新杂合双突变致遗传性抗凝血酶缺陷症的分子机制  

Molecular mechanisms of antithrombin deficiency caused by novel double heterozygous mutations

作　　者：邓昊昱[1] 陈佳佺[1] 谢辉[1] 顾怡 张纪蔚[1] 张皓[1] 周兆熊[1] 梁卫[1] 薛冠华[1] 王鹏[1] 张岚[1] 

机构地区：[1]上海交通大学医学院附属仁济医院血管外科,200127 [2]检验科

出　　处：《中华医学杂志》2013年第1期61-64,共4页National Medical Journal of China

基　　金：上海市科委自然科学基金（10ZR1418800）

摘　　要：目的探讨抗凝血酶（AT）新杂合双突变致遗传性抗凝血酶缺陷症的分子机制。方法将野生型质粒（ATwt）及突变型质粒（AT—C．134G〉A、AT-c．342T〉G、AT—C．134G〉A和c．342T〉G）瞬时转染HEK293T细胞进行体外表达。Western印迹检测质粒转染细胞裂解液中AT抗原；同源模建构建AT三维空间结构，细胞免疫荧光染色检测AT蛋白在细胞内的分沁情况。结果Western印迹：AT—C．342T〉G和AT-C．134G〉A和C．342T〉G质粒转染细胞裂解液AT蛋白表达趋势明显增加。同源模建显示第82位碱基突变为Arg后致碱基侧链明显延长，与周围碱基距离缩短。第13位碱基突变为Gin后致局部空间结构改变，与肝素间距离延长。激光共聚焦显示：AT突变体质粒表达蛋白在内质网中聚集增加。结论新杂合双突变（AT-C．134G〉A和C．342T〉G）致遗传性抗凝血酶缺陷症的分子机制为突变体AT在细胞内潴留，并且AT-C．342T〉G突变起主导作用。Objective To explore the molecular mechanisms of antithrombin （AT） deficiency caused by novel double heterozygous mutations. Methods Wild-type and mutant AT cDNA expression plasmids （ATwt, AT-c. 134G 〉 A, AT-c. 342T 〉 G, AT-c. 134G 〉 A and AT-c. 342T 〉 G） were transfected into HEK293T cells. Western blot was used to detect the AT： Ag in cell lysates. Homology was used to reestablish 3-D spatial structure of AT. Laser confocal assay was utilized to analyze the distribution of AT in endoplasmic reticulum （ER）. Results Compared to the wild-types, the AT expression of HEK293T cells sharply increased when they were transfected by AT-c. 342T 〉 G or AT-c. 134G 〉 A and c. 342T 〉 G. Homology modeling showed that the mutation （ AT-c. 342T 〉 G） caused a decreased distance between Arg and surrounding bases as Arg＇s side chain was significantly longer than Ser＇s. The mutation of 13th base pair decreases the distance between AT and heparin. Laser confocal assay showed that the AT protein concreted in HEK293T cells when they were transfected by mutant plasmids （ AT-c. 134G 〉 A and c. 342T 〉 G） and aggregated in ER. Conclusions The main molecular mechanism of AT deficiency in this pedigree is the disturbed AT secretion as a result of the mutation of AT-c. 342T 〉 G.

关 键 词：静脉血栓栓塞 突变 抗凝血酶缺陷症 

分 类 号：R543.6[医药卫生—心血管疾病]