Genomewide association studies have reproducibly identified rs2200733T on chromosome 4q25 as a risk factor for the most common sustained arrhythmia, atrial fibrillation (AF). The biologic basis for cardiac electrical instability conferred by this noncoding variant is unknown. The aim of this study was to investigate potential relations between rs2200733 versus clinical and electrocardiographic traits in a cohort of patients with early-onset AF who lack traditional risk factors. Congruent with previous reports, the minor T allele was overrepresented in subjects with lone AF. All genotype groups were statistically similar for age at diagnosis, gender distribution, family history, body mass index, AF type, ventricular rate, QRS duration, corrected QT interval, and use of PR interval–prolonging medications. However, a novel association was identified between the TT genotype and duration of the PR interval. The TT group had a mean PR interval of 189.5 ± 35.8 ms in comparison to mean PR intervals of 172.0 ± 29.0 and 171.0 ± 27.1 ms for the CT and CC groups, respectively (p = 0.013 and p = 0.0056). In conclusion, PR interval length, an established risk factor for AF, represents an rs2200733-associated intermediate phenotype.
Atrial fibrillation (AF) is the most common sustained arrhythmia, with a lifetime risk of 25%. Risk factors include advancing age, hypertension, structural heart disease, and congestive heart failure, yet a subset of younger subjects develop AF in the absence of established risk factors. Familial cases among these patients with lone AF underscore a genetic basis for disease, and research implicates pathogenic mutations and risk-conferring functional polymorphisms in AF development. In particular, hypothesis-free genomewide linkage analysis has revealed unanticipated genes for familial AF. Additionally, genomewide association studies have identified a single-nucleotide polymorphism, rs2200733T, located on chromosome 4q25 that predisposes to AF, a discovery replicated in numerous distinct populations. The implicated 4q25 locus exists in an expanse of approximately 1.5 million base pairs devoid of known genes, providing little insight to the fundamental mechanism driving the association of rs2200733T with AF. It is well known that identifying associations between common sequence variants and intermediate phenotypes facilitates discovery of biologic disease mechanisms and can have clinical significance. Because electrocardiographic (ECG) measurements are profoundly influenced by genetic contribution and represent intermediate traits of disease states, we sought to identify whether or not patients with AF possessing 1 or 2 rs2200733T alleles exhibit an intermediate phenotype distinct from patients with AF with the wild-type allele. We studied a large cohort of patients with lone AF, thus minimizing potentially confounding variables inherent in acquired AF study populations.
Methods
The study protocol was approved by the institutional review board of the Mayo Clinic, and participants were enrolled after providing informed written consent. From November 2000 to June 2009, patients referred to the Heart Rhythm Center and diagnosed with lone AF were identified. 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 electrocardiography, rhythm strip, event monitor, or Holter monitor recording was required. Lone AF was defined as AF in subjects aged <60 years without hypertension or overt structural heart disease by clinical examination, electrocardiography, and echocardiography.
Paroxysmal AF was defined as AF lasting >30 seconds that terminated spontaneously. AF was classified as persistent when it lasted >7 days and required either pharmacologic therapy or electrical cardioversion for termination. AF that was completely refractory to cardioversion or was allowed to continue was classified as permanent. Questionnaires and medical records were reviewed to identify potentially PR interval–prolonging drugs, including antiarrhythmic agents in Vaughan Williams classes IA (disopyramide, quinidine), IB (flecainide, propafenone), II (acebutolol, atenolol carvedilol, metoprolol, pindolol), III (amiodarone, dofetilide, sotalol), IV (diltiazem, verapamil), and V (digoxin).
We recruited 219 unrelated subjects with lone AF and procured whole blood for genomic deoxyribonucleic acid extraction and analysis. Primer pairs for the intronic region surrounding rs2200733 were designed using OLIGO version 6.71 Primer Analysis Software (National Biosciences, Plymouth, Minnesota). Genotyping of the rs2200733 polymorphism was performed by restriction fragment length polymorphism analysis, as previously described by Chen et al. A 702 base pair fragment harboring the variant was amplified by polymerase chain reaction. Genotypes were distinguished by restriction digestion of the polymerase chain reaction product with BclI and resolution of fragments by agarose gel electrophoresis.
ECG data were obtained for the 219 patients in our lone AF cohort; 171 had paroxysmal AF with periods of sinus rhythm allowing measurement of the PR interval. We genotyped all 219 patients and performed statistical analyses on each clinical characteristic for which data were available. Associations between the 3 genotypes and continuous variables were tested using 1-way analysis of variance; chi-square or Fisher’s exact tests were used to compare associations between genotypes and categorical variables. Statistical significance was accepted for p values <0.05.
Results
Baseline characteristics of the 219 unrelated patients with lone AF are listed in Table 1 . The genotype frequencies for rs2200733 were 51% CC, 36% CT, and 14% TT. Ninety-nine percent were white; 1 subject was Asian, and 1 subject was American Indian. These patients did not have hyperthyroidism, hypertension, diabetes, histories of coronary artery disease, or structural heart disease.
| Characteristic | CC | CT | TT | p Value ⁎ |
|---|---|---|---|---|
| (n = 111 [51%]) | (n = 78 [36%]) | (n = 30 [14%]) | ||
| Age at AF diagnosis (years) | 44.1 ± 11.9 | 44.3 ± 11.9 | 44.4 ± 11.1 | 0.99 |
| Men | 79 (72%) | 57 (73%) | 25 (83%) | 0.41 |
| Positive family history | 57 (51%) | 46 (59%) | 22 (73%) | 0.18 |
| Body mass index (kg/m 2 ) | 29.0 ± 4.5 | 28.2 ± 3.9 | 29.1 ± 4.6 | 0.41 |
| AF type | 0.71 | |||
| Paroxysmal | 77 (71%) | 49 (68%) | 19 (63%) | |
| Persistent | 20 (19%) | 18 (25%) | 7 (23%) | |
| Chronic | 11 (10%) | 5 (7%) | 4 (13%) | |
| ECG characteristics | ||||
| Ventricular rate (min −1 ) | 74.7 ± 23.0 | 74.9 ± 28.3 | 66.5 ± 12.3 | 0.22 |
| QRS duration (ms) | 99.1 ± 19.2 | 99.8 ± 21.9 | 100.8 ± 15.9 | 0.89 |
| Corrected QT interval (ms) | 426.2 ± 31.4 | 427.2 ± 30.3 | 433.6 ± 33.3 | 0.51 |
| PR interval–prolonging drug use | 90 (81%) | 64 (82%) | 24 (80%) | 0.97 |
| Antiarrhythmic use by Vaughan Williams class | ||||
| Class IA | 0 (0.0%) | 3 (4%) | 2 (7%) | † |
| Class IC | 21 (19%) | 21 (27%) | 11 (37%) | 0.10 |
| Class II | 49 (44%) | 30 (39%) | 11 (37%) | 0.64 |
| Class III | 22 (20%) | 14 (18%) | 4 (13%) | 0.71 |
| Class IV | 28 (25%) | 17 (22%) | 8 (27%) | 0.82 |
| Digoxin | 16 (14%) | 11 (14%) | 4 (13%) | 0.99 |
| Number of antiarrhythmic agents used | 0.89 | |||
| 0 | 22 (20%) | 13 (17%) | 6 (20%) | |
| 1 | 48 (43%) | 37 (47%) | 11 (37%) | |
| 2 | 36 (32%) | 25 (32%) | 11 (37%) | |
| 3 | 4 (4%) | 3 (4%) | 1 (3%) | |
| 4 | 1 (1%) | 0 (0%) | 1 (3%) |
⁎ Comparing CC, CT, and TT groups.
The major finding of this study was a significant difference in PR interval duration with respect to genotype in a lone AF cohort ( Figure 1 ). The TT group had a longer mean PR interval of 189.5 ± 35.8 ms in comparison to the mean PR intervals of 171.0 ± 27.1 and 172.0 ± 29.0 ms in the CC and CT groups, respectively. Of note, there was no statistically significant difference in use or type of PR interval–prolonging medications among genotype groups.
