Introduction

Asthma is considered as a heterogeneous disease, which is characterized by chronic airway inflammation and respiratory symptoms [1]. It affects around 300 million people all over the world (1–18% of the population in different countries) and is associated with 250,000 deaths per year and its prevalence varies worldwide with a range from 0.2% in China to 21.0% in Australia [2]. As a genetically related disease, its heritability estimates range between 48 and 79% [3]. In Africa, its incidence is 13.9% among children < 15 years, 13.8% among people aged < 45 years, and 12.8% in the total population [4]. In Egypt, two studies estimated that the prevalence of asthma in rural and urban schools was 5.34 and 6.58%, respectively, with a total prevalence of 6.09% and 6.2% [5, 6].

The multiple molecular mechanisms underlying the complex pathological processes involved in disease development and progression hindered the elucidation of polymorphisms in the β2 adrenergic receptor (β2-AR) that are causal of asthma [7]. The β2-AR is the molecular target for β-agonists used in the treatment of asthma. In the human population, 4 polymorphisms of the β2-AR which are (Arg16→Gly, Gln27→Glu, Val34→Met, and Thr164→Ile) have been found [8]. The most frequent two polymorphisms in the β2-ARgene are Arg16Gly (+46A>G; rs1042713) and Gln27Glu (+79C>G; rs1042714) [4]. These polymorphisms change at the β2-AR amino acids sequence where Arg16Gly polymorphism results in that the arginine amino acid at position 16 of the protein is changed into glycine while Gln27Glu changes the glutamine amino acid at position 27 into glutamate [4]. These β2-AR polymorphisms may change asthmatic phenotype and the response to β-agonist therapy providing the mechanism for individualized therapy in asthma [8].

The most common type of genetic variation in the human genome is single-nucleotide polymorphisms (SNPs) which results in the variation in one nucleotide at a given position. The prevalence, functional, and clinical significances of SNPs in the β2-AR have received a lot of attention over the last few decades [9]. It is believed that explaining the consequences of genetic diversity in different races and ethnic groups will be critical in the development of genetic profiles for personalized or precision medicine approaches tailored to asthmatics from different ethnic groups [10].

There are two common SNPs in β2-AR, Arg16Gly, and Glu27Gln polymorphisms. To date, little research work has been done concerning β2-AR polymorphisms in Egypt especially the Upper Egypt; thus, our study aims to find the genotypic frequencies of these polymorphisms among the Upper Egyptian people, furthermore to detect the relationship between β2-AR polymorphisms and both severity of asthma as well as patients’ response to β2-agonist therapy [10].

Holloway et al. and Turki et al. [11, 12] found that Gly16 allele is considered as a marker for asthma severity. Also, a meta-analysis of 28 studies concluded that Gly16Gly had a much higher risk for nocturnal asthma and asthma severity than the Arg16Arg [13]. Recent data showed an association between Arg16Arg genotype and mild asthma when compared with moderate/severe asthma while Gly16Gly genotype was associated with moderate/severe asthma when compared with mild asthmatics [14]. On the other hand, some authors found that Arg16 allele predisposes to exacerbations in asthmatic children and young adults, particularly in those exposed to regular Salmeterol treatment [15]; others added that there is an association between Arg16Gly genotype with severe asthma while Gly16Gly genotype was found with lower frequency in severe asthma when compared with mild/moderate asthma [16].

The aim of this study was to determine the association between β2-adrenergic receptor gene polymorphisms and asthma risk, severity, and response to therapy in Upper Egypt.

Subjects and Methods

This study was conducted at the Respiratory Medicine Department and Tissue Culture and Molecular Biology Center, Assiut University. Patients and control were selected according to the selection criteria that met the GINA Guidelines 2017 criteria for asthma from the Chest in-patient and out-patient Clinics [1]. They were randomly selected by 1:1 cross over from the data base of patients with family history of asthma.

Patients and Controls

The sample power was calculated considering the minor allele frequency (MAF) to establish the sample size, according to the NCBI (National Center for Biotechnology Information—http://www.ncbi.nlm.nih.gov/) [17]. A total of 96 subjects were included in this study. They were divided into two groups:

  • 1–58 Asthma patients (37 females and 21 males) who met the GINA Guidelines 2017 criteria for asthma. Their mean age was 38.1 (± 12.1) years (range 17–62 years old). Patients were treated with salbutamol 100 µg/puff and beclomethasone dipropionate 50 µg/puff with a dose of 2 puffs, 4 times daily. The duration of therapy is the duration of disease ranged from 2 to 12 years.

  • 2–38 Control subjects (21 females and 17 males) were recruited. Their mean age was 34.1 ± 10.1 (range 23–62 years old).

After receiving signed informed consent forms from patients and controls, peripheral blood samples from patients and controls were collected and genotyped for the presence or absence of Arg16Gly and Gln27Glu polymorphisms in the β2-AR gene.

Methods

For both patients and controls, the following steps were done:

  1. 1.

    Historic details included the age, sex, and frequency of attacks. Also, the details included the time lost from school or work, history of allergy as (allergic rhinitis, atopic dermatitis, gastro-esophageal reflux), family history of bronchial asthma and what causes exacerbation of symptoms while the environmental survey included occupation, residence, and indoors or/and outdoors pollution.

  2. 2.

    Physical examination chest examination was done to ensure the diagnosis and evaluate the severity and chronicity of asthma and to exclude other pulmonary complications and diseases.

  3. 3.

    Plain chest X-Ray P-A view was done to exclude any associated pulmonary and cardiovascular diseases.

  4. 4.

    Pulmonary function testing was done for all patients and the following parameters were recorded, FEV1 (forced expiratory volume in 1st second) pre-bronchodilator, FEV1 post-bronchodilator and FEV1/FVC (forced vital capacity).

  5. 5.

    Complete blood count peripheral blood samples were obtained in a heparin coated Wassermann tube for a complete blood picture, specially the absolute eosinophilic count.

  6. 6.

    Genotyping Genomic DNA was extracted from the venous blood samples using the GF-1 Tissue Blood Combi DNA Extraction Kit (Vivantis Technologies Sdn. Bhd. 47600 Subang Jaya, Selangor DarulEhsan, Malaysia).The DNA concentration was determined using a SPECTROstar Nano Spectrophotometer (BMG LABTECH GmbH, Allmendgruen 8-77799 Ortenberg, Germany), and 50 ng/µL of each sample was used for genotyping of both polymorphisms, Arg16Gly and Gln27Glu. Polymorphism detection was done using Genotyping technique StepOnePlus™ Real-Time PCR (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA).

    All reactions were performed under the same conditions. Each 10 µL reaction contained 5 µL TaqMan Genotyping Master Mix (2 ×), (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA), 0.5 µL TaqMan genotyping assay mix (20 ×), (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA), 1.0 µL (approximately 50 ng) of genomic DNA and 3.5 µL DNase-free water.

Statistical Analysis

The results were analyzed using the IBM SPSS Statistics software program, Version 19.0. (IBM Corp., Armonk, NY, USA). Data are presented as numbers and percentages. A P-value < 0.05 was considered statistically significant while, P value of < 0.001 indicates a highly statistically significant result.

Results

The study included two groups, 58 asthmatic cases, and 38 age-matched normal controls. Asthmatic cases showed significant higher eosinophilic count (P < 0.01) and lower FEV1/FVC, FEV1 (P < 0.01) than controls (Table 1).

Table 1 Demographic and descriptive data of studied asthma cases and control group
Full size table

The allelic frequencies for the Arg16Gly polymorphism were 15.5%, 48.3%, and 36.2% for the homozygous A wild (lowest frequency), heterozygous (most frequent), and homozygous G mutant alleles, respectively, in the asthma group (P < 0.01) and 5.3%, 47.4%, and 47.4%, respectively, in the healthy subjects (P < 0.01) (Fig. 1a). For the Gln27Glu polymorphism, the allelic frequencies for the homozygous C wild (most frequent), heterozygous and homozygous G mutant (least frequent) alleles were 51.7%, 41.4%, and 6.9%, respectively, in the asthma group (P < 0.01) and 44.7%, 39.5%, and 15.8% respectively, in the healthy subjects (P < 0.01) (Fig. 1b). There was no significant difference in the genotypic frequencies of both polymorphisms between the two groups.

Table 2 shows that the frequency of allergic history was similar in all groups with different genetic polymorphisms.

Fig. 1
figure 1

Frequency of a Arg16Gly and b Glu27Gln genotype polymorphism in asthma (inner circle) and control group (outer circle)

Full size image
Table 2 Arg16Gly and Gln27Glu genetic polymorphisms in asthmatic patients with and without history of allergy
Full size table

When asthma severity was taken into account, the heterozygous Arg16Gly and Gln27Glu were found in most of severe asthma cases (7/13, 53.8% each). While homozygous wild and mutant seemed to be protective and associated with mild disease in both alleles (Table 3).

Table 3 Frequency of Arg16Gly and Gln27Glu genetic polymorphism in mild, moderate, and severe asthma
Full size table

Finally based on response of asthmatics to β2 agonist drugs, 75% of Arg16Gly heterozygous group were good responders (P < 0.01); 81% of homozygous G mutant were bad responders. For Gln27Glu polymorphism, 60% of C wild were good responders and 75% of G mutant group were bad responders (Table 4).

Table 4 Genetic Arg16Gly polymorphism and Gln27Glu in good and bad responders to β2-agonist therapy
Full size table

Figure 2 summarizes the data and the comparisons in both types of polymorphisms. The most frequent were Gly16Gly and Gln27Gln; heterozygotes A and C were associated with severe asthma while homozygous types were protected, and mutant Gly16Gly and Glu27Glu were associated with bad response to B2 agonists while others were good responders.

Fig. 2
figure 2

Summary of Arg16Arg and Gln27Gln genetic polymorphism frequency and relations to allergy, asthma severity, and bad B2 agonist response, homozygos wild (inner circles 1–4), heterozygos (middle circles 2–5), Homozygos mutant (outer circle 3–6)

Full size image

Discussion

In the current study, the analysis of genotypic frequencies for both Arg16Gly and Gln27Glu polymorphisms revealed no statistically significant difference between asthmatics and control group which may indicate that β2-AR polymorphisms have no role in the pathogenesis of asthma and not considered a risk factor but it may cause disease modification as regards severity and response to β2-agonist therapy. These results are in consistent with those obtained many authors [3, 18]. The absence of association between the Arg16Gly or Gln27Glu polymorphisms and susceptibility to asthma was also reported in a southwest Chinese Han population [19]. On the contrary, De Pavia and colleagues showed that Arg16Arg, Gln27Gln, and Gln27Glu genotypes were associated with the occurrence of asthma [2]. In a study on Egyptian children, it was found that genotype Gly16Gly significantly increased the risk of nocturnal asthma (P = 0.003) [20].

Regarding the genotypic distribution among the two polymorphisms, the present study revealed that Arg16Gly had the highest frequency followed by Gly16Gly and the least frequent genotype was Arg16Arg. To best of our knowledge, this is the first published data on genotype distribution in our community and more work is needed to confirm these frequencies. These results were in consistent with those reported in a study conducted on a North Indian Population where Arg16Gly genotype is the most frequent followed by Gly16Gly then Arg16Arg [21]. Nevertheless, many studies were contradictory as Arg16Arg genotype was more frequent than Gly16Gly [22,23,24]. Salama et al., found that genotype Arg16Arg had the highest frequency in control group (52.6%) while in the asthmatic patients group the heterozygous Arg16Gly was the most frequent genotype (45%) [24].

In this study, the genotype Gln27Gln was the most frequent in both asthmatics and control group followed by heterozygous Gln27Glu then Glu27Glu. These results are in consistent with those found by previous studies [25,26,27]. On the contrary, the heterozygous patients were more frequent in other studies [28,29,30]. These contradictory results between our study and the other studies conducted on different populations indicate that the distribution of β2-AR polymorphisms (Arg16Gly and Gln27Glu) shows a lot of variations between different populations and this may be explained by the wide range of genetic–environmental interactions in different populations. More data will help in the development of personalized medicine according to the different results obtained from studying the relationship and effects of these polymorphisms on the disease among different populations.

Concerning severity of asthma, we found that Arg16Gly and Gly16Gly genotypes (53.8% and 38.5% of severe cases) were more related to severe asthma attacks than Arg16Arg genotype (7.7% of severe cases) which was mostly found in mild and moderate groups (17.2% and 18.8%, respectively). The current study indicates that the β2-AR polymorphism at codon 16 may be a possible determinant of asthma severity. The cause of the different effects of Arg16Gly polymorphism in different geographic regions still remains to be determined and it is possible that the balance between the various pathogenic factors that determine the severity of asthma differs in various regions [31].

This present study shows that Gln27Glu and Gln27Gln genotypes were more related to severe asthma (53.8% and 38.5% of the severe cases, respectively) while Glu27Glu genotype represented only 7.7% of the severe cases. These results are in consistent with studies showing that Gln27Glu polymorphism was associated with asthma severity [2] and that there is a possible protective relation between the Glu27Glu allele and asthma severity for subjects of Asian descent. Hall et al. have shown that asthmatic patients with Glu27Glu had less reactive airways than those with the Gln27Gln form of the β2-adrenoceptor [32]. So, we can conclude that Glu27Glu may have a role in the protection against the severe attacks of asthma.

Regarding the response to β2-agonist therapy, this study revealed higher response in patients with Arg16Arg and Arg16Gly genotypes (77.8% and 75%, respectively) compared to Gly16Gly genotype (19% response) which indicate that Gly16Gly may be involved in β2-adrenoceptor down-regulation process. The influence of Arg16Gly polymorphism on β2-adrenoceptor desensitization was determined in vitro by Green et al., who found that the Gly16 allele was associated with more rapid receptor desensitization in response to β2-agonists when compared to the Arg16 allele [33]. Lima et al., showed that albuterol-evoked FEV1 was higher with more rapid response in Arg16 homozygotes [34]. This means that patients with Gly16Gly had a little response to the β2-agonists with higher rates of severe attacks which make it more difficult to control the disease among those people. Concerning Arg16Gly polymorphism, it is better for the patient to be normal than being mutant with Gly16Gly genotype. Summerhill et al. suggested that the β2-AR Arg16Arg genotype influences either growth of the lung or the rate of decrease in lung function with age since they reported that the Arg16Arg children with asthma may constitute a significant population which is likely to show better asthma control [35]. In a study conducted on Spanish, asthmatic patients showed that Arg16 allele was slightly more frequent within the group with tachyphylaxis; however, Gly16 allele carriers were highly represented within the group of good responders [36]. The clinical response to salmeterol did not differ among β2-ARArg16Gly SNPs during chronic dosing in the presence of an ICS [34].

In this study, both Gln27Gln and Gln27Glu showed higher response for β2-agonist therapy with a percentage of 60% and 54.2%, respectively. However, Glu27Glu genotype showed the least response to β2-agonist therapy with a percentage of 25% of the cases which may indicate that Glu27Glu genotype has a role in β2-adrenoceptor down-regulation process unlike Gln27Gln and Gln27Glu genotypes. That high response rate can be considered as a better chance for patients with Gln27Gln and Gln27Glu genotypes who suffered more from severe asthma attacks. Our results were inconsistent with the study conducted by Green et al. on the functional consequences of β2-adrenergic receptor gene polymorphism in Chinese hamster fibroblasts, and showed that the Glu27 form was resistant to down-regulation [30]. Also, in Spanish population, the proportion of Gln27 carriers was higher among patients with tachyphylaxis while the presence of the Glu27 allele seems to be a protective factor against tachyphylaxis [33]. Study performed by Chong et al. revealed that Glu27 form of the receptor is resistant to desensitization compared to wild-type Gln27 form [35]. However, Kay et al. observed no statistically significant influence on desensitization of Gln27Glu polymorphism [36].

The different findings in this study may be attributed to (a) difference in approach for diagnosis and clinical management between countries, (b) the included patients were mostly atopic, (c) environmental and ethnic differences that can alter the genotypic frequency of polymorphisms, (d) presence of non-reported risk factors or comorbidities.

In conclusion, the findings suggest that the Arg16Gly and Gln27Glu polymorphisms in the β2-AR gene are associated with asthma severity and response to therapy. The high response rate of Gln27Gln and Gln27Glu patients to β2-agonist therapy can be considered as a prognostic marker for patients suffering from severe asthma attacks while patients with Gly16Gly may have a little response to the β2-agonists with higher rates of severe attacks which make it more difficult to control the disease among those people. Several studies with larger samples and more advanced research methods are needed to further confirm correlation of these genetic changes with asthma incidence, severity, response to treatments and to individualize treatment for these patients in the future.