Abstract
Abstract 2268
Introduction:
Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. There is poor correlation between FV activity in plasma and symptoms of bleeding tendency. In the present study, we identified a novel compound heterozygous mutation in the FV gene (F5) in a Japanese patient with severe bleeding symptoms.
Patient and methods: The patient was a young boy in his teens. He suffered from intracranial hemorrhage after birth and he also experienced joints and muscle hemorrhage, afterwards. His plasma FV activity was less than 1% and FV antigen was 2%. Sequence analysis of F5, FV protein analysis, and recombinant mutant protein expression experiments were performed. And to evaluate the relation between bleeding symptoms and FV phenotypes, we analyzed the FV level of platelets in the patient and his family members. Our present and previous results were verified.
Results:
Sequence analysis of F5 in this patient revealed a novel compound heterozygous mutation, G1617V missense mutation in exon 14 and a 1-bp insertion in exon 16 (p.G1645V and c.5255–5259 ins A). The patient's father had a G1617V mutation and his mother had a 1-bp insertion for the heterozygote state, respectively. The patient's two sisters each had also a 1-bp insertion for the heterozygote state. We analyzed the expression of platelet F5 mRNA by semi-quantitative RT-PCR, which showed that platelet F5 mRNA from the patient and his family members were equal to the amount in healthy subjects. Platelet FV protein from family members examined by western blotting detected equal to those of healthy subjects, although the FV bands from the patient were detected only weakly. Platelet FV antigen and activity were measured with an ELISA and a functional assay based on prothrombin time. The patient's platelet FV activity was 1.6% and antigen was 1%, compared with 100% for those of normal subjects. Platelet FV activity and antigen of the patient's father, mother, elder sister and younger sister were 26% and 37.1%, 39.6% and 51.0%, 23.3% and 38.0%, and 35.4% and 56.6%, respectively. In the expression study of pMT2/FV-wild-type and FV-G1617V mutant recombinant proteins in HEK293 cells, the FV antigen levels produced by the FV-G1617V mutant in cell lysates was approximately 73% of wild-type. In conditioned media of the study, FV antigen and specific activity were reduced to approximately 4% and 6% of wild-type, respectively.
Discussions:
Although there was similar intracellular synthesis of the FV-G1617V mutant and wild-type FV, the results in the conditioned media suggested that secretion of the FV-G1617V mutant was impaired. Platelet FV protein was detected very low by Western blot and ELISA, though platelet F5 mRNA was confirmed to be normal by RT-PCR. Our previous results raise the possibility that in the case of severe FV deficiency, such as that with a 1 % or less plasma FV level, the amount of FV in platelets is important to cope with local bleeding. This patient's platelet FV activity and antigen levels were the lowest of those of a number of FV-deficient patients whom we have analyzed so far, and this patient had severe bleeding symptoms.
Conclusion:
Our previous studies support the concept that the severity of bleeding is related to FV protein in platelets. Taken together, we believe the severity of this patient's bleeding tendency is caused by a reduction in both platelet and plasma FV, especially by the highly significant decrease in platelet FV.
Disclosures:
No relevant conflicts of interest to declare.
A novel nonsense mutation of ERCC2 in a Vietnamese family with xeroderma pigmentosum syndrome group D
