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目的:对遗传性非综合征型耳聋患者进行中国人常见的耳聋相关基因的突变分析,以明确其分子病因。方法:门诊收集非综合征耳聋患者22名的外周血样本,常规方法提取基因组DNA,PCR扩增GJB2基因和线粒体12SrRNA基因全序列,以及SLC26A4基因7+8外显子和19外显子序列。所有PCR扩增产物经DNA测序分析,测序结果提交NCBIGenBank数据库进行比对,从而对耳聋相关基因的突变进行分析。结果:22名非综合征耳聋患者中共检测到3种GJB2基因的突变:235delC纯合突变2例;235delC杂合突变3例及176del16bp和299delAT复合突变1例;mtDNA12SrRNA基因检出有2种已知致病突变:A1555G2例和C1494T2例;SLC26A4基因有IVS7-2A>G杂合突变2例,纯合突变1例。结论:PCR扩增结合DNA测序是耳聋基因诊断的经典和有效的方法,能确诊非综合征型耳聋患者的分子病因,为临床诊断和治疗提供依据。但该方法存在不能定性、耗时费力、所需成本昂贵且不能同时对不同基因的多个突变位点进行检测等缺点。因此,临床的耳聋基因诊断,需要操作简便、结果准确、通量高、价格低廉的新手段。
OBJECTIVE: To analyze the common deafness-related genes in Chinese patients with hereditary non-syndromic deafness so as to clarify the molecular etiology. METHODS: Twenty-two peripheral blood samples from non-syndromic deafness patients were collected at clinic. Genomic DNA was extracted by conventional methods. The full-length GJB2 and mitochondrial 12SrRNA sequences and the exons 7 + 8 and exon 19 of SLC26A4 gene were amplified by PCR. All PCR amplification products were sequenced by DNA sequencing and the results were submitted to the NCBIGenBank database for analysis of mutations related to deafness. Results: Twenty-two non-syndromic deaf patients detected three GJB2 gene mutations: 235delC homozygous mutation in 2 cases, 235delC heterozygous mutation in 3 cases and 176del16bp and 299delAT complex mutation in 1 case; mtDNA12SrRNA gene detection of two known Pathogenic mutations: A1555G2 cases and C1494T2 cases; SLC26A4 gene has IVS7-2A> G heterozygous mutation in 2 cases, homozygous mutation in 1 case. Conclusion: PCR amplification combined with DNA sequencing is a classic and effective method for gene diagnosis of deafness. It can diagnose the molecular etiopathogenisis of nonsyndromic deafness and provide the basis for clinical diagnosis and treatment. However, this method has some shortcomings, such as unidentifiable method, time-consuming and labor-intensive method, high cost, and inability to detect multiple mutation sites of different genes at the same time. Therefore, clinical deafness gene diagnosis, the need for easy operation, accurate results, high throughput, low prices and new means.