F9基因Arg327Ile新突变导致血友病B的分子发病机制研究

目的探讨F9基因Arg327Ile(R327I)突变导致血友病B的分子发病机制研究。方法对1名血友病B患者作实验室和基因诊断,定点突变法构建F9基因R327I突变的表达质粒;瞬时转染HEK293细胞,一期法检测细胞上清液中凝血因子Ⅸ活性(FⅨ∶C),ELISA法检测细胞上清液和裂解中FⅨ抗原(FⅨ∶Ag),Western blotting检测R327I突变蛋白的分子量及表达量;免疫荧光共定位染色法检测突变蛋白在内质网和高尔基体内分布。结果瞬时表达显示该患者突变细胞标本上清液中R327I突变型FⅨ∶C为野生型的4.49%,明显降低;细胞上清液和裂解液FⅨ∶Ag分别为野生型的31%和129%,为交叉反应物质减低型(CRMR)。Western blotting显示细胞上清液中R327I突变蛋白分子量与野生型相同,但含量比野生型明显降低;免疫荧光共定位染色显示R327I突变蛋白在内质网中分布较野生型多,而在高尔基体中较野生型少。结论 F9基因R327I突变影响蛋白质合成和分泌,突变蛋白较野生型表达量偏低,同时R327I突变蛋白存在凝血功能缺陷从而导致血友病B。

Studies on the molecular mechanism of haemophilia B caused by the Arg327Ile novel mutation in F9 gene

Objective To investigate the molecular mechanism of haemophilia B caused by a novel mutation of Arg327Ile(R327I) in F9 gene.Methods Laboratory and genetic analysis were performed in a haemophilia B patient.The R327I mutation expression plasmid was constructed with site-directed mutagenesis method based on the wild-type(WT) FⅨ expression plasmid.After the HEK293 cell was transiently transfected,the activity of FⅨ(FⅨ:C) was assayed by one stage method in the conditioned medium,while the FⅨ:Ag was measured by ELISA in both the conditioned media and the cell lysates.The molecular weight and the quantity of expressed FⅨ were analyzed by Western blot.Immunofluorescence co-localization analysis was used to examine the synthesis and secretion of the mutant protein.Results FⅨ:C of the R327I mutant protein was 4.49% of the WT in the conditioned medium,and the FⅨ:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31% and 129% respectively,compared to the WT.The mutation was characterized as cross-reaction material reduced(CRMR).Western blot analysis showed that the molecular weight of R327I protein was the same as the WT,but the amount was much less compared with WT in the conditioned medium.Immunofluorescence co-localization assays showed that there was much R327I protein in ER and less in Golgi compared with WT,implying abnormal secretion from ER to Golgi.Conclusion The abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein caused haemophilia B.