The rs6771157 C/G polymorphism in SCN10A is associated with the risk of atrial fibrillation in a Chinese Han population

A recent genome wide associated study in European descent population identified the association of Atrial fibrillation (AF) risk with a single nucleotide polymorphism (SNP) in SCN10A. The aim of this study was to evaluate whether SCN10A polymorphisms are associated with AF risk in the Chinese Han population. A total of 2,300 individuals of Chinese Han origin were recruited and three potentially functional SNPs were genotyped. Logistic regression models were utilized to calculate odds ratios (ORs) at a 95% confidence intervals (CIs). Logistic regression analysis in an additive genetic model revealed that one SNP in SCN10A (rs6771157) was associated with an increased risk of AF (adjusted OR = 1.20, 95% CI: 1.06 - 1.36, P = 0.003). Stratification analysis of several main AF risk factors indicated that the risk associations with rs6771157 were not statistically different among different subgroups. In summary, our study suggests the possible involvement of the SCN10A variant in AF development in Chinese Han populations. Further biological function analyses are required to confirm our finding.

Scientific RepoRts | 6:35212 | DOI: 10.1038/srep35212 and controls are summarized in Table 1. In brief, the variants of gender and age in two groups were comparable (P > 0.05). Compared with the non-AF subjects, AF patients were more likely to have the percentages of hypertension, diabetes and coronary artery disease (CAD) (45.1% vs. 20.8%, P < 0.001 for hypertension, 10.5% vs. 7.5%, P = 0.011 for diabetes and 10.3% vs. 0%, P < 0.001 for CAD, respectively).
Associations between SCN10A variants and AF risk. The genotype distributions of the three SNPs and their associations with AF risk are shown in Table 2. The genotype frequencies of these SNPs were all observed in agreement with Hardy-Weinberg equilibrium in the control group. The logistic regression analyses revealed that the rs6771157 C allele was significantly associated with the increased risk of AF in an additive model (adjusted OR = 1.20, 95% CI: 1.06-1.36, P = 0.003). We further calculated P values for false discovery rate (P-FDR) to perform multiple comparisons, which found that rs6771157 still remained association with the risk of AF (P-FDR = 0.010). There was no obvious evidence of significant association between the other two SNPs and AF risk.

Stratification Analysis.
We assessed the effect size of rs6771157 with AF by stratification analyses based on the main AF risk factors such as age, gender, hypertension, diabetes. As shown in Table 3, no significant difference for the association of rs6771157 with AF risk between subgroups was detected (P > 0.05 for heterogeneity test).

Discussion
This case-control study investigated the relationship between the common genetic variants of SCN10A and their potential function in the risk of AF in the Chinese Han population. Only SNP rs6771157 was identified to be significantly associated with Chinese Han AF risk.
SCN10A encodes the voltage-gated sodium channel, Nav1.8 channels. The protein product of SCN10A is predominant tetrodotoxin-resistant sodium channel in primary sensory neurons 19,20 . Although the exact role of Nav1.8 in cardiac electrophysiology remains currently unclear 21 , SCN10A is expressed both in intracardiac neurons and human cardiomyocytes 22,23 . Further, blockade of Nav.18 channels suppresses the impact of vagus nerve stimulation (VNS) on both cardiac conduction and AF inducibility [23][24][25] . Thus, SCN10A plays an important role in cardiac electrophysiology and modulating susceptibility to arrhythmias 24 .
In a previous GWAS. A tag SNP (rs6800541) in SCN10A has been found to have a strong association with PR-interval duration and AF 16 . Most recently, a function study by Jabbari et al. revealed two common nonsynonymous variants in SCN10A (rs6795970, rs12632942) result in a gain-of-function of Nav1.8 channel 21 . In the present study, we found an association between another common variant rs6771157 and AF risk in Chinese Han populations. We analyzed the linkage disequilibrium of SNP rs6771157 with the two significant SNPs via haploview software, which showed r 2 = 0.1 and 0.171 for rs6800541 and rs6801957 respectively (Fig. 1). Thus, the rs6771157, a synonymous variant located in the 19 exon of SCN10A. is a signal different from the two SNPs reported in European individuals. It was predicted as a functional SNP in an ESE (exon splicing enhancer) by the online tool SNPinfo (http://snpinfo.niehs.nih.gov/index.html), which may be responsible for aberrant splicing of pre-mRNA of SCN10A by binding to the SR proteins of splicing activators 26 . A possible explanation is that rs6771157 causes the exon skipping by activating the creation of the ESE.
Several limitations of the present study need to be considered. Firstly, the sample size is relatively small compared to GWAS, which may predispose to the failure to detect effects of another two potential functional SNPs in our study. Secondly, echocardiography results and N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) levels were not available for all the individuals to evaluate cardiac functions, which may have resulted in information bias. We did not evaluate cardiac functions by left ventricular ejection fraction (LVEF) and NT-proBNP. Furthermore, we selected the potential functional SNP by using on-line tools, which may result in positive or negative errors. Thus, further replication in larger sample sets and mechanism researches are required to confirm our findings.
In conclusion, the current investigation confirmed that one functional common variation (rs6771157) in SCN10A was significantly associated with the risk of AF in a Chinese Han population. However, the role of this variant in the AF susceptibility is warranted to be further evaluated by function studies.  Table 3. Stratified analysis on the associations of SNP in SCN10A with AF. a Obtained in logistic regression models with adjustment for age, gender, hypertension, diabetes and coronary artery disease (the stratified factor in each stratum excluded). b P for heterogeneity test using the Chi-square-based Q test. c Age was divided into two subgroups according to its median (59 years). of distinct repeating P waves, and irregular atrial activity 27 . We classified AF as paroxysmal AF (terminates spontaneously or with intervention within 7 days of onset), persistent AF (episodes that sustain beyond 7 days). We defined "Lone AF" as young AF individuals (< 60 years) without clinical or echocardiographic evidence of cardiopulmonary disease 3 . The controls were recruited from other departments of the First Affiliated Hospital of Nanjing Medical University, and they were all confirmed to be free of AF based on medical files or ECG at the time of enrollment. The general and clinical information of all participants was collected from medical recording files in the hospital system. We excluded patients with hyperthyroidism, severe cardiac dysfunction (NYHA Class IV), valvular heart disease and advanced age (beyond 90 years) in AF group. All individuals were Chinese Han origin. SNP selection. We first used public HapMap SNP database (phase II+ III Feb 09, on NCBI B36 assembly, dbSNP b126) to search SNPs that localized within the gene region of SCN10A (including 10 kb upstream of the gene), with MAF (Minor Allele Frequency) ≥ 0.05 in Chinese Han population. Then, a web-based analysis tool was used to predict the function of these SNPs (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm). After function prediction analysis, a total of six potentially functional SNPs were selected. We conducted linkage disequilibrium (LD) analysis to exclude SNPs with strong LD (r 2 ≥ 0.8), then three (rs9827941, rs7630989, and rs6771157) functional SNPs were selected for further genotyping. SNP genotyping. Peripheral venous blood samples were drawn from study participants. Genomic DNA was extracted from EDTA-preserved whole blood using a standard phenol-chloroform method 28 . The genotyping was conducted using the method of the improved multiple ligase detection reaction (iMLDR) with the technical assistance from Shanghai Genesky Bio-Tech Genetic Core Lab. All SNPs were genotyped successfully with call rates at least of 99.3%. Statistical analysis. The comparisons of clinical characteristic differences between cases and controls were identified using Student's t test for continuous variables and the χ 2 test for categorical variables. The deviation of genotype distribution from Hardy-Weinberg equilibrium was tested by χ 2 test in the control group. ORs and 95% CIs were calculated to assess the relationships between the SNPs and the risk of AF using logistic regression. The heterogeneity of associations among subgroups was evaluated using the χ 2 -based Q-test. All statistical analyses were performed with STATA 12.0 software (Stata Corp., College Station, TX, USA). P < 0.05 was the criterion of statistical significance and all statistical tests were two-tailed. LD analysis was applied by using Haploview 4.2 software.