Association Study Of KCNE Gene Family Single Nucleotide Polymorphisms With Atrial Fibrillation And Function Study Of KCNE4(E145D) On Potassium Channels

BackgroudAtrial fibrillation (AF) is a common cardiac arrhythmia with heritable components. The prevalence is about 0.77% in Chinese population, and increases to～7.5% in those＞80 years of age. ²AF may cause strokes and heart failures, and is a source of considerable morbidity and mortality.^3,4According to a large survey performed in the arrhythmia clinic at Mayo Clinic, about 36% patients with AF had no evident pathogeny, and 5% had a positive family history.⁸ It points to the fact that a subset of AF has an important genetic background. Factors that abbreviate atrial repolarization are recognized to promote AF by shortening the wavelength for reentry and favouring the occurrence of multiple wavelet reentry. It is supported by reports of gain-of-function mutations in genes encoding cardiac I_Ks (KCNQ1/KCNE1/KCNE2) and I_K1 (KCNJ2) channels predicted to decrease action potential duration (APD) and promote atrial reentry.^11,14,16,52Delayed rectifier K⁺ current (I_K) is an important determinant of cardiac repolarization. I_K is composed of two distinctive components: slow delayed rectifier potassium current (I_Ks) and rapid delayed rectifier potassium current (I_Kr). KCNQ1 and HERG encodeαsubunits of I_Ks and I_Kr channel respectively in human heart. The KCNE gene family encodes one kind ofβsubunits that associate with pore-formingαsubunits to form a complex with unique characteristics. There are five types of KCNE genes(KCNE1～KCNE5). In Chinese population, Chert et al¹¹ found that the S 140G mutation of KCNQ1 gene on chromosome 11p15.5 might account for hereditary AF. Lai et al.¹⁴ reported that KCNE1 (MinK) 38G allele was possibly linked to patients with AF phenotype. KCNE2(R27C), KCNE3(R53H) and KCNE5(T97C) substitution was also found in AF family. ^16,18,24Single mucleotide polymorphisms (SNPs) represent the natural variability in the human genome and determine the genetic predisposition to disease and the drug response. KCNE1 and KCNE4 are the most abundant KCNE transcripts in the heart. Lai et al.¹⁴ reported that KCNE1 (MinK) gene 38G allele was linked to patients with atrial fibrillation phenotype. In contrast to literature published by Lai et al., Zeng et al.^27,28 failed in detecting any connection between this SNP and AF phenotype in their study population. The possible reasons account for this difference may include the different nature of the study populations. Zeng et al.^27,28also amplified and sequenced the whole length plus 1000 bases from the upper stream of KCNE4 gene and found a non-synonymous KCNE4 SNP: G1057T (E145D). In a case-control study, the X₂ test was not significant but logistical regression analysis revealed a difference in the distribution of KCNE4 (E145D) in atrial fibrillation group and control group (OR=1.66, P=0.044). The functional consequences of this polymorphism remained unclear. There are few studies on the SNPs of KCNE2, KCNE3 and KCNE5 in Asian.^{30, 31}Amiodarone is an effective drug for the treatment of AF and has been referred to as a classâ…¢antiarrhythmic agent, but its pharmacological action is in fact very complex. ^34,35 In the present study, we examined the short-term effects of amiodarone on HERG and KCNQ1 channels expressed heterologously in cultured cells. Moreover, we made a further study of the effects of SNP on the acute electropharmacology roles of amiodarone.ObjectivePart 1.Because of the difference findings among the association studies of KCNE1 (G38S) and AF, we made a further test on their connection in a larger Chinese population. Moreover, the association of KCNE4 (E145D) and AF was also been investigated in our larger representative Chinese population. We also made a SNP discovering of KCNE2, KCNE3 and KCNE5 gene.Part 2.Because the functional effect of the SNP KCNE4 (E145D) is unknown, We constructed KCNE4 (145E/D) expression plamids and performed the whole-cell patch-clamp recording on cells co-transfected with KCNQ1 or HERG to identify potential functional consequences. Moreover, we investigated the acute effects of amiodarone on KCNQ1 or HERG channel co-transfected with SNP of KCNE family and analyzed the roles of SNP in the acute electrophysiology effects of amiodarone.MethodDNA was extracted from white blood cells with phenol-chloroform method. To determine the genotype, polymerase chain reaction (PCR) was conducted for each DNA samples before restriction fragment length polymorphism (RFLP) analysis was performed. Wide-type plasmids were amplified and site-directed mutagenesis was created by overlap extension PCR. Transient transfections were performed with Lipofectamine reagents and cells were studied with the use of whole-cell patch-clamp recording 24-48h after transfection. Amiodarone hydrochloride was diluted to the desired concentration with the bath solution, and ion currents were recorded before and after the acute application of amiodarone. Data were collected and analyzed with Clampfit 10.0 software.ResultsPart 1. 320 patients with atrial fibrillation and 404 controls were enrolled. AF group and control group were well matched in frequency of hypertension, coronary arterial disease, diabetes mellitus and age, sex et al. There was no significant association between KCNE1(G38S) and AF, but the X² test revealed a significant difference in the distribution of KCNE4(E145D) in AF and control group, which further proved the association of KCNE4(E145D) and AF. It is regretted that we did not find any SNP of KCNE2, KCNE3 and KCNE5 gene in our study population.Part 2. The presence of KCNE4 resulted in a dramatic decrease of the KCNQ1 current and produced almost undetectable currents between -80mV to +60mV. For CHO-K1 cells expressing only KCNQ1 channels, the average current density recorded at +60mV potential was 24±2.9pA/pF. In KCNQ1 +KCNE4 expressing cells, the current density was 7.3±1.1pA/pF. The co-transfection of KCNQ1 with KCNE4(145D) increased the current amplitude at +60mV potential by nearly two fold as compared to KCNQ1 alone. KCNE4(145D) lost the inhibitory property and increased the KCNQ1 current density. KCNE4(145E/D) had no effect on HERG channels. There was no difference on the plasma membrane fluorescence pattern of cells transfected with KCNQ1 plus KCNE4(145D) or plus KCNE4(145E). After acute application of 10μM amiodarone for 5min, the KCNQ1 current was inhibited slightly with the current density reduction of 7.8±1.2%, and the HERG current was significantly blocked with the current density reduction of 35.8±4.5%. However, the SNP KCNE4(145E/D) had no effect on the short-term electrophysiology properties of amiodarone.Conclusion1. In a case-control study of a larger sample, we failed to show that the KCNEl(G38S) was associated with AF, but we made a further identification on the association between KCNE4(E145D) polymorphism and AF.2. We did not fine any SNP sites of KCNE2, KCNE3 and KCNE5 in our sample.3. KCNE4 dramatically decreased KCNQ1 current density at physiologically relevant potentials and showed an inhibitory property on KCNQ1 channel. However, KCNE4(145D) polymorphism lost the inhibitory property and increased the KCNQ1 current density. Moreover, KCNE4(145D) resulted in the kinetic change of KCNQ1 channel. Subcellular localization study showed the same plasma membrane fluorescence pattern of KCNQ 1 when co-expressed with KCNE4(145E) or KCNE4(145D). It implied KCNE4(145D) polymorphism had no effect on the protein trafficking and localization of KCNQ1.4. Neither of the KCNE4 and the SNP KCNE4(E145D) had effects on the ??HERG current.5. In the experiment of acute application of amiodarone, KCNE4(145E/D) had no effect on the short-term electrophysiology properties of amiodarone, which implied the SNP did not affect the individual response to amiodarone.6. It is necessary to make a further study on the effects of this KCNE4(E145D) polymorphism on action potential and atrial electrophysiological property in the animal model.