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

Shaojuan Gu,Qing Huang,Yong Qi,Le Zhang,Hao Deng,Xiaoliu Shi,Xiong Deng,Zhi Song,Huarong Yang,Yi Guo,Jing Li,Matias A Avila

doi:10.1371/journal.pone.0066526

Abstract

Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu2+ transporting beta polypeptide gene (ATP7B). The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B 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 ATP7B 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 ATP7B 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 ATP7B 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 P<0.01). In vitro 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 ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.

Highlights

Wilson’s disease (WD) is an autosomal recessive disorder of copper metabolism
Patients were classified phenotypically into hepatic (n = 11, 55%) and neurological (n = 9, 45%) forms of WD based on initial symptoms or features
Western blot showed that B-cell CLL/ lymphoma 2 (BCL2) decreased to 54.6%, 19.3% and 3.1%, and BCL2-associated X protein (BAX) increased to1.8-fold, 1.7-fold and 3.2-fold for 24 h, 48 h and 72 h (Figure 3A, C, D). These results indicate that ATP7B small interfering RNA (siRNA) silencing induced a decrease in BCL2 expression, and an increase in BAX expression, indicative of apoptosis

Summary

Introduction

Wilson’s disease (WD) is an autosomal recessive disorder of copper metabolism. The disease is caused by mutations in the ATPase Cu2+ transporting beta polypeptide gene (ATP7B), a cellular copper transporting ATPase. Copper accumulation causes severe morphological and functional changes, including cirrhosis, hepatitis and liver failure. Genetic analysis reveals at least 506 distinct mutations, including missense and nonsense mutations, deletions and insertions Knowledge of the distribution of particular mutations may help to design shortcuts for genetic screening strategies of WD. To evaluate the frequency of the ATP7B mutations in Chinese Han patients with WD, to explore genotype-phenotype correlations and to possibly unveil the pathways involved in the ATP7B deficiency, we screened 18 families with WD and inhibited the ATP7B gene expression in human liver carcinoma (HepG2) cells

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Conclusion