**2. Renin angiotensin aldosterone gene polymorphism**

The renin angiotensin aldosterone system (RAAS) plays an important role in the regulation of humoral regulation. RAAS, which is also important in the regulation of blood pressure, cardiovascular homeostasis, fluid and electrolyte balance such as hypertension, heart failure and arrhythmia, plays an important role in the pathophysiology of various cardiovascular diseases. Renin, an acid protease synthesized by renal juxtaglomerular cells, is involved in circulation through the renal vein. A decrease in renal blood flow or a decrease in plasma sodium levels leads to an increase in renin secretion. Renin plays a key role in the production of angiotensin I in plasma or tissues. It is provided that renin is converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensinogen (AGT), the original subtype of renin, is an important source of angiotensin II. Angiotensin II functions by binding to the angiotensin II receptor on fibroblasts. Angiotensin II plays an important role in enhancing the synthesis and secretion of collagen types I and III in the regulation of proliferation of fibroblasts. Angiotensin II induces aldosterone release, resulting in myocyte necrosis and susceptible fibrosis. RAAS is functioning via angiotensin II. Angiotensin II is involved in the elevation of blood pressure in the systemic arterial and venous systems and in the increase of blood return to the heart. It increases the central sympathetic activity by increasing the oscillation from the sympathetic nerve endings. Thus, synthesis and release of aldosterone is regulated. RAAS, which plays a role in atrial remodeling and pathogenesis of AF, is an important regulator. There are not many studies aiming to investigate the relationship between RAAS gene polymorphisms and the risk of developing AF. In a study conducted by Tsai et al., it was determined that the polymorphisms occurring in RAAS genes increased the susceptibility to AF development as a result of association with environmental factors leading to elevated atrial pressures. RAAS gene polymorphisms include ACE insertion/deletion (I/D), AGT (G-217A, A-20C, G-7A, M235T and T174M) and ATR1 A1166C gene polymorphisms. In a study aiming to investigate the association of these polymorphisms with AF, in exon 2 of the AGT gene, the M235 allele, a significant relationship was found between haploids associated with the G-6 and G-217 alleles in the promoter region and AF development risk [4, 5].

#### **2.1. ACE (I/D) gene polymorphism**

The underlying mechanisms in the development of AF are still not fully understood, but a heterogeneous model plays an important role in the pathophysiology of this disease. This heterogeneous model is based on the interaction of multiple substrates and triggers [3].

There are many studies showing that genetic factors play an important role in the pathogenesis of AF. Monogenic mutations known to be associated with AF have been identified. A total of 25 gene mutations proven to be associated with AF have been identified. Genome-wide association studies (GWAS) have been conducted to investigate AF genetics, and these studies have shown that single nucleotide polymorphisms play a very important role in the development of AF. Several single nucleotide polymorphisms associated with AF predisposition have

AF is an electrical disease caused by defects in ionic currents, and a variety of studies have been undertaken to determine the genetic causes of these electrical illnesses. Studies conducted to investigate the hereditary predisposition of AF found that the development of AF in pups with AF detected in their parents was found. Even though disorders such as hypertension, myocardial infarction and diabetes mellitus, which are important risk factors for the development of AF, are regulated, they still have the risk of developing fourfold AF [3].

In many genetic studies, variants known to be associated with AF have emerged. These variants are formed as a result of abnormalities in genes encoding cardiac gap junctions, signaling molecules, ion channels and auxiliary subunits. In addition, gene polymorphisms may cause loss of function in genes that encode proteins contributing to cardiac depolarization or repolarization leading to AF's increased sensitivity, are also genetic risk factors that play an

The purpose of this chapter is to give general information about AF and compiling the studies made with the aim of determining the gene polymorphisms that can play an important role

The renin angiotensin aldosterone system (RAAS) plays an important role in the regulation of humoral regulation. RAAS, which is also important in the regulation of blood pressure, cardiovascular homeostasis, fluid and electrolyte balance such as hypertension, heart failure and arrhythmia, plays an important role in the pathophysiology of various cardiovascular diseases. Renin, an acid protease synthesized by renal juxtaglomerular cells, is involved in circulation through the renal vein. A decrease in renal blood flow or a decrease in plasma sodium levels leads to an increase in renin secretion. Renin plays a key role in the production of angiotensin I in plasma or tissues. It is provided that renin is converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensinogen (AGT), the original subtype of renin, is an important source of angiotensin II. Angiotensin II functions by binding to the angiotensin II receptor on fibroblasts. Angiotensin II plays an important role in enhancing the synthesis and secretion of collagen types I and III in the regulation of proliferation of fibroblasts. Angiotensin II induces aldosterone release, resulting in myocyte

been identified in these GWAS studies [3].

4 Cardiac Arrhythmias

important role in the development of AF [3].

**2. Renin angiotensin aldosterone gene polymorphism**

in the development of AF.

The 21-kilobase pair (kbp) long ACE gene locates on chromosome 17q23. This gene consists of 26 exons and 25 introns. The ACE (I/D) gene polymorphism is characterized by I/D of 287 base pairs in the 16th intron of the ACE gene. The genotypes of ACE (I/D) gene polymorphism differ in terms of ACE plasma and tissue levels. The DD genotype of the ACE (I/D) gene polymorphism is associated with high cellular ACE activity, which leads to myocardial fibrosis, so myocardial fibrosis develops. There are studies showing that ACE (I/D) gene polymorphism is associated with the risk of developing AF. There is a positive relationship between DD genotype and ACE activity of ACE (I/D) gene polymorphism. As a result of this relationship, angiotensin II level increases and myocardial hypertrophy, arrhythmia can develop. In the study carried out by Zhang and colleagues found a significant association between DD genotype of the ACE (I/D) gene polymorphism and increased AF. In another study conducted by Topal et al., a significant relationship was found between the incidence of ACE Alu D and increased AF [2].

#### **2.2. ACE 2350G/A (rs4343) gene polymorphism**

One of the ACE gene polymorphisms from the AF associated genes is the ACE 2350G/A (rs4343) polymorphism, and this polymorphism has a significant effect on the plasma ACE concentration. The ACE 2350G/A (rs4343) gene polymorphism is a synonymous mutation that is accepted as silent. There is insufficient study to investigate the relationship between ACE 2350G/A (rs4343) gene polymorphism and the risk of developing AF. In a study conducted by Jiang et al. in a Chinese population, the A allele of ACE 2350G/A (rs4343) gene polymorphism has been associated with the risk of developing AF in patients with essential hypertension. ACE 2350G/A (rs4343) polymorphic locus do not effect expression directly of ACE mRNA or it has not functional variant. It is assumed that there may be link imbalance between this fragment and an unknown DNA fragment acting as a muffler. In order to be able to identify gene loci in this linkage disequilibrium, a large number of studies have to be performed [1].

## **2.3. Angiotensin II type 1 receptor and angiotensin-converting enzyme 2 gene polymorphisms**

RAAS, which plays an important role in the pathophysiology of AF in the structural and electrical remodeling of the atrium, contains ACE/angiotensin II/AGTR1 and ACE2/ angiotensin (1–7)/ MAS axes. These axes regulate myocardial hypertrophy, fibrosis and remodeling. ACE/angiotensin II/AGTR1 and ACE2/angiotensin (1–7)/MAS axes have been found to play an important role in AF pathogenesis. Angiotensin II is the most vasoactive component of RAAS, and angiotensin II, which causes increased myocardial fibrosis and hypertrophy, may contribute to AF development. Angiotensin II, an important signaling molecule of RAAS, plays a role in cardiovascular effects via AGTR1. AGTR1, G-protein is a bound receptor and has been associated with some disorders such as heart failure, prehypertension and stroke. There are studies showing that in AF patients AGTR1 levels increase in the left atrium. In a study conducted with Chinese Han population, the roles of AGTR1 rs1492100, rs1492099, rs1492097 and rs3772616 gene polymorphisms in AF development were investigated. A significant correlation was found between rs1492099 gene polymorphism from these polymorphisms and the development of structural AF. The ACE2 gene shows the X chromosome. In a study conducted by Freg et al., ACE2 expression was found to be significantly reduced in patients with chronic AF. In contrast, it is observed that atrial tissue angiotensin II levels were also significantly elevated. In another study with Chinese Han population, the effects of AGTR1 and ACE2 gene polymorphisms development of structural AF were examined. It is thought that polymorphisms occurred in this gene may be genetic risk factors in the development of structural AF in the Chinese Han male population. Also, it has been shown that ACE2 and AGTR1 genes are associated in patients with structural AF [6].

found to be associated with an increase in C allele binding to steroidogenic transcription factor 1 and thus an increase in CYP112B2 activity. In a study conducted by Amir et al., CYP112B2-344 C/T gene polymorphism CC genotype was found to be an independent risk factor for AF in patients with heart failure. In a study in China Han population, conducted by Huang et al., found that CYP112B2-344 C/T gene polymorphism is not a genetic risk factor in the development of AF in patients with hypertensive heart disease. In a study performed by Zhang et al., the significant relationship is not also found between CYP112B2-344 C/T gene polymorphism and AF development [2]. It is presented primer sequences that used to determine AGTR1, ACE2, AGT, ACE (I/D) and CYP112B2-344C/T gene polymorphisms

**Table 2.** Primer sequences used in PCR and amplification product size for AGT, ACE (I/D) and CYP112B2-344C/T.

Reverse 5′-CCTCCACCCTGTTCAGCC-3′ PCR, polymerase chain reaction; AGT, angiotensinogen; ACE (I/D), angiotensin-converting enzyme (insertion/deletion);

PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; AGTR1, angiotensin II receptor 1; ACE2,

Gene Polymorphisms Associated with Atrial Fibrillation http://dx.doi.org/10.5772/intechopen.76920 7

Reverse 5′-AGGGTGCTGTCCACACTGGACCC-3′

Reverse 5′-GATGTGGCCATCACATTCGTCAGAT-3′

**Forward primer 5′-3′ Reverse primer 3′–5′**

rs1492100 TTCAATAACAGATTCCCAGAG CCACCTCAACTTGCCTGTG rs1492099 TTCAATAACAGATTCCCAGAG CCACCTCAACTTGCCTGTG rs1492097 TTCAATAACAGATTCCCAGAG CCACCTCAACTTGCCTGTG rs3772616 TGATAATTTATGTACTCCCTC CAAAGCATAAGTGTCAACAGA rs6632677 CTGACTTGTTGCAGCAAGATGC TAGGAGTCCAGGCACAGTTCAG

AGT M235T 163 bp Forward 5′-CGTTTGTGCAGGGCCTGGCTCTC-3′

ACE AluI/D 490 bp Forward 5′-CTGGAGACCACTCCCATCCTTTCT-3′

CYP112B2-344C/T 537 bp Forward 5′-CAGGAGGAGACCCCATGTGAC-3′

angiotensin-converting enzyme 2; Amp, amplification.

CYP112B2, aldosterone synthase; Amp, amplification.

**Table 1.** Primer sequences used in PCR for AGTR1 and ACE2.

**GENES amp. size (bp) Primer Primer sequences**

The major products of cellular metabolism are reactive oxygen species (ROS) and reactive nitrogen products (RNS) and they have sources in the myocardium. Redox homeostasis is disturbed when oxidant species overcome the capacity to reduce of the cell. While excessive ROS results in oxidative stress; excessive RNS results in nitrosative stress. Potentially reactive species such as the mitochondrial electron transport chain, xanthine oxidase, NADPH oxidases and nitric oxide synthases (NOS) are present in the myocardium. There are three NOS isoforms: NOS1 (neuronal NOS = nNOS), NOS2 (inducible NOS = iNOS) and NOS3 (endothelial NOS = eNOS). These isoforms are named according to the first description of the tissues. It

in **Tables 1** and **2**.

**AGTR1 and ACE2**

**3. Nitric oxide synthase gene polymorphisms**

#### **2.4. Aldosterone synthase 344 C/T gene polymorphism**

Aldosterone synthase (CYP112B2) is an enzyme that plays an important role in the synthesis of aldosterone. CYP112B2 is the mitochondrial P450 oxidase found in the adrenal cortex of the zona glomerulosa. Aldosterone plays an important role in regulation of ion motions and collagen expression, including myocardial remodeling. Delayed or reversed myocardial remodeling is achieved by the aldosterone inhibitor, thus can prevent AF. In a study by Goette et al., there was a positive relationship between elevation of AF and aldosterone levels. The CYP112B2 gene is 7 kilobases long and locates on chromosome 8q22. This gene consists of 9 exons and 8 introns. The CYP112B2-344 C/T gene polymorphism is characterized by a C/T substitution in the −344 position in the promoter region of the CYP112B2 gene. Several studies have been conducted to investigate the relationship between CYP112B2-344 C/T gene polymorphism and hypertension. In some studies, CYP112B2-344 C/T gene polymorphism has been identified as a genetic risk factor for hypertension and myocardial hypertrophy. However, a limited number of studies have been conducted to investigate the relationship between CYP112B2-344 C/T gene polymorphism and AF. In a study conducted by Lu et al., no significant relationship was found between CYP112B2-344 C/T gene polymorphism and AF development risk. In the study conducted by Shuxin Hou et al., there were no significant differences in CYP112B2-344 C/T gene polymorphism genotype distributions between AF patients and healthy control groups. The CYP112B2-344 C/T gene polymorphism has been


PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; AGTR1, angiotensin II receptor 1; ACE2, angiotensin-converting enzyme 2; Amp, amplification.


**Table 1.** Primer sequences used in PCR for AGTR1 and ACE2.

CYP112B2, aldosterone synthase; Amp, amplification.

**2.3. Angiotensin II type 1 receptor and angiotensin-converting enzyme 2 gene** 

RAAS, which plays an important role in the pathophysiology of AF in the structural and electrical remodeling of the atrium, contains ACE/angiotensin II/AGTR1 and ACE2/ angiotensin (1–7)/ MAS axes. These axes regulate myocardial hypertrophy, fibrosis and remodeling. ACE/angiotensin II/AGTR1 and ACE2/angiotensin (1–7)/MAS axes have been found to play an important role in AF pathogenesis. Angiotensin II is the most vasoactive component of RAAS, and angiotensin II, which causes increased myocardial fibrosis and hypertrophy, may contribute to AF development. Angiotensin II, an important signaling molecule of RAAS, plays a role in cardiovascular effects via AGTR1. AGTR1, G-protein is a bound receptor and has been associated with some disorders such as heart failure, prehypertension and stroke. There are studies showing that in AF patients AGTR1 levels increase in the left atrium. In a study conducted with Chinese Han population, the roles of AGTR1 rs1492100, rs1492099, rs1492097 and rs3772616 gene polymorphisms in AF development were investigated. A significant correlation was found between rs1492099 gene polymorphism from these polymorphisms and the development of structural AF. The ACE2 gene shows the X chromosome. In a study conducted by Freg et al., ACE2 expression was found to be significantly reduced in patients with chronic AF. In contrast, it is observed that atrial tissue angiotensin II levels were also significantly elevated. In another study with Chinese Han population, the effects of AGTR1 and ACE2 gene polymorphisms development of structural AF were examined. It is thought that polymorphisms occurred in this gene may be genetic risk factors in the development of structural AF in the Chinese Han male population. Also, it has been shown that ACE2 and AGTR1 genes are associated in patients with

Aldosterone synthase (CYP112B2) is an enzyme that plays an important role in the synthesis of aldosterone. CYP112B2 is the mitochondrial P450 oxidase found in the adrenal cortex of the zona glomerulosa. Aldosterone plays an important role in regulation of ion motions and collagen expression, including myocardial remodeling. Delayed or reversed myocardial remodeling is achieved by the aldosterone inhibitor, thus can prevent AF. In a study by Goette et al., there was a positive relationship between elevation of AF and aldosterone levels. The CYP112B2 gene is 7 kilobases long and locates on chromosome 8q22. This gene consists of 9 exons and 8 introns. The CYP112B2-344 C/T gene polymorphism is characterized by a C/T substitution in the −344 position in the promoter region of the CYP112B2 gene. Several studies have been conducted to investigate the relationship between CYP112B2-344 C/T gene polymorphism and hypertension. In some studies, CYP112B2-344 C/T gene polymorphism has been identified as a genetic risk factor for hypertension and myocardial hypertrophy. However, a limited number of studies have been conducted to investigate the relationship between CYP112B2-344 C/T gene polymorphism and AF. In a study conducted by Lu et al., no significant relationship was found between CYP112B2-344 C/T gene polymorphism and AF development risk. In the study conducted by Shuxin Hou et al., there were no significant differences in CYP112B2-344 C/T gene polymorphism genotype distributions between AF patients and healthy control groups. The CYP112B2-344 C/T gene polymorphism has been

**polymorphisms**

6 Cardiac Arrhythmias

structural AF [6].

**2.4. Aldosterone synthase 344 C/T gene polymorphism**

**Table 2.** Primer sequences used in PCR and amplification product size for AGT, ACE (I/D) and CYP112B2-344C/T.

found to be associated with an increase in C allele binding to steroidogenic transcription factor 1 and thus an increase in CYP112B2 activity. In a study conducted by Amir et al., CYP112B2-344 C/T gene polymorphism CC genotype was found to be an independent risk factor for AF in patients with heart failure. In a study in China Han population, conducted by Huang et al., found that CYP112B2-344 C/T gene polymorphism is not a genetic risk factor in the development of AF in patients with hypertensive heart disease. In a study performed by Zhang et al., the significant relationship is not also found between CYP112B2-344 C/T gene polymorphism and AF development [2]. It is presented primer sequences that used to determine AGTR1, ACE2, AGT, ACE (I/D) and CYP112B2-344C/T gene polymorphisms in **Tables 1** and **2**.

## **3. Nitric oxide synthase gene polymorphisms**

The major products of cellular metabolism are reactive oxygen species (ROS) and reactive nitrogen products (RNS) and they have sources in the myocardium. Redox homeostasis is disturbed when oxidant species overcome the capacity to reduce of the cell. While excessive ROS results in oxidative stress; excessive RNS results in nitrosative stress. Potentially reactive species such as the mitochondrial electron transport chain, xanthine oxidase, NADPH oxidases and nitric oxide synthases (NOS) are present in the myocardium. There are three NOS isoforms: NOS1 (neuronal NOS = nNOS), NOS2 (inducible NOS = iNOS) and NOS3 (endothelial NOS = eNOS). These isoforms are named according to the first description of the tissues. It


sympathetic or parasympathetic activation play an important role in AF triggering. In a study conducted by Thomson et al., hypertension was reported to induce triggering in developing of AF in patients with hypertrophic cardiomyopathy. Since myocardial hypertrophy is present in patients with hypertrophic cardiomyopathy, the left ventricular space is small in these patients. Thus, a decrease occurs in venous conversion and intravascular volume. As a result of this, in the patients with hypertrophic cardiomyopathy, low heart debit and various symptoms arise. Cheung et al. suggested that AF could be induced in the study they performed. Endothelin 2, which constricts the systemic vessels, protects venous return and prevents hypertension that may develop. Acute hypertension causes an increase in sympathetic nerve activity. Hypertension can occur in hypertrophic cardiomyopathy. A vasoconstrictor may show protective effect against AF in hypertrophic cardiomyopathy. Proximal AF is more common in hypertrophic cardiomyopathy than in other structural heart diseases. This monogenic disorder is a disorder affecting left ventricular hypertrophy in patients with hypertrophic cardiomyopathy. These disorders result from mutations in genes encoding the sarcomeric proteins. In a study conducted by Sharma et al., it has been shown that the endothelin 2 gene may be effective in the development of hypertension, and that this gene is expressed to in human atrial tissue. Endothelin 2 gene is localized on chromosome 1p34. It has been suggested that there is a significant relationship between hemodynamic changes and polymorphisms occurring in endothelin 2 gene in patients with essential hypertension. The functional role of endothelin 2 A985G gene polymorphism is not known precisely. mRNA stability is affected by variations in 3′-UTR. Thus, endothelin 2 transcription and translation may be affected in the endothelin 2 A985G gene polymorphism. Differences in A985 allele frequencies are observed in studies with different populations. Endothelin 2 A985G gene polymorphism plays a protective role for A985 allelic cardiovascular diseases, but this allele may trigger AF development in hypertrophic cardiomyopathic patients. In a study conducted by Nagai T et al., The endothelin 2 A985T allele has been shown to be a genetic risk factor for the development of AF in hypertrophic cardiomyopathic patients [10]. It is presented primer sequences that used to

Reverse 5′-AGGGAATGAGGGTGCAAGAA-3′ G allele-specific probe 5′-VIC-CCCTGGAGACTGGA-MGB-3′ A allele-specific probe 5′-FAM-CCGGAGGCTGGAT-MGB-3′

Gene Polymorphisms Associated with Atrial Fibrillation http://dx.doi.org/10.5772/intechopen.76920 9

**Genes Primer Primer sequences**

**Table 4.** Sequence of primers for endothelin 2 A985G gene polymorphism.

PCR, polymerase chain reaction.

Endothelin 2 A985G gene Forward 5′-ACAAACCAGGAGCAACCGTG-3′

determine Endothelin 2 A985G gene polymorphism in **Table 4**.

AF can also occur when there is or no structural heart disease. Most of the foci that cause AF are at the site where combine the cardiomyocytes and vascular smooth muscle cells are located near the pulmonary venules. Connexins (Cx) are gap junction proteins and play an important role in direct cell-cell interactions in the majority of the tissues of the body in electrical conduction in the heart. It is known that there are 20 different Cxs in humans, and each Cxs create channels with different

**5. Connexins gene polymorphisms**

**Table 3.** Sequence of primers, size of the PCR products eNOS T-786C, G894T, Intron 4a/4b gene polymorphisms.

is known that enzymes that occur in NOS1 and NOS3 are expressed in the heart. NOS2 is expressed in inflammatory and pathological conditions such as hypertrophy or heart failure. While in cardiac myocytes, NOS1 and NOS3 were present in intracellular compartments, NOS2 is present in the cytosol of cardiac myocytes. NOS plays an important role in stimulating effects of NO on guanylate cyclase, or in arising and mediating effects of nitrosation of tyrosine, cysteine residues. NO, which a highly reactive radical, is spreadable and its life is very short. l-arginine is converted to citrulline by NO production and is a substrate for NOS. NOS2 is expressed in macrophages, neutrophils, endothelial cells, vascular smooth muscle cells and cardiomyocytes. The competitive inhibition of endogenous methylarginine regulates the substrate level in NOS isoforms. Oxidative stress plays an important role in AF pathogenesis. NOS enzymes can be decomposed and transferred from NO production to superoxide anion, strong free radicals and oxidation. Therefore, NOSs that are associated with oxidative stress are important in AF pathogenesis. In the case development of AF, left atrial endocardial NOS reduction occurs. Thus, a significant reduction in NO production occurs. Clinical cohorts were performed to investigate the relationship between AF development and eNOS gene polymorphisms. In a study with a Caucasian population that developed AF, it was determined that eNOS T-786C, G894T and 4a/4b gene polymorphisms did not have genetic risk factors in the development of AF. In another study, while CC genotype of eNOS T-786C polymorphism was found to be a genetic risk factor for homocysteine concentrations, there was no significant relationship between this polymorphism and the risk of developing AF. In another study conducted with heart failure and AF patients, 894TT genotype of G894T gene polymorphism was determined as a genetic risk factor in development of AF. In a study conducted by Giusti et al., eNOS T-786C gene polymorphism was found to be associated with a decrease in eNOS gene promoter activity. Furthermore, in the same study, this polymorphism was found to be an independent risk factor for plasma homocysteine concentrations [7–9]. It is presented primer sequences that used to determine eNOS T-786, G894T, Intron 4a/4b gene polymorphisms in **Table 3**.
