CCC2基因缺陷型酵母对中国人Wilson病突变热点致病性的研究  

Study on Pathogenesis of Chinese Hot-Spot Mutation in Wilson Disease with CCC2 Gene Destructed Yeast Cells

作　　者：丰岩清[1] 李洵桦[1] 国宁[1] 梁秀龄[1] 黄帆[1] 王莹[1] 陈曦[1] 

机构地区：[1]中山大学附属第一医院神经科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2005年第5期541-544,577,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(31694和21894)

摘　　要：【目的】应用CCC2基因缺陷型酵母研究中国人Wilson病(Wilsondisease,WD)突变热点Arg778Leu的致病性,在蛋白水平上探讨中国人Wilson病的发病机制。【方法】应用RT-PCR和TOPOTA克隆技术分段克隆ATP7B的全长cDNA,USE定点诱变技术制备突变体,应用酵母功能互补分析的方法研究Arg778Leu的致病性。【结果】本研究应用USE定点诱变技术成功制备了ATP7B的Arg778Leu突变体,在CCC2缺陷型酵母内表达人的野生型ATP7B可以弥补酵母内CCC2蛋白的运铜功能,使CCC2缺陷型酵母在铜、铁及亮氨酸缺陷型培养基上生长良好;对突变体的研究表明Arg778Leu只能部分代偿CCC2蛋白的运铜功能。【结论】本试验证明CCC2基因缺陷型酵母是研究人ATP7B的一种良好的细胞模型,证明了中国人WD的常见突变Arg778Leu是一种致病性的突变。[Objective]To study the pathogenesis of Chinese WD hot-spot mutation-Arg778Leu, and to explore the molecular mechanism of Wilson disease at protein level. [Methods] To clone the full length of WD cDNA with RT-PCR and TOPO-TA clone techniques, using this as a template we prepared the Arg778Leu with USE sitedirected mutagenesis technique. We used the functional complementation assay, expressed the normal and mutated ATPTB in CCC2 yeasts and studied the function of Arg778Leu. [Results] We successfully cloned the full-length WD cDNA, and constructed ATP7B mutants-Arg778Leu which was the hot-spot mutations of ATP7B in Chinese patients. CCC2 destructed yeast grew well in the SD medium lacking copper, iron and leucine under the condition that normal ATPTB was expressed in the yeasts, but grew slowly in the same medium with Arg778Leu mutant protein expression. [Conclusion] Our experiment confirms that CCC2 yeast is a quite good model to analyze the function of ATPTB, normal ATP7B can completely complement the function of Ccc2p to deliver copper. The complementation assay shows Arg778Leu can only partly complement the function of Ccc2 protein, demonstrating the mutation is disease-causing.

关 键 词：WILSON病 定点诱变 CCC2缺陷型酵母 

分 类 号：R742.4[医药卫生—神经病学与精神病学]