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P.D1690N Sodium Voltage-Gated Channel Α Subunit 5 Mutation Reduced Sodium Current Density and Is Associated with Brugada Syndrome

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P.D1690N Sodium Voltage-Gated Channel Α Subunit 5 Mutation Reduced Sodium Current Density and Is Associated with Brugada Syndrome 5216 MOLECULAR MEDICINE REPORTS 13: 5216-5222, 2016 p.D1690N sodium voltage-gated channel α subunit 5 mutation reduced sodium current density and is associated with Brugada syndrome ZHIPENG ZENG1,2*, QIANG XIE2*, YUAN HUANG3, YUANYUAN ZHAO3, WEIHUA LI2 and ZHENGRONG HUANG2 1Laboratory of Cardiovascular Immunology, Key Laboratory of Biological Targeted Therapy of The Ministry of Education, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430000; 2Department of Cardiology, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003; 3Key Laboratory of Molecular Biophysics of The Ministry of Education, Cardio-X Institute, College of Life Science and Technology and Center of Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430000, P.R. China Received May 14, 2015; Accepted March 7, 2016 DOI: 10.3892/mmr.2016.5162 Abstract. Brugada syndrome (BrS) is an inherited primary transiently over-expressed in HEK293 cells and analyzed via arrhythmia disorder, leading to sudden cardiac death due to the whole-cell patch clamp technique. The p.D1690N mutation ventricular tachyarrhythmia, but does not exhibit clinical significantly reduced the peak sodium current density to 23% cardiac abnormalities. The sodium voltage-gated channel α of WT (at -20 mV; P<0.01), shifted steady-state activation by subunit 5 (SCN5A) gene, which encodes the α subunit of 7 mV to increasingly positive potentials (P<0.01). Furthermore, the cardiac sodium channel, Nav1.5, is the most common prolonging of the recovery from inactivation was observed in pathogenic gene, although ≥22 BrS-susceptibility genes have the p.D1690N mutant. No significant change was identified in previously been identified. In the present study, a novel genetic steady-state inactivation. Thus, the mutant-induced changes variant (p.D1690N) localized in the S5-S6 linker of domain IV contributed to the loss of function of Nav1.5 channels, which of the Nav1.5 channels was identified in a Chinese Han indicates that the p.D1690N variant may have a pathogenic family. Wild-type (WT) and p.D1690N Nav1.5 channels were role in BrS. Introduction Brugada syndrome (BrS) is a life-threatening, inherited, Correspondence to: Professor Zhengrong Huang or Professor primary arrhythmia disorder characterized by ST-segment Weihua Li, Department of Cardiology, The First Affiliated Hospital elevation in the right precordial leads (V1-V3) of the of Xiamen University, 55 Zhenhai Road, Xiamen, Fujian 361003, electrocardiogram (ECG), which does not exhibit any clinical P. R. Ch i na cardiac abnormality (1,2). BrS patients may experience E-mail: [email protected] syncope and sudden cardiac death (SCD) from episodes of E-mail: [email protected] polymorphic ventricular tachycardia (PVT) and ventricular *Contributed equally fibrillation (VF), particularly in healthy young males (3). BrS accounts for 4% of all SCD and ≤20% of sudden deaths Abbreviations: INa, depolarizing inward sodium current; ICa, in patients without obvious structural heart disease (3). BrS depolarizing inward calcium current; IKs, repolarizing outward is an inherited disease that shows an autosomal dominant slow rectifying potassium current; Ito, transient outward potassium pattern with incomplete penetrance and an incidence ranging current; IKr, repolarizing outward rapid rectifying potassium current; from ~5 per 10,000 in Western countries to 12 per 10,000 in IKATP, ATP-sensitive inward rectifying potassium current; If, funny Southeast Asia (3-5). current; ChIP, channel-interacting protein; BrS, Brugada syndrome; To date, although 22 susceptibility genes have been ECG, electrocardiogram; SCD, sudden cardiac death; PVT, identified in BrS, SCN5A, which encodes the α subunit of polymorphic ventricular tachycardia; VF, ventricular fibrillation; the major cardiac sodium channel (Nav1.5), is the most RBBB, right bundle branch block; ER, endoplasmic reticulum common pathogenic gene and is responsible for 11-28% of BrS Key words: Brugada syndrome, sodium voltage-gated channel α patients (3,6-8). However, mutations in other genes have rarely subunit 5, sudden cardiac death, genetics, patch-clamp technique, been observed in BrS patients and account for the minority channelopathies (<25%) of BrS genotype-positive cases (8,9). Furthermore, >370 mutations in the SCN5A gene have been associated with BrS using a web database, The Gene Connection For The Heart ZENG et al: p.D1690N SCN5A MUTATION REDUCES Na CURRENT DENSITY AND IS ASSOCIATED WITH BrS 5217 (http://triad.fsm.it/cardmoc/). Many of these mutations, which Affiliated Hospital of Xiamen University. All subjects have been characterized in cell lines expressing BrS mutant provided written informed consent following counseling. channels revealed a loss-of-function effect on the sodium Genomic DNA was isolated from leukocyte nuclei using current by certain mechanisms, such as reduced current density a TIANamp Blood DNA kit (Tiangen Biotech Co., Ltd., or disrupted biophysical properties (10-12). Thus, the genetic Beijing, China), and the cardiac sodium channel gene, SCN5A etiology of BrS remains unclear. Identifying novel suscepti- (transcript, NM_198056.2) was directly sequenced following bility genes will provide clinical benefit for early diagnosis, polymerase chain reaction (PCR) amplification, with the use risk stratification and personalized treatment of BrS. of an ABI PRISM 3730xl DNA sequencer (Thermo Fisher The present study investigated a Chinese Han patient Scientific, Inc., Waltham, MA, USA) performed by Tsingke presenting with BrS carrying a novel heterozygous mutation, Biological Technology Co., Ltd. (Beijing, China), as previously p.D1690N [found in the S5-S6 linker of domain IV (DIV) described (12). All DNA-identified variants were compared of the Nav1.5 channel] in the SCN5A gene. In addition, the with a control group of 150 healthy and unrelated Chinese functional outcomes of the mutated Nav1.5 channel proteins Han individuals (300 alleles) after obtaining written informed were examined in HEK293 cells. The results demonstrated consent. that the mutation reduced sodium current density and altered the biophysical sodium channel characteristics. Site‑directed mutagenesis and heterologous expression. The p.D1690N mutation was introduced into pcDNA3.1-hH1 using Materials and methods a PCR-based mutagenesis method, as previously described (12). The mutated plasmids were sequenced (Tsingke Biological Clinical characteristics. A 37-year-old male was admitted to Technology Co., Ltd.) to ensure the presence of the p.D1690N the emergency department of The First Affiliated Hospital mutation and the absence of spurious mutations. of Xiamen University (Xiamen, China) in September 2009, The human embryonic kidney 293 cell line, HEK293 was due to sudden syncope from an episode of VF (Fig. 1A). cultured in an incubator in Gibco Dulbecco's modified Eagle's A subsequent 12-lead ECG at rest was consistent with a medium (Thermo Fisher Scientific, Inc.) supplemented with type-1 Brugada pattern, defined as a prominent coved-type Gibco 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), ST-segment elevation, displaying ST-segment elevation 4 mmol/l glutamine (Invitrogen; Thermo Fisher Scientific, >2 mm at its peak followed by a negative T wave (3) and Inc.), 100 IU/ml penicillin (Amresco LLC, Solon, OH, USA) incomplete right bundle branch block (RBBB; Fig. 1B). The and 100 µg/ml streptomycin (Amresco LLC) at 37˚C in a BrS patient demonstrated no evidence of structural heart humidified atmosphere of 5% CO2. Prior to over-expression, disease by exercise stress test, electrophysiological study and the cells were seeded in 6-well plates and, upon reaching 80% echocardiography. Subsequently, the family of the patient (four confluence, were co-transfected with 0.8 µg pcDNA3.1-hH1 males and three females; mean age, 38.9±17.6 years) under- or p.D1690N mutant and 0.8 µg pIRES2-DsReD-sodium went physical examination, 12-lead ECG, 24-h Holter ECG voltage-gated channel β subunit 1 (SCN1B; which served as a monitoring (Nihon Kohden, Tokyo, Japan), echocardiography reporter gene) with Invitrogen Lipofectamine 2000 (Thermo and genetic testing. As ajmaline, flecainide and procainamide, Fisher Scientific, Inc.), according to themanufacturer's instruc- recommended by the consensus report (13), are unobtainable tions and as previously reported (12). in China, propafenone (Shanghai Xinyi Jinzhu Pharmaceutical Co., Ltd., Shanghai, China) was administered, which has been Electrophysiological analysis. Twenty-four to 48 h after demonstrated to reveal BrS. Propafenone challenges were transfection, the sodium current (INa) was recorded in cells performed and evaluated on two of the family members [the displaying red fluorescence at room temperature (23-25˚C) sister (II.3) and nephew (III.2) of the proband] as previously under whole cell patch-clamp technique, as previously described (13). The subjects provided written informed consent described (12). Briefly, the cells were continuously superfused and, following a fasting period (~10 h overnight), propafenone with a bath solution containing 70 mM NaCl, 80 mM CsCl, (1 mg/kg body weight; 10 mg/min) was intravenously adminis- 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES tered while the patient was continuously monitored by 12-lead and 10 mM glucose (pH adjusted to 7.3 using CsOH). The ECG and blood pressure. After 20 min, if the reaction was patch pipette
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