An Investigation of the Association of the H558R Polymorphism of the SCN5A Gene with Idiopathic Cardiac Conduction Disorders

Abstract

Aim: The aim of this study was to investigate the predictive value of the rs1805124 polymorphism of the SCN5A gene with regard to idiopathic cardiac conduction disorders. Results: The AG genotype frequency was significantly higher in patients with an atrioventricular block (61,2%±6,0%) compared with healthy control subjects (34,8%±2,3%), p<0.0001. The AG genotype frequencies among patients with only idiopathic complete right bundle-branch block (CRBBB) (54,2%±5,5%) and those with both CLBBB and LAH (50%±5,1) were significantly higher than in the control group (34,8%±2,3%), p<0.005. Conclusions: The AG genotype of the H558R (rs1805124) polymorphism of the SCN5A gene is a genetic predictor of idiopathic disorders of atrioventricular and intraventricular conduction.

Introduction

Cardiac conduction disorders represent one of the most important issues facing cardiology due to their high prevalence and their adverse effect on the course and prognosis of many cardiovascular diseases. Having emerged, they usually persist throughout life, which leads in most cases to a significant work decrement, and often disability of patients. Considering a high morbidity rate of cardiac conduction disorders, studying the genetic bases of these diseases seems to be undoubtedly relevant.

The SCN5A gene is a transmembrane protein of the α-subunit of voltage-gated type 5 sodium channels. It is localized at the short arm (p) of chromosome 3 (3p21 locus). Disturbance of the channel structure caused by the SCN5A gene mutation prevents the gradual phase change of the action potential and makes it impossible to conduct the impulse in the atria cardiomyocytes, the atrioventricular node, and the His-Purkinje system (Ye et al., 2003; Tan et al., 2005; García-Castro et al., 2010; Cheng et al., 2011).

The SCN5A gene encodes the proteins of the sodium ion channels of cardiomyocytes. Diseases associated with SCN5A mutations manifest themselves as electrophysiological abnormalities in cardiomyocytes and refer to a group of cardiac channelopathies. The primary channelopathies comprise the long QT syndrome, Brugada syndrome, Lev-Lenegre syndrome, idiopathic ventricular tachycardia, familial forms of atrial fibrillation, and sick sinus syndrome (Towbin and Vatta, 2001; Makielski et al., 2003; McNair et al., 2004).

It has been proved that SCN5A mutations change some amino acids of this protein and influence the development of Brugada syndrome, as well as sudden unexplained nocturnal death syndrome (Tian, 2004). These heterogeneous diseases are similar in the clinical and electrocardiographic (ECG) patterns and are associated with the disturbance of the sodium ion current or disturbance of sodium ionic channel formation, leading to abnormal heart rhythm (Juang and Huang, 2004; Tian et al., 2004; Smits et al., 2005).

Smits et al. (2005) identified a new E161K mutation in the SCN5A gene in patients with sick sinus syndrome, cardiac conduction disorder, and Brugada syndrome in two unrelated families, assuming that the E161K missense mutation in the G481A nucleotide in the DI/S1-S2 region leads to the formation of progressive cardiac conduction disorder.

Kyndt et al. (2001) revealed the GLY1406ARG mutation between the DIII-S5 and DIII-S6 domain of the SCN5A sodium channel protein in a large French family with a combination of two syndromes, sick sinus syndrome and Brugada syndrome, inherited in an autosomal dominant manner.

Four children from a German family with a history of early cardiac rhythm and conduction disorders, including sick sinus syndrome, conduction disorders, and severe ventricular tachycardia, were homozygous carriers of SCN5A missense mutation (p.I230T). Yet, heterozygous carriers had neither clinical nor electrocardiographic abnormalities (Neu et al., 2010).

Schott et al. (1999) revealed that cardiac conduction disorders were associated with SCN5A mutation. Examination of eight members of a Chinese family with a long QT syndrome in the family history found the delQKP1507-1509-mutated SCN5A (Shi et al., 2009).

Molecular genetic studies of four generations of the family with hereditary cardiac conduction disorders and several cases of sudden cardiac death revealed a mutation (W1421X) in the SCN5A gene (Niu et al., 2006).

A large Finnish family was examined for the presence of mutations in the SCN5A gene. The D1275N mutation was revealed in five members of the family suffering from atrial arrhythmias and cardiac conduction disorders (Laitinen-Forsblom et al., 2006).

The D1275N mutation in the SCN5A gene was revealed in the majority of the examined individuals of the treatment group. Moreover, among persons with a mutation in the SCN5A gene, 38% suffered from dilated cardiomyopathy, 27% had initial signs of the disease, and 43% had atrial fibrillation (Tan et al., 2001).

A G-to-A replacement in the SCN5A gene was found in two children suffering from atrioventricular block. The mutation resulted in the substitution of serine for glycine (G298S) in the domain I S5-S6 loop and asparagine for aspartic acid (D1595N) within the S3 segment of domain IV (Wang et al., 2002).

The data mentioned above generate interest in identification of the SCN5A gene association with different idiopathic cardiac conduction disorders.

According to the literature, the interconnection of polymorphism H558R (rs1805124) of the SCN5A gene was revealed in patients with atrial fibrillation (Chen et al., 2007, 2011), Brugada syndrome (Marangoni et al., 2011), and ventricular fibrillation (Olszak-Waśkiewicz et al., 2008). Park et al. (2012) revealed the interconnection of this polymorphism in patients with atrioventricular block (AVB) in the Korean population. The aim of the present study is to investigate the predictive role of the rs1805124 polymorphism of the SCN5A gene in the genesis of idiopathic cardiac conduction disorders.

Materials and Methods

A total of 246 patients with idiopathic cardiac conduction disorders were examined.

The examination of the patients was performed on the premises of the cardiovascular care unit No. 2 and the ultrasonic and functional diagnostics unit of the Krasnoyarsk Interdistrict Clinical Hospital No. 20 named after I.S. Berzon. The inclusion criteria for the treatment group included the following.

1. Confirmed diagnosis of idiopathic cardiac conduction disorders (AVB, complete right bundle-branch block [CRBBB], and complete left bundle-branch block/left anterior hemiblock [CLBBB/LAH]).

2. Krasnoyarsk as a permanent place of residence.

3. Belonging to a Caucasian race.

4. The ability of the patient to perform necessary treatment procedures.

5. Signed informed consent.

The exclusion criteria comprised the following.

1. Unspecified diagnosis of idiopathic cardiac conduction disorders (AVB, CRBBB, and CLBBB/LAH).

2. Postprimary cardiac conduction disorders.

3. Permanent place of residence is out of Krasnoyarsk.

4. Non-Caucasian in race.

5. Patients' inability to perform necessary treatment procedures.

6. Patient's refusal to participate in the study.

According to the research objective, patients with idiopathic cardiac conduction disorders were divided into the following three groups.

Group 1—patients with idiopathic AVB (n=67).

Group 2—patients with idiopathic CRBBB (n=83).

Group 3—patients with idiopathic CLBBB/LAH (n=96).

Table 1 contains information about the age and gender of the patients with idiopathic cardiac conduction disorders.

Table 1.

Age and Gender of the Patients with Idiopathic Cardiac Conduction Disorders

		CCD (n=246)
Group	Gender	n	M±ζ	Min-max	[Me; Q_25-Q₇₅]
AVB (n=67)	Men	32	43.44±16.82	14-76	43 [27; 55]
	Women	35	37.2±18.94	8-73	32 [22; 53]
	All probands	67	40.49±18.01	8-76	40.5 [23.75; 54.25]
CRBBB (n=83)	Men	41	32.53±17.12	6-85	39.0 [20; 43.5]
	Women	42	36.79±17.53	2-78	37.5 [23; 47.25]
	All probands	83	34.59±17.35	2-85	33 [21; 45]
CLBBB/left anterior hemiblock (n=96)	Men	49	42.63±19.52	7-80	44.5 [27; 59.75]
	Women	47	49.38±17.41	4-78	31.0 [37; 60]
	All probands	96	45.71±18.8	4-80	49 [33; 60]

AVB, atrioventricular block; CCD, cardiac conduction disorders; CLBBB, complete left bundle-branch block; CRBBB, complete right bundle-branch block; M, arithmetic mean value; Me, median value in case of non-normal distribution.

A molecular genetic analysis was performed on the premises of the laboratory of molecular genetic investigations of therapeutic diseases of the Therapy and Preventive Medicine Research Institute of the Siberian branch of the Russian Academy of Medical Sciences (Novosibirsk). When evaluating the rs1805124 polymorphism of the SCN5A gene in patients with idiopathic cardiac conduction disorders, we used the control group, which consisted of healthy residents of the Oktyabrsky district of Novosibirsk, n=411 with the average age of 37 years [17.0; 54.0]. The information about the age and gender of the normal healthy control subjects is presented in Table 2.

Table 2.

Age and Gender of the Normal Healthy Control Subjects (for Molecular Genetic Analysis)

		Control group (n=411) (molecular genetic analysis)
Group	Gender	n	M±ζ	Min-max	[Me; Q_25-Q₇₅]
All groups members	Men	348	31.12±17.21	14-69	28 [15; 47]
	Women	63	39.72±16.33	14-68	40 [28; 55]
	All probands	411	37.16±17.05	14-69	37 [17; 54]

The control group was examined as part of the WHO MONICA Project (Multinational Monitoring of Trends and Determinants in Cardiovascular Disease). The main screening examinations within the WHO MONICA Project included the following methods of revealing cardiovascular disease and cardiovascular risk factors: measurement of blood pressure, anthropometry (body height and mass), creation of a demographic profile, interviews for the amount and frequency of cigarette smoking and alcohol use, physical activity level, estimation of blood lipid composition, (total cholesterol, triglycerides, high-density lipoprotein cholesterol), the Rose angina questionnaire, and the 12-lead ECG evaluation using the Minnesota code (Kuznetsova et al., 2004).

The molecular genetic testing data were provided by the Therapy and Preventive Medicine Research Institute of the Siberian branch of the Russian Academy of Medical Sciences (Novosibirsk) and complied with the terms of the cooperation agreement dated December 1, 2008. In accordance with the principles of the Declaration of Helsinki, a Research Permit from the Local Ethics Committee affiliated with KrasSMU named after prof. V.F. Voino-Yasenetsky and informed consent for molecular genetic testing (protocol No. 16 dated June 22, 2009) were obtained.

All patients with idiopathic cardiac conduction disorders were exposed to the following clinical instrumental examination: clinical examination, electrocardiography, echocardiography, Holter monitoring, cycle ergometry, and molecular genetic testing. For the purpose of the differential diagnostics between primary and postprimary cardiac conduction disorders, the patients underwent selective coronary angiography and myocardial scintigraphy.

We extracted DNA from peripheral blood leukocytes by standard methods. To detect the H558R (rs1805124) single-nucleotide polymorphism of the SCN5A gene, the following primers were used: 5′-ccagggcaccagcagtgatgcg-3′ (upstream) and 5′-aagccacgttccagccgcgg-3′ (downstream). The PCR product 135 bp in length is cut into 116 and 19 bp fragments with the restriction enzyme AspLEI in the presence of the G-allele. This original method was validated through the use of direct automatic sequencing.

Statistical data analysis included standard statistical procedures (Johnson and Leone, 1964; Kenui, 1979; Glantz, 1980), provided that the statistical evaluation methods were chosen depending on the character of the index signs and the number of the control groups. For revealing the quantitative indices distribution pattern, the Shapiro-Wilk test was used. In the absence of normal distribution, descriptive statistics are presented in the form of median and percentiles. To value the significance of the found differences, the Kruskal-Wallis test was used in case of multiple comparison and the Mann-Whitney test in case of pairwise comparison. In case of normal distribution, we used descriptive statistics represented in the form of mean value and mean difference. The statistical significance of normally distributed data in the compared groups was calculated with the use of the Student's t-test.

Qualitative criteria are represented by the percentage with the standard error of the proportion. To calculate the statistical significance of the found distinctions between qualitative characters, the chi-square (χ²) test was used. In case when the expected frequencies fell short of five, the Fisher's exact test was used. To calculate the correlation between identified characters, we used the Pearson criterion and in case of nonparametric distribution, the Spearman's rank correlation coefficient. Distinctions in the distribution of allele and genotype frequencies of the investigated genes between the groups were calculated using a χ² test.

In case when a sample size fell short of 5 cases, the Fisher's exact test was used. The relative risks of the disease for a particular allele or genotype were estimated by odds ratios (OR) (Bland and Altman 2000). The OR was calculated using the following formula: OR=(a×d)/(b×c), where a—allele (genotype) frequency in the patient sample, b—allele (genotype) frequency in the hold-out sample, c—a total of other allele (genotypes) frequencies in the patient sample, d—a total of the other allele (genotypes) frequencies in the hold-out sample. The OR is presented with 95% confidence interval (CI) (Fleiss, 1989; Lakin, 1990). Distinctions were considered as statistically significant when the significance level was p<0.05. Statistical data manipulation was performed using Excel and SPSS 19 (Zaytsev et al., 2003).

The methods of examination of the patients with idiopathic cardiac conduction disorders are shown in Table 3.

Table 3.

Methods of Diagnosis of Idiopathic Cardiac Conduction Disorders in the Index Group

	AVB (n=74)		CRBBB (n=87)		CLBBB (n=22)		LAH (n=81)
Method	Abs. value	%	Abs. value	%	Abs. value	%	Abs. value	%
ECG tracing	74	100	87	100	22	100	81	100
EchoCG	74	100	87	100	22	100	81	100
Video EEG	68	91.89	85	97.70	—	—	76	93.83
Holter monitor	72	97.30	83	95.40	22	100	79	97.53
Isotope scanning	5	6.76	4	4.60	8	36.36	4	4.94
Coronary angiography	26	35.14	37	42.53	16	72.72	31	38.27

Abs. value, absolute value; ECG, electrocardiography; EchoCG, echocardiography; LAH, left anterior hemiblock.

Results

The results of the molecular genetic analysis of the SCN5A gene rs1805124 polymorphism among patients with idiopathic cardiac conduction disorders and normal healthy control subjects are presented in Table 4.

Table 4.

Genotype and Allele Frequency Distribution of Polymorphism rs1805124 of the SCN5A Gene Among the Patients with Idiopathic Cardiac Conduction Disorders and the Normal Healthy Control Subjects

	Atrioventricular blockade (n=67)		Control (n=411)
Genotypes	N	%±m	N	%±m	p
AA	21	31.3±5.7	253	61.6±2.4	0.0001
AG	41	61.2±6.0	143	34.8±2.3	0.0001
GG	5	7.5±3.2	15	3.6±0.9	0.264^a
Alleles
A-allele	83	61.9±4.2	649	79±1.4	0.0001
G-allele	51	38.1±4.2	173	21±1.4	0.0001
OR; 95% CI of OR	2.304; 1.565-3.390
AA genotype	21	31.3±5.7	253	61.6±2.4	0.0001
Genotypes AG+GG	46	68.7±5.7	158	38.4±2.4	0.0001
OR; 95% CI of OR	3.636; 2.016-6.098

Alpha level reached with the use of Fisher's exact test.

p, Alpha level in genotype distribution comparison with the control group indices.

CI, confidence interval; OR, odds ratio.

Our results demonstrated that the frequency of the homozygous genotype AA for the dominant allele among patients with idiopathic cardiac conduction disorders (31.3%±5.7%) was significantly lower compared to the control group (61.6%±2.4%, p=0.0001). The frequency of the heterozygous genotype AG was significantly higher in patients with idiopathic AVB (61.2%±6.0%) in comparison with normal healthy control subjects (34.8%±2.3%), p=0.0001. We did not reveal any significant distinctions in the homozygous genotype GG between patients with idiopathic AVB and normal healthy control subjects, p=0.264.

The analysis of the SCN5A gene alleles allowed us to reveal the following distinctions between patients with idiopathic AVB and normal healthy control subjects. The frequency of the carriers of the SCN5A gene dominant allele A among the patients with idiopathic AVB was 61.9%±4.2% and among normal healthy control subjects was 79%±1.4%, p=0.0001. The frequencies of the carriers of the SCN5A gene mutant allele G were the following: patients with idiopathic AVB—38.1%±4.2% and normal healthy control subjects—21%±1.4% (OR=2.304; 95% CI 1.565-3.390; p=0.0001).

The total value of the frequencies of the heterozygous genotype AG and the homozygous genotype GG for the rare allele was significantly higher in patients with idiopathic AVB (68.7%±5.7%) in contrast with the control group (38.4%±2.4%) (OR=3.509; 95% CI 2.016-6.098; p=0.0001).

Therefore, there is a significant increase in the frequency of the homozygous genotype for the dominant allele A accompanied with the reduction of heterozygous AG carriers of the SCN5A gene in the group of patients with idiopathic AVB (Table 4).

We also performed a molecular genetic analysis of the rs1805124 polymorphism of the SCN5A gene in 83 patients with idiopathic CRBBB and in 411 persons of the control group. The results of this analysis are presented in Table 5.

Table 5.

Genotype and Allele Frequency Distribution of Polymorphism rs1805124 of the SCN5A Gene Among the Patients with Complete Right Bundle-branch Block and the Normal Healthy Control Subjects

	CRBBB (n=83)		Control (n=411)
Genotypes	N	%±m	N	%±m	p
AA	38	45.8±5.5	253	61.6±2.4	0.011
AG	45	54.2±5.5	143	34.8±2.3	0.001
GG	0	0	15	3.6±0.9	0.151^a
Alleles
A-allele	121	72.9±3.5	649	79.0±1.4	0.106
G-allele	45	27.1±3.5	173	21.0±1.4	0.106
OR; 95% CI of OR	1.395; 0.953-2.041
AA genotype	38	45.8±5.5	253	61.6±2.4	0.011
Genotypes AG-GG	45	54.2±5.5	158	38.4±2.4	0.011
OR; 95% CI of OR	1.898; 1.179-3.049

Alpha level reached with the use of Fisher's exact test.

p, Alpha level in genotype distribution comparison with the control group indices.

During the analysis of the group of patients with idiopathic right bundle-branch block and of normal healthy control subjects, the SCN5A gene genotypes were distributed in the following way.

Our results indicated that the frequency of the carriers of the homozygous genotype AA for the dominant allele of the SCN5A gene among patients with idiopathic CRBBB (45.8%±5.5%) was significantly lower compared to the control group (61.6%±2.4%, p=0.011). The frequency of the heterozygous genotype AG of the SCN5A gene was significantly higher in patients with idiopathic CRBBB (54.2%±5.5%) in comparison with normal healthy control subjects (34.8%±2.3%), p=0.001.

We did not reveal any significant distinctions in the homozygous genotype GG for the rare allele of the SCN5A gene between patients with idiopathic CRBBB and normal healthy control subjects.

The alleles in the group of patients with idiopathic CRBBB and in normal healthy control subjects were distributed as follows.

The dominant allele A of the SCN5A gene was revealed in 72.9%±3.5% of persons with idiopathic CRBBB and in 79.0%±1.4% of normal healthy control subjects. The mutant allele G was revealed in 27.1%±3.5% of persons with idiopathic CRBBB and in 21.0%±1.4% of normal healthy control subjects, these data being not significant (p=0.106).

The total value of the frequencies of the heterozygous genotype AG and the homozygous genotype GG for the rare allele of the SCN5A gene was significantly higher in patients with idiopathic CRBBB (54.2%±5.5%) in contrast with the control group (38.4%±2.4%) (OR=1.898; 95% CI 1.179-3.049; p=0.011) (Table 5).

Hence, there is a significant increase in the frequency of the heterozygous AG genotype accompanied with the reduction of the homozygous AA genotype carriers for the dominant allele of the SCN5A gene in the group of patients with idiopathic CRBBB compared to the control set.

The results of the analysis of the rs1805124 polymorphism of the SCN5A gene in the patients with idiopathic CLBBB and in normal healthy control subjects are shown in Table 6.

Table 6.

Genotype and Allele Frequency Distribution of Polymorphism rs1805124 of the SCN5A Gene Among the Patients with Complete Left Bundle-Branch Block/Left Anterior Hemiblock and the Normal Healthy Control Subjects

	CLBBB/LAH (n=96)		Control (n=411)
Genotypes	n	%±m	n	%±m	p
AA	48	50±5.1	253	61.6±2.4	0.05
AG	48	50±5.1	143	34.8±2.3	0.008
GG	0	0	15	3.6±0.9	0.117^a
Alleles
A-allele	144	75±3.1	649	79.0±1.4	0.272
G-allele	48	25±3.1	173	21.0±1.4	0.272
OR; 95% CI of OR	1.250; 0.867-1.805
AA genotype	48	50±5.1	253	61.6±2.4	0.05
Genotypes AG+GG	48	50±5.1	158	38.4±2.4	0.05
OR; 95% CI of OR	1.600; 1.025-2.500

Alpha level reached with the use of Fisher's exact test.

p, Alpha level in genotype distribution comparison with the control group indices.

Our findings show that the frequency of carriers of the homozygous AA genotype for the dominant allele among patients with idiopathic CLBBB/LAH (50%±5.1%) was significantly lower in comparison with the control group (61.6%±2.4%, p=0.05). The frequency of the heterozygous AG genotype of the SCN5A gene was significantly higher in patients with idiopathic CLBBB/LAH (50%±5.1%) compared to the control group (34.8%±2.3%), p=0.008.

It therefore appears that in the group of patients with idiopathic cardiac conduction disorders in the left crus of atrioventricular bundle (left bundle-branch block and LAH), there is a significant prevalence of the heterozygous AG genotype of the SCN5A gene and a significant reduction of the carriers of the AA genotype for the dominant allele of the SCN5A gene.

Discussion

The etiology of cardiac conduction disorders varies greatly. Some of the causes of this pathological condition include various cardiac and noncardiac diseases and common multifactorial diseases such as coronary heart disease and hypertension. However, in a certain number of cases, it is impossible to find a clear cause of cardiac conduction disorder. It is possible, nevertheless, to trace the hereditary component of this disease accurately (Rajesh et al., 2004).

There are such traditional strategies for analysis of genetic components in the etiopathogenesis of multifactorial diseases as clinical—genealogical and genetic—epidemiological methods. It is important to state that in recent years there has been a development in genomic technology, such as a DNA analysis in case of multifactorial diseases (Pulley et al., 2010). The initial genetic findings that were based mainly on statistical hypotheses need further verification and search for new and more effective approaches in the study of this problem.

The study of multifactorial diseases in genomic medicine is now in its initial stage. There will come a time when all human genes are known and then the scientific community will have to develop the Periodic Table of Life similar to the Periodic Table of Mendeleev (McKusick, 1997, 1998).

These approaches are likely to provide opportunities for primordial prevention of cardiac conduction disorders for individuals with a hereditary load or abnormalities. Primary prevention of cardiovascular diseases is considered to be a strategic task in cardiology as well as one of the social tasks of the state. It is known that primary prevention of cardiovascular diseases for the whole population is not economically viable. This is why it is necessary to identify risk groups in the population for primary prevention needs. Family history seems to be one of the important criteria for the formation of such groups. In this regard, it is essential to identify risk factors in each family; in our situation, this refers to genetic risk factors of the development of the disease.

Thus, further study of the etiology and pathogenesis of idiopathic cardiac conduction disorders and the study of the molecular basis of cardiovascular pathology give us opportunities for more focused, individualized, and accurate therapy and prevention.

Footnotes

Acknowledgments

We thank our colleagues N. Gogolashvili, G. Matyushin, L. Nazarenko, S. Popov, and V. Kozlov for offering valuable suggestions to improve this article.

Author Disclosure Statement

No competing financial interests exist.

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