FUNCTIONAL CHARACTERIZATION OF HUMAN FACTOR VII BY MOLECULAR ANALYSIS OF INHERITED DYSFUNCTIONAL FACTOR VII VARIANT

1.对12个无血缘关系的遗传性第VII因子(FVII)家系中的30例进行了功能研究，其中2个家系先证者存在交叉反应物质阴性(CRM-)型缺陷，10个家系先证者存在交叉反应物质阳性(CRM+)型缺陷。SSCP分析发现，每例患者的CRM+型外显子2、1例外显子4、1例外显子5、1例外显子8、4例均存在相对于正常对照的异常迁移率。一个外显子4的异常迁移率的病例的特征是使用不同来源的组织因子具有不同的促凝血活性。该病例在第6055位发生G-A转换，导致第一个表皮生长因子样区中的Arg79被Gln取代，与我们先前报道的情况相同(Biochem，33：14162-149,1994)。在4例外显子8迁移率异常的病例中，第一例在11514位核苷酸发生了C到T的转换，这导致了TH…在催化区有更多的Thr359被Met取代，与以前的报告相同(布拉德，89：5085,1997)。第2例在11429位核苷酸发生A-G转换，导致Gly331被Ser取代，与以前的报道相同(血液纤维蛋白原，7：93,1996)。第3例在11267位核苷酸发生C-T转换，导致Arg277被半胱氨酸取代。第4例在11487位碱基发生T-G转换，导致His348被Gln取代。我们从Hep G2细胞中提取RNA，通过RT-PCR法制备Fvii基因，并将其插入表达载体pEE14。转染CHO-K1细胞。用ELISA法检测培养基中FVII：Ag的表达，加入维生素K诱导培养基中Fvii：C的表达，用去Fvii的血浆进行APTT检测。通过体外定点突变，将密码子331、271和260的单个碱基改变引入野生型FVII基因的cDNAs序列中。较少

1. Thirty cases from 12 unrelated families with inherited dysfunctional factor VII (FVII) were studies in order to investigate the function of factor VII.The probands from 2 families had a cross reactive material negative (CRM-) type deficiency, and the probands from 10 familie had a cross reactive material positive (CRM+) type deficiency.2. SSCP analysis identified an aberrant mobility relative to the normal control in each to the CRM+ type, exon 2 ; one, exon 4 ; one, exon 5 ; one, exon8 ; 4 cases.3. A case with an aberrant mobility in exon 4 was characterized by variable procoagulant activity using tissue factor from different sources. This case had G to A transition at nucleotide position 6055, which resulted in the substitution of Arg 79 by Gln in the 1st EGF-like domain, and was same case to our previous report (Biochem, 33 : 14162-14169,1994).4. In 4 cases with an aberrant mobility in exon 8, the first case had C to T transition at nucleotide position 11514, which resulted in th … More e substitution of Thr359 by Met in the catalytic domain, and was same to the previous report (Blood, 89 : 5085,1997). The 2nd case had A to G transition at nucleotide position 11429, which resulted in the substitution of Gly331 by Ser, and was same to the previous report (Blood Coag Fibrionl, 7 : 93,1996). The 3rd case had C to T transition at nucleotide position 11267, which resulted in the substitution of Arg277 by Cys. The 4th case had T to G transition at nucleotide position 11487, which resulted in the substitution of His348 by Gln.We prepared FVII cDNA by RT-PCR using RNA fron Hep G2 cell, in order to express each recombinant variant FVII.The wild-type FVII cDNA was inserted into the expression vector pEE14. CHO K1 cells were transfected. FVII : ag was detected in the medium by ELISA.After incuvated to add vitamin K into the medium, FVII : c was detected in the medium by APTT using FVII depleted plasma.6. A single base change at each codon 331,271 and 260 are introducing into the wild-type FVII cDNA sequence by oligonucleotide in vitro site-directed mutagenesis. Less