遗传性蛋白C缺陷症四个家系的分子发病机制研究

目的 对4个遗传性PC缺陷症家系进行临床表型诊断和基因型分析.方法 分别用发色底物法和凝固法测定4个家系先证者及家系成员的血浆PC:A、TPS:A和FPS:A;PC:Ag和FPS:Ag的测定采用ELISA法.采用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和PS基因的全部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.结果 先证者1的PC:A为36%,PC:Ag 为57%,TPS:A为48%,FPS:A为18%,FPS:Ag为23.7%,基因分析显示其PC基因2号、7号和8号外显子分别存在L-34P、K150del和A209V 的杂合突变,其中L-34P和A209V来自父亲,K150del来自母亲;先证者2的PC:A为46%,PC:Ag为64.4%,TPS:A为36%,FPS:A为19.5%,FPS:Ag为20.9%,其PC基因的2号和7号外显子分别存在T66C的多态性和R147W的杂合突变,PS基因的14号外显子存在Tyr519stop的杂合突变,其中PC突变来自母亲,PS突变来自父亲.凝血酶生成试验显示先证者2及其父母的抗凝功能减弱,其中PS缺陷的先证者和其父亲的外源性活化蛋白C抑制凝血酶生成的能力下降,而其母亲没有明显变化.先证者3的PC:A为33%,PC:Ag为48.42%,其PC基因同时存在R147W和R178W突变;先证者4的PC:A为21%,PC:Ag为18.36%,基因检测结果显示先证者4是R178W和D255H的双杂合子.结论 遗传性PC缺陷症或PC、PS联合缺陷症是导致4例先证者出现静脉血栓的原因.杂合PC基因突变(L-34P、A209V、R147W、R178W和D255H)是导致4例先证者遗传性PC缺陷症的原因.

Study of molecular mechanisms of hereditary protein C deficiency in four Chinese families

Objective To investigate the phenotype, genotype and molecular mechanisms in four Chinese pedigrees with venous thrombosis caused by hereditary PC deficiency. Methods The plasma activity of PC: A, TPS: A and FPS: A of the probands and their family members were detected with chromogenic and coagulation assay. The antigen of PC and FPS were identified with ELISA. Thrombin generation tests were applied to indicate the coagulation status. All of the nine exons and intron-exon boundaries of PC gene and PS gene were amplified by PCR and directly sequenced for mutaiton investigation. Results Compound heterozygous mutations of L-34P, K150del and A209V with 36% of PC: A and 57% of PC: Ag were identified in proband 1. PC: A was 46% , PC: Ag was 64. 4% while TPS: A, FPS: A and FPS: Ag were 36% , 19.5% and 20.9% respectively in proband 2. Two independent heterozygous mutations of R147W in PC gene inherited from his mother and T519stop in PS gene inherited from his father were identified. The anticoagulant activity of Proband 2 and his parents were declined in thrombin generation assay. In proband with PS defeciency and his father, the inhibition of thrombin generation capacity was decreased with exogenous APC, while his mother did not have significant difference. In Proband 3, PC: A was 32% while PC: Ag was 48.42% . Two independent mutations of R147W and R178W in Exon 7 were detected. Compound heterozygous mutations of R178W and D255H,with 21% of PC : A and 18. 36% of PC: Ag were identified in the Proband 4. Conclusions Hereditary PC deficiency or combined PC and PS deficiency result in venous thrombosis in four Chinese families. Mutants of L-34P, A209V, R178W, R147W and D255H might be the molecular mechanisms of PC deficiency.