遗传性听神经病的基因定位及候选基因筛查研究

目的进行与遗传性听神经病相关的新致病基因的定位克隆研究以期发现新的听神经病基因;进行中国散发听神经病患者分子流行病学研究.方法基因定位克隆研究对象是一个5代相传的X-连锁听神经病家系;OTOF及WFS1基因的突变筛查及分子流行病学研究对象是105名听力下降患者,其中明确诊断为听神经病的散发患者31例,男女比例16:15.患者最小年龄8岁,最大年龄42岁.门诊对照组共43人(其中各种原因导致的耳聋患者24人,包括药物性耳聋、前庭导水管扩大综合征等;听力正常19人);低频听力减退家系成员31人.研究方法包括连锁分析基因定位法和候选基因突变筛查方法,引物设计应用在线引物设计软件-Primer3,采取PCR扩增,直接测序的方法进行OTOF和WS1基因的突变检测.序列分析采用DNAStar软件.结果在国际上首次将X-连锁遗传性听神经病定位在X染色体上Xq23-27.3,并将其命名为AUNX1基因座.在WFS1基因的突变检测中发现两个新的突变位点:2766G/A杂合866D＞N(天冬氨酸＞天冬酰氨),2328A/G杂合,A→G 720I＞V(异亮氨酸＞缬氨酸),为听神经病散发成员所特有.在OTOF基因的突变检测中发现听神经病散发患者有一个可以引起氨基酸改变的新的突变位点:3447G/T错义突变(1075D/Y天冬氨酸变成酪氨酸).这个突变与国外报道的听神经病相关的OTOF基因突变位点不同,在我们初筛的31例病人中有6例为此种突变(～20%),为一种新的突变形式.结论本研究发现与中国听神经病具有特异和相关的致病基因座位并建立了与听神经病相关基因的检测手段,为完善听神经病的分子遗传机制和进行临床听神经病的分子诊断提供了进一步的理论依据.

Investigations of genetic basis of auditory neuropathy with gene mapping and candidate gene screening approach

Objective To carry out the study of positional cloning for finding novel causative genes in hereditary auditory neuropathy; to perform the molecular epidemiological investigations and mutation screenings in the sporadic patients with auditory neuropathy. Methods The subjects include a Chinese family with 5-generations X-linked auditory neuropathy for positional cloning study; and 105 hearing impairment affects which include 31 sporadic patients with auditory neuropathy (male to female 16:15), the youngest is 8 years old and the oldest is 42 years old. The control group is 43 individuals from out-patient department which 24 patients induced by drugs, large vestibular aqueduct syndrome et al; 31 patients with low frequency hearing loss and 19 normal hearings for epidemiological investigations and mutation screenings in OTOF and WFS1 genes. The primers were designed with online Primer3 software. PCR products bidirectional sequencing was subsequently applied in the study subjects. Results In our study, we first mapped the Chinese X-linked hereditary auditory neuropathy in a novel locus Xq23-27 and nominated as AUNX1 locus. In the screening of WFS1 gene mutations, nine mutations located on exon 8 were found in all subjects(2264C/A ?2328A/G?2328A>G?2603G>A ?2735A>G ?2766G/A?2890T>C 2933C/T?2933C>T ?2934C>T?2992G>A ?2992G/A). Of those, two novel mutations of 2328A>G (866D>N) and 2766A>G (720I>V) were the only characteristic for the patients with auditory neuropathy. The mutations cause the amino acid changes (720I>V and 866D>N) respectively. In OTOF gene mutations, five point mutations were found and 3447G/T caused the amino acid changed 1075D/Y and six of 31 patients carried this kind of novel mutation (~20%) which implied the contribution to the auditory neuropathy. Conclusions The investigations of genetics basis of auditory neuropathy in Chinese patients (hereditary and sporadic) revealed a novel locus as well as de novo mutations in WFS1 gene and OTOF gene contribute to Chinese auditory neuropathy. The primary detection system was established in our study for gene diagnosis. The molecular pathogenesis of auditory neuropathy was considered and the further theoretical basis for clinical diagnosis and understanding was developed in our study.