has 1,242 base pairs and is located in the long arm of chromosome 5 (5q 31-32). More than 80 variants of the ADRB2 gene have been described. (7,8,(10)(11)(12) Single nucleotide variants such as c.46A>G, p.Arg16Gly (rs1042713), c.79C>G, p.Gln27Glu (rs1042714) and c.-47C-T, p.Arg19Cys (rs1042711) may interfere with the downregulation of beta-2 receptor expression. In turn, the c.491C>T, p.Thr164Ile (rs1800888) variant is associated with reduced binding affinity for bronchodilators. (13)(14)(15) Evidence suggested all four variants interfere with responses to bronchodilators in terms of lung function and other clinical outcomes, such as hospital admission and length of stay at intensive care unit (ICU), among others. (7,8,11,(16)(17)(18) ❚ OBJECTIVE To investigate whether specific genotypes of the p.Arg16Gly, p.Gln27Glu, p.Arg19Cys and p.Thr164Ile variants interfere in response to treatment in children and adolescents with moderate to severe acute asthma.

❚ METHODS
Variants c.46A>G, p.Arg16Gly (rs1042713); c.79C>G, p.Gln27Glu (rs1042714); c.491C>T, p.Thr164Ile (rs1800888) and c.-47C-T, p.Arg19Cys (rs1042711) were investigated. (19) Data of patients in this sample were extracted from a prior study investigating the efficacy and safety of increasing doses of albuterol in children with acute asthma. (5)  Patients aged 2 to 17 years with a history of at least two wheezing episodes and current moderate to severe exacerbation according to the Pediatric Respiratory Assessment Measure (PRAM score ≥5) were included. (20) Exclusion criteria were as follows: preexisting chronic disease, initial clinical presentation requiring immediate ventilatory support or subcutaneous or intravenous administration of bronchodilators in the first hour of treatment, decreased level of consciousness and use of beta-agonists 4 hours or systemic corticosteroids 24 hours prior to arrival.
Patients received multiple doses of albuterol and ipratropium bromide delivered via pressurized metereddose inhalers with holding chambers. A minimum of three doses of albuterol and ipratropium were given within the first hour, followed by subsequent doses as needed. Nominal doses per treatment varied according to body weight and group (study or control) to which patients were allocated in the original study that gave rise to this trial (data published elsewhere). (5) Patients received systemic corticosteroids (2mg/kg of prednisolone or methylprednisolone; maximum dose, 60mg) and were reassessed within 1 hour, then every 30 minutes. Patients who failed to meet hospital discharge criteria (PRAM ≤3 and pulse oximetry ≥92%) (5) were hospitalized.
Need of hospital admission was defined as the primary outcome. Secondary outcomes were changes in forced expiratory volume in the first second (FEV 1 , measured at baseline and after 1 hour of treatment) and length of stay in the emergency room (ER) (in the case of outpatients).
Guardians and subjects aged over 12 years signed an informed consent term. This protocol was approved by the einstein (São Paulo). 2022;20:1-7 Ethics Committee of institutions involved. This study was registered on the Clinical Trials platform (NCT01323010) https://clinicaltrials.gov/ct2/show/NCT01323010 and conducted in compliance with Helsinki Declaration principles.

Statistical analysis
Qualitative variables were analyzed using the test χ 2 or the Fisher's exact test. The adherence test χ 2 was used to compare the frequency of selected alleles in the study and the general population. Normally and asymmetrically distributed quantitative variables were analyzed using the t test for mean comparisons and the Kruskal-Wallis test, respectively. Repeated measures analysis of variance (ANOVA) was used whenever there were multiple measures over time. The level of significance was set at 5% (alpha=0.05). Statistical analyses were performed using SPSS, version 20.0 (IBM, Amonk, NY, United States).
In the protocol that gave rise to this study, a sample size of 124 patients was initially estimated to detect a difference of 30 minutes in length of stay in the emergency room between patients in the study and the Control Group, with a statistical power of 80% (data published elsewhere). (5) ❚ RESULTS Genotyping was carried out in 60 out of 119 patients in this sample. In remaining patients, extraction attempts failed to yield viable DNA for ADRB2 gene sequencing. Demographic characteristics of patients included in this study are shown in table 1.   The distribution of variant p.Arg16Gly was relatively uniform among three genotypes. In contrast, genotypes CC, TT and CC were clearly predominant in variants p.Gln27Glu, p.Arg19Cys and p.Thr164Ile, respectively. The frequency of variant p.Arg16Gly, p.Gln27Glu and p.Arg19Cys alleles differed between the study and the general population (p<0.05 in all three cases), whereas the frequency of variant p.Thr164Ile alleles was similar (p=0.09). This may have been due to the small sample size or the fact that this study was based on a selected sample (patients aged 2 to 17 years with moderate to severe acute asthma) with high levels of miscegenation, among other reasons.
No other variants were found. Ethnic characterization of patients according to different genotypes of each variant is shown in table 3.
For the four variants investigated in this study, the majority of patients were mixed. Ethnicity was not associated with different genotypes of the same variant.
Increase in FEV 1 after 1 hour and ER length of stay did not differ according to genotype for ther different variants studied (comparison of different genotypes within variants).
However, hospital admission rates differed significantly (p=0.03; χ 2 test) between genotypes whitin the p.Arg16Gly variant. Of nine hospitalized patients, seven were homozygous for the allele corresponding to arginine in position 16 (genotype AA), one was heterozygous (genotype AG) and one was homozygous for the allele corresponding to glycine (genotype GG). Hospital admission rates did not differ between genotypes for the other variants. einstein (São Paulo). 2022;20:1-7 ❚ DISCUSSION In this study, the AA genotype (p.Arg16Gly) was clearly associated with higher hospitalization rates. In spite of almost equal distribution across the three genotypes, seven out of nine hospitalized patients were homozygous for the arginine allele. Different findings have been reported by Martinez et al., in a large longitudinal asthma study investigating responses to a single dose of albuterol in children. In that study, the odds of positive response to the bronchodilator, in the case of p.Arg16Gly variant, were 5.3-fold higher in patients with the AA genotype, and 2.3-fold higher in heterozygous individuals, relative to those with the GG genotype. (11) Studies investigating associations between ADRB2 gene variants and response to bronchodilators or disease severity in patients suffering from asthma and chronic obstructive pulmonary disease yielded conflicting results in different populations. (7,8,(22)(23)(24) In a national study conducted by de Paiva et al., in the matter of the p.Arg16Gly variant, who were homozygous for the arginine allele had a higher risk of asthma development. However, this variant alone was not associated with asthma severity. (25) In contrast, in an investigation carried out by Scichilone et al., with 84 asthma patients, the frequency of genotype AA of this same variant was significantly higher in patients with severe relative to patients with mild or moderate asthma (55% and 22%, respectively). (26) In other studies, genotype AA (p.Arg16Gly) was associated with poor chronic obstructive pulmonary disease control and greater need of antimicrobial treatment due to exacerbations. (10) Genotype AA (p.Arg16Gly) may also be associated with higher risk of acute attacks, particular in regular users of long-acting bronchodilators. (27,28) Along the same lines, Turner et al. conducted a meta-analysis of data from five crosssectional studies reporting on ADRB2 genotyping, type of treatment and exacerbations in 4,226 children. In that analysis, the odds ratio of exacerbation was 1.52 (95% confidence interval -95%CI -1.17-1.99; p=0.0021) for each copy of the allele A, in 624 children treated with inhaled corticosteroids and long-acting bronchodilators in the case of the variant p.Arg16Gly. (29) Finally, in a similar investigation of patients with acute asthma, Carrol et al. reported significantly longer in-hospital stay (including ICU) in patients with severe asthma who were genotype AA or AG carriers relative to genotype GG carriers (p.Arg16Gly). (18) Findings of this study are in keeping with those of prior investigations associating the genotype AA (p.Arg16Gly) with greater asthma severity or risk of exacerbation. Allele A (p.Arg16Gly) may also be related to poorer response to bronchodilators in patients exposed to multiple doses of such drugs, as observed in this study. (18) Given only patients with moderate to severe acute asthma treated with multiple doses of albuterol were included, higher hospital admission rates among genotype AA carriers (p.Arg16Gly) in this sample is expected.
Different allele frequencies between the study and the general population do not invalidate findings presented, since outcomes were compared between genotypes within each variant.
Conflicting results regarding the effect of ADRB2 variants across different studies may be related to several factors, such as different therapeutic approaches to asthma at different locations, and use of different criteria for asthma diagnosis and allocation of subjects to referral services, which lead to non-random allocation of patients with different degrees of asthma severity, among others. (25) The frequency of ADRB2 variants varies according to the ethnic composition of different populations. Given the high degree of miscegenation in this sample, more studies are warranted not only to better characterize the frequency of these different variants, but also to investigate association with different factors, such as asthma severity, response to bronchodilators and higher risk of unfavorable clinical outcomes in cases of acute asthma (hospital or ICU admission, need of mechanical ventilation, etc.). Tools capable of providing a deeper understanding of phenotypic and genotypic aspects of asthma patients will certainly contribute to personalized treatment and optimization of strategies aimed to prevent unfavorable outcomes. Short-acting bronchodilators are not the only therapeutic alternative for management of acute asthma. Should identification of patients with poor response to bronchodilators be possible, other effective adjuvant medications could be more often indicated. The following examples can be given: anticholinergic agents (i.e., inhaled ipratropium bromide), early administration of systemic corticosteroids and intravenous administration magnesium sulfate in more severe cases. (1)(2)(3)(30)(31)(32) Other alternatives, such as use of intravenous bronchodilators, theophylline and helium-oxygen mixture, are controversial. In contrast, use of inhaled magnesium sulfate is being increasingly reported, with a good safety profile. (33)(34)(35) Finally, regardless of genetic profile, the significance of appropriate therapeutic maintenance of asthma patients, wich basis is the use of inhaled corticosteroids alone or combined with long-acting bronchodilators, must be emphasized. Excessive, isolated use of einstein (São Paulo). 2022;20:1-7 bronchodilators is a well-established risk factor for asthma exacerbation. (1) Some limitations of this study must be acknowledged, especially regarding sample size. Larger samples would certainly enable better characterization of the prevalence of different genotypes of ADRB2 variants in pediatric patients with acute asthma. A large proportion of patients in this sample was aged less than 6 years. Therefore, spirometry could only be performed in 23 children. This may have interfered with the investigation of potential effects of different genotypes on spirometric parameters. As previously alluded to, constraints related to DNA extraction from blood samples precluded ADRB2 genotyping in a large number of patients, with negative impacts on sample size. Lack of data on prior maintenance treatment must also be emphasized.

❚ CONCLUSION
Hospital admission rates were significantly higher in carriers of the genotype AA relative to those with genotypes AG or GG (within the p.Arg16Gly variant). Hospital admission rates did not differ between genotypes for the remaining variants.
Changes in forced expiratory volume in the first second and length of stay in the emergency room did not differ between genotypes of variants investigated. Further studies are warranted for better characterization of the frequency of different genotypes, and the impact of beta 2 adrenergic receptor gene variants on therapeutic management of acute asthma in Brazilian children and adolescents.
❚ AUTHORS´ CONTRIBUTION Fábio Pereira Muchão: conception and planning of work, recruting patients and data collection, interpretation of evidence, writing and revision of the manuscript; approval of the final version. Andréa Vieira de Souza: genotyping of beta-2 adrenergic receptor, interpretation of evidence, writing and revision of the manuscript; approval of the final version. Juliana Miguita e Souza: conception and planning of work, recruting patients and data collection, writing of the manuscript; approval of the final version. Luiz Vicente Ribeiro Ferreira da Silva Filho: conception and planning of work, interpretation of evidence, writing and revision of the manuscript; approval of the final version.