Evaluating Common Variants in NOS1AP in Patients with Implantable Cardioverter Debrillator for Secondary Prevention

Background Contemporary researchers found single nucleotide polymorphisms (SNPs) in nitric oxide synthase 1 adaptor protein (NOS1AP) gene are associated with altered QT intervals and SCD. However, the clinical utility and implications of these SNP have not been described. This study aims to explore the clinical utility and implications of SNPs in nitric oxide synthase 1 adaptor protein (NOS1AP) in patients with implantable cardioverter debrillator (ICD) for secondary prevention. We rstly conducted a case-control study to evaluate the associations between hot-spot SNPs in NOS1AP (rs12143842, rs10494366, rs12567209 and rs16847548) and patients with ICD for secondary prevention. Then the clinical values of the positive SNPs were further evaluated in these patients. All patients were divided into three groups according to different genotypes of the positive SNPs. ICD interrogation data at 3, 12 months, and 3 years after implantation, which include rapid ventricular arrhythmia episodes and appropriate therapies, were analyzed in three genotypes.


Results
Case-control study revealed signi cant allelic association between rs10494366 and ICD recipients who experienced appropriate therapies. After a mean follow-up time of 31.70 ± 9.15 months, we detected not only signi cant difference among three genotypes on the distribution of ICD shocks and appropriate therapies, but apparently also the correlation of rs10494366 and ICD shocks. Furthermore, under kaplan-meier and cox regression analysis, TT genotype both showed higher risk for sudden cardiac death (SCD) compared with GG genotype.

Conclusions
The present study revealed that SNP rs10494366 was associated with appropriate therapies and SCD in patients with ICD for secondary prevention for the rst time.

Background
Sudden cardiac death (SCD) is a common clinical form of death and presumably due to a cardiac arrhythmia or hemodynamic catastrophe. Robust data supported implantable cardioverter de brillation (ICD) is highly effective in terminating life-threatening ventricular arrhythmia (VA). ICD has been applied in various patients including survivors of cardiac arrest, patients with VT and structural heart disease, and patients with signi cant LV dysfunction, for more than 3 decades. Recently, many studies found allelic variants in nitric oxide synthase 1 adaptor protein (NOS1AP) gene, which encodes a cytosolic ligand of neuronal nitric oxide synthase, are associated with altered QT intervals and SCD [1][2][3][4]. Among these allelic variants, rs12143842, rs10494366, rs12567209 and rs16847548 have attracted the most attention. However, the clinical utility and implications of these hot-spot SNPs have not been described. We rstly conducted a case-control study in patients receiving ICD for secondary prevention and healthy people aim to nd the relation between these SNPs and patients with ICD. And then an evaluation of the relativity of positive SNPs and ICD events was performed to further explore its clinical value in ICD recipients.

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 brillation (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 nally. 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 de ned 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 nal classi cation 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 Puri cation 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 le 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 ddH 2 0 Water. The multiplex-touchdown PCR ampli cation 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 nished with a nal 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 ampli cation. 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 de ned 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 followup 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 c 2 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 gured out and compared using one-way ANOVA and bivariate correlation separately. Bivariate correlation of these variables and genotypes were evaluated by spearman correlation coe cient. 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 classi ed 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.

Patient characteristics and allelic associations
A total of 86 patients and 348 control subjects were enrolled in this study. There were 39 patients with CHD, 34 patients with DCM and 8 cases of HCM, 3 cases of ARVC, 2 cases of long QT syndrome. Over a median follow-up of 31.70 ± 9.15 months, 32 ICD recipients experienced appropriate therapies and 24 patients dead. The baseline characteristic of patients were shown in Table 1. Genotype distribution of the four SNPs in controls were all in accordance with HWE. Signi cant allelic association was detected between rs10494366 and ICD recipients who suffered appropriate therapies (P = 0.024, OR = 1.25), while the association with ICD patients were not signi cant ( Table 2). Association analysis of rs12143842, rs12567209 and rs16847548 under allele, dominant, recessive model did not show signi cant.

Comparisons Of Clinical Characteristics In Three Genotypes of Rs10494366
Subsequently, all patients were divided into three groups according to genotypes of the signi cant SNP rs10494366. The number was 17, 35, 34 for TT, GT, GG genotype, respectively. There was no signi cant difference in CHD and DCM in three genotypes. The distribution of other cardiovascular diseases such as HCM, ARVC, and LQT syndrome also did not show signi cant difference in each group. However, the distribution of DM displayed differently in three groups (11,5,13 for TT, GT, GG genotype, respectively).
Other clinical characteristic data revealed no signi cant difference as listed in Table 3. Abbreviation as Table 1.

Comparisons of the ratio of patients suffered from VA episodes and ICD therapies in different genotypes
At rst, we compared the ratio of patients suffered from VA episodes and ICD therapies in different genotype under different genotype model (Table 4). The ratio showed signi cant different in shocks at 3, 12 months after implantation with P of 0.020 and 0.042, respectively. The signi cant results also emerged under recessive model of TT genotype (P = 0.023, P = 0.024, respectively). At the end of the follow-up, TT genotype showed signi cant association with ICD shock under recessive model (P = 0.038) and correlation trend under other two models. Distribution of VA episodes and ICD therapies in three genotypes and the correlation of them with genotypes Table 5 displayed the distribution of VA episodes and ICD therapies in three genotypes and the correlation of them with different genotypes. Frequencies of VA episodes and ATP after 3 months implantation in three genotypes showed no signi cant difference (P = 0.103), while the distributions of shocks and appropriate therapies in three groups were different (P = 0.011; P = 0.030, respectively). The correlation of TT genotype and ICD shock was signi cant (P = 0.004) and the correlation coe cient was 0.308. Similarly, the distributions of shocks and appropriate therapies in three genotypes at 1 year showed signi cant (P = 0.006; P = 0.012, respectively) and TT genotype correlated with ICD shock (P = 0.003). The distributions of shocks at the end of follow-up time also showed signi cant with P = 0.007, while the distributions of appropriate therapies were not signi cant(P = 0.064). ICD shocks at the end of follow-up were correlate with TT genotype with the correlation coe cient of 0.321(P = 0.003). But the appropriate therapies did not show correlation with rs10494366 correspondingly. Table 5 Distribution of rapid ventricular arrhythmias and ICD therapies in three genotypes and the correlation of them with genotypes at three follow-up times. End up   RVA  ATP  Shock  APT  RVA  ATP  Shock  APT  RVA  ATP  Shock  APT   TT  61  60  14  74  105  117  25  142  152  134  40  174   GT  54  23  4  27  115  98  15  83  199  119  29  148   GG  19  14  0  14  82  59  7  66  144  102  16

Comparison Of Cumulative Probability of Survival Classi ed By Genotypes
Furthermore, patients with TT genotype underwent worse survival outcome than other two genotypes revealed by Kaplan-meier curve of cumulative probability of survival classi ed by genotypes of rs10494366 (Fig. 1). The P value was 0.022 assessed by Log Rank method. At the end of follow-up time, the overall mortality of the present study population was 24.41%, while in patients with TT genotype was 47.06% as revealed by the Kaplan-meier curve. Moreover, after adjusting for CHD, DCM, diabetes mellitus, QRS duration and LVEF, HR of TT genotype for SCD was higher (HR = 2.956, P = 0.037) compared with GG genotype.

Discussion
The present study was designed to assess the effect of common variants in NOS1AP on patients receiving an ICD for secondary prevention. We rstly performed a case-control study to investigate the relation between hot-spot SNPs (rs12143842, rs10494366, rs12567209 and rs16847548) in NOS1AP and ICD patients. We found SNP rs10494366 show signi cant association with ICD patients who suffered appropriate therapies assuming allele model. Then we performed a prospective study to evaluate the clinical value of rs10494366 in these patients. The results of this study indicated that TT genotype of rs10494366 may play an important role in RVA episodes and shocks recorded by ICD as well as SCD in ICD-treated patients for the rst time.
Patients with ICD for secondary prevention yield great risk in recurrence of live-threatening malignant arrhythmia [9]. In the present research, 23.26% patients suffered from RVA after 3 months of implantation, and among them, 80% experienced ATP or shock treatment (Fig. 2). At the end of follow-up, the overall RVA episodes were 495 and 54.65% patients underwent RVA, among which, 68.09% delivered appropriate therapies. There were 26.74% patients experienced 85 shocks totally. At the three scheduled follow-up times, we detected not only signi cant difference among three genotypes on the distribution of ICD shocks and appropriate therapies, but apparently also the correlation of rs10494366 and shocks. Rapid sustained VT failed to termination after ATPs and VF, which represent more unstable status of electrophysiology of ventricular myocytes [10], will both trigger shocks delivered by ICD. Obviously, cardiac death frequently occurred accompanied with ICD therapies. Prior studies showed that most sudden deaths among ICD patients resulted from electromechanical dissociation occurring after appropriately detected and treated VAs [11]. Small size sample clinical data also displayed VA episodes and unsuccessful de brillations prior to death as interrogated at the time of autopsy [12,13]. In the present study, patients who carried TT genotype suffered more appropriate therapies including ICD shocks than other genotypes, thus with poor prognosis.
The total cardiovascular mortality at the end of follow-up time was identi ed as 24.41%, which was a little higher than previous studies performed in Veterans Administration trial [14] after 3 years of implantation. These different outcomes probably partly attributed to low proportion of optimal medical therapy for VT, heart failure and coronary artery disease. Furthermore, the present study revealed the probability of survival (52.94%) was lower in TT genotype. TT genotype showed greater risk for SCD under both kaplan-meier analysis and cox regression analysis. From all the above, TT genotypes of rs10494366 may play a part role in the occurrence and maintenance of RVAs and showed correlation to shocks in ICD recipients with different etiologies.
Rs10494366, which located in the intron region of NOS1AP, is a regulator of neuronal NOS [1,15]. Many researches veri ed rs10494366 in NOS1AP gene was associated with cardiac repolarization including prolonged QT interval and sudden death in independent populations [16][17][18][19][20][21][22][23][24]. SNPs, which located in noncoding region, could in uence transcriptional by exerting cis-acting elements. Vitro experiments has elucidated potential transcriptional effects of certain common variants in NOS1AP on schizophrenia [25]. Besides, Chang et al [26] found that overexpression of the NOS1AP gene product in isolated guinea pig myocytes causes attenuation of L-type calcium current, a slight increase in rapid delayed recti er current I Kr , and shortening of action potentials. Though the potential transcriptional effects of NOS1AP variants on gene expression in heart are not clearly known, these observations might explain our ndings to a certain extent.

Study Limitations
The present study is subject to several limitations. First, in some patients who have poor compliance of optimal treatment, electric storm might readily deliver without adjusting the ICD parameters in time, which could increase the weight of statistical validity slightly. But in other words, the susceptibility to electric storm in these patients revealed the potential arrhythmogenic of this allelic variant. Second, there is hitherto no standard de nition of VT/VF detection and therapy zone for ICD. Though we set a broad criterion, VT below the lower limit frequency, which is unable to detected, may inevitably dilute the impact of rs10494366 on VT episodes or arrhythmic death. Besides, to provide optimal therapies delivered by ICD, we adjusted the parameters of ICD in four patients according to documented arrhythmias resulted from deterioration of physical condition, which probably in uence the ATP or shock but would achieve more appropriate therapies on the other hand. However, considering the relatively small number of patients in these set, the in uence could be negligible. Moreover, the retrospective analysis in Japanese found left ventricular diastolic diameter ≥ 60 mm and the presence of non-sustained ventricular tachycardia before implantation were independent predictors of appropriate ICD therapy [27]. As the lack of these data, we are unable to bring into these indicators for analysis.

Conclusion
To our knowledge, the present study evaluated SNPs in NOS1AP in patients with ICD for secondary prevention for the rst time.
The association analysis revealed that minor allele of rs10494366 was associated with appropriate therapies in ICD patients.
The correlation between rs10494366 and clinical events detected by ICD revealed that TT genotype of NOS1AP may play a role on ICD shock and SCD. Thus, TT genotype of rs10494366 may be clinically useful for risk strati cation of RVA in patients receiving ICD for secondary prevention. However, researches focus on the functional test and large sample population are absolutely needed. Kaplan-meier curve of the cumulative probability of survival classi ed by genotypes of rs10494366.