PROC基因突变与静脉血栓栓塞的相关性
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摘要
目的:通过对静脉血栓栓塞(venous thromboembolism,VTE)患者的PROC基因进行测序,研究是否存在有义突变通过影响血浆蛋白C(PC)水平增加罹患VTE的风险。方法:以人PROC基因序列设计引物,特异性扩增编码PC-Gla结构域的第三外显子区,测序后找寻有义的SNVs位点,构建稳定表达突变型PC的细胞株进行细胞水平研究,同时利用ELISA法研究PC水平的分布情况。结果 :在我国VTE病患中发现3个单核苷酸突变,在HUVEC突变型PC的稳定表达株中皆可见PC的合成减少。通过检测正常标本、VTE患病率>2次的病患标本和突变标本中的PC水平,同样发现发生突变的样本PC水平低于VTE组(P=0.035 3)和正常水平值(P<0.000 1)。结论 :PCArg-1Cys、PCArg9Cys、PCVal34Met三个突变位点很可能是导致血浆PC水平明显降低以及VTE患病风险增加的重要遗传学因素。
Objective To study the effect of protein C mutation on venous thrombosis(VTE) by PROC gene sequencing in patients with VTE. Methods Human PROC gene sequence was designed to amplify the third exon region of the PC-Gla domain, and then the PCR products were sequenced to search for a single nucleotide mutation(SNVs). The SNVs was constructed into eukaryotic expression system and a stable expression of wild-type and mutant PC cells were also constructed. At the same time, the distribution of PC levels in normal and VTE patients were detected with ELISA. Results Three single nucleotide mutations were found in different patients. In HUVEC cells, the synthesis of PC decreased in each mutant strain. The PC level in the normal patients, VTE patients, and the mutant samples were detected, which were significantly lower in the mutant samples than that of the VTE group(P = 0.035 3) and the normal level(P < 0.000 1). Conclusion Three mutation sites PCArg-1Cys, PCVal34 Met and PCArg9 Cys are important genetic factors lead to a significant decrease in plasma PC levels and the increase of VTE risk.
Objective To study the effect of protein C mutation on venous thrombosis(VTE) by PROC gene sequencing in patients with VTE. Methods Human PROC gene sequence was designed to amplify the third exon region of the PC-Gla domain, and then the PCR products were sequenced to search for a single nucleotide mutation(SNVs). The SNVs was constructed into eukaryotic expression system and a stable expression of wild-type and mutant PC cells were also constructed. At the same time, the distribution of PC levels in normal and VTE patients were detected with ELISA. Results Three single nucleotide mutations were found in different patients. In HUVEC cells, the synthesis of PC decreased in each mutant strain. The PC level in the normal patients, VTE patients, and the mutant samples were detected, which were significantly lower in the mutant samples than that of the VTE group(P = 0.035 3) and the normal level(P < 0.000 1). Conclusion Three mutation sites PCArg-1Cys, PCVal34 Met and PCArg9 Cys are important genetic factors lead to a significant decrease in plasma PC levels and the increase of VTE risk.
引文
[1]朱铁楠,赵永强.易栓症诊断中国专家共识(2012年版)[J].中华血液学杂志,2013,26(2):156-157.
[2]SIMIONI P,TORMENE D,SPIEZIA L,et al.Inherited thrombophilia and venous thrombo embolism[J].Semin Thromb Hemost.2006,32:700-708.
[3]李秋月,李奕辰,张玉泉.2003-2013年1136例静脉血栓栓塞症住院患者临床资料分析[J].实用医学杂志,2015,31(6):1006-1008.
[4]SHEN MC,LIN J S,TSAY W.High prevalence of antithromb III,protein C and protein S deficiency,but no factor VLeiden mutation in venous thrombophilic Chinese patients in Taiwan[J].Thromb Res,1997,87:377-385.
[5]白春梅,潘家绮,范连凯,等.静脉血栓患者抗凝蛋白缺陷研究[J].中华内科杂志,2000,39(11):746-748.
[6]MILETICH JP,BROZE GJ JR.Beta protein C is not glycosylated at asparagine 329.The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites[J].J Biol Chem,1990,265:11397-11404.
[7]NEYRINCK AP,LIU KD,HOWARD JP,et al.Protective mechanisms of activated protein C in severe inflammatory disorders[J].Br J Pharmacol,2009,158(4):1034-1047.
[8]LIU H,WANG HF,TANG L,et al.Compound heterozygous protein C deficiency in a family with venous thrombosis:Identification and in vitro study of p.Asp297His and p.Val420Leu mutations[J].Gene,2015,563(1):35-40.
[9]STEARNS-KUROSAWA DJ,KUROSAWA S,MOLLICA JS,et al.The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex[J].Proc Natl Acad Sci U S A,1996,93:10212-10216.
[10]TAYLOR FB JR,PEER GT,LOCKHART MS,et al.Endothelial cell protein C receptor plays an important role in protein C activation in vivo[J].Blood,2001,97:1685-1688.
[11]CORRAL J,VICENTE V.Inherited thrombophilic conditions[J].Hematology,2012,17(1):163-1661.
[12]BAGLIN T.Inherited and acquired risk factors for venous thrombo embolism[J].Semin Respir Crit Care Med,2012,33:127-137.
[13]IKEJIRI M,SHINDO A,II Y,et al.Frequent association of thrombophilia in cerebral venous sinus thrombosis[J].Int JHematol,2012,95:257-262.
[14]GRINNELL BW,WALLS JD,GERLITZ B.Glycosylation of human protein C affects its secretion,processing,functional activities,and activation by thrombin[J].J Biol Chem,1991,266:9778-9785.
[15]SIMIONI P,KALAFATIS M,MILLAR DS,et al.Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma[J].Blood,1996,88:2101-2108.
[16]HEIT JA.Predicting the risk of venous thromboembolism recurrence[J].Am J Hematol,2012,87(1):63-67.
[2]SIMIONI P,TORMENE D,SPIEZIA L,et al.Inherited thrombophilia and venous thrombo embolism[J].Semin Thromb Hemost.2006,32:700-708.
[3]李秋月,李奕辰,张玉泉.2003-2013年1136例静脉血栓栓塞症住院患者临床资料分析[J].实用医学杂志,2015,31(6):1006-1008.
[4]SHEN MC,LIN J S,TSAY W.High prevalence of antithromb III,protein C and protein S deficiency,but no factor VLeiden mutation in venous thrombophilic Chinese patients in Taiwan[J].Thromb Res,1997,87:377-385.
[5]白春梅,潘家绮,范连凯,等.静脉血栓患者抗凝蛋白缺陷研究[J].中华内科杂志,2000,39(11):746-748.
[6]MILETICH JP,BROZE GJ JR.Beta protein C is not glycosylated at asparagine 329.The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites[J].J Biol Chem,1990,265:11397-11404.
[7]NEYRINCK AP,LIU KD,HOWARD JP,et al.Protective mechanisms of activated protein C in severe inflammatory disorders[J].Br J Pharmacol,2009,158(4):1034-1047.
[8]LIU H,WANG HF,TANG L,et al.Compound heterozygous protein C deficiency in a family with venous thrombosis:Identification and in vitro study of p.Asp297His and p.Val420Leu mutations[J].Gene,2015,563(1):35-40.
[9]STEARNS-KUROSAWA DJ,KUROSAWA S,MOLLICA JS,et al.The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex[J].Proc Natl Acad Sci U S A,1996,93:10212-10216.
[10]TAYLOR FB JR,PEER GT,LOCKHART MS,et al.Endothelial cell protein C receptor plays an important role in protein C activation in vivo[J].Blood,2001,97:1685-1688.
[11]CORRAL J,VICENTE V.Inherited thrombophilic conditions[J].Hematology,2012,17(1):163-1661.
[12]BAGLIN T.Inherited and acquired risk factors for venous thrombo embolism[J].Semin Respir Crit Care Med,2012,33:127-137.
[13]IKEJIRI M,SHINDO A,II Y,et al.Frequent association of thrombophilia in cerebral venous sinus thrombosis[J].Int JHematol,2012,95:257-262.
[14]GRINNELL BW,WALLS JD,GERLITZ B.Glycosylation of human protein C affects its secretion,processing,functional activities,and activation by thrombin[J].J Biol Chem,1991,266:9778-9785.
[15]SIMIONI P,KALAFATIS M,MILLAR DS,et al.Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma[J].Blood,1996,88:2101-2108.
[16]HEIT JA.Predicting the risk of venous thromboembolism recurrence[J].Am J Hematol,2012,87(1):63-67.