Author + information
- Received August 25, 2009
- Revision received October 20, 2009
- Accepted November 10, 2009
- Published online February 23, 2010.
- Daniela Husser, MD*,* (, )
- Volker Adams, PhD†,
- Christopher Piorkowski, MD*,
- Gerhard Hindricks, MD* and
- Andreas Bollmann, MD, PhD*
- ↵*Reprint requests and correspondence:
Dr. Daniela Husser, Department of Electrophysiology, Heart Center, Leipzig University, Strümpellstrasse 39, 04289 Leipzig, Germany
Objectives This study tested the hypothesis that chromosome 4q25 single-nucleotide polymorphisms (SNPs) associate with atrial fibrillation (AF) recurrence after catheter ablation.
Background Recent genome-wide association studies identified 2 SNPs on chromosome 4q25 associated with AF. Although the mechanisms underlying this increased risk are unknown, the closest gene, PITX2, is critical for myocardium development in the pulmonary veins.
Methods A total of 195 consecutive patients (mean age 56 ± 12 years, 73% male) with drug-refractory paroxysmal (78%) or persistent (22%) AF who underwent AF catheter ablation were included. Two SNPs, rs2200733 and rs10033464, were genotyped using real-time polymerase chain reaction and fluorescence resonance energy transfer. Serial 7-day Holter electrocardiographic recordings were acquired to detect AF recurrences.
Results Early recurrence of atrial fibrillation (ERAF) (within the first 7 days) was observed in 37%, whereas late recurrence of atrial fibrillation (LRAF) (between 3 and 6 months) occurred in 21% of the patients. None of the clinical or echocardiographic baseline characteristics were associated with ERAF or LRAF. In contrast, the presence of any variant allele increased the risk for both ERAF (odds ratio [OR]: 1.994, 95% confidence interval [CI]: 1.036 to 3.837, p = 0.039) and LRAF (OR: 4.182, 95% CI: 1.318 to 12.664, p = 0.011). In patients with ERAF, 45% had LRAF, as opposed to 8% in patients without ERAF (OR: 9.274, 95% CI: 3.793 to 22.678, p < 0.001).
Conclusions Polymorphisms on chromosome 4q25 modulate the risk for AF recurrence after catheter ablation. This finding points to a potential role for stratification of AF ablation therapy or peri-interventional management by genotype.
Atrial fibrillation (AF) is a heterogeneous arrhythmia at both the clinical and the molecular levels. Association studies have reported that common single-nucleotide polymorphisms (SNPs) in genes encoding cardiac ion channels (1–3), the renin–angiotensin system (4), or connexin 40 (5) may predispose to AF development. Recently, a genome-wide association study identified a haplotype block on chromosome 4q25 containing 2 SNPs, rs2200733 and rs10033464, that predisposes to AF (6). This association was replicated in several independent cohorts (7,8).
Although the molecular mechanisms underlying this increased risk are unknown, future studies to determine whether this SNP is associated with certain phenotypes or outcomes have been suggested (7). In that respect it is interesting to note that the closest gene, PITX2, is a transcription factor critical for determining left–right asymmetry (9) and the differentiation of the left atrium, and for the development of the pulmonary myocardium (10). Pulmonary myocardial sleeves are the source of ectopy associated with AF initiation and maintenance in many cases, and their electrical disconnection from the left atrium is the therapeutic target of pulmonary vein ablation procedures. Accordingly, this study examined the relationship between the 2 AF risk alleles on chromosome 4q25 (rs2200733, rs10033464) and AF recurrence after catheter ablation of AF.
In this study, 195 consecutive Caucasian patients of German descent who underwent left atrial catheter ablation for drug-refractory paroxysmal or persistent AF were included. In all patients, transthoracic and transesophageal echocardiography was performed prior to catheter ablation. Left atrial diameter and left ventricular ejection fraction were determined using standard measurements, and a left atrial thrombus was excluded. All class I or III antiarrhythmic medications with the exception of amiodarone were discontinued at least 5 half-lives before the procedure. The study was approved by the local ethics committee, and patients provided written informed consent for participation.
Left atrial catheter ablation was performed using a previously described approach (11). In brief, patients were studied under deep propofol sedation with continuous invasive monitoring of arterial blood pressure and oxygen saturation. The NavX-Ensite system (version 7.0, Endocardial Solutions, Inc., St. Paul, Minnesota) was used for nonfluoroscopic 3-dimensional catheter orientation, computed tomographic image integration, and tagging of the ablation sites with the coronary sinus lead 5/6 serving as system reference. Transseptal access and catheter navigation were performed with a steerable sheath (Agilis, St. Jude Medical, Inc., St. Paul, Minnesota). Patients presenting with AF at the beginning of the procedure were electrically cardioverted, and ablation was performed during sinus rhythm (i.e., AF termination with ablation was not attempted). In all patients, circumferential left atrial ablation lines were placed around the antrum of the ipsilateral pulmonary veins (irrigated-tip catheter, pre-selected tip temperature of 48°C, and maximum power of 30 to 50 W). In patients with persistent AF, additional linear lesions were added at the left atrial roof, the basal posterior wall, and the left atrial isthmus. Ablation of complex fractionated electrograms was not performed.
After circumferential line placement, voltage and pace mapping along the ablation line were used to identify and close gaps. The isolation of all pulmonary veins with bidirectional block was verified with a multipolar circular mapping catheter and was defined as the procedural end point.
Class I and III antiarrhythmic drugs were not reinitiated after ablation. Oral anticoagulation was prescribed for 6 months, and proton pump inhibitors were added for 4 weeks. All patients were followed up in the outpatient clinic for 6 months after the ablation. During this follow-up period, 7-day Holter recordings were performed immediately after the ablation and at 3 and 6 months after the ablation. Additional electrocardiograms and Holter recordings were obtained when patients' symptoms were suggestive of AF. An AF recurrence was defined as a documented AF episode lasting longer than 30 s. Early recurrence of atrial fibrillation (ERAF) was defined as an AF episode during the first week after the ablation, which is in alignment with previous definitions (12,13). This definition was also chosen because continuous Holter monitoring was available for all patients for this time period. Late recurrence of atrial fibrillation (LRAF) was defined as any AF episode between 3 and 6 months after the ablation (thus including a 3-month blanking period). All patients with sustained early recurring AF underwent direct-current cardioversion. Additional drug administration was left to the discretion of the treating physician.
The deoxyribonucleic acid was extracted from blood using a commercially available isolation kit (peqGOLD Blood DNA Mini Kit, PecLab, Erlangen, Germany). For rapid genotyping of the 2 SNPs, real-time polymerase chain reaction using fluorescence resonance energy transfer followed by the analysis of the melting curve was applied as previously described (14). In brief, allele-specific, commercially synthesized primers and fluorescent probes were used (TibMolBiol, Berlin, Germany) (Table 1).All samples were run in duplicates. A melting curve was detected for each sample with distinctive peaks at different temperatures. Based on these differences, a classification into wild-type, heterozygous, or homozygous variant was performed (Fig. 1).A call rate of ≥98% was achieved with this method for both variants. Genotypes were confirmed with direct sequencing.
Continuous variables are reported as mean ± 1 SD, and categorical variables are reported as frequencies. Continuous variables were compared using the unpaired Student ttest, and categorical variables were compared using the chi-square test.
For genotype–rhythm outcome correlations, 3 different models were applied. The variant alleles were assumed to have dominant, recessive, or additive effects in these models. In the dominant model, an identical effect is expected in heterozygous and homozygous variant carriers. In the recessive model, an effect is only seen in homozygous variant carriers, whereas in the additive model, heterozygous variant carriers have an intermediate effect in relation to the homozygotes.
Logistic regression analysis was used, and odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated. Multivariable analysis that included pre-procedural variables (i.e., age, sex, AF duration, AF type, left atrial diameter, left ventricular ejection fraction, antiarrhythmic drug use, and genotypes) with a p value <0.15 found in univariate analysis was performed to identify independent predictors of ERAF and LRAF.
A sample size calculation was performed assuming an AF recurrence rate of 20% and a minor allele frequency of 0.3. In a dominant model, a sample size of 180 patients is needed to detect an OR of ≥2 for AF recurrence in variant carriers with a power of 80%. A p value of <0.05 was considered statistically significant without adjustment for multiple comparisons.
Patient characteristics and AF recurrence
Patient characteristics are summarized in Table 2.The frequencies of the rs10033464 and rs2200733 genotypes were GG in 68%, GT in 31%, and TT in 1%, and CC in 55%, CT in 35%, and TT in 10%, respectively. At least 1 variant allele was present in 68%, whereas no variant allele was found in 32% of the study population (Table 3).There were no significant differences in clinical and echocardiographic variables among different genotypes (Table 2). Complete pulmonary vein isolation as a procedural end point was achieved in 191 patients (98%). All patients completed the 6-month follow-up, including the 7-day Holter study. ERAF was observed in 37%, whereas LRAF occurred in 21% of the patients. Symptomatic AF requiring cardioversion between 3 and 6 months was observed in 4%, whereas the remaining symptomatic and asymptomatic AF recurrences were detected with sequential 7-day Holter recordings.
None of the clinical or echocardiographic baseline characteristics were significantly associated with ERAF or LRAF (Table 4).In particular, ERAF and LRAF rates were 37% and 22% for paroxysmal AF and 41% and 17% for persistent AF, respectively (p = NS for paroxysmal vs. persistent AF). Pre-procedural amiodarone was used in 17% of the patients and was not associated with ERAF (42% vs. 38%, p = NS).
Chromosome 4q25 variants and AF recurrence
In univariate analysis, the dominant and additive models for both variants showed a trend to be associated with ERAF, whereas the dominant and additive model for the rs10033464 variant was significantly associated with LRAF (Table 5).
Multivariable analysis revealed that both variants were independently predictive of ERAF and LRAF (Table 6).The presence of any variant allele increased the risk for both ERAF (OR: 1.994, 95% CI: 1.036 to 3.837, p = 0.039) and LRAF (OR: 4.182, 95% CI: 1.318 to 12.664, p = 0.011) (Fig. 2)compared with the wild type. In patients with ERAF 45% had LRAF, as opposed to 8% in patients without ERAF (OR: 9.274, 95% CI: 3.793 to 22.678, p < 0.001).
To the best of our knowledge, this study is the first to explore the association between AF risk alleles and AF recidivism after catheter ablation. Based on the analysis of 195 patients, it was clearly shown that 2 variants on chromosome 4q25 were independently associated with an increased risk for ERAF and LRAF. The presence of ERAF was associated with LRAF.
ERAF after catheter ablation
An AF recurrence within 48 h (12) to 1 month (13) after catheter ablation is a common observation, with a reported prevalence of 35% to 52% (12,13,15,16), which is in alignment with our observed event rate. There is general agreement that ERAF is associated with worse rhythm outcome during the long-term follow-up, although the proportion of delayed cure varies substantially (12,13,15,16). A recent study reported freedom from AF in as low as 9% of patients with early recurrence (13), whereas previous investigators found freedom from AF in 31% to 46% of cases despite early recurrence (12,15), which is in close agreement to what we have observed in this study.
The pathophysiology of ERAF is complex and poorly understood. Left atrial radiofrequency ablation has a proinflammatory effect (17) and modifies the autonomic nervous system by reducing vagal activity and increasing sympathetic activity (18), thereby being a stimulus for AF initiation early after ablation. However, a delayed beneficial effect secondary to scar consolidation may explain later freedom from AF despite early AF recidivism. Although 1 study identified older patients' age, the presence of cardiovascular disease, the presence of AF foci (especially from the left atrial free wall), and left atrial enlargement as contributing factors for ERAF (16), more research on its potential predictors and prognostic implications clearly is warranted (13).
Genetic variants, AF phenotypes, and response to therapy
Over the last several years, data have emerged to support a genetic contribution to AF. Although several genetic loci for familial forms of AF have been identified, the genes responsible for AF at these loci remain unknown (19,20). Mutations in the cardiac sodium (21) and potassium (22) channel complexes and gap junction proteins (23) have been identified to cause AF, although this accounts for only a small fraction of AF cases. In addition to several candidate-gene association studies (1–4), recent genome-wide association studies identified 2 common variants on chromosome 4q25 that increase the risk for AF development (6–8).
The prevalence of 4q25 variants has been analyzed in different AF populations, and ranges between 26% and 41% for the rs2200733 SNP and between 19% and 22% for the rs10033464 SNP (7). Interestingly, the prevalence was somewhat higher for both variants (45% and 32%, respectively) in our cohort. This may be because we included a highly selected AF population, with most patients suffering from drug-refractory, paroxysmal lone AF. However, most patients were referred from a localized geographic area, and the prevalence of the 4q25 variants in the general population in this area may also be higher but is currently not known.
In contrast to the aforementioned research avenue, there have been only very few studies that have evaluated the correlation of genetic variants, AF phenotypes, and response to AF therapies. For instance, Firouzi et al. (5) demonstrated increased dispersion of atrial refractoriness in patients with structurally normal hearts who carried the AA genotype of the connexin 40-promoter polymorphism. Ehrlich et al. (24) found that the KCNE138G isoform was associated with reduced IKs, which in turn leads to longer atrial action potential duration and refractoriness. Data from our group are in keeping with these findings. We found lower AF rates on the surface electrocardiogram in individuals carrying the KCNE1GG genotype (25). Finally, 1 single study has assessed the relationship between an AF-associated variant and the response to antiarrhythmic medication (26). In that study of 213 AF patients, it was shown that the ACE I/D polymorphism modulates the response to antiarrhythmic drugs, with the ID/DD genotype being a strong predictor for drug failure.
In the present study, we analyzed the effects of 2 common AF-associated SNPs (rs2200733, rs10033464) and found that they modulate the response to catheter ablation. Both variants were independently predictive of ERAF and LRAF using dominant genetic models. Carriers of at least 1 variant allele had a 4-fold increased risk for LRAF. However, mechanisms by which these variants exert functional effects and consequently why they predict rhythm outcome after catheter ablation remain elusive. Initially, it has been speculated that the close proximity of these variants to the PITX2gene, which has a critical function in cardiac development, may be one underlying mechanism for AF development (6). The PITX2expression identifies the left atrium during embryonic development, whereas other transcription factors (e.g., Nkx2-5) determine its electrophysiological properties, including the capability of automatic impulse formation (10).
In a recent series of animal experiments, it has been shown that PITX2also plays a key role in the differentiation, proliferation, and expansion of pulmonary myocardial cells (10). Of particular interest is the finding that PITX2-deficient mice did not develop pulmonary myocardial sleeves as one distinctive pulmonary vein phenotype. Considering this finding together with the data of the current study, it is now tempting to speculate that chromosome 4q25 variant carriers may differ in their pulmonary vein phenotype (i.e., amount, extension, and arrhythmogenicity of pulmonary vein myocardium) and consequently on AF mechanisms, which will impact on catheter ablation of the pulmonary veins. In other words, although catheter ablation restricted to the pulmonary veins (with or without limited linear lesions) may be sufficient in patients with the wild type, other ablation strategies or more aggressive post-ablation management may be warranted in variant carriers in whom other atrial areas may be more important for AF induction and sustenance. This hypothesis is even further substantiated by the fact that 4q25 variants also increase the risk for atrial flutter, which is confined to the right atrium (6,8). Nevertheless, it must be pointed out that on the one hand, no direct mechanistic relationship between the chromosome 4q25 variants and PITX2has been established; on the other hand, the variants may also simply be a marker for other currently unidentified mechanisms independent of PITX2and consequently of the pulmonary veins.
ERAF also was associated with late recurrence. However, this outcome parameter can only be acquired after ablation during early follow-up. Although adjustment of antiarrhythmic therapy or early reablation (13) is possible when this event occurs, one would like to predict the individual risk or anticipated success rate before the procedure and to plan the ablation strategy and post-ablation management accordingly.
This study included a rather homogeneous patient population with a high proportion of lone paroxysmal AF, and a standardized ablation approach was applied. Consequently, the generalizability of our findings to other populations and the comparison with different ablation approaches, such as ablation of complex fractionated electrograms (that we did not attempt), is uncertain. Only 17% of our patients were on amiodarone before the ablation (which was not reinitiated after ablation). Although amiodarone treatment did not affect ERAF rates, our sample size is too small to draw conclusions regarding its effects on AF recidivism after ablation and consequently regarding the predictive value of the 4q25 variants in the presence of antiarrhythmic drugs.
The follow-up period of our study was short, but later AF recurrences may be due to different mechanisms that are not captured with the analyzed variants. The presence of 1 variant clearly increased the risk for AF recurrence, but homozygous variant carriers represent only a minority of the AF population. To fully appreciate their risk, especially with the rs10033464 variant, a larger sample is needed.
Although we have speculated that the variants point to different pulmonary vein phenotypes and consequently distinct AF mechanisms, they also may be associated with pulmonary vein reconduction, which is known to be the prevailing mechanism for AF recurrence (27). However, because we did not perform re-ablations within the 6-month follow-up interval, reconduction could not be assessed.
Using the candidate gene approach, this study was limited to the investigation of 2 AF-associated SNPs. On the one hand, initial testing was performed with different genetic models with all inherent statistical limitations due to the lack of previous data. On the other hand, other genes may also modulate atrial development, electrophysiology, and structure and consequently the response to AF therapies. Most importantly, our findings need to be validated in different AF populations undergoing catheter ablation.
The noncoding variants on chromosome 4q25, rs2200733 and rs10033464, modulate the risk for AF recurrence after catheter ablation. This finding points to a potential role for stratification of ablation therapy or post-ablation management by genotype.
The authors thank Mrs. Angela Kricke and Mr. Markus Lehmann for their help with laboratory work.
Dr. Husser was supported by the Volkswagen Foundation, Germany, and research grants HU 1679/1-1, Deutsche Forschungsgemeinschaft, and NBL Formel.1-109, University of Leipzig, Germany.
- Abbreviations and Acronyms
- atrial fibrillation
- confidence interval
- early recurrence of atrial fibrillation
- late recurrence of atrial fibrillation
- odds ratio
- single-nucleotide polymorphism
- Received August 25, 2009.
- Revision received October 20, 2009.
- Accepted November 10, 2009.
- American College of Cardiology Foundation
- Fatini C.,
- Sticchi E.,
- Genuardi M.,
- et al.
- Tsai C.T.,
- Lai L.P.,
- Lin J.L.,
- et al.
- Firouzi M.,
- Ramanna H.,
- Kok B.,
- et al.
- Kaab S.,
- Darbar D.,
- van Noord C.,
- et al.
- Viviani Anselmi C.,
- Novelli V.,
- Roncarati R.,
- et al.
- Mommersteeg M.T.,
- Brown N.A.,
- Prall O.W.,
- et al.
- Piorkowski C.,
- Kircher S.,
- Arya A.,
- et al.
- Oral H.,
- Knight B.P.,
- Ozaydin M.,
- et al.
- Pappone C.,
- Santinelli V.,
- Manguso F.,
- et al.
- Darbar D.,
- Herron K.J.,
- Ballew J.D.,
- et al.
- Darbar D.,
- Kannankeril P.J.,
- Donahue B.S.,
- et al.
- Chen Y.H.,
- Xu S.J.,
- Bendahhou S.,
- et al.
- Ehrlich J.R.,
- Zicha S.,
- Coutu P.,
- Hebert T.E.,
- Nattel S.
- Husser D.,
- Stridh M.,
- Sörnmo L.,
- Roden D.M.,
- Darbar D.,
- Bollmann A.
- Ouyang F.,
- Ernst S.,
- Chun J.,
- et al.