Genetics and Genomics in Sports

Any individual can enhance his/her performance by engaging in deliberate practice and training during optimal periods of development. Nonetheless, the initial level of performance at first exposure to a sport varies greatly between individuals, as some individuals show a higher response to training than others. Furthermore, some athletes are repeatedly afflicted with injuries, but some athletes are not injured at all. Numerous twin and family studies have demonstrated that genetic factors contribute significantly to the variation in physical performance. Further, it has been demonstrated that several genetic variants significantly affect the susceptibility of sports-related injuries. Since an insertion (I)/deletion (D) polymorphism in the angiotensin I-converting enzyme gene ( ACE ) was first reported to have an impact on human physical performance, numerous studies have attempted to identify genetic variants influencing sports performance. To date, at least 100 genetic markers have been reported to be linked to the status of an athleteʼs endurance, and at least 69 genetic markers have been reported to be linked to the status of an athleteʼs power. Nevertheless, there is limited evidence of the genetic factors involved in sports performance and vulnerability to sport-related injuries in Asian populations. In order to perform genetic testing for individual training to improve sport performance and reduce injury risk, reliable and valid evidence is required, in addition to careful consideration of ethical issues. To achieve such individualized training in Japanese athletes, we need to develop a strong scientific foundation on this topic in the Asian population through large-scale collaborative projects.


Introduction
Individuals can enhance their performance by engaging in deliberate practice and training during optimal periods of development. However, the initial level of performance at first exposure to a sport varies greatly between individuals; some individuals show a higher response to training than others. Furthermore, some athletes are repeatedly afflicted with injuries, but some are not injured at all. What make these individualistic variabilities in sports performance and injury susceptibility?
Numerous twin and family studies have demonstrated that genetic factors significantly contribute to the variability of physical performance. Our recent systematic review and meta-analysis studies revealed that the estimated heritability of endurance-related phenotypes and muscle strength-related phenotypes is 44%-68% 1) and 49%-56% 2) , respectively. Moreover, De Moor et al. (2007) estimated that athlete status heritability is at 66% 3) . These results indicate that both genetic and environmental factors contribute to the individual differences in sport performance. Although estimates of heritability for sport-related injuries are currently unknown, several genetic variants have been shown to significantly affect the susceptibility of sport-related injuries. Though the"nature vs.
nurture"argument has been ongoing, existing literature indicates that elite sports performance is the result of both training and genetic factors. Therefore, it is important to explore the nature of genetic factors and how we can/should use this genetic information for athletes. In this review, we summarize the current knowledge on sports genetics.

Genetic Polymorphism Associated with Sports Performance
Since an insertion (I)/deletion (D) polymorphism in the angiotensin I-converting enzyme gene (ACE) was first reported in 1998 to have an impact on human physical performance 4) , numerous studies have attempted to identify genetic variants affecting sports performance. According to recent reviews, at least 100 genetic markers have been reported to be linked to the endurance of athletes 5) , and at least 69 genetic markers have been reported to be linked to the power of athletes 6) . It is commonly understood that sport performance is a polygenic phenotype, and therefore, multiple genetic variants are considered to have a complex effect on sports performance. In 2008, Williams and Folland 7) proposed a"total genotype score (TGS)" genetic algorithm to quantify the combined influence of endurance-associated polymorphisms. Subsequently, based on this algorithm, many studies have attempted to identify a polygenic profile that determines the potential for individuals to become elite athletes 8)-12) ; several studies have reported that various TGSs differ significantly between elite athletes and non-athletic individuals in the Caucasian population 8)-10) 12) . Such TGSs, however, were based on less than seven polymorphisms, and there are many other polymorphisms that can explain sport performance discrepancies. We, therefore, aimed to investigate the associations of TGS, based on the largest number of potential polymorphisms affecting sprint/power performance, with elite athlete status in the Japanese population 13) . Briefly, we searched for genetic polymorphisms correlated with sprint/power performance and related phenotypes (e.g., sprint/power athlete status, sprint performance, muscle strength/power, muscle volume, and trainability of these parameters) using PubMed (this search was performed until September 2012), and finally selected 21 polymorphisms based on several criteria 13) . We then analyzed these polymorphisms in 649 Japanese non-athletic individuals and 211 Japanese sprint/power track and field athletes (77 regional-level, 72 national-level, and 62 international-level athletes) and calculated TGS for sprint/power performance based on 21 polymorphisms. To our knowledge, this TGS included the largest number of polymorphisms in the literature with actual genotype data of elite athletes. Guilherme and Lancha 14) showed that in TGSs computed with more polymorphisms (≥8 polymorphisms), the standard deviation tended to be lower and more stable compared to that in TGSs computed with fewer polymorphisms (≤4 polymorphisms). Furthermore, as the number of polymorphisms included in the TGS increased, the statistical significance of the TGS differences between the athletes and controls also increased. However, in our study there was no significant association between TGSs based on 21 polymorphisms and sprint/power athlete status (i.e., all groups showed similar TGSs) in the Japanese population 13) . Although we also examined the effect of TGS for endurance performance on athlete status in the Japanese population, we found no significant differences in the TGS for endurance performance between non-athletic individuals and elite endurance runners 15) . These results suggest that TGSs based on previously published performance-associated polymorphisms are not capable of predicting athlete status in Japanese track and field athletes.
One of the possible explanations for the lack of association between TGSs and elite athlete status in our studies is the ethnic differences between previous studies (Caucasians) and our studies (Asians). It is known that there are differences in genotype frequencies and haplotype networks between ethnic groups. Therefore, differences in genetic background between Caucasians and Asians may contribute to the differences in genotype-phenotype associations between these two ethnic groups. Indeed, different alleles of the This manuscript was submitted for the Special Issue "Tokyo 2020 Olympic and Paralympic Games, and Sportology", prior to the decision to postpone the Tokyo 2020 Summer Olympics to 2021. ACE I/D polymorphism have been associated with elite swimmer status in Caucasians and East Asians 16) . Namely, D allele has been associated with short-distance swimmer status among the Caucasians, while I allele has been associated with shortdistance swimmer status among East Asians. Other studies support the pattern of the ACE I/D polymorphism associations across ethnic groups 17) 18) . In view of these findings, in order to identify TGSs capable of predicting elite athlete status in the Japanese (Asian) population, data on the association between genetic variants and elite athlete status in Asian populations must be accumulated.
When we tested the effect of each polymorphism separately, six of the 21 polymorphisms were significantly associated with sprint/power athlete status in the Japanese population 13) . Table-1 displays the list of polymorphisms associated with sprint/power athlete status in Japanese track and field athletes. Such polymorphisms could be potential candidates for predicting sprint/power performance in the Japanese population. Future studies should confirm the association between TGS based on these six polymorphisms and elite sprint/ power athlete status in Asian populations.
To date, the most commonly used method in sports genetics is the candidate gene association study, which requires a prior hypothesis that particular genes of interest should contain variants that may be associated with a trait 19) . However, in the last few years, a hypothesis-free genome-wide association study (GWAS) approach has been applied in the sports genetics field 20)-22) . The first GWAS for elite athlete status was published in 2016 21) . In that study, genome-wide association analyses were conducted on two cohorts of elite endurance athletes and their controls (GENATH-LETE and Japanese endurance runners), from which 45 candidate polymorphisms were identified. Furthermore, these polymorphisms were tested for replication in seven additional cohorts of endurance athletes and controls (Australia, Ethiopia, Japan, Kenya, Poland, Russia and Spain). As a result, these analyses did not identify a panel of genomic variants specific to these elite endurance athlete groups, while meta-analysis of all available cohorts revealed a single statistically significant marker (a rs558129 polymorphism located in the polypeptide N-acetylgalactosaminyltransferase like 6 gene (GALNTL6)) even after adjustment for multiple testing. Additionally, all cohorts showed the same direction of association with the GALNTL6 rs558129 polymorphism. It is known that GALNTL6 is highly expressed in brain and testis tissues (https://www. ncbi.nlm.nih.gov/gene/442117) but the functional role of the gene is not fully elucidated. Clarifying the mechanism of the association between the GALNTL6 rs558129 polymorphism and elite endurance athlete status will help understand the biology of world-class endurance performance and therefore can contribute to the development of new training methodology for endurance performance.

Genetic Polymorphism Associated with Injury Risk
Sports participation is accompanied by an increased risk of acute and chronic sports-related injuries. The incidence of sports-related injuries negatively affects athletic performance in both individual and team sports 23)-25) . Therefore, effective preventive approaches are required. There are inter-individual differences in susceptibility to sport-related injuries, and these differences are affected by several factors, both intrinsic and extrinsic. Genetic variations also play a role in the risk profile for sports-related injury as an intrinsic factor. The Australian Institute of Sport, Athlome  In the Japanese Human Athlome Project (J-HAP) which is part of"Athlome Project Consortium" 28) , we are attempting to elucidate genetic variants in Japanese athletes associated with the risk of sport-related injuries, such as muscle injury, ligament injury, and fatigue fracture. In this project, saliva samples for DNA analyses and history of sports-related injuries were collected from > 3,000 Japanese athletes who participated in various sports, and were not limited to athletes from a specific discipline. The incidences of muscle injury and fatigue fracture were highest in track and field athletes, while the incidence of ligament injury was highest in soccer players in the J-HAP. Based on this project, to date, we found that the estrogen receptor 1 gene (ESR1) rs2234693 polymorphism is associated with risk of muscle injury 29) . It is known that muscle stiffness is a risk factor for muscle injury, and is lower in females than in males 30) . This implies that sex-related genetic polymorphisms influence muscle injuries that are associated with muscle stiffness. We therefore focused on two functional polymorphisms in the ESR1 gene, namely, rs2234693 C/T and rs9340799 G/A, and examined the association between these polymorphisms and a history of muscular injury and muscle stiffness. The results demonstrated that the rs2234693 C allele of ESR1 is associated with low incidences of muscular injury and reduced muscle stiffness, compared to those associated with the T allele, even among athletes from different disciplines 29) . These results suggested that the rs2234693 C allele of ESR1 protects against muscle injury by lowering muscular stiffness. Muscle stiffness is influenced by intramuscular collagenous connective tissues 31) , and it has been reported that estrogen suppresses collagen synthesis 32) . It is therefore possible that the rs2234693 polymorphism of ESR1 is associated with muscular stiffness and muscle injury via alteration of the properties of collagenous tissues.
Recently, several GWAS for injuries (e.g., ankle injury 33) , Achilles tendon injury, anterior cruciate ligament rupture 34) , medial collateral ligament injury 35) , and rotator cuff injury 36) ) have been published. Although these injuries are related to sports, these GWAS were conducted in the general population. On the other hand, athletes have an increased risk for various injuries because of extremely difficult training and dynamic movement in sports. It is therefore possible that GWAS in athletes reveal genetic effects on injuries more clearly. We are currently conducting GWAS for sports-related injuries in Japanese athletes from a J-HAP cohort. If these studies could identify the genetic variants associated with sports-related injuries, we will be able to identify the risk of sports-related injuries in advance, and conduct a focused injury prevention program. Additionally, understanding the mechanisms underlying the susceptibility to sports-related injuries will contribute to the development of prevention, treatment, and management programs for these injuries.

Future Directions
The current use and opinions of elite athletes and support staff in relation to genetic testing in elite sport within the UK have been investigated 37) . The survey revealed that ≤17% athletes and ≤8% support staff had experiences of genetic testing related to sports performance and injury susceptibility. Although the majority of athletes and staff had never engaged in genetic testing for sports performance and injury susceptibility, most of the athletes (> 80%) were willing to engage in genetic testing for individualized training to improve sport performance and reduce injury risk. On the other hand, > 47% of athletes and > 64% of staff answered that genetic information should not be considered to determine selection/eligibility/employment in sports. Future studies in sports genetics/genomics field should consider these opinions from athletes and their staff.
Genetic testing raises a number of important issues on ethics. The Australian Institute of Sport has developed a position statement on the ethics of genetic testing and research in sport 26) . In their statement, it is mentioned that"Direct-toconsumer (DTC) genetic testing in relation to sports performance is strongly discouraged" . Many private companies are now offering DTC genetic tests in sports. DTC companies commonly offer advice on the predisposition to athletic success in power/endurance sports, despite the lack of evidence to support such advice 38) . Japanese DTC companies often offer advice on the suitability of sports, requiring either endurance or power, based on genotypes of a few polymorphisms (usually, ACTN3 R577X and ACE I/D polymorphisms). Nevertheless, given that different alleles of the ACE I/D polymorphism have been associated with elite athlete status in Caucasians and East Asians 16) as mentioned above, we need to verify whether the test refers to evidence in the Asian population.
Further robust and credible evidence is needed to carry out genetic testing for individualized training to improve athlete performance and reduce injury risk. For application on Japanese athletes, we need to develop a strong scientific foundation on this issue in the Asian population through largescale collaborative projects.