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INTRODUCTION
Syncope is a frequent symptom in the general population and is characterized by sudden-onset unconsciousness and absence of postural tonus1. In the general population, 40% of people have experienced at least one episode of syncope2. Although syncope is considered to have a benign prognosis, it is sometimes associated with mortal clinical conditions3. The most common cause of syncope is vasovagal syncope (VVS)4. Although the underlying pathophysiologic mechanism of VVS is unclear, it is believed that abnormal and excessive mechanoreceptor response, which causes hypotonia or bradycardia, is the primary etiology of VVS. Increased autonomous nervous system activity before the syncopal episode may be due to altered function or structure of the cardiac adrenergic receptor (AR)5. Several gene polymorphisms that affect the function or structure of cardiac AR have been described recently. Notably, the Arg389Gly beta-1 adrenoreceptor gene polymorphism has been shown to cause syncope through AR dysfunction6,7. Furthermore, beta-1 receptor gene polymorphism affects the response to beta-blocker therapy in patients with coronary artery disease, hypertension, and heart failure8-12.
Beta-blockers were the most commonly prescribed therapy in VVS previously. Beta-blockers are thought to suppress peripheral vasodilatation and inhibit ventricular mechanoreceptors. Although recent clinical trials have failed to demonstrate the benefits of beta-blocker therapy over placebo13,14, these trials did not consider beta-1 gene polymorphisms. This study aimed to investigate beta-1 gene polymorphisms in patients with VVS and assess the favorable effects on the prognosis with beta-blocker therapy selection based on gene polymorphisms.
METHODS
We included 123 patients who were admitted to our outpatient clinic between 2014 and 2017 with the complaint of at least three episodes of syncope in a year and who were diagnosed with VVS after the tilt-table test. All patients were followed up at a specialized arrhythmia outpatient clinic staffed with an expert team comprising one academician and one cardiology resident. Patients with structural heart disease, sick sinus syndrome, orthostatic hypotension, atrial fibrillation, metabolic or neurological syncope, or on any prescription medicines were excluded from the study. All participants gave their written informed consent for the study, which was approved by the Institutional Ethical Committee, per the Declaration of Helsinki.
Syncope is defined as the sudden, temporary, and complete loss of consciousness accompanied by the inability to sustain postural tonus that is associated with quick and spontaneous recovery. When none of the underlying etiologies of syncope is present, such as cardiac, reflex, neurologic, or metabolic effects, it is defined as pseudosyncope15.
Demographics, clinical characteristics, and 12-lead electrocardiogram findings of the participants were recorded. The standard evaluation included M-mode, two-dimensional, and Doppler studies according to the recommendations of the American Society of Echocardiography. Transthoracic echocardiography was carried out using a Philips iE33 echocardiography machine and X5 transducer (Philips Healthcare, Andover, Massachusetts, USA) with the patient in the left lateral decubitus position. Beta-blocker treatment was started using the lowest dose (metoprolol succinate 25 mg) and titrated up to the maximum dose until the patient tolerated the dose (200 mg for metoprolol succinate). Dose titration was made 3-5 days after the changes. Overall, 34 patients were excluded due to intolerance to the beta-blocker treatment. The number of syncopes 18 months before the diagnosis was recorded. Patients were followed up at 1, 3, 6, 12, and 18 months after treatment and were asked regarding treatment compliance, the occurrence of syncope, or any other symptom. The primary outcome was recurrent syncopal episodes. Presyncope was not considered as a treatment failure. Additional visits were scheduled to evaluate patients clinical status, if necessary. The patients and treating physicians were fully blinded to the genotype.
Head-up tilt (HUT) protocol
A HUT test was performed using an electrically controlled tilt-table with a footboard for weight-bearing. Blood pressure, heart rate (HR), heart rhythm, and right forearm blood flow were closely monitored and recorded. Blood pressure was automatically assessed every 1 min. The test was performed after the initial observation of 15 min with the patient in the supine position. Patients were tilted at 70° for up to 45 min without drug provocation16,17. If a presyncope with hypotension occurred during the test, the tilt table was rapidly lowered to return the patient to the supine position, and the study was terminated.
Test results were categorized as vasodepressor type in patients who had hypotension, syncope, or presyncope; cardioinhibitory type if bradyarrhythmia occurred; and mixed type when both hypotension and bradyarrhythmia occurred. Bradycardia was defined as a 20% decrease from the baseline HR or a decrease of more than 20 beats in a minute. Hypotension was defined as a decrease in systolic blood pressure (SBP) to more than 20% of baseline or more than 30 mmHg decrease of maximum systolic pressure.
Sample collection and biochemical analysis
Blood samples were collected from patients enrolled in the study, as well as healthy volunteers. A 400-µl sample of uncoagulated blood was placed in a 1.5-ml centrifuge tube and 800 µl of LB buffer was added. The sample was centrifuged at 6000 rpm for 3 min and the supernatant was removed. In addition, the LB buffer step was repeated when a larger sample of DNA was required. The remaining pellet was white or slightly pink. The pellet was vortexed until a homogeneous appearance was obtained.
Approximately 20 µl protein-C and 220 µl BB buffer were then added to the sample and incubated for 10-15 min at 65°C. After incubation, 220 µl of ethanol was added. The mixture was transferred to filtered tubes and centrifuged for 2 min at 12,000 rpm. The liquid and the filtered portion of the centrifuged mixture were poured and 500 µl of wash solution was poured over a filter. The filter was centrifuged without any reactive material at 12,000 rpm for 3 min. The filter was placed in a new microfuge tube, 30-100 µl elution buffer was added, and the sample was incubated at room temperature for 4-5 min at room temperature and centrifuged for 2 min at 12,000 rpm. The filter was then displaced and the microfuge tube now contained the DNA that was measured using a NanoDrop. Samples with concentrations higher than 50 ng/µl and between 1.8 and 2.0 OD were stored at −20°C. The above protocol was repeated for samples that did not meet these requirements.
Analysis of Arg389Gly polymorphisms
In this study, we used the amplification refractory mutation system technique to determine polymorphic alleles using real-time polymerase chain reaction. We used this technique because it is reliable, easy to assess, and produces quality results. This technique depends on the DNA polymerase enzyme that starts reactions according to the last nucleotide coupling. The reaction uses specific primers designed to integrate with mutated primers that contain an altered nucleotide. The reactions were prepared in two distinct tubes with primers designed with appropriate nucleotides to integrate with the polymorphic alleles. Control primers were used to check the occurrence of the reaction. Alleles located on the DNA were determined based on the reaction product. If the reaction occurred in one tube, the allele was defined as homozygous and called heterozygous if the reaction occurred in both tubes.
In this study, we screened for the Arg389Gly polymorphism (rs1801253) in the beta-1 adrenoceptor gene (ADRB1). The C allele encoded the amino acid arginine and the G allele encoded glycine. Thus, the CC genotype was replaced with Arg/Arg, the GG genotype was replaced with Gly/Gly, and the CG genotype was replaced with Arg/Gly.
Statistical analysis
HardyWeinberg equilibrium (HWE) was computed for the expected genotype distribution. The HWE was tested by using FINETTI Program (http://ihg.gsf.de/cgi-bin/hw/hwa2.pl). Data were analyzed using SPSS software (version 18, SPSS Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation for quantitative variables and as number (valid percent) for categorical variables. The distribution of continuous variables was evaluated using the KolmogorovSmirnov or ShapiroWilk tests. A Chi-square test was used to assess the intergroup differences in categorical variables. Normally distributed data of independent groups were compared using the independent samples t-test. The MannWhitney U test was used for comparison of non-normally distributed data. Paired t-tests were used to evaluate quantitative data before and after the treatment. p < 0.05 was considered statistically significant.
RESULTS
Of the 123 patients diagnosed with VVS, 64 (52%) had the Arg389Arg genotype and 59 (48%) had the Arg389Gly genotype. No Gly/Gly genotype was noted among the patients with VVS. No differences related to age or sex were identified between the Arg389Arg genotype and Arg389Gly genotype groups. The average age of the Arg389Arg genotype group was 24.9 years, and that of the Arg389Gly genotype group was 25.6 years. Intergroup comparison revealed no statistically significant differences related to left ventricular ejection fraction (66.1 ± 4.9 vs. 67.2 ± 4.1, p = 0.176). Compared with Arg389Gly genotype group, the baseline SBP and HR of the Arg389Arg genotype group were higher (SBP: 120.5 ± 7.0 vs. 116.5 ± 9.7, p = 0.011; HR: 81.3 ± 5.2 vs. 74.3 ± 5.2, p < 0.001). The pretreatment syncopal episodes in patients with Arg389Arg genotype were more frequent compared with those with Arg389Gly genotype (total syncopal episodes [TSE]: 7.9 ± 3.7 vs. 6.4 ± 3.0; p = 0.012). Intergroup comparison revealed no statistically significant differences related to beta-blocker dose (108.2 ± 46.0 vs. 101.6 ± 43.7, p = 0.493). Table 1 demonstrates the baseline characteristics of patients with Arg389Arg genotype and Arg389Gly genotype.
Table 1 Baseline characteristics of patients with Arg389Arg and Arg389Gly polymorphisms
| Gene polymorphism (n = 123) | Arg389Arg (64) | Arg389Gly (59) | p |
|---|---|---|---|
| Age, years | 24.9 ± 4.2 | 25.6 ± 4.4 | 0.392 |
| Female sex (n, %) | 40 (67) | 40 (62) | 0.538 |
| Left ventricular ejection fraction (%) | 66.1 ± 4.9 | 67.2 ± 4.1 | 0.176 |
| Systolic blood pressure (mmHg) | 120.5 ± 7.0 | 116.5 ± 9.7 | 0.011 |
| Diastolic blood pressure (mmHg) | 74.1 ± 7.1 | 72.4 ± 8.5 | 0.237 |
| Heart rate (beats per minute) | 81.3 ± 5.2 | 74.3 ± 5.2 | <0.001 |
| Total syncope episodes | 7.9 ± 3.7 | 6.4 ± 3.0 | 0.012 |
| Beta-blocker dose (mg) (after diagnosis) | 108.2 ± 46.0 | 101.6 ± 43.7 | 0.424 |
SBP, diastolic blood pressure (DBP), and HR before treatment and after 1 month of treatment were compared in patients with both groups. SBP, DBP, and HR after treatment was statistically lower in both groups, and more marked in patients with the Arg389Arg genotype (120.5 ± 7.0 vs. 115.7 ± 6.7, p < 0.001; 74.1 ± 7.1 vs. 71.7 ± 8.7, p = 0.030; 81.3 ± 5.2 vs. 78.4 ± 3.5, p < 0.001). The SBP, DBP, and HR in Arg389Gly genotype group before and after 1 month of treatment were as follows: 116.5 ± 9.7 vs. 112.9 ± 7.9, p = 0.025; 72.4 ± 8.5 vs. 70.3 ± 10, p = 0.044; and 74.3 ± 5.2 vs. 72.6 ± 4.5, p = 0.026, respectively. At 3, 6, 12, and 18 months after treatment, SBP, DBP, and HR were not statistically different when compared with the values obtained at 1 month.
The TSE of patients before and after 18 months of diagnosis was compared individually in the groups. The number of TSE in patients with Arg389Arg genotype decreased significantly after beta-blocker treatment (7.9 ± 3.7 vs. 3.0 ± 1.4, p < 0.001). However, no significant change was noted in patients with Arg389Gly genotype (6.4 ± 3.0 vs. 6.8 ± 3.2, p = 0.107) (Fig. 1).
Figure 1 Box-and-whisker plot comparing syncope numbers of patients with Arg389Arg (Group 1) and Arg389Gly (Group 2) polymorphisms 18 months before (blue boxes) and 18 months after (red boxes) treatment. Numbers above bars are means ± S.D.
A further intergroup comparison was performed regarding TSE after beta-blocker treatment. After 18 months of beta-blocker treatment, patients with the Arg389Arg genotype had significantly fewer episodes than patients with the Arg389Gly genotype (3.0 ± 1.4 vs. 6.8 ± 3.2, p < 0.001) (Fig. 2).
Figure 2 Box-and-whisker plot showing the frequency of syncope according to gene polymorphism in patients with vasovagal syncope diagnosis post-treatment. Numbers above bars are means ± S.D.
The HWE distribution was detected using control groups in two similar studies conducted in our population since there was no control group in our study. Genotype frequencies of ADRB1 gene Arg389Gly polymorphism of 205 patients in two studies were evaluated. HWE balance was examined and genotype frequencies do not match HWE distribution. However, this polymorphism showed significant deviation from HWE (p < 0.05) in the study group (Table 2 and Supplemental Table 1).
Table 2 Genotype of the control group of two studies and genotype of our patients
| Genotypes of control groups | (n = 205) | Genotype frequencies (%) | Arg allele frequency |
|---|---|---|---|
| Arg/Arg | 7 | 3 (41) | 0.356 |
| Arg/Gly | 132 | 64 (39) | Gly allele frequency |
| Gly/Gly | 66 | 32 (19) | 0.644 |
| Genotypes of our patients | (n = 123) | Genotype frequencies (%) | Arg allele frequency |
| Arg/Arg | 64 | 52 (03) | 0.760 |
| Arg/Gly | 59 | 47 (97) | Gly allele frequency |
| Gly/Gly | 0 | 0 | 0.240 |
DISCUSSION
In this study, we investigated the effect of beta-1 gene polymorphism on beta-blocker therapy in patients with VVS. Our principal findings were as follows:
The SBP and HR in patients with Arg389Arg genotype were significantly higher than in patients with Arg389Gly genotype
TSE in patients with Arg389Arg genotype was significantly higher than in patients with Arg389Gly genotype before treatment
After treatment, TSE in patients with Arg389Arg genotype was significantly lower than before treatment
No significant differences were noted regarding TSE in patients with Arg389Gly genotype after treatment
After the treatment, the TSE in patients with Arg389Arg genotype was significantly lower compared with patients having Arg389Gly genotype.
VVS is the most frequent type of syncope and requires a multidisciplinary treatment approach4. Conventional treatment of VVS, which includes both pharmacological and non-pharmacological treatments, is often insufficient. In general, beta-blockers are prescribed initially as the pharmacological treatment of VVS based on results of animal studies18. Adrenergic stimulation, which eventually activates cardiac mechanoreceptors and increases epinephrine levels during syncopal episodes19,20, led to the thought that beta-blocker therapy could be beneficial21,22. It was hypothesized that beta-blockers could prevent cardiac mechanoreceptor activation by exerting negative inotropic effects and blocking peripheral effects of beta-receptor stimulation21.
Beta-1 AR is highly expressed in the cardiac myocyte plasma membrane. Stimulation of this receptor results in chronotropic, inotropic, and dromotropic effects through G-protein associated signal activation23. Human ADRB1 encodes a functional protein with 477 amino acids through at least nine different nucleotide sequences24,25. Two major gene loci have been described that may alter the function of this protein. Notably, per the nucleotide variant #145, the amino acid at position 49 encodes a serine or glycine (Ser49Gly), and per the nucleotide variant #1165, the amino acid at position 389 encodes an arginine or glycine (Arg389Gly). The Arg389 variant is more potent than Gly389 concerning adenylyl cyclase stimulation26 and causes 3-4 times more signal transition capacity. The Arg389 variant demonstrates more potent inotropic effects on human heart preparations27. Among family members with different alleles, members with the Arg389 allele have shown a higher HR than members with the Gly389 allele. Furthermore, the increase in contractility and fractional shortening following dobutamine infusion is markedly higher in members with the Arg389 allele compared with those having Gly3896,28,29. In the present study, SBP and HR of patients with the Arg389Arg genotype were higher than those with Arg389Gly genotype (SBP: 120.5 ± 7.0 vs.116.5 ± 9.7, p = 0.011; HR: 81.3 ± 5.2 vs. 74.3 ± 5.2, p < 0.001). In addition, it has been demonstrated that cells transfected with Arg389 and human ventricular myocardial cell membrane have more affinity for agonist agents, such as norepinephrine, compared with Gly38926. Similarly, patients with the Arg389 genotype exhibited markedly decreased HR, blood pressure, and plasma renin activity after beta-blocker therapy compared with patients having the Gly389 genotype24. Likewise, our study observed that SBP, DBP, and HR were statistically lower in both groups after treatment, and more marked in patients with the Arg389Arg genotype (the before and after 1 month of treatment values for SBP, DBP, and HR were 116.5 ± 9.7 vs. 112.9 ± 7.9, p = 0.025; 72.4 ± 8.5 vs. 70.3 ± 10, p = 0.044; and 74.3 ± 5.2 vs. 72.6 ± 4.5, p = 0.026, respectively). Our study findings revealed that patients with Arg389Arg genotype have an excellent response to beta-blocker therapy due to the higher inotropic, chronotropic, and dromotropic effects.
Most studies that investigated the efficacy of beta-blocker treatment in patients with VVS have revealed conflicting results. Previous studies had demonstrated the beneficial effects of beta-blocker therapy21,30,31, whereas recent studies have failed to demonstrate any such benefits14,32,33, especially the randomized trials. However, no studies have investigated the effect of beta-1 adrenoceptor polymorphism on beta-blocker therapy response. Nevertheless, recent clinical trials have advocated that beta-1 adrenoceptor polymorphism results in signal activation of the receptors and alters the treatment response to beta-blocker therapy5,34. Such variations have also been demonstrated in patients with coronary artery disease, such as in patients with ADRB1 gene polymorphism, who exhibit a different treatment response to beta-blocker therapy35,36. Moreover, in patients with heart failure, it has been demonstrated that beta-1 adrenoceptor polymorphism alters the treatment response to beta-blocker therapy. Similarly, mice with the Arg389 gene polymorphism showed an excellent treatment response to beta-blocker therapy37, probably because beta-blockers inhibit beta-1 adrenoceptor function relatively more. In the present study, patients with VVS treated with beta-blocker therapy were compared regarding the number of syncopal and presyncopal episodes. During the 18-month follow-up after treatment, patients with the Arg389Arg genotype experienced significantly fewer syncope episodes compared with patients with the Arg389Gly genotype.
A primary limitation of our study is that it was a single-center study and that there was no control group. Since there was no control group in our study, the HWE distribution was determined using the control groups in two similar studies previously conducted in the Turkish population38,39. Furthermore, the relatively short follow-up period, the absence of repeat tilt-table test to verify treatment efficacy, and the lack of patients with Gly/Gly genotype are the other limitations.
In conclusion, when treating patients with VVS, the significance of genetics should be considered, besides the environmental and physiological factors. This theory is aptly supported by our study findings, which demonstrated that VVS is more frequent in patients with Arg389Arg genotype. Furthermore, our results also indicated that ADRB1 gene polymorphism plays a crucial role in response to beta-blocker therapy.
