Title: Studies on the molecular mechanism of haemophilia B caused by the Arg327Ile novel mutation in F9 gene
Abstract: 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.
Publication Year: 2011
Publication Date: 2011-01-01
Language: en
Type: article
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