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1. Introduction
The α-actinin-3 structural protein is a fast skeletal muscle protein that is encoded by the α-actinin-3 gene (ACTN3, MIM#102574; chromosomal location: 11q13-q14) [1, 2]. ACTN3 has attracted considerable attention due to a common nonsense polymorphism (R577X) that influences muscular performance [3]. A loss-of-function nonsense mutation (C>T) at nucleotide position 1747 in the ACTN3 coding sequence is responsible for the transition of an arginine (R) to a premature stop codon (X) at residue 577 (R577X; dbSNPrs1815739). 
This single nucleotide polymorphism is responsible for the distribution of two different types of ACTN3, both of which are common in general population [4]. The 577R allele is the normal, operational version of this gene, whereas 577X allele contains a changed sequence that prevents the production of a complete functional α-actinin-3 protein [5–7]. It is noteworthy that α-actinin-3 has relatively specialized expression and is limited to the fast, glycolytic muscle fibers that are responsible for quick force generation [6]. 
It is estimated that 16% of the individuals worldwide, or approximately one billion people, are deficient in α-actinin-3. Our previous study conducted on Iranian normal individuals showed that the allelic distribution of ACTN3 in Iranian population is closer to its distribution in the Caucasian population with 11% homozygosity for the common R577X polymorphism; this is concurrent with the route of migration of the ancient human population from the Iran Plateau toward Europe. ACTN3 knockout mouse model analysis has shown that α-actinin-3 deficiency brings about a “slowing” of fast fiber attributes conjoined with a dramatic fall in grip strength, muscle mass and fast 2B fiber size, a rise in muscle aerobic capacity, and slowing of muscle contractile properties [8].
In an Australian athlete cohort [9], highly significant associations between ACTN3 genotype and athletic performance was revealed. Both male and female elite sprint athletes had significantly higher frequencies of the 577R allele than that of control participants. Furthermore, in a number of studies conducted on European, Israeli, and North American athletes, it has been shown that 577XX genotype is underrepresented in sprint athletes and slightly overrepresented in endurance athletes compared with nonathletic controls [10–15]. These data, in addition to other recent studies, provide strong evidence that α-actinin-3 deficiency is detrimental to sprint and power performance.
The effect of ACTN3 genotype has primarily been studied in elite athletes as its effect on muscle can be most readily discernible at extremes of human performance. In connection with this, although soccer is considered to be a long-duration exercise [16, 17], it is well known that matches are won in high intensity, short burst efforts such as sprinting or jumping. Therefore, in addition to technical and tactical skills, muscle strength and “explosive” leg power are very important factors that contribute toward achieving successful results during elite soccer competitions. ACTN3 is vital in producing high-power and high-velocity muscle compression [16].
Due to the lack of data according to ACTN3 R577X polymorphism in Iranian athletes, in this study, we aimed to examine the associations between ACTN3 polymorphisms and athletic performance in a group of elite soccer players and compare their genotypic distributions with nonathletic controls.
2. Methods
Study participants
A total of 290 participants comprising of 90 elite male soccer players as case group and 200 unrelated healthy males as control group were recruited in this case–control study. All of the individuals in case group had represented Iran in their sport at the international level. The individuals in control group were volunteer males recruited from the Genetics Research Center (GRC). All the participants in this study were all of Iranian ancestry to overcome any potential issues with population stratification. Composed written assent was acquired from all participants under conventions affirmed by the institutional survey leading group of the GRC at the University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Genotyping
After obtaining written informed consent from all the participants, 10 mL of blood was collected in tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA from all the samples was extracted according to the established protocols [18]. Exon 16 of ACTN3 was expanded through polymerase chain reaction (PCR) under standard conditions by using the following primers: forward 5′-CTGTTGCCTGTGGTAAGTGGG-3′, reverse 5′-TGGTCACAGTATGCAGGAGGG-3′. PCR products were digested with DdeI enzyme (New England Biolabs, Ipswich, MA, USA). The 577R and 577X alleles (CGA and TGA codons, respectively) were recognized by the presence (577X) or absence (577R) of a DdeI restriction site (Figure 1).

Molecular genetic and statistical analysis
Chi-squared tests were used to compare genotypic distributions and allelic frequencies between the two groups and also to assess the presence of Hardy–Weinberg equilibrium. Data were analyzed using SPSS 18 (SPSS Inc, Chicago, IL, USA). P values less than 0.05 were considered to be statistically significant.
3. Results
ACTN3 genotype data from the elite athlete and control groups are outlined in Table 1. There were significant diversities in genotypic frequency between the elite athlete group and the control group. Although the differences in allelic frequencies did not reach statistical significance, participants from elite athlete group did have a lower frequency of the 577RX genotype than that of 577RR and 577XX genotypes. The frequency of 577RX genotype was also lower in elite athlete group than that of control group (P<0.001). Genotypic frequencies of ACTN3 (XX, RX, and RR) for elite soccer players and normal nonathletic population (P<0.001) are shown in Figure 2. 

4. Discussion
To the best of our knowledge, this is the first study to demonstrate a higher proportion of the ACTN3 577RR and 577RX genotypes in elite level speed-power athletes from Iran compared with the normal nonathletic population. Our findings revealed that Iranian elite athletes had a lower frequency of the heterozygous RX genotype (37% vs. 57%) compared with controls. Elite athletes had higher frequencies of RR and XX genotypes (41% and 25%, respectively) than that of control participants (22% and 17%, respectively) (P<0.001).
A strong positive association of ACTN3 577R polymorphism and athletes’ performance was first reported by Yang et al. in an Australian population [9] Since then, many studies have been performed in various populations and different athletic groups which have provided conclusive evidence of a correlation. Several cross-sectional studies to date have reliably shown that the 577XX genotype is underrepresented in sprint-related 

athletes [9, 12, 13] or is associated with inferior performance [19]. Two recent meta analyses [20, 21] and a revision [22] of the literature with regards to the effect of ACTN3 on athletic performance indicate that the 577RR genotype is affiliated with sprint-power athletes among Caucasians. A large cohort study of about 300 elite Japanese athletes indicated that the ACTN3 577RX genotype was associated with sprint-power performance among track and field contestants [23].
A study on Turkish male soccer players revealed that the frequencies of ACTN3 genotypes were 20, 36, and 44 for XX, RX, and RR, respectively, which agrees with the results of this study for each genotype [24]. In the Turkish study, an allelic frequency was found to be 31 (62%) and 19 (38%) for R and X alleles of ACTN3, respectively, which is similar to our findings (107 (59.4%) R and 73 (40.6%) X alleles). Similar results were obtained in Russian football players in which 240 male athletes were examined among which 46.25% showed the RR genotype [15]. 
Santiago et al. analyzed the periodicity of the ACTN3 genotype in 60 top-level professional soccer players from Brazil and showed that the distribution of 577RR and 577RX genotypes (48.3% and 36.7%, respectively) was notably higher and lower, as in the order, than those in controls (n=123; 28.5% and 53.7%, respectively) (p=0.041) [10]. The result of this study is also in accordance with the Brazilian study [10]. Another recent study by Pimenta et al., in which 37 professional soccer players were examined, showed a prevalence of 40.5% for the RR genotype [25], which agrees with our results.
The results of this study indicate that the ACTN3 RR genotype is overrepresented in Iranian elite male soccer players compared to control participants. Moreover, we showed that the distribution of the ACTN3 genotypes and alleles in the Iranian population is similar to those observed in several reported groups of Russian, Brazilian, and Turkish populations [10, 24, 26]. There were no statistically significant differences noted in the allelic frequencies between elite soccer players and control group participants, though sample size limitations prevented a conclusive analysis in this regard. 
The ACTN3 discoveries reported here broaden past reports demonstrating higher 577RR genotype recurrence in elite speed/power-oriented athletes and competitors than that of normal participants. This supports to the speculation that α-actinin-3 protein deficiency (XX genotype) impairs muscle performance. The ACTN3 RR genotype, alone or in combination with additional polymorphisms such as the angiotensin converting enzyme insertion/deletion genotype, may be useful predictors of athletic performance at the elite level.
Acknowledgments
We are deeply grateful to the participants of this study, and we greatly appreciate Dr. Mohammad Dadkan, Dr. Amin Norouzi, Dr. Abdol Reza Allahverdi, and Mr. Alireza Mansourian for their collaboration which made this study possible. This project has not received any financial assistance. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of Interest
The authors declared no conflicts of interest.

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