**4. Endothelin 2 A985G gene polymorphism**

AF is an important complication of hypertrophic cardiomyopathy and is observed in approximately 20% of patients with hypertrophic cardiomyopathy. Hemodynamic changes following


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

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

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

Antisense 5′-GCCTCCACCCCACCCTGTC-3′

Antisense 5′-TCCATCCCACCCAGTCAA-3′

Antisense 5′-TCTCTTAGTGCTGTGGTCAC-3′

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

8 Cardiac Arrhythmias

eNOS T-786 180 bp Sense 5′-TGGAGAGTGCTGGTGTACCCCA-3′

eNOS G894T 200 bp Sense 5′-AACCCCCTCTGGCCCACTCCC-3′

Intron 4a/4b 393 (4a) 420 (4b) Sense 5′-AGGCCCTATGGTAGTGCCTTT-3′

PCR, polymerase chain reaction; eNOS, endothelial nitric oxide synthase; Amp, amplification.

AF is an important complication of hypertrophic cardiomyopathy and is observed in approximately 20% of patients with hypertrophic cardiomyopathy. Hemodynamic changes following

polymorphisms in **Table 3**.

**4. Endothelin 2 A985G gene polymorphism**

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

## **5. Connexins gene polymorphisms**

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


Thromboembolism or bleeding may develop as a result of inadequate or excessive intake of warfarin. Discomforts such as stroke and systemic thromboembolism can be reduced with anticoagulant treatments. Factors such as age, body size, environment, interacting drugs and gene polymorphisms are effective at warfarin dose requirements. Stable warfarin dose is affected by gene polymorphisms such as single nucleotide gene polymorphism. These polymorphisms play a role in the modulation of warfarin pharmacodynamics and pharmacokinetics. Gamma carbon carboxylation occurs on gamma glutamic acids. Gamma-glutamyl carboxylase (GGCX) found in the endoplasmic reticulum membrane oxidizes vitamin K-2,3 epoxite reduced vitamin K. Therefore, functional vitamin K-dependent clotting factors (II, VII, IX and X) are produced by this enzyme. GGCX catalyzes the biosynthesis of vitamin K-dependent clotting factors. Thus, this enzyme affects warfarin metabolism. Warfarin metabolism, one of the most frequently used anticoagulants in clinical therapy, is affected by the GGCX enzyme. GGCX is a gene that plays an important role in the individual differences of warfarin response. Warfarin is a common anticoagulant that a narrow therapeutic range. Genetic factors that play an important role in warfarin dose requirements include GGCX gene polymorphisms. GGCX gene that consisted of 15-exon is located on human chromosome 2p12. It has been reported that there is a relationship between polymorphisms occurring in the GGCX gene and warfarin dose variability. GGCX rs11676382, rs12714145, rs10654848 and rs699664 gene polymorphisms are the most common polymorphisms of the GGCX gene. GGCX rs11676382 (C>G) gene polymorphism found in intron 14 was found to be associated with low Warfarin dose requirements in the Caucasus. In intron 2, GGCX rs12714145 (3261G>A) gene polymorphism was found to have more warfarin dose requirements in Chinese patients with the AA genotype. In Caucasians and African Americans, there is a significant relationship between GGCX rs10654848 microsatellite (DNA repeats) gene polymorphism in intron 6 and high warfarin dose requirements. GGCX rs699664 gene polymorphism, characterized by a G/A base substitution at the 8th exon. This displacement results in the arginine/glutamine amino acid exchange at position 325. In Japanese and Chinese patients, a significant relationship was determined between this polymorphism and high warfarin dose requirements. In contrast, in Caucasians or African Americans, this gene polymorphism was found not to be associated with warfarin dose. In AF patients, it was determined that GGCX rs699664 gene polymorphism was significantly correlated with GA, AA genotypes and high warfarin dose requirements. Another polymorphism associated with warfarin dose in patients with AF is the GGCX rs2592551 gene polymorphism. The effect of GGCX rs2592551 gene polymorphism on the warfarin dose was investigated in a study conducted by Kamali et al. in a population living in the Xinjiang region (region of multiple ethnic communities of Khan, Uyghur, Kazakh, Hui, Kyrgyz, Mongol and Tajik). In this study, CT and TT genotypes of GGCX rs2592551 gene polymorphism were found to be associated with higher warfarin dose requirements than CC genotype in patients with AF [14, 15]. It is presented primer sequences that used to determine

Gene Polymorphisms Associated with Atrial Fibrillation http://dx.doi.org/10.5772/intechopen.76920 11

**Genes Forward primer (5′–3′) Reverse primer (5′–3′)**

**Table 6.** Primer sequences used in PCR for GGCX.

rs699664 AGTGGCCTCGGAAGCTGGT ACACAGGAAACACTGGGCTGAG rs2592551 GGACTTAGAAAGGAACGGATGA CTTGAGAAAAGGCAAAGCAGAC PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; GGCX, gamma-glutamyl carboxylase.

GGCX rs699664, rs2592551 gene polymorphisms in **Table 6**.

**Table 5.** Sequence of primers for the connexins.

characteristics and specific expression patterns. The polymorphisms occur in gap junction channels and in Cx proteins that play a role in action potential spread. Variants that occur in genes encoding variants that occur in genes encoding Cx40 and Cx37 that contribute to pulmonary vein-arrhythmogenic affect gene expression and function. Cx40 and Cx37 that contribute to pulmonary veinarrhythmogenic affect gene expression and function. Variants that occur in genes encoding Cx40 and Cx37 that contribute to pulmonary vein-arrhythmia affect gene expression and function. The Cx40 gene is encoded by GJA5 and is expressed in endothelial cells, coronary vascular smooth muscle cells, atrial cardiomyocytes and cardiac conduction systems. In GJA5, the TATA box sequence also changes is the result of the single nucleotide polymorphism found in the promoter region. Cx40 gene modulates broad mRNA levels and is known to be associated with AF. In a previous study, Cx40-26G>A gene polymorphism-26G allele was identified as a genetic risk factor in patients with cardiomyopathy AF. In a study performed by Carballo et al., Cx40-26G>A gene polymorphism was found to affect protein expression levels in cardiomyocytes and this polymorphism was associated with structural AF. There are significant relationships between polymorphisms occurring in GJA5 in the Cx40 gene and susceptibility to AF. Somatic mutations in the Cx40 gene have also been associated with idiopathic AF. The Cx43 gene is also encoded by GJA1 and is expressed by ventricular, atrial cardiomyocytes, vascular smooth muscle cells, endothelial cells, monocytes and macrophages. Other genes and polymorphisms associated with polymorphisms in the CX43 gene have also been reported to be effective in the development of AF. The Cx37 gene is encoded by GJA4 and is found in endothelial cells, pulmonary and vascular smooth muscle cells, monocytes/ macrophages and platelets. Polymorphisms occurring in GJA4 in the Cx37 gene are associated with atherosclerosis and coronary heart disease, and these polymorphisms have an effect on monocyte adhesion. Thus, they are important in the regulation of local inflammation. Systemic and local inflammation may play a role in the development of AF before or after surgery in some cases. The 1019 C>T gene polymorphism in the CX37 gene in GJA4 is characterized by proline/serine (P319S) substitution at position 319 in the cytoplasmic tail of the Cx37 gene. As a result, channel conductivity and permeability change. Cx37 1019 C>T gene polymorphism is also associated with platelet aggregation or monocyte adhesion. Due to the effect of this polymorphism on monocyte adhesion, sensitivity to non-structural AF may change [11–13]. It is presented primer sequences that used to determine Cx37 1019 C>T, Cx40 G-44A gene polymorphisms in **Table 5**.

## **6. Gamma-glutamyl carboxylase gene polymorphism**

Warfarin, an oral anticoagulant, is used in the correction of various thromboembolitic disorders such as prosthetic heart valves, deep vein thrombosis and pulmonary embolism.


**Table 6.** Primer sequences used in PCR for GGCX.

characteristics and specific expression patterns. The polymorphisms occur in gap junction channels and in Cx proteins that play a role in action potential spread. Variants that occur in genes encoding variants that occur in genes encoding Cx40 and Cx37 that contribute to pulmonary vein-arrhythmogenic affect gene expression and function. Cx40 and Cx37 that contribute to pulmonary veinarrhythmogenic affect gene expression and function. Variants that occur in genes encoding Cx40 and Cx37 that contribute to pulmonary vein-arrhythmia affect gene expression and function. The Cx40 gene is encoded by GJA5 and is expressed in endothelial cells, coronary vascular smooth muscle cells, atrial cardiomyocytes and cardiac conduction systems. In GJA5, the TATA box sequence also changes is the result of the single nucleotide polymorphism found in the promoter region. Cx40 gene modulates broad mRNA levels and is known to be associated with AF. In a previous study, Cx40-26G>A gene polymorphism-26G allele was identified as a genetic risk factor in patients with cardiomyopathy AF. In a study performed by Carballo et al., Cx40-26G>A gene polymorphism was found to affect protein expression levels in cardiomyocytes and this polymorphism was associated with structural AF. There are significant relationships between polymorphisms occurring in GJA5 in the Cx40 gene and susceptibility to AF. Somatic mutations in the Cx40 gene have also been associated with idiopathic AF. The Cx43 gene is also encoded by GJA1 and is expressed by ventricular, atrial cardiomyocytes, vascular smooth muscle cells, endothelial cells, monocytes and macrophages. Other genes and polymorphisms associated with polymorphisms in the CX43 gene have also been reported to be effective in the development of AF. The Cx37 gene is encoded by GJA4 and is found in endothelial cells, pulmonary and vascular smooth muscle cells, monocytes/ macrophages and platelets. Polymorphisms occurring in GJA4 in the Cx37 gene are associated with atherosclerosis and coronary heart disease, and these polymorphisms have an effect on monocyte adhesion. Thus, they are important in the regulation of local inflammation. Systemic and local inflammation may play a role in the development of AF before or after surgery in some cases. The 1019 C>T gene polymorphism in the CX37 gene in GJA4 is characterized by proline/serine (P319S) substitution at position 319 in the cytoplasmic tail of the Cx37 gene. As a result, channel conductivity and permeability change. Cx37 1019 C>T gene polymorphism is also associated with platelet aggregation or monocyte adhesion. Due to the effect of this polymorphism on monocyte adhesion, sensitivity to non-structural AF may change [11–13]. It is presented primer sequences that used to

**Genes Primer Primer sequences**

PCR, polymerase chain reaction; Cx, connexin.

10 Cardiac Arrhythmias

**Table 5.** Sequence of primers for the connexins.

Cx37 1019 C>T Forward 5′-CTGGACCCACCCCCTCAGAATGGCCAAAGA-3′

Cx40 G-44A Forward 5′-CCCTCTTTTTAATCGTATCTGTGGC-3′

Reverse 5′-AGGAAGCCGTAGTGCCTGGTGG-3′

Reverse 5′-GGTGGAGGGAAGAAGACTTTTAG-3′

determine Cx37 1019 C>T, Cx40 G-44A gene polymorphisms in **Table 5**.

**6. Gamma-glutamyl carboxylase gene polymorphism**

Warfarin, an oral anticoagulant, is used in the correction of various thromboembolitic disorders such as prosthetic heart valves, deep vein thrombosis and pulmonary embolism. Thromboembolism or bleeding may develop as a result of inadequate or excessive intake of warfarin. Discomforts such as stroke and systemic thromboembolism can be reduced with anticoagulant treatments. Factors such as age, body size, environment, interacting drugs and gene polymorphisms are effective at warfarin dose requirements. Stable warfarin dose is affected by gene polymorphisms such as single nucleotide gene polymorphism. These polymorphisms play a role in the modulation of warfarin pharmacodynamics and pharmacokinetics. Gamma carbon carboxylation occurs on gamma glutamic acids. Gamma-glutamyl carboxylase (GGCX) found in the endoplasmic reticulum membrane oxidizes vitamin K-2,3 epoxite reduced vitamin K. Therefore, functional vitamin K-dependent clotting factors (II, VII, IX and X) are produced by this enzyme. GGCX catalyzes the biosynthesis of vitamin K-dependent clotting factors. Thus, this enzyme affects warfarin metabolism. Warfarin metabolism, one of the most frequently used anticoagulants in clinical therapy, is affected by the GGCX enzyme. GGCX is a gene that plays an important role in the individual differences of warfarin response. Warfarin is a common anticoagulant that a narrow therapeutic range. Genetic factors that play an important role in warfarin dose requirements include GGCX gene polymorphisms. GGCX gene that consisted of 15-exon is located on human chromosome 2p12. It has been reported that there is a relationship between polymorphisms occurring in the GGCX gene and warfarin dose variability. GGCX rs11676382, rs12714145, rs10654848 and rs699664 gene polymorphisms are the most common polymorphisms of the GGCX gene. GGCX rs11676382 (C>G) gene polymorphism found in intron 14 was found to be associated with low Warfarin dose requirements in the Caucasus. In intron 2, GGCX rs12714145 (3261G>A) gene polymorphism was found to have more warfarin dose requirements in Chinese patients with the AA genotype. In Caucasians and African Americans, there is a significant relationship between GGCX rs10654848 microsatellite (DNA repeats) gene polymorphism in intron 6 and high warfarin dose requirements. GGCX rs699664 gene polymorphism, characterized by a G/A base substitution at the 8th exon. This displacement results in the arginine/glutamine amino acid exchange at position 325. In Japanese and Chinese patients, a significant relationship was determined between this polymorphism and high warfarin dose requirements. In contrast, in Caucasians or African Americans, this gene polymorphism was found not to be associated with warfarin dose. In AF patients, it was determined that GGCX rs699664 gene polymorphism was significantly correlated with GA, AA genotypes and high warfarin dose requirements. Another polymorphism associated with warfarin dose in patients with AF is the GGCX rs2592551 gene polymorphism. The effect of GGCX rs2592551 gene polymorphism on the warfarin dose was investigated in a study conducted by Kamali et al. in a population living in the Xinjiang region (region of multiple ethnic communities of Khan, Uyghur, Kazakh, Hui, Kyrgyz, Mongol and Tajik). In this study, CT and TT genotypes of GGCX rs2592551 gene polymorphism were found to be associated with higher warfarin dose requirements than CC genotype in patients with AF [14, 15]. It is presented primer sequences that used to determine GGCX rs699664, rs2592551 gene polymorphisms in **Table 6**.
