The rs3807989 G/A Polymorphism in CAV1 is Associated with the Risk of Atrial Fibrillation in Chinese Han Populations YAOWU LIU, M.D.,* BIXIAN NI, M.D.,† YUAN LIN, M.D.,† XIN-GUANG CHEN, M.D.,* MINGLONG CHEN, M.D., PH.D.,* ZHIBIN HU, M.D., PH.D.,† and FENGXIANG ZHANG, M.D., PH.D.* From the *Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China; and †Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
Background: A recent meta-analysis of several genome-wide association studies identified six new susceptibility single nucleotide polymorphisms (SNPs) for atrial fibrillation (AF) in individuals of the European ancestry. We aimed to replicate the associations between these SNPs and the risk of AF in a Chinese Han population. Methods: We genotyped six SNPs (rs3903239 in PRRX1, rs3807989 in CAV1, rs10821415 in C9orf3, rs10824026 in SYNPO2L, rs1152591 in SYNE2, and rs7164883 in HCN4) using the middle-throughput iPLEX Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in logistic regression models. Results: We enrolled a total of 1,593 Chinese Han origin individuals in the study, including 597 AF patients and 996 non-AF controls. Among the six SNPs analyzed in the study, the SNP rs3807989 in CAV1 on chromosome 7q31 was found to be significantly associated with a decreased risk of AF (crude OR = 0.76, 95% CI: 0.64–0.89, P = 0.001; adjusted OR = 0.75, 95% CI: 0.63–0.89, P = 0.001). There were no significant associations between the other five loci and AF risk. Conclusion: Our results confirmed that CAV1 rs3807989 may contribute to a decreased AF risk in Chinese Han populations. However, further validation studies with different ethnic backgrounds and biological function analyses are warranted to confirm our finding. (PACE 2015; 38:164–170) atrial fibrillation (AF), single nucleotide polymorphism (SNP), rs3807989, CAV1, 7q31
Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia, accounting for approximately one-third of hospitalizations for cardiac rhythm disturbances.1 With characteristics of uncoordinated atrial activation and consequent deterioration of atrial mechanical function, this
Author Contributions: Yaowu Liu and Bixian Ni are co-first authors, they contributed equally to this work. Financial support: This work was supported by grants from the National Natural Science Foundation of China (Grant 81170160), the Program for Development of Innovative Research Team in the First Affiliated Hospital of Nanjing Medical University (No.IRT-004), the Six Peak Talents Foundation of Jiangsu Province (No. 2011-WS-071), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Address for reprints: Fengxiang Zhang, M.D., Ph.D., Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, China. Fax: 86-25-8371-7168; e-mail: [email protected] Received February 15, 2014; revised May 18, 2014; accepted June 29, 2014. doi: 10.1111/pace.12494
arrhythmia is closely related to an increased risk of stroke and a threefold congestive heart failure, and higher all-cause mortality.2,3 AF is frequently observed as a complication of multiple cardiovascular risk factors, including advancing age, male sex, hypertension, diabetes, ischemia, valvular heart disease, heart failure, and hyperthyroidism.4–6 However, clinicians may notice a phenomenon that some patients maintain sinus rhythm regardless of various risk factors for AF such as valvular disease, hypertension, diabetes, and heart failure, while some other patients develop AF without any clinical or echocardiographic evidence of cardiopulmonary disease.7 Epidemiologic studies have shown that first-degree relatives of AF patients were 1.77fold to 4.67-fold more likely to have AF than the general population, and offspring with one parent suffering from AF had approximately a twofold increase in the risk of developing AF.8,9 These facts indicate predisposing genetic factors may contribute to the development of AF. Scientific studies performed in the last decade have shown that AF is heritable and identified genetic variants associated with AF.10 Since
©2014 Wiley Periodicals, Inc. 164
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CAV1 rs3807989 AND AF RISK
first reported in three families with autosomal dominant model,11 the heritability of AF has been demonstrated continuously on several monogenic studies in familial AF patients by linkage analysis.8,9,12 Although traditional methods such as linkage analysis can be applied to familial AF studies, the pathogenesis of more common sporadic nonfamilial AF remained unknown until the emergence of genome-wide association studies (GWASs). Gudbjartsson et al. performed the first GWAS on AF and identified two loci (rs2200733 and rs10033464 in proximity of the PITX2 gene on chromosome 4q25) which were highly associated with the risk of AF.13 Subsequently, a metaanalysis of several GWASs for AF confirmed a single nucleotide polymorphism (SNP) rs2106261 (in the intron region of the gene ZFHX3 on chromosome 16q22), which had a moderate correlation with AF.14 Also in ZFHX3, a genomewide scan for sequence variants detected another SNP rs7193343 that was significantly related to AF.15 After that, a recent GWAS conducted by Ellinor et al. showed significant correlation between an intronic SNP rs13376333 in the gene KCNN3 on chromosome 1q21 and lone AF.16 Most of the SNPs mentioned above have been validated in European and/or Asian populations.15,17–19 More recently, Ellinor et al. performed a largescale meta-analysis of multiple GWAS samples of the European ancestry and they identified six new AF susceptibility loci for AF, namely, rs3903239 on 1q24, rs3807989 on 7q31, rs10821415 on 9q22, rs10824026 on 10q22, rs1152591 on 14q23, and rs7164883 on 15q24.20 To our knowledge, the six loci have not been validated in the Chinese population. Therefore, we conducted a study to investigate if associations of these six SNPs with the risk of AF could be replicated in Chinese Han populations. Methods Study Population For this study, patients with AF were recruited from the Section of Pacing and Electrophysiology, Division of Cardiology, the First Affiliated Hospital of Nanjing Medical University. Evaluation of AF is based on diagnostic criteria according to 2011 American College of Cardiology Foundation/American Heart Association/Heart Rhythm Society focused updates incorporated into the American College of Cardiology/American Heart Association/European Society of Cardiology 2006 guidelines for the management of patients with AF. Clinical examinations were carried out by using routine 12-lead electrocardiography (ECG), and/or ambulatory ECG recordings. According to clinical characteristics, AF can be classified into paroxysmal AF (episodes
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that generally last 7 days or less), persistent AF (episodes that sustain beyond 7 days), and permanent AF (ongoing long-term episodes, in which cardioversion has failed or has not been attempted). AF patients who are under 60 years of age without clinical or echocardiographic evidence of cardiopulmonary disease, including hypertension, were considered as lone AF. The controls were unrelated inpatient individuals who had been confirmed to be free of AF based on ECG or medical files at the time of enrollment from multiple departments of the First Affiliated Hospital of Nanjing Medical University. We also collected general and clinical information including age, gender, and history of hypertension, diabetes, coronary artery disease (CAD), and hyperthyroidism from medical recording files in the hospital system. We excluded patients with hyperthyroidism, severe cardiac dysfunction (New York Heart Association Class IV), and advanced age (beyond 90 years) in both AF and control groups. All the enrolled individuals are of the ethnic Chinese Han origin by selfreport. Written informed consent was obtained from all participants. The study was approved by the ethical committee review board of Nanjing Medical University, China. SNP Genotyping Blood samples were drawn from study participants and genomic DNA was extracted from ethylene diamine tetraacetic acid-preserved whole blood, using the standard phenol-chloroform method.21 Genotyping analyses were performed by using the middle-throughput iPLEX Sequenom MassARRAY platform (Sequenom, Inc., San Diego, CA, USA). The primers and probes are available upon request for amplification of templates for single base extensions. Multiple methods were used to guarantee the quality of genotyping: (1) samples of case and control groups were mixed on each plate; (2) genotyping was performed without knowing the case or control status; (3) two NTCs (no template controls) were set in each plate; (4) 5% of the samples were randomly selected for genotyping repeatedly. Statistical Analysis The standard two independent samples t-test was used to compare continuous variables. The χ 2 test was used to compare categorical variables. The Hardy-Weinberg equilibrium of the genotype distribution of polymorphisms in the control group was determined by the χ 2 test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression analyses in the additive model to assess the strength of the associations. The heterogeneity of associations between subgroups was assessed
February 2015
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LIU, ET AL.
Table I. Clinical Characteristics in AF Cases and AF-Free Controls
Variables Male gender (%) Age (years) Paroxysmal AF (%) Persistent AF (%) Permanent AF (%) Lone AF (%) Hypertension (%) Diabetes (%) CAD (%)
Cases (N = 597)
Controls (N = 996)
P Value
397 (66.5%)
674 (67.7%)
0.630
58.4 ± 11.5 383 (64.2%)
59.0 ± 10.2 NA
0.278 –
196 (32.8%)
NA
–
18 (3.0%)
NA
–
71 (11.9%) 260 (43.6%)
NA 267 (26.8%)
–
Background: A recent meta-analysis of several genome-wide association studies identified six new susceptibility single nucleotide polymorphisms (SNPs) for atrial fibrillation (AF) in individuals of the European ancestry. We aimed to replicate the associations between these SNPs and the risk of AF in a Chinese Han population. Methods: We genotyped six SNPs (rs3903239 in PRRX1, rs3807989 in CAV1, rs10821415 in C9orf3, rs10824026 in SYNPO2L, rs1152591 in SYNE2, and rs7164883 in HCN4) using the middle-throughput iPLEX Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in logistic regression models. Results: We enrolled a total of 1,593 Chinese Han origin individuals in the study, including 597 AF patients and 996 non-AF controls. Among the six SNPs analyzed in the study, the SNP rs3807989 in CAV1 on chromosome 7q31 was found to be significantly associated with a decreased risk of AF (crude OR = 0.76, 95% CI: 0.64–0.89, P = 0.001; adjusted OR = 0.75, 95% CI: 0.63–0.89, P = 0.001). There were no significant associations between the other five loci and AF risk. Conclusion: Our results confirmed that CAV1 rs3807989 may contribute to a decreased AF risk in Chinese Han populations. However, further validation studies with different ethnic backgrounds and biological function analyses are warranted to confirm our finding. (PACE 2015; 38:164–170) atrial fibrillation (AF), single nucleotide polymorphism (SNP), rs3807989, CAV1, 7q31
Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia, accounting for approximately one-third of hospitalizations for cardiac rhythm disturbances.1 With characteristics of uncoordinated atrial activation and consequent deterioration of atrial mechanical function, this
Author Contributions: Yaowu Liu and Bixian Ni are co-first authors, they contributed equally to this work. Financial support: This work was supported by grants from the National Natural Science Foundation of China (Grant 81170160), the Program for Development of Innovative Research Team in the First Affiliated Hospital of Nanjing Medical University (No.IRT-004), the Six Peak Talents Foundation of Jiangsu Province (No. 2011-WS-071), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Address for reprints: Fengxiang Zhang, M.D., Ph.D., Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, China. Fax: 86-25-8371-7168; e-mail: [email protected] Received February 15, 2014; revised May 18, 2014; accepted June 29, 2014. doi: 10.1111/pace.12494
arrhythmia is closely related to an increased risk of stroke and a threefold congestive heart failure, and higher all-cause mortality.2,3 AF is frequently observed as a complication of multiple cardiovascular risk factors, including advancing age, male sex, hypertension, diabetes, ischemia, valvular heart disease, heart failure, and hyperthyroidism.4–6 However, clinicians may notice a phenomenon that some patients maintain sinus rhythm regardless of various risk factors for AF such as valvular disease, hypertension, diabetes, and heart failure, while some other patients develop AF without any clinical or echocardiographic evidence of cardiopulmonary disease.7 Epidemiologic studies have shown that first-degree relatives of AF patients were 1.77fold to 4.67-fold more likely to have AF than the general population, and offspring with one parent suffering from AF had approximately a twofold increase in the risk of developing AF.8,9 These facts indicate predisposing genetic factors may contribute to the development of AF. Scientific studies performed in the last decade have shown that AF is heritable and identified genetic variants associated with AF.10 Since
©2014 Wiley Periodicals, Inc. 164
February 2015
PACE, Vol. 38
CAV1 rs3807989 AND AF RISK
first reported in three families with autosomal dominant model,11 the heritability of AF has been demonstrated continuously on several monogenic studies in familial AF patients by linkage analysis.8,9,12 Although traditional methods such as linkage analysis can be applied to familial AF studies, the pathogenesis of more common sporadic nonfamilial AF remained unknown until the emergence of genome-wide association studies (GWASs). Gudbjartsson et al. performed the first GWAS on AF and identified two loci (rs2200733 and rs10033464 in proximity of the PITX2 gene on chromosome 4q25) which were highly associated with the risk of AF.13 Subsequently, a metaanalysis of several GWASs for AF confirmed a single nucleotide polymorphism (SNP) rs2106261 (in the intron region of the gene ZFHX3 on chromosome 16q22), which had a moderate correlation with AF.14 Also in ZFHX3, a genomewide scan for sequence variants detected another SNP rs7193343 that was significantly related to AF.15 After that, a recent GWAS conducted by Ellinor et al. showed significant correlation between an intronic SNP rs13376333 in the gene KCNN3 on chromosome 1q21 and lone AF.16 Most of the SNPs mentioned above have been validated in European and/or Asian populations.15,17–19 More recently, Ellinor et al. performed a largescale meta-analysis of multiple GWAS samples of the European ancestry and they identified six new AF susceptibility loci for AF, namely, rs3903239 on 1q24, rs3807989 on 7q31, rs10821415 on 9q22, rs10824026 on 10q22, rs1152591 on 14q23, and rs7164883 on 15q24.20 To our knowledge, the six loci have not been validated in the Chinese population. Therefore, we conducted a study to investigate if associations of these six SNPs with the risk of AF could be replicated in Chinese Han populations. Methods Study Population For this study, patients with AF were recruited from the Section of Pacing and Electrophysiology, Division of Cardiology, the First Affiliated Hospital of Nanjing Medical University. Evaluation of AF is based on diagnostic criteria according to 2011 American College of Cardiology Foundation/American Heart Association/Heart Rhythm Society focused updates incorporated into the American College of Cardiology/American Heart Association/European Society of Cardiology 2006 guidelines for the management of patients with AF. Clinical examinations were carried out by using routine 12-lead electrocardiography (ECG), and/or ambulatory ECG recordings. According to clinical characteristics, AF can be classified into paroxysmal AF (episodes
PACE, Vol. 38
that generally last 7 days or less), persistent AF (episodes that sustain beyond 7 days), and permanent AF (ongoing long-term episodes, in which cardioversion has failed or has not been attempted). AF patients who are under 60 years of age without clinical or echocardiographic evidence of cardiopulmonary disease, including hypertension, were considered as lone AF. The controls were unrelated inpatient individuals who had been confirmed to be free of AF based on ECG or medical files at the time of enrollment from multiple departments of the First Affiliated Hospital of Nanjing Medical University. We also collected general and clinical information including age, gender, and history of hypertension, diabetes, coronary artery disease (CAD), and hyperthyroidism from medical recording files in the hospital system. We excluded patients with hyperthyroidism, severe cardiac dysfunction (New York Heart Association Class IV), and advanced age (beyond 90 years) in both AF and control groups. All the enrolled individuals are of the ethnic Chinese Han origin by selfreport. Written informed consent was obtained from all participants. The study was approved by the ethical committee review board of Nanjing Medical University, China. SNP Genotyping Blood samples were drawn from study participants and genomic DNA was extracted from ethylene diamine tetraacetic acid-preserved whole blood, using the standard phenol-chloroform method.21 Genotyping analyses were performed by using the middle-throughput iPLEX Sequenom MassARRAY platform (Sequenom, Inc., San Diego, CA, USA). The primers and probes are available upon request for amplification of templates for single base extensions. Multiple methods were used to guarantee the quality of genotyping: (1) samples of case and control groups were mixed on each plate; (2) genotyping was performed without knowing the case or control status; (3) two NTCs (no template controls) were set in each plate; (4) 5% of the samples were randomly selected for genotyping repeatedly. Statistical Analysis The standard two independent samples t-test was used to compare continuous variables. The χ 2 test was used to compare categorical variables. The Hardy-Weinberg equilibrium of the genotype distribution of polymorphisms in the control group was determined by the χ 2 test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression analyses in the additive model to assess the strength of the associations. The heterogeneity of associations between subgroups was assessed
February 2015
165
LIU, ET AL.
Table I. Clinical Characteristics in AF Cases and AF-Free Controls
Variables Male gender (%) Age (years) Paroxysmal AF (%) Persistent AF (%) Permanent AF (%) Lone AF (%) Hypertension (%) Diabetes (%) CAD (%)
Cases (N = 597)
Controls (N = 996)
P Value
397 (66.5%)
674 (67.7%)
0.630
58.4 ± 11.5 383 (64.2%)
59.0 ± 10.2 NA
0.278 –
196 (32.8%)
NA
–
18 (3.0%)
NA
–
71 (11.9%) 260 (43.6%)
NA 267 (26.8%)
–
