[Molecular mechanisms of two novel mutations of factor XIII gene resulting in hereditary coagulation deficiency].

ABSTRACT

OBJECTIVE: To investigate the mechanisms of two novel missense mutations of factor XIIIA subunit gene (Arg77-->Cys,Ser413-->Trp) in the pathogenesis of hereditary factor XIII deficiency. METHODS: Site-directed mutagenesis was conducted to obtain 2 mutant human XIII A recombinant plasmids, mut-PCI/FXIIIA. Normal wild type factor XIII A recombinant plasmid, wt-PCI/FXIIIA, and mut-PCI/FXIIIA, were transfected into cultured COS7 cells line, renal fibroid cell of African green monkey using Superfect reagent respectively, The expression levels of DNA, RNA and protein of human factor XIII, both wild type and mutant, were detected by PCR, RT-PCR and Western blotting. Pulse-chase experiment was used to look into the changing of factor XIII A in the cytoplasm. Factor XIIIA activity was assayed by Biotin-pentylamine incorporation technique. RESULTS: The mRNA levels of the two mutants in transfected cells were similar to that of the wild type factor XIIIA. But the amount of mutant factor XIIIA protein and its activity in cells decreased markedly, even disappeared. Pulse-chase experiment revealed that at the two mutants existed chase time 0.5 h and 1 h considerable amounts in cells and then disappeared rapidly later. CONCLUSION: The 2 mutations of the factor XIIIA cause the instability, degradation, and rapid disappearance of FXIIIA in cytoplasm, thus resulting in hereditary factor XIII deficiency.