Comparison of Elite Athletes and Essential Hypertension Patients for Angiotensin-Converting Enzyme ( ACE ) I/D and ACE G2350A Gene Polymorphisms of Elite Athletes, Essential Patients for

Thickening of the ventricular wall due to an increase in ventricular volume and hypertrophy of myocardium is an example of cardiac structural changes, besides it is known to show an increase in stroke and decrease in resting as to ABSTRACT Objective: Angiotensin-converting enzyme ( ACE ) gene produces an ACE protein which is the key component of the renin-angiotensin system and contains several polymorphisms which affects the physical performance that is significantly important for athletes. Insertion/deletion (I/D) and G2350A polymorphisms which have one of the most powerful influences in the ACE gene is known to regulate cardiovascular mechanisms by taking a role in angiotensin-system. Methods: The present study aimed to investigate the genetic association and haplotypes formed by rs1799752 ( ACE I/D) and rs4343 ( ACE G2350A) polymorphisms in essential hypertension patients, elite athletes and healthy controls in total 79 subjects (27 hypertension patients, 27 elite athletes and 25 control group individuals) from Turkish population. In this study, ACE I/D and G2350A polymorphisms were genotyped by using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method and identified by bioanalyzer after restriction digestion. Results: The prevalence of genotype frequencies of the ACE rs1799752 II/ID/DD was 3.7%, 44.44%, 51.85% in hypertension patients; 3.7%, 33.33%, 62.96% in elite athletes and 12%, 24%, 64% in healthy controls, respectively. Also, the prevalence of genotype frequencies of the ACE rs4343 GG/GA/AA was 7.41%, 77.78%, 14.81% in hypertension patients; 0%, 77.78%, 22.22% in elite athletes and 4%, 84%, 12% in healthy controls, respectively. Conclusion: Our data suggest that “D” allele for I/D and “A” allele for G2350A polymorphisms of ACE gene may have a potential role with a large number of patients and control groups in Turkish population.


Introduction
The people who make regular and professional sports have different structured and worked of hearts which is called the athlete's heart. This difference in the cardiac structure and function of the heart causes cardiovascular diseases such as hypertension.
Thickening of the ventricular wall due to an increase in ventricular volume and hypertrophy of myocardium is an example of cardiac structural changes, besides it is known to show an increase in function [1,2]. The hearts of those who use muscle strength such as runners, aerobic exercisers and wrestlers are changing and evolving. These changes occur not only by exercise but also by the influence of genetic factors. There are a variety of genes related to sport performance and athlete's heart structure. Moreover, they are likely to be polygenic because there are hundreds of genetic variance combinations among individuals. Genetic diversity is the reason why individuals are different in developing species. In genes, polymorphisms appear as minor genetic variations that have led to genetic diversity in the genetic code. These changes should be seen in more than 1% of the population to be called polymorphism [3]. The polymorphisms affect the sport performance via changing the significant gene functions especially the angiotensin-converting enzyme gene.
The ACE gene is one of the most studied candidate genes related to hypertension. ACE is a zinc-dependent dipeptidyl carboxypeptidase that plays an important role in the regulation of blood pressure by converting "angiotensin I" peptide into a potent vasoconstrictor peptide "angiotensin II", while it also inactivates the vasodepressor bradykinin to eventually promote the growth of cardiac cells, the biggest number of studies have been performed to change in cardiac structure and function such as left ventricular hypertrophy caused by a complication of cardiovascular diseases such as essential hypertension as well as left ventricular hypertrophy shown for athlete groups [2,4]. ACE gene that encodes the angiotensin-converting enzyme is located in the q arm of the chromosome 17, and it has 26 exons and 25 introns. According to NCBI records, ACE gene has more than 160 polymorphisms which are mainly single nucleotide polymorphisms (SNP) [5]. Among the polymorphisms of the ACE gene, the G2350A polymorphism (rs4343) is known to have the most significant effects on the plasma angiotensin-converting enzyme (ACE) concentration, although the most widely studied polymorphism of ACE gene is insertiondeletion of 287 bp existing in intron 16 of this gene [2]. It has been demonstrated for an athlete group that G2350A polymorphism of ACE gene can cause alterations in cardiac structure and function [6]. Although there are several studies have been conducted to evaluate the correlation between ACE G2350A polymorphism and hypertension, it was found conflicting results in those previous studies [7][8][9]. Therefore, we aimed to perform an association study to evaluate the allelic and genotypic frequencies of both (ACE I/D, ACE G2350A) polymorphisms and their prevalence on essential hypertension and sports performance.

Ethical Approval
The study was approved by the Ethics Committee of Istanbul University (Istanbul, Turkey; Project no. 2011/2115-897). The study protocol was performed according to the Declaration of Helsinki and the institutional review board-approved protocols for human study participants at the Istanbul University. All the participants provided written informed consent.

Study Design
The study group was designed according to the three different experimental groups for a total of 79 subjects consisting of 27 essential hypertensive patients (H, group 1), 27 elite athletes (E, group 2) and 25 unrelated healthy individuals as controls (C, group 3). The mean age range of hypertension patients, elite athletes and controls were 39 ± 6.2, 15 ± 3.1 and 28 ± 5.5, respectively.

Statistical Analysis
Statistical analysis of the study was conducted with R Console 3.4.1., RGui platform (i386, 64-bit). The frequency of genotypes and alleles was determined by direct gene counting method. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated for the association between ACE (I/D) and ACE (G2350A) polymorphisms in hypertension patients, elite athletes and controls, respectively.
Pearson's chi-square ( 2 ) test was performed to compare the genotype distribution and allele frequency between groups. Chisquare independence test was used to investigate the significance of the distribution of hypertension patients, elite athletes and healthy controls. All P-values were two-sided and P-value < 0.05 was considered to represent a statistically significant result.

Study Group
The study group consisted of a total of 79 subjects with H (n = 27), E (n = 27) and C (n = 25). Samples with age 15-39 years were obtained in this study and there was no statistically significant differences between age, gender and age of onset distributions (p > 0.05). The average blood pressure parameters of the subjects and controls are summarized in (Table 1).

G2350A Polymorphisms in H, E and C Groups
The genotype and allele frequencies of the ACE I/D (rs1799752) polymorphism in the study groups are presented in (Table 2). ( Table 3) indicates the genotype and allelic frequencies of the ACE G2350A (rs4343) polymorphism in the study group.

Polymorphisms in H, E and C Groups
The haplotype frequencies of the ACE rs1799752 (I/D) and ACE rs4343 (G2350A) polymorphisms in the study groups are presented in Table 5. Haplotype analysis showed that, using rs1799752-rs4343 haplotype I-G as reference, the frequency of respectively, Table 5).   Table 6).

Discussion
Hypertension is an important risk factor for a variety of cardiovascular, renal and neurological diseases. Elevated blood pressure (BP) is a precursor to excessive morbidity and premature mortality. Although systemic hypertension is a risk factor for disease burden, the risk is uneven, heterogeneous and unpredictable. Also, genetic factors contribute to 30-50% of causation to essential hypertension [10]. Hypertension may be identified as a polygenic hereditary disease simultaneously influenced by a variety of environmental factors [11]. Therefore, it raises the possibility of genetics in the development of hypertension and/or related complications [12]. The renin-angiotensin-aldosterone system hypertension and its complications [13].
In this study, it was investigated the role of ACE I/D and ACE G2350A polymorphisms on hypertension and sport performance in the Turkish population. The subjects with already known health problem other than essential hypertension were excluded from the study for avoiding any potential confounding effects.
Insertion/deletion (I/D) and G2350A polymorphisms of the ACE gene have been studied about cardiovascular diseases, especially hypertension [14,15]. ACE genotype studies have revealed several different results due to variable frequency of the related ACE alleles (I/D, G/A) among populations. In past, ACE I/D polymorphism has been extensively studied in association with hypertension [16]. A large meta-analysis by Staessen et al. [17] revealed that homozygous D allele causes 10% increased risk for hypertension when compared to that of homozygous I allele in women and Asians.
For the Hispanic population, DD genotype was found independent risk factor of hypertension [18]. Similar results were observed in the population of China, the male population of Bangladesh, Japan and Argentina [19][20][21][22]. These results were not replicated in other similar investigations. For example, a meta-analysis restricted to Caucasians showed no association of ACE I/D polymorphism with hypertension [23]. Similar reports were submitted by Miyama et al. [24] and Glavnik and Petrovic [25]. For the Pakistani population, two studies were noted in this regard. Ismail et al. [26] reported that ACE II genotype is associated with essential hypertension in young Pakistani population, but this association was not observed by Alvi and Hasnain [15].
The polymorphism in exon 17, ACE G2350A has also reported being associated with plasma ACE concentration. Zhu et al. [27] have observed its association with hypertension. In the population of Emirati, similar results were noted by Saeed Mahmood et al. [9] using a sample population of 254 individuals. Nawaz and Hasnain also observed an association between ACE G2350A polymorphism and hypertension in a different research project.
Also, Nawaz and Hasnain investigated the role of ACE I/D and ACE G2350A polymorphisms in increasing the blood pressure of subjects exposed to different sound levels. Their findings suggest that noise has no prominent role in changing the effects of ACE I/D polymorphism for increasing the blood pressure [28]. It was observed that different allele carriers of the ACE I/D and ACE G2350A gene polymorphisms have different chances of hypertension on exposure to noise [12].
In this study, the distribution of the II, ID and DD genotypes  [34] and activity [35]. Also, it was demonstrated that G allele of ACE G2350A was associated with an elevated ACE level in the circulation [9,12]. According to the Fajar's pooled data, D allele of ACE I/D was associated with 1.26-fold and A allele of ACE G2350A was correlated with 1.67-fold increasing the risk of left ventricular hypertrophy. Compared to "D" allele of ACE I/D, "A" allele of ACE G2350A had a greater risk for left ventricular hypertrophy.
In our study, we showed "D" allele of ACE I/D was associated with 0.902-fold for (H) and 1.234-fold for (E) subjects, respectively.

Ethics Approval and Consent to Participate
The study was approved by the Ethics Committee of Istanbul University (Istanbul, Turkey; Project no. 2011/2115-897).