[Molecular genetics of hemophilia A]

Hemophilia A (HemA), an X linked genetic disease, is the most common coagulation disorder with an incidence of about 1-2 in 10,000 males and is caused by mutations in the factor VIII (FVIII) coagulation gene. Firstly, some clinical aspects of the HemA are presented: the current methods to assess both the amount and activity of FVIII, the severity range observed and the presence of inhibitor antibodies against the therapeutic FVIII. Follows a discussion of the relationship of the structural domains of the FVIII protein (Figure 1), the aminoacid sequence and their functions. An activation-inactivation model of the successive peptide bonds cleavages of the FVIII is also presented (Figure 2). After the cloning of the FVIII gene in 1984, almost all types of HemA causing mutations have been characterized. However, the size and complexity of this gene prevented a screening of the full range of mutations for an accurate molecular diagnosis. Moreover, most of the patients with moderate and mild disease have missense mutations whereas approximately half of severe patients have nonsense, frameshift, and some missense mutations. There are also less frequently mutations such as deletions and insertions leading to severe phenotype and mutations affecting mRNA splicing and duplications causing both severe and mild HemA. In order to give genetic counselling in HemA families, studies at the DNA level using intragenic and/ or extragenic polymorphism analysis have been used. But this approach is not entirely satisfactory because it fails in several situations. Most of the causing mutations described above are private, and they have been found in only a few unrelated families. Recently, a common molecular inversion of the FVIII gene was identified in 50% of unrelated patients with severe HemA. The copies of a particular DNA sequence (termed F8A gene). One copy is located within intron 22 of the FVIII gene and the other two, 500 kb upstream. An homologous recombination mechanism was proposed for the inversion between an intragenic copy of the F8A gene and either the distal (80% of the inversion) or the proximal copy (20%). Both of these inversions lead to severe HemA because no intact FVIII is produced and can be easily diagnosed by Southern blot analysis. This inversion originates almost exclusively in male germ cells, because pairing Xq with its homologous in female meiosis would probably inhibit the proposed intrachromosome recombination. The molecular analysis of the inversion of intron 22 is now considered as the first line for families with severe HemA patients. In recent years the treatment of patients with hemophilia A and B has been intravenous injection of FVIII or FIX concentrates, respectively. This regimen of regular injection of plasmatic proteins bears a high risk of infection by contaminating viruses (HIV, HBV, etc). Future treatment for patients with hemophilia may include the use of either gene therapy or recombinant coagulation factors. Both strategies would completely avoid the infection risk offering a safe and effective treatment for the disease. Recombinant factors, obtained by genetic engineering methods, provide a renewable and unlimited source of FVIII or FIX. The clinical trials of recombinant factors have already started in mid-1995 giving positive results. On the other hand, gene therapy for hemophilia is now in the pre-clinical stage but offers the prospect of a cure for the disease, thus potentially freeing patients from regular injections of the lacking protein. However, experiments in animal models suggest that it may be difficult to obtain adequate therapeutic levels of factors for long periods of time. Recently, a retroviral-mediated gene delivery of human FVIII in mice has been reported using the ex vivo strategy of gene therapy. Therapeutic levels of FVIII in the circulation were obtained for > 1 week and it was also observed that the capacity of primary cells to deliver FVIII in blood was strongly dependent on