Genetic Characteristics and Transcriptional Regulation of Sodium Channel Related Genes in Chinese Patients With Brugada Syndrome

Objective: To investigate the genetic characteristics and transcriptional regulation of the SCN5A gene of Brugada syndrome (BrS) patients in China. Methods: Using PubMed, Medline, China National Knowledge Internet (CNKI), and Wanfang Database, Chinese patients with BrS who underwent SCN5A gene testing were studied. Results: A total of 27 suitable studies involving Chinese BrS patients who underwent the SCN5A gene test were included. A total of 55 SCN5A gene mutations/variations were reported in Chinese BrS patients, including 10 from southern China and 45 from northern China. Mutations/variations of BrS patients from southern China mostly occurred in the regions of the α-subunit of Nav1.5, including DIII (Domain III), DIV, DIII-DIV, C-terminus regions, and the 3'UTR region. Furthermore, we analyzed the post-transcriptional modifications (PTMs) throughout the Nav1.5 protein encoded by SCN5A and found that the PTM changes happened in 72.7% of BrS patients from southern China and 26.7% from northern China. Conclusions: SCN5A mutations/variations of BrS patients in southern China mostly occurred in the DIII-DIV to C-terminus region and the 3'-UTR region of the SCN5A gene, different from northern China. PTM changes were consistent with the mutation/variation distribution of SCN5A, which might be involved in the regulation of the pathogenesis of BrS patients.


INTRODUCTION
Brugada syndrome (BrS) is an inheritable arrhythmogenic disease. The typical electrocardiographic manifestations include ST segment elevation ≥2 mm and T wave inversion on the right thoracic lead (V1-V3) of ECG. BrS is prone to polymorphic ventricular tachycardia, ventricular fibrillation, and sudden cardiac death while the heart structure is normal (1,2). It is more prevalent in Asian population with an onset age of 30-40 years old, and the ratio of male to female is 8-10:1 (3,4). The mortality accounted for 4-12% of sudden death each year and even for 20% sudden death without organic heart disease in Southeast Asia (5).
Up to date, 23 genes have been confirmed to be related to BrS (5)(6)(7), including gene mutations/variations that lead to ion channel dysfunction such as sodium, calcium, and potassium ions. About 30-35% of BrS patients were identified with pathogenic mutant genes, while SCN5A mutations/variations encoding the Nav1.5 α-subunit of the cardiac sodium channel accounted for 20 to 30% (5,(8)(9)(10). Currently, the recommended treatment for BrS is ICD implantation and medication (quinidine, isoproterenol, etc.); however, therapeutic effect is unsatisfactory.
The mutations/variations from BrS patients can significantly reduce the inward Na + current by delaying activation, accelerating inactivation, delaying reactivation, or reducing the membrane expression of ion channels (11,12). Furthermore, the decrease in the inward Na + current has influence on the depolarization and repolarization of the cardiac action potential, thus causing the generation of typical ECG of BrS (2). Genetic distribution characteristics of BrS on the SCN5A mutations/variations from the world (9) and Japan (13) had been reported. However, there are no genetic distribution analyses on the SCN5A mutation/variation location sites in Chinese BrS patients until now. In the present study, we aim to analyze the reported SCN5A mutation/variation location sites of Chinese BrS patients and predict the PTMs affected by the mutations/variations.

Information Retrieval and Inclusion Criteria
Two investigators searched Medline, PubMed, CNKI, and Wanfang Database. The query terms were "Brugada syndrome" "China" and "SCN5A." Articles published in Chinese or English in peer-reviewed journals that met the following criteria were included in our study. A. Inclusion of subjects with BrS were as previously defined (14). B. Chinese patients. C. Who underwent SCN5A gene DNA sequencing.
In addition, we also contacted the corresponding author of several studies in order to obtain more specific experimental data that were not included in the article. In order to resolve any difference or uncertainty between the two investigators, a third investigator was responsible for reexamining the source data and consultation.
Predictive Analysis the PTMs of SCN5A First, we retrieved the amino acid sequence and analyzed the domain details of the α-subunit of SCN5A protein in the following website: https://www.uniprot.org/uniprot/Q14524, then predicted the PTM sites with the software on the amino acid sites and mapped on the mutation/variation sites reported from the literature in China. Furthermore, we analyzed the change of PTMs in the natural variant.

Statistical Analysis
Categorical variables were expressed as percentage and analyzed using the chi-square test. All analyses were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05.

Studies Retrieved and Information
Extraction, and Distribution of the SCN5A Mutations/Variations on Nav1.5 Protein A flow chart of the data research is shown in Figure 1. We excluded 306 unqualified studies that did not match the inclusion criteria or were duplicates. A total of 27 suitable studies were included, and details are shown in Table 1. Duplicated loci were removed, and a total of 55 mutations/variations including 45 sites from northern China and 10 sites from southern China (Guangdong, Guangxi, Hong Kong, Hainan, Jiangxi, Fujian,   Taiwan) were included. Further analysis of these loci was performed as detailed below. The Nav1.5 channel has four highly conserved homologous transmembrane-spanning domains (DI-DIV) that are connected by an interdomain linker (IDL), and each domain consists of six transmembrane α spins (S1-S6). In order to visualize the distribution of these loci, we marked the mutation/variation sites on Nav1.5 protein (excluding introns and 3'UTR, which were not translated into amino acids), as shown in Figure 2.   Furthermore, the locations of SCN5A gene mutations/variants were divided into five parts as follows: N-Term, Transmembrane regions, IDL, C-Term, and others as shown in Figure 4A. The mutation/variation site distributions of SCN5A in southern China and northern China were 0, 50, 0, 30, and 20%, and 11.1, 24.5, 33.3, 8.9, and 22.2%, respectively (p = 0.000).
On the other hand, the locations of SCN5A gene mutations/variants were divided into three parts as follows: before-DIII, after-DIII, and others as shown in Figure 4B. The mutation/variation site distributions of SCN5A in southern China and northern China were 10, 80, and 10% and 60, 20, and 20%, respectively (p = 0.001).
Then, we compared the distributions of mutations/variations among China (southern China and northern China), Japan, and the world, as shown in Figure 5A. Japanese data refer to a Japanese multicenter register (42), and the global data are from the website http://triad.fsm.it/cardmoc/. We distinguished the SCN5A mutation sites on the protein Nav1.5 structure by Nterm, Transmembrane regions, IDL, and C-term and found 0, 62.5, 0, and 37.5% for each part in southern China; 14.3, 31.4, 42.9, and 11.4% in northern China; 4.4, 66.7, 20, and 8.9% in Japan; and 4.8, 71, 18.4, and 5.8% in the world, respectively (p = 0.000).
In addition, the structure of Nav1.5 protein was divided into two parts: DI, IDL I-II, DII, and IDL II-III were set as the first half part (before-DIII), and DIII, IDL III-IV, DIV, and C-Term were set as the second half part (post-DIII) as shown in Figure 5B. The results indicated that 88.9% of the mutation sites were located in the post-DIII region in southern China, while only 22.9% in northern China, 42.2% in Japan, and 47% in the world (p = 0.000).

PTMs Prediction for Nav1.5 Protein Change
As mutations that cause changes in amino acids may have influences on protein modification, we predictively analyzed the PTMs of SCN5A with software and mapped on the mutations/variations in China ( Table 2), and found a tendency for amino acids to acquire more modification sites after mutation. PTM change was likely to occur in 72.7% of BrS patients from southern China and 26.7% from northern China (p = 0.000).

DISCUSSION
Our main findings in the study of the genetic characteristics of SCN5A in Chinese BrS patients were as follows: (1) More SCN5A gene mutations/variations were found in northern China than in southern China. (2) SCN5A mutations/variations of BrS patients in southern China mostly occurred in the DIII-DIV to C-terminus region and the 3 ′ -UTR region of the SCN5A gene.
(3) PTM changes were consistent with the mutation/variation distribution of SCN5A, which might be involved in the regulation of the pathogenesis of BrS patients.
BrS can be found all over the world, and the prevalence of BrS can reach 0.5‰ in high-prevalence areas. BrS is the leading cause of death for men less than 40 years old, only second to the death rate of traffic accidents in Southeast Asian countries (42,43). In southern China, BrS patients were anticipated to have a relatively high incidence rate. However, our study revealed that SCN5A gene mutations published were found to be more in northern China than southern China. The possible reasons may be that BrS is a rare disease, and the total number of cases reported at present was not large and patients in many studies did not undergo DNA sequencing, which results in data bias. We will continue to pay attention to relevant reports and continue to collect cases to further confirm the data.
Further analysis showed that the locations of mutation sites had their own characteristics in southern China. Most mutation sites were clustered in the transmembrane regions in southern China statistically different from northern China. Mutation sites were mostly located in the second half part of the protein structure (post-DIII) in southern China, while in the first half part positions (before-DIII) in northern China, Japan, and the world.
The SCN5A gene, located on chromosome 3p21, contains 28 exons with a total length of about 80 kb and encodes the α-subunit protein Nav1.5. Some mutations lead to a decrease in current density, others do not lead to a decrease in I Na , while some location-specific SCN5A mutations resulted in poorer outcomes during follow-up (44). As different mutation FIGURE 5 | (A) The translation comparison of the SCN5A mutation/variation distribution on the Nav1.5 protein structure between southern China and northern China. Nav1.5 protein was distinguished by N-term, Transmembrane regions, IDL, C-term, and other parts. The light blue columns represent the N-terminus region. The orange columns represent the transmembrane region. The gray columns represent the IDL region. The yellow columns represent the C-terminus region. (B) The translation comparison of the SCN5A mutation/variation distribution on the Nav1.5 protein structure between southern China and northern China. Nav1.5 protein was distinguished by before-DIII and after-DIII. The blue columns represent before-DIII parts. The orange columns represent after-DIII parts.

Mutations
The potential PTMs before mutation The potential PTMs after mutation Structural position Location