The role of novel genetic variants and DNA methylation as risk factors for tendon pathology in physically active individuals

Along with the many health benefits of regular physical activity, there is also the risk of injury. Around 30-50% of all sporting injuries involve damage to tendons and in long-distance runners the lifetime risk of developing Achilles tendinopathy exceeds 50%. Patellar tendinopathy (PT) is likewise common among sporting populations, with prevalence rates higher than 30% in both volleyball and basketball players. The main aims of this thesis were to investigate whether novel genetic variants (copy number variation (CNV) and single nucleotide polymorphisms (SNPs)) in candidate genes were associated with Achilles tendon pathology (ATP) and to investigate whether DNA methylation status was altered in patellar tendinopathy, within the promoter regions of candidate genes. Copy number variation was investigated in the ADAM8, CASP8, COL4A1, COL5A1, MMP23B, MMP3 and TNC genes across all ATP cohorts. The British and South African ATP cohorts were genotyped for the CASP3 rsl049253 and TNFRSF1A rs4149577 single nucleotide polymorphisms. These studies were carried out using fluorescence based TaqMan qPCR. DNA methylation was measured in the ADAMT54, MMP11 and TIMP1 genes by Pyrosequencing, using DNA isolated from healthy and tendinopathic patellar tendon tissue. On the whole, the genetic association studies on copy number variation within this thesis showed that CNV within the loci we investigated does not appear to be a risk factor for ATP. Nevertheless, significant differences in discrete copy number were reported within the COL5A1 gene between the AUS CON and ATP groups (p = 0.012 using the Hs00180523_cn assay) and within the COL4A1 gene between the combined CON and ATP groups (p = 0.014 using the Hs00739915_cn assay). There was also a significant difference in copy number within the MMP3 gene between the AUS CON and ATP groups (p = 0.035 using the Hs02908568_cn). However, these significant findings were not replicated when data were analysed as continuous copy number, therefore further investigation would be needed to replicate these findings in other cohorts. Similarly, the genetic association studies on the single nucleotide polymorphisms TNFRSF1A rs4149577 and CASP3 rsl049253 found no associations with ATP within combined and individual South African and UK cohorts. The DNA methylation studies within this thesis showed that the methylation status of the promoter regions of the ADAMTS4, MMP11 and TIMP1 genes was altered at certain CpG sites in patellar tendinopathy compared to controls. There were significant differences in methylation status reported between the CON and PT groups for CpG site 3 of the ADAMT54 gene (p = 0.016), CpG site 4 of the MMP11 gene (p = 0.045) and CpG site 2 of the TIMP1 gene (p = 0.012). The results from this thesis provide evidence that copy number variation within the genes studied may not be as important in the risk of developing ATP. This thesis has also excluded two SNP variants as risk factors for ATP in the cohorts investigated. The preliminary studies on DNA methylation status within the ADAMT54, MMP11 and TIMP1 genes suggest that DNA methylation within these genes may be an important risk factor for patellar tendinopathy.