单位：[1]Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China医技科室北京市临床医学研究所实验中心首都医科大学附属北京友谊医院[2]National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China首都医科大学附属北京友谊医院[3]Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China临床科室国家中心肝病分中心首都医科大学附属北京友谊医院
Background: Wilson's disease (WD) is a rare autosomal recessive disorder characterized by the deposition of copper mainly in the liver or nerve system that leads to their dysfunction. Mutations in the gene encoding ATPase, Cu+ transporting, beta polypeptide (ATP7B) are causative for WD. The aim of this study was to develop a rapid and convenient assay for detection of the three most common causative ATP7B mutations, p.R778L, p.P992L, and p.V1106I. Methods: Plasmids containing DNA fragments harboring each of the three ATP7B mutations were constructed. High-resolution melting (HRM) analysis was conducted by asymmetric polymerase chain reaction (PCR) amplification with paired primer and unlabeled probe, performed in a 96-well plate formatted LightCycler 480 Real-Time PCR System. The assay was evaluated for accuracy and reproducibility by genotyping of 41 WD cases. Results: The unlabeled probe HRM assays performed on constructs with the p.R778L, p.P992L, and p.V1106I mutations in the ATP7B gene resulted in additional melting peaks. According to the unlabeled probe HRM molecular signature, we could differentiate homozygous mutations from wild-type with the Delta Tm (difference between melting temperatures) >4 degrees C, and the coefficient of variation in repeatability tests was <5%. In the validation assay using our method to examine clinical samples, a 100% accuracy rate was achieved. Conclusions: The newly developed assay to rapidly genotype the ATP7B mutations is convenient, accurate, and reproducible, and represents a favorable alternative to Sanger sequencing in the identification of specific ATP7B mutations.
基金：
National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81071973]; Wang Bao-En Liver Fibrosis Foundation [20100013]
第一作者单位：[1]Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China[2]National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China
通讯作者：
通讯机构：[1]Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China[2]National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China[*1]Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
推荐引用方式(GB/T 7714)：
Xu Anjian,Lv Tingxia,Zhang Bei,et al.Development and evaluation of an unlabeled probe high-resolution melting assay for detection of ATP7B mutations in Wilson's disease[J].JOURNAL of CLINICAL LABORATORY ANALYSIS.2017,31(4):doi:10.1002/jcla.22064.
APA：
Xu, Anjian,Lv, Tingxia,Zhang, Bei,Zhang, Wei,Ou, Xiaojuan&Huang, Jian.(2017).Development and evaluation of an unlabeled probe high-resolution melting assay for detection of ATP7B mutations in Wilson's disease.JOURNAL of CLINICAL LABORATORY ANALYSIS,31,(4)
MLA：
Xu, Anjian,et al."Development and evaluation of an unlabeled probe high-resolution melting assay for detection of ATP7B mutations in Wilson's disease".JOURNAL of CLINICAL LABORATORY ANALYSIS 31..4(2017)
