Further evidence for the importance of an androgen response element in the factor IX promoter

Previous work involving the characterizing of a factor IX promoter mutation (-26 G-->C) of a 21-year-old patient with severe haemophilia B suggested that an androgen response element (ARE) was present in the wild-type factor IX promoter but was disrupted in this patient. However, other theories not involving this ARE have been suggested for the mechanism of recovery in the more typical (Leyden) promoter patients, so that the ARE hypothesis requires further evidence if it is to be accepted. We now present a case history and functional data on another 48-year-old severe haemophilia B patient (UK232) with a different (G-->A) mutation at the same position, -26. This mutation impairs transactivation of the minimal factor IX promoter region (-220-->+43) by HNF4 in transient transfection experiments in HepG2 and HeLa cells. It disrupts binding of both androgen receptor (AR) and HNF4 to oligonucleotides spanning this region (-40-->-9) in competition gel mobility shift assays. It impairs AR/testosterone transactivation of these oligonucleotides (-40-->-9) when tetramerized upstream of a CAT reporter gene in cotransfection assays in HeLa cells. And, finally, no clinical recovery has occurred since puberty. These results strengthen the evidence for the importance of nucleotide -26, both for the normal transcription of the gene in response to HNF4 and for the proposed Leyden recovery mechanism in response to AR and testosterone acting directly through the factor IX ARE.