Novel ATPase Cu²⁺ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

<div><p>Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu²⁺ transporting beta polypeptide gene (<i>ATP7B</i>). The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the <i>ATP7B</i> deficiency were documented. The <i>ATP7B</i> gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of <i>ATP7B</i> small interfering RNA (siRNA)-treated human liver carcinoma (HepG2) cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), sterol regulatory element binding protein 1 (SREBP1), and minichromosome maintenance protein 7 (MCM7) of <i>ATP7B</i> siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR) and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X) in the <i>ATP7B</i> gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val) existed in these families. Transfection of HepG2 cells with <i>ATP7B</i> siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All <i>P</i><0.01). <i>In vitro</i> study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and <i>ATP7B</i> deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.</p>