联合应用高分辨率熔解曲线和多重连接依赖探针扩增技术对PAH基因突变筛查研究  

The research of combining high resolution melting with multiplex ligation-dependent probe amplification technology in the mutation scanning for PAH gene

作　　者：季春燕[1] 孙莉莉[2] 曹丽华[1] 胡煜[1] 黄宏[2] 王述森[1] 罗阳[1] 

机构地区：[1]中国医科大学基础医学院医学基因组学教研室,教育部医学细胞生物学重点实验室,沈阳110001 [2]沈阳市妇女儿童保健中心

出　　处：《中华医学遗传学杂志》2011年第6期649-653,共5页Chinese Journal of Medical Genetics

基　　金：基金项目：国家科技部“863”项目（2007AA022440）;国家自然科学基金（30971164）

摘　　要：目的联合应用高分辨率熔解曲线（highresolutionmelting，HRM）和多重连接依赖探针扩增（multiplexligation—dependentprobeamplification，MLPA）技术检测苯丙酮尿症（phenylketonuria，PKU）患者基因突变，并探讨两种技术联合运用对于突变筛查的价值。方法采用HRM和MLPA技术对26例临床诊断为PKU的患者苯丙氨酸羟化酶基因（phenylalaninehydroxylase，PAH）进行检测，并通过测序验证；针对未报道的基因改变进行聚合酶链反应一限制性片段长度多态性分析。结果在52个等位基因中检测到44个突变等位基因（共21种不同突变），包括第4外显子拷贝数的增加，总检出率达84．62％（44／52），其中C．584—585insA和IVSl0＋1G〉T突变在国内外未见报道。此外，R243Q（25％）突变为中国最常见的突变种类。结论应用HRM和MLPA技术相对提高了PAH基因突变筛查的检出率，检测结果丰富了基因突变数据库，并为临床诊断与产前诊断提供实验室依据。Objective To explore the value of combining high resolution melting （HRM） with multiplex ligation-dependent probe amplification （ MLPA ） for detecting mutations underlying phenylketonuria. Methods HRM was used for detecting small mutations in phenylalanine hydroxylase gene （PAH） of 26 phenylketonuria patients. The results were verified with DNA sequencing. MLPA was used for detecting potential deletions/duplications in the PAH gene. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） analysis was performed for additional potential mutations. Results A total of 21 mutations were found in 44/52 alleles （84.62%）, which included a dupEx4. Among the 21 types of mutation, 19 were reported previously, and the remaining two were novel mutations： c. 584 _585insA and IVS10q-IG〉T. In addition, the mutation of R243Q （25%） was the most common type in China. Conclusion The study showed that the combination of HRM and MLPA could increase the detection rate for mutation in PKU. The study has added new information to the human mutation database of PAH and provided a basis for clinical diagnosis and prenatal counseling.

关 键 词：苯丙酮尿症 苯丙氨酸羟化酶 基因突变 高分辨率熔解曲线 多重连接依赖探针扩增 

分 类 号：R440[医药卫生—诊断学]