Expression analysis of osteoarthritis susceptibility loci

Abstract

Osteoarthritis (OA) is a common disease characterised by the progressive loss of the articular cartilage of synovial joints. It is multifactorial and polygenic. Candidate genebased association studies and genome-wide association scans (GWAS) have been used to identify OA risk alleles with over 10 regions of the human genome so far reported as harbouring OA susceptibility. Expression quantitative trait loci (eQTLs) demonstrate a correlation between gene expression levels and DNA polymorphism, the assumption being that the polymorphism resides within a regulatory element of the gene. The functional polymorphism can be cis-acting (within or close to the gene) or trans-acting (located a distance from the gene). It has become apparent for many common diseases that the majority of risk alleles are eQTLs rather than polymorphisms that alter amino acid sequences. The aim of this thesis was to investigate whether genes proximal to or harbouring OA-associated polymorphisms identified by GWAS are subject to cisregulation, which could therefore be contributing to the association signal. The expression levels of the genes were analysed in joint tissues from OA patients who had undergone joint replacement. Overall gene expression was measured by quantitative PCR whilst allelic expression was assessed using assays that can distinguish mRNA output from each allele of a transcript SNP. The functional effect of SNPs was tested in vitro using luciferase reporter assays. Expression analysis of genes at the OA-associated chromosome 7q22 locus identified HBP1 as the likely target of the OA susceptibility mapping to this region. Expression analysis of SMAD3 and GNL3 identified eQTLs active in OA joint tissues for both genes, whilst luciferase reporter assays identified two 3´UTR SNPs as potential mediators of the SMAD3 eQTL. Overall, this thesis demonstrates that OA tissues are subject to a range of regulatory elements that can influence disease during development or ageing.