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Title: Functional analysis of polymorphisms associated with osteoarthritis susceptibility that affect cis-regulation
Author: Wilkins, James
ISNI:       0000 0001 3568 3268
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2008
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Osteoarthritis (OA) is a common, multifactorial disease that is characterized by focal degeneration of the smooth articular cartilage in any of the synovial joints. Although the underlying molecular mechanisms for OA are still not fully understood, epidemiological studies have evidenced a significant genetic component to OA susceptibility. Genome-wide linkage scans and large-scale association studies have had success in unraveling the genetic architecture underlying OA with the identification of a number of susceptibility genes. In this work, functional analyses are reported of OA associated polymorphisms within two susceptibility genes: BMP5 and GDF5, both members of the TGF-β superfamily of secreted proteins. The extent of differential allelic expression (DAE) of BMP5 in human mesenchymal tissues was first examined with significant differences in BMP5 allelic output observed (allelic ratios exceeding 4:1 in the tissues of some donors). Significant variability in allelic expression within the different tissues of donors was also observed, suggesting that polymorphism in cis-regulation of BMP5 expression is common and that there is a considerable effect of tissue specific elements on BMP5 expression. DAE was then used as a phenotype to map tissue-specific cis-regulatory polymorphisms with the identification of a single nucleotide polymorphism (SNP) located downstream of BMP5 that was significantly associated with DAE as well as OA, suggesting that variability in cis-regulation of BMP5 is important in OA susceptibility. Moreover, the functional effect of a previously identified OA associated microsatellite within intron 1 of BMP5 was investigated using luciferase reporter assays and electrophoretic mobility shift assays (EMSAs) with significant differences observed both in the ability of various microsatellite alleles to modulate BMP5 promoter activity and to bind GATA-3 nuclear proteins, further suggesting a role for variability in BMP5 expression in OA susceptibility that may in part be due to altered GATA-3 binding. Finally, functional characterization of a previously reported OA associated SNP in the 5′ UTR of GDF5 is presented in which EMSAs show differential binding of nuclear factors between the two SNP alleles, strengthening the possible functional contribution of this SNP to OA susceptibility. Overall, this work demonstrates that polymorphism in cis-regulation is likely to play a role in OA susceptibility.
Supervisor: Loughlin, John Sponsor: Marshall Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medical Sciences ; Biology (medical sciences) ; Genetics (medical sciences) ; Orthopaedics ; Rheumotology ; Molecular genetics ; osteoarthritis ; genetics ; differential allelic expression