Effect of ACTN3 R577X Genotype on Injury Epidemiology in Elite Endurance Runners

polymorphism (rs1815739) in theACTN3gene causes individuals with the ACTN3XX genotype to be de cient in functional -actinin-3. Previous investigations have found that XX athletes are more prone to suffer non-contact muscle injuries. This investigation aimed to determine the in uence of theACTN3R577X polymorphism in the injury epidemiology of elite endurance athletes. Using a cross-sectional experiment, the epidemiology of running-related injuries was recorded for one season in a group of 89 Spanish elite endurance runners.ACTN3R577X genotype was obtained for each athlete using genomic DNA samples. From the study sample, 42.7% of athletes had the RR genotype, 39.3% had the RX genotype, and 18.0% had the XX genotype. A total of 96 injuries were recorded in 57 athletes. Injury incidence was higher in RR runners (3.2 injuries/1000 h of running) than in RX (2.0 injuries/1000 h) and XX (2.2 injuries/1000 h;p= 0.030)

using genomic DNA samples. From the study sample, 42.7% of athletes had the RR genotype, 39.3% had the RX genotype, and 18.0% had the XX genotype. A total of 96 injuries were recorded in 57 athletes. Injury incidence was higher in RR runners (3.2 injuries/1000 h of running) than in RX (2.0 injuries/1000 h) and XX (2.2 injuries/1000 h;p= 0.030) runners. RR runners had a higher proportion of injuries located in the Achilles tendon, RX runners had a higher proportion of injuries located in the knee, and XX runners had a higher proportion of injuries located in the groin (p= 0.025). TheACTN3genotype did not affect the mode of onset, the severity, or the type of injury. TheACTN3genotype slightly affected the injury epidemiology of elite endurance athletes with a higher injury rate in RR athletes and differences in injury location. However, eliteACTN3XX endurance runners were not more prone to muscle-type injuries. Keywords: athletic performance; exercise-related injury; single nucleotide polymorphism; track and eld athlete; -actinin-3 de ciency 1. Introduction The status of elite athlete requires many hours of strenuous training per week that can impose severe physiological and mechanical stress on the human body, ultimately leading to sports injuries. For this reason, injury is an inherent feature of elite sport [1], and injury prevention has become a key aspect of every conditioning program. Because injuries may inhibit regular training, negatively impact sports performance, and even shorten an athlete's career, the study of all the risk factors that predispose to injury is essential to design effective injury prevention programs. Endurance running is a sports discipline with an elevated injury incidence due to the high mechanical load produced by weight-bearing and the high running mileage necessary to prepare for endurance competitions [2]. For this reason, a high proportion of injuries in elite endurance runners are muscle or tendon overuse injuries, and the main sites of injury location are lower leg and knee [3,4]. Age, training volume, history of previous injuries, and running kinematics have been considered as the main determinants for the likelihood Genes2021,12, 76.

reason, a high proportion of injuries in elite endurance runners are muscle or tendon overuse injuries, and the main sites of injury location are lower leg and knee [3,4]. Age, training volume, history of previous injuries, and running kinematics have been considered as the main determinants for the likelihood Genes2021,12, 76.

Genes2021,12, 76 2 of 9 of suffering endurance running injuries [4,5]. However, genetics might also play a role in the predisposition to injury in some athletes [6], particularly the variations in genes associated with muscle and tendon proteins [7]. -Actinin-3 is a key component of the skeletal muscle Z-disk in fast-twitch muscle bers. Hence, it is believed that -actinin-3 is important for the production of forceful muscle contractions or to resist muscle damage induced by eccentric contractions [8]. A single nucleotide polymorphism (p.R577X; rs1815739) in theACTN3gene (the gene that codi es -actinin-3) results in the replacement of an arginine (R) with a premature stop codon (X) [9]. Homozygosity for this stop codon (XX genotype) produces an -actinin-3 de ciency, as opposed to individuals with RX or RR genotypes that express functional - actinin-3 [10]. Although an -actinin-3 de ciency is compensated for by a higher expression of -actinin-2, signi cant evidence has shown that XX individuals are underrepresented in elite power-oriented athletes, which might be indicative of a negative effect of this genotype on the function of fast-twitch muscle bers [11]. Recent investigations have found that XX athletes might also be more prone to suffer sports-related muscle injuries when compared to RR counterparts [12–14], although this is not always the case [15]. There is greater consensus on the higher predisposition of XX individuals to ankle sprains [16–18] and high levels of muscle damage during endurance competitions [19,20]. All this information suggests an a priori predisposition of XX athletes to sports-related injuries, but a lack of replication is present for most current ndings that associate genetics with a predisposition to sports injury, as recently suggested [21]. For this reason, we aimed to determine the in uence of theACTN3R577X polymorphism on the injury epidemiology of elite endurance athletes, following the methodology of a previous investigation carried out on amateur endurance runners [12]. We obtained information to characterize the in uence of this polymorphism on injury epidemiology, such as incidence, conditions, severity, mode of onset, body location, and cause that led to each injury in elite runners with differentACTN3genotypes. We hypothesized that elite

injury epidemiology of elite endurance athletes, following the methodology of a previous investigation carried out on amateur endurance runners [12]. We obtained information to characterize the in uence of this polymorphism on injury epidemiology, such as incidence, conditions, severity, mode of onset, body location, and cause that led to each injury in elite runners with differentACTN3genotypes. We hypothesized that elite endurance runners with the XX genotype would have a higher incidence of muscle and ligament injuries when compared to RX and RR athletes. 2. Materials and Methods 2.1. Participants Initially, 97 elite Spanish Caucasian endurance athletes volunteered to participate in the study. Participants were cataloged as elite endurance athletes because they competed in national and international events of middle- and long-distance modalities (from 800 m to the marathon). Among them, there were 5 medallists and 20 nalists in International Championships, and 12 champions and 23 medallists in National endurance running competitions. For this sample, two participants were excluded by age (>45 years), and six were excluded because theirACTN3genotype was not clearly identi ed in the genotyping analysis. Age, anthropometric characteristics, running experience, and training status of the nal study sample of 89 elite endurance runners are depicted in Table. The study protocol conformed to the Declaration of Helsinki for Human Research of 1974 (last modi ed in 2013) and was approved by the Camilo Jos²Cela University Ethics Committee. Written informed consent was obtained from all participants.

Genes2021,12, 76 3 of 9 Table 1. Age, anthropometric characteristics, running experience, and training status of Spanish elite endurance athletes with differentACTN3R577X genotypes. Variable (units) RR RX XX p-Value Number (frequency, %) 38 (42.7) 35 (39.3) 16 (18.0) - Men (frequency, %) 16 (42.1) 24 (68.6) 8 (50.0) 0.072 Women (frequency, %) 22 (57.9) 11 (31.4) 8 (50.0) Age (years) 22.8 4.2 24.5 10.5 26.6 7.0 0.422 20 years (frequency, %) 13 (34.2) 11 (31.4) 5 (31.3) 0.078>20 years 30 years (frequency, %) 23 (60.5) 21 (60.0) 6 (37.5) <30 years (frequency, %) 2 (5.3) 3 (8.6) 5 (31.3) Height (cm) 170.2 7.2 172.3 8.4 172.2 6.7 0.353 Body mass (kg) 56.4 8.3 58.1 8.6 57.1 6.3 0.639 Experience (years) 11.5 4.9 12.6 4.9 14.7 6.8 0.098 Endurance running (hours/year) 639 248 650 174 623 237 0.770 Resistance training (hours/year) 152 72 148 67 129 64 0.461 Competitions (number/year) 16.1 6.6 17.5 8.5 16.7 7.1 0.780 Data are mean standard deviation (SD) for each genotype. 2.2. Experimental Design This investigation was a cross-sectional experiment to determine the effect of the ACTN3R577X genotype (RR vs. RX vs. XX) on the injury epidemiology of endurance running-related injuries suffered by elite endurance athletes. For this investigation, par- ticipants completed an ad hoc questionnaire between September and November of 2019, seeking to record non-contact injuries sustained during the previous season retrospectively. Only injuries resulting from their training routines or competitions in endurance running activities were recorded. The questionnaire was based on the consensus statement on injury de nitions and data collection in epidemiological studies in athletics [22]. 2.3. Experimental Protocol In the questionnaire, a recordable injury was de ned as a physical complaint or visible damage to any part of the lower limbs sustained by the athlete and assessed by a quali ed medical/healthcare practitioner. The injury was recorded irrespective of whether it produced a time loss from training and/or competition or whether it was only a medical attention injury that did not impede normal training. All traumatic injuries, such as the ones caused by a fall or due to contact with an obstacle

by the athlete and assessed by a quali ed medical/healthcare practitioner. The injury was recorded irrespective of whether it produced a time loss from training and/or competition or whether it was only a medical attention injury that did not impede normal training. All traumatic injuries, such as the ones caused by a fall or due to contact with an obstacle or another athlete, were discarded as they are not potentially affected by theACTN3genotype. Training exposure was de ned as any physical activity conducted under the control or guidance of the coach, with the role of maintaining or improving the athlete's physical condition. Competition exposure was de ned as physical activities conducted in of cial endurance running competitions. The questionnaire gathered information about the number of injuries sustained in the previous season. Afterward, the information on type, severity, body location, exposure, recurrence, mode of onset, and the possible cause that led to the injury was obtained independently for each recordable injury, following the methodology described by Moreno-P²rez et al. [12]. The questionnaire also gathered information about training routines (endurance running hours per week, resistance training hours per week, number of training weeks per year, number of competitions). Hence, injury incidence was calculated for each athlete as number of injuries per year and as number of injuries per 1000 h of endurance running. 2.4. Genetic Testing Once participants had completed the questionnaire, they were asked to provide two buccal swab samples using self-guided instructions. The samples were stored in envelopes, and genomic DNA was isolated afterward using an organic-based DNA extraction method adapted to Amicon ® (Sigma–Aldrich, Madrid, Spain). DNA was extracted within 30 days after sample collection, and the sample was eluted in 50 L. Positive controls for all geno- types were obtained from the Mexican branch of the CANDELA Consortium. Genotyping

Genes2021,12, 76 4 of 9 of theACTN3rs1815739 polymorphism (c.1858C>T; p.R577X) was conducted using a Taq- Man SNP Genotyping Assay (Assay ID: C___590093_1_; Applied Biosystems, Foster City, CA, USA), and the reaction was performed in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). 2.5. Statistical Analysis Data on injury epidemiology were transferred from the questionnaire to an ad hoc database. The normality of each variable was initially tested with the Kolmogorov–Smirnov test and parametric/non-parametric statistics were performed for normally/non-normally distributed variables, respectively. For the continuous variables, genotype comparisons (RR vs. RX vs. XX) were performed using a one-way analysis of variance (ANOVA; followed by Tukey's post-hoc comparisons) or the Kruskal–Wallis test. For the variables presented as frequency, the differences in distribution among genotypes were identi ed with crosstabs and 2tests, including adjusted standardized residuals. Compliance of Hardy–Weinberg Equilibrium (HWE) in the genotype distribution of the sample was tested using 2tests. Spearman's rho was used to calculate the association between training volume and the number of injuries per year. All statistical analyses were performed with statistical software (SPSS Statistics 22, IBM Armonk, NY, USA). Descriptive data are presented as means and standard deviations. Statistical signi cance was set atp< 0.05. 3. Results Genotyping forACTN3R577X was successful in 89 out of 95 participants (93.7% of successful genotyping). From the nal study sample, 38 (42.7%) athletes had the RR genotype, 35 (39.3%) athletes had the RX genotype, and 16 (18.0%) had the XX genotype (Table ). The genotype distribution met the HWE. There was no difference in the propor- tion of men/women elite endurance athletes among genotypes nor in age, anthropometric characteristics, or training variables. Of the study participants, 32 athletes reported no injury during the preceding season, while the remaining 57 athletes reported a total of 96 injuries. There were no differences in the distribution of athletes with/without injury across the differentACTN3genotypes in the whole sample or when analyzing the subsamples of male and female elite endurance athletes (Table). The injuries were recorded by a physician (62.1%), by a physiotherapist (32.6%), or by other healthcare providers (5.3%). From the total, 37.9%

athletes reported a total of 96 injuries. There were no differences in the distribution of athletes with/without injury across the differentACTN3genotypes in the whole sample or when analyzing the subsamples of male and female elite endurance athletes (Table). The injuries were recorded by a physician (62.1%), by a physiotherapist (32.6%), or by other healthcare providers (5.3%). From the total, 37.9% of injures were diagnosed by magnetic resonance imaging, 28.7% by echography, 4.6% by X-ray, and 28.7% by other clinical testing. Table 2. Distribution of athletes with/without an injury reported in the preceding season according to theirACTN3R577X genotype. Variable (units) RR RX XX p-Value Total Athletes with injury (frequency, %) 23 (60.5) 24 (68.6) 10 (62.5) 0.766 Athletes without injury (frequency, %) 15 (39.5) 11 (31.4) 6 (37.5) Males Athletes with injury (frequency, %) 8 (50.0) 19 (79.2) 5 (62.5) 0.153 Athletes without injury (frequency, %) 8 (50.0) 5 (20.8) 3 (37.5) Females Athletes with injury (frequency, %) 15 (62.8) 5 (45.5) 5 (62.5) 0.449 Athletes without injury (frequency, %) 7 (31.8) 6 (54.5) 3 (37.5) Data are numbers and frequencies (in percentage) of athletes with/without injury reported in the preceding season from the total number of athletes or the number of athletes in each sex. There were no differences in the mean value of injuries per year, nor in the distribution of the number of injuries per year among genotypes (Table). However, the median value for injury incidence was higher in RR (3.2, range from 0.8 to 7.4 injuries/1000 h of running) runners than in RX (2.0, range from 0.9 to 7.4 injuries/1000 h of running) and XX (2.2, from 0.9 to 6.3 injuries/1000 h of running;p= 0.030) runners. Table epidemiological information about injury conditions in the sample of elite endurance

Genes2021,12, 76 5 of 9 runners. TheACTN3genotype did not affect the proportion of time loss/medical attention injuries, the severity of the injury, the distribution of training and competition injuries, the proportion of recurrent injuries, the mode of onset, or the possible cause that led to the injury. However, RR runners had a higher proportion of injuries located in the Achilles tendon, RX runners had a higher proportion of injuries located in the knee, and XX runners had a higher proportion of injuries located in the groin (Table, p= 0.025). Finally, the ACTN3 genotype did not affect the type of injury with a similar distribution of muscle, bone, tendon, and ligament injuries. Table 3. Injury incidence, distribution of athletes according to the number of injuries, and distribution of injuries according to severity, exposure, recurrence, mode of onset, and possible cause in elite endurance runners with differentACTN3R577X genotypes. Variable All RR RX XX p-Value Incidence /per year 1.0 1.0 1.0 1.0 0.321 /1000 h or running 2.8 3.2 2.0 2.2 0.030 Number No injury (%) 36.0 39.5 31.4 37.5 0.177 1 injury (%) 31.5 21.1 40.0 37.5 2 injuries (%) 21.3 18.4 22.9 25.0 3 injuries (%) 11.2 21.1 5.7 0.0 Time loss Medical attention (%) 9.2 7.1 10.3 11.8 0.819 Time loss (%) 90.8 92.9 89.7 88.2 Severity Minor (%) 13.4 12.9 12.7 16.0 0.993Moderate (%) 51.4 51.6 52.7 48.0 Serious (%) 35.2 35.5 34.5 36.0 Exposure Training (%) 94.9 95.3 94.9 94.1 0.981 Competition (%) 5.1 4.7 5.1 5.9 Recurrence New onset (%) 61.2 59.5 66.7 52.9 0.598 Recurrent (%) 38.8 40.5 33.3 47.1 Mode of onset Sudden (%) 46.9 45.2 43.6 58.8 0.552 Gradual (%) 53.1 54.8 56.4 41.2 Possible cause Excessive load (%) 62.2 61.9 61.5 64.7 0.526 Surface (%) 11.2 11.9 15.4 0.0 Shoe (%) 8.2 4.8 7.7 17.6 Biomechanics (%) 4.1 7.1 2.6 0.0 Unknown (%) 14.3 14.3 12.8 17.6 Data are frequencies (in percentage) from the total of injuries recorded in each genotype. Table 4. Distribution of injuries according to body location and type of injury in elite endurance runners with different ACTN3R577X genotypes. Variable

Surface (%) 11.2 11.9 15.4 0.0 Shoe (%) 8.2 4.8 7.7 17.6 Biomechanics (%) 4.1 7.1 2.6 0.0 Unknown (%) 14.3 14.3 12.8 17.6 Data are frequencies (in percentage) from the total of injuries recorded in each genotype. Table 4. Distribution of injuries according to body location and type of injury in elite endurance runners with different ACTN3R577X genotypes. Variable All RR RX XX p-Value Body location Groin (%) 13.5 4.8 * 13.2 37.5 * 0.025 Hip (%) 1.0 2.4 0.0 0.0 Thigh (%) 15.6 14.3 13.2 25.0 Knee (%) 10.4 4.8 21.1 * 0.0 Lower leg (%) 15.6 21.4 13.2 6.3 Achilles tendon (%) 11.5 19.0 * 7.9 0.0 Ankle (%) 9.4 4.8 13.2 12.5 Foot (%) 16.7 19.0 13.2 18.8 Other (%) 6.3 9.5 5.3 0.0 Type of injury Strain/muscle rupture (%) 19.6 22.7 15.0 22.2 0.295 Stress fracture/other bone injury (%) 13.7 11.4 10.0 27.8 Tendinosis/tendinopathy (%) 33.3 31.8 40.0 22.2 Sprain/ligament injury (%) 7.8 2.3 12.5 11.1 Other (%) 25.5 31.8 22.5 16.7 Data are frequencies (in percentage) from the total of injuries recorded in each genotype. (*) Different from expected value atp< 0.05.

Genes2021,12, 76 6 of 9 4. Discussion In an attempt to increase the scarce knowledge available about the in uence of genetics on elite athlete's susceptibility to injury, we designed a cross-sectional investigation to assess the in uence of theACTN3R577X polymorphism on injury epidemiology in elite endurance athletes. This investigation replicates the methods used in a previous study [12], with the only difference being the characteristics of the study sample (elite vs. amateur endurance runners). The main conclusions of this investigation indicate that theACTN3 genotype had a slight but interesting in uence on injury epidemiology of elite endurance athletes. Speci cally, there was a higher injury rate in RR athletes than in RX and XX genotypes, and there were differences in injury location. However, our main hypothesis related to a higher incidence of muscle injuries in XX athletes has not been con rmed with the current data. The overall injury incidence was 2.8 injuries per 1000 h of endurance running, and 64% of the sample reported at least one injury during the preceding season, which is in agreement with previous data on elite samples [23,24]. Additionally, the number of injuries per year was associated with endurance running volume/year (Spearman's rho = 0.259; p= 0.014), suggesting that a high running mileage was an important injury risk factor in this sample of elite endurance runners as previously found in other types of endurance runners [25,26]. However, the number of injuries per 1000 h of endurance running was 45–60% higher in RR athletes than in RX and XX counterparts. This higher incidence was produced because 21% of the RR athletes sustained three injuries or more (Table), while no XX runner sustained 3 injuries. This is not a unique nding in the literature as a higher injury incidence of RR vs. RX-XX has also been found in amateur runners [12] and in women college athletes [15]. The higher injury incidence among the individuals with a full expression of -actinin-3 (RR genotype) may be explained by higher levels of muscle strength and a lower range of motion [27,28], which could be translated into more physical

The study examines the impact of ACTN3 genotype on injury rates in elite runners.