Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by very low plasma levels of blood coagulation factor IX (fIX) during childhood. After puberty, plasma fIX levels gradually rise to a maximum of 60% of normal, probably under the influence of testosterone. Single point mutations in the fIX promoter region of hemophilia B Leyden patients have been reported at -20, -6, -5, +8 and +13. In addition, one promoter mutation (G----C at -26) has been detected that abolishes fIX expression throughout life (M. Ludwig, personal communication). We examined how one of the hemophilia B Leyden mutations (T----A at -20) and the G----C mutation at -26 interfere with fIX gene transcription. We report that the wild-type promoter of the human fIX gene contains a binding site (at nucleotides -34 to -10) for hepatocyte nuclear factor 4 (HNF-4), a member of the steroid hormone receptor superfamily of transcription factors. The binding of HNF-4 is disrupted by both the T----A mutation at -20 and the G----C mutation at -26. Whereas HNF-4 transactivates the wild-type promoter sequence in liver (HepG2) and non-liver (HeLa) cell types quite well, it transactivates the -20 mutated promoter to only a limited extent and the -26 mutated promoter not at all. These data suggest that HNF-4 is a major factor controlling fIX expression in the normal individual and that its inability to bind efficiently to the -20 T----A and the -26 G----C mutated promoter sequence results in hemophilia. Further, the severity of the hemophilia phenotype appears to be directly related to the degree of disruption of HNF-4 binding and transactivation.