Factor V Mutations, Arg2174Leu, a 5-bp Deletion and Val1813Met, Are Associated with Factor V Deficiency and A Bleeding Tendency

Abstract number: P1832

Shinozawa¹ K, Inaba¹ H, Amano¹ K, Suzuki¹ T, Takahashi² H, Iijima³ K, Tanaka⁴ A, Arai¹ M, Fukutake¹ K

¹¹Department of Laboratory Medicine, Tokyo Medical University, Tokyo, Japan ¹¹Department of Laboratory Medicine, Tokyo Medical University, Tokyo, Japan ²²Department of Internal Medicine, Niigata Prefectural Kamo Hospital, Niigata, Japan ³³Division of Clinical Laboratory, Tottori University Hospital, Tottori, Japan ⁴⁴Department of Laboratory Medicine, Tokyo Medical University Hachioji Medical Center, Tokyo, Japan

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. Although genetic defects underlying this disease have been reported recently, they are fewer than those reported for other coagulation factor deficiencies such as hemophilia. We performed genetic analysis of the FV gene in three unrelated Japanese patients with FV deficiency by direct sequencing. One of the three patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (6247–6251delACCCT) that creates a premature stop codon at residue 2032 in the C1 domain of FV and Val1813Met, which is located in the region that encodes the A3 domain of FV. The other two patients were homozygous for the Val1813Met or Arg2174Leu mutation, caused by a G to T transversion in exon 25 which is located in the C2 domain of FV. The 5-bp deletion and Arg2174Leu substitution are newly identified mutations. These 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 HEK293 cells. In transiently transfected cells, the FV-Arg2174Leu and Val1813Met mutants showed lower antigen and activity levels in culture media than were shown by wild-type. Furthermore, both the antigen and activity levels of the 5-bp deletion mutant of FV in the culture media were lower than those of the two missense mutants. The truncated protein was collected from conditioned media, and phospholipid binding was analyzed. The 5-bp deletion mutant of FV was unable to bind to phospholipid vesicles. Thus, we identified and characterized a deletion mutation and two missense mutations that cause FV deficiency.