Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594561
Title: Functional analysis of the OA associated variant rs143383
Author: Syddall, Catherine
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2013
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Abstract:
Osteoarthritis (OA) is a common, multifactorial musculoskeletal disease that is characterised by joint pain and reduced joint function. It is a polygenic disease and progresses as a result of the focal loss of the articular cartilage of the synovial joint. rs143383 is a C to T transition single nucleotide polymorphism (SNP) located in the 5ʹ untranslated region (UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with an increased risk of OA and of a number of other common musculoskeletal diseases. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of my study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and; 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15 and DEAF-1 were identified as interacting with the GDF5 5ʹUTR. Knockdown and over expression of the factors demonstrated that they were repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. The genotype of a second GDF5 5ʹUTR SNP, rs143384, which is located downstream of rs143383, has been previously found to impact upon the DAE at rs143383. Thus protein binding to rs143384 was also investigated using EMSAs. In conclusion, I identified four trans-acting factors that modulate the expression of GDF5 via the OA susceptibility locus rs143383.
Supervisor: Not available Sponsor: Nuffield Foundation ; Oliver Bird Rheumatism Programme
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.594561  DOI: Not available
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