用DHPLC技术进行单纯性先天性心脏病易感基因SORBS2的突变分析

发布时间：2018-04-05 01:21

本文选题：变性高效液相色谱　切入点：SORBS2基因　出处：《南方医科大学》2012年硕士论文

【摘要】：背景与目的：随着现代社会科学技术的发展,以及医学模式的进化,人类疾病谱和死亡谱已经发生了很大改变,出生缺陷已逐渐成为我国婴幼儿死亡的主要原因。先天性心脏病(Congenital Heart Disease, CHD)是胎儿时期心血管发育异常而导致的先天畸形,是出生缺陷中最常见的类型,几乎占据了主要出生缺陷的三分之一,在新生儿非感染性死亡中占首位。广东省优生优育协会2009年对广东省0-5岁儿童出生缺陷情况调查结果显示,先天性心脏病是广东地区最常见的出生缺陷,已成为严重影响儿童身心健康及人口生存质量的重大公共卫生问题,给社会和家庭带来严重的经济和精神等方面的负担。先天性心脏病中仅表现为心脏畸形而不伴有其他系统的先天异常,称之为单纯性先天性心脏病(non-syndromic CHD),约占80%。随着分子遗传学和分子生物学技术的发展,近些年来的研究证实,该病是与环境因素相关的多基因遗传病,其遗传背景复杂,致病机制尚不明确,且其遗传因素在疾病发生过程中存在着明显的地区差异和种族差异。由于发病率极高且患病后果严重,因此,研究该病的致病机制与易感基因试图建立行之有效的疾病筛查和诊断技术可及早预防重型先天性心脏病患儿的出生,以期降低该病的发病率,不仅可以减少患儿家庭负担,而且对提高我国人口素质和人口质量具有重要意义。心脏发育是胚胎发育过程中一个及其复杂的事件,许多基因按照时空顺序的表达调控着心脏的正常发育,其中任何一个基因的异常都有可能导致先天性心脏病的发生。单纯性CHD的致病基因很多,它们之间存在相互影响和协同调节,在心脏发育的不同时期有着不同的表达与调节。并且与许多和机体生长发育、能量代谢、细胞周期、细胞骨架以及细胞黏附、细胞迁移、LIM蛋白、锌指蛋白等相关的蛋白基因在心脏发育中相互协调、相互作用,形成有序的网络系统。因而,心脏并不是一个静态的器官,将心脏的发育与身体其他器官分开来看是错误的；把儿童与成人的心脏疾患完全区分开也是有失公允的。心脏从开始发育,到最终成熟,再到成熟后的功能维护,是一个动态的、整体的过程。目前,对先天性心脏病的研究分为四个方面：配体受体如NOTCH1.NODAL等；转录因子如GATA4、NKX2.5、TFAP2B等心脏发育早期的重要转录因子；收缩蛋白如MYHl.ACTC等,其他复杂基因如FLNA.ELN等。尤以转录因子的研究最多,涉及到其他遗传学机制的分析报道较少,存在很大空白。心脏的发育涉及多种基因的相互调控与影响,而且与这些基因在不同时间和不同空间的先后表达和相互作用有关,其中任何基因表达的质或量的异常也可能会影响心脏的发育。在心脏表达的蛋白很多,其中衔接蛋白就属于其中一类,而我们对于这一类蛋白在心脏中的表达调控功能还知之甚少。衔接蛋白由两个或两个以上的没有酶活性的蛋白质相互作用组成,调节各种细胞功能。VInexin,CAP/ponsin,与SORBS2(ArgBP2)构成一个新的衔接蛋白家族,它们有与活性肽sorbin同源的SoHo保守区域,以及3个SH3结构域(Src同源3)。与该衔接蛋白家族相联系的一部分蛋白已经确定。有越来越多的证据表明,该蛋白家族在调节细胞扩增,迁移,黏附,凋亡,细胞骨架和信号转导方面发挥重要作用。其中的soRBS2蛋白在心脏大量表达,它的蛋白产物表达在心肌肌原纤维的Z带中,对于装配和维持心肌肌原纤维的稳定性、附着性有重要作用。 sORBS2又名ArgBP2,是一个70kD的在非肌肉细胞中表达于应力纤维,在心脏表达于Z带的蛋白质。SORBS2定位于4q35.1,共有八种亚型,是通过不同剪接方式获得的,分别由492到1100aa的氨基酸组成。其中大量表达于心脏的亚型1全长226794bp,cDNA为4939bp,编码666个氨基酸,由23个外显子,22个内含子组成。 SORBS2除了在心肌纤维的Z带发挥作用之外,还有很多其他功能。通过羧基末端的SH3结构域与非受体酪氨酸蛋白激酶家族成员c-Abl相互作用,通过氨基末端的SoHo同源结构域与脂筏蛋白相互作用。由该蛋白在上皮和心肌亚细胞定位可知,其作为衔接蛋白在应力纤维中装配信号复合体；在肌动蛋白细胞骨架和细胞运动性中发挥重要调控作用；在锚定连接中,与桥粒黏着斑蛋白协同作用；结合泛素连接酶Cbl,非受体酪氨酸激酶c-Abl可磷酸化Cbl,激活的Cbl可以促进Abl的降解,发挥信号调节作用；或者与ATK1复合介导PAK1通路的激活；通过抑制细胞黏附,细胞迁移等作用抑制肿瘤细胞转移。 SORBS2基因在细胞骨架的稳定中有重要作用,作为支架蛋白,通过调节多种集中在细胞骨架的细胞信号通路控制细胞黏附和运动的平衡性。微管、微丝、中间丝及其结合蛋白构成了心肌细胞的骨架系统,它们在维持心肌细胞正常形态,调节心肌细胞等一系列生理活动中发挥着重要作用。在细胞内,细胞骨架成分将肌小节和细胞外基质连接起来,构成一个收缩力的传导环路,起着传导收缩力以及机械支撑的作用。心肌细胞骨架系统作为一种动态结构,不仅在维持心肌细胞形态,参与收缩活动中发挥作用,而且对心肌离子通道的锚定作用和功能方面有着重要的调控作用。细胞骨架变化往往会引起心肌缺氧、心肌肥厚及心力衰竭等病理性进程。细胞骨架结合蛋白种类繁多,功能复杂,而衔接蛋白恰是其结合蛋白中的一种,其确切功能大部分还不清楚。研究细胞骨架,骨架结合蛋白及其与心肌离子通道的关系对于阐明多种心律失常的发生机制有重要的理论和实际意义。因为心脏是一个动态的、整体的器官,大量不同种类的基因表达在其中,调控着心脏的正常发育与成熟,以及成熟后的功能维护。SORBS2作为衔接蛋白,在信号转导、心肌肌原纤维的稳定和细胞骨架调控等方面的作用,让我们将之与心脏的发育联系起来,并且进一步与先天性心脏病的形成联系到一起。近年来,一种具有高效、精确、高通量、半自动化、价格低廉等优点的用于检测已知突变及未知突变筛查,敏感性和特异性高达96%-100%的方法,变性高效液相色谱(Denaturing High Performance Liquid Chromatography, DHPLC)技术被广泛的应用。与同样是用于突变筛查的其他变异检测技术相比,变性高效液相色谱有着明显的优势。本研究主要是通过定点诱变技术与基因克隆技术来构建SORBS2基因的已知变异,并针对SORBS2基因的所有编码区及剪接位点来设计变性高效液相色谱检测方法,并用该检测方法筛检中国人群单纯性先天性心脏病病例以期找到中国人SORBS2基因的相关数据。本研究旨在为进一步研究单纯性先天性心脏病分子机制,建立一种快速、准确、经济且适于中国人群中国南方地区单纯性先天性心脏病相关基因突变的研究方法,并为临床上该类疾病的诊断、预防和治疗提供参考,同时也为SORBS2基因突变导致的其他衔接蛋白异常疾病的分子机制的研究提供借鉴和参考。材料与方法： 1.标本：共收集209例先天性心脏病病例(男性121例、女性84例、未知4例,年龄为0-6个月)和120例正常人的全血标本,这些标本大多来自于中山博爱医院和珠江医院等。采用标准的饱和酚／氯仿法从外周血中提取基因组DNA。 2.分子分析方法： (1).针对NCBI数据库中SORBS2的基因变异采用大引物PCR方法定点诱变和基因克隆方法：首先通过设计定点诱变引物,采用PCR技术直接从人基因组DNA样品中获取目的基因——含某一变异的基因,然后运用经典的基因工程技术将其克隆至pMD20-T载体,转化的宿主菌为大肠杆菌DH5α。每个克隆重组子中插入的目的基因进行DNA测序以验证基因变异型。 (2).针对SORBS2基因的所有编码区及剪接位点设计变性高效液相色谱分析的引物,构建SORBS2基因的变性高效液相色谱分析方法。 3.统计学分析：用建立的SORBS2基因变性高效液相色谱分析方法对构建的包含SORBS2基因已知变异的人工变异体进行稳定性分析,确定该体系的稳定性和可靠性。 4.标本筛检：对所收集的标本进行SORBS2基因突变筛查。 5.实验结果的综合分析、总结。结果：运用大引物PCR定点诱变方法人工构建包含SORBS2基因已知变异的变异体,并将诱变得到的片段克隆到pMD20-T-载体后进行测序,所有克隆均鉴定为所需变异,诱变成功率达100%。建立的针对SORBS2基因的变性高效液相色谱分析方法能够快速、准确地区分已知变异序列和野生型序列,并且灵敏性和稳定性都好。收集209例单纯性先天性心脏病和120例正常人标本,对这些标本进行SORBS2基因的变性高效液相色谱分析检测,未检测到定点诱变的已知变异,也未发现新的致病突变,只检测到4个已知的单核苷酸多态性(SNP)和两个未报道过的位于内含子的基因变异。结论：变性高效液相色谱分析技术是近几年来被广泛应用的基因突变检测技术。这种检测方法不受突变碱基位点与类型的局限,无需序列特异性探针,无需纯化,在PCR结束后直接进行变性高效液相色谱检测,即可完成对样品基因型的分析。这种方法因其操作简便、快速,使用成本低,结果准确,并且灵敏度高而受到普遍的关注和广泛应用。本课题研究中建立的针对SORBS2基因的变性高效液相色谱分析检测方法能够快速、准确地将SORBS2基因变异序列与野生型序列区分,并且实验的重复性和再现性好、稳定性高,体系可靠。本研究建立的基于PCR的变性高效液相色谱分析SORBS2基因突变方法是一种高通量、高灵敏度、半自动化、快速、准确、经济的突变检测技术。近年来,衔接蛋白家族的功能不断被发掘,其中的SORBS2因在心脏中大量表达,而被考虑是否与先天性心脏病的形成相联系,SORBS2基因的致病突变还未有被报道,也没有对SORBS2基因进行人群突变筛查的研究。本研究中,利用变性高效液相色谱分析技术对中国人先天性心脏病病例标本进行突变筛查,没有发现新致病突变,这也说明了中国人SORBS2基因突变的携带率相对较低。先天性心脏病作为一种与环境因素相关的多基因疾病,虽然已经有超过40种基因被报道与先天性心脏病的形成有关,然而对于它的致病机理,我们仍然只有浅显而初步的认识。已经报道过的可能与先天性心脏病形成有关的致病基因中有很大一部分,都只在一些零星的散发病例中被发现。目前,国内外对先天性心脏病的病因分析主要认为是转录因子突变造成的,涉及到其他遗传学病因分析的报道较少。根据PubMed等文献检索数据库的检索结果,未见有关中国人群SORBS2基因突变导致先天性心脏病的报道。因此本研究对先天性心脏病的遗传学病因的研究具有重要的意义和价值。综上所述,虽然SORBS2在心脏中大量表达,但其对单纯性先天性心脏病的病因影响不大。我们猜测,SORBS2可能对心肌病变和细胞骨架变化引起的其他心脏疾病可能有着一定程度的影响,这将是我们后续研究中的一个重要方向。本研究不仅为SORBS2基因的检测建立了一种简便、经济、快速、高效和灵敏的检测方法,而且对进一步阐明中国人先天性心脏病的分子机制,对临床上该病的诊断、预防和治疗具有重要的参考意义,同时也为SORBS2基因突变导致的其他衔接蛋白异常疾病的分子机制的分析提供了借鉴。
[Abstract]:Background and Purpose :

With the development of modern society science and technology , and the evolution of medical model , the human disease spectrum and death spectrum have changed greatly . birth defects have become the main cause of infant death in China . Congenital heart disease ( CHD ) is the most common type in the birth defect , which accounts for the most common birth defects in Guangdong region .

With the development of molecular genetics and molecular biology , it is proved that the disease is a multi - gene genetic disease associated with environmental factors .

Cardiac development is one of the most complex events in the development of the embryo . Many genes regulate the normal development of the heart according to the expression of space - time sequence .
It is also unfair to distinguish children from the heart of adults . The heart starts to develop , eventually mature , and then to mature functional maintenance is a dynamic , integral process .

At present , the study of congenital heart disease is divided into four aspects : ligand receptor such as NOTCH1 . NODAL , etc . ;
Transcription factors such as GATA4 , NKX2.5 , TFAP2B and other important transcription factors in the early stages of cardiac development ;
There are many other complex genes , such as MYHl . ACTC , etc . , and other complex genes such as FLNA , ELN , etc .

SORBS2 , also known as ArgBP2 , is a 70 kD protein expressed in non - muscle cells in non - muscle cells and expressed in Z - band in the heart .

SORBS2 , in addition to its role in the Z - band of cardiac muscle fibers , has a number of other functions . By interacting with the non - receptor tyrosine protein kinase family member c - Abl through the SH3 domain at the carboxy terminus , the SoHo homology domain at the amino terminus interacts with the lipid raft protein . It is known from the localization of the protein in the epithelial and myocardial sub - cells , which act as an adapter protein in the stress fiber assembly signal complex ;
plays an important role in regulating the cytoskeletal and cell motility of actin ;
in that anchor connection , the bridge particle has a synergistic effect with the protein spot ;
In combination with ubiquitin ligase Cbl , non - receptor tyrosine kinase c - Abl can phosphorylate Cbl , and activated Cbl can promote the degradation of Abl .
or the activation of PAK1 pathway is mediated by combination with ATK1 ;
inhibiting tumor cell metastasis by inhibiting cell adhesion , cell migration , and the like .

Microtubules , microfilaments , intermediate filaments and their binding proteins play an important role in maintaining normal morphology of myocardial cells , regulating cardiac muscle cells and other physiological activities .

Because the heart is a dynamic , integral organ , a large number of different kinds of genes are expressed in it , regulate the normal development and maturation of the heart , as well as the functional maintenance after maturation . SORBS2 plays a role in signal transduction , stabilization of cardiac muscle fiber and regulation of cytoskeletal muscle , etc . , and links it with the development of the heart , and further relates to the formation of congenital heart disease .

In recent years , a method for detecting known mutations and unknown mutation screening , sensitivity and specificity up to 96 % -100 % , which has the advantages of high efficiency , accuracy , high flux , semi - automation , low price and so on , is widely used in the detection of known mutation and unknown mutation screening , sensitivity and specificity up to 96 % -100 % .

The aim of this study is to study the molecular mechanism of simple congenital heart disease and to establish a rapid , accurate , economical and suitable method for the diagnosis , prevention and treatment of congenital heart disease in southern China .

Materials and Methods :

1 . Specimens : 209 cases of congenital heart disease ( 121 male , 84 female , 4 unknown , 0 - 6 months old ) and 120 normal controls were collected from peripheral blood by standard saturated phenol / chloroform method .

2 . Molecular analysis method :

( 1 ) For the gene mutation of SORBBS2 , the gene mutation and gene cloning method of SORBS2 were determined by PCR . The gene was cloned into pMD20 - T vector directly from human genomic DNA sample by PCR . The transformed host bacteria was E . coli DH5.alpha . The gene inserted in each clone was sequenced to verify the gene variant .

( 2 ) A denaturing high - performance liquid chromatography analysis method for SORBS2 gene was constructed for all coding regions and splice sites of SORBS2 gene .

3 . Statistical analysis : The stability and reliability of the system were determined by analyzing the stability of the constructed artificial variants containing the known variation of SORBS2 gene by means of the established SORBS2 gene denaturing high - performance liquid chromatography .

4 . Screening of specimen : The SORBS2 gene mutation screening was performed on the collected specimen .

5 . Comprehensive analysis and summary of experimental results .

Results :

The variants of the known mutation of SORBS2 gene were constructed artificially by using a large - primer PCR constant - point mutation method , and the fragment obtained from the mutation was cloned into pMD20 - T - vector for sequencing . All the clones were identified as the required variation , and the success rate of mutagenesis was 100 % .

The established denaturing high - performance liquid chromatography analysis method for SORBS2 gene can rapidly and accurately distinguish the known mutation sequence and the wild - type sequence in the region , and has good sensitivity and stability .

209 cases of simple congenital heart disease and 120 normal human specimens were collected . The denaturing high performance liquid chromatography ( HPLC ) analysis of SORBS2 gene was performed on these specimens . No known mutation was detected and no new pathogenic mutation was detected . Only 4 known single nucleotide polymorphisms ( SNPs ) and two unreported genetic variants were detected .

Conclusion :

denaturing high performance liquid chromatography ( HPLC ) is a widely used technique for gene mutation detection in recent years . This method is not limited by mutation base site and type . It does not need to be purified . After PCR is completed , it can be directly subjected to denaturing high performance liquid chromatography detection to complete the analysis of the genotype of the sample .

The method of denaturing high - performance liquid chromatography analysis for SORBS2 gene which was established in this research was able to quickly and accurately distinguish SORBS2 gene mutation sequence from wild - type sequence , and the repeatability and reproducibility of experiment were good , stability was high , and the system was reliable . The research was based on PCR - based denaturing high - performance liquid chromatography SORBBS2 gene mutation method was a high - throughput , high - sensitivity , semi - automatic , fast , accurate and economical mutation detection technique .

In recent years , the function of the adaptor protein family has been continuously explored , among which SORBS2 is expressed in large quantities in the heart , and is considered whether it is associated with the formation of congenital heart disease . The pathogenic mutation of SORBS2 gene has not been reported , nor does the SORBS2 gene have been screened for mutation screening . In this study , the mutation screening of Chinese congenital heart disease cases by denaturing high - performance liquid chromatography is not reported , and no new pathogenic mutation is found , which also shows that the mutation rate of the Chinese SORBBS2 gene is relatively low .

Congenital heart disease is a multi - gene disease associated with environmental factors , although more than 40 genes have been reported to be associated with the formation of congenital heart disease , yet we still have only a superficial and preliminary understanding of the pathogenesis of congenital heart disease . It has been reported that a large proportion of the pathogenic genes that may be associated with congenital heart disease have been reported to be found only in sporadic sporadic cases .

At present , the etiology of congenital heart disease at home and abroad is mainly considered to be caused by the mutation of the transcription factor , and the report of other genetic etiology analysis is less . According to the search results of the literature search database such as literature search , there are no reports about congenital heart disease caused by gene mutation of SORBBS2 in Chinese population . Therefore , the study has important significance and value for the study of genetic cause of congenital heart disease .

In conclusion , although SORBS2 is abundantly expressed in the heart , it has little impact on the etiology of simple congenital heart disease . We suspect that SORBS2 may have some degree of impact on the changes in cardiomyopathy and other heart diseases caused by changes in cellular skeleton , which will be an important direction in our subsequent study .

This study not only establishes a simple , economical , rapid , efficient and sensitive detection method for the detection of SORBS2 gene , but also has important reference significance for further clarifying the molecular mechanism of Chinese congenital heart disease , and provides reference for the diagnosis , prevention and treatment of the disease .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

【相似文献】