Ser234Leu missense mutation in the A1 domain of factor V causing moderate factor V deficiency in a Chinese family

To investigate the molecular defects in a Chinese pedigree with inherited factor V (FV) deficiency.

Laboratory studies including activated partial thromboplastin time (APTT), prothrombin (PT), and thrombin time (TT) were tested in a patient and his family members. FV antigen (FV:Ag) and FV activity (FV:C) were measured by both ELISA and one-stage clotting assays. All the exons, exon-intron boundaries and promoter regions of FV gene were analysed by direct sequencing. The detected mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA and the wild-type and mutant FV proteins were expressed in COS-7 and CHO cells.

The proposita, a 52-year-old Chinese man, had no spontaneous bleeding syndrome. It was found that he had prolonged APTT and PT, 52 s and 22.8 s, respectively, a FV:C of 5.5% and a FV:Ag of 33.1%. Gene analysis showed the proposita was a compound heterozygote of FV mutations, carrying Ser234Leu and Arg413Cys. The FV antigen and activity levels of the Ser234Leu and Arg413Cys mutants are lower than wild type both in cell lysates and in culture media. Protein degradation inhibitor experiment in transfected COS-7 cells showed that Ser234Leu and Arg413Cys degraded intracellularly through the lysosomal pathway. CHO cells expressing either the wild-type or the mutant FV were subjected to immunofluorescence staining with the indicated antibodies and organelle markers, indicating that Ser234Leu and Arg413Cys can be transported to Golgi partially.

We identified the molecular pathological mechanism of the novel C785T mutation causing type I inherited FV deficiency for the first time.