一中国汉族有汗型外胚叶发育不良家系的GJB6基因突变研究

发布时间：2018-03-24 01:06

本文选题：有汗型外胚叶发育不良　切入点：常染色体显性遗传　出处：《南京大学》2016年硕士论文

【摘要】：背景人体皮肤是由胚胎的两个胚层发育而来：表皮及其附属器是由外胚层分化而来,而真皮结缔组织是由中胚层发育而来。外胚叶发育不良是一类外胚叶来源的两个或多个组织结构性或功能性异常的遗传性疾病,其受累的器官主要包括头发、汗腺、牙齿、甲、皮脂腺以及粘液腺等。此类疾病可分为多种亚型,而不同亚型之间又可以通过排汗是否正常、受累部位以及遗传方式进行区分。有汗型外胚叶发育不良(hidrotic ectodermal dysplasia, HED) (OMIM 129500;)又称为Clous ton综合征,最早在一个法国-加拿大家系中发现并报道。有汗型外胚叶发育不良是一种常染色体显性遗传病,其发病率较低,为十万分之一,其主要的临床表现为毛发缺陷、甲营养不良和掌跖角化三联征,患者的牙齿及汗腺不受累,有些患者可以合并口腔黏膜白斑。这种常染色体显性遗传病的致病基因为位于人类第13号染色体上的GJB6 (gap junction beta 6)基因,其编码的蛋白质为缝隙连接蛋白Cx30。我们收集了一个中国汉族有汗型外胚叶发育不良的家系,该家系共有252名成员,其中包含患者45人,是迄今为止文献报道的最大的家系。我们收集并分析了该家系患者的临床信息,并采集了该家系中25名患者和14名正常者的外周血标本进行基因检测来确定致病突变。目的通过临床信息的采集整理分析该家系中有汗型外胚叶发育不良患者的临床表现的典型性与特殊性,并确定遗传方式。通过对该家系患者的GJB6基因检测确定致病突变。探讨该有汗型外胚叶发育不良家系患者基因型与表型的关系。方法收集该中国汉族有汗型外胚叶发育不良家系所有成员的临床信息,绘制家系图并分析遗传方式。留取自愿参与本研究的来自该家系的25名具有临床表现的患者以及14名无临床表现的正常成员的临床照片,采集39名自愿参与者的外周血样。并收集218名汉族未罹患有汗型外胚叶发育不良的正常人作为对照组。用TIANamp Blood DNA Kit (DP318)试剂盒提取DNA,对GJB6基因的外显区进行PCR扩增并测序。用Trizol试剂提取先证者头皮组织中的mRNA,然后用TaKaRa TaqTM Version2.0 plus dye试剂盒进行反转录成cDNA,对cDNA进行扩增测序以验证突变结果。对对照组218名汉族未罹患有汗型外胚叶发育不良的正常人进行GJB6基因突变的检测以排除致病突变的普遍性。结果通过对该有汗型外胚叶发育不良家系25名有临床表现的患者进行GJB6基因检测发现该基因第263位存在一个错义突变,胞嘧啶(C)被胸腺嘧啶(T)置换,由此,第88位氨基酸密码子GCG突变成GTG,导致丙氨酸(Ala)被缬氨酸(Val)替代,即A88V。这一突变通过对先证者组织中mRNA的反转录PCR扩增测序得到验证。在该家系参与本研究的14名无临床表现的成员和218名健康对照中均没有发现相同的碱基改变。因此认为GJB6基因的c.263 C→T(p.A88V)为该有汗型外胚叶发育不良家系的致病突变。通过对基因型及表型的分析,我们发现了一例该突变所致的HED特殊临床表现。结论GJB6基因的p.A88V突变为该中国汉族有汗型外胚叶发育不良家系的致病突变,并且在该家系中发现了该病的一个特殊表现患者。由于有汗型外胚叶发育不良的临床表现存在复杂性和多样性,因此基因检测是诊断该病最确切的证据。
[Abstract]:The background of the human skin is made up of two layers: the development of embryo and appendages is composed of ectodermal epidermal differentiation, and dermal connective tissue is composed of mesoderm and ectodermal dysplasia. Two or more organizations structural or functional class of ectodermal origin of different genetic diseases often. The involved organs including hair, teeth, sweat glands, sebaceous glands and mucous glands, etc.. This kind of disease can be divided into several subtypes, between different subtypes can be distinguished through perspiration is normal, the affected area and genetic. Hidrotic ectodermal dysplasia (hidrotic ectodermal, dysplasia, HED) (OMIM 129500; Clous) also known as ton syndrome, the earliest found and reported in a French Canadian family. Hidrotic ectodermal dysplasia is an autosomal dominant genetic disease, its incidence is low, one hundred thousand One, the main clinical manifestations of hair defects, nail dystrophy and palmoplantar keratoderma triad, teeth and sweat glands of patients are not affected, some patients with oral leukoplakia. This disease genes of autosomal dominant genetic disease is located on human chromosome thirteenth GJB6 (gap junction beta 6) gene, the encoding protein connexin Cx30. we collected a China Han hidrotic ectodermal dysplasia of the family, the family has 252 members, including 45 patients, is by far the largest family reported in the literature. We collected and analyzed the clinical information of this pedigree, and collected the pedigree in 25 patients and 14 normal peripheral blood samples were detected to identify the causative mutation. To analysis of sweat type in this family embryo Ye Fa through clinical information collection Dysplasia in patients with clinical manifestations of typical and special, and to determine the mode of inheritance. Through the detection of GJB6 gene in patients with this family identified pathogenic mutations. To explore the relationship between genotype and phenotype in patients with a poor family sweat type ectodermal. Methods the clinical information of Han Chinese sweat type ectodermal all members of poor families, drawing the pedigree map and genetic analysis. The clinical pictures taken voluntarily participated in this study from the family of 25 patients and 14 patients had no clinical manifestations of normal members, collecting 39 voluntary participants in peripheral blood samples were collected from 218 Han did not. With normal hidrotic ectodermal dysplasia as the control group. TIANamp Blood DNA Kit (DP318) DNA extraction kit, amplified by PCR and sequencing of GJB6 gene exon region. Using Trizol reagent from the proband head The skin tissue in mRNA, and then use the TaKaRa TaqTM Version2.0 plus Dye Kit for reverse transcription into cDNA, the cDNA mutation was amplified and sequenced to verify the results. The 218 patients in the control group were not Han with detection of GJB6 gene mutation to exclude common pathogenic mutations in normal human hidrotic ectodermal dysplasia results. The hidrotic ectodermal dysplasia family of 25 patients with clinical manifestations of GJB6 gene was detected and found that the 263rd genes have a missense mutation, cytosine (C) by thymine (T) replacement, thus, eighty-eighth amino acid codon mutation from GCG to GTG, leading to alanine (Ala) valine (Val) instead of A88V., the mutation based on the first card mRNA in reverse transcription PCR sequencing verified. In the study in the family of 14 without clinical manifestations and 218 healthy members are not as Find the same base change. So that the GJB6 gene of c.263 C, T (p.A88V) mutation for the hidrotic ectodermal dysplasia family disease. Through the analysis of genotype and phenotype, we found a case of the mutations of HED special clinical manifestations. Conclusion GJB6 gene mutation is the p.A88V Chinese Han hidrotic ectodermal dysplasia family mutation, and found a special manifestation in patients with the disease in this family. The clinical manifestations of hidrotic ectodermal dysplasia is complexity and diversity, so the diagnosis of the disease gene detection is the most definitive evidence.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R596.1

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