采用SNaPshot技术对苏南地区耳聋患者进行基因突变热点筛查

Genetic screening of mutation hot-spots for nonsyndromic hearing loss in southern Jiangsu province with SNaPshot

目的明确苏南地区非综合征型耳聋患者7个耳聋致病基因突变热点的突变频率，验证中国人群耳聋遗传病因筛查的SNaPshot技术平台的效能。方法以125例非综合征型耳聋（non—syndromic hearing loss，NSHL）患者为研究对象，应用SNaPshot技术，针对GJB2基因235delC和299—300delAT，SLC26A4基因IVS7—2A〉G和2168A〉G，线粒体DNA（mtDNA）1555A〉G、7445A〉G和3243A〉G共7个突变热点在一个反应管中进行多重PCR后，单碱基荧光延伸标记，再采用ABI3130遗传分析仪行毛细管电泳进行基因分型，并利用直接测序和聚合酶链反应-限制性片段长度多态（polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP）方法验证基因分型结果。结果（1）125例样本中，GJB2基因235delC突变频率为24．0％，299-300delAT突变频率为5．6％；SLC26A4基因IVS7—2A〉G突变频率为15．2％，2168A〉G突变频率为3．2％；线粒体DNA1555A〉G突变频率为4．8％，7445A〉G突变频率为0．8％，未发现线粒体DNA3243A〉G位点突变，7个热点综合突变频率为53．6％。（2）SNaPshot结果与直接测序或PCR—RFLP结果完全吻合，检测的特异性和敏感性均为100％。结论（1）苏南地区耳聋患者7个位点突变频率超过半数；（2）用SNaPshot技术筛查耳聋基因，在一个反应管中同时检测到了7个突变热点，其检测效能高，具有临床应用价值。

Objective To investigate the mutation frequency in 7 mutation hot-spots of deafness gene in southern Jiangsu province and verify the performance of the SNaPshot technology platform, designed for genetic screening of non-syndromic hearing loss （NSHL） in Chinese. Methods One hundred and twentyfive NSHL patients were enrolled. Amplification of 235delC, 299-300delAT in GJB2 gene, IVS7-2A〉G, 2168 A〉G in SLC26A4 gene, and 1555A〉G, 7445 A〉G and 3243 A〉G in mitochondrial DNA （mtDNA） was performed using multiplex polymerase chain reaction （PCR） technology. Afterwards, the sequence-specific probe interrogated each locus and labeled it at the 3′ end using fluorescent dideoxynucleotide chemistry by the SNaPshot Multiplex Kit, the resulting products were then separated electrophoretically in ABI PRISM 3130 Genetic Analyzer and analyzed in the presence of a fifth-dye-labeled size standard. Finally, the genotyping results were verified by direct sequencing or PCR-restriction fragment length polymorphism （PCR-RFLP）. Results （1） The total mutation frequency for the 7 mutation hot-spots was 53.6%. The mutation frequency of 235delC was 24.0%, 299-300delAT was 5.6% in the GJB2 gene, IVST-2A〉G was 15.2 %, 2168A〉G was 3.2 % in the SLC26A4 gene. The mutation frequency of 1555A〉 G and 7445 A〉G in mtDNA was 4. 8% and 0. 8% respectively. The mutation 3243 A〉G was not detected. （2） The SNaPshot results were consistent with that from direct sequencing or PCR-RFLP, and the specificity and sensitivity of detection were 100%. Conclusion （1） More than half of the patients with deafness in southern Jiangsu province carry the mutations of the seven hot-spots. （2） The genetic screening technology platform based on SNaPshot can detect 7 mutations in one reaction, and is efficient and suitable for clinical practice.