title: Development and evaluation of an unlabeled probe high-resolution melting assay for detection of ATP7B mutations in Wilson's disease.
abstract: 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 ΔTm (difference between melting temperatures) >4°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.
authors.affiliation: Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Anjian
authors.initials: A
authors.lastname: Xu
authors.affiliation: Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Tingxia
authors.initials: T
authors.lastname: Lv
authors.affiliation: Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Zhang
authors.affiliation: National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Ou
authors.affiliation: Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China.|National Clinical Research Center for Digestive Disease, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
mesh_terms: D000073840:Copper-Transporting ATPases / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D006527:Hepatolenticular Degeneration / Q000235:genetics*; D006801:Humans; D015335:Molecular Probes / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D015335:Molecular Probes; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases
keywords: 
  ATP7B
  ; Wilson's disease; genotype; mutation detection; unlabeled probe HRM