目标序列捕获及平行测序在临床耳聋基因诊断中的应用  

Targeted gene capture and massively parallel sequencing to identify causative genes mutations in clinical genetic testing for hereditary hearing loss

作　　者：苏钰[1,2] 汤文学 代志瑶[4] 高雪 王国建[1,2] 黄莎莎[1] 康东洋[1] 林曦 戴朴[1,2] 

机构地区：[1]解放军总医院耳鼻咽喉头颈外科,北京100853 [2]解放军总医院海南分院耳鼻咽喉头颈外科,海南572000 [3]美国Emory大学耳鼻咽喉科 [4]解放军总医院第一附属医院耳鼻咽喉头颈外科,北京100048 [5]解放军二炮总医院耳鼻咽喉科,北京100088

出　　处：《中华耳科学杂志》2014年第1期45-49,共5页Chinese Journal of Otology

基　　金：十二五国家科技支撑计划重点项目(No.2012BAI12B00/2012BAI12B01);中国博士后基金及特别资助(No.20120481482&201104779);北京市自然科学基金(No.7122172)

摘　　要：目的利用目标序列捕获(Targeted sequence capture)、生物编码(Barcode)及大规模平行测序(Massivelyparallel sequencing,MPS)技术,对遗传性耳聋患者进行分子病因学研究,探索低成本、多基因、多样本的耳聋基因检测在临床应用的可行性。方法利用Illumina测序平台对来自解放军总医院聋病分子诊断中心88例(来自61个家系)具有明确家族史且常见耳聋基因检测阴性的耳聋患者,以及8例阳性对照患者进行42种基因的目标序列捕获、Barcode和MPS,并对测序结果进行生物信息学分析。结果利用上述方法在88个个体中大于一人入组家系中发现了10个家系8个基因(TECTA、DFNA5、CDH23、USH1C、MYO7A、POU3F4、SLC26A4、EYA4)14个位点的致病性突变。准确验证了8个阳性对照。大于一人入组家系的致病基因检出率明显高于单人入组家系。结论高通量测序的发展为遗传性耳聋的诊断带来了巨大的机遇。目标序列捕获、Barcode、MPS的结合进一步提高了测序的准确性、降低了测序成本,同时生物信息学的进步,将促进低成本、多基因、多样本的临床耳聋基因检测成为可能。Objectives To explore the possibility of incorporating Targeted sequence capture, Barcode and MPS into clinical deafness genes diagnosis. This new strategy will allowed a much greater number of samples to be examined with saved the cost and shorten turnaround time. Methods The 96 patients with sensorineural hearing loss were enrolled through the Otolaryngology Department of Chinese PLA General Hospital .We have carried out 42 targeted gene capture and sequenced with MPS for 96 samples, including 8 positive control and 88 samples from 61 families. No patient was found to have any obvi- ous signs of syndromie symptoms and patients with GJB2 mutations, SLC26A4 hot spot mutations, mtDNA1494,1555 muta- tions had been excluded. Detailed family histories, clinical evaluations, temporal bone imaging results, audiograms, and other relevant clinical manifestations were collected for each patient. Results Fourteen variants in 8 different genes were identi- fied, which might have led to SNHI in 4 families compatible with autosomal dominant inheritance, 4 families with autosomal recessive inheritance, and 2 families with X-linked inheritance, including TECTA, EYA4, DFNA5,USH1C, CDH23, MYO7A, SLC26A4. Eight positive control were verified accurately. The families with more than one deaf member which has been enrolled in this genetic research studies, have a higher probability to find causitive mutations. Conclusion The new NGS approach delivers a step change in the diagnosis of inherited hearing loss, a continued refinement of the Targeted genomic capture, MPS and Barcode technology will further improve the sequencing accuracy and reduce the cost. These maybe broaden the availability of clinical genetic testing for the individuals with undiagnosed deafness in the future.

关 键 词：遗传性耳聋 目标序列捕获 生物编码 平行测序 生物信息学 

分 类 号：R764.04[医药卫生—耳鼻咽喉科] R764.43[医药卫生—临床医学]