Association of Myostatin Gene Polymorphisms with Strength and Muscle Mass in Athletes: A Systematic Review and Meta-Analysis of the MSTN rs1805086 Mutation

Polymorphism (rs1805086), c.458A>G, p.Lys(K)153Arg(R), (K153R) of the myostatin gene (MSTN) has been associated with a skeletal muscle phenotype (hypertrophic response in muscles due to strength training). However, there are not enough reliable data to demonstrate whether MSTN rs1805086 K and R allelic variants are valid genetic factors that can affect the strength phenotype of athletes’ skeletal muscles. The aim is to conduct a systematic review and meta-analysis of the association of MSTN rs1805086 polymorphism with the strength phenotype of athletes. This study analyzed 71 research articles on MSTN and performed a meta-analysis of MSTN K153R rs1805086 polymorphism in strength-oriented athletes and a control (non-athletes) group. It was found that athletes in the strength-oriented athlete group had a higher frequency of the R minor variant than that in the control group (OR = 2.02, P = 0.05). Thus, the obtained results convincingly demonstrate that there is an association between the studied polymorphism and strength phenotype of athletes; therefore, further studies on this association are scientifically warranted.


History of Discovery
In addition to sports training, environmental exposure, nutrition, and professional activity of a person, genetic factors also have a great influence on the strength indicators of an athlete's skeletal muscles [1]. The study of genetic foundations, including gene polymorphisms and their connections with a body's resistance to physical load as a whole, and their contribution to an athlete's strength abilities and development should reasonably be considered as one of the most important and significant areas of modern sports science [2].
Myostatin (MSTN) protein was discovered in 1997 and was encoded by the MSTN gene, located on chromosome 2 2q32.2; it encodes 375 amino acids in three exons and occupies a site of approximately 8 kb [3]. This discovery was considered a significant success in the study of genetic factors for increasing muscle mass and developing strength abilities.
This gene was named MSTN because of its ability to inhibit muscle differentiation and growth [4], whereas the overexpression of MSTN is associated with muscle atrophy [5]. However, these studies have confirmed the central and critical role of MSTN in suppressing muscle growth [6,7].
Special attention is paid to MSTN because the very first publications on this factor concluded its absence to affect an increase in muscle mass due to hypertrophy and hyperplasia of the muscle fibers [8]. The increase in the detailed scientific studies of MSTN and Special attention is paid to MSTN because the very first publications on this factor concluded its absence to affect an increase in muscle mass due to hypertrophy and hyperplasia of the muscle fibers [8]. The increase in the detailed scientific studies of MSTN and the possibilities of using the published data for various biomedical and sports purposes, including gene doping [9], has increased interest in the subject.
The ability of MSTN to limit the growth of muscle mass immediately attracted the attention of researchers, as it can be used in sports and sports medicine.
MSTN, also known as the growth differentiation factor-8 (GDF-8), is a protein-based hormone that acts as a negative regulator of muscle growth. This was first mentioned by McPherron et al. [10]. The authors found that a mutation in MSTN leads to an increase in the size of muscle tissue. In the initial stages, these were primarily conducted on animals followed by those on humans. MSTN is particularly of interest in sports, wherein one can monitor its correlation with performance, especially in sports that require muscle strength and mass [10].
Mutations in MSTN lead to a significant increase in muscle mass [11]. It is an important gene that affects myogenesis as its role is to regulate the growth and differentiation of muscle cells [12]. In particular, the genetic predisposition to gain muscle mass is due to the low expression of MSTN, which is advantageous in the improvement of strength [13].
As MSTN is the most common type of skeletal muscle protein, it is of interest in studies related to sports science [14]. However, its expression is also noted in the heart and adipose tissues [15,16].
The growing interest in MSTN has led to a large number of scientific publications that can be found in the Web of Science and PubMed database ( Figure 1). A review of the publications confirms that MSTN is an endogenous negative regulator of skeletal muscle mass, which acts as an antianabolic agent that suppresses the activation, replication, DNA, and protein synthesis of muscle satellite cells affecting myogenic differentiation [17]. Researchers from Taiwan found that MSTN was negatively correlated with age and the percentage of fat mass in healthy young men [18]. The results of the experiments proved that the reference value of MSTN concentration in blood serum in healthy young men is 12.3 ± 3.6 ng/mL and that it negatively correlates with age [19].
Considerable attention should be paid to the factors contributing to the inhibition of MSTN expression. According to scientific sources, such factors include hypodynamia, various origin diseases, state of weightlessness, and aging [9,20]. The level of MSTN in skeletal muscles is also influenced by power-oriented physical exercise [21]. Researchers from Taiwan found that MSTN was negatively correlated with age and the percentage of fat mass in healthy young men [18]. The results of the experiments proved that the reference value of MSTN concentration in blood serum in healthy young men is 12.3 ± 3.6 ng/mL and that it negatively correlates with age [19].
Considerable attention should be paid to the factors contributing to the inhibition of MSTN expression. According to scientific sources, such factors include hypodynamia, various origin diseases, state of weightlessness, and aging [9,20]. The level of MSTN in skeletal muscles is also influenced by power-oriented physical exercise [21].
One of the main factors in sports that significantly affects the level of MSTN secreted is power-oriented physical activity, hypoxia, and dietary supplements. Moreover, the production of MSTN is influenced by essential amino acids, which are often consumed by athletes after intensive training [24].
Recent studies have shown that essential amino acids suppress MSTN expression in human skeletal muscles [30,31].
MSTN is a potential genetic marker of the athletic abilities in strength sports because of the involvement of a large number of skeletal muscles and the functions of myokines. Some research related to the study of MSTN and its role in hypertrophy and skeletal muscle strength seemed contradictory [20,32,33]. Therefore, in this study, attention was given to the influence of the K153R rs1805086 polymorphism on the manifestation of skeletal muscle strength in athletes.

Mechanism of Effect of MSTN on Skeletal Muscle Mass and Strength
Physical activity causes muscle hypertrophy and performing physical power-oriented exercises clearly demonstrated this. This type of exercise causes mechanical damage to sarcomeres and sarcolemmas. After a certain period of time, the balance shifts toward protein synthesis and, as a result, phenotypic changes increase the volume and strength of skeletal muscles. These processes release active MSTN, which affects satellite cells and fibroblasts located near the damaged area. MSTN can cause protein degradation in myofibrils, which are important for the normal functioning of muscle fibers as they remove unnecessary, wasted proteins from the muscle cells [34].
MSTN is one of the main factors associated with muscle atrophy. In studies involving humans, it was found that by the 25th day of the sedentary regime, the level of MSTN increased by 12% [9]. MSTN can regulate the function of muscle fibers and nearby cells, which include fibroblasts and satellite cells or satellites. Mature muscle fibers are the products of final differentiation [28].
An increase in muscle size is achieved by the fusion of satellite-proliferating cells with fibers. Primarily, microtrauma in a single muscle fiber acts as a stimulus for the proliferation of satellite cells in adult organisms. When these cells are activated and emerge from a dormant state, genes characteristic of myoblasts are also activated. Therefore, satellite cells become myoblasts that migrate to the damaged areas of muscle tissue and depending on the degree of damage, either merge with the damaged muscle fiber (hypertrophy) or merge with each other, thus creating new fibers (hyperplasia). Therefore, satellite cells regulate the functional state of skeletal muscles in the adult body ( Figure 2). They are necessary for the restoration of damaged muscle fibers and are a source of additional nuclei in case of muscle hypertrophy after training sessions. MSTN negatively affects the proliferation of satellite cells [35]. Power-oriented training sessions result in mechanical stretching of the muscle and lead to microdamage. There is also evidence that MSTN negatively regulates the activation of resting satellite cells, hindering their development. Such inhibitory effects are necessary for normal muscle regeneration as a premature fusion of satellite cells with myofibrils can impair muscle fiber functions.  In general, the mechanism by which MSTN controls the number of muscle fibers is not well studied. It is synthesized as an inactive protein and undergoes changes to turn into a mature active form in two stages [36]. It enters the bloodstream as a latent precursor protein and undergoes a proteolytic process, turning into a mature peptide that binds to the extracellular type II receptor (ActRIIB) activin. The binding of MSTN to ActRIIB induces the intracellular activation of proteins, by which MSTN modulates the proliferation and differentiation of myoblasts, and ultimately, the muscle mass [30,37,38].

Effect of MSTN on Tendons and Bones
Tendons are an important component in the manifestation of the maximum strength of the skeletal muscle. Weightlifting and speed-strength sports athletes with high indicators of skeletal muscle strength often have tendon injuries as their muscle strength exceeds their endurance. During strength training, fibroblasts proliferate, collagen synthesis increases and the cross-sectional area of the tendons increases to make them stiffer. This allows the tendons to withstand high-intensity physical loads and reduce the risk of damage to them [39].
MSTN can change the mechanical properties of tendons by impairing their ability to stretch, increasing the risk of damage. Such data cast doubt on the feasibility of inhibiting MSTN expression for sports purposes [35]. The exact mechanisms of the effect of MSTN on tendons and ligaments are still unknown, and further studies are needed to assess its regulatory role in these processes [40]. When studying the regeneration of muscles and tendon fibroblasts, it is assumed that MSTN affects the expression of type 1 collagen. Recent studies have reported that local injections of exogenous MSTN during tendon healing increase the cross-sectional area of the tendon [41].
In both human and animal studies, there is evidence that MSTN is an important regulator of muscle mass as well as bone density. The mechanisms by which MSTN regulates bone formation are not completely understood, but it is clear that it has a direct effect on the proliferation and differentiation of stem cells [42,43]. Since MSTN and its receptor are expressed during bone regeneration, it affects bone density [43]. It is likely that MSTN directly affects bones, increasing bone mineral density. Some features of different phenotypes may be associated with increased biomechanical load, e.g., in weightlifters or under the influence of other factors, such as mechanical growth factors or growth hormones. These issues have yet to be studied in more detail, but if a number of studies prove that MSTN does have an effect on bones, then it can be assumed that MSTN inhibitors will be useful not only for increasing muscle mass but also for bone density. This assumption is supported by recent data showing that MSTN significantly increases bone volume during fibular healing [44]. In general, the mechanism by which MSTN controls the number of muscle fibers is not well studied. It is synthesized as an inactive protein and undergoes changes to turn into a mature active form in two stages [36]. It enters the bloodstream as a latent precursor protein and undergoes a proteolytic process, turning into a mature peptide that binds to the extracellular type II receptor (ActRIIB) activin. The binding of MSTN to ActRIIB induces the intracellular activation of proteins, by which MSTN modulates the proliferation and differentiation of myoblasts, and ultimately, the muscle mass [30,37,38].

Effect of MSTN on Tendons and Bones
Tendons are an important component in the manifestation of the maximum strength of the skeletal muscle. Weightlifting and speed-strength sports athletes with high indicators of skeletal muscle strength often have tendon injuries as their muscle strength exceeds their endurance. During strength training, fibroblasts proliferate, collagen synthesis increases and the cross-sectional area of the tendons increases to make them stiffer. This allows the tendons to withstand high-intensity physical loads and reduce the risk of damage to them [39].
MSTN can change the mechanical properties of tendons by impairing their ability to stretch, increasing the risk of damage. Such data cast doubt on the feasibility of inhibiting MSTN expression for sports purposes [35]. The exact mechanisms of the effect of MSTN on tendons and ligaments are still unknown, and further studies are needed to assess its regulatory role in these processes [40]. When studying the regeneration of muscles and tendon fibroblasts, it is assumed that MSTN affects the expression of type 1 collagen. Recent studies have reported that local injections of exogenous MSTN during tendon healing increase the cross-sectional area of the tendon [41].
In both human and animal studies, there is evidence that MSTN is an important regulator of muscle mass as well as bone density. The mechanisms by which MSTN regulates bone formation are not completely understood, but it is clear that it has a direct effect on the proliferation and differentiation of stem cells [42,43]. Since MSTN and its receptor are expressed during bone regeneration, it affects bone density [43]. It is likely that MSTN directly affects bones, increasing bone mineral density. Some features of different phenotypes may be associated with increased biomechanical load, e.g., in weightlifters or under the influence of other factors, such as mechanical growth factors or growth hormones. These issues have yet to be studied in more detail, but if a number of studies prove that MSTN does have an effect on bones, then it can be assumed that MSTN inhibitors will be useful not only for increasing muscle mass but also for bone density. This assumption is supported by recent data showing that MSTN significantly increases bone volume during fibular healing [44].

Myostatin Mutations
In previous studies, it was found that a number of missense substitutions in exons 1 and 2 of MSTN are of great interest to researchers to confirm MSTN connection with athletes' strength abilities, muscle hypertrophy [45], and recovery after intensive strength exercises. The polymorphisms K153R, A55T, E164K, P198A, I225T, and c.373 + 5 [20,[46][47][48] are of particular interest to the gene as well.

MSTN Mutation (rs397515373, c.373 + 5 G>A)
This mutation is very rare, with an average prevalence of 0.0004% in the population. It was necessary to obtain 500,000 samples to detect a mutation once. In 2004, a paper describing a case of MSTN mutation in a child was published. In both the allelic copies of MSTN, the newborn boy had mutations that suppressed the synthesis of the functioning MSTN protein. The child was observed to have enlarged muscles of the thighs and upper extremities at birth. Ultrasonography of this child showed that the cross-section of the quadriceps femoris muscle was 7.2 SD, which was higher than the average (± standard deviation) value for 10 persons matched for age and gender. Moreover, the thickness of his subcutaneous fat was 2.88 SD below the average value of that of his peers. All reflexes of the child were normal, except for those associated with tendons. Interestingly, this mutation was also present in other members of this family. One of the relatives was extraordinarily strong, and the 24-year-old mother of the child was a professional athlete and had developed muscles, although to a lesser extent than her son. This study showed for the first time that the MSTN rs397515373 mutation (c.373 + 5 G>A) leads to an increase in muscle mass and strength [49].
A55T is important for the stability of the inhibitory activity of MSTN and affects the mature MSTN [50].
A study devoted to physical exercise reported that after 8 weeks of exercise with weights the subjects with A55T polymorphism AT and TT genotypes had greater muscle hypertrophy than those with AA genotype [51]. Studies have shown that MSTN polymorphisms can affect the skeletal muscle phenotype after exercise with weights. However, previous studies of MSTN SNPs associated with muscle hypertrophy in response to prolonged power-oriented strength exercises have not confirmed pronounced muscle hypertrophy after strength physical load [52].
Studies on an Asian sample set (n = 500) showed that the A55T polymorphism can affect the activity of MSTN, mass of skeletal muscles, and the amount of fat in the body. The results have shown that the A55T polymorphism determines the genetic predisposition to the development of excessive obesity and low muscle mass in Asians [53].
Chinese scientists found that people with the MSTN A55T AT + TT genotype showed a significant increase in the thickness of biceps (0.292 ± 0.210 cm, P = 0.03) but not of quadriceps (0.254 ± 0.198 cm, P = 0.07) compared to those of AA genotype carriers. Thus, the obtained results suggest a possible association between A55T polymorphism and muscle hypertrophy caused by strength training in Chinese individuals [51].
Korean researchers have found that the A55T polymorphism is associated with skeletal muscle recovery after strength training. The study sample included 48 young, healthy college students (age 24.8 ± 2.2 years, height 176.7 ± 5.3 cm, weight 73.7 ± 8.3 kg) who performed 50 repetitions in strength exercises. After strength exercises subjects with heterozygous AT showed significantly faster muscle recovery than those in the AA group (P = 0.042). These results prove that the A55T polymorphism AT genotype is associated with a faster recovery of skeletal muscle strength after intense strength exercise [54].
Turkish scientists failed to identify the relationship between the A55T polymorphism and the morphological data of arm wrestlers [51,54,55]. Moreover, no statistically significant relationships have been found in highly qualified athletes in endurance sports [56,57].
In a number of studies related to the influence of this genotype on the phenotype of athletes and people not engaged in sports, the results of experiments showed no statistically Genes 2022, 13, 2055 6 of 16 significant differences [58]. According to the website http://www.ensembl.org (accessed on 15 October 2022), the average frequency of a rare allele was 1%.
There are only indirect assumptions that this mutation can affect the manifestation of muscle mass and strength in humans. These assumptions are based on the fact that this polymorphism can make a significant contribution to the biochemical variability of mature MSTN, and accordingly, affect the state of the vertebrate muscular system. However, this assumption requires further study [9,47].
The MSTN rs1805086 RR genotype gene is more common in top-class weightlifting athletes [59]. Some researchers found a positive association between the K153R rs1805086 polymorphisms and the manifestation of strength abilities and muscle hypertrophy [13,46,51,60], whereas other researchers did not find any significant connection [33,46,61]. Some studies have proven a connection with high performance in high jumps (P < 0.05) [46]. Studies on the relationship between K153R and skeletal muscle phenotypes in elderly Caucasian women have shown that the heterozygote MSTN rs1805086 KR is a favorable polymorphism for the increased muscle mass in the biceps of the shoulder [62].
In the studies conducted with 16 women and 34 men of Caucasian, African-American, and Afro-European ethnicities that participated in the European Championships and the Olympic games in sports, such as football (n = 4), basketball (n = 10), tennis (n = 6), volleyball (n = 6), canoeing (n = 2), rugby (n = 10), baseball (n = 6), and track-and-fields (sprint, javelin, and shot put) (n = 6), who were compared with a control group of 100 people, including 40 women and 60 men not involved in sports, the authors failed to find statistically significant differences between the elite athletes and people in the control group in terms of the K153R polymorphism occurrence frequency and the success in competitions [60].
Studies on the relationship between MSTN and muscle pathologies in healthy elderly people are contradictory [63]. The association between low MSTN levels and low skeletal muscle mass was observed only in men but not in women. The authors point to the need for further research on MSTN as a biomarker of muscle mass and strength [20].

MSTN K153R (rs1805086) Polymorphism Frequency
According to the Ensembl database, the frequency of the rare variant K153R is on an average 7% (3% in Caucasians and 22% in Africans), and large sample sizes are necessary to reliably identify the association of this polymorphism with strength abilities and muscle hypertrophy ( Figure 3).  The problem with studying mutations in MSTN is the low frequency of some alleles. Obtaining the required number of subjects and statistically significant data would need very specific subjects, for example highly qualified athletes of weightlifting sports or people with an exceptional proportion of skeletal muscles [56]. Such subjects, for instance, can also include some ethnicities, considering their residence [33]. The conducted studies could not always prove a connection between the athletes' skeletal muscle strength, muscle mass, and competitive performance [32,61]. Due to the low frequency of K153R polymorphism in Caucasian athletes of cyclic sports, the authors The problem with studying mutations in MSTN is the low frequency of some alleles. Obtaining the required number of subjects and statistically significant data would need very specific subjects, for example highly qualified athletes of weightlifting sports or people with an exceptional proportion of skeletal muscles [56]. Such subjects, for instance, can also include some ethnicities, considering their residence [33].

Materials and Methods
The purpose of this study was to summarize the relationship between K153R polymorphism and athletes' strength indicators by conducting a systematic review and metaanalysis, which can potentially reveal more statistically reliable data compared to individual studies.

Search for Publications
The search for scientific publications was carried out using PubMed, Web of Science, eLIBRARY.ru, SNPedia, Wiley Online Library, and Europe PMC resource databases. For this, the following keywords were used: myostatin, MSTN, GDF-8, K153R, and rs1805086. A list of publications on MSTN was compiled to include 71 scientific articles published prior to April 2021 from the above databases. For the analysis and systematization of publications, the EndNote Viever X9.2 bibliographic managers of Clarivate Analytics and the Zotero application were used. In addition to analyzing the content of the publications, their references were also studied. After selecting all potentially relevant articles, all the information on the MSTN K153R rs1805086 polymorphism was carefully studied in the control and experimental groups.

Inclusion and Exclusion Criteria
Out of 71 scientific articles in PubMed, Web of Science, eLibrary.ru, SNPedia, Ensembl, Wiley Online Library, and Europe PMC databases, the publications directly related to the study of the K153R (rs1805086) polymorphism were selected. The studies used were marked with the following statement: "The study was approved by the ethics committee." To be included in this review, the studies had to meet the following criteria: 1.
Be published from 1997 to April 2021, 2.
Sample size should not be less than 10 people, and a control group in the study is mandatory, 3.
Full text of the study should be available, 4.
Participants must be adults who are not elderly, 5.
Subjects had to be healthy at the time of the study, 6.
No studies should be conducted on animals.
Of the 71 scientific articles, 61 were excluded after the first stage of working with databases. The criteria for rejecting the work included discrepancy between the title and subject of the study, experiments on animals, and experiments with small samples. The main criterion for dropping out of publications in the first stage was that the studies were not related to the MSTN K153R polymorphism.
After analyzing ten full-text publications, six were excluded after the second stage. These articles were devoted either to the expression of MSTN or they had an inadequate methodological quality of experiments. As a result, four publications were included in the meta-analysis. A flowchart showing the algorithm for selecting publications for the meta-analysis is shown in Figure 4. main criterion for dropping out of publications in the first stage was that the studies were not related to the MSTN K153R polymorphism.
After analyzing ten full-text publications, six were excluded after the second stage. These articles were devoted either to the expression of MSTN or they had an inadequate methodological quality of experiments. As a result, four publications were included in the meta-analysis. A flowchart showing the algorithm for selecting publications for the metaanalysis is shown in Figure 4.

Data Extraction
To include the publication in the meta-analysis, the full text of each study was analyzed for the general content and its compliance with the aforementioned acceptance criteria. From each eligible article, the following data were obtained: authors, publication year, study organization, study population (number of subjects, ethnicity, gender), polymorphism number and name, and muscle phenotype.
The phenotypic data included in this analysis were skeletal muscle mass and muscle strength.

Statistical Analysis
The Review Manager 5.4.1 (RevMan) computer program was used to perform the meta-analysis proposed by the Cochrane Community in 2014. The meta-analysis used data on the number of genotypes in the control and experimental groups to check for "publication error" (a systematic error associated with the predominant publication of positive results), and the asymmetry of the funnel graph [64]. The relationship between the K153R polymorphism and phenotypic data and the strength abilities of the subjects were evaluated using the odds ratio (OR) criterion and 95% confidence interval (CI) by comparing the control and experimental groups. The heterogeneity of the obtained data was estimated with the heterogeneity index I2 [65]. In the test for the overall effect, which was set according to the Z-criterion, the two-sided value of P was considered significant.
The statistical significance of each study included in the meta-analysis was also analyzed to compare the significance of this study with the generalized indicators. The degree of statistical significance of each study was assessed by χ 2 , in which P values < 0.05, were considered statistically significant. Statistical analysis according to the χ 2 -criterion was carried out using SPSS 23.0.
Based on the fact that in some publications, the results of the genotyping of subjects were expressed in nucleotides, whereas in others as amino acids, the following designations were used: the amino acid "Lys" was designated by the letter "K", and "Arg" by the letter "R". Thus, the mutant variant was designated by the letter "R".
Problems with Sample Collection Because the occurrence of the MSTN K153R rs1805086 polymorphisms in populations is 7% on average, it creates problems in identifying people with the rare genotype [32]. Two of the factors that significantly affect the association of MSTN genotypes with muscle mass and skeletal muscle strength are sex and age. Experiments to identify the effect of MSTN on muscle mass and strength showed different results depending on these two factors [66,67].
Contradictory data were also obtained in cases where the subjects were representative of different sports [56]. The impact of MSTN on skeletal muscle strength depends on the sport. In the type of sport requiring the ability to maintain a given physical load for a long time, no statistically significant data were found on the association of MSTN polymorphisms with muscle mass and strength [61].
Some studies have reported that estrogen affects the change in the expression of MSTN caused by power-oriented physical exercises [66]. In addition, differences in ethnicity, sample size, body weight, and level of physical activity may be potential reasons for the different results in studies related to MSTN [68]. The authors pointed out that nutritional factors should be considered when assessing the level of MSTN. Sex is also an important factor in the reduction of muscle strength and age-related decrease in muscle mass. Men usually begin to lose muscle mass after 40 years of age, when the level of testosterone in serum drops. Women can gradually lose 10-15% of their muscle mass from age 25 until the onset of menopause, after which it increases at a rate of 2% annually. Therefore, the amount of muscle mass is also affected by sex and diet.
In addition, studies often consider a specific human muscle as an object, such as the biceps or quadriceps, and this is also a limiting factor for the full assessment of the relationship between MSTN and the muscle mass and strength of the entire body as a whole.
The absence of a control group in some studies does not allow the solution to the problem of the statistical validity of the obtained data [55]. To obtain the most reliable data, larger sample sizes are required.
Finally, after testing for strength exercises, researchers considered only some indicators of muscle fatigue; therefore, they were limited to confirming the connection mechanism between specific strength indicators, MSTN genotypes, and muscle strength. It should be noted that most scientific publications are primarily based on previous research [68][69][70].
There are studies in which the authors concluded that the MSTN K153R polymorphism does not affect muscle phenotypes in women, wherein their sample of subjects was 33 people aged between 90-97-years-old. Considering that this polymorphism is very rare, the results of such studies seem very doubtful [20].
Thus, in this manuscript, the systematic review of publications related to the influence of the MSTN K153R polymorphism helped conclude that the data obtained can be regarded as contradictory. With such a discrepancy, the question arises whether the allelic variants K and R of the MSTN rs1805086 gene are genetic factors that can affect human strength abilities and skeletal muscle hypertrophy.
Meta-analysis overcomes the limitation of a small sample size by combining the results of a number of individual studies to obtain a single best estimate.

Results
In this study, it was found that athletes with the R MSTN variant had significantly greater muscle strength and mass due to the power-oriented physical training compared with that of the carriers of the K MSTN variant. This indicates that the presence of the R variant in MSTN rs1805086 can be considered a genetic marker associated with increased skeletal muscle strength and muscle mass (OR = 2.02, P = 0.05).

Case-Control Study
In the control group of African American athletes, the frequency of the RR rs1805086 genotype was higher in 2-3% of cases than in the control groups in Russian and Caucasus natives. The KR heterozygote was also more common in the African-American control group than in the Russian control group (Table 1). In addition, the rs1805086 KR/RR genotypes were also significantly more common in the African-American athletes (35% and 14% vs. 13.0% and 7.1%, respectively) ( Table 2).  Checking the statistical significance of the obtained data by conducting a Chi-square test (χ 2 ) for the analysis of each sample separately did not show any statistically significant results, with the exception of the Russian subjects of 2017 [71]; however, the generalization of the data yielded statistically significant results (P = 0.030) for the analysis of genotypes KK, KR, and RR, and P = 0.0011 for the genotypes KR and KK/RR.
The frequency of rs1805086 KR/RR genotypes was significantly higher in the group of athletes than that of those in the control group (Table 2). African-American athletes were an exception. This may be due to the fact that, according to the Ensembl resource, the frequency of K153R polymorphism worldwide is significantly higher among African-Americans (average of 22%) than in other populations (Figure 3).
In general, five ethnic sub-groups were used for the meta-analysis, in which 416 athletes and 357 subjects from the control group participated. The frequencies of the KR/RR genotypes compared to that of the KK genotype were significantly higher in the group of athletes (12.5%) compared to the control group (95% CI, P = 0.011). The results of the meta-analysis were as follows: the random effects model: OR = 2.02, P = 0.05, Z = 1.94 ( Figure 5) and the fixed-effects model: OR = 2.15, 95% CI, P = 0.05; (Figure 6). The coefficient of heterochrony between the studies was I2 = 33% (P = 0.20). These results show that the mutation of the R variant (i.e., the KR/RR genotypes) has a statistically significant relationship with the phenotype of athletes with respect to the development of skeletal muscle strength abilities and muscle mass. The odds ratio in the meta-analysis was assessed by the Mantel-Haenszel (M-H) test. The funnel plot for the M-H criterion is shown in Figures 5 and 6.
Checking the statistical significance of the obtained data by conducting a Chi-square test (χ2) for the analysis of each sample separately did not show any statistically significant results, with the exception of the Russian subjects of 2017 [71]; however, the generalization of the data yielded statistically significant results (P = 0.030) for the analysis of genotypes KK, KR, and RR, and P = 0.0011 for the genotypes KR and KK/RR.
The frequency of rs1805086 KR/RR genotypes was significantly higher in the group of athletes than that of those in the control group (Table 2). African-American athletes were an exception. This may be due to the fact that, according to the Ensembl resource, the frequency of K153R polymorphism worldwide is significantly higher among African-Americans (average of 22%) than in other populations ( Figure 3).
In general, five ethnic sub-groups were used for the meta-analysis, in which 416 athletes and 357 subjects from the control group participated. The frequencies of the KR/RR genotypes compared to that of the KK genotype were significantly higher in the group of athletes (12.5%) compared to the control group (95% CI, P = 0.011). The results of the meta-analysis were as follows: the random effects model: OR = 2.02, P = 0.05, Z = 1.94 ( Figure 5) and the fixed-effects model: OR = 2.15, 95% CI, P = 0.05; (Figure 6). The coefficient of heterochrony between the studies was I2 = 33% (P = 0.20). These results show that the mutation of the R variant (i.e., the KR/RR genotypes) has a statistically significant relationship with the phenotype of athletes with respect to the development of skeletal muscle strength abilities and muscle mass. The odds ratio in the meta-analysis was assessed by the Mantel-Haenszel (M-H) test. The funnel plot for the M-H criterion is shown in Figures 5 and 6.  The hypothesis that the effectiveness of strength training is significantly higher in athletes with the R MSTN rs1805086 variant was confirmed by a meta-analysis ( Figures 5  and 6).

Discussion
As mentioned earlier, the frequency of mutant homozygotes (RR) is below 1% among the general population, which limits the possibility of studying large groups of people with variant R. On the other hand, according to Ensembl base, the frequency of the mutant variant R is approximately 3-4% on average among the general population across the globe. Among the athletes of power and weightlifting sports, the frequency of the minor variant R and the homozygote RR was significantly higher, reaching 10% [73].
The K153R polymorphism is significant in the development of muscle mass and strength. Previously, a number of studies have also shown that the rare R variant increases the inhibition of MSTN synthesis, thereby leading to an increase in skeletal muscle mass and muscle strength [37].
When searching for publications in several databases, four suitable studies were found in which athletes were compared with subjects in a control group. The feature of comparison was the MSTN K153R polymorphism. After combining the data from the The hypothesis that the effectiveness of strength training is significantly higher in athletes with the R MSTN rs1805086 variant was confirmed by a meta-analysis ( Figures 5 and 6).

Discussion
As mentioned earlier, the frequency of mutant homozygotes (RR) is below 1% among the general population, which limits the possibility of studying large groups of people with variant R. On the other hand, according to Ensembl base, the frequency of the mutant variant R is approximately 3-4% on average among the general population across the globe. Among the athletes of power and weightlifting sports, the frequency of the minor variant R and the homozygote RR was significantly higher, reaching 10% [73].
The K153R polymorphism is significant in the development of muscle mass and strength. Previously, a number of studies have also shown that the rare R variant increases the inhibition of MSTN synthesis, thereby leading to an increase in skeletal muscle mass and muscle strength [37].
When searching for publications in several databases, four suitable studies were found in which athletes were compared with subjects in a control group. The feature of comparison was the MSTN K153R polymorphism. After combining the data from the selected studies, the group of athletes with the rare R variant comprised 52 subjects, and the control group comprised 25 subjects (12.5% vs. 7.9%, respectively). The generalized data showed statistically significant differences (P = 0.011) according to the χ 2 -criterion. The rare occurrence of this allele did not allow the obtaining statistically significant differences separately, with the exception of a sample of Eastern Russians [71,72]. Combining the samples into a single general population made it possible to increase the level of statistical significance of the analyzed data.
The data reported were not included in the meta-analysis, in which the author studied 79 Turkish athletes (arm wrestlers) aged 24 years in comparison with a control group consisting of 34 people. Associative studies were conducted on two polymorphisms of MSTN, A55T, and K153R. The authors failed to find statistically significant relationships between the studied polymorphisms and anthropometric indicators. Perhaps the reason for such data was the absence of genotypes with the R variant among the group of athletes [55].
In another study, the authors found that the currently published data on the MSTN K153R polymorphism and the human muscle phenotypes show contradictory results [66]. A number of studies have reported a significant effect of MSTN variants, and the reaction of muscle mass in response to strength training regardless of sex, which confirms the hypertrophic response to strength training in adults of both sexes. The K153R polymorphism is associated with a greater muscle hypertrophic response to exercise [74].
A study of the relationship between the MSTN K153R polymorphism and "explosive" leg strength in untrained men proved that the MSTN K153R polymorphism is associated with the ability to generate "peak" power during muscle contractions, evaluated using the vertical jump test [46]. The authors indicated that the polymorphisms Lys (K) and 153 Arg (R) located in exon 2 (replacement rs1805086, 2379 A>G) affect the phenotype of skeletal muscles. The replacement of the amino acid Lys (K) with 153 Arg (R) was found in the active mature peptide of the MSTN protein, and this replacement can affect proteolytic processing due to its propeptide or the ability to bind to ActRIIB, which in turn, induces myoblast proliferation and muscle mass differentiation.
A study conducted in China on 94 healthy untrained men of the age group 8-22 years, convincingly demonstrated that the increase in the thickness of biceps (X = 0.300 ± 0.131 cm) and quadriceps (X = 0.421 ± 0.281 cm) (P < 0.01 for both muscle groups) was significantly higher among individuals with the KR genotype than among those with the KK genotypes of the MSTN K153R polymorphism. Thus, the obtained results proved that this polymorphism leads to larger skeletal muscle size in the absence of training and is also associated with a more noticeable increase in muscle mass after strength training in subjects with the R variant [51]. This is the first study to demonstrate the results of a meta-analysis of the MSTN K153R polymorphisms with the phenotype and functions of skeletal muscles in athletes. In particular, it was found that the frequency of genotypes that contribute to an increase in muscle volume and skeletal muscle strength in athletes (KR and RR genotypes) was significantly higher in the experimental group than in the control group. It was also confirmed that the K153R mutation is more common in the African-American group than that in other groups. In addition, a meta-analysis of five groups (two African-Americans, one Caucasus native, and two Russian), including a total of 773 test athletes and 357 general people in the control group, showed a significantly higher prevalence of KR/RR genotypes in the athletes than that in the control group.
The R variant is favorable for sports in which muscle strength and mass are important, such as bodybuilding, powerlifting, weightlifting, arm wrestling, kettlebell lifting, shot put, and bobsleigh. It can be assumed that the strong effect of this allele on the ability to become a successful athlete in weightlifting and speed-power sports is based on the inhibition of MSTN synthesis, as reported in some previous studies. In their respective studies, researchers have established a trend of MSTN K153R polymorphisms to influence the skeletal muscles' hypertrophic response to strength training in women with the heterozygotic genotype [74]. The experiments have shown an increase in leg muscle mass in subjects with the KR genotype in response to strength training; this was 68% higher than in women with the KK genotype (P = 0.056) [74]. These data also indicate a significant role of rare variant R in MSTN in the hypertrophic response of the muscles of the subjects. The authors note that the MSTN K153R polymorphism has not been sufficiently studied and needs further research, particularly in women with high body weight. In addition, of interest is the reaction of the muscular system in response to power-oriented physical load, taking into account the MSTN genotypes.
It should also be noted that in almost all of the publications found, the authors indicated that the data obtained in the conducted studies may be limited by the levels of statistical significance of the statistical processing methods used. This is due to the low frequency of the R MSTN variant. Therefore, to solve this problem, further studies with larger sample sizes are needed. In addition, as in most studies related to the K153R MSTN polymorphism in athletes, it seems necessary to conduct experiments aimed at identifying associations between other MSTN polymorphisms and the expression of the MSTN protein in order to obtain additional information about the mechanisms by which MSTN polymorphisms affect the effectiveness of training process for increasing muscle mass and developing athletes' strength abilities.

Conclusions
Meta-analysis of data on the MSTN K153R (rs1805086) polymorphisms convincingly demonstrated that the KR and RR genotypes are statistically associated with the strength abilities of athletes and their muscle mass when performing power-oriented training. Combining efforts in a search for subjects with the rare R MSTN variant will allow us to obtain more significant information about the magnitude of the effect of this polymorphism during strength training. Other polymorphisms of MSTN and its molecular mechanisms should also be studied, which will allow us to understand the factors that contribute to an increase in muscle strength and mass much better.
A deeper understanding of the mechanisms that control the maintenance of strength abilities of skeletal muscles will help to increase the effectiveness of sports selection, add to the list of molecular markers of sports inclinations, and develop more effective methods for the development of athletes' strength abilities.
It is well-known that the inhibition of MSTN expression leads to an increase in muscle mass and improves muscle regeneration. Perhaps future studies will need to be performed to explore the relationship between MSTN and stem cells, which will allow new data to be obtained on the molecular mechanisms by which MSTN affects the manifestation of the abilities of weightlifting athletes. A better understanding of the molecular mechanisms of MSTN inhibition, including power-oriented physical load, is likely to be a promising area for improving the professional skills of weightlifting athletes.