Searching the effect of mutations on ATP7B gene that caused wilson disease to the copper transport on saccharomyces cerevisiae model system

Objective : It was aimed to detect the change amount of copper by transforming wild type ATP7B gene and T788I, V1036I and R1038G-fsX83 mutations in ATP7B gene which detected on Wilson Disease patients that has different clinical findings to Saccharomyces cerevisiae that has mutant CCC2gene (BY4741?CCC2) which obtained from wild type BY4741 (MAT? his3?1 leu2?0 met15?0 ura3?0) strain.Method : Total RNA was isolated from HepG2 cell line that belongs to hepotecellular carcinoma and cDNA synthesis reaction was performed. ATP7B gene was amplified with specific primers by PCR and it was cloned into plasmid DNA. T788I, V1036I and R1038G-fsX83 mutations were obtained by site directed mutagenesis on wild type ATP7B plasmid DNA. Plasmid DNAs that have wild type ATP7B; T788I, V1036I and R1038G-fsX83 mutations were verified by DNA sequencing. Wild type ATP7B, T788I, V1036I and R1038G-fsX83 mutants were transformed to Saccharomyces cerevisiae (BY4741?CCC2). Conventional and real time PCR was performed. Complementation assay was performed. In different medium condition copper and iron were measured in supernatant and pellet by ICM MS analysis.Results : In BY4741?CCC2 strain, ATP7B gene complemented CCC2 gene in yeast and it has been demonstrated that it is functional. On iron limited medium it has been demonstrated that T788I, V1036I and R1038G-fsX83 mutants have decreased copper in supernatant and increased copper in cell pellet comparing with wilde type ATP7B. In control media, no difference was detected on amount of copper both in supernatants and pellets. On the same experiement conditions, comparing with wild type ATP7B and mutants, no difference was found on amount of iron that has been measured in both supernatants and pellets.Conclusion: In iron limited medium it has been demonstrated that T788I, V1036I and R1038GfsX83 mutants have decreased copper in supernatant and increased copper in cell pellet comparing with wilde type ATP7B. The difference of copper transport between wild type ATP7B and three mutants were detected by ICP-MS and the hypothesis was confirmed. ATP7B gene was transformed to BY4741?CCC2 background yeast strain for the first time and complementation was shown. A model system was established for the mutations that belong to Wilson Disease patients that have different clinical findings.Key Words : Wilson disease, copper, ATP7B , mutation, CCC2, yeast