摘要目的 探讨4个遗传性凝血因子Ⅻ(FⅫ)缺陷症家系基因突变及分子发病机制.方法 检测凝血酶原时间(PT)、APTT、FⅫ促凝活性(FⅫ∶C)和FⅫ抗原(FⅫ∶ Ag)等凝血指标;PCR扩增FⅫ基因的全部外显子及侧翼序列,PCR产物纯化后测序;在野生型pIRES2-EGFP FⅫ表达质粒上构建突变体FⅫ表达质粒,瞬时转染COS7细胞,测定细胞裂解液和培养上清中FⅫ∶Ag水平和FⅫ∶C.结果 4例先证者APPT均明显延长,FⅫ∶C均＜2％,FⅫ∶Ag亦同步下降至1％.4个家系均检出常见的FⅫ46C/T多态性.家系A先证者检出5741-5742delCA(His101Gln)及7142insertC(Lys346Gln)双重杂合突变;家系B先证者检出6800-6808de19bp杂合缺失突变.FⅫ6800-6808de19bp瞬时转染结果显示,在细胞裂解液中突变体FⅫ∶Ag为野生型的85.6％;在细胞培养上清中突变体FⅫ∶Ag为野生型的51.9％,FⅫ∶C为野生型的56.4％;家系C先证者检出8699G＞ A(Gly542Ser)杂合突变;家系D先证者检出8699G＞A(Gly542Ser)纯合突变(其父母为近亲结婚).结论 4个遗传性FⅫ缺陷症家系除有46C/T多态性外,共发现g.5741-5742delCA、g.7142insertC、g.6800-6808de19bp、g.8699G＞A 4种基因突变.其中g.5741-5742delCA和g.6800-6808de19bp为两种新的突变.FⅫ6800-6808de19bp体外表达显示,FⅫ6800-6808de19bp突变蛋白合成基本正常,但存在蛋白分泌障碍.

abstractsObjective To identify the genotype and pathogenesis in four Chinese pedigrees with Factor Ⅻ deficiency.Methods Activated partial thromboplastin time (APTT),F Ⅻ procoagulant activity (FⅫ∶ C),FⅫ antigen(FⅫ∶ Ag)and other coagulant parameters were detected.The FⅫ deficiency Pedigree members,all exons,boundary introns including the splice junctions of the FⅫ gene were amplified with Polymerase chain reaction (PCR).Expression plasmids were constructed by mutagenesis based on the wild-type and transfected into COS7 cells.FⅫ∶ C and FⅫ∶ Ag of the expression levels were tested in the supernatant and cell lysate.Results The four probands presented prolonged APTT with all the values of F Ⅻ∶ C and FⅫ∶ Ag were low to 2％ and 1％,respectively.There were common 46C/T polymorphism in the promoter regions of FⅫ gene in four pedigrees.Proband A was heterozygous for two mutations,g.5741-5742delCA (His101Gln) and g.7142insertC (Lys346Gln).Proband B was a heterozygous deletion mutation g.6800-6808de19bp.The results of the transfection revealed that FⅫ∶ Ag in cell lysates and conditioned media protein FⅫ6800-6808de19bp were 85.6％ and 51.9％.The FⅫ∶ C in the conditioned media was 56.4％.Proband C was a heterozygous mutation g.8699G ＞ A(Gly542Ser).Proband D was a homozygous mutation 8699G ＞ A,whose parents with consanguineous marriage.Conclusions Four mutations,g.5741-5742delCA,g.7142insertC,g.6800-6808de19bp and g.8699G ＞ A with 46C/T polymorphism in the promoter regions of FⅫ gene,were identified in the four Factor Ⅻ deficiency pedigrees.The two mutations g.5741-5742delCA and g.6800-6808de19bp were first found in China.FⅫ 6800-6808de19bp expressed in vitro suggested that almost normal proteinum synthesis but defect proteinum secretion.