Liu Hui, Wang Hua-Fang, Tang Liang, Yang Yan, Wang Qing-Yun, Zeng Wei, Wu Ying-Ying, Cheng Zhi-Peng, Hu Bei, Guo Tao, Hu Yu
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Collaborative Innovation Center of Hematology, Huazhong University of Science and Technology, China.
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Collaborative Innovation Center of Hematology, Huazhong University of Science and Technology, China.
Gene. 2015 May 25;563(1):35-40. doi: 10.1016/j.gene.2015.03.002. Epub 2015 Mar 4.
Hereditary protein C deficiency (PCD) is an autosomal inherited disorder associated with high risk for venous thromboembolism (VTE). This study aimed to explore the functional consequences of two missense mutations, p.Asp297His and p.Val420Ile, responsible for type I/II PCD and recurrent deep vein thrombosis (DVT) in a Chinese family. The plasma protein C activities (PC:A) of the proband and his sister were reduced to 4% and 5% of normal activity. However, protein C antigen (PC:Ag) concentrations were not equally decreased, with levels of 90.5% and 88.7%, respectively. Two missense mutations p.Asp297His and p.Val420Leu were identified in the protein C gene (PROC). The PC:A and PC:Ag levels in heterozygous state for p.Asp297His were 66% and 64.8%, whereas in heterozygous state for p.Val420Leu, these levels were 67% and 145%, respectively. Wild type (WT) and two mutant PROC cDNA expression plasmids were constructed and transfected into HEK 293T cells. Western blot analysis revealed that both p.Asp297His and p.Val420Leu showed a normal intracellular protein level. The extracellular protein level and specific activity of p.Asp297His were equally reduced to 37.7 ± 4.3% and 22.1 ± 2.5%, respectively. Mutant p.Val420Leu showed a relatively higher PC:Ag level and undetectable PC:A. Immunofluorescence staining revealed that WT and p.Val420Leu proteins were largely co-localized with both the protein disulfide isomerase (PDI) and cis-Golgi Marker (GM130), while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130. The thrombosis symptom in this family was associated with the two missense mutations in the PROC gene.
遗传性蛋白C缺乏症（PCD）是一种常染色体显性遗传病，与静脉血栓栓塞症（VTE）的高风险相关。本研究旨在探究两个错义突变p.Asp297His和p.Val420Ile在中国一个家族中导致I/II型PCD和复发性深静脉血栓形成（DVT）的功能后果。先证者及其姐姐的血浆蛋白C活性（PC:A）降至正常活性的4%和5%。然而，蛋白C抗原（PC:Ag）浓度并未同等降低，分别为90.5%和88.7%水平。在蛋白C基因（PROC）中鉴定出两个错义突变p.Asp297His和p.Val420Leu。p.Asp297His杂合状态下的PC:A和PC:Ag水平分别为66%和64.8%，而p.Val420Leu杂合状态下，这些水平分别为67%和145%。构建野生型（WT）和两个突变型PROC cDNA表达质粒并转染至HEK 293T细胞。蛋白质印迹分析显示，p.Asp297His和p.Val420Leu的细胞内蛋白水平均正常。p.Asp297His的细胞外蛋白水平和比活性分别同等降至37.7±4.3%和22.1±2.5%。突变型p.Val420Leu显示出相对较高的PC:Ag水平且未检测到PC:A。免疫荧光染色显示，WT和p.Val420Leu蛋白主要与蛋白二硫键异构酶（PDI）和顺式高尔基体标志物（GM130）共定位，而PC p.Asp297His突变蛋白主要与PDI共定位，与GM130的共定位较少。该家族中的血栓形成症状与PROC基因中的两个错义突变相关。
