European Heart Journal Advance Access originally published online on January 16, 2007
European Heart Journal 2007 28(3):305-309; doi:10.1093/eurheartj/ehl460
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The common non-synonymous variant G38S of the KCNE1-(minK)-gene is not associated to QT interval in Central European Caucasians: results from the KORA study
1 Institute of Human Genetics (IHG), GSF National Research Centre of Environment and Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany
2 Institute of Human Genetics (IHG), Technical University of Munich, Klinikum rechts der Isar, Trogerstr. 32, D-81675 Munich, Germany
3 Department of Medicine I, Ludwigs-Maximilians University Munich, Klinikum Grosshadern, Marchioninistr 15, D-81377 Munich, Germany
4 Institute of Medical Informatics (IMEI), GSF National Research Centre of Environment and Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany
5 Institute of Epidemiology (EPI), GSF National Research Centre of Environment and Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany
Received 12 October 2006; revised 19 November 2006; accepted 14 December 2006; online publish-ahead-of-print 16 January 2007.
* Corresponding author. Tel: +49 89 3187 3545; fax: +49 89 3187 3474. E-mail address: arne.pfeufer{at}web.de
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Abstract |
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Aims The QT interval in the general population is a complex trait with 30–50% heritability. QT prolongation is associated with an increased risk of sudden death. A recent family-based study found an association between QT interval and the common non-synonymous Glycin 38 Serine variant (G38S, rs1805127) of the KCNE1 gene coding for the minK-potassium channel subunit. We intended to replicate this finding in a large population sample of central European Caucasian ancestry as part of our ongoing search for genetic variants predisposing to arrhythmias.
Methods and results We studied 3966 unrelated individuals from the KORA S4 population-based study without atrial fibrillation, pacemaker implant, or pregnancy. Individuals were genotyped by MALDI-TOF mass spectrometry. We did not detect any significant association between the genotypes of the G38S variant and the QT interval in the entire population or in any gender.
Conclusion Unlike the common Lysine 897 Threonine variant of KCNH2 (K897T, rs1805123) the G38S variant of KCNE1 does not appear to have a strong modifying effect on QT interval. However, we cannot rule out an effect of G38S on QT in other ethnic groups, under exercise or medications or on the risk for arrhythmias and sudden death.
Key Words: Cardiac repolarization • Genetic association study • Single nucleotide polymorphism (SNP) • Genetic epidemiology
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Introduction |
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The analysis of genetic variants modifying cardiac electrophysiological traits has met increased attention in recent years.1 Beyond cellular electrophysiology of ion channels and other candidate proteins2 and molecular cloning of disease genes in families affected by mendelian arrhythmogenic diseases,3 it is believed to provide a third and independent route to the identification of genes and gene products involved in cardiac electrophysiology.4 One of the prerequisites of this approach is the use of population samples that are large, free of underlying population stratification, have been carefully phenotyped, and have biosamples available. In addition, these studies are motivated to enable tests that can identify individuals predisposed to arrhythmias useful in primary and secondary prevention.
One trait finding particular attention of researchers is the QT interval. This is mostly because of its relatively high heritability,5,6 accurate measurability,7 and its associated predisposition to sudden cardiac death.8 Several previous association studies have analysed whether non-synonymous SNPs in cardiac ion channel candidate genes modify the QT interval. One of these SNPs is the Lysine 897 Threonine variant of the KCNH2 gene (K897T, rs1805123).9 The rarer T897 allele was reproducibly found to be associated with a shortened QT interval following an additive model of allelic effects.4
A recent study of 441 men and women sampled within families reported association of another common non-synonymous SNP in another cardiac ion channel gene, the Glycine 38 Serine variant of the KCNE1 gene (G38S, rs1805127).10 The KCNE1 gene encodes the MinK protein, which forms the beta-subunit of the cardiac IKs channel. The allele frequency of the minor S38 allele has been reported to be between 16.4 and 48.5% in different ethnic groups.11 In the study, however, G38S had an exceptionally low minor allele frequency of 3.3%. It was found to be associated to QT interval only in men, in whom it accounted for 2.2% of QT variance in a multivariate linear regression model (P < 10e–4). Male probands with heterozygous GS38 genotype had on an average 21.7 ms longer QT intervals compared with GG38 homozygotes, equaling to a difference of almost one standard deviation (SD) (
= 23.7 ms). In our study, we intended to replicate the previous result in 3966 probands from the KORA S4 survey, a large population-based sample of Central European Caucasian origin.
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Individuals
Between 1999 and 2001, we conducted an epidemiological survey of the general population living in or near the city of Augsburg, Southern Germany (KORA S4). This was the fourth in a series of population-based surveys originating from our participation in the WHO MONICA project. The study population consisted of unrelated residents of German nationality born between 1 July 1925 and 30 June 1975 identified through the registration office. A sample of 6640 subjects was drawn with 10 strata of equal size according to gender and age. Following a pilot study of 100 individuals, 4261 individuals (66.8%) agreed to participate in the survey, who were ethnic Germans with very few exceptions (>99.5%). From 4115 probands, a positive consent, a DNA sample as well as an electrocardiogram (ECG) recording were available. After the application of exclusion criteria, atrial fibrillation, pacemaker or defibrillator implant, or ongoing pregnancy, 3966 individuals were used for association analysis. A detailed description of probands and phenotypic measurements is given in Table 1. The same study population has been previously used to screen for associations in ion channel candidate genes4 and in a genome-wide approach.12 Blood samples were drawn after informed consent had been obtained. All studies involving humans were performed according to the declarations of Helsinki and Somerset West and were approved by the local medical Ethics Committee.
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ECG recording
In the S4 survey, 12-lead resting ECGs were recorded using a digital recording system (Bioset 9000, Hörmann Medizinelektronik, Germany). QT intervals were determined with the Hannover ECG analysis software (HES-Version 3.22).13 Computerized analysis of an averaged cycle was performed from all cycles of the 10 s recording after exclusion of ectopic beats as previously described. The QT interval determined by this algorithm represents the earliest beginning of depolarization until the latest deflection of repolarization between any two leads. In an international validation study, the HES-software was among the best performing digital ECG systems.14 Reproducibility of HES QT measurements over short- and long-term time intervals has been investigated.7
QT interval correction
For the purpose of this replication study, we analysed the raw QT interval as well as the corrected QT interval according to Bazett's formula,4 which corrects QT only for heart rate in a nonlinear fashion. In addition, we used a linear correction formula for QT as has been suggested from Framingham Heart Study data15 with the correction parameters derived from a multivariate linear regression model including the covariates heart rate (RR interval), sex, and age within KORA S4 as previously described.4 Correction factors were determined separately for each sex. The QT interval corrected for rate-, age-, and sex was determined for men:
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DNA extraction and genotyping
DNA was extracted from EDTA anticoagulated blood using a salting out procedure.16 The G38S variant of the KCNE1 gene was determined using PCR, primer extension, and MALDI-TOF mass spectrometry in a 384-well format (Sequenom, San Diego, USA) as previously described.4 Hardy–Weinberg equilibrium (HWE) P-values were calculated using the STATA statistical software package.17
Genotype phenotype association analysis
SNPs were tested for association to QT, QTc-Bazett, and QTc-RAS as the dependent variables by applying two-tailed one-degree-of-freedom linear regression test (1df) and two-tailed two-degree-of-freedom ANOVA analysis (2df). The 1df test has a relatively higher power to detect weak effects, whereas the 2df test accounts for dominance and recessivity by allowing the trait increase of each genotypic change to take an individual value. To determine gender specific differences of SNP-phenotype associations, we performed sex-specific regression analysis in the total sample. Sample sizes of males (n = 1959) and females (n = 2007) were similar and therefore comparable for effect size. Although this study was intended to replicate a previous significant finding, in light of the grossly different allele frequency we did not use one-tailed but two-tailed statistics. All reported significance levels have not been adjusted for multiple testing.
We designed our study using n = 3966 individuals to be able to detect the effect of the GS38 heterozygous genotype described to prolong QT interval by about one standard deviation at the published Caucasian allele frequency of 30% with near certainty. In fact, our study had >99% power in the entire sample and >98% in men to detect with 
< 0.05, an effect prolonging QT by only 0.2 standard deviations at the given allele frequencies (Table 2).
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Results |
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In the total sample of 3966 individuals, QT interval had a mean value of 407.9 ms and a standard deviation of ±28.1 ms, QT corrected according to Bazett's formula had a mean value of 423.0 ms and a standard deviation of ±21.6 ms, and linearly corrected QTc-RAS had a mean value of 417.6 ms and a standard deviation of ±17.2 ms when corrected to a 60-year-old man with a heart rate of 60 b.p.m. (Table 1).
The G38S variant of the KCNE1 coding sequence was genotyped with a call rate of 98.7%. Genotypes showed no significant deviation from HWE (P = 0.08). The minor allele frequency (MAF = 36%) was well in common with those in other Caucasian samples (Table 3).
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Genotyping results revealed no significant association between G38S and the uncorrected or corrected QT interval. For the uncorrected QT interval, we found all P-values to be >0.16. For QT corrected according to Bazett's formula, P-values were >0.64 and for QTc-RAS >0.16 (Table 3). The associations between QT and G38S stratified by gender and age were also not significant (all P > 0.05). Applying the same model as in the initial publication, the QTc-Bazett difference between GG38 homozygous and GS38 heterozygous men was –0.7 ms (P = 0.53).
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Discussion |
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We could not confirm the previously published strong effect of the non-synonymous G38S variant of the KCNE1 gene in men or any other effect of this variant on QT interval. Our non-replication is in line with a previous investigation that also could not find any evidence of association between G38S and QT interval length18 but was less powered to do so.
Power simulations showed that we would have been adequately powered to detect an effect if it existed in the range of 1 SD as in the initial publication as well as down to the range of 0.2 SD, which equals the magnitude of the effect of K897T in KCNH2.
It appears unlikely that the non-replication can be explained by a lack of precision of the QT interval measurement or another unsuitability of the samples as we have successfully replicated the association of the KCNH2 K897T SNP to QT interval in the same sample in a previous publication.4 In addition, a novel QTL for QT interval in the promoter of the NOS1AP (CAPON) gene could be identified in this sample and reproducibly confirmed in others.12 The population-representative recruitment of individuals from one geographic area with limited recent immigration increases the homogeneity of the sample and thus increases the power to detect true positives and likewise reduces the probability of false positives due to population stratification.
Non-synonymous variants are generally considered to be likely causal variants themselves and not just markers associated by linkage disequilibrium to causal variants in their vicinity. Therefore, also in individuals from other ethnic groups we would expect no association between this variant and the QT interval. However, we cannot rule out the possibility that G38S may be a causal variant only on certain genetic backgrounds or that it may be in linkage disequilibrium to neighbouring causal variants only in some ethnic groups. Independent replication studies of similar size in individuals of such groups will be the only way to resolve the issue whether this negative association result is dependent on ethnicity or is universally valid.
Similarly, the non-replication does not rule out the possibility that the two alleles of G38S may still exert subtle differences on the repolarization process. It makes it likely that such an effect does not exist on repolarization at rest, but an effect limited to exercise, intake of medications, or other conditions may well exist. We likewise cannot rule out a modification of the risk of arrhythmias or sudden cardiac death by G38S via more complex repolarization- or non-repolarization-driven effects.
Effects of other variants within the KCNE1 gene may also be present. Two other QT-modifying variants have been previously described, the Intron 2 variant IVS2–128 G>A19 and the promoter variant rs727957.4 Both of them await independent replication.
This finding necessitates the use of large sample sizes for future studies of QT interval and sudden cardiac death in order to obtain statistically significant and reproducible associations as—despite the relatively high heritability of the QT interval—the contribution of individual variants is recognized to be rather low.
The QT interval is a valuable endophenotype to investigate the predisposition to complex arrhythmias but can nevertheless replace disease phenotypes such as VT/VF or sudden cardiac death in future investigations. We are now starting to anticipate the substantial size of study samples carrying these phenotypes that will have to be recruited in future to obtain reliable and reproducible genetic associations for complex arrhythmias.
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This work was funded by the German Federal Ministry of Education and Research (BMBF) in the context of the German National Genome Research Network (NGFN), the Competence network on atrial fibrillation (AFNet), and the Bioinformatics for the Functional Analysis of Mammalian Genomes programme (BFAM) by grants to S.K. (01GS0109, 01GS0499), H.-E.W. and A.P. (01GI0204), and to T.M. (01GR0103). The KORA platform is funded by the BMBF and by the State of Bavaria. This work was performed at the Technical University of Munich, Klinikum rechts der Isar, Trogerstr. 32, D-81675 Munich, Germany; Ludwigs-Maximilians University Munich, Klinikum Grosshadern, Marchioninistr 15, D-81377 Munich, Germany; GSF National Research Center, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany.
Conflict of interest: none declared.
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Footnotes |
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These authors contributed equally to the work. 
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