This work has been submitted to NECTAR, the Northampton Electronic Collection of Theses and Research. Article R A T C E N Title: ELN and FBN2 gene variants as risk factors for two sports­related musculoskeletal injuries Creators: El Khoury, L., Posthumus, M., Collins, M., van der Merwe, W., Handley, C. J., Cook, J. and Raleigh, S. M. DOI: 10.1055/s­0034­1390492 Example citation: El Khoury, L., Posthumus, M., Collins, M., van der Merwe, W., Handley, C. J., Cook, J. and Raleigh, S. M. (2015) ELN and FBN2 gene variants as risk factors for two sports­related musculoskeletal injuries. International Journal of Sports Medicine. 36(4), pp. 333­337. 0172­4622. It is advisable to refer to the publisher's version if you intend to cite from this work. Version: Accepted version Official URL: https://www.thieme­ connect.com/products/ejournals/abstract/10.1055/s­0034­1390492 Note: © 2016 Georg Thieme Verlag KG http://nectar.northampton.ac.uk/8000/ 1 ELN and FBN2 gene variants as risk factors for two sports related musculoskeletal 2 injuries 3 Abstract 4 The ELN and FBN2 proteins are important in extracellular matrix function. The ELN 5 rs2071307 and FBN2 rs331079 gene variants have been associated with soft tissue 6 pathologies. We aimed to determine whether these variants were predisposing factors for 7 both Achilles tendinopathy (AT) and anterior cruciate ligament (ACL) ruptures. 8 For the AT study, 135 cases (TEN group) and 239 asymptomatic controls were recruited. 9 For the ACL rupture study our cohort consisted of 141 cases (ACL group) and 219 controls. 10 Samples were genotyped for both the ELN rs2071307 and FBN2 rs331079 variants using 11 TaqMan assays. Analysis of variance and chi-squared tests were used to determine whether 12 either variant was associated with AT or ACL rupture with significance set at p<0.05. 13 The GG genotype of the FBN2 variant was significantly over-represented within the TEN 14 group (p=0.035; OR=1.83; 95% CI 1.04–3.25) compared to the CON group. We also found 15 that the frequency of the G allele was significantly different between the TEN (p=0.017; 16 OR=1.90; 95% CI 1.11–3.27) and ACL groups (p=0.047; OR=1.76; 95% CI 1.00–3.10) 17 compared to controls. The ELN rs207137 variant was not associated with either AT or ACL 18 rupture. In conclusion, DNA sequence variation within the FBN2 gene is associated with both 19 AT and ACL rupture. 20 Keywords Gene; Achilles Tendinopathy; Tendon rupture; Ligament rupture; Injury 21 prevention. 22 1 23 Introduction 24 Injury to the Achilles tendon and the anterior cruciate ligament are severe traumas typically 25 sustained during sports activities. Achilles tendon injuries, including chronic Achilles 26 tendinopathy (AT) and acute Achilles tendon rupture, are prevalent within athletic populations 27 [26]. Indeed, the lifetime incidence of AT is approximately 10% in the general population and 28 as high as 50% within competitive runners [12]. Chronic AT may be due, in part, to excessive 29 exposure of the Achilles tendon to acute or repetitive mechanical loading forces experienced 30 during exercise [15]. Anterior cruciate ligament (ACL) ruptures have low lifetime prevalence 31 in the general population but have been reported to be nearly 80% among netball players [7]. 32 The most common mechanism of ACL rupture involves a sudden change in an athlete’s 33 direction or rapid deceleration [19]. Both AT and ACL ruptures are complex multifactorial 34 phenotypes with several intrinsic and extrinsic risk factors. However, the exact aetiology is 35 not yet fully understood [3]. 36 Among the intrinsic risk factors, several genetic sequence variants have been shown to 37 increase the risk (predispose individuals) to AT and ACL ruptures. Variants within the TNC 38 [3], MMP3 [29], GDF5 [23] and TIMP2 genes [5] are associated with the risk of AT. 39 Furthermore, variants within the COL1A1 [22] and COL12A1 genes [24] have also been 40 associated with ACL ruptures. Interestingly, a variant within the COL5A1 gene was found to 41 be associated with both AT [16] and ACL ruptures [25]. These findings show that both 42 chronic AT and ACL ruptures have a partial polygenic basis where complex interactions 43 between genes and the environment are likely to exacerbate the risk of both types of injuries 44 [3]. All the genes described above encode proteins with either a structural or regulatory role 45 in maintaining the homeostasis of the soft tissue extracellular matrix (ECM). Therefore, it is 46 fair to assume that other genes, which code for additional regulatory components of the ECM 47 might also be candidates for AT and ACL rupture. 2 48 Elastin (ELN) is an insoluble polymer composed of several tropoelastin molecules covalently 49 bound to each other by cross-links [31]. ELN proteins contribute to tendon and ligament 50 elasticity by allowing them to stretch and return to their original state. These proteins have an 51 important load-bearing role in musculoskeletal tissues and are expressed in places where 52 mechanical energy is stored [8]. The ELN rs2071307 gene variant has been shown to be 53 associated with other multifactorial conditions of the extracellular matrix, such as aortic 54 stenosis [6] and aortic aneurysm [33]. Interestingly, the ELN rs2071307 variant is located 55 within exon 20 of the gene and is a non-synonymous SNP. It is predicted to be deleterious 56 (Queen’s University. http://compbio.cs.queensu.ca/F-SNP/) since it substitutes a hydrophobic 57 amino acid glycine with a hydrophilic serine residue (National Center for Biotechnology 58 Information. http://www.ncbi.nlm.nih.gov/projects/SNP/). This substitution may disrupt the 59 integrity of the microfibrils rendering them more prone to damage [18] and therefore this 60 variant may predispose to soft tissue damage during sports performance. 61 Fibrillins are large glycoproteins present in the extracellular matrix of tendons and ligaments 62 [2]. Both fibrillin-1 (FBN-1) and fibrillin-2 (FBN-2) share high amino acid homology and are 63 involved in providing strength and flexibility to various soft tissues. FBN-2 is abundant in 64 elastic tissues, such as tendons and ligaments [35] where it plays an important role in the 65 assembly of elastic fibres [2]. Mutations within the FBN2 gene are known to associate with 66 musculoskeletal 67 Furthermore, the rs331079 variant located within intron 7 of the gene (University of Florida. 68 www.snpper.chip.org) has previously been associated with intracranial aneurysms [32]. 69 As both the FBN2 rs331079 and the ELN rs2071307 variants associate with other conditions 70 related to the extracellular matrix we considered them as possible risk determinants for both 71 AT and ACL rupture. Accordingly, the aim of this study was to test that hypothesis. pathologies such as congenital contractural arachnodactyly [9]. 72 3 73 Material and Methods 74 One hundred and thirty five (60 Australian (AUS) and 75 South African (SA)) Caucasian 75 participants with Achilles tendinopathy (TEN group) were recruited to this study from the 76 Musculoskeletal Research Centre at La Trobe University in Melbourne, and from the Medical 77 Practice at the Sports Science Institute of South Africa. Furthermore, 239 (143 AUS and 96 78 SA) asymptomatic Caucasian controls (CON groups) were recruited to this study from 79 recreational sports clubs within the Melbourne area in Australia, and within the Cape Town 80 area in South Africa. Chronic AT was clinically diagnosed as described by Mokone et al.[17] 81 in the first manuscript describing the South African AT cohort. The Australian cohort used the 82 same clinical diagnosis described by Mokone et al. In addition, diagnosis was confirmed with 83 soft tissue ultrasound examination in all the AUS and 40 of 75 SA participants. In addition, 84 141 South African Caucasian participants with surgically diagnosed ACL ruptures (ACL 85 group) and 219 apparently healthy (CON group), unrelated, physically active, gender 86 matched South African Caucasian participants without any self-reported history of ligament 87 or tendon injury were recruited for this study as previously described [22]. Seventy four 88 participants sustained the injury through a non-contact mechanism and were analysed as a 89 separate subgroup (NON subgroup). 90 Previous injury data was used as inclusion criteria in the various cohorts analysed. In the 91 AUS Achilles cohort, the CON group had no history of any tendon injury, whereas in the SA 92 Achilles cohort, the CON group merely had no previous history of Achilles tendon injuries. In 93 the case of ACL rupture, the first ACL rupture was documented as the specific inclusion 94 injury. Therefore, by definition, no participant in the ACL group had a previous ACL rupture 95 None of the participants included in this study had symptoms or signs of Ehlers-Danlos 96 syndrome (EDS), hypermobility or benign hypermobility joint syndrome or other monogenic 97 connective tissue disorders when their medical examinations were reviewed by the medical 98 practitioner [16,17,34]. 4 99 Physical activity data was recorded for the South African Achilles tendinopathy cohort (SA 100 CON and SA TEN), but not for the Australian Achilles tendinopathy cohort (AUS CON and 101 AUS TEN). In addition physical activity data was also recorded for the South African ACL 102 cohort (SA ACL and SA CON). The data recorded for the SA CON and SA TEN groups 103 included total years participated in running and high impact sports, as well as hours per week 104 of participation in the last 2 years. The data reported for the ACL cohort included years of 105 participation in contact sports, non-contact jumping sports, non-contact non-jumping sports 106 and skiing sports, .Data were collected as previously described [16, 25]. 107 Based on our earlier work, this study had a large enough sample size to detect associations 108 with an OR of 2.0 at p<0.05 with 80% power [28]. All participants gave informed written 109 consent, in accordance with the journal’s recommendations [10,11], and all completed a 110 medical and injury history questionnaire. Ethical approval was obtained from the Research 111 Ethics Committees at the University of Cape Town, South Africa, La Trobe University, 112 Australia, Monash University, Australia and the University of Northampton, United Kingdom 113 prior to initiating this work. 114 For the Australian cohort, DNA was extracted from whole blood using Qiagen DNA extraction 115 kits (Flexigene DNA kit, Qiagen P/L, Valencia, California, USA) as per the manufacturer’s 116 recommendations. DNA from the South African individuals was extracted from blood using 117 the method described by Lahiri and Nurnberg [14] and modified by Mokone et. al. [16,17]. 118 Upon extraction, DNA was frozen at -20 °C for long-term storage, and smaller aliquots were 119 stored at 4 °C for short term usage. 120 DNA from all participants was genotyped for the FBN2 rs331079 and ELN rs2071307 gene 121 variants using fluorescence-based TaqMan assays (Applied Biosystems, Foster City, CA, 122 USA). PCR reactions contained allele-specific probes and primers in a PCR mastermix 123 containing AmpliTaq DNA Polymerase Gold (Applied Biosystems, Foster City, CA, USA) in a 124 total reaction volume of 12 μL. PCR was performed on an Applied Biosystems 5 125 StepOnePlusTM real-time PCR system (Applied Biosystems, Foster City, CA, USA). 126 Genotypes were called according to output clustering profiles using Applied Biosystems 127 StepOnePlusTM real-time PCR software Version 2.1 (Applied Biosystems, Foster City, CA, 128 USA). Rox was used as a passive reference to normalise fluorescence signal intensity 129 relative to the amount of sample used. 130 The 131 (http://hydra.usc.edu/gxe). The initial calculations were done using a recessive model and a 132 disease population prevalence of 10%. Assuming a risk allele frequency of 60%, a matched 133 case-control population of 136 individuals per group was adequate to detect an allelic OR of 134 2.0 at a power of 80% and a significance level of 5%. 135 Data were analysed using SPSS Version 20 (SPSS Science Inc, Chicago, Ill, USA) statistical 136 program. A one-way analysis of variance was used to establish if any significant difference 137 existed between the characteristics of the TEN and CON groups within the Australian and 138 South African cohorts as well as between the ACL rupture and CON groups. A chi-squared 139 (2) analysis or Fisher’s exact test was used to determine if significance differences existed 140 between genotype and/or allele frequencies, as well as other categorical data between the 141 groups. In all analysis significance was accepted when p<0.05. Adjustments for multiple 142 testing were not conducted as it has been previously described [21] that no appropriate 143 method exists. Furthermore, the Bonferroni adjustment was considered too conservative [21] 144 and inappropriate for a situation like this where there is prior evidence that the gene of 145 interest is associated with a trait [20]. Hardy-Weinberg equilibrium was determined using the 146 program Genepop web version 3.4 (Curtin University. http://genepop.curtin.edu.au/). statistical power of the study was determined using Quanto v1.2 147 6 148 Results 149 Running was the predominant sporting activity resulting in Achilles tendon injuries (63.1%, 150 N=65) in the SA cohort. The SA groups were matched for the mean number of years 151 participating in running (CON, 8.7 ± 8.2 yrs, n=95; TEN, 10.0 ± 11.0 yrs, n=62; p=0.402). 152 However, there was a significant difference in hours of training between the two groups 153 (CON, 3.6 ± 3.0 hrs/week, n=91; TEN, 2.4 ± 2.7 hrs/week, n=55; p=0.011), where the SA 154 CON group trained for more hours per week. The SA TEN participants participated in more 155 years of high impact sports compared to the SA CON group in the past (CON, 9.4 ± 8.4 yrs, 156 n=95; TEN, 13.1 ± 11.1 yrs, n=62; p=0.018), however, the SA CON group performed a 157 greater amount of high impact sports during the last 2 years (CON, 3.6 ± 3.1 yrs, n=95; TEN, 158 2.5 ± 12.9 yrs, N=62; p=0.029. Although all AUS participants were physically active 159 individuals, the type of sporting activity involved in, the hours of training and the frequency of 160 activity were not recorded. 161 The SA ACL and SA CON groups were matched for years of participation in contact sports 162 (SA CON, 11.7 ± 7.1 yrs, n=219; SA ACL, 11.5 ± 8.0 yrs, n=141; p=0.892), non-contact 163 jumping sports (SA CON, 27.8 ± 19.9 yrs, n=190; SA ACL, 25.7 ± 22.6 yrs, n=141; p=0.398), 164 non-contact non-jumping sports (SA CON, 11.5 ± 7.1 yrs, n=219; SA ACL, 10.5 ± 8.5 yrs, 165 n=141; p=0.575), and skiing sports (SA CON, 19.1 ± 16.9 yrs, n=219; SA ACL, 8.6 ± 8.5 yrs, 166 n=141; p=0.094). 167 Since the ELN rs2071307 and FBN2 rs331079 allele and genotype frequencies in both of the 168 South African (SA) and Australian (AUS) TEN and CON groups were similar (Supplementary 169 table 1), the data was collectively analysed. The CON and TEN groups were similarly 170 matched for age and gender (Table 1). 171 similarly matched for height. Furthermore, when co-varied for sex and age at recruitment, the 172 TEN group was found to be significantly heavier (p<0.001) with larger BMIs (p<0.001) (Table 173 1). The TEN group was recruited on average 5.1 years after the initial injury. When co-varied for sex, the two groups were 7 174 Participants in the AUS TEN group carrying the ELN rs2071307 AA (53.1 ± 11.6, n=10) 175 genotype were significantly (p=0.005) older when they reported their initial Achilles tendon 176 injury when compared to those with a GG (37.2 ± 12.6, n=16) or GA (37.8 ± 13.6, n=32) 177 genotype. There were, however, no significant differences in the mean ages of the three 178 genotype groups in the CON AUS group (GG: 40.7 ± 11.8, n=48; GA: 37.4 ± 12.2, n=68; AA: 179 40.1 ± 12.1, n=24; p=0.323). There were no other significant genotype effects of either 180 variants with respect to height, weight, BMI, or sex in the AT group (data not shown). 181 Furthermore, the investigated variants did not show any interaction with age, height, weight, 182 BMI and sex in the ACL population (data not shown). 183 The genotype frequency distributions of the FBN2 rs331079 and the ELN rs2071307 variants 184 within the AT and the ACL rupture groups are shown in table 2. In the combined TEN cohort, 185 the FBN2 rs331079 genotype frequency was significantly different (p=0.035) between the 186 CON (GG, 76.9%; GC + CC, 23.1%) and TEN (GG, 85.9%; GC + CC, 14.1%) groups (Table 187 2). The GG genotype was significantly over-represented within the TEN group (p=0.035; 188 OR=1.83; 95% CI 1.04 – 3.25). We also found a significant (p=0.017; OR=1.90; 95% CI 1.11 189 – 3.27) allele frequency distribution difference for the FBN2 rs331079 variant between the 190 CON (G, 87.4%; C, 12.6%) and TEN (G, 93.0%; C, 7.0%) groups (Table 2). Similarly, we 191 also found a significant (p=0.047; OR=1.76; 95% CI 1.00 – 3.10) allele frequency distribution 192 difference of the rs331079 locus between the CON (G, 89.3%; C, 10.7) and ACL (G, 93.6%; 193 C, 6.4%) groups. Also, in the AT population, there were no significant ELN rs2071307 194 genotype (p=0.795) or allelic (p=0.741) frequency differences between the CON and TEN 195 groups (Table 2). 196 Although not significant, we found a tendency towards an allelic (p=0.064) association for the 197 ELN rs2071307 variant and a tendency towards a genotypic (p=0.075; p=0.112) association 198 between the CON and ACL groups for the FBN2 rs331079 and ELN rs2071307 variants 199 respectively. There were no genotypic or allelic associations between the CON and NON 8 200 subgroup. Furthermore, these gene variants did not show any significant distribution 201 difference when participants were grouped into genders (data not shown). 202 Discussion 203 We have shown that the FBN2 rs331079 variant is significantly associated with the risk of 204 both AT and ACL rupture. Specifically, the GG genotype was over-represented in 205 participants with chronic AT and the G allele was over-represented in both pathologies. 206 Therefore, it appears that individuals carrying the G allele or the GG genotype are 207 approximately twice as likely to develop either of the two injuries. Interestingly this same 208 variant has recently been shown to associate with intracranial aneurysms in a Dutch 209 population [32]. However, in the Dutch study it was the C allele that was found to be the risk 210 factor as opposed to the G allele. It is noteworthy that FBN2 mRNA levels have been shown 211 to be elevated in rat Achilles tendon undergoing repair with expression of FBN2 reported to 212 be increased for ten days post injury [13]. Similarly, an increase in the expression of FBN2 213 has been found in other pathologies such as mitral valve prolapse [27]. 214 ELN and FBN-2 are known to form a network of microfibrils that maintains the tendon 215 architecture [31]. An increase in FBN-2 levels might be expected to increase the density of 216 the tendon and lead to an increase in tendon stiffness and rigidity possibly affecting the 217 compliance of the tendon to muscle movement [4]. On the other hand, a decrease in FBN-2 218 levels could result in weaker tendons caused by structural deficiencies in the microfibril 219 network [30]. Impairment of the function of FBN-2 is believed to be a major determinant of 220 microfibrillopathy [30] which is speculated to precede a tendinopathy. Furthermore, the 221 increase in FBN2 expression levels observed during tendon repair [13] is consistent with an 222 important role for FBN-2 in maintaining the tendon’s architectural integrity. 223 Mutations such as the G3532T and G3590A substitutions have been found within the FBN2 224 gene that lead to the development of connective tissue disorders such as congenital 225 contractural arachnodactyly [9]. The rs331079 variant that we investigated in this study 9 226 resides 227 www.snpper.chip.org). Although intronic variants do not determine the primary sequence of 228 a protein molecule [1], they may have other, hitherto, undiscovered roles that are necessary 229 for appropriate expression of protein molecules. However, at present the functionality of this 230 variant has not been described and therefore we do not know why it predisposes individuals 231 to AT and ACL rupture. The rs331079 variant is known to be part of a linkage block in 232 Caucasians and is in high linkage disequilibrium (D'=1) with the FBN2 rs331081, rs331082, 233 and rs331085 variants (Wellcome Trust Sanger Institute. www.ensembl.com). All three of 234 these additional variants are also located within intron 7 of the FBN2 gene (University of 235 Florida. www.snpper.chip.org). The linkage disequilibrium between the rs331079 variant that 236 we investigated and rs331081, rs331082, and rs331085 means that it is conceivable that one 237 of these linked variants may also have a role in predisposing to AT or ACL. 238 Our data do not support an association between the ELN rs2071307 variant and either AT or 239 ACL ruptures. It is interesting to note however, that although we found no relationship 240 between this variant and either pathology; the rs2071307 SNP is a non-synonymous and 241 possibly deleterious polymorphism (Queen’s University. http://compbio.cs.queensu.ca/F- 242 SNP/) which results in a change of amino acid from hydrophobic glycine to hydrophilic serine 243 (University of Florida. www.snpper.chip.org). It is possible of course, that other variants 244 within this gene may be associated with either AT or ACL ruptures. 245 Although our study found a significant association between the FBN2 rs331079 G allele and 246 the risk of AT and ACL rupture, the work has some limitations. Firstly, although our SA 247 cohorts (both TEN and ACL rupture groups) were matched for some aspects of physical 248 activity there were some differences in training behaviour and previous exposure to high 249 impact sports for the TEN cohort.. Secondly, we did not have detailed information on sports 250 history for the Australian cohort. Levels of physical activity should be accurately documented 251 in future studies.Furthermore, although the study was sufficiently powered to detect 252 associations with relatively large effects it should be repeated in bigger cohorts. Likewise, within an intronic region of the FBN2 gene (University of Florida. 10 253 additional association studies should be carried out in populations of different ethnicities 254 showing different minor allele frequencies for the rs331079 (African, 3%; European, 10%; ad- 255 mixed American, 28%; East Asian, 7%) and the rs2071307 (African, 26%; European, 39%; 256 ad-mixed 257 www.1000genomes.org). 258 Finally, the findings from this study advance our understanding of the polygenic basis of 259 musculoskeletal injuries. 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J Cell Biol 1995; 129: 1165-1176. 358 359 List of legends 360 Table 1: General characteristics of the Achilles tendinopathy group (TEN), the anterior 361 cruciate ligament rupture group (ACL), and the ACL subgroup with the non-contact (NON) 362 mechanism of injury as well as their respective control groups. 363 Table 2: The genotype and allele frequency distribution of the two selected candidate 364 variants within the Achilles tendinopathy (TEN), ACL ruptures (ACL) and their respective 365 asymptomatic control (CON) groups. 366 367 368 369 16