Identification and Cloning of Novel Mutations In a Compound Heterozygous Factor V Deficient Patient

Abstract 2210 Introduction: Congenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients normally present with very low or unmeasurable levels of functional and/or immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the F5 gene. Heterozygotes typically have approximately half-normal levels of FV and are asymptomatic, whilst compound heterozygotes may show mild to severe bleeding diathesis. Patient Description: A proband now aged 73, was clinically diagnosed since childhood as having severe FV deficiency (<3% FV activity by clinical lab analysis), exhibiting severe bleeding tendency with surgery or dental extraction. Other symptoms included frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in mid-thirties. Additional routine clinical assays for clotting factors were normal. There was no history of parental bleeding tendency. Methods: DNA was isolated from peripheral blood leucocytes. Exonic and flanking intronic sequences of F5 were amplified by PCR and subjected to automated nucleotide sequencing. To investigate the role of identified mutations in FV function and protein secretion, wildtype (WT) FV and FV variants consistent with mutations identified for the patient using the QuikChange Site-Directed Mutagenesis Kit were cloned into the pED-FV-1033 expression vector. This produced recombinant single chain FV that requires proteolytic activation to express cofactor function. Plasmids encoding WT FV, and the mutation identified in the patient were transfected into baby hamster kidney cells (BHK) using Lipofectamine and stable clones were established. Secretion of FV protein was quantified by commercial enzyme immunoassay (EIA), and FV activity was evaluated using conventional prothrombin time (PT) and activated partial thromboplastin time (APTT) clotting assays. Western blot analysis using monoclonal antibodies to both FV heavy (FVaH) and light chains (FVaL) detected FV antigen and fragmentation profiles. Results: Based on the lack of parental bleeding, compound heterozygosity was probable for this FV deficient patient because the DNA sequence analysis revealed two novel mutations; the first changed the codon for Leu1821 to Ser (L1821S), the second changed Gly2192 to Cys (G2192C). Clotting assays showed low patient plasma FV activity of 0.5±0.015% for PT and 2.0±0.10% for APTT compared to normal pooled plasma. Western blot analysis demonstrated the patient FV banding pattern was comparable to normal plasma, whilst densitometric analysis of the specific bands showed that the patient had 9% of the normal FV antigen level. Recombinant FV secretion detected by EIA from various clones ranged from 0 to 16.6ng/mL. L1821S (28%) and G2192C (25%) mutants secreted lower FV protein compared to WT. Clotting assays (n=2) showed that Leu1821S activity levels were 15% that of WT in contrast to G2192C where no FV clotting activity was detectable. Compared to WT, L1821S had 55% and G2192C had only 1.4% of the specific activity.Western blot analysis demonstrated the L1821S banding pattern was comparable to WT whilst G2192C lacked several fragments. Prolonged thrombin (FIIa) pretreatment of FV clones confirmed WT and both mutants were cleaved to FVaL (∼74k Da) and FVaH (∼105 kDa). In addition, FIIa pre-treatment enhanced clotting activity for most of the clones evaluated. Interestingly, the inactive G2192C mutant acquired modest FV function after pre-incubation with FIIa. Conclusions: Two novel FV mutations that affect both function and secretion have been identified in a FV deficient patient and have been cloned. The L1821S mutation is located close to a FV metal ion binding site that may affect function. G2192C introduces a thiol into FV, reported in the literature to have deleterious effects on protein folding, which may explain attenuated secretion and lack of clotting activity. Only after prolonged pre-treatment with FIIa, G2192C function was acquired, which may suggest that this mutation stabilizes the pro-cofactor conformation of FV. Low in vitro secretion of mutant FV compared to WT corresponds to low patient plasma FV antigen, which is compounded by profound loss of function in G2192C. Further biochemical studies are underway to understand the effect of G2192C on FV activity. Disclosures: No relevant conflicts of interest to declare.