遗传性血管性血友病7例实验诊断和分子发病机制研究  

Laboratory diagnosis and molecular characterization of seven Chinese von Willebrand's disease families

作　　者：谢飞[1] 王鸿利[1] 王学锋[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海血液学研究所,上海200127

出　　处：《诊断学理论与实践》2006年第5期384-389,共6页Journal of Diagnostics Concepts & Practice

摘　　要：目的:建立系统的血管性血友病(vWD)实验诊断和研究的方法,对7个遗传性vWD家系进行系列的实验诊断和分子发病机制研究。方法:自行建立瑞斯托霉素诱导的血小板凝集试验(RIPA)、血管性血友病因子(vWF)瑞斯托霉素辅因子(vWF:RCo)、vWF抗原(vWF:Ag)、vWF胶原结合试验(vWF:CB)和多聚体分析方法,对vWD患者及家系成员进行测定。采用PCR产物测序以鉴定vWF基因缺陷。采用定点突变、剪接位点分析、序列复杂性分析和核基质结合区评分等生物信息学方法,研究基因突变对蛋白功能的影响。结果:明确诊断vWD患者1型1例,2A型2例,2B型1例,2M型1例及3型2例,其中1型、2M型和2B型vWD为国内首次报道。鉴定到10个基因突变,其中6个为国际上首次发现。体外表达实验显示,R1374S突变引起细胞分泌下降;R1308C突变导致蛋白功能降低;C2327S突变影响多聚体形成。结论:本研究建立的方法和推荐的组合实验适合于绝大多数vWD患者的诊断和分型需要。R1374S、R1308C和C2327S突变分别导致患者出现1型、2B型和2A型vWD表型。插入、缺失和无义突变诱发无义介导的mRNA衰变(NMD),导致vWF合成减少,是本研究中3型患者的发病机制。Objective To develop and employ systematic methods for clinical laboratory diagnosis of seven von Willebrand＇s disease（vWD） families, and to elucidate the molecular mechanisms. Methods RIPA, vWF：RCo, vWF：Ag, vWF：CB and muhimer assay were developed and various phenotypes were determined on the members of the seven vWD families. PCR products were sequenced to locate the potential gene defects. Mutant expression vectors were constructed for the detection of mutant protein expression. In silico analysis, including splice site, sequence complexity and MAR assay were performed. Results Seven vWD patients including one case of type 1, two of type 2A, one of type 2B, one type 2M and two of type 3 were diagnosed. Ten causative mutations of vWF gene were identified. The study in vitro showed a significant decrease of vWF levels for R1374S, a drop of vWF：CB for R1308C and an impaired muhimerization for C2327S. Conclusions Combination of the tests is qualified for the diagnosis of vWD. R1374S, R1308C and C2327S mutation are shown to be associated with the type 1, type 2B and type 2A vWD, respectively. Gene deletion, insertion, mutation in the acceptor site of intron 14 and associated nonsense-mediated mRNA decay are responsible for type 3vWD families in our study.

关 键 词：血管性血友病因子 血管性血友病 基因突变 实验室诊断 

分 类 号：R554.1[医药卫生—血液循环系统疾病]