复合杂合蛋白C基因突变致静脉血栓的分子发病机制研究  

Molecular Mechanisms of Venous Thrombosis Caused by Compound Heterozygous Mutants in the Protein C Gene

作　　者：吴瑛婷[1,2] 丁秋兰[2] 戴菁[2] 陆晔玲[2] 奚晓东[1] 王学锋[2] 王鸿利[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院血液学研究所,200025 [2]上海交通大学医学院附属瑞金医院临床输血科,200025

出　　处：《血栓与止血学》2010年第5期198-205,215,共9页Chinese Journal of Thrombosis and Hemostasis

摘　　要：目的对2个复合杂合的蛋白C(PC)基因突变致静脉血栓的家系进行临床表型、基因型和分子发病机制的研究。方法对血浆蛋白C活性(PC:A)和抗原(PC:Ag)分别用发色底物法和ELISA法进行测定,以凝血酶生成试验检测患者凝血功能的变化。PCR法扩增先证者PC基因的9个外显子及其侧翼序列,PCR产物纯化后直接测序,检测其基因突变。RT-PCR法结合定点突变法构建PC基因野生型和突变型表达质粒(PCwt、PCR178W、PCD255H、PCL-34P、PCT295I),瞬时转染COS-7细胞,检测细胞内、外PC:Ag的含量。利用细胞免疫荧光染色观察内质网和高尔基体中PC蛋白的定位。结果先证者1,28岁青年男性,临床诊断为下肢深静脉血栓和肺栓塞,PC:A21%,PC:Ag18.36%,为I型蛋白C缺陷症,基因分析显示在其PC基因7号和9号外显子分别存在R178W和D255H的复合杂合突变,其中D255H来自其父亲;先证者2,男,16岁,PC:A21%,PC:Ag20.04%,Ⅰ型PC缺陷症,在其PC基因的2号和9号外显子分别存在L-34P和T295I的复合杂合突变,其中L-34P来自其母亲,T295I来自其父亲。凝血酶生成试验显示其父母的抗凝功能减弱,凝血酶调节蛋白(TM)抑制凝血酶生成的能力降低。体外表达研究显示,PCL-34P只有7%分泌至细胞外,细胞内PC:Ag也只有8.72%,说明存在严重的分泌障碍和细胞内降解加速现象;PCR178W只有极少量(8%)从细胞内分泌,而PCD255H和PCT295I分别有57%和34%分泌至细胞外,细胞内PC:Ag含量分别是44.38%、38.05%和61.57%。细胞免疫荧光染色显示,PCT295I突变蛋白在内质网中滞留,在高尔基体中定位减少导致分泌减少。结论复合杂合性PC基因突变(R178W和D255H、L-34P和T295I)是导致此两例先证者1型遗传性PC缺陷症的原因。R178W、D255H、L-34P是国内首次报道的PC基因突变,T295I是国际首次报道的PC基因突变,分泌障碍和细胞内降解是R178W、D255H、L-34P和T295I导致PC缺陷症的分子机制。Objective Objective To study the phenotype, genotype and molecular mechanisms in two Chinese pedigrees with hereditary protein C （PC） deficiency caused by compound mutations. Methods The plasma level of protein C activity （PC： A） and protein C antigen （PC： Ag） of the probands and their family members were detected using chromogenic assay and ELISA, respectively. Thrombin generation tests were used to detect coagulation status. All of the nine exons and intron-exon boundaries of PROC were amplified by PCR and directly sequenced. Wild type and mutant PC cDNA expression plasmids（PC wt,PC R178W,PC D255H,PC L-34P,PC T295I） were constructed and transfected into COS-7 cells for in vitro expression study. Immunofluorescence assays were performed to investigate the cellular localization of PC. Results Compound heterozygous mutations of R178W and D255H, with 21% of PC： A and 18.36% of PC： Ag compared with normal levels, were identified in a 28-years old male who was diagnosed with DVT and PE. In Proband2, a 16- years old male who had DVT at the age of 14, whose PC ： A was 20% while PC ： Ag was 20.04%. Two independent mutations of L-34P in Exon 2 and T295I in Exon 9 detected in PROC were inherited from his mother and father, respectively. The anticoagulant activity of parents of Proband 2 was declined in throm- bin generation assay initiated with TM. In vitro study, PC R178W and PC L-34P were not secreted from the cells essentially and gradually degraded inside the cells while the secretion of PC D255H and PC T295I was reduced to 57% and 34% as compared with PC wt. Immunofluorescence assays showed that a very small a- mount of PC T295I was transported to the Golgi, suggesting that trace amount of mutant PC was secreted. Conclusion Compound heterozygous mutations cause hereditary protein C deficiency in these two Chinese families. Impaired secretion and degradation intracellularly of the mutants might be the molecular mechanisms of PC deficiency caused by R178W, D255H, L-3dP and T295I mutations. T

关 键 词：蛋白C 复合杂合基因突变 蛋白C缺陷症 

分 类 号：R596[医药卫生—内科学]