一种致遗传性凝血因子Ⅶ缺陷症的新突变（Ser250Phe）的鉴定与功能分析  

Identification and functional analysis of a novel missense mutation Ser250Phe underlying congenital coagulation factor Ⅶ deficiency

作　　者：江明华[1] 王兆钺[2] 余自强[2] 苏健[2] 曹丽娟[2] 张威[2] 

机构地区：[1]浙江省温州医学院附属第二医院检验科,325027 [2]苏州大学附属第一医院、江苏省血液研究所、卫生部血栓与止血重点实验室

出　　处：《中华医学遗传学杂志》2013年第2期152-156,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（81070395）

摘　　要：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F7Ser250Phe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36．5S、4．0％及130．2ng／mL；测序发现先证者F7基因存在g．15975G〉A（IVs6—1G〉A）与g．16750C〉T（Ser250Phe）双杂合突变，其父亲为g．16750C〉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土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 （FT） 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 VⅦ-250Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO ceils was performed to clarify the molecular mechanism of F VⅦ deficiency caused by the FⅦ-250Phe mutation. Results The patient had a prolonged prothrombin time （PT：36.5 s） and low levels of both FVg 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 （172.45Ⅶ2.25） 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.

关 键 词：凝血因子Ⅶ缺乏 基因突变 亚细胞定位 

分 类 号：R5[医药卫生—内科学]