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Abstract
Objective To determine the extent to which genetic variation in the potassium channel gene KCNQ1 causes atrial fibrillation (AF).
Design Case–control study.
Setting National University Hospital, Singapore.
Patients Han Chinese patients (n=111) with lone AF (onset <60 years and lacking risk factors) and 265 Han Chinese controls.
Interventions Blood draw, 12-lead electrocardiogram and transthoracic echocardiogram were performed on patients with AF at enrolment.
Main outcome measures DNA sequence variants in the coding region and exon–intron boundaries of KCNQ1 as detected by direct sequencing.
Results Four previously reported coding variants were identified: I145I, S546S, P448R and G643S. An additional 19 non-coding variants were identified, nine of which are newly reported. None were predicted to create a cryptic splicing site. The allele frequencies of the two non-synonymous variants did not differ significantly in the AF cases compared with 265 Han Chinese controls (P448R: 10.8% in cases vs 8.6% in controls, p=0.41; G643S: 1.4% in cases vs 0.8% in controls, p=0.43).
Conclusions Comprehensive mutation scanning of KCNQ1 did not identify novel pathogenic mutations or risk-conferring polymorphisms. As in Caucasians, genetic variation in KCNQ1 is not a common cause of AF in Han Chinese. Routine genetic testing of KCNQ1 for AF is, therefore, not warranted.
- Atrial fibrillation
- potassium channel
- KCNQ1
- genetics
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In cardiac myocytes, the KCNQ1 gene encodes the pore-forming α subunit of the channel that conducts the slow component of the delayed rectifier potassium current (IKs). Missense mutations in KCNQ1 have been found to underlie familial forms of atrial fibrillation (AF).1 2 In fact, the first mutation reported for AF was a gain-of-function mutation, S140G-KCNQ1, discovered in a Han Chinese family with autosomal dominant AF.1 Another missense KCNQ1 mutation, R14C, was identified in a family with a high prevalence of hypertension.2 AF was present in individuals who harboured the R14C-KCNQ1 mutation and who also had atrial dilation. In patch-clamp studies, when R14C-KCNQ1 was expressed with KCNE1 in CHO cells that were exposed to hypotonic solution, a marked increase in potassium current was observed.
Both of these gain-of-function KCNQ1 mutations are predicted to shorten the atrial action potential duration and refractory period, thus promoting AF. These mutations, however, are most likely ‘private mutations’ and unlikely to account for the vast majority of AF cases. In an attempt to determine the importance of KCNQ1 mutations in causing AF, Ellinor and colleagues screened the coding region of KCNQ1 in 141 unrelated subjects with lone AF (ie, disease onset at <60 years old in the absence of traditional risk factors).3 They found 15 polymorphisms, but no disease-causing mutations. The screened cohort was largely Caucasian and thus may not be representative of all cohorts with lone AF. To date, KCNQ1 has not been systematically studied as a cause of AF in cohorts of other ethnic origins.
We hypothesised that mutations in KCNQ1 may underlie some cases of lone AF in Han Chinese patients. To test this hypothesis, we used direct sequencing to analyse the coding region and exon–intron boundaries of KCNQ1 in 111 unrelated Han Chinese patients with lone AF.
Materials and methods
Patient evaluation
Consecutive patients with lone AF who were referred to the arrhythmia clinic at the National University Hospital, Singapore between June 2006 and December 2008 were enrolled in our study following informed, written consent. Inclusion criteria were AF onset at <60 years of age and lack of risk factors for AF such as hypertension, diabetes, hyperthyroidism or significant structural or valvular abnormalities that included moderate or severe valvular regurgitation, mitral stenosis of any severity, left ventricular ejection fraction <50% and clinically apparent atrial septal defect. Patients were also excluded if they had a history of coronary artery disease, chronic heart failure, primary cardiomyopathy or myocardial infarction.
AF was defined as replacement of sinus P waves by rapid oscillations or fibrillatory waves that varied in size, shape and timing, and were associated with an irregular ventricular response when atrioventricular conduction was intact. Documentation of AF on an electrocardiogram (ECG), rhythm strip, event monitor or Holter monitor recording was required. ‘Paroxysmal’ AF was defined as AF lasting more than 30 s that terminated spontaneously. AF was classified as ‘persistent’ if it lasted more than 7 days and required either pharmacological therapy or electrical cardioversion for termination. AF that was completely refractory to cardioversion or was allowed to continue was classified as ‘permanent.’
Detailed two-dimensional and Doppler transthoracic echocardiographic studies were performed to measure indexes of left atrial and left ventricular size, to assess left ventricular systolic and diastolic function, and to exclude significant structural heart and valvular abnormalities. Echocardiographic studies were performed during sinus rhythm for patients with paroxysmal or persistent AF. Additionally, each patient filled out a structured questionnaire for collection of data on patient demographics, symptomatology, family history, other comorbid illnesses and treatment (drugs, pacemaker, catheter ablation).
Genetic analysis
Genomic DNA was isolated from peripheral leucocytes of study subjects using a Gentra Puregene Blood Kit (Qiagen, Hilden, Germany). The coding region and exon–intron boundaries of KCNQ1 were screened by direct sequencing. Primer pairs for PCR (PCR)-amplification of the 16 exons and ≥50 base-pairs of the flanking intron sequence of KCNQ1 were designed with the use of Primer3 (http://frodo.wi.mit.edu/) (funded by Howard Hughes Medical Institute, National Institutes of Health, National Human Genome Research Institute). All of the PCR reactions were run on the Biometra T-Gradient Thermal Cycler (LABREPCO, Horsham, Pennsylvania). With the exception of exon 1, the following PCR-amplification conditions were used: following an initial denaturation step at 95°C for 4 min, the reactions were cycled 30 times through denaturation at 94°C for 30 s, variable annealing temperatures for 30 s and extension at 72°C for 40 s. The reactions were terminated by an additional extension step at 72°C for 5 min. A touchdown PCR condition was used for the amplification of exon 1: following an initial denaturation step at 95°C for 4 min, the reactions were cycled 10 times through denaturation at 94°C for 30 s, an annealing temperature at X°C (about 5°C higher than the melting temperature of primer used) for 30 S and extension at 72°C for 40 S with an annealing temperature decrement of 1°C/cycle until the final annealing temperature reached (X–9)°C. Next, the reactions were cycled 25 times through denaturation at 94°C for 30 s, an annealing temperature (X–9)°C for 30 S and extension at 72°C for 40 s. The reactions were terminated by an additional extension step at 72°C for 5 min. Amplified PCR products were analysed on a 2% agarose gel, purified using the QIAquick PCR purification kit (Qiagen) and then sequenced by the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California) with use of an ABI GeneAmp PCR system 9700 (Applied Biosystems). The sequences of genomic fragments were analysed on the automated 16-capillary DNA Sequencer, ABI PRISM 3100 Genetic Analyser (Applied Biosystems).
The Ensembl (http://www.ensembl.org/index.html) and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) online databases were queried for identified sequence variants. Potential effects on mRNA splicing were analysed using NetGene2 (http://www.cbs.dtu.dk/services/NetGene2). Conservation of amino acids altered by missense variants was investigated by aligning human KCNQ1 (NP_000209.2) to dog (XP_540790.2), cattle (XP_001252338.1), mouse (NP_032460.2) and rat (NP_114462.1) KCNQ1, using the HomoloGene link on the NCBI website.
Statistical analysis
For continuous variables, summary data are presented as mean±SD. Categorical variables are presented as number (%). Allele frequencies of identified variants in AF cases were compared with allele frequencies in a previously analysed cohort of 265 normal Han Chinese controls4 using the χ2 test. The control subjects were 18–35 years old, free of cardiac conditions and of Han Chinese descent for at least two previous generations by self-report. Deviation of genotype proportions from Hardy–Weinberg equilibrium expectations was tested using the χ2 test. Stata version SE 9.2 (StataCorp) was used for all statistical analyses. Two-sided p values were reported. Statistical significance was evaluated at the 5% level.
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the National University of Singapore's human research committee. The authors have full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Results
During the study period, 111 unrelated probands with lone AF were enrolled. Table 1 summarises the baseline characteristics of the study cohort. The mean age at diagnosis was 48.7±10.8 years, and the mean age at enrolment was 54.0±10.1 years. The cohort was predominantly male (81.1%) and was entirely Han Chinese. Ten patients (9.0%) reported a family history of AF in at least one first-degree relative. With the exception of enlarged left atria (mean left atrial volume index: 34.3 ml/m2), all echocardiographic parameters were normal. ECGs showed normal durations and intervals in sinus rhythm with no evidence of pre-excitation.
Baseline characteristics of the study cohort (n=111)
The coding region and exon–intron boundaries of KCNQ1 were screened by direct sequencing. Four previously reported coding single nucleotide polymorphisms (SNPs) were identified; two were synonymous (I145I and S546S), and two were non-synonymous (P448R and G643S) (table 2). In addition, 19 non-coding SNPs were identified, nine of which had not been previously reported. None of these SNPs was predicted to create a cryptic splicing site. All identified variants did not deviate significantly from Hardy–Weinberg equilibrium expectations.
KCNQ1 sequence variants identified in 111 Chinese patients with lone atrial fibrillation
The allele frequencies of P448R and G643S variants in the AF cohort were not significantly different from the allele frequencies in a previously analysed cohort of 265 Han Chinese controls4 (P448R: 10.8% in AF cases vs 8.6% in controls, p=0.41; G643S: 1.4% in AF cases vs 0.8% in controls, p=0.43).
Discussion
This study systematically screened KCNQ1 as a candidate gene for AF in Han Chinese. We did not find any novel pathogenic mutations or risk-conferring polymorphisms from comprehensive mutation scanning of KCNQ1.
The first mutation reported for monogenic AF was in KCNQ1 in a Han Chinese family.1 Since the initial report, to our knowledge, there have been no attempts to comprehensively screen the coding region of KCNQ1 in a Han Chinese AF cohort to identify novel disease-causing KCNQ1 variants. To increase the likelihood of identifying disease-causing variants, we restricted our analysis to patients with lone AF, as opposed to acquired AF. In this study, we used direct sequencing to analyse the coding region and exon–intron boundaries of KCNQ1 in 111 unrelated Han Chinese patients with lone AF. We analysed the exon–intron boundaries to exclude variants that might have created cryptic splicing sites. We did not find any disease-causing or risk-conferring variants for AF in KCNQ1 in this comprehensive mutation scanning.
The main limitation of this study was the relatively small sample. It is possible that by screening a larger sample, novel pathogenic mutations may be found. Nonetheless, our interpretation remains the same: genetic variation in KCNQ1 is not a common cause of AF.
Conclusion
Our findings suggest that consistent with prior observations in Caucasians, genetic variation in KCNQ1 is not a common cause of AF in Han Chinese. Routine genetic testing of KCNQ1 for AF is, therefore, not warranted.
Footnotes
Funding National Medical Research Council of Singapore (NMRC/1141/2007).
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the National University of Singapore Institutional Review Board.
Provenance and peer review Not commissioned; not externally peer reviewed.