title: Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.
abstract: Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Amvrosiadou
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
authors.forename: Margarita
authors.initials: M
authors.lastname: Petropoulou
authors.affiliation: Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
authors.forename: Myrto
authors.initials: M
authors.lastname: Poulou
authors.affiliation: Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Tzetis
authors.affiliation: Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
authors.affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece; Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece.
authors.forename: Theodore K
authors.initials: TK
authors.lastname: Christopoulos
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece. Electronic address: ioannou@chem.uoa.gr.
authors.forename: Penelope C
authors.initials: PC
authors.lastname: Ioannou
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D000483:Alleles; D015374:Biosensing Techniques; D027682:Cation Transport Proteins / Q000235:genetics*; D000073840:Copper-Transporting ATPases; D004252:DNA Mutational Analysis / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D006527:Hepatolenticular Degeneration / Q000235:genetics*; D006801:Humans; D008274:Magnesium; D009154:Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D027682:Cation Transport Proteins; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D008274:Magnesium
keywords: ATP7B gene mutations; Genotyping; Multi-allele DNA biosensor; Primer extension reaction; Visual detection; Wilson disease