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Title: The transcriptional regulation of the gene for human clotting factor IX
Author: Morgan, Gareth Edwin
ISNI:       0000 0001 3420 6574
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1997
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Haemophilia B Leyden is a rare form of haemophilia B in which patients suffer from a bleeding disorder in childhood but completely recover in adulthood. These patients all have point mutations in the gene promoter between nucleotides -21→+13 which cause this altered expression pattern. An unusual patient with a -26 G→C mutation, haemophilia B Brandenburg, did not recover from his bleeding disorder during puberty. It was suggested that this was due to the disruption of the binding of the androgen receptor (AR) to an androgen response element (ARE) in the promoter (-36→- 22). To obtain further evidence for the importance of the ARE in the Leyden recovery, a patient with a -26 G→A mutation, UK 232, was studied here. This mutation, like the -26 G→C, also disrupted the ARE. This patient is now aged 47 and has not recovered from his bleeding disorder, confirming the importance of this nucleotide in the ARE. To determine the nucleotides important for the response to AR and testosterone in the factor IX ARE, mutations were introduced at each position in the ARE, some spacer positions, and in some flanking positions. These mutant ARE sequences were then tested firstly, in vitro, in a competition gel mobility shift assay with an E.coli produced protein A-AR DNA binding domain fusion protein, and secondly, in vivo in transient transfection assays. In these assays nucleotides -35, -32, -26, -25, and -23 were critical for the response to AR and testosterone, and -36, -31, and -27 impaired this response to a lesser degree. Conflicting evidence was obtained in the two assays concerning the importance of nucleotides -33 and -22. These data were used to predict the adult phenotypes of two children with mutations in the factor IX promoter (HB 396 and HB 388). It was predicted that one of them, HB 396, would recover from his bleeding disorder as his mutation (-19 G→C) does not disrupt the ARE, whilst the other, HB 388 would not recover as his mutation ( - 23 C→T) does disrupt this element. Patient Liverpool 2 with a mild haemophilia B Leyden phenotype was found to have a -6 G→A promoter mutation at a CpG dinucleotide which has been identified in 12 other patients. A haplotype study of 9 of these patients revealed that it is probable both recurrent mutation and a founder effect are responsible for the mutation's high frequency.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry