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Title: Transcriptional regulation of PRPF31 : the role of variable gene expression in determining phenotype in retinitis pigmentosa
Author: Rose, A. M.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2012
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Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP) for over a decade, yet the molecular basis underlying the observed phenotypic non-penetrance in families remains unknown. In the population, there are differentially expressed alleles of PRPF31: both high-expressivity and a low-expressivity alleles. Inheritance of a higher-expressivity allele alongside a mutant allele provides protection against the clinical manifestations of the disease. The aim of the research was to investigate phenotypic non-penetrance associated with PRPF31 mutations, through the understanding of transcriptional regulation of PRPF31 in both health and disease. Firstly, it was investigated whether the non-penetrant effect was caused by a cis- or trans-acting factor. Microsatellite data was used to show that inheritance of the wildtype chromosome 19q13 determined affection status of individuals harbouring a mutation in PRPF31. It was, therefore, concluded that a cis-acting element controls the non-penetrant phenotype. A family affected by adRP with a history of non-penetrance was identified and a large deletion (112kb) in the 19q13.4 region encompassing PRPF31 was fully characterized. Importantly, the deletion encompassed all introns of PRPF31 and also a large region upstream, therefore including putative regulatory elements of the gene. Dual-luciferase reporter assay was performed to define the core promoter of PRPF31 and also the core promoter of TFPT, a gene lying in a head-to-head arrangement with PRPF31, with partially shared exon 1. Core promoters were defined for both genes. A patient with a history of isolated adRP was identified, in whom a single base pair deletion in the PRPF31 core promoter was present. Functional studies were performed, demonstrating that the mutant allele had a serious adverse affect on transcription of PRPF31- this patient represents the first report of functional haploinsufficiency as a disease mechanism in adRP. Comparison of the PRPF31 core promoter sequence in an asymptomatic and a symptomatic individual lead to the identification of one polymorphism that significantly affected transcriptional activation of the gene. The duplication of a 38bp repeat element increased transcription, and it was found that this element was statistically under-represented in a cohort of symptomatic individuals compared to the general population. This represents the first identification of a molecular factor controlling non-penetrance in PRPF31-associated adRP. Finally, the evolution of the PRPF31 and TFPT core promoters was studied. The conservation of the region was analysed and regions homologous to the human core promoter fragments were sought in monkey, dog and mouse. Dual-reporter luciferase assay was performed to characterise the promoter elements in these three species, and demonstrated that conservation of a gene was not always accompanied by conservation of regulatory elements.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available