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 investigate the molecular pathogenesis of hereditary coagulation factor Ⅻ (FⅫ) deficiency. Methods Clinical phenotypes and gene diagnosis were analyzed in two hereditary FⅫ pedigrees: coagulation and ELISA were used to detect plasma F Ⅻ procoagulant activity (F Ⅻ C) and antigen content (F Ⅻ:Ag) ; Direct sequencing for gene sequence analysis, for the proband’s mutation sites, detection of their family members corresponding gene mutations; using thrombin generation test to detect changes in coagulation function in patients with wild-type pIRES2-EGFP / FⅫ expression plasmid On the basis of site-directed mutagenesis, a mutant FⅫ expression plasmid was constructed and transiently transfected into 293T cells by Polyfect transfection reagent. FⅫ:Ag was measured in cell lysate and culture supernatant, and FⅫ:C was determined in supernatant. Results F Ⅻ: C was 4.68% and 12.08% in the two hereditary FⅫ deficiency pedigrees and 4.6% and 2.2% in F Ⅻ:Ag respectively. Exon 13 and exon 14 of F12 gene in proband 1 (G8489A, Glu502Lys) and (C8833A, Tyr586Stop), respectively. Two homozygous heterozygous mutations were found in exon 13 of F12 of proband 2 (G8489A; Glu502Lys). Some members of the pedigree detected corresponding Of heterozygous mutations. There was no significant difference in thrombin generation potential between two hereditary FⅫ deficiency patients and normal controls. Transient transfection results showed that FⅫ:C and FⅫ:Ag in FⅫ mutein E502K supernatant were 27.2% and 14.4% of the wild type, respectively; FⅫ:Ag in the cell lysate was 36.7% of the wild type. Conclusions In vitro experiments further confirmed that F50 was significantly reduced by the disruption of the synthesis and secretion of the mimic protein E502K. The proband 1 was caused by the two-hybrid mutation of the F12 gene (Glu502Lys; Tyr586Stop); the proband 2 was the inherited FⅫ deficiency caused by the G8489A homozygous mutation (Glu502Lys); the two mutations of E502K and Y586Stop were the first in the world Reported.