1 INTRODUCTION

Brugada syndrome (BrS) (OMIM: 600163) is an inherited disorder of cardiac electrical conduction, characterized by a specific ST-segment elevation in the right precordial leads (V1-V3) on the electrocardiogram (ECG), strongly associated with the high risk of ventricular arrhythmias and sudden death (Antzelevitch, 2006). Clinical presentation of BrS also involves sudden infant death syndrome and sudden unexpected nocturnal death syndrome. About 4% of all sudden death cases and at least 20% of sudden death cases with typical heart structures are associated with BrS (Kusano, 2013). The overall prevalence of BrS is globally estimated at 0.05% and found to be of different incidences in different regions. The highest prevalence of BrS is verified in Southeast Asia (3.7 per 1000 population), followed by the Middle East and South Asia, with both rates of 1.8 per 1000 population (Vutthikraivit et al., 2018). Males show a higher prevalence and more severe clinical manifestations than females (Benito et al., 2008). Mutations in SCN5A gene are associated with BrS, accounting for approximately 30% of BrS cases (Brugada et al., 2018). Variety of genes have been identified to be related to BrS, but SCN5A gene is the most dominant when occupied by >5% of positive genotyped patients (Ackerman et al., 2011). According to The European Society of Cardiology guidelines, SCN5A genetic testing is recommended in cases of Class I: the family with carriers of SCN5A mutation in BrS, Class IIa: the patients suspected with BrS based on clinical features, family history, and electrocardiographic (ECG) testing, Class IIb: the patients with ECG type 2 and type 3 of BrS (Priori et al., 2015). The genetic testing in affected family members plays a pivotal role in treatment classification which comprises at-risk (+) and precaution (−) groups.

SCN5A gene is located on chromosome 3p21 and has 28 exons that encode for the cardiac voltage-gated sodium channel (Na_v1.5). Na_v1.5 contains four transmembrane domains (DI-DIV) connected by inter-domain linkers. Each domain includes six transmembrane segments (S1-S6) joined by intra-extracellular loops (Rook et al., 2012). Segments S1-S4 in each domain are essential to forming a voltage-sensing domain (VSD) that is activated to generate sodium current (Chen-Izu et al., 2015). Na_v1.5 induces the depolarization of atrial and ventricular action potentials, thus creating the up-stroke of the cardiac action potential (Jiang et al., 2020). To date, more than 900 SCN5A variants are associated with cardiac disorders and scattered throughout different gene regions. The variants induce the diversity of BrS phenotypes as well as causing loss-of-function or gain-of-function of sodium channel via altered voltage- and time-dependent activation/inactivation and decelerated recovery from inactivation (Savio-Galimberti et al., 2018). Therefore, identifying SCN5A variants and their functional assessment are vital in optimizing and personalizing patient treatment.

In Vietnam, data regarding the molecular characteristics of SCN5A variation are still lacking. Very few studies have determined the association between the genotype and phenotype of SCN5A variants in Vietnamese patients. Recently, computation tools have become widely used to assess the consequence of variants potentially causing disease (Thusberg et al., 2011). Thus, we conducted this study to screen for SCN5A variants and evaluated the genotype–phenotype correlation of BrS on 117 probands. The results will support risk stratification of BrS for treatment and provide detailed information to clinicians for genetic counseling.

2 MATERIALS AND METHODS

3 RESULTS

3.3 Novel SCN5A variants

In our study, eight of 21 SCN5A variants were found as novel variants and were not previously recorded in the databases (ClinVar: https://www.ncbi.nlm.nih.gov/clinvar/; LOVD: https://www.lovd.nl/; dbSNP: https://www.ncbi.nlm.nih.gov/snp/). All SCN5A variants are displayed in Table 2 and Figure 1. The location on the protein sequence was followed by the UniprotKB (Q14524), as described: the N-terminus (aa 1–131), domain I (DI S1-S6, aa 132–410), inter-domain linker I-II (aa 411–717), domain II (DII S1-S6, aa 718–938), inter-domain linker II-III (IDL II-III, aa 939–1206), domain III (DIII S1-S6, aa 1207–1466), inter-domain linker III-IV (IDL III-IV, aa 1467–1529), domain IV (DII S1-S6, aa 1530–1771), and the C-terminus (aa 1772–2016). The novel variants were located mainly in the DI, accounting for 15.15%, compared to 6.06% in both N-terminal and C-terminal regions. Among SCN5A variants, a frameshift variant was determined in two patients, such as c.325_327delAAC (N109del) in exon 3. Also, genomic duplication was observed in two patients who showed a duplicate of exon 2 and 3 by MLPA. Five missense variants were identified along 28 exons of SCN5A: c.551A>G (Exon 5), c.754G>A (Exon 7), c.797T>A (Exon 7), c.980G>A (Exon 8), c.5389A>T (Exon 28), produced the amino acid change at His184Arg, Asp252Asn, Leu266His, Gly327Glu, and Ile1797Phe, respectively. A synonymous variant c.5484C>T was found in a single patient at nucleotide position 5484 with no effect on the wild-type 1828 Alanine amino acid residue (Ala1828Ala).

4 DISCUSSION

BrS is a rare inherited cardiac disorder and is generally related to sudden death during sleeping in South East Asia. Most BrS patients are diagnosed without clarifying clinical manifestations, leading to the challenge of early diagnosis. SCN5A gene encodes for the cardiac voltage-gated sodium channel (Na_v1.5) and is responsible for the incidence of BrS. The SCN5A variants are estimated in up to 30% of the patients. Loss-of-function mutations on SCN5A have been investigated to cause dilated cardiomyopathy and lower expression levels of SCN5A or produce defective Na_v1.5 proteins (Wilde & Amin, 2018). Therefore, genetic screening of SCN5A is necessary for the clinical diagnosis of BrS patients, identification of non-genetic carriers, and selection of promising strategies to prevent genetic carriers in the offspring.

BrS is well known to be more distributed and severe in males than in females. The vast frequency of affected males in this study was marked at 97.43%, in agreement with prevalence in Asia (Milman et al., 2019). Several hypotheses have been postulated to clarify the gender discrepancy. Variable sex receptors containing estrogen, progesterone, and testosterone are discovered in the cardiac myocytes; thus, the alteration of sex hormone concentration can cause fluctuation in the performance and expression of myocardial ion channels (Lizotte et al., 2009).

In our study, the prevalence of SCN5A variants was determined at 25.6%, higher than Taiwanese and Hispanic populations, which accounted for 8% and 16%, respectively (García-Castro et al., 2010; Juang et al., 2015). Syncope and SCD are common clinical characteristics in BrS-related SCN5A variants. We found that syncope and a risk family history of SCD were presented in 38.46% and 31.62% of our BrS patients, respectively. The syncope symptom of those who carried SCN5A variants (66.33%) has a significantly higher risk than those of non-carriers (RR = 3.147, [95% CI: 1.668–5.982], p = 0.004). A similar rate was obtained in a meta-analysis, which was evaluated at 35.9% in the occurrence of syncope related to SCN5A (+) (RR = 1.12, [95% CI: 0.87–1.45], p = 0.37) (Raharjo et al., 2018). Moreover, our data contrast with the result of Yamagata et al. (2017), who did not observe a statistical difference in the family history of SCD (p = 0.5); however, a significantly higher rate of cardiac events was recorded between SCN5A (+) and SCN5A (−) groups (HR = 1.1, 95% CI: 1.1–3.8, p = 0.02) (Yamagata et al., 2017). In addition, BrS could be related to the major arrhythmic events (MAEs), comprising VT and VF (RR = 3.406, [95% CI: 1.722–5.4], p = 0.0035). Sommariva et al. (2013) indicated that the patients harboring SCN5A variants had an increased risk of MAEs than none of those (p = 0.024) (Sommariva et al., 2013). Nishii et al. (2010) determined the existence of SCN5A variants related to early and frequent recurrence of VF in symptomatic BrS patients, not in the first onset of VF (21.7% vs. 20.0%, p = 0.373) (Nishii et al., 2010). The variable correlation of BrS clinical phenotype may be produced by inter-individual genetic differences, regulating excitation-contraction physiology and affecting arrhythmia susceptibility among distinct ethnicities (Splawski et al., 2002; Suh & Vijg, 2005).

In the current study, 21 different SCN5A variants were successfully identified. Interestingly, one genomic duplication (Ex2-Ex3 dup) (2/117) was detected in our patients. To date, very few publications have shown the large rearrangement in SCN5A. Eastaugh et al. (2011) first described a major deletion in SCN5A. The c.999-424_1338 + 81del causes loss of exon 9 and 10 of SCN5A, resulting in haploinsufficiency (Eastaugh et al., 2011). The deletions of SCN5A were expressed in the other regions such as promoter, exons, and even the whole SCN5A gene (Hertz et al., 2015; Jenewein et al., 2017; Trujillo-Quintero et al., 2019). It is a fact that genomic instability always leads to the complete loss of function of the affected gene; therefore, these variants are more likely to induce the BrS phenotype. Several studies have been performed to identify the genomic imbalance in SCN5A, but the frequency rearrangement was 0% (García-Molina et al., 2013; Koopmann et al., 2007). These findings suggest that MLPA technique should be included when conducting genetic screening for BrS, even though the rearrangement rate is infrequent.

The novel variant c.325_327delAAC (N109del) was located on the N-terminal of Na_v1.5 protein. N-terminal is a conserved region and plays a crucial role in regulating the cardiac sodium channel. The alteration of N-terminal can cause the increase or decrease of function on the channel, involving a quality control mechanism (Clatot et al., 2012). Following this mechanism, cells carried mutants use endoplasmic reticulum-associated degradation (ERAD) as a protective role to degrade proteins that fail to acquire their native conformation by the ubiquitin–proteasome system (Brodsky & Scott, 2007). The majority of our novel missense variants were discovered scattered from S2 to S6 segments of DI, consisting of c.551A>G (H184R), c.754G>A (D252N), c.797T>A (L266H), and c.980G>A (G327E). Most of these variants were predicted as pathogenic variants, except c.551A>G (H184R) as benign (Figure 1; Table 4). The S1-S4 components of each domain are considered as the voltage sensor domain (VSD), which plays a central function in controlling the activity of the channel, while S5-S6 segments assemble to form the pore domain and are responsible for the ion selectivity of Na_v1.5 (Chen-Izu et al., 2015). Thus, minor structure modifications can initiate the dysregulation functioning of the channel. Additionally, two novel variants, c.5389A>T (I1797F) and c.5484C>T (A1828A), were found on the C-terminal of Na_v1.5 and predicted as disease-causing and benign, respectively. The substitutions in the C-terminal domain have been explored to modulate channel inactivation (Glaaser et al., 2006). Although the synonymous c.5484C>T (A1828A) variant did not change the amino acid sequence, it may disrupt mRNA transcription, translation, and splicing (Goymer, 2007; Stergachis et al., 2013).

Together with novel variants, 13 known variants also were identified. The c.1100G>A (R367H, rs28937318) variant has been evaluated to cause loss-of-function in Na_v1.5 channel and is also associated with atrial standstill and early repolarization in idiopathic ventricular fibrillation (Kapplinger et al., 2010; Takehara et al., 2004; Watanabe et al., 2011). The c.1890+14G>A variant was found in the intronic region and was predicted benign on the ClinVar database (rs145427253) (Duzkale et al., 2013). Other variants were determined to occur at 28.57% (6/21) on the internal domain linker, such as DI-II (c.1712G>C (S571T), c.1975C>T (R659W)), DII-III (c.2893C>T (R965C), c.3338C>T (A1113V), c.3578G>A (R1193Q)), and DIII-IV (c.4534C>T (R1511W)) (Abe et al., 2018; Chimparlee et al., 2021; Kapplinger et al., 2009, 2010; Meregalli et al., 2009; Shen et al., 2022). Of these variants, c.3338C>T (A1113V) and c.3578G>A (R1193Q) were predicted as benign, and damage was evaluated in the other. The mutation rate on the linker domains on Na_v1.5 was relatively high, accounting for 35% in the study of Kapplinger et al. (2009) Several studies have reported the affection of SCN5A variants on internal domain linkers. They indicated that these linkers play a valuable function in pathogenesis (Smits et al., 2005; Wehrens et al., 2003; Yamamura et al., 2010). Variant c.2236G>A (E746K) (DII/ S1-S2) was a hot spot for BrS and related to the reduced activation process on Na_v1.5 channel (Huang et al., 2017; Zaytseva et al., 2022). An alteration c.2678G>A caused the change of amino acids from Arg to His at position 893, also detected on DII. This variant induced loss-of-function, showing a significant decrease in the current density peak than the wild-type through patch clamp (Ishikawa et al., 2021). The c.4171G>A (G1391R) variant was first reported by Szperl et al. (2018) in the Polish population. Genotype and phenotype analysis showed that the carriers with G1391R variant presented a higher risk of cardiac events in females than in males (Szperl et al., 2018). The minor deletion c.4850_4852delTCT (F1617del) variant was found to be associated with LQT3 syndrome, which induced the change of fast inactivation by decreasing gating charges (Chen et al., 2005). According to te Riele et al. (2017), the missense variant c.5693G>A (R1898H) led to a decrease at 36% in peak sodium current (p = 0.002), also in the abundance of Na_v1.5 (p = 0.005) on fluorescence microscopy and N-Cadherin (p = 0.026) clusters at the intercalated disk (te Riele et al., 2017).

Our study's genotype–phenotype correlation and the functional prediction of all variants were only approached by statistical analysis and bioinformatic tools. Further, follow-up studies should be performed to provide insight into the role of SCN5A variants in the severity of BrS and promptly specify the most appropriate treatment for each patient.

5 CONCLUSION

Our study initially evaluated the genotype–phenotype correlation of SCN5A variants with patients of BrS in Vietnam. The presence of SCN5A variants shows a significantly higher risk in the family history of sudden death, syncope, and arrhythmia. Aside from published SCN5A variants, seven additional variants were predicted to cause functional alteration in cardiac voltage-gated sodium channel (Na_v1.5). Follow-up studies should be conducted to clarify the impact of SCN5A variants on the clinical characteristics of BrS.

AUTHOR CONTRIBUTIONS

Van-Khanh Tran, Hung Manh Pham, Thinh Huy Tran, Thanh Van Ta, and The-Hung Bui: Conceived and designed the study and analysis. Thi Phuong Le, Dinh Phong Phan, Hoai An Trinh, Thanh Van Ta, and Ha Minh Nguyen: Contributed to data collection and carried out the experiments, performed analysis, and finalized the results. Hung Manh Pham, Duy Phuong Dang, Thanh Dat Ta, and Van-Khanh Tran: Contributed to the drafting of the manuscript. All authors have read and approved the final version for publication.

FUNDING INFORMATION

This study was supported by the Ministry of Health of Vietnam, grant number 2723/QĐ-BYT.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no conflict of interest regarding the publication of this article.

ETHICS STATEMENT

This study was approved by the Ethics Committee of Hanoi Medical University (Hanoi Medical University Institutional Review Board, Hanoi, Vietnam, reference number: 48/HĐĐĐĐĐHYHN).

CONSENT

Written informed consent was obtained from the patient for publication.