两个遗传性蛋白C缺陷症家系的分子致病机制研究  

Analysis of two cases of hereditary protein C deficiency causing venous thrombosis

作　　者：温梦珍 芦一凡 刘媚娜[1] 秦朗译 金艳慧[1] 王明山[1] 杨丽红[1] Wen Mengzhen;Lu Yifan;Liu Meina;Qin Langyi;Jin Yanhui;Wang Mingshan;Yang Lihong(Department of Laboratory Medicine,the First Affiliated Hospital of Wenzhou Medical University,Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江省检验诊断及转化研究重点实验室,温州325015

出　　处：《中华血液学杂志》2025年第3期244-251,共8页Chinese Journal of Hematology

基　　金：浙江省检验诊断及转化研究重点实验室(2022E10022);温州市基础性医疗卫生科技项目(Y20210111)。

摘　　要：目的探究两个遗传性蛋白C(PC)缺陷症家系的分子致病机制。方法两位先证者均因静脉血栓栓塞症(VTE)分别于2021年6月和2022年10月就诊于温州医科大学附属第一医院,收集先证者和家系成员临床资料及血样本,检测血浆蛋白C活性(PC∶A)和抗原(PC∶Ag)含量及其他相关凝血指标。以凝血酶生成试验(TGT)评估其抗凝能力。采用DNA直接测序法确定PROC基因突变位点。用生物信息学软件分析突变基因的保守性和致病性。用PyMOL软件分析蛋白质三维模型以及突变氨基酸之间的相互作用。构建野生型和两个突变型表达载体,并瞬时转染HEK293T细胞,提取阳性转染细胞内的细胞总RNA进行突变PROC基因转录水平的研究。采用ELISA法和Western blot和细胞免疫荧光实验进行PC突变蛋白翻译水平的研究。结果家系1、2先证者的PC∶A分别降至35%、40%,PC∶Ag分别降至44%、39%,D-二聚体分别升高至4.42 mg/L、0.83 mg/L,其他相关凝血指标无明显异常。测序分析显示,家系1、2先证者的PROC基因第9外显子分别存在c.833T>C(p.Leu278Pro)、c.1330T>C(p.Trp444Arg)杂合错义突变。凝血酶生成试验表明两位先证者及其携带以上PROC基因突变家系成员的抗凝功能均受损。保守性分析显示Leu2278和Trp444在同源物种间呈高度保守。致病性分析显示p.Leu278Pro和p.Trp444Arg均为有害突变。蛋白质模型分析显示该两种突变均能导致蛋白质结构发生改变。体外表达实验显示,与野生型相比,p.Leu278Pro和p.Trp444Arg两种突变在mRNA表达层面没有明显差异,但两种突变导致细胞培养上清液PC∶Ag含量和PC蛋白表达量明显低于野生型,而在细胞培养裂解液中高于野生型。结论PROC基因第9外显子杂合错义突变p.Leu278Pro和p.Trp444Arg导致PC蛋白分泌障碍可能是遗传性PC缺陷症的分子致病机制。ObjectiveTo investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C(PROC)genes through laboratory phenotype analysis,genetic mutation analysis,and in vitro expression experiments.MethodsTwo probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University.Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C(PC)activity(PC∶A),PC antigen(PC∶Ag)levels,and other relevant coagulation parameters.The anticoagulant capacity was assessed using the thrombin generation test(TGT).The mutation sites of the PROC gene were identified using direct DNA sequencing.Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene.PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids.Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected.Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene.Enzyme-linked immunosorbent assay,Western blot,and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein.ResultsProbands 1 and 2 exhibited PC∶A levels of 35%and 40%and PC∶Ag levels of 44%and 39%,with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L,respectively.Meanwhile,other coagulation parameters revealed no significant abnormalities.TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers.Sequencing analysis revealed heterozygous missense mutations c.833T>C(p.Leu278Pro)in proband 1 and c.1330T>C(p.Trp444Arg)in proband 2 within exon 9 of the PROC gene.Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species.Pathogenicity analysis indicated that both p.Leu278Pro and p.Trp

关 键 词：蛋白C 基因突变 体外表达 血栓形成 

分 类 号：R73[医药卫生—肿瘤]