Haemophilia B mutations in a complete Swedish population sample: a test of new strategy for the genetic counselling of diseases with high mutational heterogeneity

Abstract

Carrier and prenatal diagnosis based on the identification of the gene defect (direct diagnosis) increases the proportion of haemophilia B families that can be offered precise genetic counselling from the 50-60% attainable by DNA markers, to 100% and they also provide information on the molecular biology of the disease. We propose that in order to maximize the practical and scientific benefits of direct diagnosis the gene defect of complete (possibly national) populations of patients should be characterized and the information stored in appropriate confidential databases. We demonstrate the feasibility of such a strategy by characterizing the mutations of all the patients registered with the Malmö haemophilia centre. These patients (44 male and 1 female) are from 45 unrelated families and 24 (53%) have negative family history. The 25 patients with similar reduction of factor IX:C and factor IX:Ag (24 male + 1 female) have: two gross deletions, three frameshifts, four translation stops, six mutations expected to affect pre-mRNA splicing and 10 amino acid substitutions. The six patients with greater reduction of factor IX:C than factor IX:Ag and the seven with reduced IX:C and normal IX:Ag have only amino acid substitutions. Patients with inhibitors have: one complete deletion, one frameshift and three translation stops. One patient has both a translation stop and a functionally neutral amino acid substitution (His257----Tyr). Characterization of the factor IX mutation was successful in every case, usually entailed 4 person-days work, and has led to the identification of 12 amino acid residues essential for the factor IX structure and function.

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

“…39,40 Other limiting factors included the low efficiency of skeletal muscle at executing certain critical posttranslational modifications of the F.IX protein, 3 and the fact that vector uptake is receptor mediated, which sets an upper limit to the amount of vector that can gain access to cells at a single injection site. Using the experimentally determined constraints on vector dose per site (2 ϫ 10 12 vg/site), the procedure required an excessive number of injections merely to achieve a dose of 2 ϫ 10 12 vg/kg, still substantially short of the target dose of 1 ϫ 10 13 vg/kg.…”

Novel hemophilia B mouse models exhibiting a range of mutations in the Factor IX gene

“…Analysis of the database has been informative in a number of respects, and has shown that the underlying mutation predicts certain features of the clinical disease. Elegant work by Giannelli and colleagues, 9 and later by other groups as well, 34,35 showed that mutations that lead to extensive loss of coding information (large gene deletions, frameshifts, nonsense mutations) are more likely to be associated with inhibitory antibody formation following exposure to F.IX concentrates. Thus, in a study of 18 children with severe hemophilia B who developed inhibitors accompanied by severe allergic reactions, 10 of 17 analyzed had a complete deletion of the F.IX gene, and the remaining 7 had smaller deletions or nonsense mutations.…”

“…By contrast, such deletions account for only a small fraction (Ͻ 5%) of mutations in humans with hemophilia B. 9 This is a substantial shortcoming in the hemophilia B disease model, because mice and humans with large deletions of F.IX have a propensity for formation of inhibitory antibodies on exposure to F.IX protein. 10,11 The formation of these antibodies makes it impossible to track the results of a therapeutic intervention.…”

Functional Analysis of the Factor IX Epidermal Growth Factor-Like Domain Mutation Ile66Thr Associated with Mild Hemophilia B

“…To induce the Ile66Thr mutation, a Quick-Change TM site-directed mutagenesis kit from Stratagene (La Jolla, Calif., USA) was used with the following primers (mutations are italicized): 5 GGC AGT TGC AAG GAT GAC A C T AAT TCC TAT GAA TGT TGG and 5 CCA ACA TTC ATA GGA ATT A G T GTC ATC CTT GCA ACT GCC. Thereafter, sequence analysis of the entire FIX fragment was performed to verify the presence of the desired mutations; the objective was achieved using a modified dideoxy method to sequence genomic DNA of exons a-h [22,23] . Human kidney 293 cells were transfected with the vectors mentioned above.…”