title: [Molecular mechanisms of Arg77Cys missense mutation and Arg174stop nonsense mutation of factor XIII A gene causing severe factor XIII deficiency].
abstract: The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor XIII (FXIII) deficiency. Site-directed mutagenesis was conducted to obtain human FXIIIA expression plasmids bearing the mutations. Wild type FXIIIA recombinant plasmid (pcDNA3.1-FXIIIA-wt) and 2 mutant FXIIIA recombinant plasmids (pcDNA3.1/FXIIIA/77mut, pcDNA3.1/FXIIIA/174mut) were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent, respectively. FXIII activities were measured by the Berichrom(®) FXIII chromogenic assay. The expression levels of FXIIIA mRNA were detected by real-time RT-PCR. The recombinant FXIIIA mutants were determined by using Western blot and ELISA. The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82 ± 0.21 and 0.76 ± 0.17, respectively. The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type (1.06 ± 0.51). FXIII activity and FXIIIA antigen levels in concentrated media of cell expressing the wild type protein were (24.0 ± 2.9)% and (13.2 ± 2.3)%, respectively. FXIII activity and FXIIIA antigen levels in cell lysates containing the wild type recombinant protein were (61.6 ± 30.4)% and (32.8 ± 14.5)%, respectively. However, the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type. It is concluded that both mutations severely disturb the normal expression of FXIIIA protein. The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.
authors.affiliation: Department of Pathology and Laboratory Medicine, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, China. zwdin@126.com
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Zheng
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Luo
authors.forename: Zhi-Bin
authors.initials: ZB
authors.lastname: Yao
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense; D005177:Factor XIII Deficiency / Q000235:genetics*; D026122:Factor XIIIa / Q000235:genetics*; D005838:Genotype; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018389:Codon, Nonsense; D026122:Factor XIIIa