目的 探讨Ser250Phe突变致遗传性凝血因子Ⅶ(coagulation factorⅦ,FⅦ)缺陷的分子机制.方法 用STA-R全自动血凝分析仪检测先证者及家系成员的凝血酶原时间(prothrombin time,PT)、活化部分凝血活酶时间(activated partial thrombinoplastin time,APTT)、纤维蛋白原(fibrinogen,FIB)及凝血酶时间(thrombin time,TT)；一步法及ELISA分别检测先证者及家系成员凝血因子Ⅶ活性及抗原；PCR扩增先证者及其家系成员因子7基因(factor 7 gene,F7)所有外显子及侧翼序列,直接测序分析；PCR介导质粒DNA定点诱变法构建F7 Ser250Phe突变表达质粒,将表达质粒瞬时转染HEK293细胞,一步法测定培养上清FⅦ活性及ELISA及Western印迹测定培养上清及细胞裂解液FⅦ抗原；同时将F7表达质粒与高尔基体或内质网定位的荧光质粒共转染CHO细胞进行亚细胞定位分析.结果 先证者及家系成员APTT、TT及FIB均正常,而先证者PT、FⅦ活性及抗原分别为36.5 s、4.0％及130.2 ng/mL；测序发现先证者F7基因存在g.15975G＞A(IVS6-1 G＞A)与g.16750 C＞ T(Ser250Phe)双杂合突变,其父亲为g.16750 C＞T杂合子,母亲为g.15975G＞A杂合子,妹妹不携带这两种突变；HEK293细胞上清中FⅦ250Phe活性为(4.12±0.61)％(以FⅦ250Ser载体转染细胞培养上清FⅦ活性作为100％),培养上清中FⅦ250Ser与FⅦ250Phe抗原水平分别为(37.77±2.30)ng/mL和(4.02±0.52) ng/mL;而细胞裂解液中FⅦ250Ser与FⅦ250Phe抗原水平分别为(172.45±2.25) ng/mL和(130.51±2.32) ng/mL;Western印迹分析示转染了FⅦ250Phe载体的HEK293细胞培养上清无可检测FⅦ抗原,而在细胞裂解液中检测到FⅦ抗原,与ELISA检测结果一致；在CHO细胞表达的重组FⅦ250Phe绿色荧光信号能与核周内质网红色荧光信号重合,而且也与高尔基体红色信号重合,与重组FⅦ250Ser一致.结论 复合IVS6-1G＞A与Ser250Phe突变是导致患者遗传性凝血因子Ⅶ缺乏的原因；FⅦSer250Phe突变未见报道,其能正常合成,并转运至内质网及高尔基体,但不能有效分泌至细胞外,可能于细胞内部分降解或由于半衰期缩短而分泌后迅速降解.%Objective To identify and characterize a missence mutation Ser250Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family.Methods The FⅦ gene (F7) was analyzed by DNA sequencing,and the FⅦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method.In addition,a F Ⅶ-250Phe mutant corresponding to the identified mutation was expressed in HEK293 cells,and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of FⅦ deficiency caused by the FⅦ-250Phe mutation.Results The patient had a prolonged prothrombin time (PT:36.5 s) and low levels of both FⅦ antigen and activity (130.2 ng/mL and 4.0％,respectively).Two heterozygous mutations were identified in the F7 gene (NG-009262.1),which included a g.15975 G＞A mutation at the splice receptor site of intron 6 (IVS6-1G＞A) and a novel g.16750 C＞T mutation in exon 8,which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system.By gene expression experiments,the antigen and activity levels of FⅦ-250Ser and FⅦ-250Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL,respectively.ELISA and Western blotting analyses indicated that expression of the mutant FⅦ-250Phe and wild type FⅦ-250Ser was (130.51±2.32) ng/mL and (172.45±2.25) ng/mL,respectively.FⅦ-250Phe was found in endoplasmic reticulum and Golgi apparatus,suggesting that the mutant F Ⅶ-250Phe could be normally synthesized in the cells but was inefficiently secreted.Conclusion Compound heterozygous mutations in F7 gene (g.15975 G＞A and g.16750 C＞T) may be responsible for the FⅦ deficiency in this patient.The novel FⅦ 250Phe can be transported from endoplasmic reticulum to Golgi apparatus,but may be degraded or inefficient.
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