功能学研究在心脏钠通道疾病诊断中的作用

目的:探讨心脏钠通道变异功能学研究在临床疑诊遗传性心律失常诊断中的作用。方法:分析疑诊长QT综合征(LQTS)和Brugada综合征(BrS)家系的临床表现;通过基因筛查和体外功能学研究明确临床诊断。结果:家系1,先证者有心悸病史,无晕厥事件,家族猝死史阴性。常规心电图无QTc延长,Holter监测提示室性期前收缩,心率100次/min时QTc 460ms,疑诊LQTS;基因筛查发现SCN5A A1870T变异。家系2,先证者有心悸病史,无晕厥事件,家族猝死史阳性,Holter监测提示频发室性期前收缩和短阵室上性心动过速。常规心电图QTc正常,无brugada心电图样特征,氟卡氨激发试验阴性。基因筛查发现SCN5AL1410P变异。家族中变异携带者均未达到BrS诊断标准。体外膜片钳研究显示,与野生型通道比较,A1870T变异稳态激活曲线有-4.0mV的移位;突变稳态失活曲线有2.8mV的移位。提示A1870T增加窗电流,为功能增强型突变。而SCN5AL1410P变异未检出电流,提示为功能完全缺失型突变。结论:体外功能学研究证实SCN5A A1870T变异为LQTS突变,而L1410P变异为BrS突变,为临床疑诊病例提供分子功能学诊断依据。这表明体外功能学研究能为临床疑诊遗传性心律失常患者提供更多的诊断信息。     

长QT综合征KCNH2基因突变L413P和L559H的致病机制

目的研究长QT综合征致病基因之一KCNH2基因L413P和L559H突变的致病机制。方法DNA定点突变技术构建L413P和L559H表达载体,脂质体介导法转染人胚胎肾细胞表达。用膜片钳技术记录KCNH2通道电流变化情况,观察HERG蛋白表达和细胞内定位情况。结果电生理学显示L413P和L559H突变后完全无电流。在转染L413P或L559H的细胞中只观察到未成熟HERG蛋白,而转染WT的细胞可观察到成熟和未成熟两种HERG蛋白形式。L413P和L559H突变蛋白主要分布在细胞核周围的局部区域,提示突变蛋白滞留于内质网中而研蛋白均匀分布在细胞膜核细胞质中。L413P或L559H与等量野生型质粒共转染后电流相比无显著变化。低温与E-4031不能纠正HERG突变的异常。结论L413P和L559H突变可引起突变蛋白转运障碍,但基因表达和电生理学结果显示突变对野生型无负显性作用,突变的致病机制可能是单倍体不足。     

HERG基因Y475C突变致2型长QT综合征的机制探讨

目的研究HERG基因Y475C突变的致病机制。方法利用DNA定点突变技术构建Y475C表达载体,脂质体介导法转染人胚胎肾细胞进行表达。转染后48h用膜片钳技术记录KCNH2(亦称HERG)通道电流变化情况,明确Y475C突变导致2型长QT综合征的机制。结果电生理学研究显示与野生型通道电流相比,Y475C突变单独或与等量野生型质粒共转染时电流密度均显著降低,并且通道激活特征,包括V1/2(半激活电压)和K斜率因子也有显著改变。Y475C对通道失活特征无显著影响。结论Y475C突变通过负显性抑制效应以及通道门控特性的改变导致该基因编码的快速激活延迟整流钾电流显著降低是引起患者QT间期延长的机制。     

先天性长QT综合征KVLQT1和HERG基因新突变位点的检测

目的：研究中国人先天性长QT综合征（long QT syndrome,LQTS)HERG和KVLQT1的基因突变情况。方法,利用聚合酶链反应和DNA测序对11个LQTS家系HERG跨膜编码区S1－S6和KVLQT1跨膜编码区S2－S6进行基因检测。结果（1）11个LQTS患者在国外已知突变点均无突变;（2）发现4个新的错义突变位点,分别为T1515G（HERG）,C682T,C934T,G983A（KVLQT1）。其对应的氨基酸改变为E505D,R228C,S230L,P312S和R328C。结论：在中国人LQTS患者HERG和KVLQT1上发现了4个新的基因突变位点。     

先天性长QT综合征HERG基因A561V突变的功能研究

目的在收集的一个先天性长QT综合征家系中发现HERG基因A561V突变,探讨HERG基因A561V突变在真核细胞中的功能表达。方法采用克隆载体快速PCR法及限制性内切酶法将突变体克隆到真核表达载体pcDNA3中,用Superfeet转染试剂将野生型及突变型HERG质粒与荧光载体pRK5．GFP共转染至HEK293细胞,用免疫荧光化学法及蛋白免疫印迹法检测蛋白质表达,用全细胞膜片钳法检测HERG通道的电流表达。结果构建的突变体经DNA直接测序示HERG基因cDNA1682位点碱基C变为T,突变体蛋白质位于细胞膜上及细胞质中,野生型通道蛋白显示135000和155000的2条蛋白条带,而杂合型和突变型通道蛋白仅有135000的1条蛋白条带,野生型通道记录到尾电流而杂合型通道及突变型通道均未检测到电流表达。结论成功构建并表达了HERG基因A561V突变的功能,中国患者具有与欧美患者相同的HERG基因突变热点。     

长QT综合征家系KCNQ1 S145L和KCNH2 Y475 C基因新突变

目的研究中国遗传性长QT综合征(LQTS)患者的临床特点及LQTS最常见的基因KCNQ1和KCNH2突变.方法应用聚合酶链反应和测序分析77个遗传性LQTS家系,筛查了LQTS致病基因KCNQ1和KCNH2,观察临床表现和心电图改变.结果77例先证者心电图表现为LQT1者24例、LQT2者42例、LQT3者3例,8例心电图表现不典型.年龄(27.6±16.4)岁.QTc(561±70)ms,发病年龄(17.6±14.7)岁.晕厥触发因素包括运动、情绪激动和铃声刺激等.目前已经发现了4KCNQ1突变和7 KCNH2突变,其中6个为首次发现.结论LQT2为中国最常见的LQTS;本组发现KCNQ1和KCNH2各1个新突变;中国LQTS患者心电图表现和临床特点与欧美LQT患者有所不同.     

先天性长QT间期综合征KVLQT1基因新的同义突变位点

目的 :研究中国人先天性长QT间期综合征 (LQTS)患者KVLQT1和HERG基因突变位点。方法 :采用聚合酶链反应和DNA测序对 11个LQTS家系KVLQT1和HERG基因跨膜编码区进行突变检测。结果 :①15例患者在国外已知的突变位点上 ,均未发现有突变。②发现KVLQT1基因 2个新的同义突变 (C6 36T ,G912A) ,位置分别在KVLQT1基因的S4、Pore跨膜片段。结论 :这 2个新的同义突变可能通过影响延迟性整流钾通道的跨膜电流而与LQTS相关     

长QT综合征的危险度分层

长QT综合征(long QT syndrome,LQTS)最常见的病因为:K~+ 通道基因 KCNQ1(LQT1 位点),KCNH1(LQT2 位点)和 Na~+ 通道基因SCN5A(LQT3 位点)发生突变。该文按照基因型与其他临床变量进行不同层次危险度分析。 方法 评价分析了193个确诊为LQTS的家系,其中104个家系在LQT1位点发生突变,68个家系在LQT2位点发生突变,21个家系在     

国人长QT综合征基因筛查及分子致病机制研究状况

自1995年发现第一个长QT综合征(LQTS)致病基因至今,目前已在13个致病基因上发现了950多个突变。已公开发表的中国LQTS患者特异基因突变点有47个,包括KCNQ1上17个、KCNH2上19个、SCN5A上4个、KCNE1上1个、KCNJ5上1个。在对病人进行基因筛查研究的基础上,对突变位点的分子致病机制进行了研究的有:KCNQ1上的突变L191P、F275S和G314S,KCNH2上的突变L413P、F463L、Y475C、E505D、L539fs/47、P559H、A561V、G604S、V630A、N633S、R863X,KCNE1上的G52R,KCNJ5上的Kir3.4-Gly387Arg。致病机制包括负显性或单倍体不足导致的通道功能缺失。     

发现KCNH2基因上移码突变所致遗传性长QT综合征一例及家系分析

目的应用基因筛查技术对1个遗传性长QT综合征(LQTS)患病家系进行12个已知致病基因的突变分析,明确致病突变。方法在符合伦理要求和获得知情同意的情况下,经过详细的病史采集及临床检查后,采集该家系中7名患者和1名表型正常个体外周血并提取基因组DNA。利用Complete Genomics测序平台对先证者进行全基因组测序,分析已知致病基因:KCNQ1、KCNH2、SCN5A、ANK2、KCNE1、KCNJ2、CACNA1C、CAV3、SCN4B、AKAP9、SNTA1、KCNJ5。对于定位于先证者中的突变,用聚合酶链式反应和直接测序法在家系中其他成员中进行测序,最终确定致病基因突变位点。结果在该家系患者的KCNH2基因上发现1个移码突变c.2400delC(p.Gly800fs*10),在家系内正常成员和正常人群中均未发现该突变。结论在1个中国LQTS家系中发现了一个LQTS相关的KCNH2基因新突变(del D1790)。     

HERG mutation predicts short QT based on channel kinetics but causes long QT by heterotetrameric trafficking deficiency

OBJECTIVE: Mutations in the KCNH2 (hERG, human ether-à-go-go related gene) gene may cause a reduction of the delayed rectifier current I(Kr), thereby leading to the long QT syndrome (LQTS). The reduced I(Kr) delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques. METHODS AND RESULTS: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power. CONCLUSION: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels.     

Characterization of novel KCNH2 mutations in type 2 long QT syndrome manifesting as seizures

BACKGROUND

Long QT syndrome (LQTS) is characterized by corrected QT interval prolongation leading to torsades de pointes and sudden cardiac death. LQTS type 2 (LQTS2) is caused by mutations in the KCNH2 gene, leading to a reduction of the rapidly activating delayed rectifier K⁺ current and loss of human ether-à-go-go-related gene (hERG) channel function by different mechanisms. Triggers for life-threatening arrhythmias in LQTS2 are often auditory stimuli.

OBJECTIVES

To screen KCNH2 for mutations in patients with LQTS2 on an electrocardiogram and auditory-induced syncope interpreted as seizures and sudden cardiac death, and to analyze their impact on the channel function in vitro.

METHODS

The KCNH2 gene was screened for mutations in the index patients of three families. The novel mutations were reproduced in vitro using site-directed mutagenesis and characterized using the Xenopus oocyte expression system in voltage clamp mode.

RESULTS

Novel KCNH2 mutations (Y493F, A429P and del234–241) were identified in the index patients with mostly typical LQTS2 features on their electrocardiograms. The biochemical data revealed a trafficking defect. The biophysical data revealed a loss of function when mutated hERG channels were coexpressed with the wild type.

CONCLUSIONS

In all families, at least one patient carrying the mutation had a history of seizures after auditory stimuli, which is a major trigger for arrhythmic events in LQTS2. Seizures are likely due to cardiac syncope as a consequence of mutation-induced loss of function of the rapidly activating delayed rectifier K⁺ current.     

Functional assessment of compound mutations in the KCNQ1 and KCNH2 genes associated with long QT syndrome

BACKGROUND: Long QT syndrome (LQTS) is a cardiovascular disorder characterized by prolonged QTc time, syncope, or sudden death caused by torsades de pointes and ventricular fibrillation. We investigated the clinical and electrophysiologic phenotype of individual mutations and the compound mutations in a family in which different genotypes could be found. OBJECTIVES: The purpose of this study was to determine the impact of genotype-based diagnostic assessment in LQTS. METHODS: We used cascade screening and functional analyses to investigate the phenotype in a family with LQTS. The contributions of the compound mutations in the KCNQ1 and KCNH2 genes (KCNQ1 R591H, KCNH2 R328C) were analyzed by heterologous expression in Xenopus laevis oocytes using two-electrode voltage clamp and by confocal imaging. RESULTS: KCNH2 R328C did not show any functional phenotype whereas KCNQ1 R591H resulted in severe reduction of current. Neither wild-type nor mutant channels affected each other functionally in coexpression experiments. Therefore, a direct interaction between KCNQ1 and KCNH2 was ruled out under these conditions. CONCLUSION: Assessment of novel mutational findings in LQTS should include accurate genetic and functional analysis. Notably, appropriate studies are needed if two or more mutations in different genes are present in one proband. Our findings prompt reconsideration of the impact of compound mutations in LQTS families and reinforce the need for thorough functional evaluation of novel ion channel mutations before assignment of pathogenic status.     

Additional gene variants reduce effectiveness of beta-blockers in the LQT1 form of long QT syndrome

INTRODUCTION: Beta-blockers are widely used to prevent the lethal cardiac events associated with the long QT syndrome (LQTS), especially in KCNQ1-related LQTS (LQT1) patients. Some LQT1 patients, however, are refractory to this therapy. METHODS AND RESULTS: Eighteen symptomatic LQTS patients (12 families) were genetically diagnosed as having heterozygous KCNQ1 variants and received beta-blocker therapy. Cardiac events recurred in 4 members (3 families) despite continued therapy during mean follow-up of 70 months. Three of these patients (2 families) had the same mutation [A341V (KCNQ1)]; and the other had R243H (KCNQ1). The latter patient took aprindine, which seemed to be responsible for the event. By functional assay using a heterologous mammalian expression system, we found that A341V (KCNQ1) is a loss-of-function type mutation (not dominant negative). Further genetic screening revealed that one A341V (KCNQ1) family cosegregated with S706C (KCNH2) and another with G144S (KCNJ2). Functional assay of the S706C (KCNH2) mutation was found to reduce the current density of expressed heterozygous KCNH2 channels with a positive shift (+8 mV) of the activation curve. Action potential simulation study was conducted based on the KYOTO model to estimate the influence of additional gene modifiers. In both models mimicking LQT1 plus 2 and LQT1 plus 7, the incidence of early afterdepolarization was increased compared with the LQT1 model under the setting of beta-adrenergic stimulation. CONCLUSION: Multiple mutations in different LQTS-related genes may modify clinical characteristics. Expanded gene survey may be required in LQT1 patients who are resistant to beta-blocker therapy.     

N- and C-terminal KCNE1 mutations cause distinct phenotypes of long QT syndrome

BACKGROUND: Long QT syndromes (LQTS) are inherited diseases involving mutations to genes encoding a number of cardiac ion channels and a membrane adaptor protein. The MinK protein is a cardiac K-channel accessory subunit encoded by the KCNE1 gene, mutations of which are associated with the LQT5 form of LQTS. OBJECTIVE: The purpose of this study was to search for the KCNE1 mutations and clarify the function of those mutations. METHODS: We conducted a genetic screen of KCNE1 mutations in 151 Japanese LQTS patients using the denaturing high-performance liquid chromatography-WAVE system and direct sequencing. In two LQTS patients, we identified two KCNE1 missense mutations, located in the MinK N- and C-terminal domains. The functional effects of these mutations were examined by heterologous coexpression with KCNQ1 and KCNH2. RESULTS: One mutation, which was identified in a 67-year-old woman, A8V, was novel. Her electrocardiogram (ECG) revealed marked bradycardia and QT interval prolongation. Another mutation, R98W, was identified in a 19-year-old woman. She experienced syncope followed by palpitation in exercise. At rest, her ECG showed bradycardia with mild QT prolongation, which became more prominent during exercise. In electrophysiological analyses, R98W produced reduced I(Ks) currents with a positive shift in the half activation voltages. In addition, when the A8V mutation was coexpressed with KCNH2, this reduced current magnitude, which is suggestive of a modifier effect by the A8V KCNE1 mutation on I(Kr). CONCLUSION: KCNE1 mutations may be associated with mild LQTS phenotypes, and KCNE1 gene screening is of clinical importance for asymptomatic and mild LQTS patients.     

Functional characterization of the common amino acid 897 polymorphism of the cardiac potassium channel KCNH2 (HERG)

OBJECTIVE: To determine whether the amino acid 897 threonine (T) to lysine (K) polymorphism of the KCNH2 (HERG) potassium channel influences channel performance or patient phenotype. METHODS: The phenotypic effects of this polymorphism were investigated in vitro by electrophysiological experiments in HEK-293 cells and in vivo by exercise electrocardiography in a group of LQTS patients carrying the same genetically proven KCNQ1 mutation. RESULTS: When expressed in HEK-293 cells, the 897T isoform of the KCNH2 channel exhibited changes in inactivation and deactivation properties, and a smaller current density than the more common 897K isoform. Western blot experiments indicated that the decreased current density associated with 897T was caused by reduced channel expression. During a maximal exercise test in 39 LQT1 patients carrying an identical KCNQ1 mutation (G589D) and showing a prolonged QT interval (>440 ms), QT intervals were longer in patients carrying the 897T allele than in those homozygous for the 897K allele. CONCLUSIONS: The K897T variation has an effect on channel function and clinical phenotype. Our data warrant further investigations into the significance of this polymorphism in drug-induced and inherited LQTS.     

Nav1.5/R1193Q polymorphism is associated with both long QT and Brugada syndromes

BACKGROUND: Brugada syndrome (BS) and long QT syndrome (LQTS) are electrical disorders with a genetic background. They are revealed on surface electrocardiograms as either right bundle branch block and ST segment elevation in the right pericardial leads (V1, V2 and V3) in BS or as a long QTc interval on all 12 leads of the electrocardiogram in LQTS. Both BS and LQTS can lead to syncope and even sudden death. The R1193Q SCN5A variant was recently associated with LQTS, BS and cardiac conductance disease. OBJECTIVE AND METHODS: The aim of the present study was to screen the SCN5A gene from two patients -- one with BS and the other showing signs of a BS/LQTS phenotype -- for mutations, and to characterize the effect of the mutations on channel function using the patch clamp technique. RESULTS: A heterozygous mutation (R1193Q) was identified on the SCN5A gene in both patients. The R1193Q polymorphism was absent in 100 unrelated control alleles, suggesting that it has a low frequency in the French Canadian population. Mutant R1193Q expressed in tsA201 cells, which was studied using the patch clamp technique, had a persistent sodium current that could account for the QTc prolongation. A shift of steady-state inactivation toward more hyperpolarized voltages was observed that could explain the BS phenotype. CONCLUSION: The R1193Q polymorphism is definitively associated with cardiac electrical abnormalities.     

HERG错义突变V630A和N633S引起QT延长综合征的分子机制

目的 HERG基因突变可引起遗传性QT延长综合征（10ng QT syndroms,LQTS）。研究探讨HERG通道孔区的错义突变导致LQTS的分子机制。方法 采用Megapfimer方法制备HERGV630A、N633S突变体,并亚克隆到pSP64和pcDNA3．1表达载体。用T7RNA聚合酶体外合成cRNA,经Ribogreen荧光定量后,将相应的cRNA注入卵母细胞,在18℃培养箱中培养3d后,用标准双电极电压钳技术测量卵母细胞的表达电流。所有数据均用pCLAMP软件采集,并应用Kaleidagraph 3．5和Igor软件进行数据处理。结果 当V630A、N633S分别同HERG野生型在卵母细胞共同表达时,这两种突变体均具有明显的负显性抑制效应,其抑制效应达到50％～70％。同野生型HERG通道相比,这两种异源多聚体的失活显著加快,半失活电压负向偏移。结论 该研究首次阐明了两个位于HERG通道孔区外口的错义突变V630A、N633S所引起的通道电生理学功能改变,从分子水平证明HERG通道孔区的错义突变与致命性心律失常的关系。     

6个长QT综合征家系的分子遗传学检测

目的对6个先天性长QT综合征(long QT syndrom e,LQTS)家系成员进行基因检测。方法运用位于KCNH2和SCN5A基因内和临近的短串联重复(short tandem repeat,STR)(D7S1824、D7S2493、D7S483、D3S1298、D3S1767、D3S3521)位点确定染色体单体型。6个家系的所有成员进行单倍型连锁分析,确定基因型。1个家系经直接测序确定其基因型。结果家系1的致病基因位于染色体LQT3位点,而家系2~6的致病基因位于LQT2位点,家系6经直接测序确定该家系的先证者为散发病例,突变基因为KCNH2。确诊LQTS患者22例,其中6例为无症状基因携带者,排除6例可疑患者。结论LQTS的遗传学研究检测不但能确定LQTS基因分型,而且可进行LQTS的症状前诊断。从而为临床的基因靶向治疗提供依据。     

Mutations in the HERG K+-ion channel: a novel link between long QT syndrome and sudden infant death syndrome

In a 7-week-old infant who experienced sudden infant death syndrome (SIDS), a novel missense mutation was identified in KCNH2, causing a lysine-to-glutamic acid amino acid substitution at position 101 (K101E). KCNH2 codes for the HERG ion channel and mutations in the gene are associated with congenital long-QT syndrome (LQTS), and in the family of this case of SIDS, the mutation was associated with Torsades de pointes tachycardia, making SIDS the most likely outcome of congenital LQTS.