2例遗传性凝血因子Ⅻ缺陷症分子发病机制研究

The molecular mechanisms of inherited factor Ⅻ deficiency in two unrelated families

目的探讨遗传性凝血因子Ⅻ(FⅫ)缺陷症的分子发病机制。方法对2例遗传性FⅫ家系作临床表型检测及基因诊断:采用凝固法及ELISA法分别检测先证者及其家系成员血浆FⅫ促凝活性(FⅫ∶C)和抗原含量(FⅫ∶Ag);直接测序法作基因序列分析,针对先证者的突变位点,检测其家系成员相应的基因突变;采用凝血酶生成试验,检测患者凝血功能的变化;在野生型pIRES2-EGFP/FⅫ表达质粒基础上,定点突变法构建突变型FⅫ表达质粒,以Polyfect转染试剂介导瞬时转染293T细胞,分别测定细胞裂解液及培养上清液中FⅫ∶Ag,测定上清液中FⅫ∶C。结果 2个遗传性FⅫ缺陷症家系的先证者的FⅫ:C分别为4.68%和12.08%,FⅫ∶Ag分别为4.6%和2.2%;先证者1的F12基因第13、14号外显子分别发现了(G8489A、Glu502Lys)和(C8833A、Tyr586Stop)2种复合杂合突变;先证者2的F12的第13号外显子发现(G8489A;Glu502Lys)纯合突变,其家系部分成员检测到相应的杂合突变。2名遗传性FⅫ缺陷症患者的凝血酶生成潜力与正常人比较无明显差异。瞬时转染结果显示,FⅫ突变蛋白E502K上清液中FⅫ∶C和FⅫ∶Ag分别为野生型的27.2%和14.4%;细胞裂解液中FⅫ∶Ag为野生型的36.7%。结论体外表达的实验进一步证实了突变蛋白E502K合成和分泌障碍导致FⅫ明显降低。先证者1由罹病F12基因(Glu502Lys;Tyr586Stop)双杂合突变所致;先证者2为G8489A纯合突变(Glu502Lys)所致遗传性FⅫ缺陷症;E502K和Y586Stop 2种突变均为国际首次报道。

Objective To study the pathogenesis of the 2 Chinese pedigrees of Factor Ⅻ deficiency mutation.Methods The diagnosis was made by coagulant parameters.FⅫ coagulation activity（FⅫ：C）and antigen（FⅫ：Ag）were detected by clotting test and ELISA respectively.Gene mutations were analyzed in the probands and their family members with PCR and DNA sequencing.Thrombin generation tests were used to detect coagulation status of the probands.Expression plasmids were constructed by site-directed mutagenesis based on the wild-type and transiently transfected into 293T cells.FⅫ：C and FⅫ：Ag of the expression products were tested in the supernatant and cell lysate.Results Plasma FⅫ：C and FⅫ：Ag of probands 1 and 2 were 4.6% and 4.68%,2.2% and 12.08%,respectively.Combined heterozygous mutations of Glu502Lys and Tyr586Stop were identified in proband 1,while homozygous mutation of Glu502Lys was found in proband 2.The results of the transfection revealed that FⅫ antigens in conditioned media and cell lysates of mutant protein Glu502Lys were 27.2% and 36.7%,respectively.The activity of FⅫ in cell lysate was 14.4% of the normal plasma level.Conclusion The mutation of Glu502Lys influenced the synthesis and secretion of mutant protein,leading to low level of FⅫ.Mutation of Glu502Lys and Tyr586Stop might be the molecular pathogenesis in proband 1,while the mutation of Glu502Lys might be the molecular mechanism in proband 2.Both Glu502Lys and Tyr586Stop mutations were first reported.