ABCC2基因剪切突变影响MRP2蛋白C端功能在Dubin-Johnson综合征中的致病作用及其机制

Dubin-Johnson综合征(DJS)是以直接胆红素升高为主要特征的常染色体隐性遗传病， ABCC2基因突变导致MRP2蛋白功能缺陷是其致病因素。本项目前期研究发现，中国DJS病人中存在较高频率的ABCC2剪切突变，且大部分会导致MRP2蛋白C端变异，可能与MRP2蛋白的成熟运输、膜插入回收及转运活性相关。本项目拟研究ABCC2基因剪切突变在DJS中的致病作用及其机制。首先明确ABCC2不同剪接突变影响可变剪接导致的MRP2蛋白C端序列改变；然后通过构建不同MRP2蛋白C端突变体的细胞模型研究剪切突变造成的MRP2蛋白成熟运输差异、检测不同C端突变体对NHERF1、Radixin相互作用导致其膜插入回收的改变、对底物诱导ATP水解功能及MRP蛋白转运活性的影响。最后从动物疾病表型和临床层面分析MRP2蛋白C端变异与DJS的关系，对我国DJS病人的临床诊疗具有重要意义。

Dubin Johnson syndrome (DJS) is an autosomal recessive genetic disease characterized by the increase of direct bilirubin. The functional defect of MRP2 caused by ABCC2 gene mutation is the pathogenic factor. Our previous studies indicated that there are high frequency of ABCC2 splicing mutations in Chinese patients with DJS. Most of the gene mutation may lead to C-terminal variation of MRP2 protein, which may be related to membrane location and transport activity of MRP2 protein. This project aims to study the pathogenicity and mechanism of ABCC2 gene splicing mutation in DJS. First, we intend to clear that different splicing mutations of ABCC2 may lead to the C-terminal variable changes of MRP2 protein. Besides, the difference of post-translational processing and trafficking caused by the mutation will be studied by constructing cell models such as gene knockout. Co-IP will be performed to detect the interactions between wild-type/mutant MRP2 and NHERF1-Radixin as well as detect the Insertion and endocytosis of MRP2. Photoaffinity labeling will be used to detect the ATP hydrolysis induced by substrate and ATP dependent substrate transportation. Finally, the relationship between the variation of C end of MRP2 and DJS was analyzed from the animal models and clinical level. This study will provide valuable reference for clinical diagnosis and treatment of DJS patients in China.