A Heterozygous Missense hERG Mutation Associated

Physiol Biochem 2018;51:1301-1312 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000495549 DOI: 10.1159/000495549 © 2018 The Author(s) online:27 27November November2018 2018 www.karger.com/cpb Published online: Published by S. Karger AG, Basel and Biochemistry Published www.karger.com/cpb Cheng et al.: A Heterozygous Missense hERG Mutation Associated with Early Accepted: 19 November 2018

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Original Paper

A Heterozygous Missense hERG Mutation Associated with Early Repolarization Syndrome Yun-Jiu Cheng Hao Yao Li-Juan Liu Su-Hua Wu

Cheng-Cheng Ji

Xu-Miao Chen

Jun Fan

Department of Cardiology, the First Affiliated Hospital, Sun Yat-Sen University, Key Laboratory on Assisted Circulation, NHC, Guangzhou, China

Key Words Early repolarization syndrome • Early repolarization pattern • KCNH2 • Gene mutation • Sudden cardiac death Abstract Background/Aims: Early repolarization syndrome (ERS) has been recently recognized as early repolarization pattern with idiopathic ventricular fibrillation. However, the genetic background of ERS has not been fully understood. Methods: A Chinese family with sudden cardiac death associated with ERS was investigated. Direct sequencing of ERS susceptibility genes was performed on the proband and family members. Whole-cell patch-clamp methods were used to characterize the mutant channel expressed in HEK 293 cells. Results: One missense mutation (p. K801T) was found in the hERG (KCNH2 gene) by the direct sequencing of candidate genes. Whole cell voltage clamp studies of the K801T mutation in HEK 293 cells demonstrated a 1.5fold increase in maximum steady state current (37.2±7.3 vs 20.3±4.4 pA/pF) that occurred at a 20 mV more positive potential compared to the wild type channels. The voltage dependence of inactivation was significantly shifted in the positive voltage direction (WT -59.5±1.4 vs K801T -44.3±1.2 mV). Kinetic analysis revealed slower inactivation rates of K801T, but faster rates of activation and deactivation. The hERG channel blockers tested inhibited K801T-hERG channel in concentration response, and the potencies of these drugs can be rank-ordered as follows: quinidine> disopyramide> sotalol> flecainide. Conclusion: Our study indicated that the K801T mutation caused the gain of function of hERG channels that may account for the clinical phenotype of ERS. Quinidine and disopyramide could improve the function of K801ThERG mutant channel, and may be therapeutic options for patients with the K801T hERG mutation. © 2018 The Author(s) Published by S. Karger AG, Basel

Y.-J. Cheng, H. Yao and C.-C. Ji contributed equally to this work. Su-Hua Wu and Li-Juan Liu

Department of Cardiology, the First Affiliated Hospital, Sun Yat-Sen University and Key Laboratory on Assisted Circulation, NHC, No. 58 Zhongshan Rd II, Guangzhou 510080 (China) E-Mail [email protected]; [email protected]

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Physiol Biochem 2018;51:1301-1312 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000495549 and Biochemistry Published online: 27 November 2018 www.karger.com/cpb Cheng et al.: A Heterozygous Missense hERG Mutation Associated with Early Repolarization Syndrome

Introduction

Early repolarization pattern (ERP) on the electrocardiogram (ECG) is defined as J point elevation ≥0.1 mV or QRS notching or slurring in at least 2 contiguous inferior and/or lateral leads [1]. It is prevalent in 2.3% to 29.3% of the population depending on race, age and gender category [2, 3]. Several recent reports and our previous studies indicated that ERP was associated with increased risk for ventricular fibrillation (VF) and sudden cardiac death (SCD) in the general population and in patients with structural heart disease [2, 4-6]. The 2015 ESC Guidelines for the management of patients with ventricular arrhythmias defined early repolarization syndrome (ERS) as the presence of ERP with resuscitation from a documented episode of unexplained VF or polymorphic ventricular tachycardia (VT) [7]. The autosomal dominant inheritance nature of the ERP has been suggested by case– control studies as well as by studies in the general population, but the familial inheritance of malignant ERS has not been clearly demonstrated [4, 8]. So far, rare genetic variants in genes governing cardiac repolarization, including SCN5A, KCNJ8, and L-type calcium channel subunits, have been shown to be causally associated with ERS [9]. Human hERG (KCNH2 gene) encodes α-subunit of the rapid component of the delayed rectifier K+ channel, which plays an important role in regulating the repolarization of the cardiac action potential [10]. In the present study, a Chinese family with the clinical phenotype of ERS was found to have a novel mutation in the hERG gene, K801T, and we report herein that this newly discovered mutation in hERG causes a substantial gain-of-function. We also tested the effects of antiarrhythmic drugs which could prolong cardiac repolarization on the mutant K801T hERG channels expressed in HEK 293 cells. Materials and Methods

Clinical investigation The proband with a strong family history of sudden cardiac death was referred to us for further evaluation. A complete battery of studies including medical history, physical examination, electrolytes, 12lead ECG, 24-hour Holter monitoring, echocardiography, coronary angiography and genetic testing were done. The study conformed to the principles outlined in the Declaration of Helsinki. This study was approved by the institutional review board, and the patients provided written informed consent for participation.

Genetic analysis Genomic DNA was extracted from peripheral lymphocytes using a TIANamp Blood DNA isolation kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. All exons of the hERG, KCNE1, KCNE2, SCA5A, KCNJ8, KCNA4, KCND2, KCND3, CACNA1C, CACNB2 and CACNA2D1 genes were amplified by polymerase chain reaction (PCR) and were analyzed by direct sequencing. Sequencing results were compared to 150 healthy subjects of the same ethnic background with normal ECGs to determine the distinction between mutation and polymorphism.

Site-directed mutagenesis and hERG constructs Nucleotide change identified in ERS proband was introduced into hERG cDNA cloned in pGFPires as described previously [11]. Human embryonic kidney (HEK) 293 cells were transfected with the WT- or K801T-HERG constructs using an Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The day after transfection, cells were released with trypsin using standard methods and distributed into six well plates, and those cells exhibiting green fluorescence 12-20 h posttransfection were selected for subsequent electrophysiological study.

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Electrophysiological analysis in transfected HEK 293 cells Functional characterization of WT- and mutant hERG-channels was performed by voltage clamp techniques using transfected HEK 293 cells. Patch clamp recordings were made using low-resistance electrodes (C) was found in the hERG gene of the proband (Fig. 3A), resulting in an amino acid change from lysine to threonine at position 801 (p.K801T) in the hERG potassium channel. The schematic topology in Fig. 3C depicts location of this mutation which is located in C-terminus of hERG protein, and is therefore likely to influence function. The same mutation was present in his younger sister and two cousins. However, this mutation was not observed in the proband’s father, indicating the K801T mutation of the proband could have been inherited from his decreased mother. The K801T substitution 1 ethnic background, and has not was not present in any of 150 control subjects of the same been reported according to the NHLBI Exome Sequencing Project, Exome Variant Server (http://evs.gs.washington.edu/EVS/), suggesting that the mutation is associated with the phenotype.

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Cellular electrophysiology To determine the effects of the K801T mutation on hERG channel function and its contribution to the clinical phenotype, we expressed WT- and K801T-hERG constructs in HEK 293 cells for electrophysiological analysis. Whole-cell recordings showed that both WTand K801T-hERG currents elicited by sequential depolarizing pulses exhibit slow initial rates of activation with peak current increasing with voltage to a maximum. With further increases in voltage, the current decreases because of the rapid onset of inactivation (rectification). WT and K801T recordings also displayed the typical large tail currents generated by inactivated channels rapidly reopening on repolarization. Current from the cells expressing the two hERG constructs was measured at the end of the first voltage clamp step, normalized by cell capacitance and averaged. A comparison of the steady current density-voltage relations between the WT- and K801T-hERG channels is shown in Fig 4B. The peak current occurred at about +10 mV for WT-hERG and at about +30 mV for K801ThERG, and was approximately 1.5-fold greater for K801T- (37.2±7.3 pA/pF, n=25) compared to WT-hERG (20.3±4.4 pA/pF, n=18, P