Beta2-adrenergic receptor variants in children and adolescents with bronchial asthma
The region encoding the N-terminal of human β2-adrenergic receptor gene (ADRB2) shows several polymorphisms. To this end, we studied change in susceptibility and/or response to therapy in 175 asthmatic children and adolescents by the two most common variants of the ADRB2 gene namely, rs1042713 (Gly16Arg) and rs1042714 (Gln27Glu). Although, the variants did not correlate with risk of development nor with the severity of the asthma, Gly16/Glu27 haplotype in homozygous individuals conferred protection against development of asthma and was associated with a lower frequency of dyspnea and sputum production. In contrast, the Arg16/Gln27 haplotype was associated with a better response to treatment. These findings show that the risk of development of asthma or response to treatment can be, respectively, deciphered by the detection of both rs1042713 and rs1042714 variants in ADRB2 gene.
Beta2-Adrenergic Receptor Gene, Single Nucleotide Polymorphism, Haplotype, Bronchial Asthma, Egyptian, Review
Bronchial asthma is the most common chronic respiratory disease in childhood and adolescence. The prevalence of asthma in developed countries is increasing to epidemic proportions (1). In Egypt, asthma affects 15-16 % of children aged 3-15 years and one in four children with asthma fails to attend school regularly because of poor asthma control (2). Asthma is a complex multifactorial disease for which a strong genetic element has been firmly established (3). The identification of genes and molecular pathways involved in its pathogenesis will be of great value in predicting disease outcome and establishing targeted therapeutic approaches (3,4).
Bronchial asthma is characterized by chronic inflammation of the airways, mucus hypersecretion and bronchial hyperresponsiveness (BHR) to various stimuli, resulting in intermittent airflow obstruction that is often reversible either spontaneously or with treatment (5-7). The β2-adrenergic receptor (β2-AR), a member of G protein coupled receptor family, is abundantly expressed in the respiratory tract (in bronchial smooth muscle cells, airway epithelial cells, mononuclear cells, and presynaptic cholinergic nerve terminals) (8). It mediates various physiological airway responses, including bronchodilation, vasodilatation and improvement of mucociliary clearance, as well as various anti-inflammatory actions (e.g., mast cell stabilization, cytokine production, reduction of protein extravasation in the airways) (9, 10). Inhaled β2-AR agonists are therefore used as first line bronchodilators in asthma (11, 12) (Figure 1).
The β2-AR gene (ADRB2) (MIM#109690) is located on chromosome 5q31-32 (13). Numerous single nucleotide polymorphisms (SNPs) exist in the amino terminal region of ADRB2, the most common of which are rs1042713 (c.46A>G) and rs1042714 (c.79C>G) (14). These SNPs have been found to alter the amino acid sequence of the β2-AR in the area flanking the receptor ligand site (Arg16Gly and Gln27Glu, respectively) causing receptor down-regulation and desensitization (10). Our research group has demonstrated that these ADRB2 polymorphisms are associated with a higher risk of myocardial infarction and of chronic obstructive pulmonary disease, as well as an increased bronchodilator response in Egyptian patients (10, 14). It has been reported that these polymorphisms may result in an increased susceptibility of asthma and bronchodilators response heterogeneity (15, 16). However, inconsistent and conflicting results were reported across various ethnic populations in Asia, South America, and Europe (17).
Due to lack of consistent data regarding the influence of ADRB2 polymorphisms on different asthma-related phenotypes and the unique genetic make-up of Egyptians due to several colonization events, we aimed at studying the association of these two SNPs with asthma risk and severity and with bronchial hyper-responsiveness in a group of Egyptian asthmatic children and adolescents. We also explored the potential structural and the functional impact of the aforementioned variants by in silico computational tools.
3. Materials and Methods
3.1. Study participants
This study was conducted in two groups: asthma individuals: 75 patients diagnosed with bronchial asthma, and control group: 100 age-matched healthy controls. Asthmatic patients (3-15-years-old) were recruited from the Pediatrics and the Allergy and Immunology clinics of the Suez Canal University Hospital, Ismailia, Egypt, from March 2017 to August 2017. Asthmatic patients were diagnosed according to the Global Initiative for Asthma (GINA). Comorbidities, levels of asthma symptom control over the last 4 weeks, disease severity, therapeutic level and response, and adherence to treatment were assessed based on GINA guidelines (18). Chest radiographs were acquired to exclude other causes of airflow limitation. Asthmatic patients with chronic comorbidities, with manifestations of acute respiratory infection within 3 weeks, on systemic steroids within 2 weeks, or non-compliant to treatment were excluded from the study. Control subjects were healthy children who accompanied their sibs to the Pediatrics Clinic and had no previous history of atopy or chronic airway disease. The study was approved by the Medical Research Ethics Committee of the Suez Canal University Faculty of Medicine (Ismailia, Egypt). Written informed consents were obtained from parents/guardians of the participants.
3.2. Pulmonary function tests
Spirometry was performed in accordance with the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines (19). Basal pulmonary parameters, including forced expiratory volume in first second (FEV1), forced vital capacity (FVC), and peaked expiratory flow rate (PEFR) were determined. A bronchial airway provocation test using methacholine at different dose gradients was performed to assess airway hyper-responsiveness of patients (7). For each subject, the methacholine dose causing a 20% decline in FEV1 (in mg/mL) was recorded. The positive cutoff value of Methacholine challenge test is defined as a PC20 < 8 mg/ml (20).
3.3. Anthropometeric measurements
3.4. Biochemical analysis
Whole venous blood samples were collected in EDTA vacutainers. Serum total IgE was measured by enzyme-linked immunosorbant assay (ELISA), whereas, absolute eosinophil count (AEC) was counted by Coulter counter. Absolute esinophilic count <0.1. x 103/µl and total IgE concentrations <90 IU/ml were considered normal (24).
3.5. Allelic discrimination of ADRB2 variants
Genomic DNA was extracted from peripheral blood leukocytes using the Wizard® Genomic DNA Purification Kit (Promega Co., USA), quantified by absorbance at 260/280 nm using NanoDrop-1000 spectrophotometer (NanoDrop Tech., Wilmington, USA), and stored at -20°C. Genotypes of the ADRB2 rs1042713 and rs1042714 SNPs were assayed using Real-Time polymerase chain reaction allelic discrimination TaqMan assays (Applied Biosystems, assay ID C_2084764_20 and C_2084765_20). Alleles for rs1042713 are G and A (Gly16 and Arg16, respectively) and for rs1042714 are C and G (Gln27 and Glu27, respectively). PCR reaction was carried out in a 25-µl reaction volume containing 20 ng sample DNA, 12.5 µl TaqMan Universal PCR Master Mix (containing AmpErase UNG, AmpliTaq Gold enzyme, dNTPs, and reaction buffer) and 1.2.5 µl of TaqMan SNP genotyping assay Mix. Positive and negative controls were run for each plate. PCR amplification was carried out in StepOne™ Real-Time PCR System (Applied Biosystems, USA) according to the following conditions: two initial holds (50°C for 2 min and 95°C for 10 min) followed by a 40-cycle two-step PCR (95°C denaturation for 15 s and annealing/extension 60°C for 1 min). 10% of the randomly selected samples were re-genotyped in separate runs to exclude the possibility of false genotype calls with a 100% concordance rate.
3.6. In silico data analysis
The workflow adopted in this study followed that of a prior publication by this research group (22). In brief, genomic sequence data was retrieved from NCBI. Functional and structural analysis of the promoter region was performed via Eukaryotic Promoter Database. Genome annotation for ADRB2 transcripts was performed using ECgene and ensemble software. Several databases were used for protein analysis (peptide full sequence identification, secondary structure prediction conserved domains and essential domains identification) including Ensemble, Protein Data Bank, UNiProt/SwissProt, and Protter. Functional annotation was performed using pFam, Prosite, and eMotif. Gene and protein expression in the respiratory tract was identified using GEO. Subcellular localization was determined using Compartment program. Protein-protein interaction data was retrieved using STRING database version 10. ADRB2 variations were obtained from Ensembl. Functional prediction of mutation consequence was determined by polymorphism phenotyping (PolyPhen) version 2.0. Finally, the haplotype block structure of ADRB2 was compared in different populations using HapMap project.
3.7. Statistical analysis
Statistical analysis was carried out using the “Statistical Package for the Social Sciences (SPSS) for windows” software, version 23. The Hardy-Weinberg equilibrium (HWE) was calculated Online (http://www.oege.org/software/hwe-mr-calc.shtml) and tested by χ2 test to compare the expected versus observed distribution of genotypes. The allele frequency and carriage rate was calculated as previously described (22). Haplotype analysis was performed using Haploview software version 4.0. Odds ratios (OR) with a 95% confidence interval (CI) were calculated to determine the effect of each genotype according to the genetic association models. Adjustment of the effect of potential confounders was performed by binary logistic regression analysis. A two-tailed P-value of 0.05 was considered statistically significant.
4.1. Characteristics of the study participants
Demographic, clinical, and laboratory characteristics of the study participants are shown in Table 1. Asthmatic patients had a significantly higher frequency of positive family history of bronchial asthma (P = 0.037).
|Mean age (y)||9.7 ± 3.05||9.8 ± 3.09||0.870|
|Age categories||≥5y||42 (56.0)||55 (55.0)||0.983||1.00|
|≥10y||26 (34.7)||36 (36.0)||0.94|
|≥15y||7 (9.3)||9 (9.0)||1.01|
|Sex||Female||33 (44.0)||58 (58.0)||0.878||1.0|
|Male||42 (56.0)||42 (42.0)||1.08|
|Rural||28 (37.3)||39 (39.0)||0.93|
|BMI percentile||<85th||44 (58.7)||55 (55.0)||0.881||1.0|
|<95th||23 (30.7)||34 (34.0)||0.84|
|≥95th||8 (10.7)||11 (11.0)||0.90|
|Pubertal status||Females||40 (53.3)||53 (53.0)||0.965||1.00|
|Males||35 (46.7)||47 (47.0)||0.98|
|FH of asthma||Negative||42 (56.0)||72 (72.0)||0.037||1.00|
|Positive||33 (44.0)||28 (28.0)||2.02|
|Risk factor||Allergic||70 (93.3)|
|Non allergic||71 (94.6)|
|Onset||Early onset||37 (49.3)|
|Late onset||38 (50.7)|
|Mean duration (y)||5.5 ± 2.04|
|Total IgE (IU/mL)||80 (25-126)|
|AEC (x106/L)||122 (34-422)|
|Pulmonary function test|
|FVC||77.5 ± 6.95|
|Pre-FEV1||76.9 ± 7.40|
|Post-FEV1||55.3 ± 8.23|
n: number of subjects; BMI: body mass index; FH: family history; AEC: absolute eosinophil count; FVC: forced vital capacity; FEV1: forced expiratory volume in first second; PEFR: peaked expiratory flow rate; BHR: bronchial hyperresponsiveness. Values are shown as number (%) and mean ± standard deviation. Odds ratio (OR) was calculated at a 95% confidence interval (CI). P-value (p) <0.05 was considered significant.
4.2. Molecular analysis of ADRB2 SNPs
Genotype frequencies of the ADRB2 SNPs in both asthma patients and controls were in agreement with frequencies expected by HWE (P > 0.05), Table 2. The G allele of the rs1042713 SNP (Gly16) and the A allele of the rs1042714 SNP (Gln27) were more frequent in the study population. However, no significant differences in genotype frequencies or allele frequencies were observed for either SNPs when comparing asthmatic patients and controls under all genetic association models, Table 3.
Haplotype analysis of the study population demonstrated the presence of four haplotype allele combinations. The frequencies of Gly16/Gln27, Gly16/Glu27, Arg16/Gln27, and, Arg16/Glu27 haplotypes were 27.6 %, 34.9 %, 35.9 % and 1.7 % respectively, Table 4. Homozygote carriers of the Gly16/Glu27 haplotype were less likely to develop asthma (P < 0.05), Table 5.
|Genotype frequency (rs1042713)|
|GG||27 (36.0)||36 (36.0)||0.989||0.022||1.0|
|GA||40 (53.3)||54 (54.0)||0.9|
|AA||8 (10.7)||10 (10.0)||1.1|
|G||94 (62.7)||126 (63.0)||0.949||0.004||1.0|
|A||56 (37.3)||74 (37.0)||1.0|
|Genotype frequency (rs1042714)|
|CC||29 (38.7)||40 (40.0)||3.438||0.179||1.0|
|CG||41 (54.6)||45 (45.0)||1.2|
|GG||5 (6.7)||15 (15.0)||0.4|
|C||99 (66.0)||125 (62.5)||0.455||0.499||1.0|
|G||51 (44.0)||75 (37.5)||0.8|
n: number of subjects; p HWE: p value of Hardy-Weinberg equilibrium. Values are shown as number (%). Chi square (c2) for trend was used. Odds ratio (OR) was calculated at a 95% confidence interval (CI). P-value (p) <0.05 was considered significant. The allele frequency within each group was determined as the number of occurrences of an individual allele divided by the total number of alleles.
|Homozygote comparison||AA vs. GG||1.1|
|GG vs. CC||0.4|
|Heterozygote comparison||GA vs. GG||0.9|
|CG vs. CC||1.2|
|Dominant model||GA+AA vs. GG||1.0|
|CG+GG vs. CC||1.0|
|Co-dominant model||GA vs. GG+AA||0.9|
|CG vs. CC+GG||1.4|
|Recessive model||AA vs. GG+GA||1.1|
|GG vs. CC+CG||0.4|
|Allelic model||A vs. G||1.0|
|G vs. C||0.8|
Odds ratio (OR) was calculated at a 95% confidence interval (CI).
|Gly16/Gln27||GC||83 (27.6)||39 (29.3)||44 (26.2)||1.100||0.294||1.37 (0.75-2.51)|
|Gly16/Glu27||GG||105 (34.9)||46 (34.6)||59 (35.1)||0.097||0.755||1.10 (0.59-2.03)|
|Arg16/Gln27||AC||108 (35.9)||45 (33.8)||63 (37.5)||0.163||0.686||0.88 (0.47-1.62)|
|Arg16/Glu27||AG||5 (1.7)||3 (2.3)||2 (1.2)||0.618||0.432||2.04 (0.33-12.5)|
n: number of subjects. Values are shown as number (%). Chi square (c2) test was used. Odds ratio (OR) was calculated at a 95% confidence interval (CI). P-value (p) <0.05 was considered significant. OR for alleles was calculated as presence versus absence of this particular allele.
|Haplotype Copy Number|
|0 copy||36 (48.0)||56 (56.0)||1.185||0.553||1.0|
|1 copy||32 (82.1)||35 (35.0)||1.4|
|2 copies||7 (17.9)||9 (9.0)||1.2|
|0 copy||29 (38.7)||41 (41.0)||8.602||0.014||1.0|
|1 copy||44 (58.7)||44 (44.0)||1.4|
|2 copies||2 (2.7)||15 (15.0)||0.18|
|0 copy||30 (40.0)||37 (37.0)||0.403||0.818||1.0|
|1 copy||37 (49.3)||54 (54.0)||0.8|
|2 copies||8 (10.7)||9 (9.0)||1.1|
|0 copy||72 (96.0)||98 (98.0)||0.618||0.432||1.0|
|1 copy||0 (0.0)||0 (0.0)||NA|
|2 copies||3 (4.0)||2 (2.0)||2.0|
n: number of subjects. Values are shown as number (%). Chi square (c2) test was used. Odds ratio (OR) was calculated at a 95% confidence interval (CI). P-value (p) <0.05 was considered significant. OR for alleles was calculated as presence versus absence of this particular allele.
4.3. ADRB2 SNPs and disease characteristics
When asthma patents were stratified according to disease characteristics and laboratory findings, no significant association was found with any of the ADRB2 genotypes, Table 6. However, asthma patients with the Arg16/Gln27 haplotype had higher post-bronchodilator FEV1 (65.3 ± 12) indicative of a better response to therapy, as compared to non-carriers of the same haplotype (58.9 ± 11) (P < 0.05). Moreover, patients with the Gly16/Gln27 haplotype had less frequent dyspnea and sputum formation (P = 0.011 and 0.019, respectively), Table 7.
|Clinical characteristics||rs1042713 (Gly16Arg) genotypes|
|p||rs1042714 (Gln27Glu) genotypes|
|Early onset (<3y)||9 (33.3)||24 (60.0)||4 (50.0)||0.101||14 (48.3)||19 (46.3)||4 (80.0)||0.360|
|Late onset (>3y)||18 (66.7)||16 (40.0)||4 (50.0)||15 (51.7)||22 (53.7)||1 (20.0)|
|Asthma duration (y)||5.4 ± 2.2||6 ± 2.9||6.2 ± 2.1||0.121||6.2 ± 2.7||6.3 ± 2.6||5.8 ± 3.5||0.723|
|Cough||27 (100.0)||39 (97.5)||7 (87.5)||0.155||27 (93.1)||41 (100.0)||5 (100.0)||0.196|
|Wheezes||22 (81.5)||28 (70.0)||6 (75.0)||0.570||21 (72.4)||32 (78.0)||3 (60.0)||0.639|
|Dyspnea||10 (37.0)||25 (62.5)||5 (62.5)||0.105||14 (48.3)||23 (56.1)||3 (60.0)||0.774|
|Sputum||11 (40.7)||28 (70.0)||4 (50.0)||0.054||14 (48.3)||26 (63.4)||3 (60.0)||0.448|
|Tightness||15 (55.6)||24 (60.0)||7 (87.5)||0.257||19 (65.5)||24 (58.5)||3 (60.0)||0.838|
|Intermittent||12 (44.4)||17 (42.5)||5 (62.5)||0.649||17 (58.6)||16 (39.0)||1 (20.0)||0.341|
|Mild persistent||7 (25.9)||15 (37.5)||2 (25.0)||8 (27.6)||14 (34.1)||2 (40.0)|
|Moderate persistent||8 (29.6)||8 (20.0)||1 (12.5)||4 (13.8)||11 (26.8)||2 (40.0)|
|Total IgE (IU/mL)||81.4 ± 92.1||86.0 ± 91.3||50.3 ± 40.2||0.710||77.5 ± 92.3||78.7 ±85.9||117 ± 79.0||0.280|
|AEC (x106/L)||249 ± 295||165 ± 247||102 ± 119||0.553||182 ± 256||194 ± 270||178 ± 201||0.826|
|Pulmonary function test|
|FVC||78.2 ± 8.08||78.1 ± 7.34||80.0 ± 8.88||0.818||80.4 ± 7.3||76.8 ± 8.0||79.0 ±2.5||0.160|
|Pre-FEV1||76.9 ± 8.53||77.0 ± 8.49||78.7 ± 8.46||0.860||79.1 ±7.7||76.4 ± 8.6||71.6 ±8.1||0.127|
|Post-FEV1||59.2 ± 11.92||64.2 ± 12.5||66.8 ± 11.6||0.167||64.8 ± 13.4||62.4 ± 11.6||53.6 ±8.9||0.169|
|PEFR||76.4 ± 12.8||75.1 ± 11.1||80.7 ± 10.1||0.461||78.6 ± 11.6||75.2 ± 11.7||69.8 ±8.7||0.217|
|Positive BHR||15 (55.6)||17 (42.5)||3 (37.5)||0.495||13 (44.8)||18 (43.9)||4 (80.0)||0.302|
n: number of patients; y: years;AEC: absolute eosinophil count; FVC: forced vital capacity; FEV1: forced expiratory volume in first second; PEFR: peaked expiratory flow rate; BHR: bronchial hyperresponsiveness. Values are shown as number (%) and mean ± standard deviation. Chi square test was used for categorical variables. One way ANOVA or Kruskal-Wallis tests were used for quantitative data followed by post-hoc Dunnett t (2-sided) test. P-value (p) <0.05 was considered significant.
|Early onset (<3y)||22 (61.1)||15 (38.5)||0.066||14 (48.3)||23 (50.0)||0.884||12 (40)||25 (55.6)||0.240||34 (47.2)||3 (100)||0.115|
|Late onset (>3y)||14 (38.9)||24 (61.5)||15 (51.7)||23 (50.0)||18 (60)||20 (44.4)||38 (52.8)||0 (0.0)|
|Asthma duration (y)||5.8 ± 2.0||5.2 ± 2.0||0.185||5.4 ± 2.1||5.6 ± 2.0||0.691||5.0 ± 1.5||5.8 ± 2.2||0.065||5.5 ± 20||5.6 ± 2.0||0.909|
|Cough||35 (97.2)||38 (97.4)||0.954||27 (93.1)||46 (100.0)||0.071||30 (100)||43 (95.6)||0.514||70 (97.2)||3 (100)||0.770|
|Wheezes||24 (66.7)||32 (82.1)||0.184||21 (72.4)||35 (76.1)||0.788||24 (80.0)||32 (71.1)||0.430||54 (750)||2 (66.7)||0.745|
|Dyspnea||25 (69.4)||15 (38.5)||0.011||14 (48.3)||26 (56.5)||0.635||13 (43.3)||27 (60.0)||0.167||37 (51.4)||3 (100)||0.243|
|Sputum||26 (72.2)||17 (43.6)||0.019||14 (48.3)||29 (63.0)||0.238||13 (43.3)||30 (66.7)||0.058||41 (56.9)||2 (66.7)||0.739|
|Tightness||24 (66.7)||22 (56.4)||0.477||19 (65.5)||27 (58.7)||0.631||17 (56.7)||29 (64.4)||0.629||44 (61.1)||2 (66.7)||0.846|
|Intermittent||15 (41.7)||19 (48.7)||0.815||17 (58.6)||17 (37.0)||0.152||12 (40.0)||22 (48.9)||0.458||34 (47.2)||0 (0.0)||0.253|
|Mild persistent||12 (33.3)||12 (30.8)||8 (27.6)||16 (34.8)||9 (30.0)||15 (33.3)||22 (3.6)||2 (66.7)|
|Moderate persistent||9 (25.0)||8 (20.5)||4 (13.8)||13 (28.3)||9 (30.0)||8 (17.8)||16 (22.2)||1 (33.3)|
|Total IgE (IU/mL)||77.6±72||83.8±100||0.758||77.5±92||82.9±85||0.798||89.3±92||75.2±84||0.496||77.5±86||160±68||0.107|
|Pulmonary function tests|
|Positive BHR||16 (44.4)||19 (48.7)||0.818||13 (44.8)||22 (47.8)||0.817||17 (56.7)||18 (40.0)||0.167||33 (45.8)||2 (66.7)||0.596|
Number of patients=75.y: years; AEC: absolute eosinophil count; FVC: forced vital capacity; FEV1: forced expiratory volume in first second; PEFR: peaked expiratory flow rate; BHR: bronchial hyperresponsiveness. Values are shown as number (%) and mean ± standard deviation. Chi square test was used for categorical variables. Student-t and Mann-Whitney U tests were used for comparison. P-value (p) <0.05 was considered significant.
4.4. Structural genomic analysis of ADRB2
The gene encoding β2-AR (ADRB2) is located on chromosome 5q31-q32 from position 148825245 to position 148828687 (3443 nucleotide long) (homo sapiens assembly; GRCh38.p2:CM000667.2). It is intronless; consisting of only one exon. The gene has a single transcript on the forward strand (ENST00000305988) 2041 nucleotides long, including the 5’-and 3’-untranslated regions (UTR). The protein-coding region spans 1242 nucleotides within the full-length transcript; these encode the 413 amino acid residues forming the receptor (Met-1 to Leu-413). Promoter analysis revealed the presence of GC-boxes at positions -484, -428, -367, and -120.
4.5. Genetic variants of ADRB2
ADRB2 is highly polymorphic, particularly in the coding region. Most of the variants are benign missense mutations. We studied two common missense mutations in the sequence coding for the N terminal extracellular domain of the protein: c.46G>A (G16R) and c.79C>G (Q27E) at 5:148826877 and 5:148826910, respectively, Figure 2. Online computational tools (Polyphen and SIFT) predicted Gly16Arg and Gln27Glu variants to be benign with a score of 0.043 (sensitivity: 0.94; specificity: 0.83), and 0.009 (sensitivity: 0.96; specificity: 0.77), respectively. However, analysis of nearby miRNA binding sites in ADRB2 illustrated complementary sites for miR-26a, miR-26b, miR-1297, and miR-590-3p at the 5’ end of the gene.
4.6. Structural and functional analysis of β2-AR
The β2-AR is a single polypeptide chain consisting of 413 amino acid residues with a molecular weight of 46.5 KDa. It is composed of an extracellular amino terminus, seven transmembrane-spanning domains, 3 intracellular and 3 extracellular loops, and an intracellular carboxyl terminus. The amino acid sequence and predicted membrane topography for the human β2-AR is presented in Figure 3. After formation of the polypeptide chain, the receptor undergoes three types of post-translational modifications that alter protein structure and molecular weight: glycosylation, palmitoylation, and disulfide bond formation, Table 8. The N-linked glycosylation at the amino acids 6 and 15 in the extracellular space, is responsible for membrane insertion and agonist-induced receptor trafficking. Palmitoylation occurs at the cysteine residue of amino acid 341, and is responsible for the reversible anchoring of the carboxy-terminus to the membrane. Palmitoylation has been found to stimulate agonist-induced adenylyl cyclase activity and affects β2-AR phosphorylation and desensitization. The region between the seventh transmembrane-spanning domain and the palmitoylated cysteine is sometimes denoted as the fourth intracellular loop. Disulfide bond formation has been found to occur at cysteine residues 106 ↔ 191 and 184 ↔ 190. These bonds stabilize the β2-AR ligand-binding pockets. β2-AR also contains phosphorylation sites in the third intracellular loop and proximal cytoplasmic tail. Phosphorylation of these sites directly interferes with receptor coupling to stimulatory G proteins and triggers the agonist-promoted desensitization, internalization, and degradation of the receptor (Figure 4).
|N terminus||1-34||34||Glycosylation||Asn-6, -15|
|Transmembrane 2||72-95||24||Asp-79||Proper receptor folding & transmembrane assembly of the receptor||Asp-79||Affects binding to catecholamine & uncoupling with Gs protein|
|Extracellular loop 1||96-106||11||Disulfide bond||Cys-106||Generating the thermodynamically stable conformation of the receptor required for agonist or antagonist binding||Cys-106 substitution could reduce binding affinities 14- to 1400-fold for agonist and 4- to 16-fold for antagonist|
|Transmembrane 3||107-129||23||Asp113, Thr-118||Agonist or antagonist binding site|
|Intracellular loop 2||130-150||21||Phosphotyrosine||Tyr-141||Supersensitization of the receptor||Tyr-141||Abolishes insulin-induced tyrosine phosphorylation & insulin-induced receptor supersensitization|
|Extracellular loop 2||175-196||22||Disulfide bond||Cys-184, -190, -191||Generating the thermodynamically stable conformation of the receptor required for agonist or antagonist binding||Cys-184, -190, -191 substitution could reduce binding affinities 14- to 1400-fold for agonist and 4- to 16-fold for antagonist|
|Transmembrane 5||197-220||24||193-207||Agonist and antagonist binding|
|Intracellular loop 3||221-274||54||Phosphoserine by PKA||Ser-246||Mediates agonist-promoted desensitization, internalization, & degradation of the receptor|
|Transmembrane 6||275-298||24||Phosphoserine by PKA||Ser-261, -262||Mediates agonist-promoted desensitization, internalization, & degradation of the receptor|
|286-293||Agonist and antagonist binding|
|Extracellular loop 3||299-305||7|
|Transmembrane 7||306-329||24||312-316||Agonist and antagonist binding|
|C terminus||330-413||84||Palmitoylation||Cys-341||Anchoring to the plasma membrane, reduce accessibility of Ser-345 and -346. Agonist stimulation promotes depalmitoylation and further allows Serine phosphorylation.||Cys-341||Uncoupled receptor|
|Phosphoserine by PKA||Ser-345, -346||Mediates agonist-promoted desensitization, internalization, & degradation of the receptor||Delayed agonist-promoted desensitization|
|Phosphoserine by BARK||Ser--355, -356||Mediates agonist-promoted desensitization, internalization, & degradation of the receptor|
|410-413||PDZ-binding: (1) anchoring receptor proteins in the membrane to cytoskeletal components, (2) when endocytosed to prevent degradation in lysosomes & promote recycling to the plasma membrane|
4.7. Functional enrichment analysis for β2-AR
Pathway enrichment analysis in the KEGG pathways identified eight pathways: calcium signaling (hsa04020), cGMP-KG signaling (hsa04022), cAMP signaling (hsa04024), neuroactive ligand-receptor interaction (hsa04080), adrenergic signaling in cardiomyocytes (hsa04261), regulation of lipolysis in adipocytes (hsa04923), renin secretion (hsa04924), and salivary secretion (hsa04970). The β2-AR transmembrane protein is predicted to be located in various components (plasma membrane, lysosomes, endosomes, nucleus, and extracellular matrix). Activated receptors are internalized into endosomes prior to their degradation in lysosomes (UniProt.org), Figure 5. The gene ontology enrichment analysis identified several GO terms for ADRB2. Main molecular functions were adenylate cyclase binding, beta2-adrenergic receptor activity, epinephrine and norepinephrine binding, and potassium channel regulator activity. Protein-protein interaction network is depicted in Figure 5 revealed physical and functional associations with other proteins, and demonstrated some enriched biological processes which are related to asthma pathogenesis; activation of transmembrane receptor protein tyrosine kinase activity, cell-cell signaling, G-protein coupled receptor signaling pathway, negative regulation of smooth muscle contraction, positive regulation of mitogen activated protein kinase (MAPK) cascade, positive regulation of mini excitatory postsynaptic potential, positive regulation of protein ubiquitination, regulation of sodium ion transport, and response to psychosocial stress and cold.
Though bronchodilators are the main drug of choice for the management of bronchial asthma, multiple clinical trials have showed that 70% of asthmatic patients receiving β2-AR agonists partially lose their drug-induced protection (25, 26). Human ADRB2 is located in a region that has been linked to allergic phenotypes and BHR (27, 28). SNPs in the ADRB2 gene have been linked to pathogenesis of and therapeutic responses in bronchial asthma (29).
In the current study, we investigated the relationship of ADRB2 gene variants at codons 16 and 27 with susceptibility and clinical outcomes in asthmatic children and adolescents. These two polymorphisms are located within the amino-terminal extracellular domain of the receptor near the ligand-binding site (15). They are proposed to alter the functional properties of the receptor and its behavior after agonist exposure (30). Both Arg16Gly and Gln27Glu polymorphisms have been linked with a range of disorders, including chronic obstructive pulmonary disease (11), cystic fibrosis (31), congestive heart failure (32), coronary heart disease (14), hypertension (33), rheumatoid arthritis (34), Graves’ disease (35), and atopic dermatitis (36), thus highlighting the putative role of these SNPs in immune system-related diseases.
Our results revealed that the distribution of genotype frequencies at position 16 and 27 did not deviate from HWE among patients and controls. The minor allele frequency (MAF) at Arg16Gly variant was identical (0.37 for Arg16) in controls and patients, while at Gln27Glu the frequency of Glu27 was 0.37 in controls and 0.44 in the patient group. Overall and stratified analysis revealed no evidence of an association of Arg16Gly and Gln27Glu genotypes with asthma risk or severity under any genetic association models. In addition, both variants did not show associations with clinical features or treatment response. Consistently, previous studies reported no significant differences in genotype and allele frequencies of the two variants with bronchial asthma (30, 37-39). Subsequent meta-analysis studies did not detect association between ADRB2 polymorphisms and risk of asthma across various ethnic groups (40, 41). In comparison with other various populations and other studies conducted in different regions of Egypt (Table 9), our findings were strikingly consistent with most studied populations and with other studies conducted in the same region, which correlated the study variants with other disorders (10, 14), but differ from that of other regions of Egypt (42, 43) (Table 9). As we speculated previously (44), the heterogeneous genetic profile noted in the current region could be attributed to the historical and geographical background of Suez Canal area where the current research has been executed. This area which links the three continents was controlled by a succession of powerful empires which could affect the genetic profile signatures of the common variants. Additionally, the different study design and type of patients will contribute to the apparent genetic heterogeneity.
vs. current study
vs. current study
|G allele||A allele||C allele||G allele|
|Current study||0.63 (126)||0.37 (74)||0.63 (125)||0.37 (75)|
|Hussein et al., 2017 (10)||0.61 (66)||0.39 (42)||0.770||0.63 (68)||0.37 (40)||1.000|
|Toraih et al., 2014 (13)||0.63 (94)||0.37 (56)||1.000||0.55 (82)||0.45 (68)||0.250|
|Karam et al., 2013 (42)||0.37 (82)||0.63 (138)||<0.001||0.74 (164)||0.26 (56)||0.094|
|Hamdy et al., 2002 (43)||0.43 (103)||0.57 (137)||0.004||0.76 (182)||0.24 (58)||0.045|
|1000 Genome project|
|ALL||0.52 (2626)||0.47 (2382)||0.134||0.79 (3985)||0.20 (1023)||0.008|
|AFR||0.48 (634)||0.52 (688)||0.032||0.86 (1142)||0.13 (180)||<0.001|
|AMR||0.54 (377)||0.45 (317)||0.226||0.75 (526)||0.24 (168)||0.050|
|EAS||0.45 (455)||0.54 (553)||0.012||0.92 (934)||0.08 (74)||<0.001|
|EUR||0.61 (618)||0.38 (388)||0.840||0.59 (594)||0.41 (412)||0.562|
|SAS||0.55 (542)||0.44 (436)||0.286||0.80 (789)||0.19 (189)||0.005|
|ALL||0.57 (142444)||0.42 (103492)||0.434||0.68 (168082)||0.31 (78116)||0.397|
|NFE||0.62 (69972)||0.37 (41472)||0.956||0.57 (63973)||0.42 (47689)||0.434|
|ALL||0.57 (17683)||0.42 (13197)||0.434||0.67 (20731)||0.33 (10203)||0.553|
|NFE||0.62 (9326)||0.37 (5638)||0.956||0.56 (8523)||0.43 (6455)||0.354|
|TSI||0.66 (117)||0.33 (59)||0.588||NA||NA||NA|
|NHLBI Exome Sequencing|
|African-American||0.51 (2259)||0.48 (2147)||0.105||0.81 (3597)||0.18 (809)||0.002|
|European-American||0.62 (5351)||0.37 (3249)||0.956||0.58 (4987)||0.42 (3613)||0.469|
Allele frequency (count) is shown for each polymorphism. AFR: African; AMR: American; EAS: East Asian; EUR: European; SAS: South Asian; NFE: Non-Finnish European; TSI: Toscans in Italy (Data source: Ensembl.org). P-value (p) <0.05 was considered significant
It is becoming a necessity that populations with diverse ancestral histories and ethnic backgrounds be included in genetic association studies. Minor allele frequencies of common SNPs in a genomic region greatly differ among study populations. Populations of African descent were formed prior to Asian or European populations, thus are supposedly more prone to genetic variations and diversity. Additionally, this heterogeneity could be also a reflection of the diversity of the mutation pattern among Arabs, as most of the Egyptian population is now of Arab origin. Thus haplotype analysis of more SNPs in the gene and linkage disequilibrium patterns should be undertaken rather than single polymorphism analysis.
Therefore, we performed a haplotype analysis which revealed that the frequencies of Gly16/Gln27, Gly16/Glu27, Arg16/Gln27, and Arg16/Glu27 haplotypes in the current study population are 27.6%, 34.9%, 35.9% and 1.7% respectively. Haplotype distributions showed that carrying two copies of the Gly16/Glu27 haplotype conferred protection against the development of asthma. A possible explanation is that the Glu27 variant is more resistant to down-regulation as has been demonstrated in a previous in vitro study, and it is consequently possible that increasing numbers of β2-ARs are produced (45), while the Gln27 allele has been shown to be associated with higher IgE profile (46, 47). Moreover, it has been demonstrated that the Gly16 receptor has a higher alveolar-capillary membrane conductance and a better ability to clear fluid from the lungs compared to the Arg16 allele (48).
Regarding the clinical outcomes of asthma patients, the Gly16/Gln27 haplotype was associated with a significantly lower frequency of dyspnea and sputum production, while carriers of the Arg16/Gln27 haplotype exhibited a better response to treatment in the form of a higher post-bronchodilator FEV1. The results of recent genetic association studies are quite paradoxical. Lee et al. (49) reported that children with the Gly16/Glu27 haplotypes had a lower risk of wheezing illness with a clear dose-response relationship. The study by Matheson et al. (50) showed that the Arg16/Gln27 haplotype is associated with severe respiratory symptoms and frequent exacerbations. In contrast, other clinical studies have supported the possibility that the Gly16 form of the receptor is associated with markers of more severe asthma (51, 52), while other studies reported the association of the Glu27 allele with asthma severity in Asian patients (53).
The precise impact of the Arg16Gly and Gln27Glu polymorphisms on the pathophysiology of bronchial asthma remains unclear. Some studies have demonstrated that these SNPs do not affect the binding capacity of catecholamines to the receptors, the adenylate cyclase activity of the receptor, receptor synthesis rates or agonist-promoted internalization (54). Other studies, however, suggested that these SNPs play a role in the down-regulation phenomenon and functional desensitization of the receptor due to alterations in receptor degradation after the internalization step (55-57) (Figure 4). Further functional studies are required to clarify this issue. In addition, exploring other genetic determinants for BHR is necessary for selecting and adjusting the appropriate medications.
The authors thank the Center of Excellence in Molecular and Cellular Medicine and the Oncology Diagnostic Unit, Suez Canal University, Ismailia, Egypt for providing the facilities for performing the molecular work of the current study.
3. Kaneko, Y., H. Masuko, T. Sakamoto, H. Iijima, T. Naito, Y. Yatagai, H. Yamada, S. Konno, M. Nishimura, E. Noguchi, and N. Hizawa: Asthma Phenotypes in Japanese Adults – Their Associations with the CCL5 ADRB2 Genotypes. Allergol Int 62, 113-121 (2013)
4. Hussein, M. H., E. A. Toraih, N. M. Aly, E. Riad, and M. S. Fawzy: A passenger strand variant in miR-196a2 contributes to asthma severity in children and adolescents: A preliminary study. Biochem Cell Biol 94, 347-357 (2016)
7. Toraih, E. A., M. H. Hussein, E. Al Ageeli, E. Riad, N. B. AbdAllah, G. M. Helal, and M. S. Fawzy: Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma. Respir Res 18, 169 (2017)
8. Litonjua, A. A., L. Gong, Q. L. Duan, J. Shin, M. J. Moore, S. T. Weiss, J. A. Johnson, T. E. Klein, and R. B. Altman: Very important pharmacogene summary ADRB2. Pharmacogenet Genom 20, 64–69 (2010)
9. Staus, D. P., L. M. Wingler, R. T. Strachan, S. G. Rasmussen, E. Pardon, S. Ahn, J. Steyaert, B. K. Kobilka, and R. J. Lefkowitz: Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol 85, 472–481 (2014)
10. Hussein, M. H., K. E. Sobhy, I. M. Sabry, A. T. El Serafi, and E. A. Toraih: Beta2-adrenergic receptor gene haplotypes and bronchodilator response in Egyptian patients with chronic obstructive pulmonary disease. Adv Med Sci 62, 193-201 (2017)
12. Plazinska, A., W. Plazinski, and K. Jozwiak: Agonist binding by the β2-adrenergic receptor: an effect of receptor conformation on ligand association–dissociation characteristics. European Biophysics Journal 44, 149-163 (2015)
15. de Paiva, A. C., F. A. Marson, J. D. Ribeiro, and C. S. Bertuzzo: Asthma: Gln27Glu and Arg16Gly polymorphisms of the beta2-adrenergic receptor gene as risk factors. Allergy Asthma Clin Immunol 10, 8 (2014)
16. Gao, G., S. Wang, and J. Zhang: Study on beta 2 adrenergic receptor genetic polymorphisms in asthmatics in the people of the Han nationality of northern China. Zhonghua Jie He He Hu Xi Za Zhi 23, 93–97 (2000)
17. Contopoulos-loannidis, D.G., E. N. Manoli, and J. P. Ioannidis: Meta-analysis of the association of beta-adrenergic receptor polymorphisms with asthma phenotypes. J Allergy Clin Immunol 115, 963–972 (2005)
18. Global Initiative for Asthma (GINA). Global Strategy for Asthma Management and Prevention 2015. Available from :http://www.ginasthma.org/ (accessed 26 April, 2018)
19. Soriano, B., I. Alfageme, P. Almagro, C. Casanova, C. Esteban, J. J. Soler-Cataluña, J. P. de Torres, P. Martinez-Camblor, M. Miravitlles, B. R. Celli, and J. M. Marin: Distribution and Prognostic Validity of the New Global Initiative for Chronic Obstructive Lung Disease Grading Classification. Chest 143, 694–702 (2013)
20. Mazi, A., and L. C. Lands: A PC20 Cutoff Concentration Of 4mg/ml Can Be Used To Define A Positive Methacholine Challenge Test In Children. Am J Respir Crit Care Med 187, A4561 (2013) Avialable at http://www.atsjournals.org/doi/abs/10.1.164/ajrccm-conference.2013.1.87.1._MeetingAbstracts.A4561
21. Badawi, N. E., A. Abo Barakat, S. A. El Sherbini, and H. M. Fawzy: Prevalence of overweight and obesity in primary school children in Port Said city. Egyptian Pediatric Association Gazette 61, 31-36 (2013)
22. Fawzy, M. S., O. Alhadramy, M. H. Hussein, H. M. Ismail, N. M. Ismail, N. M. Biomy, and E. A. Toraih: Structural and functional impact of ATP-Binding Cassette Transporter A1 R219K and I883M Polymorphisms within obese Children and Adolescents. Mol Diagn Ther 19, 221–234 (2015)
24. Fischbach, F.T., and M. B. Dunning Nurses’ Quick Reference to Common Laboratory & Diagnostic Tests. Lippincott Williams & Wilkins, 2006. Available at https://books.google.com.sa/books?id=dfy9QgAACAAJ
26. Gupta, M. K., K. Asosingh, M. Aronica, S. Comhair, G. Cao, S. Erzurum, R. A. Jr. Panettieri, and S. V. Naga Prasad: Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma. PLoS ONE 10, e0125803 (2015)
27. Meyers, D. A., D.S. Postma, C. I. Panhuysen, J. Xu, P. J. Amelung, R. C. Levitt, and E. R. Bleecker: Evidence for a locus regulating total serum IgE levels mapping to chromosome 5. Genomics 23: 464–470 (1994)
28. Xu J., D. A. Meyers, C. Ober, M. N. Blumenthal, B. Mellen, K. C. Barnes, R. A. King, L. A. Lester, T. D. Howard, J. Solway, C. D. Langefeld, T. H. Beaty, S.S. Rich, E. R. Bleecker, N. J. Cox, and Collaborative Study on the Genetics of Asthma: Genome wide screen and identification of gene-gene interactions for asthma-susceptibility loci in three U.S. populations: collaborative study on the genetics of asthma. Am J Hum Genet 68, 1437–1446 (2001)
29. Saadi, A.V., H Gupta, A. Angural, S. K. Dhanya, S. Mony, D. Oberoi, S. C. D’Souza, R. C. Sahoo, M. H. Hande, P. M. Gopinath, and K. Satyamoorthy: Single nucleotide polymorphisms of ADRB2 gene and their association with susceptibility for Plasmodium falciparum malaria and asthma in an Indian population. Infect Genet Evol 20, 140-147 (2013)
30. Litonjua, A. A., L. Gong, Q. L. Duan, J, Shin, M. J. Moore, S.T. Weiss, J. A. Johnson, T. E. Klein, and R. B. Altman: Very important pharmacogene summary ADRB2. Pharmacogenet Genomics 20, 64–69 (2010)
32. Shin, J., M. T. Lobmeyer, Y. Gong, I. Zineh, T. Y. Langaee, H. Yarandi, R. S. Schofield, J. M. Jr. Aranda, J. A. Hill, D. F. Pauly, and J. A. Johnson: Relation of beta(2)-adrenoceptor haplotype to risk of death and heart transplantation in patients with heart failure. Am J Cardiol 99, 250-255 (2007)
33. Lou, Y., J. Liu, Y. Huang, J. Liu, Z. Wang, Y. Liu, Z. Li, Y. Li, Y. Xie, and S. Wen: A46G and C79G polymorphisms in the β2-adrenergic receptor gene (ADRB2) and essential hypertension risk: a meta-analysis. Hypertens Res 33, 1114-2113 (2010)
34. Malysheva, O., M. Pierer, U. Wagner, M. Wahle, U. Wagner, and C. G. Baerwald: Association between beta2 adrenergic receptor polymorphisms and rheumatoid arthritis in conjunction with human leukocyte antigen (HLA)-DRB1 shared epitope. Ann Rheum Dis 67, 1759-1764 (2008)
35. Chu, X., Y. Dong, M. Shen, L. Sun, C. Dong, Y. Wang, B. Wang, K. Zhang, Q. Hua, S. Xu, and W. Huang: Polymorphisms in the ADRB2 gene and Graves disease: a case-control study and a meta-analysis of available evidence. BMC Med Genet 10:26 (2009)
36. Roguedas, A. M., M. P. Audrezet, V. Scotet, D. Dupré-Goetghebeur, C. Ferec, and L. Misery: Intrinsic atopic dermatitis is associated with a beta-2 adrenergic receptor polymorphism. Acta Derm Venereol 86, 447-448 (2006)
37. Szczepankiewicz, A., A. Breborowicz, P. Sobkowiak, L. Kramer, and A. Popiel: Role of ADRB2 gene polymorphism in asthma and response to beta(2)-agonists in Polish children. J Appl Genet 50, 275-281 (2009)
40. Liang, S. Q., X. L. Chen, J. M. Deng, X. Wei, C. Gong, Z. R. Chen, and Z. B. Wang: Beta-2 Adrenergic Receptor (ADRB2) Gene Polymorphisms and the Risk of Asthma: A Meta-Analysis of Case-Control Studies. PLoS One 9, e104488 (2014)
41. Guo, X., H. Zheng, C. Mao, E. Guan, and H. Si: An association and meta-analysis study of 4 SNPs from beta-2 adrenergic receptor (ADRB2) gene with risk of asthma in children. Asian Pac J Allergy Immunol 34, 11-20 (2016)
42. Thakkinstian, A., M. McEvoy, C. Minelli, P. Gibson, B. Hancox, D. Duffy, J. Thompson, I. Hall, J. Kaufman, T. F. Leung, P. J. Helms, H. Hakonarson, E. Halpi, R. Navon, and J. Attia: Systematic Review and Meta-Analysis of the Association between β2-Adrenoceptor Polymorphisms and Asthma: A HuGE Review. Am J Epidemiol 162, 201-211 (2005)
43. Karam, R. A., N. A. Sabbah, H. E. Zidan, and H. M. A. Rahman: Association between Genetic Polymorphisms of ß2 Adrenergic Receptors and Nocturnal Asthma in Egyptian children. J Investig Allergol Clin Immunol 23, 262-266 (2013)
44. Hamdy, S. I., M. Hiratsuka, K. Narahara, M. El-Enany, N. Moursi, M. S. Ahmed, and M. Mizugaki: Allele and genotype frequencies of polymorphic DCP1, CETP, ADRB2, and HTR2A in the Egyptian population. Eur J Clin Pharmacol 58, 29-36 (2002)
45. Ibrahim, G. H., F. A. Khalil, F. Mostafa, M. S. Fawzy, M. Said, A. E. Omar, and T. B. El-Abaseri: Analysis of common MEFV mutations in Egyptian patients with familial Mediterranean fever: molecular characterisation of the disease. Br J Biomed Sci 67, 202-207 (2010)
52. Naghan, P., F. Fanak, A. Seyed, N. Nima, and R. Mohammad: A pilot study of polymorphism of adrenergic beta-2 receptor and mild asthma: A clinical and pharmacogenetic study. Iran J Pharm Res 12, 199–204 (2013)
57. Adriani, K., M. Brouwer, F. Baas, A. Zwinderman, A. van der Ende, and D. van de Beek: Genetic variation in the β2-adrenocepter gene is associated with susceptibility to bacterial meningitis in adults. PLoS One 7, e37618 (2012)