Hemophilia B Leyden is an X chromosome-linked
bleeding disorder characterized by an altered developmental expression of
blood coagulation factor IX. This form of
hemophilia B has been found to be associated with a variety of single point mutations in the
factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the
hemophilia B Leyden phenotype. This mutation lies within the
factor IX promoter region (-40 to -9) that contains overlapping binding sites for
hepatocyte nuclear factor 4 (HNF-4) and
androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in
factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the
androgen-responsive
element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the
hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe
hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant
androgen receptor protein for
oligonucleotides containing wild-type and -21 or -20 mutated
DNA. The binding affinity for the
oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the
bleeding disorder in these two patients. This study adds further support for the notion that the recovery from
hemophilia at puberty may not only be related to an intact
androgen-responsive
element but also to the degree of disruption of the HNF-4-binding site.