Abstract
Background: Common genetic variants in the nitric oxide synthase 1 adaptor protein gene (NOS1AP) and in the HERG potassium channel gene (KCNH2) have been associated with cardiac repolarization in middle-aged and elderly subjects.
Aim: We examined the relation between these variants and QT interval duration in a population of healthy young adults.
Methods: We measured QT interval duration and genotyped rs10494366 T>G (NOS1AP gene, n=1,842) and rs1805123 A>C (KCNH2 gene, n=1,894) in subjects aged 24–39 years.
Results: The NOS1AP variant was significantly related with heart rate-corrected QT interval duration (QTc). Additive regression model adjusting for age, sex, systolic blood pressure, body mass index, alcohol use, and smoking indicated that the G allele was associated with a 3.2 ms (95% confidence interval (CI) 1.7–4.6 ms, P<0.0001) increase in QTc interval duration for each additional copy. The KCNH2 variant was not significantly related with QTc interval duration in the study sample.
Conclusion: These findings provide evidence from a population of healthy young adults that a common variation in the NOS1AP gene influences cardiac repolarization within the normal physiological range. Further studies are warranted to investigate the effects of this variant on sudden cardiac death and ventricular arrhythmias.
Introduction
QT interval duration in the electrocardiogram is a measure of cardiac repolarization time. Prolonged repolarization is a risk factor for sudden cardiac death Citation1 and increased cardiovascular mortality in apparently healthy men and women Citation2, Citation3. The QT interval duration is a complex trait with many environmental and genetic determinants Citation4–7. Extremely long or short QT intervals are rare Mendelian disorders associated with increased risk of sudden death caused by ventricular arrhythmias Citation8.
Approximately 35%–40% of the variation in QT interval duration is heritable Citation4, Citation9, Citation10. Previous studies have revealed common genetic variants that influence QT interval duration in population samples. In a recent genome-wide association study, Arking et al. Citation11 identified a common genetic variant (rs10494366 T>G) in the NOS1AP gene that has been associated with QT interval duration in several populations Citation11–13. Laitinen et al. Citation14 reported a common amino acid-exchanging polymorphism in the KCNH2 gene (K897T, rs1805123 A>C) of the potassium channel that was associated with the QT interval duration in long QT syndrome type 1 patients. In a population sample of Finnish middle-aged adults, women with the AC/CC genotypes had longer QT interval duration than women with the AA genotype Citation15. Other population studies have also examined the relation between the KCNH2 gene variant and QT interval duration, but the results have been inconsistent Citation7, Citation16–19. The majority of the available evidence indicates that an association may exist between increased QT interval duration and genotypes containing the A allele, i.e. suggesting an opposite effect initially reported in the Finnish sample.
Key messages
In healthy young adults, the common variation in the NOS1AP gene influences cardiac repolarization within the normal physiological range.
The difference in QTc interval duration was 6.4 ms between minor homozygotes and major homozygotes—magnitude of this effect is similar to the effect of drugs that prolong cardiac repolarization and increase the risk of ventricular arrhythmias.
To gain a better understanding of the effects of these two common variants on cardiac repolarization in healthy young adults, we examined the relation of rs10494366 (NOS1AP gene) and rs1805123 (potassium channel KCNH2 gene) on QT interval duration in the Cardiovascular Risk in Young Finns Study.
Research design and methods
The Cardiovascular Risk in Young Finns Study is a multicentre study of atherosclerotic risk factors of children and young adults. The first cross-sectional study was conducted in 1980 and included 3,596 (83.2% of those invited) healthy children and adolescents, aged 3, 6, 9, 12, 15, and 18 years. Details of the study design have been presented elsewhere Citation20–22. In 2001, we re-examined 2,283 individuals, who had then reached the age of 24 to 39 years Citation23, Citation24. Of these subjects, 2,151 participated in the electrocardiogram (ECG) data collection, and 2,002 had high-quality QT interval data (of these 246 were excluded because of QT prolonging drugs, data problems, or ectopic beats, see details below). Of these subjects, 1,896 had data on NOS1AP or KCNH2 and did not use drugs that prolong the QT interval. Thus, in the present analysis, we included 1,842 subjects with data on NOS1AP gene variant (rs10494366) and 1,894 subjects with data on KCNH2 gene variant (rs1805123). Subjects taking drugs listed at www.qtdrugs.org that may alter the QT interval were excluded (n=97). These subjects had 7.4 ms (95% confidence interval (CI) 2.6–12.2 ms) longer heart rate-corrected QT (QTc) interval duration compared to others (P=0.002). The investigation conforms with the principles outlined in the Declaration of Helsinki Citation25. Written informed consent was obtained from each study participant to perform genetic studies.
Clinical characteristics
Height and weight were measured and body mass index calculated. Blood pressure was measured using a random zero sphygmomanometer. The average of three measurements was used in the statistical analysis. Smoking habits and alcohol consumption were obtained from a self-administered questionnaire. Subjects were classified as smokers if they indicated smoking on a daily basis. Details of methods have been described previously Citation24, Citation26, Citation27.
QT interval duration
In 2001, a single channel electrocardiogram (ECG) was recorded during a 3-minute period. The ECG signal was collected after the participants had remained comfortably in a supine position for at least 15 minutes for ultrasound examination Citation23, as part of the protocol to measure short-term heart rate variability indices. The three ECG leads were positioned diagonally as follows: 1) above sternum, 2) below sternum 3) above umbilicus. The resulting QRS complex corresponds to leads V1-V2. The ECG signal was analogue-to-digital converted with a sampling rate of 200 Hz. The signal was recorded, and all R-R intervals were calculated. All ECG signals were visually examined by one operator (T.A.K.). A stationary period was identified from the 3-minute recording. The mean duration of the stationary period was 174 s (SD 12 s), and the mean number of R-R intervals was 197 (SD 34). The ECG recordings that included more than two ectopic beats were excluded (n=87); recordings that included one or two ectopic beats were manually interpolated. Inclusion of subjects with ectopic beats, however, gave identical results. Data from 46 subjects were lost because of problems with data recording or saving. The ECG recordings were available for 2,018 subjects (women 56%).
The average QT interval duration from the ECG recording was computed using a commercially available program for analysis of physiological data (WinCPRS, Absolute Aliens, Turku, Finland). Waveform detection was performed using multiple time windows of 50 ms duration. The range of ECG signal within this time window was computed for each time point, and the window was moved over the whole beat. This procedure generated the waveform curve, i.e. the average ECG within the duration of the recording. The isoelectric line was determined by joining the points 300 ms before the consecutive R-peaks in the ECG. The Q-peak was defined as the minimum between the start of the isoelectric line and the R-peak. The T-wave end was defined as the crossing point of the isoelectric line and the tangent of the steepest downward slope of the T-wave. All ECGs were visually reviewed to ensure that the computer program had correctly recognized Q- and T-waves. In 115 subjects, the computer program did not recognize the correct timing of the Q- or T-waves. In these subjects, the QT interval was manually computed. In 99 subjects, it was possible to calculate the QT interval from the averaged ECG recording. In 16 subjects, the QT interval was manually computed and averaged from printed ECG strips comprising several cycles (these 16 subjects were excluded from the present analysis). Heart rate-corrected QT interval duration (QTc) was calculated using Bazett's (QTc=QT/RR0.5) Citation28 and Fridericia's formulae (QTc-Fri=QT/RR1/3) Citation29.
To investigate the reproducibility of the ECG analysis method, we re-collected ECG recordings in 45 subjects 4 months after the first measurement. The 4-month between-visit coefficient of variation was 1.9% for QTc measurements.
DNA isolation and genotyping
Venous blood was drawn into ethylenediamine tetra-acetic acid tubes and stored at −70°C. Genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit and the BioRobot M48 Workstation (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. DNA samples were genotyped by employing the 5' nuclease assay and fluorogenic allele-specific TaqMan MGB probes Citation30 using the ABI Prism 7900HT Sequence Detection System for both polymerase chain reaction (PCR) and allelic discrimination (Applied Biosystems, Foster City, CA, USA).
PCR reactions for the single-nucleotide polymorphisms rs10494366 (NOS1AP gene) and rs1805123 (KCNH2 gene) contained genomic DNA, 1×Universal PCR Master Mix, 900 nM of each primer and 200 nM of each TaqMan probe in 384-well plates in a total volume of 5 µL. PCR cycling protocol was: 95°C for 10 min followed by 45 cycles of 92°C for 15 s and 60°C for 1 min. Genotyping was done by using a commercially available assay kit from Applied Biosystems (Custom assay kit for rs1805123, call rate 99.9%, and Assay ID C_1777074_10 for rs10494366, call rate 97.3%). The DNA and master mix were pipetted to the 384-plates using a TECAN Freedom EVO-100 instrument (Tecan Group Ltd, Männedorf, Switzerland). Negative controls (water) and random duplicates were used as quality control.
The prevalence rates of NOS1AP rs10494366 genotypes C/C, C/T, and T/T were 41%, 46%, and 13%, respectively. The prevalence rates of KCNH2 rs1805123 genotypes A/A, A/C, and CC were 70%, 27%, and 3%, respectively.
Statistical methods
Data are expressed as means and standard deviations. Statistical methods included t tests, chi-square test, and linear regression. Possible gene variant×sex interactions were tested using linear regression models. As there were no significant sex interactions in the associations of gene variants with the QT interval duration, the analyses were performed by combining sexes. Statistical tests were performed with SAS version 8.1, and statistical significance was inferred at a two-tailed P-value <0.05. The chi-square test was used to compare the frequencies of the polymorphisms between the study groups and for calculation of the Hardy-Weinberg equilibrium (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Conformity of the genotype proportion to the Hardy-Weinberg equilibrium was examined in the whole population.
Results
The genotype frequencies of rs10494366 and rs1805123 polymorphisms were both in Hardy-Weinberg equilibrium (P=0.93 and P=0.61, respectively).
The characteristics of the study subjects are shown in . Men had significantly higher blood pressure, body mass index, smoking rate, and alcohol consumption compared to women. Women had higher heart rate, higher uncorrected QT and QTc interval duration compared to men (P<0.0001 for all).
Table I. Characteristics of study subjects. Values are mean±SD, or percentages.
QT interval duration and NOS1AP gene rs10494366.
The distribution of QT interval durations across the NOS1AP rs10494366 genotypes is shown in . Mean age, sex distribution (P=0.64, chi-square) and heart rate did not differ between the genotypes. The genotype was significantly related with uncorrected QT and QTc interval duration. These relations remained significant after adjusting for covariates. Additive linear regression model adjusting for age, sex, systolic blood pressure, body mass index, alcohol use, and smoking indicated that the G allele was associated with a 3.2 ms (95% CI 1.7–4.6 ms, P<0.0001) increase in QTc interval duration for each additional copy. In sex-stratified analysis, the G allele was associated with 4.5 ms (P<0.0001) and 2.0 ms (P<0.05) increase in QTc interval duration in men and women, respectively. The genotype effect on QT interval duration was similar between sexes (genotype×sex interaction term, P=0.14).
Table II. Age, heart rate, and QT intervals across NOS1AP (rs10494366) genotypes. The values are means and standard deviations.
QRS duration did not differ between NOS1AP groups (P=0.63)
QT interval duration and KCNH2 rs1805123
The distribution of QT interval durations across the KCNH2 (rs1805123) genotypes is shown in . Mean age, sex distribution (P=0.91, chi-square), heart rate, and QTc interval duration did not differ between the genotypes. There was a non-significant decreasing trend in uncorrected QT interval duration across the genotypes. Results from the unadjusted additive regression model suggested that the A allele was associated with a 1.8 ms (95% CI 0.5–4.0 ms, P=0.13) increase in QT interval duration for each additional copy. The association was further attenuated after adjusting for covariates (P=0.47). The genotype effect on QT interval duration was similar between sexes (heart rate-adjusted genotype×sex interaction term, P=0.31).
Table III. Age, heart rate, and QT intervals across KCNH2 rs1805123 genotypes.
Discussion
We were able to replicate in the Young Finns Study, a cohort of North European ancestry, that the common NOS1AP variant rs10494366 was associated with increased QT interval duration in healthy young adults. The difference between in QTc interval duration was 6.4 ms between minor homozygotes and major homozygotes. This effect was highly statistically significant and nearly identical to that previously reported by Aarnoudse et al. Citation13 in the Rotterdam Study (7.2 ms), and by Post et al. Citation12 in the old order Amish population (6.1 ms). A magnitude of this effect is approximately similar to the effect of drugs that prolong cardiac repolarization and increase the risk of ventricular arrhythmias. The US Food and Drug Administration recommend that a 5 ms prolongation of QTc in a drug trial in healthy volunteers warrants an ECG safety evaluation in later stages of drug development Citation31. The association of this variant to sudden cardiac death was assessed in the Rotterdam study in a prospective cohort of 5,374 subjects aged ≥55 years. They found a non-significant increase in risk of sudden cardiac death per additional minor allele copy, but the study may have been underpowered Citation13. These findings indicate that NOS1AP modulates repolarization reserve. This by itself may not directly increase arrhythmia susceptibility. In addition, as the mechanism by which NOS1AP variant influences QT interval is unknown, it could even be beneficial as prolongation of refractoriness can also be antiarrhythmic. Nevertheless, our results together with previous observations imply that further investigations are warranted to assess whether this genetic marker represents a risk factor for arrhythmias and sudden cardiac death at the population level.
The mechanism by which the variation in NOS1AP influences QT interval duration is unknown. NOS1AP spans over 299 kilobases of DNA on chromosome 1 (1q23.3). The gene encodes the nitric oxide synthase 1 adaptor protein, a cytosolic protein that has been found to regulate neuronal nitric oxide synthase activation possibly by regulating its ability to associate with adaptor protein and receptor complexes Citation32. Neuronal nitric oxide synthase protein expression occurs within cardiac myocytes and may play a role in the control of basal and adrenergically stimulated cardiac contractility and in the autonomic control of heart rate Citation33. Neuronal nitric oxide synthase knock-out mice have been found to have suppressed cardiac inotropic response suggesting a role for NOS1AP in cardiac function Citation34. Overexpression of neuronal nitric oxide may influence myocardial contractility by suppressing the function of L-type calcium channels Citation35. In hypertensive rats, upregulation of neuronal nitric oxide using gene transfer was associated with reduced cardiac norepinephrine release Citation36. As reviewed by Paton et al. Citation37, defective nitric oxide synthase may lead to increased contractility, prolonged calcium transients, and reduced L-type calcium current inactivation. These changes may be reflected in prolongation of the action potential duration Citation38.
Mutations in the KCNH2 gene, which encodes for the major subunit of the rapidly activating potassium channel Citation39, cause type 2 long QT syndrome. In families with long QT syndrome and identifiable genetic mutations, most occur in the potassium channel Citation40. Binding to this channel subunit is also a major determinant of drug-induced QT prolongation. In the K897T polymorphism, the adenosine nucleotide at position 2690 is changed into a cytosine, which may change the electrical charge of the protein product and thereby potentially interfere with the function of the channel. Previous observations on this polymorphism are somewhat inconclusive. Pietilä et al. Citation15 found a longer QTc interval duration to be associated with the C allele carriage in middle-aged Finnish women. Similarly, women with CC genotype in the Finnish Cardiovascular Study tended to have longer QT intervals over the duration of an exercise test, but this difference was statistically significant only at rest Citation19. On the contrary, two large German studies found longer QTc to be associated with the A allele Citation7, Citation16. Gouas et al. Citation17 compared two groups of 200 subjects with the shortest and longest QTc from a population-based sample in France and found that A alleles were significantly more frequent in the group with the longest QTc interval. Most recently, Newton-Cheh et al. Citation18 found a marginally (1.6 ms) longer QTc interval duration per additional A allele. In the present study, we observed a non-significant trend suggesting that the A allele was associated with a 1.8 ms (95% CI 0.50–4.0, P=0.13) increase in the uncorrected QT interval duration for each additional copy. Thus, the majority of the available evidence suggests that an association may exist between increased QT interval duration and genotypes containing the A allele.
Women have longer QT interval duration than men because in males the QT interval duration shortens during puberty Citation41. Women are more likely than men to develop drug-induced arrhythmias Citation42, and women with long QT syndrome are at higher risk than men of cardiac arrest and sudden cardiac death Citation43, Citation44. Because of these biological differences, the possible sex specific genetic effects may be of interest. In this regard, the data on the NOS1AP gene are inconclusive. Arking et al. Citation11 found a stronger association in women than in men in a population of white American adults, but not in a population of German adults. Post et al. Citation12 found no significant gene-by-sex interaction in a population of Amish adults. Similarly, Aarnoudse et al. Citation13 reported no difference in the gene effect between men and women in the Rotterdam study. In the present study, we found no evidence that the NOS1AP rs10494366 variant would have a greater influence on QT interval duration in women than in men.
The method of measurement of the QT interval duration is a key issue for any interpretations of repolarization abnormalities. One limitation of our study is the relatively crude measure of QT interval duration, as it was measured from single-channel ECG recordings. Measuring QT interval across all standard 12 leads may have provided more accurate estimates of the duration. We used Bazett's formula Citation28 to correct QT interval duration for heart rate, so that the results would be comparable with previous studies. The adequacy of this formula has been questioned because it overcorrects the measured QT interval duration at fast heart rate and undercorrects it at low heart rate Citation45. A large part of the QT interval heritability is shared with genes for heart rate. Therefore, the use of correction formulas in gene finding studies may produce erroneous results Citation46. In the present study, however, the average heart rate did not differ between the studied genetic variants, and similar results were seen using the Fridericia's correction formula and the uncorrected QT interval duration adjusting for heart rate in multivariable models. Structural cardiac diseases, especially left ventricular hypertrophy, may affect QT interval duration. Lack of data on cardiac size is a limitation of our study. It is also acknowledged that our study sample may be too small to show an association between KCNH2 K897T and QT interval. We found no significant sex interactions in the associations of gene variants with the QT interval duration, therefore the analyses were performed by combining sexes. However, the sex interaction analysis might have limited interpretability in terms of power even with these numbers. Finally, we measured only one single-nucleotide polymorphism in the NOS1AP gene and one in the KCNH2 gene; other variants in these genes may also be important Citation12, Citation13, Citation18.
In summary, we were able to replicate in a well phenotyped sample of young healthy adults that variation in the NOS1AP gene is strongly associated with the QTc interval duration. This common gene variation is responsible for QT prolongation to an extent comparable with some QT-prolonging drugs that cause concern due to the risk of ventricular arrhythmias. Further studies are warranted to investigate the biochemical pathways and mechanisms how this genetic variation influences cardiac repolarization, and to assess the effects on sudden cardiac death and ventricular arrhythmias.
Acknowledgements
This study has been supported by Academy of Finland (grants no. 77841 and 210283), the Social Insurance Institution of Finland, the Turku University Foundation, the Juho Vainio Foundation, the Emil Aaltonen Foundation (T.L.), the Finnish Foundation of Cardiovascular Research, the Finnish Cultural Foundation, Federal funds to Turku University Hospital, the Maud Kuistila Foundation, and Tampere University Hospital Medical Research Fund.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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