title: Rapid diagnosis of Wilson disease by a 28-mutation panel: real-time amplification refractory mutation system in diagnosing acute Wilsonian liver failure.
abstract: BACKGROUND
  Wilson disease is one of the commonest inherited and potentially fatal yet treatable liver disorders. About 5-27% patients present with acute liver failure and require prompt chelation therapy and life-saving liver transplantation. Diagnosis during acute liver failure is particularly difficult with short time allowance. Direct molecular diagnosis remains the most decisive tool but is often hindered by demanding techniques and numerous mutations. We developed a one-step, 3-h, reproducible, and accurate real-time amplification refractory mutation system which can simultaneously detect 28 ATP7B mutations.
  
  
  METHODS
  Primers were designed to complement the mutant sequence at the 3' end. The mutations were p.S105X, p.Q511X, p.R616Q, p.S693P, p.S693C, p.R778L, p.A874V, p.T888P, p.R919G, p.T935M, p.P992L, p.M1025R, p.D1047V, p.I1148T, p.R1156H, p.T1178A, p.V1216M, p.P1273Q, p.G1281C, p.R1320S, p.V1334D, p.V176SfsX28, p.G869EfsX4, IVS3+1G>T, IVS4-1G>C, IVS4-5T>G, IVS6+9A>G, and IVS9+5G>T. Reaction was performed using QuantiTect SYBR Green PCR Master Mix on an Applied Biosystems StepOne thermal cycler. Values of the threshold cycle were compared between normal and mutant alleles.
  
  
  RESULTS
  Primers of all mutations were highly specific with absence of wild-type amplification. All the results were validated by direct DNA sequencing.
  
  
  CONCLUSIONS
  This rapid and cost-efficient method allows wide mutation coverage, rendering the SYBR-green assay feasible and attractive for large-scale routine application.
authors.affiliation: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China.
authors.forename: Chloe M
authors.initials: CM
authors.lastname: Mak
authors.forename: Ching-Wan
authors.initials: CW
authors.lastname: Lam
authors.forename: Sik-To
authors.initials: ST
authors.lastname: Lai
authors.forename: Yui
authors.initials: Y
authors.lastname: Hui
authors.forename: Sidney
authors.initials: S
authors.lastname: Tam