Wilson disease is an autosomal recessive copper metabolism disorder caused by mutations in copper-transporting ATPase (ATP7B). Our preliminary results suggested that one WD patient characterized with very mild symptoms; however, this patient is homozygous for the 2810del T mutation in the exon 12 of the ATP7B gene. The copper resistance assay showed that 2810delT mutation resulted in the complete loss of copper transporting activity; however, cells transfected with ATP7B-d12 retained approximately 80% activity in copper resistance assays compared with wild-type ATP7B. The apoptosis and absorption analysis showed that ATP7B-d12 mutant were more resistant to copper-induced cell apoptosis and was increased more than 4-fold when the cells were cultured in copper. Our minigene approach showed that the expression of exon 12 alternatively spliced ATP7B was much higher in 2810del T minigenes. In addition, we found that the expression level of exon 12 alternatively spliced ATP7B increased 3-fold upon EPIA treatment. By analyzing blood samples from 135 WD patients in the ATP7B gene, 36 mutations were identified from the sequence analysis of whole 21 exons and promoter region of ATP7B gene. In this proposal, we hypothesize that the mechanism of alternative splicing of ATP7B gene may play an important role in the potential treatment of Wilson disease. To test this hypothesis, we will use copper resistance assay, apoptosis analysis, absorption assay, minigene approach, the 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS technology to investigate the possible molecular mechanism of splicing therapeutics are known to modify or correct RNA splicing to achieve therapeutic goals. This information would facilitate the contribution of the possibility of applying splicing therapy to WD patients.
