摘要目的 应用基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser DesorptionIonization Time of Flight Mass Spectrometry,MALDI-TOF-MS)技术检测非综合征型耳聋患者突变基因,并用直接测序法进行验证,评估MALDI-TOF-MS在临床耳聋基因检测中的可行性.方法 采集454例非综合征型耳聋患者的外周血,提取基因组DNA,采用MALDI-TOF-MS检测常见的4个耳聋致病基因的20个位点,包括GJB2(35delG、167delT、176_191de116、235delC、299_300delAT),GJB3 (538C→T、547G→A),SLC26A4 (281C→T、589G→A、IVS7-2A→G、1174A→T、1226G→A、1229C→T、IVS15+5G→A、1975G→C、2027T→A、2162C→T、2168A→G),线粒体12S rRNA(1494C→T、1555A→G).同时应用直接测序法对上述20个位点进行检测,以验证MALDI-TOF MS的准确性.结果 454例耳聋患者中共检出166例存在致聋突变(36.56％).其中GJB2基因突变96例(21.15％),GJB3基因突变4例(0.88％),SLC26A4基因突变64例(14.10％),线粒体12S rRNA基因突变3例(0.66％).直接测序法结果与MALDI-TOF-MS结果一致,两者符合率达100％.结论 针对中国人群常见耳聋相关基因热点突变设计的MALDI-TOF-MS对非综合征型耳聋患者的突变检出率较高,与传统常用基因检测方法相比,具有检测位点多、覆盖率高、准确、高通量、低成本等特点,能够满足临床对常见耳聋基因检测的要求.

abstractsObjective To investigate the feasibility of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS),according to the genetic test of non-syndromic hearing loss (NSHL),and check using the direct sequencing.Methods Peripheral blood was collected from 454NSHL patients.DNA samples were extracted and 20 loci of the four common disease-causing genes were analysed by MALDI-TOF-MS,including GJB2 (35delG,167delT,176_191de116,235delC,299_300delAT),GJB3 (538C→T,547G→A),SLC26A4 (281C→T,589G→A,IVS7-2A→G,1174A→T,1226G→A,1229C→T,IVS15 +5G→A,1975G→C,2027T→A,2162C→T,2168A→G),and mitochondrial 12S rRNA (1494C→T,1555A→G).Direct sequencing was also used to analyse the aforementioned 20 loci in order to validate the accuracy of MALDI-TOF-MS.Results Among the 454 patients,166 cases (36.56％) of disease-causing mutations were detected,which included 69 cases (21.15％) of GJB2 gene mutation,four cases (0.88％) of GJB3 gene mutation,64 cases (14.10％) of SLC26A4 gene mutation,and three cases (0.66％) of mitochondrial 12S rRNA gene mutation.Moreover,the results obtained from direct sequencing and MALDI-TOF-MS were consistent,and the results showed that the two methods were consistent.Conclusions The MALDI-TOF-MS detection method was designed based on the hearing lossrelated mutation hotspots seen in the Chinese population,and it has a high detection rate for NSHL related mutations.In comparison to the conventional detection methods,MALDI-TOF-MS has the following advantages:more detection sites,greater coverage,accurate,high throughput and low cost.Therefore,this method is capable of satisfying the needs of clinical detection for hearing impairment and it is suitable for large-scale implementation.