发作性肌张力障碍性舞蹈徐动症华人大家系的分子致病机制研究

Paroxysmal dystonic choreoathetosis (PDC) is a rare autosomal dominant inheritance paroxysaml movement disorder characterized by episodes or attacks of involuntary movements of the limbs, trunk, and facial muscles. Involuntary movements precipitate some attacks, and other attacks occur when the individual has consumed alcohol or caffeine, or is tired or stressed. Attacks can last from one minute to over an hour. Chinese family with PDC has not been reported. Previous researches had linked pathogenic gene of PDC to chromosome 2q33-35, and 9 families had been confirmed with mutation of myofibrillognesis regulator 1 (MR-1) gene. MR-1 gene has been mapped to chromosome 2q35 and has 3 isoforms,and missense mutations localized in first and second exons of MR-1 gene long isoform (MR-1L) and short isoform(MR-1S). However, the pathogenic mechanism that MR-1 mutation causes PDC is lack of research, furthermore, 7 families with PDC were confirmed without MR-1 gene mutation. Our preliminary experiment was perfomed in 4 members of the first Chinese large family with PDC,and the results of genetic linkage analysis with 5 microsatellites indicated that the pathogenic gene of this family may be linkage to chromosome 2q33-35. Therefore, current research aims to exactly localize pathogenic gene for the Chinese family with PDC by further microsatellite genetic linkage analysis, and then, gene sequencing will be performed to confirm MR-1 gene mutation and find the specific mutant sites. Because MR-1 gene contains conservative glyoxalase sequence, and lactoylglutathione may be a co-factor to dystonia, we study the change of MR-1 gene function by comparing different effects on S-D-lactoylglutathione metabolize between wild type and mutation type MR-1 genes expression products in C6 glioma cells. MR-1 protein contents and protein degradation rates in C6 cells will also be measured,in order to test the impact of MR-1 gene mutation on protein stability. Moreover, subcellar localization of wild tpye and mutation tpye MR-1 protein in C6 cells will be examinated by Western Blot and autoradiograph techniques,so,the effect to MR-1 gene mutation on subcellar localization of MR-1 protein will be found by comparing the differences.Hence, pathogenic mechanism of PDC could be initially uncovered through finding the changes of enzymatic activity, protein stability and subcellar localization of mutation type MR-1 protein in C6 glioma cells.

发作性肌张力障碍性舞蹈徐动症（PDC）是一种罕见的常染色体显性遗传性疾病，此前无华人家系报道。既往研究表明PDC致病基因位于2q33-35染色体，同时9个家系证实有2q35染色体的肌原纤维调节因子1（MR-1）基因突变。然而，其致病机制缺乏研究，此外7个家系未发现MR-1基因突变。本研究旨在利用首个华人PDC大家系，对2q31-35染色体进行微卫星基因连锁分析,确定致病基因位于2q35染色体后，直接基因测序查找MR-1基因的突变位点。为进一步研究PDC可能的致病机制，利用质粒转染C6细胞开展三方面体外研究：由于MR-1基因含有保守的乙二醛酶序列，且乳酸谷胱甘肽可能与肌张力障碍相关，测定野生型和突变型MR-1蛋白对S-D乳酸谷胱甘肽的代谢差异研究酶功能变化；通过测定突变型MR-1蛋白含量和降解率的变化研究MR-1蛋白稳定性的变化；利用放射自显影技术对突变型MR-1蛋白的亚细胞定位进行研究。

本研究针对一种罕见的2009年我们发现了首个华人非运动诱发的肌张力障碍（PNKD）大家系进行研究，该家系包括5代26个患者（3例已经死亡）。. 在本基金的支持下完成了以下研究工作：. （1）对其余23例中的17例及另外9名直系非患病家系进行了随访研究，家系谱图显示为常染色体显性遗传；（2）经过连锁分析提示致病突变位于 D2S126 和 D2S377之间，通过对PNKD基因全外显子测序，确认该家系为PNKD外显子1上的第165个碱基对突变，即A7V 突变；（3）通过质粒构建、基因转染C6细胞后进行亚细胞定位及利用酶标的对细胞内对谷胱甘肽含量测定、电泳进行PNKD蛋白半定量的方法，突变后可以导致蛋白在C6胶质瘤细胞中的分布范围明显增大，且谷胱苷肽含量增加，同时，PNKD蛋白稳定性下降；（4）利用ITRAQ标记和LC-MS分析的方法对3例患者和3例家族非患病成员进行了血浆和脑脊液中的差异蛋白进行分析。结果显示PNKD 蛋白在脑脊液中无表达，脑脊液中有42个差异蛋白，包括7个分子功能蛋白、8个分子成分蛋白和21种生物过程蛋白。PNKD 蛋白在血浆中无表达，血浆中有57个差异蛋白，包括21个分子功能蛋白；（5）通过GO富集分析和PATHWAY富集分析的方法对差异蛋白功能进行了注释。结果显示脑脊液中差异蛋白参与36种GO富集功能、3种PATHWAY富集功能。血浆中差异蛋白参与55种GO富集功能、18种PATHWAY富集功能；（6）通过生物网络分析显示：脑脊液中血小板衍生生长因子α、神经前体细胞表达蛋白4与血浆中肌动蛋白β与PNKD蛋白功能密切相关，可能与PNKD发病相关。. 本研究首次报道了华人PNKD家系，并确诊了致病基因；确诊PNKD为膜蛋白，影响谷胱苷肽含量，突变后稳定性下降；首次确诊PNKD 蛋白在人血浆和脑脊液中无表达，其可能通过血小板衍生生长因子α等蛋白间接发生作用。