多重定量连接酶链反应在遗传性耳聋检测中的应用研究

Establishment of the multiplex quantitative ligase chain reaction for detecting mutations of deafness genes

目的 建立以多重定量连接酶链反应（multiplex quantitative ligase chain reaction,MQ-LCR）为基础的聋病基因诊断技术,实现廉价、快捷、准确的检测目标.方法 针对GJB2基因230delC、299-300delAT,mtDNA A1555G,SLC26A4基因IVS7-2 A＞G、2168A＞G等5种突变类型设计相应的探针和引物,建立检测体系,从复旦大学儿科医院眼耳鼻喉咽科门诊中随机选择感音神经性聋儿98例以及听力正常对照30名,遵循双盲法原则分别以MQ-LCR法和DNA测序法进行检测,结果进行比较分析.结果 98例聋儿中发现聋病基因纯合或双突变48例,杂合突变31例,阴性19例,GJB2基因突变致病的占29.6%（29/98）,mtDNAA1555G致聋的占4.08%（4/98）,SLC26A4致聋的占15.31%（15/98）.30名听力正常对照中发现杂合突变3例,阴性27例,其中GJB2基因235delC杂合突变2例,IVS7-2A＞G1例.经分析MQ-LCR和DNA测序法检测结果完全一致,未发现假阳性和假阴性.结论 所建立的MQ-LCR法检测聋病常见突变（235delC、299-300delAT、mtDNA A1555G、IVS7-2 A＞G、2168A＞G）具有价格便宜,操作快捷,结果准确可靠的特点,可应用于我国常见聋病基因的大规模筛查,为明确聋儿病因,降低聋生出生率提供一种很好的方法.

Objective To establish a low-cost, convenient and accurate multiplex quantitative ligase chain reaction （MQ-LCR） technique to detect the five common mutations in Chinese patients with deafness.Methods Primers and probes for 5 common mutations of deafness genes, i. e. , GJB2 gene 235delC and 299-300delAT, mtDNA A1555G, SLC26A4 gene IVS7-2 A 〉 G and 2168A 〉 G, were designed and synthesized. The technique for those mutations was established, and the reliability of the technique was tested in 98 patients with impaired hearing and 30 children with normal hearing, who were randomly selected from the ENT in Children＇s Hospital of Fudan University. The subjects were detected by MQ-LCR and direct DNA sequencing of PCR products, following a double-blind approach. Finally the results from the two methods were compared. Results The results revealed 48 cases carried two mutations, 31 cases carried heterozygous mutations in the 98 deaf children, and 3 had heterozygous mutation in 30 normal controls.These results were consistent with that from DNA sequencing. No false positive and false negative result was obtained. Conclusion The MQ-LCR technique established in this study is of low-cost, convenience,accuracy, high sensitivity and high specificity. It is suitable for large-scale detection and preventive diagnosis of mutations in deafness.