遗传性耳聋胚胎植入前基因诊断技术的建立应用及遗传性耳聋家系分子致聋机制研究

发布时间：2018-04-05 21:34

本文选题：胚胎植入前遗传学诊断　切入点：多重替代扩增技术　出处：《中国人民解放军军医进修学院》2011年博士论文

【摘要】：耳聋是一种严重影响人类生活质量的常见疾病,据我国2006年残疾人抽样调查结果显示听力残疾(含多重残疾)人共2,780万,并以每年新生3万聋儿的速度增长。因此,建立能够预防和减小后代耳聋再发风险的诊断方法和干预措施,提高防聋治聋的水平、降低人群中耳聋发病率对提高我国人口素质有着非常重要的现实和长远意义。高密度遗传标记的发展和应用为人类耳聋基因定位克隆和分子流行病学研究提供了强有力的工具。截止到2011年2月,共有166个非综合征型耳聋基因位点见诸报道(62个为常染色体显性遗传基因位点,96个为常染色体隐性遗传基因位点,8个位于X染色体,1个位于Y染色体),62个非综合征型耳聋基因和更多的综合征性耳聋基因成功克隆。在本研究中,我们应用单细胞全基因组多重替代扩增技术(Multiple displacement amplification, MDA)、单细胞巢氏PCR(polymerase chain reaction)、单细胞实时荧光定量PCR以及微卫星标记(microsatellite marker, STR)连锁分析等现代分子生物学技术建立了单细胞基因诊断的方法体系,并初步应用到遗传性耳聋的PGD临床工作中；另外以微卫星分子标记(STR)作为遗传标记,在一个常染色体显性非综合征型高频频感音神经性耳聋大家系中进行筛查并成功定位及筛查出致病基因；完成Treacher Collins综合征致病基因的突变筛查。本研究包括如下三部分：第一部分遗传性耳聋胚胎植入前基因诊断技术的建立和初步临床应用遗传性耳聋绝大多数为单基因特异性点突变,符合辅助生殖技术(in vitro fertilization, IVF)胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)医学指征,可有效避免传统中期妊娠异常胚胎的治疗性流产对患者本人和家属造成的身心伤害。本课题组朱玉华博士在前期研究中,鉴定了一个常染色体显性遗传非综合征型耳聋DFNA64家系的致病基因DIABLO,该家系的先证者夫妇在了解胚胎植入前遗传学诊断技术(PGD)可预防聋儿出生后,有进行此项研究的强烈意愿,并签署了知情同意书。通过对该家系先证者夫妇单淋巴细胞及他人废弃胚胎的单卵裂球进行单细胞巢氏PCR、单细胞全基因组多重替代扩增(MDA)及后续PCR测序、实时荧光定量PCR及微卫星标记连锁分析等研究,初步建立了遗传性耳聋胚胎植入前基因诊断技术体系。本课题组与北京医科大学第三医院辅助生殖中心刘平教授合作,对该夫妇进行了一次完整的PGD辅助生殖临床诊疗流程,诊断一个健康胚胎并植入母体子宫,但未获得成功妊娠。第二部分非综合征型遗传性耳聋家系致病基因的定位研究经表型及遗传方式分析,将收集的SD-L030家系判定为常染色体显性遗传非综合征型耳聋家系。选用微卫星STR分子标记作为遗传标记及经典连锁分析方法,对该家系进行了耳聋致病基因的定位、筛查和鉴定工作。SD-L030家系共4代,成员共30人(包括已故和配偶)是一个常染色体显性遗传性耳聋家系。应用微卫星标记对已知23个DFNA位点进行初步筛查,将该家系致聋基因定位于7p15处D7S629-D7S516之间约1.87cM的区域,与已知耳聋基因DFNA5所在区域重叠。直接测序在DFNA5基因的第7内含子中鉴定了一个新的DFNA5突变,IVS7-2AG,该突变与此家系表型共分离,通过提取外周血总mRNA并进行RT-PCR,发现该位点突变导致DFNA5第八外显子转录缺失。第三部分Treacher Collins综合征致病基因筛查及分子机制研究 Treacher Collins综合征(Treacher Collins syndrome, TCS)是一种常见的遗传疾病,发病率为1/50,000,主要表现为双侧颧骨骨结构发育异常造成颅面部的发育不全。本研究对两例TCS疑似病例进行了TCOF1基因的直接测序,一例患者携带TCOF1的第2外显子c.146 TC杂合突变,此突变位点将编码第49位的异亮氨酸替换为苏氨酸,对其父亲、母亲进行该位点突变筛查,未发现突变,该位点突变为一新生突变。本研究结果为该家系下一步遗传咨询和产前诊断的需求提供了资料和依据。同时对TCS发病机理及该病致病基因分子机制的研究进展做了深入的分析和总结。
[Abstract]:Deafness is a common disease seriously affect the quality of human life, according to a sample survey in 2006 showed that China's disabled hearing disability (including multiple disabilities) a total of 27 million 800 thousand, and the annual growth rate of 30 thousand new children. Therefore, diagnosis and intervention measures to establish prevention and to reduce the risk of recurrent offspring deafness, improve the anti treatment of hearing loss level, reduce the incidence of hearing loss in the crowd has a very important practical and long-term significance to improve the quality of our population.
The development and application of high density genetic markers provides a powerful tool for the research of cloning and molecular epidemiology of human deafness gene location. By the end of February 2011, there are 166 types of non syndromic deafness loci reported (62 autosomal dominant genetic loci, 96 autosomal recessive genetic loci, 8 located on the X chromosome, the 1 chromosome Y), 62 non syndromic deafness syndrome gene and gene cloning more success.
In this study, we used single cell whole genome amplification multiple alternative (Multiple displacement amplification, MDA), single cell nested PCR (polymerase chain reaction), real time fluorescent quantitative PCR single cell and microsatellite markers (microsatellite, marker, STR) and linkage analysis of modern molecular biology technology to establish the diagnosis method of single cell system gene, and its preliminary application in clinical work PGD hereditary deafness; in addition to microsatellite markers (STR) as genetic markers in an autosomal dominant non syndromic high-frequency sensorineural deafness pedigree screening and successfully mapped and identified the pathogenic gene; Treacher Collins mutation screening retardation-1. This study consists of three parts:
The first part of the establishment of genetic deafness preimplantation genetic diagnosis technology and preliminary clinical application
The vast majority of genetic deafness is a single gene specific point mutations, with assisted reproductive technology (in vitro fertilization, IVF) of preimplantation genetic diagnosis (preimplantation genetic diagnosis, PGD) medical indications, can effectively avoid the traditional mid pregnancy abortion treatment of abnormal embryos on patients and their family physical and psychological harm caused by the subject. Dr. Zhu Yuhua group in the previous study, an autosomal dominant non syndromic deafness pedigree of DFNA64 DIABLO gene were identified in the probands of couples in understanding the genetic diagnosis technology of embryo implantation (PGD) can prevent deaf children after birth, have a strong desire for the study. And signed informed consent.
Single cell nested PCR by single blastomere of the proband were single lymphocytes and others discarded embryos, whole genome in a single cell multiple displacement amplification (MDA) and subsequent PCR sequencing and linkage analysis markers real-time fluorescence quantitative PCR and microsatellite, established the technical system of preimplantation genetic deafness gene diagnosis. The research group and Beijing Medical University Third hospital assisted reproductive center Professor Liu Ping cooperation, the couple made a complete PGD assisted reproductive clinical diagnosis and treatment process, the diagnosis of a healthy embryo and implanted into the womb, but did not get a successful pregnancy.
The second part is the research orientation of non syndromic hereditary deafness pedigree
By the analysis of phenotypic and genetic methods, SD-L030 pedigrees collected to determine autosomal dominant non syndromic deafness family. Using microsatellite STR as genetic marker and classical linkage analysis of the family, the location of deafness gene, screening and identification of.SD-L030 family, a total of 4 generations members, a total of 30 people (including the deceased and spouse) is an autosomal dominant hereditary deafness pedigrees. Application of microsatellite markers was screened on 23 known DFNA loci, the family deafness gene located between 7p15 D7S629-D7S516 about 1.87cM area overlap with the known deafness gene DFNA5 region. Direct sequencing in the seventh intron of DFNA5 gene in the identification of a novel mutation of DFNA5, IVS7-2AG, and the mutation in this family phenotype were separated by extraction of peripheral blood mRNA and RT-PCR, found the point mutation In DFNA5 exon eighth deletion transcription.
Study on the syndrome of pathogenic gene screening and molecular mechanism of comprehensive third part of Treacher Collins
Treacher Collins syndrome (Treacher Collins, syndrome, TCS) is a common genetic disease, the incidence rate of 1/50000, mainly for the development of bilateral malar bone structure caused by abnormal craniofacial hypoplasia. In this study, two cases of suspected TCS were studied by direct sequencing of TCOF1 gene of second patients with TCOF1 exon c.146 TC heterozygous mutation, the mutation site of forty-ninth encoding isoleucine to threonine substitution, the father, the mother of the mutation site mutation was found, the mutation is a new mutation. The research results provide information and basis for the demand for genetic counseling and prenatal diagnosis of the family is the next step. At the same time, research progress on the molecular pathogenesis of TCS and the pathogenic mechanism of genes were analyzed and summarized in detail.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764.43

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