MOLECULAR-GENETICS OF HEMOPHILIA-A

Hemophilia A (HemA), an X linked genetic disease, is the most common c oagulation disorder with an incidence of about 1-2 in 10,000 males and is caused by mutations in the factor VIII (FVIII) coagulation gene. F irstly, some clinical aspects of the HemA are presented: the current m ethods to assess both the amount and activity of FVIII, the severity r ange observed and the presence of inhibitor antibodies against the the rapeutic FVIII. Follows a discussion of the relationship of the struct ural domains of the FVIII protein (Figure 1), the aminoacidic sequence and their functions. An activation-inactivation model of the successi ve 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 compl exity of this gene prevented a screening of the full range of mutation s for an accurate molecular diagnosis. Moreover, most of the patients with moderate and mild disease have missense mutations whereas approxi mately halt of severe patients have nonsense, frameshift, and same mis sense mutations. There are also less frequently mutations such as dele tions and insertions leading to severe phenotype and mutations affecti ng mRNA splicing and duplications causing both severe and mild HemA. I n order to give genetic counselling in HemA families, studies at the D NA 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 a bove are private, and they have been found in only a few unrelated fam ilies. Recently, a common molecular inversion of the FVIII gene was id entified in 50% of unrelated patients with severe HemA. The inversion is mediated by the presence of three copies of a particular DNA sequen ce (termed F8A gene). One copy is located within intron 22 of the FVII I gene and the other two, 500 kb upstream. An homologous recombination mechanism was proposed for the inversion between an intragenic copy o f the F8A gene and either the distal (80% of the inversion) or the pro ximal copy (20%). Both of these inversions lead to severe HemA because no intact FVIII is produced and can be easily diagnosed by Southern b lot analysis. This inversion originates almost exclusively in male ger m cells, because pairing Xq with its homologous in female meiosis woul d probably inhibit the proposed intrachromosome recombination. The mol ecular analysis of the inversion of intron 22 is now considered as the first line for families with severe HemA patients. In recent years th e treatment of patients with hemophilia A and B has been intravenous i njection of FVIII or FIX concentrates, respectively. This regimen of r egular injection of plasmatic proteins bears a high risk of infection by contaminating viruses (HIV, HBV, etc). Future treatment for patient s with hemophilia may include the use of either gene therapy or recomb inant 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, provid e a renewable and unlimited source of FVIII or FIX. The clinical trial s of recombinant factors have already started in mid-1995 giving posit ive 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 disea se, thus potentially freeing patients from regular injections of the l acking 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 g ene therapy. Therapeutic levels of FVIII in the circulation were obtai ned for > 1 week and it was also observed that the capacity of primary cells to deliver FVIII in blood was strongly dependent on the site of implantation. Although much work remains to be done, these positive r esults are encouraging for the future of gene therapy for this relativ ely common genetic disease.