第1：先天出汗性外胚层发育不良患者中GJB6基因突变筛查第2：AMPD1基因在体细胞系和神经细胞瘤细胞系中表达的初步研究

发布时间：2018-04-14 12:25

本文选题：脱发 + 中国人群　；参考：《中南大学》2013年博士论文

【摘要】：研究背景：先天性疾病是一种在出生前或出生后一个月内发生的疾病。很多先天性疾病都是遗传物质异常所导致的遗传性疾病。先天性出汗性外胚层发育不良(Hidrotic ectodermal dysplasia, HED),也称为Clouston综合症,是一种常染色体显性遗传疾病,其特点是杵状甲、脱发以及手掌和脚底角质化。先天性杵状甲(或者杵状指)是一类临床上较少见的遗传性皮肤病,由于甲床和第三指节骨部分的连接组织增生,导致指甲板扩大和手指及脚趾终端分部。目前对于杵状甲还缺少具体可行的治疗方法,但是针对其病理症状进行治疗可能会减少杵状甲表型的发生；如果在疾病发生早期进行干预还有可能逆转病情,但目前尚缺少有效的手术治疗方法。先天性脱发的临床特征为正常头皮毛发不可再生性脱落,稀疏的眉毛和睫毛以及缺少腋毛、阴毛或其他体毛。有1-2%的先天性脱发患者会出现整个头皮甚至全部体表出现无毛特征。脱发病在有家族病史的家系中更易出现,这说明遗传因素可能是脱发病发生的一个诱因。对有2个或2个以上患者的家系的研究结果表明,基因异常能提高患病风险。针对不同脱发病型可采用不同的治疗对策,如果脱发病灶较小,一些治疗可能使头发重生脱发面积变小；但是对于严重脱发的患者,注射一些糖皮质激素如氯倍他索,醋酸氟轻松及皮质类固醇或者使用药膏的治疗方法对于脱发并没有明显的效果。类固醇药物的注射经常用来治疗一些小面积的脱发及眉毛缺失,但还不能确定其具体疗效。先天性毛发和指甲疾病一直以来就是具有未知遗传基础的一组罕见疾病,并研究较少。有研究指出称编码缝隙连接蛋白connexin30的GJB基因突变能导致此类疾病的发生。间隙连接蛋白-6(GJB6),或connexin30(Cx30),在人体中是由GJB6基因编码的蛋白质,是间隙连接复合物之一。GJB6基因位于13号染色体长臂1区1号带和1区2号带1亚带(13q11-q12.1)之间。间隙连接蛋白(Connexins)是一组结构相关的跨膜蛋白家族,可以装配形成脊椎动物的间隙连接。间隙连接由两个半通道或连接子构成,每个半通道或连接子由六个间隙连接蛋白装配组成。间隙连接在多细胞生物体中能够直接传递细胞间信号,它们由成簇的连接子构成通道集群,能使相邻细胞间的离子、营养物质以及小的代谢产物自由通过。外胚层上皮细胞及内耳的上皮细胞表达大量的间隙连接蛋白,其在协调角质细胞的生长和表皮细胞分化中扮演重要的角色；在内耳的听觉上皮细胞中,它能够回收听觉转导过程中流失的钾离子。间隙连接在多种生化反应中有至关重要的作用,如协调心脏肌肉去极化过程,胚胎发育以及毛细血管的应答。基于此,间隙连接蛋白基因的突变可能导致机体功能及发育的异常。在本研究中,我们对一个来自中国人群的先天性指甲发育不良以及脱发和掌跖角化过度家系进行GJB6的突变筛查,寻找其遗传病因。我们有望通过鉴定GJB6基因的突变引入快速的分子诊断和新型药物的治疗策略,在未来,可能有助于找到治疗这种罕见遗传性疾病的方法。研究方法：一个患有杵状甲和脱发的中国家庭,其包括四个患者和两个正常人,被选为研究对象并签订知情同意书。通过家谱分析有力的证明了该疾病是常染色体显性遗传并且所有患病的成员是等位基因杂合突变。肝素化处理收集的外周血,用苯酚-氯仿提取基因组DNA,并十二烷基磺酸钠-蛋白酶K纯化DNA。用琼脂糖凝胶电泳检测DNA的提取质量。通过聚合酶链式反应(PCR)分别用引物：(1)5'-AGACTAGCAGGGCAGGGAGT-3'(上游)以及5’-GGAAAAAGATGCTGCTGGTG-3'(下游)；(2)5’-CCTCCAGCTGATCTTCGTCT-3'(上游)以及5’-GGTTGGTATTGCCTTCTGGA-3'(下游),扩增人类GJB6基因全长序列,产生1350bp和1250bp扩增片段。扩增后用浓度为1%的琼脂糖凝胶电泳分离PCR产物,纯化PCR产物,然后在ABI-PRISM3100自动测序仪上进行双向测序,所有的测序结果进行组装、分析并用SeqMan Ⅱ程序与野生型序列进行比对。结果：本研究调查了一个五代的有常染色体显性遗传性家族史的杵状指和脱发家系。所有的患者都有先天性的杵状指、脱发和掌跖角化等相似的临床症状。患者杵状指症状在出生时即发现,脱发在6岁时出现。体检发现患者头发、睫毛、眉毛及其他体毛出现缺失。没有发现患者有听觉、视觉异常,牙齿异常及精神异常。实验室各检测指标正常,患者寿命正常。我们对GJB6基因进行聚合酶链式反应扩增后,用琼脂糖凝胶电泳进行分离纯化。对这六个家系成员的GJB6基因进行测序,发现了一个杂合的错义突变c.31GA,这一变异导致了GJB6编码蛋白的第11位氨基酸从甘氨酸G变成了精氨酸R,这种变异可能使蛋白质的功能或结构发生转变从而引发了脱发和杵状指症状。这种在细胞质内存在的蛋白质CX30的氨基端的G11R的突变在4例患者中都存在。家系里的2个正常人中均未发现缺失、插入或错义突变。近年来,已发现了越来越多的连接蛋白参与的人类疾病的发生。连接蛋白的突变影响到多个器官和系统并与多种疾病的发生有关。另外,不同的连接蛋白突变能引起相同或相似的疾病。基因组研究不断地揭示与连接蛋白有关的疾病的突变,包括耳聋、皮肤病、中枢和外周神经病变、白内障以及心血管功能障碍等。GJB6基因的突变导致连接蛋白的组成和结构发生变化,它可能导致间隙连接的非正常转运活动。缝隙连接是细胞间水、离子及小分子交换的通道。它参与细胞间短程、快速的信息传递。由于这个位置上的突变在其他人群中也有过报道,所以我们没有进行后续的正常对照研究。结论：总结本研究的结果就是：“在GJB6基因(在蛋白质水平位G11R)中一个错义突变31G→A在中国人群的天生性HED患者中普遍存在”。为了对那些有患该病史的家族提供适当的诊断、遗传咨询和产前诊断,GJB6基因突变尤其是G11R等位基因是家族中患杵状甲和脱发所必须考虑的因素。GJB6基因的UTR区域和启动子区域也应该得到鉴定,研究突变体的调控效果和功能性分析将毫无疑问有助于了解GJB6基因表型变异性的机制,并综合先前对GJB6基因的研究,这些结果丰富了对连接蛋白功能特点的了解。然而,还需要研究突变的类型、突变的位置、组织学检查患病个体的候选基因以及鉴定世界范围内不同区域更多的患者。通过当前对HED疾病的分子生物学基础的了解,疾病起因于GJB6基因的突变,我们将引进一个新颖的对头发和指甲的各种疾病治疗的药物学或者基于基因的治疗方法,这类疾病的基因治疗方法在临床上可能不会立即实现,但是在此领域有效的研究无疑肯明了在未来能对该疾病有效管理。研究背景：孤独症是在一种开始于儿童36个月之前的通过其行为定义的综合征。自闭症的特点是在社会交往中存在普遍的缺陷,即语言和非语言沟通减少和刻板行为及狭隘的兴趣和活动。之前的研究中已报道过孤独症患者存在一些神经性病变包括海马体、杏仁核、小脑和大脑皮质的异常等。孤独症是一种遗传性疾病,但是它可能由多种基因缺陷造成。这些缺陷的基因可能与大脑发育、信号传导、转运或者细胞结构有关。有可靠的证据证明,孤独症的缺陷基因中有某些特定的基因能够造成患者的代谢异常,即酶分子的缺陷会造成异常的酶促反应速率,导致代谢底物或产物的浓度异常。尽管在孤独症病人中代谢异常者所占比例未知,但是已有许多研究证明一些代谢缺陷和孤独症有关,包括苯丙酮尿症、组氨酸血症、肌酸缺乏综合征、腺苷酸琥珀酸裂解酶缺陷症、5’-核苷酸酶高活性和嘌呤代谢紊乱等。腺苷酸琥珀酸裂解酶缺乏症是一种常染色体隐性遗传疾病,嘌呤合成异常导致体液中积累琥珀酰嘌呤；该疾病约有一半的患者具有孤独症的特征,80%伴有癫痫。磷酸腺苷脱氨酶(Adenosine monophosphate deaminase, AMPD)或肌腺苷酸脱氨酶,是在高等真核生物中的一种复合变构酶。单磷酸腺苷脱氨酶即AMPD1,是AMPD家族中具有肌肉组织特异性的一种酶类。AMPD1基因一共有16个外显子,在基因组中约占20kb,除了2号外显子是由12个核苷酸组成的,每个外显子大小在101至220个核苷酸之间。AMPDl基因在1号染色体短臂(p)1区3带至2区1带之间(1p13-p21)。AMPD1在小鼠中编码同工酶A,在人类中编码异构体M。在骨骼肌中,AMPD1能够催化腺苷单磷酸(AMP)的脱氨基作用,产生肌苷单磷酸(IMP)和氨(NH3)。 AMPD1也在嘌呤核苷酸循环(PNC)中起重要作用。在骨骼肌中,PNC中产生的富马酸参与三羧酸循环循环(TCA),最终导致ATP水平的上升。在我们的前期研究中,采用全基因组关联研究(GWAS)的方法来研究了孤独症易感的单核苷酸多态性位点(SNP)。在AMPD1基因相邻位点发现了一处易感SNP,这一发现表明AMPD1基因可能是孤独症易感的基因。在本研究中,我们研究了AMPD1在体细胞HEK293和神经母细胞瘤细胞SH-SY5Y中的分布。希望这个研究能够对突变或者生物工程合成蛋白在动物模型中的表达,以及其他研究中有帮助。研究方法：使用Trizol-氯仿-异丙醇的方法从成年小鼠肌肉组织中提取总RNA。用Oligo (dT)18引物逆转录合成cDNA,进而进行AMPD1基因的RT-PCR。 cDNA为模板进行AMPD1真核表达载体的构建。以AMPD1基因全长cDNA的扩增引物为：正向引物5'-ATCCGGAATTCAATGCCTCTATTCAAACTAACAGGTC-3', EcoRI为限制性酶切位点；反向引物5'-ATGCGGGATCCTCATTCTGTTGCTTTAAGACCCTCA-3', BamHI为限制性酶切位点。进行PCR之后,对DNA样本进行1%琼脂糖凝胶电泳；切下目的片段后用Qiagen胶回收试剂盒纯化。使用Qiagen质粒mini试剂盒抽p3Xflag载体。将mAMPD1的PCR片段和p3Xflag空载体用EcoRI和BamHI进行双酶切。酶切产物进行使用纯化,并将酶切后的PCR片段和空载体按照比例连接。连接产物转进感受态大肠杆菌后涂板。挑选单克隆在Amp+LB培养基中进行培养,碱裂解法抽提细菌质粒后,进行EcoRI和BamHI的双酶切鉴定。阳性克隆进行测序鉴定。重组的质粒p3Xflag-mAMPD1使用Lipo2000转染进HEK293和SH-SY5Y细胞系中,用免疫组化的方法使用Flag标记和内质网的标记蛋白的抗体对转染后代细胞进行荧光标记,研究AMPD1的表达的细胞分布。结果：在p3Xflag-mAMPD1质粒构建中,使用小鼠肌肉组织cDNA为模板进行PCR扩增mAMPDl片段。PCR产物用1%琼脂糖凝胶电泳分离,切胶纯化。p3Xflag (6.4Kb)转化进感受态细胞中,然后提取质粒。纯化的PCR产物(97. Ong/ml)以及提取的质粒(p3Xflag,237.7ng/ml)进行酶切,使用限制性内切酶EcoRI和BamHI。mAMPD1(27.lng/ml)和p3Xflag (23.4ng/ml)的酶切产物进行连接,然后转进E. coli DH5α感受态细胞进行扩增。随机选取单克隆,通过测序检查插入片段。转染进质粒的HEK293细胞免疫荧光染色发现AMPD1均匀的分布于胞浆不在细胞核分布。另一个细胞系,SH-SY5Y的转染及免疫荧光结果也证明AMPD1的胞浆细胞分布。结论：在本研究中,我们检测了AMPD1在HEK293和SH-SY5Y细胞系中的分布情况,发现AMPD1特异性地分布在胞浆中,这是我们关于孤独症相关基因的表达研究,动物以及临床研究的初步结果。
[Abstract]:Background: congenital disease is a kind of a month before birth or after birth in the occurrence of the disease. Many congenital diseases are hereditary disease genetic abnormalities caused by congenital hidrotic ectodermal dysplasia (Hidrotic ectodermal, dysplasia, HED), also known as Clouston syndrome, is a constant autosomal dominant genetic disease, which is characterized by clubbing, hair loss and the palms and soles of the feet horny. Congenital clubbed nail (or clubbing) is a clinically rare hereditary skin disease due to connective tissue hyperplasia and third knuckle bone nail bed portion of the nail plate and fingers and expand the lead terminal segment. The toes clubbed a lack of specific and feasible treatment method, but according to the pathological symptoms of the treatment may reduce the occurrence of a clubbing phenotype; if the disease occurs in early intervention and Can reverse the disease, but there is a lack of effective surgical procedures. The clinical features of congenital alopecia is normal scalp hair not renewable shedding, sparse eyebrows and eyelashes and lack of armpit hair, pubic hair or other body hair. 1-2% congenital alopecia patients may have a whole head skin surface appeared glabrous or all alopecia features. It is easy to appear in a family history of the family, suggesting that genetic factors may be a cause of hair loss disease. On 2 or more than 2 of patients with family. The results showed that the abnormal gene can increase the risk of illness. According to the different type of alopecia can be treated by different strategies, if alopecia lesions are smaller, some treatment may make the hair rebirth alopecia area smaller; but for the patients with severe alopecia, injection of glucocorticoids such as clobetasol, fluocinonide and corticosteroids or. Methods of treatment with ointment for hair loss and no obvious effect. Steroid injections are often used to treat hair loss and eyebrows missing some small area, but also can not determine the specific effect.
A group of rare congenital diseases of hair and nails has always been disease with unknown genetic basis, and less research. Studies have shown that GJB gene that encoding connexin connexin30 mutations can lead to such diseases. The gap junction protein -6 (GJB6), or connexin30 (Cx30), in the human body is composed of GJB6 gene encoding protein is one of the gap junction complex.GJB6 gene is located on the long arm of chromosome 13 in 1 District No. 1 zone and 1 Zone No. 2 with 1 sub zones (13q11-q12.1). The gap junction protein (Connexins) is a transmembrane protein family group structure, the formation of the vertebrate gap assembly connection. Gap junction structure by 2.5 channel or connector, each half channel or connector is composed of six connexin assembly. Gap junctional intercellular signal can directly transfer in multicellular organisms, they consist of clusters even Then a channel cluster, can make ion between adjacent cells, nutrients and small metabolites free passage. Ectodermal epithelial cells and inner ear epithelial cells express large gap junction protein, plays an important role in the coordination of keratinocyte growth and differentiation of epidermal cells; in the inner ear auditory epithelial cells. It can recover loss of potassium ions in the process of auditory transduction. Gap junction plays a crucial role in many biochemical reactions, such as the coordination of the heart muscle depolarization process, embryonic development and capillary responses. Based on this, the mutation of gap junction protein genes may cause the body function and developmental abnormalities.
In this study, we performed mutation screening of GJB6 from a China population of congenital hypoplastic nails and hair loss and palmoplantar hyperkeratosis pedigree, find its genetic etiology. We expected treatment strategy, the introduction of molecular rapid diagnosis and new drug through the identification of GJB6 gene mutation in the future may help find a cure for this rare genetic disease.
Methods: with a clubbed nail and hair loss Chinese family, including four patients and two normal people were selected as the research object and signed informed consent. The pedigree analysis proves that the disease is autosomal dominant and all affected members are alleles of heterozygous mutations. Heparinized peripheral blood was collected. Genomic DNA was extracted by phenol chloroform extraction, and the quality of twelve sodium dodecyl sulfate - protease K DNA. purified by agarose gel electrophoresis. The detection of DNA by polymerase chain reaction (PCR) respectively with primers: (1) 5'-AGACTAGCAGGGCAGGGAGT-3'(upstream) and 5 -GGAAAAAGATGCTGCTGGTG-3' (downstream) (; 2) 5 'and 5' -CCTCCAGCTGATCTTCGTCT-3'(upstream) -GGTTGGTATTGCCTTCTGGA-3' (downstream), amplified the full-length sequence of human GJB6 gene, 1350bp and 1250bp amplified fragment. After amplification with concentration of 1% June The separation of PCR lipid products of sugar gel electrophoresis. The purified PCR products were then sequenced on automatic ABI-PRISM3100 sequencer, all sequencing results were assembled, analyzed and compared with the sequence of SeqMan II program with the wild type.
Results: This study investigated a five generations with autosomal dominant familial history of clubbing and alopecia family. All patients had congenital clubbing, hair loss and clinical symptoms similar to patients with palmoplantar keratoderma. Acropachy symptoms at birth, alopecia in 6 when appear. Physical examination found that patients with hair, eyelashes, eyebrows and hair loss. Other patients have found no abnormal vision, hearing, and mental disorders. The abnormal dental lab index of normal patients, normal life. We were amplified by polymerase chain reaction of GJB6 gene was isolated and purified by agarose gel electrophoresis for sequencing. The GJB6 gene of the six family members, found a heterozygous missense mutation c.31GA, the mutation causing GJB6 encoding proteins of eleventh amino acids into arginine glycine from R G, this variant may The protein function or structure change, causing hair loss and acropachy symptoms. This mutation exists in the cytoplasm of the CX30 protein N-terminal G11R exists in 4 patients. 2 normal people in the Department were not found in deletion, insertion or missense mutation. In recent years, has found more and more connected proteins involved in human disease. Connecting protein mutation affects multiple organs and systems and related to many diseases. In addition, connexin mutations can cause similar disease. Different genomic studies continue to reveal and mutation, including protein related diseases connected deafness, skin disease, central and peripheral neuropathy, cataract and cardiovascular dysfunction such as mutation of.GJB6 gene leads to the composition and structure of connexin changes, it may lead to abnormal gap junction Transport activity. Gap junctions are intercellular water, ions and small molecular exchange channels. It is involved in cellular short-range, rapid transmission of information. Because of this position mutations in other populations have also been reported, so we do not have a normal follow-up study.
Conclusion: the results of this study is: "in the GJB6 gene (at the protein level G11R) mutation in a missense 31G, A exists in Chinese people born in HED patients. For those who have had the history of the family to provide the appropriate diagnosis, genetic counseling and prenatal diagnosis, GJB6 gene especially the G11R mutation allele of UTR region and the promoter region of.GJB6 gene family factors with clubbed nail and hair loss that must be considered should be identified, control effect and functional study of mutant analysis will no doubt help to understand the mechanism of GJB6 gene phenotypic variability, and prior to GJB6 genetic studies, these results enrich the understanding of connexin features. However, still need to study types of mutations, mutation location, candidate gene organization examination and identification of affected individuals worldwide Patients in different regions. The more the molecular basis of the current understanding of the HED disease, the disease causing mutation in the GJB6 gene, we will introduce a novel on the hair and nails in treating various diseases pharmacology or gene based treatment, this kind of disease gene therapy may not be immediately implemented in clinically, but effective research in this field is willing in the future to the effective management of the disease.
Background: autism is in a beginning in the 36 months before the children through syndrome definition. Its behavior characteristic of autism is a common defect in social interaction, namely, language and non language communication and reduce the stereotyped behavior and narrow interests and activities. The research has been reported before in autism there were some nerve lesions including the hippocampus, amygdala, cerebellum and cerebral cortex abnormalities. Autism is a genetic disease, but it may be caused by a variety of genetic defects. These defects of genes may be associated with brain development, signal transduction, cell or the transportation structure. There is reliable evidence of autism gene defects in certain genes can cause patients with metabolic abnormalities, including defects of enzyme molecules will cause abnormal enzymatic reaction, resulting in concentration of metabolic substrate or product as abnormal. In patients with autism tube metabolic abnormalities in the proportion of unknown, but many studies prove that some metabolic defects and autism, including phenylketonuria, histidinemia, creatine deficiency syndrome, adenylosuccinate lyase deficiency, 5 '- nucleotidase activity and high purine metabolism. Adenylosuccinate lyase deficiency disease is an autosomal recessive genetic disease, abnormal accumulation of purine synthesis of succinyl purine in body fluids; about half of the patients with autism characteristics of the disease, 80% with epilepsy.
Adenosine monophosphate deaminase (Adenosine monophosphate, deaminase, AMPD) or myoadenylate deaminase, is a complex allosteric enzyme in higher eukaryotes. Adenosine monophosphate deaminase is an enzyme that is AMPD1,.AMPD1 gene has a muscle specific AMPD in the family of a total of 16 exons in the genome of about for 20KB, in addition to exon 2 was composed of 12 nucleotides, each exon size between 101 to 220 nucleotides of.AMPDl gene on the short arm of chromosome 1 (P) 1 to 2 District 1 District 3 zone between (1p13-p21).AMPD1 code of A isoenzyme in mice and in humans encoding M. isoforms in skeletal muscle, AMPD1 can catalyze adenosine monophosphate (AMP) deamination, produce inosine monophosphate (IMP) and ammonia (NH3). AMPD1 is also in the purine nucleotide cycle (PNC) plays an important role in skeletal muscle, fumaric acid produced in the PNC in the three cm Acid circulation (TCA), resulting in increased levels of ATP. In our previous study, a genome-wide association study using (GWAS) method to study the polymorphism of autism susceptibility (SNP). The AMPD1 gene between adjacent sites found a susceptible SNP, which indicates a the AMPD1 gene may be autism susceptibility genes. In this study, we investigated the distribution of AMPD1 in HEK293 cells and neuroblastoma cells in SH-SY5Y. Hope this research can for mutations or protein synthesis of biotechnology in animal models of expression, as well as other studies in help.
Method: using Trizol- chloroform to extract the total RNA. from the muscle of adult mice with Oligo (dT) 18 primer cDNA was synthesized, and then AMPD1 gene RT-PCR. cDNA AMPD1 eukaryotic expression vector as template. The full-length cDNA of AMPD1 gene amplification primer: forward primer 5'-ATCCGGAATTCAATGCCTCTATTCAAACTAACAGGTC-3', EcoRI as the restriction enzyme site; reverse primers 5'-ATGCGGGATCCTCATTCTGTTGCTTTAAGACCCTCA-3', BamHI restriction sites. After PCR, 1% agarose gel electrophoresis of DNA samples; cut fragment with pure Qiagen Gel Extraction Kit

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R758.71;R730.264

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