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

El Khoury, Louis

Raleigh, Stuart M

Ribbans, Bill

English

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.<br/

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Investigating the ELN rs2071307 gene variant as a risk factor for Achilles Tendon Pathologies in a British CohortBackgroundLouis Y. El Khoury1,2, William J. Ribbans1, Stuart M. Raleigh11The Centre for Physical Activity and Chronic Disease, The Institute of Health and Wellbeing, University of Northampton, Northampton, UK2 School of Biological Sciences, University of Essex, Colchester UKLouis.el-khoury@essex.ac.ukMethods DiscussionResultsReferencesParticipantsA British Caucasian cohort consisting of 108 ATP cases (TEN n=84 and RUP n=24) and 131asymptomatic controls was recruited as described in our earlier study4. Participants signed aconsent form and completed a medical and injury history questionnaire. This study wasapproved by the Research Ethics Committee at the University of Northampton, UnitedKingdom.DNA Extraction and GenotypingSaliva (2 ml) was collected from each participant using the OG-500 tubes (DNA Genotek,Ottawa, Canada) and DNA was extracted according to the manufacturer’srecommendations. All participants were genotyped using custom-designed TaqMan assaystechnology (Applied Biosystems, Foster City, USA) for the ELN G/A rs2071307. Genotypecalls were automatically made using the Applied Biosystem StepOnePlus real-time PCRsoftware (Applied Biosystems, Foster City, USA).Statistical AnalysesPopulation data such as genotype and allele frequencies in addition to the Hardy-WeinbergEquilibrium were calculated using the R Genetics package. Differences in genotypic andallelic distributions were tested using chi-squared or a Fisher’s exact test. Statisticalsignificance was accepted at p<0.05.RNA Secondary StructureThe secondary RNA structures of exon 20 where the rs2071307 resides were determinedusing the SFold statistical algorithm (http://sfold.wadsworth.org) . The structures werefolded in the absence of divalent ions at 37 C and at a concentration of 1 M.There was no significant genotypic or allelic association between the ELN rs2071307 and therisk of TEN (p=0.086, p=0.119), RUP (p=0.501, p=0.243), or when both pathologies werecombined into the ATP group (p=0.413, p=0.399) respectively (table 1). Furthermore, nosignificant genotypic or allelic were reported when the dataset was split according to gender(data not shown).Elastin is known to display a remarkable durability following enormous stretch and recoil cycles. However, the substitution of the amino acid glycine to serine induced by the rs2071307 variant isdescribed to alter the mechanical properties of ELN4. We report a difference in free energy change (ΔG°) which suggests a potentially greater force driving the spontaneous forward reaction of theA allele RNA than that of the G allele RNA5. These thermodynamic properties could explain an over-expression of ELN mRNA as observed in post-injury Achilles tendons of mice6. Although theassociation of the ELN rs2071307 gene variant with soft tissue pathologies is documented in aortic stenosis and aneurysms, it appears not to be associated with the risk of ATPs in a BritishCaucasian cohort. This data is consistent with the early study in Australian and South African cohorts. It should be noted however, that the sample number is small and that these findings requirereplication in other ethnicities.Table 1. Genotype and allele frequency distribution of the ELN rs2071307 variant within the UK control (CON)and Achilles tendon pathology (ATP), as well as the pathological sub-groups Achilles tendinopathy (TEN) andAchilles tendon rupture (RUP).Injuries to the Achilles tendon (tendinopathies or ruptures) are considered as ones of themost severe musculoskeletal traumas in sports with an incidence rate of 50% in athletes and10% in the general population. A number of gene variants coding for tendon structuralproteins such as COL5A11 and FBN22 have previously been associated with Achilles tendonpathologies (ATP). These protein along with others maintain a harmonious interaction withelastin to allow tendons to respond to tensile load by stretching and returning to theiroriginal lengths. The ELN rs2071307 variant has been associated with soft tissue pathologiesand is believed to be a good candidate gene as it results in the substitution of thehydrophobic amino acid glycine with the hydrophilic serine. However, in a previous studythis variant was not associated with either Achilles tendinopathy or ACL rupture inpopulations from Australia and South Africa2. As recent evidence suggests that genetic riskfactors for tendinopathy may depend, to some extent, on geographic location3, the aim ofthis study was to determine whether the ELN rs2071307 variant was associated with the riskof ATP in a British cohort.1 Mokone GG, et al. The Guanine-Thymine Dinucleotide Repeat Polymorphism Within the Tenascin-C Gene Is Associated WithAchilles Tendon Injuries. Am J Sports Med 2005;33:1016–21.2 El Khoury L, et al. ELN and FBN2 gene variants as risk factors for two sports-related musculoskeletal injuries. Int J Sports Med2015;36:333–7.3 El Khoury L, et al. MMP3 and TIMP2 gene variants as predisposing factors for Achilles tendon pathologies: Attemptedreplication study in a British case–control cohort. Meta Gene 2016;9:52–5.4 Rickaby R, et al. Variation within three apoptosis associated genes as potential risk factors for Achilles tendinopathy in a Britishbased case–control cohort. Gene 2015;571:167–71.5 He D, et al. Polymorphisms in the Human Tropoelastin Gene Modify In Vitro Self-Assembly and Mechanical Properties ofElastin-Like Polypeptides. PLoS One 2012;7:e46130.6 Alberts B, et al. Catalysis and the Use of Energy by Cells. In: Molecular Biology of the Cell . Garland Science 2002. 47–128.7 Guerquin M-J, et al. Transcription factor EGR1 directs tendon differentiation and promotes tendon repair. J Clin Invest2013;123:3564–76.Acknowledgements This research was supported by a grant to Dr Stuart M. Raleigh from the University of Northampton. Louis El Khoury would like to thank the Genetics Society and the Biochemical Society for funding the conference attendance and travel costs.Figure 3. Comparison of the secondary structure of the RNA transcribed from exon 20 of the ELN gene. A)Secondary RNA structure when the G allele is present at the rs2071307 variant. B) Secondary structure whenthe A allele is present at the rs2071307 variant. The structural differences are magnified in the box, and thealleles are highlighted by a red circle.ELNrs2071307CON (n=131) ATP (n=108) TEN (n=84) RUP (n=24)GG 38.2 (50) 39.8 (43) 42.8 (36) 29.2 (7)GA 45.8 (60) 50.0 (54) 51.2 (43) 45.8 (11)AA 16.0 (21) 10.2 (11) 5.9 (5) 45.0 (6)P Value 0.413a 0.086b 0.501cHWE 0.722 0.400 0.074 0.676A allele 38.9 (102) 35.2 (76) 31.5 (53) 47.9 (23)P Value 0.399a 0.119b 0.243cThe values are expressed as a frequency with the number of participants (n) in parenthesis. a CON vs ATP; b CON vs TEN; c CON vs RUPWe report a structural difference between the RNA secondary structure obtained from a G and an A allele at the rs2071307 locus. This structural difference results in altering the free energy of the molecule: G allele, ΔG°=-70.6 kcal/mol; A allele, ΔG°=-71.6 kcal/mol.

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

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

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