Advances in Molecular Biology
Volume 2014 (2014), Article ID 471635, 11 pages
http://dx.doi.org/10.1155/2014/471635

Research Article

Beta₂-Adrenergic Receptor Gene Polymorphisms in Egyptian Patients with Acute Myocardial Infarction

Eman Toraih,¹ Mohammad H. Hussein,² and Dahlia I. Badran³

¹Genetics Unit, Department of Histology and Cell Biology, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt
²Department of Chest Diseases, Faculty of Medicine, Cairo University, Giza, Egypt
³Department of Medical Biochemistry, Faculty of Medicine, Suez Canal University, Ismailia 41522, Egypt

Received 31 July 2014; Revised 8 November 2014; Accepted 17 November 2014; Published 9 December 2014

Academic Editor: Piero R. Bianco

Copyright © 2014 Eman Toraih et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Background. Beta₂-adrenergic receptor (ADRB2) gene polymorphisms, Arg16Gly and Gln27Glu, have been implicated in the pathogenesis of cardiovascular diseases. The aim of this study was to determine the association of these two polymorphisms with the risk of myocardial infarction (MI) in the Egyptian population. Methods. Blood samples were collected from 68 MI patients and 75 healthy controls. They were assessed for the presence of cardiovascular risk factors and genotyped for the Arg16Gly (rs1042713) and Gln27Glu (rs1042714) polymorphisms using allelic-discrimination polymerase chain reaction. Results. There is no significant difference in genotype and allele frequencies at codon 16 between MI patients and controls (). However, at codon 27, MI risk was higher in Gln₂₇ homozygous participants than in Glu₂₇ carriers (). The haplotype frequency distribution showed significant difference among cases and controls (); homozygotes for Gly₁₆/Gln₂₇ haplotype were more susceptible to MI than Gly₁₆/Glu₂₇ carriers. Patients with Arg₁₆/Gln₂₇ haplotype had higher serum total cholesterol levels () and lower frequency of diabetes in MI patients (). However, both Glu₂₇ genotypes and haplotype showed lower frequency of hypertension (). Conclusions. Our findings suggested that the ADRB2 gene polymorphisms may play an important role in susceptibility of MI among Egyptian population.

1. Introduction

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide, accounting for up to 40% of all deaths [1, 2]. It is caused by myocardial cell death due to prolonged ischemia [3]. MI is a multifactorial, polygenic disorder driven by interactions of an individual’s genetic background and several environmental factors [4–7]. Greater understanding of MI etiology is mandatory to identify individuals at high risk and to improve prevention and therapy of this common and important condition. Cardiovascular risk factors, as smoking, obesity, hypertension, dyslipidemia, and diabetes, have long been known [8]. However these risk factors do not explain why some individuals are more susceptible to these environmental determinants in comparison to others with the same given risk factors. Several genomic association studies, family-based and twin studies, and linkage analysis provide insights into multiple susceptible genes underlying MI disease [9, 10].

The beta 2-adrenergic receptors (-AR), a member of the G-protein-coupled receptor superfamily, have attracted great attention due to their multiple physiological and health effects, particularly those involving cardiovascular phenotypes [11–16]. In vascular smooth muscle, -AR mediate vasodilation in response to adrenergic agonists, and, in healthy myocardium, they mediate chronotropic and inotropic responses to endogenous and exogenous adrenergic agents [17–21]. These functions render -AR an important target for cardiovascular disease (CVD) therapy [15, 22, 23]. Interestingly, studies reported -AR dysfunction to be implicated in the pathogenesis of several CVD including hypertension [24–27], coronary heart disease [13, 23], idiopathic dilated cardiomyopathy [28], congestive heart failure [29, 30], idiopathic ventricular arrhythmias [31], and sudden cardiac death [32].

Several single nucleotide polymorphisms (SNP) in the -AR (ADRB2) gene (MIM# 109690) have been identified in many populations [33, 34]. The most important two SNPs are located within the amino-terminal extracellular domain near the receptor’s ligand-binding site [35]. The first SNP was caused by a change of adenine to guanine (A > G) at nucleotide 46 resulting in amino acid substitution from arginine to glycine at codon 16 (Arg16Gly or R16G). The second SNP is the change of cytosine to guanine (C > G) at nucleotide 79 causing the substitution from glutamine to glutamic acid at codon 27 (Gln27Glu or Q27E) [15, 36, 37]. In vivo and in vitro functional studies suggested that these two polymorphisms have altered receptor function and behavior [38–42]. Several clinical and pharmacological studies highlighted the important role of these polymorphisms in the pathogenesis of CVD and their impact on drug response [22, 30, 39, 43]. However, evidence regarding the role of ADRB2 gene polymorphisms in patients with MI is ambiguous and inconsistent. Significant differences in the genotype frequencies and haplotype structure of ADRB2 gene were noted in different ethnic populations [13, 23, 25, 44, 45]. Thus, this case-control study was conducted to determine the prevalence of these ADRB2 gene polymorphisms in Egyptian population and to assess their genetic association with the susceptibility to MI disease.

2. Materials and Methods

2.1. Study Participants

The study population was composed of 68 patients with acute myocardial infarction (MI) and 75 age-matched healthy controls with no history of cardiovascular events, hypertension, or any other chronic diseases. Patients were recruited from Coronary Care Unit (CCU), Suez Canal University Hospital, Ismailia, Egypt, during the period between April 2013 and March 2014. The diagnosis of MI was confirmed by evidence of symptoms in the presence of either diagnostic elevations of cardiac enzymes or diagnostic changes on electrocardiograms (ECG) [46]. The cardiovascular risk factors were assessed in all participants. They underwent evaluations including measurement of blood pressure, body mass index (BMI), fasting serum lipid and glucose levels, cardiac enzymes, and ECG. Diabetic patients either had a known history of diabetes mellitus or were diagnosed according to the American Diabetic Association recommendations [47]. Hypertension was defined as the average SBP ≥ 140 mmHg and/or the average DBP ≥ 90 mmHg and/or self-reported current treatment for hypertension with antihypertensive medication [48]. The study was approved by the Medical Research Ethics Committee of Faculty of Medicine, Suez Canal University. Written informed consent was obtained from all participants.

2.2. Samples Collection and Biochemical Measurements

Fasting blood samples were collected from both cases and controls. Blood from patients was collected within 24 hours of CCU admission. Two milliliters (mL) of venous blood was withdrawn in EDTA tubes to be used for subsequent genetic analysis. Another two milliliters was collected in Vacutainer Serum Separator Tubes II (Becton Dickinson Plymouth) to obtain serum. Parameters such as fasting blood sugar (FBS), total cholesterol (TC), triglycerides (TG), high density lipoproteins-C (HDL-C), and low density lipoprotein-C (LDL-C) were analyzed within 2 h after collection. Fasting blood sugar was estimated using Roche/Hitachi analyzer kits based on the glucose oxidase and peroxidase method [49]. Serum TC and TG were measured by enzymatic colorimetric determination method using Roche/Hitachi cholesterol kit (Cat. number 1489232) [50, 51] and Roche/Hitachi triglycerides kit (Cat. number 1488872) [52]. HDL-C was measured using Roche/Hitachi HDL-C kit (Cat. number 03030024) and was estimated by phosphotungstic acid precipitation followed by enzymatic analysis in supernatant fraction [53]. LDL-C was calculated by the Friedewald formula after considering its limitations if TG > 400 mg/dL: [54]. FBS < 126 mg/dL, TC levels <200 mg/dL, TG levels <150 mg/dL, LDL-C < 130 mg/dL, and HDL-C > 60 mg/dL were considered normal [55].

2.3. Genotyping

Genomic DNA was extracted from blood leukocytes using QIAamp DNA Blood Mini kit (QIAgen, Clinilab Co., Egypt, Catalog number 51104) according to the manufacturer’s instructions [56]. Concentration and purity of the extracted DNA were measured by NanoDrop ND-1000 (NanoDrop Tech., Inc. Wilmington, DE, USA). Genotyping of the ADRB2 polymorphisms (Arg16Gly, rs1042713, and Gln27Glu, rs1042714) was performed by real-time polymerase chain reaction (RT-PCR) technology. PCR was performed in a 25-μL reaction volume containing 12.5 μL Taqman Universal PCR Master Mix, No AmpErase UNG (2x), 20 ng genomic DNA diluted to 11.25 μL with DNase-RNase-free water, and 1.25 μL 20 × TaqMan SNP Genotyping Assay Mix (Applied Biosystems, Egypt, assay ID C_2084764_20 and C_2084765_20). Internal positive controls and no-template controls (no DNA) were included in each run. PCR amplification was carried out in an AB 7500HT instrument with the Sequence Detection System (SDS) Software version 2.1.1 (Applied Biosystems, Egypt) according to the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of 92°C for 15 sec and 60°C for 1 min. Genotyping was performed blinded to case/control status. Ten per cent of the randomly selected samples were regenotyped in separate runs to exclude the possibility of false genotype calls with 100% concordance rate.

2.4. Statistical Analysis

Statistical analysis was carried out using the Microsoft Excel 2010 and the “Statistical Package for the Social Sciences (SPSS) for windows” software, version 20. Odds ratios (OR) with a 95% confidence interval (CI) were calculated. Categorical variables were compared using the chi-square (χ ²), while Mann-Whitney and Kruskal-Wallis tests were used to compare continuous variables. Due to significant differences in some cardiovascular risk factor variables between cases and control participants, binary logistic regression analysis was performed that included the ADRB2 polymorphism and potential confounders (age, gender, smoking status, family history of cardiovascular disease, prevalence of obesity, and hyperlipidemia). A two-tailed value of 0.05 was considered statistically 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. The carriage rate was calculated as the number of individuals carrying at least one of the investigated alleles divided by the total number of individuals in each group [57, 58]. The Hardy-Weinberg equilibrium was calculated using the Online Encyclopedia for Genetic Epidemiology (OEGE) software (http://www.oege.org/software/hwe-mr-calc.shtml) and tested by χ ² test to compare the expected genotype frequencies among patient and control groups.

3. Results

3.1. Molecular Analysis of ADRB2 Gene Polymorphisms

A total of 68 patients and 75 controls were included in the analysis. Characteristics of the study population are shown in Table 1. SNP analysis of Arg16Gly (R16G; rs1042713) and Gln27Glu (Q27E; rs1042714) polymorphisms of ADRB2 gene showed that the frequencies of Arg₁₆ and Gly₁₆ alleles in our study population were 0.37 and 0.63, while those of Gln₂₇ and Glu₂₇ alleles were 0.67 and 0.33, respectively. The distribution of Arg16Gly genotypes and alleles did not differ significantly between MI patients and controls. For Gln27Glu polymorphism, Gln₂₇/Glu₂₇ was significantly the most prevalent genotype in the healthy control group (50.7%) while Gln₂₇/Gln₂₇ was the most predominant genotype among patients (50%) (). The frequency of Glu₂₇ allele was significantly lower in MI patients compared to controls (33.1% versus 45.3%; ); see Table 2. The distribution of ADRB2 genotypes among patients and controls was found in accordance with those expected by the Hardy-Weinberg equilibrium (). Haplotype analysis revealed the presence of three haplotypes only: Arg₁₆/Gln₂₇, Gly₁₆/Gln₂₇, and Gly₁₆/Glu₂₇. Their frequencies were 0.30, 0.29, and 0.41, respectively. Different combinations of haplotypes existed in the study population and showed significant difference between MI patients and healthy controls (); see Table 3. The frequency of Gly₁₆/Glu₂₇ haplotype was significantly lower in MI patients than controls (). In addition, homozygote individuals for Gly₁₆/Gln₂₇ haplotype had higher risk of developing MI compared to noncarriers, with adjusted OR (95% CI) of 20.4 (1.4–296); see Table 4.

Table 1: Baseline characteristics of the study groups.

Variables
Patients
()
Controls
()
value*

Risk factors for CVD

    Age, y
53.5 ± 7.4
52.8 ± 6.1
0.203

    Male gender, %
45 (66.2)
53 (70.7)
0.252

    Smokers, %
37 (54.4)
15 (20.0)
<0.001

    FH of CVD, %
35 (51.5)
17 (22.7)
<0.001

    BMI, kg/m²
29.5 ± 3.6
27.4 ± 2.7
<0.001

    Hypertension, %
48 (70.6)
—

    SBP, mmHg
129.7 ± 22.7
122.0 ± 11.2
0.010

    DBP, mmHg
83.8 ± 13.3
77.5 ± 7.7
0.001

    Diabetic, %
32 (47.1)
—

    FBS, mg/dL
129.2 ± 45.7
98.2 ± 11.3
<0.001

Lipid profile

    Hyperlipidemia, %
62 (91.2)
38 (50.6)
<0.001

    TC, mg/dL
199 ± 40
174 ± 40
0.002

    TG, mg/dL
203 ± 63
151 ± 84
<0.001

    HDL-C, mg/dL
38 ± 7.4
47.4 ± 12
<0.001

    LDL-C, mg/dL
121 ± 41
96 ± 40
0.001

    VLDL-C, mg/dL
40.5 ± 13
30.2 ± 17
<0.001

Cardiac enzymes

    CK, ng/mL
1688 ± 1832
—

    CK-MB. ng/mL
320 ± 370
—

    LDH, IU/L
1562 ± 1223
—

Values are presented as number (percentage) or mean ± standard deviation. Chi-square () and Mann-Whitney tests were used. *Statistically significant at < 0.05; FH: family history; CVS: cardiovascular disease; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBS: fasting blood sugar; TC: total cholesterol; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol; CK: total creatine kinase; CK-MB: creatine kinase isoenzyme MB fraction; LDH: lactate dehydrogenase.

Table 2: Genotype and allele frequencies of ADRB2 gene polymorphisms in patients and controls.

Variables
Patients ()
Controls ()

value
Crude   
OR (95% CI)
Adjusted&   
OR (95% CI)

Arg16Gly polymorphism

Genotype frequency

    Arg₁₆/Arg₁₆
10 (14.7)
08 (10.7)
0.010
0.919
1.0
1.0

    Arg₁₆/Gly₁₆
30 (44.1)
40 (53.3)

0.6 (0.2–1.7)
0.3 (0.07–2.0)

    Gly₁₆/Gly₁₆
28 (41.2)
27 (36.0)

0.8 (0.2–2.4)
1.9 (0.3–9.9)

Allele frequency

    Arg₁₆ allele
50 (36.8)
56 (37.3)
0.009
0.920
1.0
0.2 (0.1–0.7)

    Gly₁₆ allele
86 (63.2)
94 (62.7)

1.0 (0.6–1.6)
0.8 (0.2–3.7)

Gln27Glu polymorphism

Genotype frequency

    Gln₂₇/Gln₂₇
34 (50.0)
22 (29.3)
4.012
0.045 *
1.0
1.0

    Gln₂₇/Glu₂₇
23 (33.8)
38 (50.7)

0.3 (0.1–0.8) *
0.5 (0.1–1.5)

    Glu₂₇/Glu₂₇
11 (16.2)
15 (20.0)

0.4 (0.1–1.2)
1.9 (0.4–8.2)

Allele frequency

    Gln₂₇ allele
91 (66.9)
82 (54.7)
4.459
0.034 *
1.0
0.2 (0.06–1.1)

    Glu₂₇ allele
45 (33.1)
68 (45.3)

0.5 (0.3–0.9) *
0.5 (0.1–1.5)

Values are shown as number (%). Chi-square () for trend was used. OR (95% CI): odds ratio and confidence interval. &Adjusted for confounding factors. Adjusted OR for alleles was calculated as presence versus absence of this particular allele. *Statistically significant at .

Table 3: Haplotype combinations of ADRB2 gene polymorphisms in patients and controls.

Haplotype combinations
Patients ()
Controls ()

value
Crude  
OR (95% CI)
Adjusted&  
OR (95% CI)

Gly₁₆/Glu27 + Gly₁₆/Glu₂₇
11 (16.2)
15 (20.0)
9.404
0.002 *
1.0
1.0

Gly₁₆/Glu₂₇ + Gly₁₆/Gln27
5 (7.40)
13 (17.3)

0.5 (0.1–1.9)
0.2 (0.02–1.5)

Gly₁₆/Glu₂₇ + Arg₁₆/Gln27
18 (26.5)
25 (33.3)

0.9 (0.3–2.6)
0.2 (0.05–1.3)

Gly₁₆/Gln₂₇ + Arg₁₆/Gln27
12 (17.6)
15 (20.0)

1.1 (0.3–3.2)
0.1 (0.01–0.8)

Arg₁₆/Gln27 + Arg₁₆/Gln₂₇
10 (14.7)
6 (8.00)

2.2 (0.6–8.1)
0.5 (0.07–3.9)

Gly₁₆/Gln27 + Gly₁₆/Gln₂₇
12 (17.6)
1 (1.30)

16.3 (1.8–145) *
9.0 (0.5–161)

Values are shown as number (%). Chi-square () for trend was used. OR (95% CI): odds ratio and confidence interval. &Adjusted for confounding factors. *Statistically significant at .

Table 4: Haplotype frequencies of ADRB2 gene polymorphisms in patients and controls according to the copy number.

Variables
Patients ()
Controls ()

value
Crude  
OR (95% CI)
Adjusted&  
OR (95% CI)

Arg₁₆/Gln₂₇

    Negative
28 (41.2)
29 (38.7)
0.146
0.703
1.0
1.0

    1 copy
30 (44.1)
40 (53.3)

0.7 (0.3–1.5)
0.4 (0.1–1.66)

    2 copies
10 (14.7)
6 (8.0)

1.7 (0.5–5.3)
0.5 (0.07–3.9)

Gly₁₆/Gln₂₇

    Negative
39 (57.4)
46 (61.4)
3.378
0.066
1.0
1.0

    1 copy
17 (25.0)
28 (37.3)

0.7 (0.3–1.4)
0.3 (0.1–1.08)

    2 copies
12 (17.6)
1 (1.3)

14.1 (1.7–113) *
20.4 (1.4–296) *

Gly₁₆/Glu₂₇

    Negative
34 (50.0)
22 (29.3)
4.012
0.045 *
1.0
1.0

    1 copy
23 (33.8)
38 (50.7)

0.3 (0.1–0.8) *
0.5 (0.17–1.5)

    2 copies
11 (16.2)
15 (20.0)

0.4 (0.1–1.2)
1.9 (0.47–8.2)

Values are shown as number (%). Chi-square () for trend was used. OR (95% CI): odds ratio and confidence interval. &Adjusted for confounding factors. *Statistically significant at .

3.2. ADRB2 Gene Polymorphisms and Disease Characteristics

Characteristics of patients according to ADRB2 genotypes and haplotypes are demonstrated in Tables 5 and 6. Diabetes mellitus (DM) frequency was significantly higher in Gly₁₆/Gly₁₆ genotype carriers (62.5%) compared to other genotypes carriers (6.2% and 31.3% for Arg₁₆/Arg₁₆ and Arg₁₆/Gly₁₆, resp.) (). The frequency of hypertension was significantly higher in MI patients with Gln₂₇/Gln₂₇ genotype (66.7%) compared to their counterparts (25% for Gln₂₇/Glu₂₇ and 8.3% for Glu₂₇/Glu₂₇) (). In addition, MI patients with the Arg₁₆/Gln₂₇ haplotype had significantly higher serum cholesterol levels compared to noncarriers ( versus , ).

Table 5: Disease characteristics of patients according to ADRB2 genotypes ().

Characteristics
Arg16Gly genotypes
value
Gln27Glu genotypes
value

Arg₁₆/Arg₁₆
Arg₁₆/Gly₁₆
Gly₁₆/Gly₁₆
Gln₂₇/Gln₂₇
Gln₂₇/Glu₂₇
Glu₂₇/Glu₂₇

Total number
10 (14.7)
30 (44.1)
28 (41.2)

34 (50.0)
23 (33.8)
11 (16.2)

Risk factors for CVD

    Age at diagnosis
52 ± 7.4
53 ± 8.1
55 ± 6.6
0.577
53 ± 7.7
55 ± 7.7
53 ± 5.3
0.367

    Male gender, %
7 (15.5)
22 (48.9)
16 (35.6)
0.412
22 (44.0)
21 (42.0)
7 (14.0)
0.059

    Smokers, %
4 (10.8)
20 (54.1)
13 (35.1)
0.185
18 (48.7)
15 (40.5)
4 (10.8)
0.278

    FH of CVD, %
7 (20.0)
16 (45.7)
12 (34.3)
0.325
21 (60.0)
10 (28.6)
4 (11.4)
0.219

    Hypertension, %
10 (20.8)
20 (41.7)
18 (37.5)
0.085
32 (66.7)
12 (25.0)
4 (8.30)
<0.001 *

    DM, %
2 (6.25)
10 (31.3)
20 (62.5)
0.003 *
14 (43.7)
11 (34.4)
7 (21.9)
0.429

    BMI, Kg/m²
32 ± 3.8
30 ± 4.0
29 ± 3.6
0.080
30 ± 3.3
29 ± 4.1
28 ± 2.9
0.195

Lipid profile

    Hyperlipidemia, %
10 (16.1)
28 (45.2)
24 (38.7)
0.336
31 (50.0)
20 (32.3)
11 (17.7)
0.455

    Total cholesterol, mg/dL
202 ± 33
208 ± 38
188 ± 43
0.085
199 ± 41
207 ± 40
181 ± 32
0.139

    Total triglyceride, mg/dL
228 ± 45
200 ± 74
195 ± 54
0.121
199 ± 56
199 ± 81
218 ± 38
0.199

    HDL-C, mg/dL
38 ± 5.2
39 ± 6.7
37 ± 8.7
0.374
38 ± 7.7
39 ± 8.0
36 ± 4.8
0.883

    LDL-C, mg/dL
119 ± 30
1129 ± 37
111 ± 46
0.125
122 ± 43
128 ± 39
101 ± 35
0.099

    VLDL-C, mg/dL
46 ± 8.9
40 ± 15
39 ± 11
0.121
40 ± 11
40 ± 16
44 ± 7.5
0.199

Cardiac enzymes

    CK, ng/mL
1624 ± 1422
1499 ± 1654
1912 ± 2149
0.746
1821 ± 2088
1717 ± 1735
1215 ± 1078
0.791

    CK-MB. ng/mL
362 ± 412
328 ± 336
294 ± 402
0.306
294 ± 320
450 ± 470
126 ± 107
0.089

    LDH, IU/L
1666 ± 603
1368 ± 1161
1733 ± 1437
0.289
1775 ± 1517
1301 ± 882
1451 ± 625
0.506

Values are shown as mean ± standard deviation or number (percentage). CVD: cardiovascular disease; DM: diabetes mellitus; BMI: body mass index; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; CK: total creatine kinase; CK-MB: creatine kinase isoenzyme MB fraction; LDH: lactate dehydrogenase. Two-sided chi-square and Kruskal-Wallis tests were used.

*Statistically significant at < 0.05.

Table 6: Disease characteristics of patients according to ADRB2 haplotypes ().

Characteristics
Arg₁₆/Gln₂₇
value
Gly₁₆/Gln₂₇
value
Gly₁₆/Glu₂₇
value

Positive
Negative
Positive
Negative
Positive
Negative

Total number
40 (59)
28 (41)

29 (42.7)
39 (57.3)

34 (50)
34 (50)

Risk factors for CVD

    Age at diagnosis
53 ± 7.9
55 ± 6.6
0.355
53.8 ± 7.8
53.3 ± 7.1
0.582
54.3 ± 7.1
52.7 ± 7.7
0.443

    Male gender, %
29 (64.4)
16 (35.6)
0.188
16 (35.6)
29 (64.4)
0.098
25 (55.6)
20 (44.4)
0.200

    Smokers, %
24 (64.9)
13 (35.1)
0.269
12 (38.7)
19 (61.3)
0.548
15 (48.4)
16 (51.6)
0.808

    FH of CVD, %
17 (51.5)
16 (48.5)
0.234
13 (39.4)
20 (60.6)
0.598
20 (60.6)
13 (39.4)
0.089

    Hypertension, %
30 (62.5)
18 (37.5)
0.340
24 (50.0)
24 (50.0)
0.058
16 (33.3)
32 (66.7)
<0.001 *

    DM, %
12 (37.5)
20 (62.5)
0.001 *
17 (53.1)
15 (46.9)
0.100
18 (56.3)
14 (43.7)
0.331

    BMI, Kg/m²
30.2 ± 4
28.5 ± 3
0.121
29.1 ± 3
29.7 ± 4
0.955
28.9 ± 4
30.0 ± 3
0.090

Lipid profile

    Hyperlipidemia, %
38 (61.3)
24 (38.7)
0.184
25 (40.3)
37 (59.7)
0.213
31 (50.0)
31 (50.0)
1.000

    Total cholesterol, mg/dL
206 ± 36
188 ± 42
0.030 *
192 ± 44
204 ± 36
0.070
198 ± 39
199 ± 41
0.731

    Total triglyceride, mg/dL
207 ± 68
196 ± 54
0.945
192 ± 59
210 ± 65
0.330
205 ± 71
199 ± 56
0.864

    HDL-C, mg/dL
38.5 ± 6.3
37.1 ± 8
0.170
38 ± 8.7
38.2 ± 6
0.471
38.0 ± 7.1
37.9 ± 7
0.917

    LDL-C, mg/dL
126 ± 36
112 ± 46
0.061
115 ± 48
124 ± 34
0.133
119 ± 39
121 ± 43
0.722

    VLDL-C, mg/dL
41.4 ± 13
39.2 ± 11
0.945
38.4 ± 11
42.1 ± 13
0.330
41.1 ± 14
39.9 ± 11
0.864

Cardiac enzymes

    CK, ng/mL
1530 ± 1583
1912 ± 2149
0.842
1978 ± 2359
1471 ± 1305
0.995
1554 ± 1554
1821 ± 2088
0.581

    CK-MB. ng/mL
337 ± 351
294 ± 402
0.159
327 ± 405
314 ± 347
0.660
345 ± 418
293 ± 320
0.835

    LDH, IU/L
1442 ± 1051
1733 ± 1437
0.852
1655 ± 1662
1493 ± 768
0.267
1350 ± 801
1774 ± 1517
0.358

Values are shown as mean ± standard deviation or number (percentage). CVD: cardiovascular disease; FH: family history; DM: diabetes mellitus; BMI: body mass index; FBS: fasting blood sugar; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; CK: total creatine kinase; CK-MB: creatine kinase isoenzyme MB fraction; LDH: lactate dehydrogenase. Two-sided chi-square and Mann-Whitney tests were used.

*Statistically significant at < 0.05.

4. Discussion

To the best of our knowledge, this is the first work examining the association of Arg16Gly and Gln27Glu polymorphisms of beta₂-adrenergic receptor with acute myocardial infarction in Egyptian population. Frequency distribution of alleles and genotypes was in Hardy-Weinberg equilibrium for both SNPs. Gly₁₆ and Gln₂₇ were the most prominent alleles, representing 0.63 and 0.67 of the population, respectively. Correspondingly, Gly₁₆/Gly₁₆ and Arg₁₆/Gly₁₆ at codon 16 and Gln₂₇/Gln₂₇ and Gln₂₇/Glu₂₇ at codon 27 were the most common genotypes in the Egyptian population. This was in agreement with another Egyptian study where Gly₁₆ and Gln₂₇ were the most frequent alleles representing 0.60 and 0.75, respectively [59]. Similar frequencies in prior studies were found in different ethnic populations (African American, Caucasian, and Saudian populations) [33, 35, 39, 60, 61]. In contrast, individuals with Asian descent reported higher frequencies of Arg₁₆ and Gln₂₇ alleles and absence of Glu₂₇/Glu₂₇ genotype [39, 59, 60, 62–64]. Haplotype analysis revealed the presence of 3 haplotypes only; their frequencies were 30% for Arg₁₆/Gln₂₇, 29% for Gly₁₆/Gln₂₇, and 41% for Gly₁₆/Glu₂₇. The absence of Arg₁₆/Glu₂₇ was consistent across several haplotype studies conducted in different ethnic populations, including African Americans, Brazilians, Europeans, Asians, and Hispanic-Latinos populations [35, 36, 63–69]. Strong linkage disequilibrium (LD) exists between these two SNPs, and, as a result, the Arg₁₆ allele is typically seen with Gln₂₇, whereas Gly₁₆ can coexist with either Gln₂₇ or Glu₂₇ [33, 59, 60, 63, 70].

In the present study, the distribution of Arg16Gly genotypes and alleles did not differ significantly between cases with MI and control subjects. This was in agreement with others who reported no effect of Arg16Gly polymorphism with the occurrence of MI disease among Americans, Europeans, and Turkish populations [15, 19, 25, 71]. However, for Gln27Glu polymorphism, our results showed higher frequency of the Gln₂₇ variants among patients and that the Glu₂₇ allele conferred protection against the occurrence of MI disease. This was similar to previous studies which reported a significant association of Gln₂₇ homozygotes with coronary events, ventricular tachyarrhythmias, and sudden cardiac death [14, 15, 27, 32, 72]. Others highlighted the protective role of Glu₂₇ allele against MI in young and elder populations [14, 19, 44, 71]. Also, Kaye et al. showed that subjects with the Glu₂₇ allele had a significant improvement in left ventricular ejection fraction compared with subjects homozygous for Gln₂₇ [73]. In contrast, one study found the opposite correlation; carriers of Glu₂₇ allele had a higher incidence of coronary artery disease and a higher likelihood of later need for coronary revascularization [74], while others did not reveal such associations with MI [11, 13, 23, 75].

The Arg16Gly and Gln27Glu polymorphisms are known to alter the functional properties of the receptor and its behavior after agonist exposure [38, 76]. Although these two amino terminal polymorphisms are located near the ligand-binding site, they do not alter the binding capacity of endogenous or exogenous catecholamines to -AR or affect further G-protein coupling and adenylyl cyclase activation [35, 77]. In addition, receptor synthesis rates and agonist-promoted internalization were not different between the receptors [78, 79]. However, studies of agonist stimulation in cultured cells demonstrated that the Glu₂₇ receptor exhibited enhanced resistance to downregulation when compared with the Gln₂₇ variant, as assessed by receptor number [77, 80]. Other in vitro studies proposed that this desensitization phenomenon might be due to differential alterations in receptor degradation after the internalization step [78, 81, 82]. Moreover, another difference was demonstrated by cell-signaling studies in two cell lines; the Glu₂₇ variant was associated with magnification of the catecholamine-induced activation of intracellular signaling transduction [83], thus likely promoting superior cardiovascular performance in human [84].

To examine possible additive or synergistic effects of the two tested polymorphisms, haplotype analysis within the ADRB2 locus was done, revealing significant difference in the distribution frequencies of haplotype combinations between MI patients and healthy controls. Homozygotes for Gly₁₆/Gln₂₇ haplotype showed genetic susceptibility for MI in our population samples, whereas heterozygote individuals with other haplotype combinations (Gly₁₆/Gln₂₇ + Arg₁₆/Gln₂₇ or Gly₁₆/Gln₂₇ + Gly₁₆/Glu₂₇) did not show a significant association with disease risk, thus highlighting a clear dose-response relationship between Gly₁₆/Gln₂₇ and MI. Moreover, carriers of Gly₁₆/Glu₂₇ haplotype had lower prevalence of MI disease and showed protection against the occurrence of the disease. On the other hand, Arg₁₆/Gln₂₇ haplotype did not influence the susceptibility for developing MI in our Egyptian population. Similarly, two clinical studies showed differential distribution of haplotype frequencies of ADRB2 gene between MI cases and controls in the American population, with evidence showing dominant protective effect of the Gly₁₆/Glu₂₇ haplotype on MI and other age-related phenotypes, whereas the presence of any detrimental effect was largely associated with the Gln₂₇ allele and Gly₁₆/Gln₂₇ haplotype [71, 85]. This was in line with other previous studies which reported significant association between Gly₁₆/Gln₂₇ haplotype and low exercise performance in heart failure patients [82]. Consistent with this hypothesis, Gly₁₆/Glu₂₇ was shown to be the optimal haplotype combination for cardiovascular response during exercise because of increased receptor numbers and enhanced stroke volume and cardiac output [84, 86] and to elicit more β-blocker drug response in postischemic heart [87]. In contrast, two studies reported a decreased frequency of Gly₁₆/Gln₂₇ haplotype in male and female Caucasian patients with MI [13, 88]. Other studies, however, did not find association between the ADRB2 haplotype and the risk of MI [25, 89, 90]. We propose two explanations underlying the association of Gly₁₆/Gln₂₇ haplotype homozygosity with MI development. Several lines of evidence indicated that ADRB2, like other G-protein-coupled receptors, form dimers. The formation of -AR dimers was shown to have functional effects on receptor stimulated adenylate cyclase activity [91]. So one possible mechanism is the interaction between two receptors with Gly₁₆/Gln₂₇ haplotype forming “homodimer” may have different agonist binding, signal transduction, or agonist-promoted desensitization properties than other types of dimers. However, there is an alternative possibility which is that Arg16Gly and Gln27Glu polymorphisms might be in LD with other polymorphic loci in ADRB2 gene or nearby inflammatory mediator genes, which have direct effect on the pathogenesis of MI [92]. Drysdale et al. described other molecular haplotypic structure of ADRB2 including our SNPs [66]. One of them, Arg19Cys polymorphism in the 5′-leader cistron, was found to be in LD with Gln27Glu polymorphism [93] and caused altered receptor translation, hence affecting -AR protein expression in vitro [32, 94, 95].

Another main finding in our study was the difference in genotype distribution of the ADRB2 Gln27Glu polymorphism between hypertensive and normotensive MI patients. Homozygotes of the Gln₂₇ variant had a significant higher frequency of hypertension compared to other genotype carriers. This was in agreement with previous studies conducted in Americans, European, and Han Chinese populations [11, 92, 95]. Binder et al. reported association between both Gln₂₇ haplotypes (Gly₁₆/Gln₂₇ and Arg₁₆/Gln₂₇) and high diastolic blood pressure in Caucasian males [96]. Subjects with the Gln₂₇ variant were shown to have attenuated vasodilatory response to infused isoproterenol, while those with Glu₂₇ variant had more robust reversal of constriction than did the Gln₂₇ in normotensive males [87, 97]. Furthermore, carriers of the allele Glu₂₇ and Gly₁₆/Glu₂₇ haplotype showed increased forearm vasodilatation during mental stress and handgrip exercise [98] and might cause arterial hypotension with thoracic epidural anesthesia [99], presumably due to its minimal downregulation by catecholamines, hence increasing -AR signaling [87]. In contrast, others demonstrated that the variation within codons 16 and 27 of ADRB2 gene were unlikely to confer genetic susceptibility for hypertension in Americans, black South Africans, and Asians suggesting LD with other unidentified genetic variants [48, 56, 64, 100–102].

Assessment of other cardiovascular risk factors in our MI patients revealed a significant difference in genotype frequencies of ADRB2 Arg16Gly polymorphism between diabetic and nondiabetic MI patients. The frequency of diabetes mellitus (DM) was significantly higher in patients with Gly₁₆/Gly₁₆ genotype than other genotypes. The -AR are known to be expressed in a variety of tissues including pancreatic beta-cells and to play a key role in the regulation of glucose metabolism as well as insulin sensitivity and secretion [103, 104]. Masuo et al. have reported significant association between the Gly₁₆ variant of the ADRB2 gene with increased insulin resistance [105]. Prior et al. suggested that ADRB2 haplotypes mediate insulin action, glucose tolerance, and potentially risk for type 2 diabetes mellitus in obese, postmenopausal women [104]. On the contrary, homozygosity of Arg₁₆ allele in the ADRB2 gene was associated with a higher frequency for development of type 2 diabetes in Taiwanese and Danish populations [106, 107], while no association with type 2 diabetes was found in Koreans [108], Tongan population [109], and young Danish males [110].

In our MI patients, Arg₁₆/Gln₂₇ haplotype had higher total cholesterol levels compared to their counterpart. Earlier studies have suggested that the Arg16Gly polymorphism may be associated with cholesterol metabolism in certain populations [39]. In Han Chinese population, Arg₁₆ homozygotes had higher serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels [111]. An inverse association was found in Saudian population with higher levels of total cholesterol, triglycerides, and LDL-C in Arg₁₆/Gly₁₆ and Gly₁₆/Gly₁₆ individuals [16]. In addition, the Gly₁₆ homozygotes had a lower HDL-C level than the Arg₁₆ homozygotes in Japanese population [27], while no association with dyslipidemia was found with ADRB2 gene polymorphisms at codons 16 and 27 in Korean population [108].

The inconsistent associations of ADRB2 variants in our results with some prior studies could be caused by the genetic heterogeneity among different ethnic groups [59, 66, 112], since the susceptibility to MI is affected by multiple genetic factors and genotype-environment interactions (multifactorial inheritance mode), in which each factor has a small influence on the development of the disease [4, 113]. Thus variations in the frequency of SNPs in different populations, the modulating effects of other SNPs or mutations within individuals, variation in the penetrance of a SNP due to other factors as age-effect, different gender of enrolled study populations, and the difficulty in matching for all known environmental factors that predispose to MI could provide other plausible explanations for the conflicting results between studies. Overall, our study had few limitations; our sample size may be considered small, there was no control for chronic pharmacological treatment which may interfere with our findings, and only two polymorphisms were analyzed. In addition, the Egyptian population is admixed and a region with a specific genotype combination associated with risk may also be associated with a peculiar environmental factor.

5. Conclusion

In conclusion, the present study provides the first preliminary evidence that Gln27Glu polymorphism of -adrenergic receptors, but not the Arg16Gly polymorphism, is related to the prevalence of myocardial infarction disease in Egyptian population. Identification of the molecular mechanisms underlying the relationship between -adrenergic receptor genes and myocardial infarction offers opportunities to identify individuals at high risk and may help to improve the prevention and treatment of this important disease.

Conflict of Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interests.

Authors’ Contribution

Eman Toraih, Mohammad H. Hussein, and Dahlia I. Badran conceived and designed the experiments. Dahlia I. Badran collected samples and data. Eman Toraih, Mohammad H. Hussein, and Dahlia I. Badran performed the experiments. Eman Toraih and Dahlia I. Badran analyzed the data. Eman Toraih, Mohammad H. Hussein, and Dahlia I. Badran contributed reagents/materials/analysis tools. Eman Toraih, Mohammad H. Hussein, and Dahlia I. Badran wrote the paper. Eman Toraih, Mohammad H. Hussein, and Dahlia I. Badran were responsible for critical revision and final approval.

Acknowledgment

The authors thank Dr. Dalia Sabry, the Lab Manager at Biotechnology Research Center, for her technical support.

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