Identification of Ten Novel Mutations Associated with Inherited Coagulation Factor V Deficiency.

Factor V is a rare bleeding disorder characterized by low levels of plasma and/or platelet factor V, with an estimated prevalence of 1/1,000,000 and results from defects in the factor V gene. Human coagulation factor V (FV), a single chain glycoprotein, is synthesized in hepatocytes and megakaryocytes with 80% circulating free in plasma and the remainder being released upon platelet activation. The factor V gene comprises 25 exons ranging in size from 72bp to 2820bp coding for a protein which is oriented as A1-A2-B-A3-C1-C2 domains. FV is activated to its active form (FVa) by thrombin or activated factor X (FXa) which removes the B domain, generating a heavy chain and a light chain that are linked together in the presence of calcium ions. FVa binds to FXa and serves as its cofactor in the prothrombinase complex to convert prothrombin to thrombin. There is a high degree of homology between the A and C domains of FV and FVIII.

We have investigated 8 unrelated patients from two centres with phenotypic and clinical charisteristics of FV deficiency. Mutation screening was carried out in these patients using Denaturing high performance liquid chromatography (dHPLC) and sequencing. Probable causative mutations were identified in all patients.

A total of 10 novel mutations were identified in 8 patients and were located in the A1, A2, A3 and B domains. No mutations were identified in the C domain, and entries on the FV mutation database support our findings that mutations in this domain are less common than elsewhere in this gene. 5/8 patients were diagnosed with mild-moderate FV deficiency, and single heterozygous mutations were identified in each of these patients. 3 missense , 1 donor splicesite and 1 nonsense mutation were identified in the A1, A3 and B domains. The remaining 3/8 patients had severe FV deficiency (FV levels <2u/dl). One was compound heterozygous for 2 missense mutations in the A3 domain; one had a missense mutation in the A2 domain and a frameshift mutation (insertion of a single base pair) in the A3 domain. We have as yet identified only a heterozygous missense mutation in the third patient with severe FV deficiency. Phenotypic data and family history are strongly suggestive of the presence of a second mutation. Quantitative DNA analysis has confirmed the presence of 2 FV alleles, and RNA analysis is in progress to identify the second mutation.

100 normal alleles were analysed by dHPLC analysis or allele specific amplification to exclude these changes from being polymorphisms. We also have examined the homology between factor V and factor VIII, and the degree of similarity, between native and mutant amino acids to support these mutations as being causative of FV deficiency.