Investigating the role of ELN rs2071307 gene variant as a risk factor for Achilles Tendon Pathologies in a British Cohort

Injuries to the Achilles tendon (tendinopathies or ruptures) are considered as ones of the most severe musculoskeletal traumas in sports with an incidence rate of 50% in athletes and 10% in the general population. A number of gene variants coding for tendon structural proteins such as COL5A11 and FBN22 have previously been associated with Achilles tendon pathologies (ATP). These protein along with others maintain a harmonious interaction with elastin to allow tendons to respond to tensile load by stretching and returning to their original lengths. The ELN rs2071307 variant has been associated with soft tissue pathologies and is believed to be a good candidate gene as it results in the substitution of the hydrophobic amino acid glycine with the hydrophilic serine. However, in a previous study this variant was not associated with either Achilles tendinopathy or ACL rupture in populations from Australia and South Africa2. As recent evidence suggests that genetic risk factors for tendinopathy may depend, to some extent, on geographic location 3, the aim of this study was to determine whether the ELN rs2071307 variant was associated with the risk of ATP in a British cohort

Genetic and epigenetic variation within extracellular matrix genes as risk factors for human tendinopathy

Background and Aims Regular physical activities have shown to have various health benefits however there is always an accompanying risk of developing musculoskeletal soft tissue injuries. Indeed, damaged tendons account for 30-50% of sports-related injuries where the lifetime risk of Achilles tendon pathology (ATP) approaches 50% in runners, and that of patellar tendon pathology (PTP) is 21% in football players. The exact aetiology and mechanisms of tendon pathologies are still under investigation, however extrinsic and intrinsic risk factors (of which genetic) have been identified. Recent genetic association studies found that gene variants within the TNC, COL5A1, MMP3, GDF5 and CASP8 were associated with ATP in a Caucasian Australian and South African population. Furthermore, epigenetic mechanisms such as DNA methylation and microRNA (miRNA) activity have been implicated in a range of diseases but were never investigated for their role in human tendinopathy. Based on the aforementioned information, this thesis aimed at investigating novel candidate genes that may be associated with ATP and to replicate previously conducted studies in a newly recruited case-control population. Additionally, this thesis aimed at investigating potential differences in DNA methylation profiles and miRNA expression levels between healthy and damaged Achilles and patellar tendons. One hundred and thirty six UK Caucasian participants with clinically diagnosed ATP and 131 asymptomatic, unrelated, physically active control participants were recruited for this study. Furthermore, the previously recruited 173 clinically diagnosed ATP participants and 238 asymptomatic, unrelated, physically active control participants from Australia and South Africa (AUS+SA) were also included in the studies of this thesis. Participants within the combined AUS+SA were genotyped for the ELN rs2071307, FBN2 rs331079, ADAM12 rs3740199, ADAMTS2 rs1054480, ADAMTS5 rs226794, ADAMTS14 rs4747096, and TIMP2 rs4789932 variants, and the UK participants were genotyped for the COL5A1 rs71746744, FBN2 rs331079, GDF5 rs143833, MMP3 rs679620, TIMP2 rs4789932 variants using fluorescent based TaqMan® technology. Furthermore, the UK cohort was genotyped for the COL5A1 rs12722 variant using polyacrylamide gel electrophoresis. Moreover, 10 healthy and 10 diseased patellar tendon tissue samples in addition to 4 healthy, and 1 diseased Achilles tendon samples were obtained for the epigenetic studies. The DNA methylation profile within the TIMP2 and GDF5 promoter regions were analysed for all samples using pyrosequencing technology. Furthermore, the expression levels of TIMP2, miR-21, miR-155, and miR-191 were determined by RT-PCR using TaqMan technology. Results and Discussion The genetic association studies conducted showed that the FBN2 rs331079 GG genotype was over-represented among the tendinopathy (TEN) group and that the ELN rs2071307 AA genotype was over-represented in the rupture (RUP) group within the AUS+SA population. Furthermore, the COL5A1 rs12722 and rs71746744 were associated with RUP (TT genotype over-represented in the RUP group, p=0.004; OR=4.2; 95% CI 1.58-11.97) and ATP (DEL allele over-represented in the CON group, p=0.046; OR=1.61; 95% CI 1.01-2.56) respectively in the male UK cohort. The GDF5 rs143833, on the other hand, was not associated with ATP (p=0.538) and showed no sign of gender-specific association (female p=0.737; male p=0.319) in the UK population. Furthermore, the CT genotype for the TIMP2 rs4789932 variant was over-represented (p=0.004; OR=1.77; 95% CI 1.20 - 2.64) in the ATP group of the combined AUS+SA population and the CC genotype was over represented (p=0.016; OR=2.36; 95% CI 1.16 – 5.81) in ATP of the UK male cohort. It was also reported that the MMP3 rs679620 GG genotype was over represented (p=0.027; OR=2.51; 95% CI 1.11 – 5.64) in the UK RUP group. The ADAM12 rs3740199 (p=0.633), ADAMTS2 rs1054480 (p=0.316), ADAMTS5 rs226794 (p=0.342), and ADAMTS14 rs4747096 (p=0.849) gene variants were not associated with ATP in the AUS+SA population. Interestingly, individuals carrying the ADAMTS14 rs4747096 GG genotype within the AUS+SA population and the ELN rs2071307 AA genotype within the AUS population developed their injuries at a significantly (p=0.024; p=0.005, respectively) later stage than other participants. Moreover, UK males diagnosed with tendinosis and carrying the GG genotype at the MMP3 rs679620 locus developed significantly (p=0.003) thicker tendons than other participants with the AA, and AG genotypes. The preliminary epigenetic DNA methylation studies showed no differences in the average methylation profiles of the investigated regions within the TIMP2 (p=0.885) and GDF5 (p=0.333) genes in the patellar tendon samples. Moreover, no DNA methylation differences (p=0.617) were observed in the investigated region of the TIMP2 gene in the Achilles tendon samples. Interestingly, the single ATP sample showed a lower GDF5 average methylation profile than the CON samples. Furthermore, the expression of TIMP2 was up-regulated, and miR-191 was down-regulated in the ATP tissue sample compared to the CON group. The expression levels of miR-21 and miR-151, however, were not different between the two groups. Conclusion This thesis provides evidence that novel genes coding for structural and ECM regulatory enzymes are associated with ATPs in Caucasians. The findings of this thesis should to be replicated in new and larger cohorts from different ethnic backgrounds before being incorporated into multifactorial risk assessment models aiming at reducing the incidence of human tendinopathy

TIMP2 and GDF-5 gene variants and achilles tendon pathology: replication study in a British case-control population

Introduction: Achilles tendon pathology (ATP) encompasses a range of tendon overuse injuries that can be sub-classified into separate pathologies [Weinfeld, 2014]. To date, a number of single nucleotide polymorphisms (SNPs) have been associated with ATP [Raleigh and Collins, 2012] but, with the exception of the COL5A1 rs12722 variant, limited work has been published on attempting to replicate these findings in cohorts other than those recruited from either South Africa or Australia. We selected variants within the TIMP2 (rs4789932) and GDF-5 (rs143383) genes, that have previously been shown to associate with ATP [El Khoury et al, 2013 and Posthumus et al, 2010], and attempted to replicate previous associations in a newly recruited British-based, case-control, Caucasian cohort. Methods: We recruited 133 ATP Caucasian patients from the County Clinic in Northampton along with 131 physically active controls from various sports clubs within the East Midlands region. DNA samples were collected from saliva (DNA genotek Ltd) and Taqman technology, using allele specific probes and primers, was used to genotype all DNA samples. Reactions were run on a StepOne Plus real-time PCR instrument (ABI). Genotypes were called according to post run cluster profiles and data were analysed using Chi-squared (c2) analysis or Fisher’s exact test. Significance was accepted at p < 0.05. All procedures were approved by the University of Northampton Research Ethics Committee. Results: For the TIMP2 rs4789932 variant we found no association between genotype and case or control status in the entire cohort (p = 0.279). However, in sex specific analysis we did find that the CC genotype was associated (p = 0.043) with male ATP cases compared to controls (Table 1). For the GDF-5 rs143383 variant, we found no association between genotype and case or control status in the entire cohort (p = 0.538) or in either male (p = 0.319) or female (p = 0.737) specific analysis (data not shown). Genotypes did not associate with any other potential confounding variables. Conclusions: The TIMP2 rs478992 CC genotype was associated with male cases of ATP. Although this locus was previously associated with ATP in cohorts recruited from the Southern Hemisphere it was the CT genotype that was the risk factor and the association was not sex specific [El Khoury et al, 2013]. This result might be related to differences in unknown environmental exposures between the cohorts investigated that may modify the effect of the genotype. We found no evidence of an association between ATP and the GDF-5 variant. These data should be viewed as preliminary findings and will need to be repeated in a larger cohort

MMP3 and TIMP2 gene variants as predisposing factors for Achilles tendon pathologies: attempted replication study in a British case–control cohort

Variants within the MMP3 (rs679620) and TIMP2 (rs4789932) genes have been associated with the risk of Achilles tendon pathology (ATP) in populations from South Africa and Australia. This study aimed to determine whether these variants were associated with the risk of ATP in British Caucasians. We recruited 118 cases with ATP, including a subset of 25 individuals with Achilles tendon rupture (RUP) and 131 controls. DNA samples were isolated from saliva and genotyped using qPCR. For the TIMP2 rs4789932 variant we found a significant (p = 0.038) difference in the genotype distribution frequency between males with ATP (CC, 39.4%; CT, 43.7%; TT, 16.9%) compared to male controls (CC, 20.7%; CT, 59.8%; TT, 19.5%). We also observed a difference in the TIMP2 rs4789932 genotype distribution between males with rupture compared to male controls (p = 0.038). The MMP3 rs679620 GG genotype was found to be overrepresented in the Achilles tendon rupture (RUP) group (AA, 24.0%; AG, 32.0%; GG, 44.0%) compared to controls (AA, 26.7%; AG, 54.2%; GG, 19.1%). In conclusion, the CT genotype of the TIMP2 rs4789932 variant was associated with lower risk of ATP in males. Furthermore, while we revealed differences for both variants in genotype distribution between the RUP and control groups, the sample size of the RUP group was small and confirmation would be required in additional cohorts. Finally, although both the TIMP2 rs4789932 and MMP3 rs679620 variants tentatively associated with ATP, there were differences in the direction of association compared to earlier work

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 3 kb 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