Epigenetic Status of The Human MMP11 Gene Promoter is Altered in Patellar Tendinopathy.

Patellar tendinopathy (PT) is a debilitating condition that often affects those who are physically active. Gene variation is known to contribute to human tendinopathy but the role of DNA methylation, as an epigenetic factor, has only recently been discovered. Using a case-control approach, we sought to determine whether differences existed between the methylation status of the MMP11 gene promoter in patellar tendinopathy compared to healthy tendon. We used PCR and pyrosequencing to interrogate the methylation profiles of 4 CpG sites (areas of the genome rich in C/G nucleotides) upstream of the MMP11 gene in DNA from males with PT (n = 10) and those with healthy tendon (n = 10). We also conducted a correlation analysis to establish whether age influenced methylation in the PT patients and controls. We found a significant (p = 0.045) difference in the methylation status of a single CpG site 65 base pairs (bp) upstream of the MMP11 promoter between the PT group and controls. There were no other differences in the extent of MMP11 promoter methylation between the two groups. Interestingly, we also found that in controls the degree of methylation at a second CpG site, 55 bp upstream of the first exon, tentatively correlated (r = 0.77, p = 0.009) with age. However, the correlation did not reach significance when a potential outlier was removed. This is the first study to show an epigenetic alteration to a member of the MMP gene family in human patellar tendinopathy. The data add to our understanding of how epigenetics should be considered when developing appropriate risk models.

Promoter methylation status of the TIMP2 and ADAMTS4 genes and patellar tendinopathy.

OBJECTIVES: Patellar tendinopathy (PT) is a debilitating and prevalent condition that tends to affect those who are physically active or engaged in jumping sports. Although tendinopathies are known to have a genetic basis, the role of DNA methylation as an epigenetic factor and risk determinant for human PT has never been described. We sought to determine whether differences existed between the methylation profiles of both the TIMP2 and ADAMTS4 gene promoter sequences in a cohort of males having undergone surgery for patellar tendinopathy compared to controls. DESIGN: Case-control epigenetic study using DNA from 10 males with PT and 10 males with healthy tendons. METHODS: We used PCR and targeted pyrosequencing to interrogate the methylation profiles of CpG sites upstream of both the TIMP2 (4 sites) and ADAMTS4 (6 sites) genes. We compared methylation differences between the two groups using t-tests. RESULTS: We report no significant (p>0.05) methylation differences within the TIMP2 gene promoter between the PT group and controls across the 4 CpG sites investigated. In contrast, we detected a significant (p=0.016) difference in the methylation status of 1 CpG site, approximately 3kb upstream of the ADAMTS4 gene between the PT group and controls. CONCLUSIONS: To our knowledge, this is the first study to investigate how DNA methylation impacts on the risk of human tendinopathy. Our data indicate that the methylation status of the ADAMTS4 gene is altered in patellar tendinopathy and we speculate on how this change might modify the patellar tendon extra-cellular matrix environment.

Variation within three apoptosis associated genes as potential risk factors for Achilles tendinopathy in a British based case-control cohort.

Achilles tendon pathology (ATP) is a degenerative condition which exhibits excessive tenocyte apoptosis. Tumour necrosis factor receptor 1 (TNFR1), caspase-3 (CASP3) and caspase-8 (CASP8) are important regulators of apoptosis. To date, the effects of variation within the genes for TNFR1 and CASP3 as risk factors for ATP have not been described. There is evidence that two single nucleotide polymorphisms (SNPs) within the CASP8 gene are associated with ATP, but only in populations from the Southern Hemisphere. The primary aim of this study was to determine whether SNPs within the TNFRSF1A and CASP3 genes were associated with ATP in British Caucasians. We additionally sought to determine whether copy number variation (CNV) within the CASP8 gene was associated with ATP. We recruited 262 (131 ATP cases and 131 asymptomatic controls) Caucasian participants for this genetic association study and used quantitative PCR with chi-squared (χ(2)) tests and ANOVA to detect significant associations. For our entire cohort, we found no association between the TNFRSF1A rs4149577 (p=0.561), CASP3 rs1049253 (p=0.643) and CASP8 variants (p=0.219) and ATP. Likewise, when we tested potential interactions between gender, genotype and the risk of ATP, we found no association with the variants investigated. In conclusion, the TNFRSF1A, CASP3 and CASP8 gene variants were not associated with ATP in British Caucasians.