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KCNJ5基因Gly387Arg突变导致家族性长QT综合征
KCNJ5 Gly387Arg Mutation Responsible for Familial Long QT Syndrome
【作者】 刘金秋;
【导师】 杨延宗;
【作者基本信息】 大连医科大学 , 生理学, 2008, 博士
【摘要】 背景:心源性猝死严重危害人类健康,发病率高达0.1%-0.2%,仅在美国每年就可导致大约30万-40万人死亡。90%猝死的患者可以找到心脏病因,由于猝死后存活的患者有限,因此至少5%患者没有潜在的心脏病原因,这些患者大多数年龄小于40岁,其中长QT综合征是引起这部分病人猝死的主要原因之一。长QT综合征又称心室复极延长综合征,其特征性表现是心电图上QT间期延长, T波异常,易产生尖端扭转型室速和室颤。长QT综合征按病因通常可分为获得性和遗传性两种类型。获得性长QT综合征虽也具有遗传易感性,但主要与心肌局部缺血、电解质紊乱和应用某些药物等因素有关,临床上祛除这些诱发因素常可有效治疗。而遗传性长QT综合征的发生机制要复杂得多,又无有效的治疗措施,因此长期以来一直是心律失常领域的研究焦点。从Jervell和Lange-Nielsen于1957年在世界上报道了第一个呈常染色体隐性遗传的长QT综合征家系,至今已经已经发现了10个基因的数百个突变可导致10种不同类型的长QT综合征,初步揭示了部分长QT综合征的分子缺陷及其作用机制。然而,已经识别出的10个长QT综合征致病基因只可以解释大约80%的长QT综合征患者的病因和发病机制,仍有20%左右的长QT综合征患者的致病基因有待识别。不仅如此,由于长QT综合征既具有显著地遗传异质性,不同患者的致病基因不一定相同,即使基因相同,致病突变也多不同;同时存在巨大的表型差异,同样是长QT综合征患者,有的毫无症状,有的则出现晕厥和猝死等表现,因而有必要对所有无血缘关系的长QT综合征尤其是家族性长QT综合征患者进行分子学遗传研究。目的:定位新的家族性长QT综合征的致病基因座,识别新的家族性长QT综合征的致病基因,并在细胞水平研究其作用机制,为长QT综合征的早期诊断、预后评估、预防咨询以及基因治疗奠定基础。方法:收集长QT综合征家系和无血缘关系的健康对照者。采集外周静脉血,常规抽提基因组DNA。在前期已经排除了所报道的10个长QT综合征相关基因KCNQ1、KCNH2、SCN5A、Ankyrin B、KCNE1、KCNE2、KCNJ2、CACNA1C、CAV3和SCN4B的基础上,进行微卫星全基因组扫描、基因分型、单倍型和连锁分析,确定候选基因在染色体上的座位。运用生物信息学知识确定所定位区域的首选候选基因,通过PCR-测序和序列比对分析以期发现家族性长QT综合征先证者的基因突变。然后,分别在发生基因突变的家系和健康对照者中筛查所发现的突变基因,以评估该基因突变在不同人群中的流行率,并对比分析突变区域氨基酸在各物种间的保守性,初步确定所发现的突变是致病突变而不是罕见的分子多态。运用分子克隆技术克隆所发现的潜在致病基因的野生型,插入到克隆载体pGEM-T,针对所发现的突变通过定位诱变获得该基因的突变体,再通过酶切反应及测序证实后转化感受态细胞,中量制备所识别出的长QT综合征基因的重组质粒,在体外逆转录制备适量的野生型和突变型长QT综合征基因的cRNA,通过显微注射装置注入爪蟾卵母细胞,运用双电极电压钳在细胞水平研究突变基因产物所发生的电生理改变。结果:获得了一个长QT综合征大家系和500例无血缘关系的健康对照者的临床资料和血标本,绘制了该家系的系谱图,抽提了基因组DNA。通过微卫星全基因组扫描获得了家系成员的基因型,构建了单倍型图。通过连锁分析,确定该家系长QT综合征的候选基因在染色体上的座位是11q23-24(在11号染色体上,两标记位点D11S990和D11S4123之间的5.34cM的间隔区域,其中在位点D11S4123在θ=0.00时得到最大的LOD值5.4183)。运用生物信息学知识确定所定位区域的首选候选基因为KCNJ1和KCNJ5,通过PCR-测序和序列比对分析发现家族性长QT综合征先证者的KCNJ5基因发生了一个改变氨基酸的杂和错义突变,即KCNJ5基因(基因登录号NM000890)第1473位的鸟嘌呤(即第387位密码子的第一个核甘酸)变成了胞嘧碇(1473 G C),相应地其所编码的离子通道蛋白的第387位的甘氨酸变成了精氨酸(Gly387 Arg)。相同的突变存在于家系中所有的患者,但500例无血缘关系的健康对照者均无此突变,也没有在该家族性长QT综合征先证者中发现KCNJ1基因存在突变。多物种序列比对显示KCNJ5基因Gly387 Arg突变具有高度保守性,初步确定所发现的突变是致病突变而不是罕见的分子多态。运用分子克隆技术成功克隆了所发现的潜在致病基因KCNJ5的野生型,插入到克隆载体pGEM-T,针对所发现的突变通过定位诱变获得该基因的突变体,再通过酶切反应及测序证实后中量制备了该基因的重组质粒,在体外逆转录制备了适量的野生型和突变型KCNJ5的cRNA,通过显微注射装置注入爪蟾卵母细胞,全细胞双电极电压钳研究发现KCNJ5的Gly387 Arg突变为功能获得性,使KCNJ5基因所编码的钾离子通道在所有电压水平的电流密度均大幅度升高,在-50mV,突变型KCNJ5和野生型KCNJ5的电流密度分别为- 23.2±1.2pA/pF( n= 11)和- 8.5±0.8pA/PF( n= 8) (p<0.05);电压为-20mV时,两者的电流密度分别为15.2±1.1pA/pF(n=11)和5.1±0.5 pA/pF(n=8)(p<0.05)。结论:确定了一个新的长QT综合征致病基因座11q23-24,识别了一个新的长QT综合征致病基因KCNJ5,KCNJ5基因Gly387 Arg突变对KCNJ5基因所编码的钾离子通道的功能具有显著影响。
【Abstract】 Background Sudden cardiac death is a major life-threatening disorder most frequently encountered in clinical practice with an overall incidence in the general population estimated to be between 0.1%and 0.2%, resulting in approximately 300,000 to 400,000 deaths annually in the United States. Sudden cardiac death in an otherwise healthy young individuals and the lack of an apparent etiology in many of those victims initially led to the classification as“sudden unexplained death syndrome”or“sudden infant death syndrome”. In most cases, idiopathic polymorphic ventricular tachycardia described as“Torsade de pointes”(TdP) which referred to the characteristic rotation of the electrical axis along an imaginary iso-electric line on the surface electrocardiogram or ventricular fibrillation (VF) was gradually recognized as the primary trigger, and the underlying pathogenic determinants resulting in TdP or VF were lengthened cardic repolarization characterized by abnormal QT-interval prolongation on the surface electrocardiogram, that is, long-QT syndrome. Long-QT syndrome predisposes subjects to syncope, seizures, and sudden death resulted from TdP or VF. Long-QT syndrome is generally classified into two categories, acquired or idiopathic. In addition to inheritable susceptiblity, acquired long-QT syndrome is often attributable to local myocardial ischemia, imbalanced electrolytes, or administered medications and may be easily cured by getting rid of such risk factors. However, idiopathic long-QT syndrome has long been focus of medical research because of more complex mechanism and no efficacious mesures. In 1957, Jervell and Lange–Nielsen made the first report on a familial disease characterized by a striking prolongation of the QT-interval, congenital deafness and a high incidence of sudden cardiac death at young age . Subsequently, an almost identical disorder whereas lacking the trait of sensorineural deafness was identified by Romano and Ward. At present, Hundreds of mutations in 10 distinct genes accountable for a hereditary form of long-QT syndrome has been identified: KCNQ1 (KvLQT1, type 1 long-QT syndrome, LQT1), KCNH2 (hERG, LQT2), SCN5A (Nav1.5, LQT3), ANK2 (Ankyrin-B, LQT4), KCNE1 (MinK, LQT5), KCNE2 (MiRP1, LQT6), KCNJ2 (Kir2.1, LQT7), CACNA1C (Cav1.2, LQT8), CAV3 (Caveolin-3, LQT9) and SCN4β(Navβ4, LQT10). Overall, 8 of these genes encode proteins that are specifically involved in cardiac action potential generation, whereas ANK2 and CAV3 encoding non-channel proteins are considered to be more general-purpose. The main mechanism by which these mutated genes give rise to long-QT syndrome is an excessive and heterogeneous lengthening of the repolarization phase of the ventricular action potential. Nevertheless, the identified genetic defects only account for approximately 80 percent of congenital long-QT syndrome and the long-QT-associated genes in about 20 percent of cases remain to be identified. Furthermore, the accumulating evidence reveals that long-QT syndrome is of remarkable both genetical and clinical heterogeneity ranging from diverse culprit genes to different mutations of an identical gene in various patients with long-QT syndrome or from asymptome to syncope, even to sudden death in patients with the same mutation of a gene responsible for long-QT syndrome. Therefore, These findings highlight the need for a comprehensive genetic screening of unrelated patients, especially of familial ones with long-QT syndrome, for a novel causal gene.Objectives The aims of the present investigation were to map a novel locus linked to long-QT syndrome to a certain region on chromosome, to identify a novel gene responsible for long-QT syndrome by performing a systematic screening of selected candidate genes in a located position, and further to explore the molecular basis for long-QT syndrome. These researches may pave the way for early diagnosis, prognostic appraisal, prophylactic consultation, and gene-specific therapy.Methods As many as possible unrelated families with congenital long-QT syndrome were identified in China according to a set of diagnostic criteria proposed by Schwartz and colleagues and a cohort of around 500 unrelated healthy subjects as control were recruited from the same Chinese population. The diagnostic criteria for long-QT syndrome principally consist of a positive family history, clinical history, and electrocardio- graphic findings, and exclusion of organic heart diseases and other identified causative factors. The clinical data including medical records, electrocardiograms and echocardiography reports were collected. The genomic DNA from all participants, including the probands and all consenting family members, and controls was extracted from peripheral venous blood lymphocytes by use of Wizard Genomic DNA Purification Kit. After ruling out the known 10 long-QT syndrome-associated genes by polymerse chain reaction (PCR)-sequencing or linkage analysis of 3 to 5 of microsatellite markers near a gene, incliding KCNQ1、KCNH2、SCN5A、Ankyrin B、KCNE1、KCNE2、KCNJ2、CACNA1C、CAV3, and SCN4B, we performed the whole-genome scanning of microsatellites mainly from ABI PRISM Linkage Mapping Sets v2.5, genotyping on MegaBACE 500, analysis of haplotypes, and linkage analysis with software SAGE in order to map a novel gene linked to long-QT syndrome to a small region on a chromosome. The preferred candidate genes in the located region were established by bioinformatical knowledge and the entire coding sequences of selected candidate genes were screened in probands for mutations by direct PCR-sequencing and sequence alignment in order to identify a novel mutated gene leading to long-QT syndrome. The resultant mutant genes identified were consequently targeted to detect in all relevant family members available and 500 unrelated subjects as controls to evaluate their prevalence in different populations. The conservation of amino acid residues altered by the identified mutations was determined by aligning protein homologs and orthologs among species. The total mRNA was extracted from human myocardium with TRIzol. The full-length wild-type cDNA of human KCNJ5 identified was obtained by reverse transcription (RT) - PCR using pfuUltraTM high- fidelity DNA polymerase and inserted into the oocyte expression plasmid pGEM-T with T4 DNA ligase. Mutation was introduced into a wild-type KCNJ5 clone with the use of a Quick Change? II XL Site-Directed Mutagenesis Kit. The wild-type and mutant KCNJ5-pGEM-T constructs were corroborated by sequencing prior to subsequent experiments. The appropriate wild-type and mutant recombinant vector KCNJ5-pGEM-T were transfected and cloned to obtain sufficient KCNJ5-pGEM-T for physiological research. The wild-type and mutant KCNJ5 cRNA was synthesized by standard in vitro run-off transcription from plasmid KCNJ5-pGEM-T using the T7 mMessage Machine kit in terms of manufacturer’s instructions. Oocytes were surgically removed from anaesthetized X. laevis frogs and defolliculated enzymatically as described previously. The wild-type and mutant KCNJ5 cRNA was mocroinjected respectively or together in equal molar ratios into the prepared oocytes (0.5-2 ng / oocyte). The injected oocytes were kept in a low K solution. Whole-cell currents were mesured 24-48 hours after KCNJ5 cRNAinjection by two-electrode voltage-clamp amplifier. Pipettes were pulled from borosilicate glass and had a final tip resistance of 0.5-2.5 MΩwhen filled with 3M KCl and submerged in low K solution. Currents were measured in a high K solution. Data were sampled with pulse and analyzed using the Igor software and GraphPad software. All data are presented as mean±standard error of the mean. For statistical analyses t tests as well as one way ANOVAcombined with Tukey’s mutiple comparison test were used. P < 0.05 was considered to be significant.Results A large kindreds with familial long-QT syndrome were identified in China and a cohort of 500 unrelated healthy subjects as ethnically matched control were recruited from the same Chinese population. Peripheral venous blood specimens were prepared and genomic DNA was extracted from lymphocytes. The clinical data including medical records, electrocardiograms and echocardiography reports were collected. The known 10 long-QT syndrome-associated genes were rule out from the present famiy with idiopathic long-QT syndrome. The genotypes of these family members were determined and the haplotype was constructed. By linkage analysis a novel locus linked to long-QT syndrome was finely mapped to a small region on the 11th chromosome, i.e., 11q23-24, roughly 5.34cM between microsatellite markers D11S990 and D11S4123, with a maximal two-point LOD score of 5.4183 for D11S4123 marker atθ=0.00. Two genes of KCNJ1and KCNJ5 located at the mapped chromosomal region were preferred by bioinfor- matical knowledge. A novel heterozygous missense mutation was identified in KCNJ5 from the proband representative of studied pedigree with long-QT syndrome. Rather, a 1473 G C mutation (accession number, NM000890) at nucleic acid level, predicting the substitution of arginine for glycine at codon 387 (Gly387 Arg) at amino acid level, was identified in the index case of the family. The mutation was located at the functionally important C-terminus of the KCNJ5-encoded ion channel protein. The same mutation was present in all the affected family members but absent in 500 matched controls. Additionally, no mutation in KCNJ1 was detected in the representative of this pedigree. A cross-species alignment of KCNJ5 - encoded Kir3.4 ion channel protein sequence displayed the mutaed amino acid was highly conserved evolutionarily among comprehensive species, which indicated rudime- ntarily that the mutation Gly387 Arg might be a pathogenic component rather than a rare benign molecular polymorphism. The human KCNJ5 gene was cloned and the mutation was introduced into it successfully by site directed PCR. The wild-type and mutant KCNJ5-pGEM-T recombine- ant constructs were constructed and substan- tiated by enzymatic analysis and direct PCR- sequencing. The appropriate recombinant constructs were amplified and sufficient cRNA of both wild-type and mutant KCNJ5 was synthesized in vitro. Enough oocytes were surgically acquired from anaesthetized toads. Electrophysiological analysis of the ion channels expressed in the oocytes surviving microinjection of cRNA revealed significant effect of the mutation on the the activity of inward rectifier potassium channel (Kir3.4) in contrast to its wild-type counterpart. At -50 mV, the average current was -23.2±1.2 pA/pF (n=11) and -8.5±0.8 pA/PF (n=8) respectively for the mutant and wild-type Kir3.4 (p<0.05); It was 15.2±1.1 pA/pF (n=11) for the mutant Kir3.4 and 5.1±0.5 pA/pF (n=8) for the wild-type Kir3.4 at -20mV (p<0.05). These experimental findings validate the pathogenic link between compromised Kir3.4 function and susceptibility to long-QT syndrome.Conclusions A novel locus linked to long-QT syndrome is mapped to 11q23-24, which is equivalent to a chromosomal region of about 5.34cM between microsatellite markers D11S990 and D11S4123. A novel gene, namely KCNJ5, responsible for long-QT syndrome was subsequently identified. KCNJ5 Gly387Arg mutation imposes remarkable effect on Kir3. 4 channel currents and may predispose individuals to long-QT syndrome. Our data establishes KCNJ5 as an 11th gene responsible for long-QT syndrome, indicating implications for genetic diagnosis, genetic counseling, and gene-specific therapy.