Abstract
Background: A novel gene Caveolin-1(CAV1) was identified to be susceptibility to PR interval and also associated with atrial fibrillation (AF) in two Genome wide associations studies (GWAS) studies in European ancestry. The purpose of this study was to determine the association of the SNPs in CAV1 gene of rs3807989 with AF in Chinese Han patients. Methods and results: We attempted a replication in a cohort of 839 Chinese AF patients and 1215 healthy controls using melting temperature shift allele-specific genotyping analysis. One SNP in CAV1 (rs3807989) was genotyped. The final study cohort consisted of 839 AF patients and 1215 healthy controls. No significant association was detected between rs3807989 and AF in a Chinese Han population (allelic P-adj = 0.828 with OR = 1.02; genotypic P-adj = 0.815, 0.405, 0.760 with a dominant model, recessive model, and additive model). After logistic regression with multiple covariates, the association remained non-significant with adjusted P value 0.828. When the AF cases were further divided into lone AF (31.5%) and other types of AF (68.5%), no significant association was found between rs3807989 and lone AF (P-adj = 0.929 with OR = 0.990) and other types of AF (P-adj = 0.597 with OR = 1.060). Conclusion: The SNP rs3807989 in CAV1 gene is not associated with AF or lone AF in our studies, which suggests that the SNP rs3807989 in CAV1 may not be a risk factor for AF in Chinese Han population.
Keywords: CAV1, atrial fibrillation, Rs3807989
Introduction
Atrial fibrillation (AF) is one of the most common cardiac arrhythmias, which affects 1-2% of the general population [1]. The prevalence of AF increases substantially with aging, from ~1% in young adults to ~10% in people older than 80 years [2]. AF has been shown to be an independent risk factor for ischemic stroke (IS), one of main causes of mortality and morbidity [3]. AF is mostly associated with cardiac risk factors, including hypertension, congenital heart disease, coronary artery disease and valvular heart disease [4]. Nearly 30% of AF cases, which have no predisposing factors, are therefore classified as lone AF [5].
Accumulating studies have indicated that genetic factors play an important role in the etiology of AF, in particular lone AF. In the last decade, a number of AF associated genes and loci, such as KCNQ1, KCNH2, SCN5A, KCNE2, KCNJ2, NUP155, NPPA, SCN3B, 4q25, ZFHX3 and KCNN3, have been identified with genetic linkage and genome-wide association studies (GWAS) [6-10]. Caveolin-1 gene (CAV1), encoding a caveolae protein, is expressed in atrial myocytes [11]. In two independent GWAS studies, CAV1 has been reported to be associated with AF [12,13]. Mice deficient in Cav1 develop dilated cardiomyopathy and pulmonary hypertension [14]. More recently, a SNP in CAV1 gene, rs3807989, has been associated with early-onset lone AF before the age of 40 years [15]. However, these study results need to be independently confirmed in other ethnic populations [16]. In the present case-control association study, we investigated the association of rs3807989, a tag SNP in CAV1, with AF in a Chinese Han population.
Materials and methods
Study subjects
The study subjects were enrolled from multiple hospitals in Dalian, Wuhan, Suizhou, Xiangfan and Shiyan cities and all were Chinese Han people. This study was approved by institutional review boards on human subject research, and was conformed to the guidelines of the Declaration of Helsinki. Informed consents were obtained from the participants or guardians.
Diagnosis criteria
AF was diagnosed by a panel of cardiologists, according to standard diagnostic criteria, including electrocardiograms (ECG) and/or Holter ECG [17]. Patients with other types of cardiac arrhythmias were excluded from this study. Patients with cardiomyopathies and valvular heart disease were also excluded by echocardiography (Echo). We recruited AF patients of younger than 80 years old with AF. Lone AF was defined as patients without history of hypertension, coronary artery disease (CAD), congenital heart disease, congestive heart failure, ischemic stroke or diabetes. All controls had no arrhythmias, coronary artery disease (CAD), congenital heart disease, cardiomyopathies or valvular heart disease by ECG, Echo, magnetic resonance imaging (MRI) and computed tomography (CT).
Genotyping
Peripheral venous blood samples were drawn from all participants and leukocytes were isolated. Genomic DNA was extracted from leucocytes following standard protocols of the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). The SNP rs3807989 was genotyped on a Rotor-GeneTM 6000 High Resolution Melt (HRM) system (QIAGEN, Hilden, Germany), with a reaction of 25 μL polymerase chain reaction (PCR), containing 1 μL of LC green dye, 5 pmol of each primer, 25 ng of genomic DNA, 2.5 μL of 10 × PCR buffer with 1.5 mmol/L MgCl2, 5 mmol deoxynucleotide triphosphates, and 1 U of Taq polymerase. Two positive controls for each genotype (A/A, A/G, and G/G) were included in each run. A total of 48 cases and controls were randomly selected for verification of genotyping results using direct DNA sequence analysis. DNA sequencing was performed in both-directions using the BigDye Terminator v3.1 Cycle Sequencing Kits on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Statistical analysis
Power analysis was carried out by using PS software v3.0 (Dupont WD, Plummer WD: “Power and Sample Size Calculations: A Review and Computer Program”, Controlled Clinical Trials 1990). SNP rs3807989 genotypes were tested for Hardy-Weinberg equilibrium in controls using PLINK v1.05. Allelic and genotypic association of rs3807989 with AF was assessed using Pearson’s 2 × 2 and 2 × 3 contingency table χ2 test (PLINK). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using the χ2 test (PLINK). Multivariate regression analysis was performed by incorporating age, sex, hypertension, CAD (for AF dataset) and ischemic stroke as covariates using multivariate logistic regression (PLINK). Empirical P values were determined using the PLINK v1.05 program with 100,000 Monte-Carlo simulations.
Results
Clinical characteristics of study subjects
The clinical characteristics of the study population were summarized in Table 1, consisting of 839 AF patients and 1215 controls. In AF patient group, the ratio of males to females was significantly higher than that in control group (P < 0.001). Prevalence of hypertension in AF patients was significantly higher than that in control group (P < 0.001). There is no age difference between the two groups. In the AF patient group, there were 495 paroxysmal AF (69.0%), 298 persistent AF (35.5%), and 46 permanent AF (5.5%). In addition, there were 264 lone AF.
Table 1.
Clinical characteristics of study population
| Characteristic | AF patients (n = 839) | Controls (n = 1215) | P |
|---|---|---|---|
| Agea | 53 ± 15 | 52 ± 15 | 0.479 |
| Male/female | 473/366 | 803/402 | < 0.001 |
| Lone AF (%) | 264 (31.5%) | N/A | * |
| Other AF (%) | 575 (80.5%) | N/A | * |
| paroxysmal AF (%) | 495 (59.0%) | N/A | * |
| persistent AF (%) | 298 (35.5%) | N/A | * |
| permanent AF (%) | 46 (5.5%) | N/A | * |
| coronary artery disease (%) | 309 (36.8%) | N/A | * |
| Hypertension (%) | 265 (31.6%) | 240 (19.8%) | < 0.001 |
| Ischemic stroke (%) | 93 (11.1%) | N/A | * |
Age for the case group refers to as the age at diagnosis of disease; age for the control group refers to as the age at which the subject was enrolled into the study.
untested P values.
For power analysis, we set the population parameters of an OR of 1.3 and rs3807989 allele A frequency of 0.260. At a type I error rate of 0.05, our study with 839 AF cases versus 1215 controls provided statistical power of more than 94.4% respectively. Therefore, the data suggested that our sample size provided sufficient power to identify the association between SNP rs3807989 and AF.
SNP rs3807989 was not associated with AF
The call rate was 100% for rs3807989 in all samples. This quality control of SNP in HRM genotyping was 100% according to direct sequencing verification of 48 random selected DNA samples. No deviation of rs3807989 genotype distribution from Hardy-Weinberg Equilibrium was found among 1,215 controls (P > 0.05, data not shown). As shown in Table 2, the association of the SNP rs3807989 with AF was not significant in our study population with a P value of 0.990. After logistic regression with multiple covariates, the association remained non-significant with adjusted P value 0.828 (Table 2). Furthermore, no significant association of the SNP rs3807989 with lone AF or other AF group was observed (Table 2). In addition, genotypic association of the SNP rs3807989 with AF was also not significant in three inheritance models, dominant, recessive and additive models (P-obs > 0.05 and P-adj > 0.05) (Table 3).
Table 2.
Analysis of allelic association of rs3807989 with AF and CAD
| Phenotype (case/control) | Allele A frequency (case/control) | P-obs | OR (95% CI) | P-adj | P-emp |
|---|---|---|---|---|---|
| Total AF (839/1215) | 0.260/0.260 | 0.990 | 1.02 (0.85-1.22) | 0.828 | 0.905 |
| Lone AF (264/1215) | 0.268/0.260 | 0.738 | 0.99 (0.75-1.23) | 0.929 | 0.995 |
| Other AF (575/1215) | 0.266/0.260 | 0.688 | 1.06 (0.86-1.23) | 0.597 | 0.758 |
| AF with CAD (309/1215) | 0.297/0.260 | 0.306 | 1.30 (0.90-1.89) | 0.162 | 0.463 |
| AF w/o CAD (530/1215) | 0.262/0.260 | 0.970 | 0.97 (0.80-1.17) | 0.720 | 0.842 |
| AF with HT (265/1215) | 0.252/0.260 | 0.744 | 0.83 (0.61-1.11) | 0.206 | 0.627 |
| AF w/o HT (574/1215) | 0.274/0.260 | 0.379 | 1.06 (0.87-1.28) | 0.573 | 0.775 |
AF, atrial fi brillation; CAD, coronary artery disease; HT, hypertension; IS, ischemic stroke. P-obs: unadjusted P values; P-adj: obtained using multivariate logistic regression analysis. P-emp: obtained by performing 100,000 Monte-Carlo simulations.
Table 3.
Analysis of genotypic association of rs3807989 with AF
| Phenotype | Numbers case/control | Model | P-obs | P-adj | P-emp |
|---|---|---|---|---|---|
| AF | 839/1215 | Dominant | 0.788 | 0.815 | 0.789 |
| Recessive | 0.588 | 0.405 | 0.628 | ||
| Additive | 0.787 | 0.760 | 0.786 |
P-obs: unadjusted P values; P-adj: obtained using multivariate logistic regression analysis. P-emp: obtained by performing 100,000 Monte-Carlo simulations.
Discussion
In the present study, we carried out a large-scale case-control association study in a Chinese cohort consisting of 839 AF patients and 1215 controls. The SNP rs3807989 in the intron of CAV1 gene was assessed. Although the SNP rs3807989 has been associated with AF in GWAS studies, this association has not been replicated in Chinese population. The results showed that the SNP rs3807989 in CAV1 gene was not significantly associated with entire AF group, lone AF group or other AF group.
In two independent GWAS with Caucasian people, the SNP rs3807989 has been associated with risk of AF with relative low OR values [12,13]. In a study with Canadian people, significant association of rs3807989 with AF has been reported (P = 0.015 and OR = 1.35). However, the association becomes insignificant when correcting for multiple testing [15]. In this study, we did observe significant association of the SNP rs3807989 with AF or lone AF. Therefore, the SNP rs3807989 in CAV1 gene may be a weak risk factor for AF.
Caveolin-1 (CAV1) is one of the major isoforms of caveolae. Caveolae are 50-100 nm cell surface plasma membrane wevaginations, which is rich in cholesterol [11]. The CAV1 gene is highly expressed in endothelial cells. Most biological functions of these endothelial cells are directly or indirectly mediated by CAV1. CAV1 has also been suggested to play an important role in the regulation of plasma lipoprotein metabolism [18]. In Cav1-deficient mice, higher plasma triglyceride (TG) levels are observed [19]. Recent studies have suggested that CAV1 is involved in endothelial function and ischemia [20]. The association of the SNP rs3807989 in CAV1 gene by GWAS studies has not been validated and reported. Recently, a GWAS study in African-American population, a SNP rs11773845 in CAV1 gene has been associated with PR interval [21]. Therefore, the roles of the CAV1 gene in AF need further investigation.
In summary, we carried out case-control study with large cohorts of AF patients and controls. The SNP rs3807989 in CAV1 gene was not associated with AF or lone AF. Our data suggested that the SNP rs3807989 in CAV1 may not be a risk factor for AF in Chinese Han population.
Acknowledgements
The authors thank Dr Bo Yan for his help. This work was supported by the National Natural Science Foundation of China (NSFC 30800378; NSFC 81270247).
Disclosure of conflict of interest
None.
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