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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→G720I>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。
Objective To study the localization and cloning of new pathogenic genes related to hereditary neuropathy in order to find new genes of auditory neuropathy and conduct molecular epidemiological studies on the distribution of auditory neuropathy in China. Methods The gene targeting clone was a 5-generation pedigree with X-linked auditory neuropathy. Mutation screening and molecular epidemiology of OTOF and WFS1 genes were performed in 105 hearing loss patients, of which patients with definite diagnosis of auditory neuropathy 31 cases, male to female ratio of 16:15. The minimum age of patients is 8 years old, the maximum age of 42 years old. Out-patient control group of 43 people (including a variety of causes of deafness in 24 patients, including drug-induced deafness, vestibular aqueduct enlargement syndrome; normal hearing 19); low frequency hearing loss family members 31 people. The research methods include linkage analysis of gene loci and mutation screening of candidate genes. The primers were designed by online primer design software-Primer3. The mutations of OTOF and WS1 were detected by PCR amplification and direct sequencing. Sequence analysis using DNAStar software. Results For the first time in the world, X-linked hereditary auditory neuropathy locates on X chromosome Xq23-27.3 and named it AUNX1 locus. Two new mutation sites were found in the mutation detection of WFS1 gene: 2766G / A heterozygote 866D> N (aspartic acid> asparagine), 2328A / G heterozygous, A → G720I> V Amino acids> Valine), specific to members of the auditory neuropathy distribution. In the mutation detection of OTOF gene, we found that there is a new mutation site in patients with auditory neurosis who can cause amino acid changes: 3447G / T missense mutation (1075D / Y aspartate to tyrosine). This mutation is different from foreign reports of neuropathy related OTOF gene mutation sites, 6 of the 31 patients we screening for the mutation (~ 20%), a new mutation. Conclusion This study found that there is a specific and relevant pathogenic gene locus in Chinese patients with auditory neuropathy and established a detection method for genes related to auditory neuropathy. This provides a further theory for improving the molecular genetic mechanism of auditory neuropathy and molecular diagnosis of clinical auditory neuropathy in accordance with.