Subjects
From September 2012 to June 2015, 97 consecutive patients underwent implantation of ICD for secondary prevention in Zhengzhou University People’s Hospital. 348 healthy people were recruited from physical examination center of our hospital as control group. The treatment of enrolled patients conformed to expert consensus on secondary prevention indications [5]. Secondary prevention was defined if the patient has survived a cardiac arrest or experienced rapid ventricular arrhythmia (RVA) including sustained ventricular tachycardia (VT) and ventricular fibrillation (VF). Exclusion criteria were as follows: 1) expectation of life was less than 1 year; 2) patients suffered pacemaker infections that need to be removed; 3) pacemaker malfunction or lead dislodgement. 9 patients were excluded after evaluated by clinicians (5 patients with a life expectancy less than 1 year, 2 patients suffered from pacemaker infections, and 2 patients had electrode displacement). 86 patients were enrolled in the present study finally. The research protocol was approved by the institutional review board of the Zhengzhou University People’s Hospital. All participants provided informed written consent in accordance with the Declaration of Helsinki prior to enrollment.
A detailed clinical characteristic, including body mass index, drug therapy, electrocardiology and echocardiography parameters were collected at baseline. Coronary heart disease (CHD) was defined by coronary angiograph showing more than 75% stenosis in at least one main vessel of the coronary artery. Ischemic cardiomyopathy was defined as left ventricular dysfunction with Left ventricular enlargement caused by previous myocardial infarction or significant CHD [6]. The diagnosis of Dilated cardiomyopathy (DCM), Hypertrophic cardiomyopathy (HCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) were based on the classification scheme presented by the European Society of Cardiology [7]. SCD was defined as a witnessed natural death attributable to cardiac causes within 1 hour of onset of acute symptoms or as an unwitnessed unexpected death of a person seen in a stable medical condition within 24 hours before death without evidence of a noncardiac cause [8]. SCD event was adjudicated independently by two investigators. A third investigator independently reviewed the event to provide final classification if disagreement existed. All healthy people were ruled out the cardiovascular disease mentioned above.
Genotyping
Blood samples were collected from participants using tubes containing EDTA and genomic DNA was isolated from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Shanghai, China). Genotyping of four common polymorphisms (rs12143842, rs10494366, rs12567209 and rs16847548) was performed on all cases and controls using polymerase chain reaction (PCR) and direct DNA sequencing. The primer sequences for PCR were designed by Primer3web version 4.0.0 (http://bioinfo.ut.ee/primer3/) and listed in Additional file 1. PCR was performed in a total reaction volume of 25 μL containing 12.5 μL of 2×Es Taq MasterMix (cwbio, Shanghai, CHINA), 0.5 μL of each primer, 1.5 μL of DNA template, and 10 μL of ddH20 Water. The multiplex-touchdown PCR amplification protocol consisted of 5 min at 94˚C for initial denaturation, 10 cycles of denaturing at 95˚C for 30s, annealing at 65˚C (with 1˚C decrements from 65˚C to 55˚C at every cycle) for 30s, and extension at 72˚C for 30s. This was followed by a further 30 cycles of denaturing at 94˚C for 30s, annealing at 55˚C for 30s, and extension at 72˚C for 30s. The reaction was finished with a final extension for 5 min at 72˚C. PCR products for SNPs were direct DNA Sanger sequenced using the ABI 3730XL (Applied Biosystems, Foster City, CA, USA) after PCR amplification. Chromas 2.6.4 software (Technelysium Pty Ltd, South Brisbane, QLD, Australia) was used to view the DNA sequences.
ICD programming and follow-up
ICD programming was conducted in Henan Provincial Pacemaker Programing Center in our hospital. Recognition program of tachycardia was divided into 3 zones as following: VF zone with tachycardia circumference length (CL) less than 250ms, fast VT zone from 250ms to 320ms, and slow VT zone from 321ms to 400ms. The diagnose of VT zone based on the stability and morphological standard of tachycardia. The algorithms for discrimination of supraventricular tachycardia were activated in the VT zones. When tachycardia CL coincided slow VT zone, anti-tachycardia pacing (ATP) conducted at the 85% CL of VT for three tracings in the form of 8 consecutive pulses. And for fast VT zone, treatment procedure was ATP at the 85% CL of VT for three tracings in the form of 8 consecutive pulses, and shocks following if ATP failure. For VF zone, only one tracing ATP followed by shocks. Follow-up started at the time of ICD implantation and device interrogations were scheduled in pacemaker programing center every 3-6 months after ICD implantation. The longest follow-up time was 36 months. Pacemaker programming was required when patients experienced symptoms like syncope, incessant palpitations, or ICD therapy. All device interrogations were independently reviewed by the third-party technician to determine whether ICD therapies were delivered appropriately or inappropriately. Appropriate therapies included those delivered in response to VT, VF. Inappropriate therapies were defined as ATP or shock for supraventricular tachycardia, non-sustained VT, and oversensing events resulted from ICD lead noise, myopotentials, electromechanical interference, and T-wave. Intracardiac electrograms were reanalyzed by at least 2 independent electrophysiologists to confirm the type of arrhythmia if episodes controversy exists. The parameters of the ICD were changed whenever necessary as dictated by the physicians during each follow-up. Participants were censored at the time of loss to follow-up or death if the cause of death was other than SCD.
Statistical analyses
Statistical analyses were performed using SPASS17.0 (SPSS Inc, Chicago, IL). The allelic association and genotypic association under dominant and recessive models were analyzed using a 2 × 2 chi-square tests. Data of baseline clinical characteristic was grouped by genotypes of the positive SNP. Continuous variables were expressed as mean ± standard deviation. One-way ANOVA was performed for comparison between three continuous variables. Categorical data was counted and expressed as percentages and c2 test was used to compare the difference among them. Frequencies of rapid ventricular arrhythmia episodes, ATPs and ICD shocks 3 months, 12 months after implantation, and at the end of follow-up time in three genotypes were figured out and compared using one-way ANOVA and bivariate correlation separately. Bivariate correlation of these variables and genotypes were evaluated by spearman correlation coefficient. The ratio of patients suffered from VA episodes and ICD therapies under different genotype model were compared by chi-square test. Fisher’s exact test should be used instead of the chi-square test when any expected frequency is less than 1 or 20% of expected frequencies are less than or equal to 5. Kaplan-meier curve of the cumulative probability of survival classified by genotypes of the positive variant was further performed using Log Rank method to analyze the impact of three genotypes on SCD. Finally, hazard ratios of different genotypes for SCD were calculated by cox regression analysis in complicate model using Forward: LR method. A P-value of <0.05 was considered statistically significant.