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. Author manuscript; available in PMC: 2013 Aug 27.
Published in final edited form as: J Am Coll Cardiol. 2012 Jun 6;60(9):841–850. doi: 10.1016/j.jacc.2012.03.031

Common variation in the NOS1AP gene is associated with drug-induced QT prolongation and ventricular arrhythmia

Y Jamshidi 1,*,✉, IM Nolte 2,*, C Dalageorgou 1, D Zheng 1, T Johnson 6, R Bastiaenen 4, S Ruddy 1, D Talbott 1, KP Norris 5, H Snieder 2, AL George 5, V Marshall 3, S Shakir 3, PJ Kannankeril 6, PB Munroe 7, AJ Camm 4, S Jeffery 1, DM Roden 5, ER Behr 4,✉
PMCID: PMC3753216  NIHMSID: NIHMS493565  PMID: 22682551

Abstract

BACKGROUND

Use of anti-arrhythmic drugs is limited by the high incidence of serious adverse events including QT prolongation and torsades de pointes. Recent studies have demonstrated a role for NOS1AP gene variants in modulating QT interval in healthy subjects and modification of the severity of presentation and the risk of arrhythmias in LQTS.

METHODS

We carried out an association study using 167 SNPs spanning the NOS1AP gene in 58 Caucasian patients experiencing dLQTS and 87 Caucasian controls from the DARE Study.

RESULTS

Association analysis identified one polymorphism significantly associated with dLQTS (rs10800397: p=3.7×10−4; OR=3.3, 99.95% CI=1.0–10.8). The associations were more pronounced in the subgroup of amiodarone users, in which three SNPs including rs10800397 were significantly associated (most significant SNP rs10919035: p=3.0×10−4; OR=5.5, 99.95% CI=1.1–27.9). We genotyped rs10919035 in an independent replication cohort of 28 amiodarone-dLQTS cases versus 173 controls (meta-analysis of both studies: OR=2.81; p=2.4×10−4; 95% CI=1.62–4.89). Analysis of QTc among 74 controls from our dataset showed a similar pattern of significance over the gene region as the case-control analysis. This pattern was confirmed in 1,480 controls from the BRIGHT cohort (top SNP DARE rs12734991 in meta-analysis: mean [SD] increase in QTc interval per C allele=9.1 [3.2]ms; p=1.7×10−4).

CONCLUSIONS

Our results provide the first demonstration that common variations in the NOS1AP gene are associated with a significant increase in the risk of dLQTS. We suggest that common variation in the NOS1AP gene may have relevance for future pharmacogenomic applications in clinical practice permitting safer prescription of drugs for vulnerable patients.

Keywords: cardiac arrhythmias, NOS1AP, drug, single nucleotide polymorphisms, genetics, risk stratification

Introduction

Currently amiodarone is the most commonly used drug for the treatment of cardiac arrhythmias, followed closely by sotalol. However, use of anti-arrhythmic drugs is limited by the high incidence of associated side-effects, including bradycardia and QT prolongation which can result in torsades de pointes (TdP) arrhythmia1–3. Amiodarone, traditionally classified as a class III antiarrhythmic agent4, 5, also possesses electrophysiological properties from all four Vaughan–Williams classes6, 7. In addition to inhibiting potassium channels including IKr (class III effect), thus delaying phase 3 depolarization and increasing the action potential duration and effective refractory period8, 9 (seen as a prolongation of the QT-interval on the ECG), amiodarone decreases conduction velocity by blocking Na+ channels (class I effect)10, reduces the number of beta-adrenergic receptors with a resultant anti-adrenergic effect (class II effect)11, 12, and suppresses Ca2+-mediated action potentials by blocking L-type calcium channels (class IV effect)13, 14. Sotalol meanwhile is an anti-arrhythmic drug with class II (beta-adrenoreceptor blocking) and class III (cardiac action potential duration prolongation) properties.

It has been suggested that every individual has a physiological “cardiac repolarization reserve”15, which may compensate for any endogenous or exogenous factors (e.g. drugs) that would either decrease repolarizing or increase depolarizing currents during the action potential. It has also been suggested that the amount of this repolarization reserve may be genetically determined. It can therefore be hypothesized that individuals with a reduced repolarization reserve are more vulnerable to developing significant and manifest QT-interval prolongation and TdP when exposed to potassium channel blocking drugs such as amiodarone and sotalol.

Recently, genome-wide analysis has identified and consistently associated common variants of the nitric oxide synthase 1 adaptor protein (NOS1AP) gene with QT-interval prolongation across independent replication studies16–21. An early study associated the rs10494366 variant with QT-interval variation, which was estimated to explain up to 1.5% of QT variance16, and this was confirmed across independent replication studies17–22. Two recent large meta-analyses of genome-wide data identified a number of additional signals in the NOS1AP gene (rs12029454, rs12143842, rs16857031, and rs4657178)23, 24. However, despite the attempts of previous studies16 to identify and validate a single functional variant in NOS1AP associated with QT interval, resequencing of all exons in NOS1AP has not yet identified any missense mutations that explain these results, suggesting that the functional variants associated with these SNPs are likely to be regulatory in nature.

NOS1AP is a regulator of neuronal NOS (nNOS encoded by NOS1), one of the isoforms of NOS. The nNOS enzyme regulates intracellular calcium levels and myocyte contraction in the heart25–27. It is thought that nNOS inhibits the inward Ca2+ current through voltage dependent calcium channels, reducing the intracellular calcium concentrations and thereby suppressing beta-adrenoreceptor stimulation of the heart. The NOS1AP protein is expressed in the heart and has been shown to interact with nNOS to accelerate cardiac repolarization by inhibition of L-type calcium channels28–31, thereby providing a rationale for the association of NOS1AP gene variants with QT-interval duration.

The appreciation that common genetic variants may well modulate both disease and response to drugs is a critical concept when trying to understand mechanisms of drug action and their variability in individuals. This variability can arise because of variation in genes encoding drug targets, genes modulating the overall activity of the complex biological systems within which the drugs act and genes that are responsible for drug metabolism and elimination. In view of the role of NOS1AP in cardiac repolarization, we hypothesized that genetic variation in the NOS1AP gene influences the incidence of drug-induced ventricular arrhythmia and QT prolongation.

Methods

Study Cohort

The Drug-Induced Arrhythmia Risk Evaluation (DARE) Study is a unique national cohort of 112 patients experiencing drug-induced ventricular arrhythmias and/or severe QT interval prolongation, referred by cardiologists around England over a 5 year period (2003-8). A case-control study was established from the DARE study consisting of 59 self-reported Caucasian cases who had experienced an arrhythmic event associated with drug-induced QT prolongation, and 91 self-reported Caucasian control subjects, all of whom had provided DNA samples.

Cases were included if they had one or more of the following diagnosed as secondary to a medication: documented classical Torsades de Pointes (TdP) defined as 3 beats or more of polymorphic ventricular tachycardia associated with QT prolongation and pauses prior to onset of the arrhythmic event; ventricular fibrillation and/or cardiac arrest associated with QTc interval prolongation; and QTc interval prolongation with a history consistent with cardiac syncope, excluding vasovagal syncope and seizures. After withdrawal of the culpable drug cessation of ventricular arrhythmia and syncope and at least partial resolution of QT prolongation were required. All QTc intervals were corrected using Bazett’s formula and values >450ms (males) or >470ms (females) were considered prolonged.

Healthy controls were provided from primary care physicians responsible for the cases to ensure geographical matching. Inclusion criteria were as follows: no history of drug induced arrhythmias, ventricular arrhythmias or the congenital long QT syndrome. Controls with abnormal resting 12 lead ECGs were excluded.

Clinical and ECG Assessment

The cases’ acute presentation with arrhythmia and/or syncope and past medical history were assessed by obtaining hospital records, interview and patient questionnaires. These evaluated the acute circumstances of the clinical event to assess for evidence of underlying structural cardiac disease, prior medical conditions, drug history and acute metabolic disturbances. Paper ECGs taken during hospital admission were analyzed to confirm arrhythmia diagnoses and to assess maximal QTc interval during drug exposure. The QT interval was measured manually at stable heart rates by averaging the QT and RR intervals of up to 5 cardiac cycles (up to 10 cardiac cycles in atrial fibrillation), and was corrected for time using Bazett’s formula to calculate the QTc interval. These data were reviewed by a panel (ERB and AJC) for inclusion or exclusion. At least several months after the event with the drug exposure removed the cases underwent resting 500Hz digital 12 lead ECGs acquired using PC based Cardionavigator recorders (Reynolds-Spacelab).

Controls underwent a similar interview and questionnaire as cases. They also underwent resting 500Hz digital 12 lead ECGs acquired using PC based Cardionavigator recorders (Reynolds-Spacelab). Each automatically calculated QT interval was checked manually. If they were similar the automatic measurement was not revised. If not then the same method described above for manual measurements was utilized.

NOS1AP Sequencing

Genomic DNA samples were extracted from peripheral lymphocytes in blood and saliva samples using standard techniques (Nucleon® Genomic DNA Extraction Kit, Tepnel PLC, Oragene® DNA Extraction Kit, respectively) and normalized to a concentration of 50 ng/ml. Gene sequencing was carried out in all cases and control subjects using the ABI 3130 System (Applied Biosystems) to investigate potentially novel mutations in exonic regions and intron/exon boundaries. DNA samples were initially amplified using standard PCR techniques with primer sequences by Sigma-Genosys (available on request) for NOS1AP (ENSG00000198929). Amplification was carried out with the use of a Touchdown Thermal Cycler (Hybaid); any product that did not amplify successfully was subjected to a different PCR method using HotStar Taq polymerase (Qiagen) following the manufacture’s protocol. In some cases it was necessary to add 4 µl of a PCR additive, Q solution (Qiagen), in a total 25 µl reaction. Products were sequenced using the Applied Biosystems kit, v3.1 for a 10µl reaction in an automated capillary DNA Sequencer (ABI3130, Biomics Centre, St George’s University of London).

NOS1AP Association Study

One hundred and ninety eight tagging SNPs, designed to be inclusive of the intronic, exonic, untranslated region and 5 kb of the proximal promoter region derived from NCBI build 35 of the NOS1AP gene, were genotyped in all cases and controls using the Infinium Human_CVD 50K Bead Array (Illumina-IBC/CVD)32 and analyzed using the Illumina platform 500GX (Biomics Centre, St George’s University of London). The ITMAT-BROAD-CARe (IBC/CVD Illumina iSELECT array is a custom, cardiovascular disease (CVD) gene-centric single nucleotide polymorphism (SNP) genotyping platform that contains greater SNP marker density and linkage disequilibrium (LD) coverage for more than 2000 CVD candidate regions than current genome-wide arrays. The tagging approach utilized on the IBC array was designed to capture maximal genetic information from the HapMap populations for both common and lower frequency SNPs. Seven SNPs covered up to 4.3kb of the upstream region of NOS1AP from the start site of the gene and after resequencing we found two more SNPs, which we added to the data set. The downstream region of the gene was not covered. Two outliers (1 case and 1 control) were excluded from the analysis as they had >10% missing data. Furthermore we checked for ethnic outliers using the complete genetic data set of the CVD chip. Multidimensional scaling analysis in PLINK v1.0733 (http://pngu.mgh.harvard.edu/purcell/plink/) revealed that three controls were not of Caucasian descent despite their self-reported Caucasian ethnicity. After these exclusions there were 58 cases and 87 controls available for analysis. The average call rate of the remaining subjects was 99.9%. Thirty three SNPs were non-polymorphic in this data-set, hence 165 SNPs from the Human_CVD 50K array could be analyzed for association with QT interval prolongation and drug-induced ventricular arrhythmia. Two additional SNPs in the 5’ region identified through direct sequencing were also included in the final analysis.

NOS1AP rs10919035 replication cohort

Cases

For validation, an independent set of 28 amiodarone-treated patients collected at Vanderbilt University Medical Center, under appropriate IRB-approved protocols was used. Patients were of European descent from North America with diLQTS, defined as documented TdP during treatment with amiodarone. The diagnosis was confirmed by clinical electrophysiology review (DR). The case definition required documented TdP, associated with any reversible QT prolongation during the drug exposure. Covariates included age, sex, self-reported ethnicity, hypokalaemia and the culprit drug at the time of the index arrhythmia.

Replication drug-exposed controls

105 self-identified European ancestry subjects derived from a clinical study at Vanderbilt University Medical Center, under an IRB-approved protocol were included as drug-exposed controls. The study uses electronic medical record-based surveillance to identify patients in whom assorted QT prolonging antiarrhythmics were being initiated. For this study, controls were defined as those who did not develop qualifying arrhythmias, had <50 msec increase in QTc (by Bazett’s formula) interval on drug exposure, and no QTc interval exceeding 500 msec during drug treatment.

Replication normal controls

68 self-identified European ancestry subjects derived from a clinical study at Vanderbilt University Medical Center, under an IRB-approved protocol were included as drug-exposed controls. Control subjects were normal healthy volunteers recruited from the general population and challenged with an antiarrhythmic drug (ibutilide)34. For this study, controls were defined by the absence of qualifying arrhythmias, <50 msec increase in QTc interval using Bazett’s formula for heart rate correction, and no QTc interval exceeding 500 msec during ibutilide challenge.

The frequency distributions of the two control samples were similar and hence the control data were pooled for analysis.

QTc replication cohort

BRIGHT

Over two thousand unrelated white European hypertensive individuals from the BRIGHT study35 were genotyped using the HumanCVD BeadChip (Illumina Inc, San Diego, CA). 1909 individuals passed quality control checks (samples with low call rate, cryptic duplicates and relatives, outliers in ancestry principle component analysis (PCA), sex X chromosome mismatch were excluded). Of these N=1628 individuals had twelve-lead ECG recordings (Siemens-Sicard440; http://www.brightstudy.ac.uk/info/sop04.html). For this analysis we excluded individuals with QRS duration >120ms (N=83), and individuals with atrial fibrillation or persistent flutter (Minnesota codes 8-3-1 or 8-3-2; N=25). No data were available for anti-arrhythmic drug consumption. We also excluded individuals with a missing covariate (age; N=40). Thus data on N=1480 individuals were tested for association. We used a normal linear model with QTc as outcome, and sex, age and 10 ancestry principal components as covariates to control for population stratification. We analyzed all SNPs within 50kb of the NOS1AP transcript, specifically from rs4657139 (chr1:160296531) to rs457879 (chr1:160602678) inclusive. We excluded SNPs that could not be called with high confidence (4 SNPs) and one SNP with call rate below 98%. The results for 195 SNPs were provided.

Statistical Analysis

Cases and controls from the DARE study were compared for population characteristics using a chi-square test (sex) and Mann-Whitney U test (age and QTc interval). Genotype frequencies were tested for Hardy-Weinberg equilibrium using the chi-square test with one degree of freedom. The genotype frequencies, assuming an additive model, were compared between the whole group of cases and controls, between subjects on amiodarone and controls, and between subjects on sotalol and controls (case-control analysis) using logistic regression analysis in PLINK v1.0733 (http://pngu.mgh.harvard.edu/purcell/plink/). Age and sex were not used as covariates because the sex distribution appeared not to be significantly different between cases and controls, and controls appeared to be older than cases, hence were more likely to develop QT prolongation or ventricular arrhythmia but nevertheless did not demonstrate either. As QTc interval appeared to be significantly longer among the cases after removal of the drug than among the controls, we also tested the model where QTc interval was included in the model to correct for possible mediating effects. Cases who had a ventricular or atrioventricular paced ECGs or a left branch bundle block were excluded from this analysis (n=12). The same analysis was performed in the replication study for our top SNP rs10919035.

In addition to the case-control study on drug-induced ventricular arrhythmia and QT prolongation, we studied QT interval as a quantitative trait in the population-based DARE control sample (74 out of 87 controls had data on QT interval available), in order to replicate earlier observed associations. Cases were not included in this analysis as they had a longer QTc interval even after removal of the drug and hence were not representative of the population. A linear regression was performed for each SNP following an additive model on QTc interval with SNP, age, and sex as covariates. A similar analysis was done in the BRIGHT cohort (n=1480) and the results of the two cohorts were meta-analyzed using the fixed-effect inverse variance method in PLINK v1.0733 (http://pngu.mgh.harvard.edu/purcell/plink/).

Since multiple SNPs were tested, a multiple testing correction was applied using SNP Spectral Decomposition (SNPSpD;36, 37). This method calculates the effective number of independent marker loci accounting for linkage disequilibrium between the SNPs. With this number a Bonferroni correction is applied to assess the significance threshold. For our data set of 167 SNPs in the NOS1AP gene the effective number was 98.8 and hence a p-value less than 0.00052 was considered statistically significant. Odds ratios (ORs) and 99.95% confidence intervals (99.95% CIs) were calculated to assess the strength of the association. Since only one SNP was genotyped in the replication study for amiodarone-induced QTc interval, a p-value <0.05 was considered significant in this cohort.

Results

Study Characteristics

Fifty eight subjects experiencing drug-induced QT prolongation and ventricular arrhythmias (50 (89%) with documented TdP), and 87 healthy control subjects from the DARE study were available for the primary analysis. Case and control characteristics are shown in Table 1. Controls were on average almost 9 years older than cases (p<0.001). No sex difference was observed. Twenty-seven (46%) cases were treated with amiodarone and 15 (27%) with sotalol, while 15 (27%) had received more than one culpable drug. Eleven cases (20%) were associated with hypokalemia. The cases also had a higher frequency of accompanying structural heart disease and, when the drug exposure was removed, demonstrated greater QTc prolongation than controls (p=1.0×10−3)

Table 1.

Characteristics of cases and controls included in the analysis

DARE Cases DARE
Controls		 Vanderbilt
Cases		 IBU
Controls		 QTSPS
Controls		 BRIGHT
N 58 87 28 68 105 1,480
Age, mean (SD) 62.5 (15.5) 71.1 (10.5)** 64 (15.16) 26.8
(5.61)		 62.2
(14.26)		 57.7
(10.18)
Sex, female, N (%) 39 (67%) 46 (56%) 21 (75%) 37 (53%) 38 (36%) 916
(61.9%)
Culpable drug exposure:a
  Amiodarone, N(%) 27 (47%) - 28 (100%) - - -
  Sotalol, N(%) 15 (26%) - - - - -
  Diuretics, N(%) 6 (11%) - 9 (32%) 0 43 (41%)
  More than one drug, N(%) 16 (28%) - 0 0 0
Presentation:
  Documented torsades de pointes, N(%) 50 (89%) - 28 (100%) na 0
  Ventricular fibrillation/cardiac arrest, N(%) 12 (21%) - 0 na 0
  Syncope only, N(%) 2 (4%) - 0 na 0
  Hypokalaemia, N(%) 11 (20%) - 7 (.25%) na 0
Other medical history:
  Prior myocardial infarction, N(%) 14 (25%) - 3 (10%) na 14 (14%) 0 (0%)
  Heart failure*, N(%) 12 (21%) 0 (0%) 9 (32%) na 1 (.009%) 0 (0%)
  Atrial fibrillation and/or flutter, N(%) 29 (52%) - 18 (64%) na 97 (92%) 0 (0%)-
  Congenital long QT syndrome*, N(%) 2 (4%) 0 (0%) 0 na 0 0 (0%)
  Hypertension, N(%) 32 (57%) - 13 (46%) na 65 (62%) 1480
(100%)
  Hypothyroidism, N(%) 11 (20%) - 0 na 18 (17%) 0 (0%)
  Diabetes mellitus, N(%) 14 (25%) - 0 na 20 (19%) 0 (0%)
Maximal QTc during drug exposure:
  mean (SD), ms
  range, ms
  median, ms		 592 (73.3)
466–850
590		 - 584
523–840
613		 434
375–492
428		 467
403–556
464		 -
QTc without drug exposure: b
  mean (SD), ms
  range, ms
  median, ms		 441 (25.9)
381–503
435		 426 (18.2)**
379–469
423		 430
344–505
428		 388
367–457
406		 443
367–565
442		 417 (12.0)
343–704
420
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ns = not significant,

*

= from questionnaire data only, na = not applicable

a

Available for 57 cases

b

Available for 44 cases and 74 controls

DNA Sequencing and Association Analysis

Sequencing of the NOS1AP exonic regions and intron/exon boundaries on chromosome 1 did not identify any novel coding mutations or polymorphisms. However re-sequencing of 2kb upstream of the ATG site identified two additional SNPs that were not included on the Human_CVD 50K Bead Array (Illumina) and therefore these were included in the association analysis.

Association analysis of the 167 SNPs spanning the NOS1AP gene identified one polymorphism that was significantly associated with drug-induced ventricular arrhythmia and QT prolongation (rs10800397: p=3.7×10−4; OR=3.3, 99.95% CI=1.0–10.8) (Figure 1; Table 2a). This association was driven by the group of amiodarone users (rs10800397: P=4.3×10−4; OR=4.5, 99.95% CI=1.0–19.8; cases 37.0%, controls 14.4%) (Figure 1; Table 2a). For this subgroup of cases, three non-coding SNPs were significantly associated with drug-induced ventricular arrhythmia and QT interval prolongation (most significant SNP rs10919035: P=3.0×10−4, OR=5.5; 99.95% CI=1.1–27.9; allele frequencies cases 27.8%, controls 7.1%). These SNPs did not include either of the SNPs rs10494366 (P=0.79, OR=1.1; allele frequencies amiodarone-induced cases 37.0%, controls 35.1%) and rs16857031 (P=0.13, OR=1.8; allele frequencies amiodarone-induced cases 27.8% and controls 19.0%) known to be associated with variation in resting QTc nor SNPs that were in strong linkage disequilibrium with them16, 23, 24. The remaining NOS1AP SNPs previously associated with resting QTc (rs12143842, rs12029454, rs4657178) were not included on the array and hence could not be investigated directly. However SNPs that were in strong LD with these in the HapMap CEU population38 and that were present on the chip, were not associated with amiodarone-induced ventricular arrhythmia and QT interval prolongation, except for SNP rs6427664, which was in LD with rs4657178 (r2=0.82 in HapMap CEU sample) and showed suggestive association (p=0.0025; OR=3.4, 99.95% CI 0.8–14.0). Including QTc interval as a covariate in the model weakened the associations, but the odds ratios did not change (Table 2b). None of the SNPs were statistically significantly associated with sotalol-induced ventricular arrhythmia and QT interval prolongation, although the top SNPs of this analysis were the same as for amiodarone (Table 2).

Figure 1.

Figure 1

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Regional association plot for NOS1AP SNPs. Statistical significance of SNPs are shown on the −log (P) scale for the whole group (red squares), the amiodarone-users (blue diamonds), and the sotalol users (green triangles). The recombination rate is shown at the right axis. ORs and 99.95%CIs of the top SNPs (p<0.01) are shown in the top of the figure (whole group in red; amiodarone-users in blue). The most significantly associated SNP is represented by a large blue diamond. The correlation of this SNP to other SNPs at the NOS1AP locus is shown on a scale from minimal (white) to maximal (bright red/green/blue). The rs-ids of SNPs previously associated with QTc interval prolongation in the general population or that where in strong LD with such SNPs are given in the lower part of the figure. Figure made using an adapted version of the R script of SNAP44,45.

Table 2.

a Top drug-induced arrhythmia associated SNPs (p<0.01) in the group of 26 amiodarone users, the group of 15
sotalol users, and the whole group of 58 cases compared with 87 controls. The SNPs are sorted according to significance
in the analysis of amiodarone users. Significant SNPs (p<0.00052) are denoted in bold.
Minor Controls Amiodarone users Sotalol-users All cases
SNP Allele MAF MAF P-value OR 99.95%CI MAF P-value OR 99.95%CI MAF P-value OR 99.95%CI
rs10919035 T 7.1% 27.8% 3.0×10−4 5.5 1.1 – 27.9 20.0% 0.022 4.1 0.5 – 34.2 20.7% 0.0011 3.7 0.9 – 14.6
rs10800397 T 14.4% 37.0% 4.3×10−4 4.5 1.0 – 19.8 30.0% 0.023 3.7 0.5 – 27.8 31.0% 3.7×10−4 3.3 1.0 – 10.8
rs10800404 T 14.4% 37.0% 4.3×10−4 4.5 1.0 – 19.8 30.0% 0.023 3.7 0.5 – 27.8 30.2% 6.9×10−4 3.1 1.0 – 10.1
rs10800352 G 14.4% 35.2% 7.1×10−4 4.4 1.0 – 20.2 30.0% 0.030 3.2 0.5 – 20.4 28.5% 0.0023 2.8 0.9 – 8.8
rs7522678 A 13.2% 33.3% 9.0×10−4 4.2 0.9 – 19.0 26.7% 0.043 3.2 0.4 – 23.3 25.9% 0.0042 2.7 0.8 – 8.8
rs10800409 T 8.6% 27.8% 0.0013 4.0 0.9 – 17.6 23.3% 0.023 3.5 0.5 – 23.3 22.4% 0.0016 3.2 0.9 – 11.3
rs6427664 A 19.0% 38.9% 0.0025 3.4 0.8 – 14.0 30.0% 0.14 2.1 0.4 – 11.9 31.0% 0.015 2.1 0.7 – 5.9
rs10918859 A 12.6% 29.6% 0.0039 3.4 0.8 – 14.8 26.7% 0.044 2.9 0.5 – 18.5 25.0% 0.0061 2.5 0.8 – 8.1
rs12403202 T 20.7% 40.7% 0.0045 2.8 0.8 – 10.0 16.7% 0.59 0.7 0.1 – 5.1 29.8% 0.077 1.7 0.6 – 4.5
rs12742393 A 36.1% 57.4% 0.0053 2.7 0.8 – 9.4 46.7% 0.24 1.7 0.4 – 7.7 48.3% 0.032 1.8 0.7 – 4.4
rs6664702 C 17.8% 35.2% 0.0058 3.1 0.7 – 12.8 33.3% 0.043 2.8 0.5 – 16.1 30.2% 0.0094 2.3 0.8 – 6.9
rs10753784 G 35.6% 57.4% 0.0061 2.5 0.8 – 8.1 40.0% 0.64 1.2 0.3 – 5.2 48.3% 0.034 1.7 0.7 – 4.1
rs4298709 G 39.1% 59.3% 0.0077 2.6 0.7 – 9.1 42.9% 0.69 1.2 0.3 – 5.5 51.8% 0.031 1.7 0.7 – 4.3
rs4531275 T 25.9% 44.4% 0.0085 2.7 0.7 – 9.6 39.3% 0.12 2.1 0.4 – 11.1 38.6% 0.016 2.0 0.7 – 5.4
rs10399680 C 21.3% 38.9% 0.0098 2.6 0.7 – 9.5 30.0% 0.28 1.7 0.3 – 8.3 31.0% 0.058 1.7 0.6 – 4.6
b Top drug-induced arrhythmia associated SNPs (p<0.01) corrected for
QTc interval in the group of 19 amiodarone users, the group of 13 sotalol users,
and the whole group of 44 cases compared with 74 controls. The SNPs are
sorted in the same order as in table 2a. Significant SNPs (p<5.2×10−4) are
denoted in bold. Other p-values <0.01 are shown in italic.
Minor
Allele		 Amiodarone users
 Sotalol-users
 All cases
SNP P-value OR 99.95%CI P-value OR 99.95%CI P-value OR 99.95%CI
rs10919035 T 0.0023 6.6 0.8 – 56.7 0.14 2.9 0.2 – 37.2 0.011 3.5 0.6 – 19.3
rs10800397 T 0.066 2.8 0.4 – 20.7 0.13 2.7 0.3 – 27.6 0.014 2.8 0.7 – 11.9
rs10800404 T 0.066 2.8 0.4 – 20.7 0.13 2.7 0.3 – 27.6 0.024 2.6 0.6 – 10.9
rs10800352 G 0.0098 4.5 0.6 – 34.8 0.035 3.7 0.4 – 31.8 0.014 2.7 0.7 – 10.7
rs7522678 A 0.016 4.0 0.5 – 29.5 0.21 2.3 0.2 – 23.2 0.069 2.2 0.5 – 9.3
rs10800409 T 0.013 4.0 0.6 – 27.1 0.10 2.8 0.3 – 23.7 0.027 2.7 0.6 – 12.3
rs6427664 A 0.21 1.9 0.3 – 12.0 0.51 1.5 0.2 – 10.7 0.22 1.6 0.4 – 5.6
rs10918859 A 0.036 3.3 0.5 – 23.6 0.046 3.4 0.4 – 28.3 0.028 2.4 0.6 – 10.0
rs12403202 T 0.044 2.5 0.5 – 12.5 0.51 0.7 0.1 – 5.9 0.37 1.4 0.4 – 4.4
rs12742393 A 0.74 1.2 0.3 – 5.3 0.33 1.6 0.3 – 8.7 0.23 1.5 0.5 – 4.5
rs6664702 C 0.078 2.6 0.4 – 16.2 0.18 2.2 0.3 – 16.3 0.12 1.8 0.5 – 6.8
rs10753784 G 0.31 1.6 0.3 – 7.2 0.82 0.9 0.2 – 5.2 0.42 1.3 0.4 – 3.8
rs4298709 G 0.55 1.3 0.3 – 6.8 0.71 0.8 0.1 – 5.6 0.34 1.4 0.4 – 4.1
rs4531275 T 0.089 2.3 0.4 – 11.9 0.18 2.2 0.3 – 15.8 0.079 1.9 0.5 – 6.3
rs10399680 C 0.47 1.4 0.3 – 7.1 0.89 1.1 0.2 – 6.9 0.56 1.2 0.4 – 4.1
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MAF: minor allele frequency; OR: odds ratio; CI: confidence interval; NA: not applicable.

MAF: minor allele frequency; OR: odds ratio; CI: confidence interval.

The most significant SNP (rs10919035) was also genotyped in the replication cohort of 28 cases with amiodarone induced prolonged QT interval and 173 controls (challenged with the antiarrhythmic drug ibutilide). The effect of this SNP showed a trend in the same direction as observed in the DARE cohort (allele T: 26.8% among cases versus 16.5% in drug-challenged controls; p=0.060). Meta-analysis of the results of rs10919035 from both studies revealed an OR of 2.81 for each T allele (p=2.4×10−4; 95% CI 1.62–4.89).

To validate the reported association between NOS1AP and QTc interval duration, the 167 SNPs spanning the NOS1AP gene were examined for association in the population-based DARE control subjects (n=74). None of the SNPs were significantly associated with QT interval after multiple testing correction (Table 3; Figure 2a). To extend this analysis we combined the data in a meta-analysis with those of the BRIGHT cohort (n=1480) (Table 3; Figure 2a). The results of the BRIGHT cohort were similar to those of the DARE cohort and, although the associations were more significant, the effect sizes were 2 to 4 times smaller. SNP rs12734991 showed the largest increase in QT interval in the DARE cohort (β=11.1 ms per C allele; P=6.4×10−4) and it was second in the meta-analysis (β=4.2 ms per C allele; P=3.5×10−6). The most associated SNPs overlap with those SNPs arising from the case-control analysis. SNP rs10800397, which showed the strongest association with drug-induced ventricular arrhythmia and QT interval prolongation, also caused an increase in QT interval in the controls (in DARE/meta-analysis: β=11.5/4.6 ms per T allele; P=0.019/4.3×10−5). In addition, SNP rs6427664, which was in LD with rs4657178, reached a significance level of 0.012/9.1×10−5 in DARE/meta-analysis (β=11.1/4.2 ms per A allele). In contrast, SNPs rs10494366 and rs16857031 were not significant (p=0.85/0.005 and 0.95/22, respectively in DARE/meta-analysis), and neither were SNPs that were in strong LD with other previously QTc-associated non-typed SNPs rs12143842 and rs12029454.

Table 3.

Top SNPs from the DARE cohort (p<0.05) associated with the continuous QTc interval in 74 controls ordered according to their significance together with the results of the BRIGHT cohort (n=1480) and of the meta-analysis of the two cohorts. Significantly associated are shown in bold (p<5.2×10−4). P-values <0.01 are shown in italic.

DARE
 BRIGHT
 Meta-analysis
SNP Position Allele Beta SE P-value Beta SE P-value Beta SE P-value
rs12734991 160461200 C 11.1 3.1 6.4×10−4 3.5 0.9 1.7×10−4 4.2 0.9 3.5×10−6
rs4657166 160427963 G 11.5 3.6 0.0023 2.6 1.0 0.010 3.2 1.0 0.00091
rs12733377 160519067 G 9.3 3.1 0.0037 3.3 0.9 4.0×10−4 3.8 0.9 2.0×10−5
rs12729882 160508661 A 8.9 3.0 0.0049 3.1 0.9 8.6×10−4 3.6 0.9 5.2 ×10−5
rs2661818 160531438 G 8.6 3.0 0.0051 4.0 0.9 1.7×10−5 4.4 0.9 6.1×10−7
rs10399680 160480119 C 11.1 3.8 0.0052 3.7 1.1 7.9×10−4 4.2 1.0 5.7×10−5
rs6664702 160471531 C 11.7 4.4 0.0099 3.6 1.1 0.0013 4.1 1.1 1.6×10−4
rs1964052 160602048 T 11.6 4.4 0.011 −1.1 1.4 0.44 0.1 1.3 0.95
rs4298709 160503206 G 8.5 3.3 0.011 3.0 0.9 0.0015 3.4 0.9 1.6×10−4
rs10918936 160469112 A 8.1 3.1 0.011 2.9 1.0 0.0025 3.3 0.9 2.5×10−4
rs6427664 160481097 A 11.1 4.3 0.012 3.7 1.1 7.6×10−4 4.2 1.1 9.1×10−5
rs905720 160600724 T 8.5 3.5 0.018 −0.4 1.0 0.73 0.4 1.0 0.72
rs4557949 160477578 A 8.0 3.3 0.019 3.8 0.9 4.9×10−5 4.1 0.9 4.8×10−6
rs4145621 160485312 C 8.0 3.3 0.019 3.9 0.9 3.7×10−5 4.2 0.9 3.5×10−6
rs10800397 160503714 T 11.5 4.8 0.019 4.2 1.1 3.0×10−5 4.6 1.1 4.3×10−5
rs10800404 160521736 T 11.5 4.8 0.019 4.4 1.2 1.4×10−4 4.8 1.1 1.9×10−5
rs12135795 160416389 A 7.6 3.2 0.019 2.3 0.9 0.016 2.7 0.9 0.0026
rs10753765 160418397 G 7.6 3.2 0.019 2.3 0.9 0.016 2.7 0.9 0.0026
rs3927640 160422437 T 7.6 3.2 0.019 2.3 0.9 0.015 2.7 0.9 0.0025
rs4657161 160422913 G 7.6 3.2 0.019 2.3 0.9 0.015 2.7 0.9 0.0025
rs12090585 160305400 A −8.9 3.9 0.024 NA NA NA NA NA NA
rs347271 160585953 A 19.6 8.7 0.027 2.4 2.5 0.34 3.7 2.4 0.12
rs4328057 160458029 G 19.7 8.8 0.028 −1.8 3.1 0.57 0.6 2.9 0.84
rs10918951 160475138 A 11.6 5.4 0.036 0.2 2.1 0.91 1.7 2.0 0.38
rs16859092 160491174 C 11.6 5.4 0.036 −0.2 2.1 0.92 1.3 2.0 0.50
rs12742393 160491210 A 6.9 3.3 0.039 3.2 0.9 7.5×10−4 3.5 0.9 1.3×10−4
rs4656364 160545479 C 10.0 4.8 0.040 3.3 1.5 0.033 3.9 1.5 0.0076
rs10753784 160515544 G 6.6 3.2 0.043 3.3 1.0 5.5×10−4 3.6 0.9 9.4×10−5
rs10919035 160510636 T 14.1 6.9 0.043 5.1 1.4 3.3×10−4 5.5 1.4 8.1×10−5
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NA: not available; SE: standard error

Figure 2.

Figure 2

Open in a new tab

(A) Association of the NOS1AP SNPs with the continuous QTc interval in the DARE controls (white diamonds) (n=74), in the BRIGHT cohort (grey diamonds) (n=1480) and in the meta-analysis of the two cohorts (black diamonds) (n=1554) and (B) association of the NOS1AP SNPs with the continuous QTc interval in the DARE-BRIGHT meta-analysis (black diamonds) (n=1554) and with dichotomous drug-induced ventricular arrhythmia and QT interval prolongation (grey squares) (n=58 cases and 87 controls).

Discussion

This study is the first to demonstrate that common variations in the NOS1AP gene are associated with a significant increase in the risk of drug-induced, and in particular amiodarone-induced, ventricular arrhythmia and QT prolongation. Prolongation of the QT interval associated with TdP is currently the most common cause of withdrawal or restriction of the use of antiarrhythmic drugs39.

We performed a comprehensive screen of 167 SNPs in and close to the NOS1AP gene in order to investigate association of NOS1AP variations with drug-induced ventricular arrhythmia and prolongation of the QT interval. SNP rs10800397 reached a significance level of p=3.7×10−4 in the overall DARE case-control study and this was predominantly explained by the subgroup of amiodarone users (p=4.3×10−4). For this subgroup of cases, three non-coding SNPs were significantly associated with drug-induced ventricular arrhythmia and QT interval prolongation. The most significant one, rs10919035, was in moderate LD with rs10800397 (r2=0.49). We carried out a replication study using amiodarone treated cases from a second study (Vanderbilt) and these were compared to a drug-challenged control group (IBU and QTSPS). Although we could not fully validate our results from the DARE cases and non-drug-challenged controls, meta-analysis of the results of rs10919035 from both studies revealed an OR of 2.81 for each T allele (p=2.4×10−4; 95% CI 1.62–4.89). It is interesting that the allele frequency among the replication cases is the same as amongst the DARE cases. The non-significance in the replication study alone appears to be caused by the unexpectedly higher frequency amongst the IBU/QTSPS controls (17% versus 7% in the DARE controls, 8% in 1000Genomes CEU, and 11% in HapMap Phase 2 CEU44) and might therefore be a population specific effect. It is also important to note that the drug-challenged controls were challenged with ibutilide and not amiodarone. Unlike most other Class III antiarrhythmic drugs such as amiodarone, ibutilide does not produce its prolongation of action potential via inihibition of potassium channels including IKr, nor does it have a sodium-blocking, antiadrenergic, and more importantly calcium blocking activity that other Class III agents possess.

In contrast to the interaction of amiodarone with NOS1AP variants, ventricular arrhythmia and QT prolongation induced by sotalol did not seem to be significantly affected by NOS1AP, although the same SNPs demonstrated the largest genotype differences with smaller odds ratios. This subgroup was too small (n=15) to draw any certain conclusions with regards to drug-specific interactions.

A common pathway between amiodarone induced ventricular arrhythmia and QT-interval prolongation and NOS1AP common variation could be the role of the NOS regulator pathway in cardiac L-type CaV currents. A recent study by van Noord et al.40 found that the presence of the minor alleles of the NOS1AP SNPs rs10494366 and rs10918594 was associated with the modification of the QTc prolonging effect of the calcium channel blocker, verapamil, in the prospective population-based Rotterdam Study. In addition, Chang et al.28 found that overexpression of the NOS1AP gene product (CAPON) in isolated guinea pig myocytes causes attenuation of the L-type CaV current, a slight increase in rapid delayed rectifier current (IKr), and shortening of action potentials.

Data from the congenital long QT syndrome (LQTS) may also support such a mechanism. Rare variants in the LQT8 gene CACNA1C, encoding a subunit of the L-type CaV channel, cause an unusual form of LQTS by increasing calcium influx into the myocyte41, 42 The recently identified LQT12 gene, α1-Syntrophin (SNTA1), has also been shown to be involved in the nNOS pathway43. A mutation in the gene causes inhibition of nNOS and is associated with increased peak and late sodium currents. Therefore genes encoding proteins interacting with nNOS have the potential to alter cardiac repolarization, perhaps by influencing calcium cycling in cardiac myocytes. NOS1AP minor allele variants have more recently been associated with modification of the severity of presentation of LQTS44 and the risk of arrhythmias in LQTS45.

In addition to the case-control study on drug-induced arrhythmia, we studied QT interval as a quantitative trait in the population-based control subjects. In the meta-analysis of the DARE controls (n=74) and the BRIGHT cohort (n=1480) 22 SNPs reached significance (p<5.2×10−4). Although the results in the BRIGHT cohort were obviously more significant as a result of the larger sample size, the effects of the SNPs were 2–4 times smaller than in the DARE controls. Furthermore, it was interesting to note that many of the top hits of this analysis overlap with the top hits of the drug-induced case-control analysis. This implies that the effect of NOS1AP on drug-induced QT interval prolongation is not independent of the effect of NOS1AP on QT interval in general. Since cases and controls already demonstrated significantly different mean QTc intervals after drug removal, we also corrected the case-control drug-induced QT interval analysis for baseline QTc interval. Although the results became less significant, the odds ratios only diminished slightly. This suggests that the investigated QT prolonging drugs and in particular amiodarone interact with NOS1AP variants. Unfortunately our study design does not allow testing for interactions directly since all cases used drugs while none of the DARE controls did and the drug- challenged controls from the Vanderbilt study were on ibulitide and assorted other anti-arrhythmic drugs and not specifically amiodarone. The findings do lend support, however, to the concept of repolarization reserve being influenced by common genetic variation.

Another explanation for the still larger QTc interval after removal of the drug among cases may at least partially be that the cases demonstrated a higher frequency of hypertension and underlying cardiac disease than the healthy controls. These are known acquired risk factors for QT interval prolongation and TdP46.

There are currently no published functional studies investigating variable expression of NOS1AP polymorphisms. Given the shared effects of NOS1AP and amiodarone on L-type calcium and potassium currents, however, one might hypothesize that individuals carrying genetic variants in NOS1AP which impair its expression, and in turn, result in increased L-type calcium currents and/or QT prolongation, may have additional arrhythmogenic risk with amiodarone therapy. These common variants may have relevance for future pharmacogenomic applications in clinical practice permitting safer prescription of amiodarone for vulnerable patients. The process of development of safer novel drugs may also benefit from this improved understanding of the biological pathways underlying the individuals’ variation in drug response.

Study Strengths

Firstly, the DARE cases were collected prospectively and nationally in a systematic manner with identical comprehensive phenotyping, a strength compared to other series. Secondly, the coverage of the NOS1AP gene was comprehensive and included 167 polymorphic SNPs in and close to the NOS1AP gene. Thirdly, the high odds ratio and level of significance despite small numbers provides compelling support for the association as does the similar frequency of SNPs in cases in the replication case-control study and the trend towards a significant association.

Study Limitations

Several limitations of this study warrant discussion. First, we were limited by the small number of subjects treated with amiodarone that presented with ventricular arrhythmia and QT-prolongation. These cases are rare and hence it is not feasible to obtain a large sample. Secondly, while the DARE controls were originally matched for age, sex and ethnicity, it was difficult to also match for drug exposure and other co-morbidity which results in difficulties in determining whether the associations identified are caused by drug exposure or by the underlying arrhythmic event. The replication case-control study was however able to utilize controls exposed to QT prolonging drugs although not amiodarone specifically and as described above ibulitide lacks the calcium blocking activity of amiodarone that we speculate is important for mediating the NOS1AP SNP effects. Thirdly, we found that SNPs within the NOS1AP locus associated with the risk of amiodarone-induced QT prolongation. As with previous resequencing efforts of all exons in NOS1AP16, we did not identify any missense mutations that explain the association results. It is most likely these SNPs are not functional variants and are only in linkage disequilibrium with the causal SNP or regulatory DNA element.

Conclusion

In conclusion, our study shows that common variants in the NOS1AP gene play a role in the pathogenesis of drug-induced, and particularly amiodarone-induced, ventricular arrhythmias and QT prolongation.

Acknowledgements

DARE: This study was funded by the British Heart Foundation, Project grant No. 06/094. The DARE study was funded under a special program grant from the British Heart Foundation. The genotyping was carried out in the SGUL Medical Biomics Centre. Thanks are due to the many British cardiologists who referred cases to the study team.

Vanderbilt: This study was supported by U01/U19 HL65962, the Pharmacogenomics of Arrhythmia Therapy site of the Pharmacogenomics Research Network and by a grant from the Fondation Leducq (Trans-Atlantic Network of Excellence “Alliance Against Sudden Cardiac Death”, 05 CVD 01).

BRIGHT: This work was supported by the Medical Research Council of Great Britain (grant number G9521010D); and by the British Heart Foundation (grant number PG/02/128). Genotyping for the HumanCVD BeadChip was supported by the British Heart Foundation (grant number PG/07/131/24254 to P.B.M.). T.J. is supported by the Wellcome Trust (grant number 093078/Z/10/Z). The BRIGHT study is extremely grateful to all the patients who participated in the study and the BRIGHT nursing team. We would also like to thank the Barts Genome Centre staff for their assistance with this project. This work forms part of the research themes contributing to the translational research portfolio for Barts and the London Cardiovascular Biomedical Research Unit, which is supported and funded by the National Institute for Health Research.

Abbreviations and Acronyms

CI

confidence interval

ECG

electrocardiogram

LQTS

long QT syndrome

MAF

minor allele frequency

nNOS

neuronal nitric oxide synthase

NOS1AP

nitric oxide synthase 1 adaptor protein

OR

odds ratio

PCR

polymerase chain reaction

SNP

single nucleotide polymorphism

TdP

torsades de pointes

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