遗传性LQT2综合征相关基因HERG的研究新进展

HERG基因编码的蛋白在心室复极过程中很重要,HERG基因突变导致遗传性长QT综合征。获得性长QT综合征也与HERG基因相关。随着分子遗传学的进展,HERG基因突变如何导致长QT综合征的分子机制正逐步被揭示。     

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.     

LQT2与癫痫发病机制相关性研究

遗传性离子通道病与可兴奋组织突发性功能紊乱密切相关,主要累及神经、肌肉、心脏、肾脏等组织器官。遗传性长QT综合征(LQT2)是由于编码钾离子通道的HERG基因突变导致的遗传性心脏疾病,临床上以反复发作的晕厥及猝死为特征。研究表明,第二信使c AMP浓度的改变可以调控HERG通道蛋白表达。目前认为,很多人类特发性癫痫是由于突变基因编码的离子通道蛋白所致神经元异常放电的一种离子通道病,临床表现有阵挛、晕厥史和猝死。本文结合国内外研究现状,对LQT2与癫痫发病机制的相关性作一综述。     

致LQT2的无义突变的HERG通道的表达和药物拯救研究

目的本研究旨在探讨氨基糖苷类抗生素庆大霉素在异源表达系统中对导致长QT综合征2型的无义突变的HERG通道的作用。方法采用聚合酶链反应法制备相关突变体——W927X、R863X和E698X,并克隆到真核细胞表达载体中。将突变体的cDNA瞬时转染至HEK293细胞,应用全细胞膜片钳技术记录通道电流。药物拯救采用将转染24h后的HEK293细胞在含庆大霉素(400μg/mL)的培养液中孵育24h。结果W927X能表达典型的HERG电流,尽管与野生型HERG相比,电流幅值明显下降;R863X和E698X仅表达与未转染细胞上类似的内生性电流,说明未能形成功能性的HERG通道。庆大霉素能增强W927X的功能性表达,使其最大尾电流密度由11.6±2.4pA/pF(n=8)增至19.5±2.7pA/pF(n=7,P<0.05)。通道激活动力学特征在野生型HERG、W927X和W927X加庆大霉素干预组均没有明显差异。然而,庆大霉素对R863X和E698X并无明显作用。结论氨基糖苷类抗生素能部分恢复无义突变的HERG通道的功能性表达,且对不同的突变体其作用效应不同。     

长QT综合征相关基因新突变R863X-HERG的功能研究

目的 R863X是在国人长QT综合征家系中发现的一个新的HERG基因无义突变,本研究旨在探讨R863X-HERG的功能。方法 采用PCR方法制备突变体R863X-HERG,并克隆到真核细胞表达载体中;用CHO细胞表达系统及全细胞膜片钳电生理技术研究突变体的功能。结果 实验表明R863-HERG基因单独表达时不能形成有功能的离子通道,而突变体R863X同野生型HERG共表达时,通道的电流密度降低约30％,且HERG WT／R863X通道的电压依赖性失活过程加速。结论突变体R863X亚基自身不能装配成有功能的通道,但可和野生型HERG亚基形成有功能的HERG通道异聚体,改变通道的门控特性。HERG蛋白C末端可能在通道装配中发挥一定的作用。     

长QT综合征相关基因新突变G52R-KCNE1的功能研究

目的 了解长QT综合征相关基因新突变G52R-KCNE1的功能。方法 采用交叠PCR方法体外制备突变体G52R-KCNE1,并克隆到原核细胞表达载体中;体外合成RNA、显微注射入卵母细胞,在卵母细胞中共表达野生型KCNQ1(WT-KCNQ1)、野生型KCNE1(WT-KCNE1)和突变体G52R-KCNE1,采用标准双电极电压钳了解突变体的功能。结果 突变体G52R-KCNE1不能放大WT-KCNQ1通道的电流强度;当WT-KCNE1和G52R-KCNE1等量注射时,G52R-KCNE1可降低WT-KCNQ1／WT-KCNE1通道约50％的电流强度,而不影响该通道的激活动力学。结论 位于跨膜区第52位的甘氨酸对维持KCNE1的功能非常重要;突变体G52R-KCNE1明显抑制WT-KCNE1通道的功能,这可能是突变基因携带者心肌细胞复极化延缓,QT间期延长的分子电生理机制。     

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。     

先天性长QT综合征相关HERG基因细胞株的建立及其蛋白质表达

目的:建立稳定的先天性长QT综合征相关HERG基因的细胞株,并观察其蛋白质表达。方法:原核克隆载体PGEM-HERG经限制性内切酶获得HERG cDNA,将HERG cDNA亚克隆到真核表达载体pcD-NA3中,用Lipofectamin2000转染试剂介导将pcDNA3-HERG及荧光真核表达载体PRK5-GFP共转染至HEK-293细胞,利用G-418进行细胞筛选,并用稀释法建立稳定的HEK-HERG细胞株,用细胞免疫荧光化学法及蛋白免疫印迹法(Western-blot)检测基因的蛋白质表达。结果:建立的HEK-HERG细胞株稳定传代,细胞免疫荧光化学法及Western-blot法检测到了HERG通道蛋白质的表达。结论:该方法可成功建立HEK-HERG细胞株并表达HERG通道蛋白质,为今后突变型HERG基因的研究提供了细胞基础。     

口服补钾治疗LQT2的新方法

先天性长QT综合征(LQTS)是心肌细胞复极异常的一种遗传性疾病。HERG编码快速激活延迟整流钾通道，HERG的变异(LQT2型)在先天性LQTS中约占45％。该文对长期口服补钾治疗使得血清中K 浓度轻度持久性增加后，是否能够持久改善LQT2亚型患者心肌复极参     

特发性长QT综合征——致病基因的发现及其与临床联系的前景

特发性长ＱＴ综合征——致病基因的发现及其与临床联系的前景戚文航特发性长ＱＴ综合征（ＬＱＴＳ）是一相对少见的疾病。１９９４年在世界范围内正式登记收编的不足５００个家族。但疾病隐袭而凶险，致命性心律失常、心脏性猝死发生率颇高。据调查未经治疗的患者，２１％...     

Not All hERG Pore Domain Mutations Have a Severe Phenotype: G584S Has an Inactivation Gating Defect with Mild Phenotype Compared to G572S, Which Has a Dominant Negative Trafficking Defect and a Severe Phenotype

Introduction: Mutations in the pore domain of the human ether-a-go-go- related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification.
Methods and Results: We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584S-hERG channels.
Conclusions: hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.     

CRP＋1444C/T多态性与2型糖尿病的关系

目的探讨C反应蛋白（CRP）＋1444C/T基因多态性与2型糖尿病（T2DM）的相关性。方法采用PCR-RFLP法对156例T2DM患者（T2DM组）和149例健康体检者（NC组）行＋1444C/T基因型检测,同时测定血清CRP水平。结果T2DM组血清CRP水平显著高于对照组;两组均未检测到TT基因型,两组CRP＋1444C/T的基因型和等位基因频率分布比较均无统计学差异,对照组CT基因型者CRP水平显著高于CC基因型者。结论CRP＋1444C/T基因多态性与T2DM无相关性,血清CRP水平升高与T2DM有关。     

Brg1在Peutz-Jeghers综合征息肉组织中的蛋白表达及基因突变

目的:研究Brg1(Brahma-related gene1)基因在Peutz-Jeghers综合征(Peutz-Jeghers syndrome,PJS)息肉组织中蛋白表达及基因突变的意义,探讨其与肿瘤发生的关系.方法:应用免疫组织化学技术分析72例PJS息肉组织Brg1蛋白的表达,同时应用PCR-DNA技术检测39例PJS息肉和2例癌变组织中Brg1基因第4和10外显子的基因突变,初步探讨其和PJS发生、发展及预后的关系.结果:Brg1蛋白在PJS息肉中的表达率为54.17%(39/72),与小肠腺癌的表达率(76.67%)相比明显降低,与正常组织的表达率(16.67%)相比明显增高;PJS息肉组和正常小肠黏膜组Brg1蛋白阳性表达差异有统计学意义(P<0.05);PJS息肉组和小肠癌组Brg1蛋白阳性表达差异有统计学意义(P<0.05).39例PJS息肉标本和2例癌变标本中,Brg1第4和10外显子的基因突变率为零.结论:Brg1蛋白在PJS息肉中高表达并对PJS的发生发展起着重要作用,但Brg1基因突变在PJS中少见,Brg1蛋白的表达可作为判断PJS息肉恶变及预后的重要指标.     

PEA15与KCNJ10基因两个单核苷酸位点在2型糖尿病家系成员中的多态性研究

我国中部地区55个T2DM家系的265名成员，检测其PEA15基因（位点rs8175359）和KCNJ10基因（位点rs2486253）的单核苷酸多态性。受检位点的基因型和等位基因频率分布，在T2DM组和家系成员组之间不存在显著差异，提示该多态性不与T2DM相关。     

Missense mutations in the pancreatic islet beta cell inwardly rectifying K+ channel gene (KIR6.2/BIR ): a meta-analysis suggests a role in the polygenic basis of Type II diabetes mellitus in Caucasians

Summary The K⁺ inwardly rectifier channel (KIR) is one of the two sub-units of the pancreatic islet ATP-sensitive potassium channel complex (I_KATP), which has a key role in glucose-stimulated insulin secretion and thus is a potential candidate for a genetic defect in Type II (non-insulin-dependent) diabetes mellitus. We did a molecular screening of the KIR6.2 gene by single strand conformational polymorphism (SSCP) and direct sequencing in 72 French Caucasian Type II diabetic families. We identified three nucleotide substitutions resulting in three amino acid changes (E23K, L270V and I337V), that have also been identified in other Caucasian Type II diabetic subjects. These variants were genotyped in French cohorts of 191 unrelated Type II diabetic probands and 119 normoglycaemic control subjects and association studies were done. The genotype frequencies of the L270V and I337V variants were not very different between Type II diabetic subjects and control groups. In contrast, analysis of the E23K variant showed that the KK homozygocity was more frequent in Type II diabetic than in control subjects (27 vs 14 %, p = 0.015). Analyses in a recessive model (KK vs EK/EE) tended to show a stronger association of the K allele with diabetes (p = 0.0097, corrected p-value for multiple testing < 0.02). The data for the E23K variant obtained here and those obtained from three other Caucasian groups studied so far were combined and investigated by meta-analysis. Overall, the E23K variant was found to be significantly associated with Type II diabetes (0.001 ≤p≤ 0.0016, corrected p-values for multiple testing p≤ 0.01). This study shows that KIR6.2 polymorphisms are frequently associated with Type II diabetes in French Caucasians. Furthermore, a meta-analysis combining different Caucasian groups suggests an significant role of KIR6.2 in the polygenic context of Type II diabetes. [Diabetologia (1998) 41: 1511–1515] Received: 22 January 1998 and in final revised form: 8 June 1998     

Islet gene expression and function in type 2 diabetes; studies in the Goto-Kakizaki rat and humans

Defective β-cell function with resulting impairment of glucose-stimulated insulin release is a critical factor in the pathogenesis of type 2 diabetes. Accumulated studies in pancreatic islets of the spontaneously diabetic Goto-Kakizaki (GK) rat suggest that this is a useful animal model of type 2 diabetes. The GK rat is non-obese, and abnormal glucose regulation develops early in life in association with impaired insulin secretion. There are some differences in islet morphology and function reported between different GK rat colonies. In addition to reduction of β-cell mass, a number of β-cell defects have been described with possible relevance for the reduced insulin secretion. Interestingly, some of these defects have also been shown in isolated islets from type 2 diabetic humans. The polygenic nature of diabetes heredity in the GK rat may well resemble the genetic basis in the majority of patients with type 2 diabetes. Here, we review studies concerning β-cell function and islet gene expression in the GK rat and compare it with the limited number of investigations on similar topics in isolated islets from patients with type 2 diabetes.     

Atypical hemolytic uremic syndrome and acute tubular necrosis induced by complement factor B gene (CFB) mutation: A case report

Rationale:Atypical hemolytic uremic syndrome (aHUS) is an uncommon and serious disease that manifests hemolytic anemia, thrombocytopenia, and acute kidney injury. Genetic complement abnormalities have been shown to be responsible. Compared with the aHUS caused by other mutated genes, aHUS secondary to CFB mutation in adults is extremely rare. We report an adult with CFB mutation developing aHUS.Patient concerns:A 56-year-old man was admitted for 4-day history of nausea and fatigue, anuria for 2 days, and unconsciousness for 10 hours.Diagnoses:The patient presented with life-threatening anemia, thrombocytopenia, acute kidney injury, and nervous system abnormalities. The patient had schistocytes on the peripheral blood smear, increased lactate dehydrogenase (LDH), and plasma-free hemoglobin levels. The patient was later found to harbor a pathogenic variant in the CFB gene (C.1598A>G), and was diagnosed with aHUS and acute kidney injury.Intervention:The patient was treated by plasmapheresis, continuous renal replacement therapy, blood transfusion, and anti-infective and antihypertensive treatment.Outcomes:After the treatment, the patient''s consciousness returned to normal, and the hemoglobin, platelet, and serum creatinine recovered. The disease activity remained quiescent during the follow-up.Lessons:A rare heterozygous variant c.1598A>G p.Lys 533Arg in the CFB gene, which was associated with adult-onset aHUS, was described and successfully treated. This case can help in understanding the early diagnosis and effective therapies of this rare disease.     

Angiotensin II type 2 receptor gene polymorphism and cardiovascular phenotypes: the GLAECO and GLAOLD studies

BACKGROUND: The angiotensin II type 2 (AT2) receptor is thought to play a role in cardiovascular disorders such as neointima formation after vascular injury, cardiac hypertrophy and myocardial infarction (MI). Recently, the biallelic polymorphism G + 1675A in intron 1 of the AT2 receptor gene has been associated with left ventricular posterior, septal and relative wall thickness, as well as left ventricular mass index in young hypertensive males. METHODS: To investigate its potential role in left ventricular hypertrophy (LVH) and other cardiovascular traits, 1968 individuals from two population samples (the Glasgow Heart Scan, GLAECO and Glasgow Heart Scan Old, GLAOLD studies) with echocardiographically and electrocardiographically assessed phenotypes, were genotyped for G + 1675A using allele-specific oligonucleotide hybridization. Both studies had a similar design, only the age-ranges differed, being 25-74 years in the GLAECO study and 55-74 years in the GLAOLD study, so that internal consistency of results could also be assessed. Since the AT2 gene is located on the X chromosome, males and females were analysed separately. RESULTS: The + 1675A allele frequency was 0.49 and 0.51, in the GLAECO and GLAOLD studies, respectively. In both studies, the genotype frequencies were similar in hypertensive and non-hypertensive individuals. In the GLAOLD study, in females with episodes of coronary ischemia and MI, the AT2 + 1675A allele was more common than in females with no episode (86.5% vs. 73.5%, respectively; P < 0.007). This effect was not observed in males. In the same study, AT2 + 1675A allele carriers were more common in males with LVH, than in those without LVH (60.3% vs. 46.0%, respectively; P = 0.047). This result was unchanged after exclusion of subjects taking antihypertensive drugs (including ACE inhibitors) (64.4% vs. 47.4%, P = 0.038). However, in the GLAECO study, these results could not be replicated, even when subjects > 55 years of age were considered separately. CONCLUSIONS: Our study gives rise to a potential implication of the AT2 G + 1675A polymorphism in LVH and coronary ischemia subgroups. Since these results were not consistent in both studies, but are partially in agreement with two independent investigations, further efforts should be made to elucidate the specific nature of these genotype/phenotype interactions.     

Angiotensin II type 2 receptor gene polymorphisms and cardioprotective role in essential hypertension

Angiotensin II type 2 receptor (AT2R) has been proved to be involved in a cardioprotective role, but only a few studies have addressed the association of AT2R-genotype with this role. Whether the AT2R genotype is associated with hypertension is controversial. The aim of the study was to explore the information of single-nucleotide polymorphisms (SNPs) of the AT2R gene in Cantonese, an essential subpopulation of Chinese, and study the association of SNPs in the AT2R gene with hypertension, and to detect the genotypes that indicate a cardioprotective role. Two hundred and sixty-two patients with essential hypertension and 75 normotensive subjects were enrolled for a case-control study. All of the subjects were Cantonese. Sixteen individuals were chosen to sequence the AT2R gene and 16 SNPs were acquired. G/T rs5193 and G/A rs5194 were two SNPs in the 3′ untranslated region which were focused on the association of the AT2R-genotype and phonotype. Polymerase chain reaction was performed to amplify the fragment spanning the two SNPs. Genotype and haplotype were identified by dot blot hybridization. Four haplotypes in males (G-G, G-A, T-A, T-G) and eight haplotype combinations in females (G-G/G-G, G-A/G-A, G-G/G-A, G-G/T-A, G-G/T-G, T-A/T-A, T-G/T-G, and T-A/T-G) were detected. G-G and G-A haplotype were predominant, while T-A and T-G were rare in Cantonese. None of these was associated with hypertension. T-A carriers with essential hypertension indicated lower levels of left ventricular mass (LVM) and left ventricular hypertrophy index (LVHI). The levels of LVM and LVHI were still significantly lower in T-A carriers with hypertension adjusted for age or body mass index for men and women separately. No episodes of coronary heart disease and heart failure were detected in T-A carriers with hypertension. Haplotypes of G/T rs5193-G/A rs5194 are not associated with essential hypertension. Among these haplotypes, T-A may be implicated in a cardioprotective role to protect hypertense subjects from left ventricular hypertrophy.