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 mut ation (-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, Howeve r, other theories not involving this ARE have been suggested for the m echanism of recovery in the more typical (Leyden) promoter patients, s o that the ARE hypothesis requires further evidence if it is to be acc epted. 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 transact ivation of the minimal factor IX promoter region (-220 --> +43) by HNF 4 in transient transfection experiments in HepG2 and HeLa cells, It di srupts binding of both androgen receptor (AR) and HNF4 to oligonucleot ides spanning region (-40 --> -9) in competition gel mobility assays, It impairs AR/testosterone transactivation of these oligonucleotides ( -40 --> -9) when tetramerized upstream of a CAT reporter gene in cotra nsfection 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 th e gene in response to HNF4 and for the proposed Leyden recovery mechan ism in response to AR and testosterone acting directly through the fac tor IX ARE.