【作者】 李建忠；

【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， 2010， 博士

【摘要】 耳聋是最常见的出生缺陷,也是最常见的感觉神经性疾病。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。要提高防聋治聋的水平,最重要的是要更深入地了解耳聋的病因和发病机理。随着导致耳聋的各种环境因素逐步得到控制,遗传因素在耳聋病因上的重要性日渐突出,研究遗传性耳聋的病因和发病机理,寻找能够预测、减少后代耳聋再发风险及新的治疗方法,对于提高防聋治聋的水平、减少人群中耳聋发病率进而提高我国人口素质有重要的现实和长远意义。本研究正是针对非综合征型遗传性耳聋进行了病因及发病机理的研究,按三个家系分成三部分。一、X连锁非综合征性聋(DFN2)致病基因(PRPS1)的鉴定及PRPS1突变后的功能研究本课题组韩冰硕士在前期研究中,鉴定了一个X连锁遗传性非综合征型耳聋家系(GZ-Z052)的致病基因PRPSl,发现第193位核苷酸由鸟嘌呤变为腺嘌呤(c.193G>A),使第65位氨基酸由天冬氨酸变为天冬酰胺(p.D65N)。该基因所在区域(Xq22)与已报道的尚未找到致病基因的三个DFN2家系的致病基因定位区域重叠。为了探索PRPSl是否就是DFN2 (DFNX1)的致病基因,本研究对上述三个家系进行了PRPS1基因的突变筛查,在每一个家系中都发现了突变,分别是：c.259G>A(p.A87T),c.869T>C(p.I290T)和c.916G>A(p.G306R),这几个突变都与各自家系的耳聋表型共分离。来自正常人的DNA样品共1025条X染色体经测序未发现PRPS1基因上述4个位点突变。对来自GZ-Z052家系8个成员的红细胞和经培养的皮肤成纤维细胞的PRPS用离子对反相HPLC法测定酶活性,发现患者的红细胞和成纤维细胞的酶活性分别是正常对照者酶活性的56%和55%。原核表达的6个PRPS1突变体酶(其中4个为本课题发现的突变,另2个为文献报道的引起PRPS酶活性下降而导致综合征性耳聋的突变位点)进行酶活性检测,发现相对于野生型酶,6个突变体的酶活性都是下降的。本课题发现的4个突变体的酶活性介于野生型和文献报道的引起PRPS1酶活性下而导致综合征性耳聋的突变位点之间。表明DFN2家系是由于PRPS1基因突变后酶活性部分下降所致。二、X连锁非综合征性聋家系(SX-Z089)致病基因的鉴定及产前基因诊断经表型及遗传方式分析,将SX-Z089家系判定为X连锁非综合征型耳聋家系。该家系男性患者的颞骨CT检查有典型的DFN3的cT影像学特征。行候选基因筛查时,发现DFN3的致病基因POU3F4基因存在c.647G>A(p.G216E)突变,与家系耳聋表型共分离。该突变在多物种间保守,在110例正常人中未发现上述突变,从而判定该突变为SX-Z089家系的致病基因。对该家系中一名孕18周的携带者取羊水对胎儿DNA进行检测时发现胎儿为c.647G>A(p.G216E)突变半合子,提示该胎儿会复制家系中男性患者的表型。为此家庭提供了明确的遗传咨询依据。对该家系进行线粒体全序列测序筛查时,发现9名母系成员携带mtDNA961de1T／insC(n)突变,但此突变不与耳聋表型共分离,且家系耳聋已归因于POU3F4基因突变,表明mtDNA 961delT/insC(n)突变不是该家系耳聋的病因。曾有报道mtDNA 961delT/insC(n)突变与药物性耳聋相关,但在SX-Z089家系中多人携带此突变,且有明确氨基糖甙类药物使用史而不出现耳聋。所以,mtDNA 961de lT/insC(n)突变的致病性需进一步确认。三、常染色体显性非综合征型遗传性耳聋家系(HN-J069)致病基因的定位研究经表型和遗传方式分析,将HN-J069家系判定为常染色体显性遗传性非综合征型耳聋家系,在该家系中首先对22个已知的常染色体显性遗传性耳聋基因位点进行了连锁分析,排除了这些已知基因为该家系耳聋的致病基因的可能。SNP芯片全基因组连锁分析发现5号染色体160-190cM之间的LOD值接近2,对应于第5号染色体长臂5q33.3-5q35.3区间。下一步拟通过STR遗传标记进行精细定位,可能缩小连锁区域的范围,有望找到一个新的致聋基因。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 was 27.8 millions. And the number of newborn with congenital hearing impairment reaches to 20-30 thousands every year. Genetic factors play more important role in the etiology of hearing impairment for the environmental factors are controlled gradually and effectively. To improve the prevention and treatment of hearing impairment and to reduce the number of cases with hearing loss, it is necessary to conduct the study on the cause and molecular mechanism of hearing impairment. In this study, we focus on the molecular cause and mechanism of the hereditary non-syndromic hearing impairment. The study divided into three parts according to three families.PART1:The confirmation of the causative gene PRPS1 of DFN2 and the functional study of all mutants.In the previous study of our research group, we found that the candidate gene of GZ-Z052 family with X-linked non-syndromic hearing impairment is PRPS1, with mutation of c.193G>A(p.D65N). The locus (Xq22) of the gene overlapped to that of three previously reported DFN2 families. Mutation screening of PRPS1 gene was carried out in the three DFN2 families and different missense mutation in PRPS1 gene was identified in each family. The mutations are c.259G>A (p.A87T), c.869T>C(p.1290T)and c.916G>A(p.G306R), faithfully segregating with the hearing loss in each family. None of the four mutations were seen in 1025 unrelated control chromosomes, supporting the pathogenicity of the mutations.The activity of the PRPS of erythrocyte and cultured fibroblast of the affected males of GZ-Z052 family were 56% and 55% of the normal controls. The activity of all of six mutants of PRPS 1 (p.D65N, p.A87T, p.I290T, p.G306R, p.M115T, and p.Q133P) expressed in E. coli BL21, were decreased. The enzyme activity of the four mutants were between that of wild type and p.M115T and p.Q133P, indicating that DFN2 is caused by mutation of PRPS1 gene.PART 2:Identification of causative gene of SX-Z089 family and prenatal diagnosis conducted in this familySX-Z089 family exhibits X-linked non-syndromic hearing impairment. The affected males had typical temporal bone CT imaging of DFN3, e.g, bilateral dilation of the internal auditory canals and with a fistulous communication between its lateral aspect and the basal turn of the cochlea. A c.647G>A (p.G216E) mutation in POU3F4 gene was found segregating with the phenotype of the family. The G216 is conserved from clawed frog to human. The mutation was absent in 110 normal controls. A hemizygotic mutation of c.647G>A in POU3F4 gene was found in the DNA sample of the amniotic fluid in prenatal diagnosis, indicating that the fetus will copy the phenotype of the affected males of the family.Nine maternal members of SX-Z089 family carried mtDNA 961delT/insC(n) mutation. This mutation do not segregate with the phenotype of the family. The mtDNA 961delT/insC(n) mutation is unlikely the causative mutation of the family. The mtDNA 961delT/insC(n) had been reported to associate with the aminoglycoside-induced hearing loss. However, in SX-Z089 family some members with mtDNA 961delT/insC(n) mutation did not have hearing loss even had a history of aminoglycoside use. The role of mtDNA 961delT/insC(n) in aminoglycoside-induced hearing loss remains to be confirmed.PART 3:Gene mapping of HN-J069 family with autosomal dominant hereditary non-syndromic hearing impairmentWe have collected a family with autosomal dominant non-syndromic hereditary hearing loss (DFNA) from HeNan province. Twenty-two DFNA loci with known genes had been ruled out by linkage analysis using the STR markers near the known DFNA genes in family HN-J069. We have mapped the disease locus to 30cM interval on chromosome 5, between 5q33.3 and 5q35.3, by genome wide SNP typing. Fine mapping will be conducted with STR markers in this interval. This will lead to identification of a new deafness gene in this family.更多还原