【作者】 金占国；

【导师】 韩东一； 袁慧军；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉—头颈外科， 2011， 博士

【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,IVS7-2A>G,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 T>C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。更多还原

【Abstract】 Hearing impairment is the most common birth defect and sensorineural disorder. Based on the national survey of disabled population in 2006, the total number of hearing impairment in China has reached 27.8 millions. The number of newborn with congenital hearing impairment is 20-30 thousands every year. Therefore, diagnostic techniques and interventions should be established to reduce the risk of future recurrence of deafness. It has very important long-term significance of improving the quality of the population in China.In this study, we have established preimplantation genetic diagnostic (PGD) technique for hereditary hearing loss by multiple displacement amplification, nested PCR, real-time quantitative PCR and microsatellite marker. Using microsatellite marker, we have successfully identified a novel hearing loss locus and the gene in a family with autosomal dominant non-syndromic deafness. We Successful identify TCOF1 gene mutation in a He-Nan family with Treacher Collins syndrome.PART1:Establishment and application of PGD technique for hereditary hearing lossThe vast majority of genetic deafness was a single-gene disease. Therefore, it is in line with the medical indications for PGD, and can be effectively prevented abnormal embryos for therapeutic abortion, which would avoid the physical and psychological harm for patients. In the previous study of our research group, we have demonstrated that mutation in DIABLO underlying non-syndromic hearing impairment in a large Chinese family. The couple of the proband had very strong demand for PGD, and signed informed consent. We applied the multiple displacement amplification of whole genome, nested PCR, real-time fluorescent quantitative PCR and microsatellite marker to establish the PGD techniques from single lymphocytes of the couple of proband and superseded single blastomere. We have successfully performed an intact PGD cycle in the couple of proband and identified a healthy fetus for implantation. Unfortunately, this fetus did not successfully get nidation due to poor endometrium environment. PART 2:Identification of gene underlying autosomal-dominant nonsyndromic hearing loss in a Chinese familyIn this study, we identified the causative gene in an autosomal-dominant nonsyndromic hearing loss Chinese pedigree named SD-L030 and analyzed DFNA5 mutation. This Chinese family spanned four generations and comprised 30 members. Application of microsatellite markers on DFNA 23 loci preliminary screening of 25 genes, data were analyzed by linkage analysis. We mapped the locus to the region between D7S629 and D7S516 (two-point lod-score of 5.39) with application of 8 microsatellite markers. By direct sequencing of best candidate genes in mapping region, we identified a novel missense mutation ivs7-2 A>G in DFNA5 gene, which was faithfully cosegregated with hearing loss in the family. This mutation causes skipping of exon 8, resulting in premature termination of the open reading frame. In order to understand DFNA5 mechanisms and to gain more insights into the function changes of protein resulting from the missense mutation, we will establish effective experimental model in near future.PART 3:Mutational screening of TCOFl gene in patients with Treacher Collins syndromeTreacher Collins syndrome (TCS) is characterized by hypoplasia of the zygomatic bones and mandible, and so on. Almost 40%-50% of individuals have conductive hearing loss attributed most commonly to malformation (including ankylosis, hypoplasia, or absence) of the ossicles and hypoplasia of the middle ear cavities. We have collected two sporadic patients with conductive hearing loss and notching of the lower eyelid recruited from clinic of Department of otolaryngology of PLA General Hospital. By direct sequencing TCOFl, we identified a novel missense mutation c.146 T>C (p.I49T) in the second exon of TCOFl gene in one patient, but was not found in the gene in his parents. Furthermore, the amine acid of isoleucine in this site is highly conservative across eight species. This result can be used to conduct prenatal diagnosis in this family.更多还原