ACTN3 R577X polymorphism is not associated with team sport athletic status in Italians

Research in the field of genetics among elite athletic performers has focused primarily on the sport at the end point of the sports performance continuum (sprint/power vs. endurance performance), yet the type of sport in which both the aerobic and anaerobic energy systems are important for achieving a high level of performance, such as team sports or endurance sports, has received little attention.

One of the most common targets of investigation has been a single nucleotide polymorphism (SNP) in the ACTN3 gene, which encodes the alpha-actinin-3 protein [1]. This polymorphism leads to a premature stop codon (X), rather than an arginine (R) at position 577. Alpha-actinin-3 deficiency (ACTN3 577XX genotype) occurs in approximately 16% of the global population [1] and is associated with variations in human muscle performance. The alpha-actinin-3 protein is almost exclusively expressed in fast, glycolytic, and type IIX fibers that are responsible for the generation of rapid forceful contractions during activities such as sprinting.

Meta-analyses [2,3] and a recent review [4] of the literature regarding the influence of ACTN3 on athletic performance suggest that the ACTN3 577RR genotype is associated with sprint/power athletes among Caucasians. Recently, Mikami et al. [5] examined a large cohort of elite Japanese athletes (n = 299) and found that the ACTN3 R577X genotype was associated with sprint/power performance among track and field competitors, especially running 100- to 200-m sprints, and that R allele was associated with relative peak power in male collegiate athletes [6]. On the contrary, a recent study of Caucasian and East Asian swimmers did not show an association between ACTN3 polymorphism and elite swimmer status [7]. The ACTN3 577X allele and the 577XX genotype were significantly associated with only certain groups of elite endurance athletes [8-17].

Seto et al. [18] provided a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and on adaptation to changing physical demands in the general population. The authors highlighted the emerging evidence of the roles of alpha-actinin-3 in the regulation of metabolic and signaling pathways, contractile properties, and muscle fiber type. They showed that alpha-actinin-3 deficiency resulted in increased calcineurin activity in mouse and human skeletal muscle and an enhanced adaptive response to endurance training. Only a few studies investigated the association between the ACTN3 R577X polymorphism and team sport performance. Santiago et al. [19] examined the frequency of this ACTN3 genotype in 60 top-level professional Spanish soccer players, showing that the percentage distributions of 577RR and 577RX genotypes (48.3% and 36.7%) were significantly higher and lower, respectively, than those in controls (n = 123; 28.5% and 53.7%) or endurance athletes (n = 52; 26.5% and 52%) (p = 0.041). In a previous study [20], we did not find a significant difference in top-level soccer players’ (n = 42) jump performance among the various genotypes.

Pimenta et al. [21] examined 200 Brazilian professional soccer players and found that 577RR carriers ran 10, 20, and 30 m faster than 577XX carriers. Furthermore, the former scored higher in jump tests compared to both 577RX and 577XX carriers.

Not all the studies, however, found an association between ACTN3 R577X polymorphism and team sport performance [22-24]. Recently, Garatachea et al. [25] did not find an association between ACTN3 genotypes and explosive leg power in elite basketball players or with the status of being this type of athlete. Finally, Eynon et al. [26] compared the genotype and allele frequencies of the ACTN3 R577X polymorphisms in a relatively large cohort of team sport athletes (n = 205), endurance athletes (n = 305), sprint/power athletes (n = 378), and non-athletic controls (n = 568) from Europe and found that ACTN3 R577X polymorphism was not associated with team sport athletic status. However, they did find that the 577RR genotype was overrepresented in sprint/power athletes compared to team sport athletes.

The aim of this study was to analyze the associations between ACTN3 R577X polymorphisms and elite team sport athletic status in a group of Italian athletes and to compare their genotype distributions with elite sprint/power athletes, endurance athletes, and non-athletic controls.

The genotype frequencies among these athletes were compared with those of the randomly selected healthy non-athletic Italian males (controls). The controls were selected if they did not participate regularly in any competitive or structured sport or physical activity.

All participants and their parent/guardian (for minors) provided written informed consent, and the study protocol and informed written consent procedure were approved by the Medical Ethics Committees of the Italian Sports Federations, in accordance with Declaration of Helsinki for Human Research of 1974 (last modified in 2000).

Genomic DNA was extracted from each athlete with a buccal swab during the years 2004 to 2014, according to the manufacturer’s directions provided with a commercially available kit (Qiagen S.r.l., Milan, Italy) [27]. Exon 16 of ACTN3 was amplified through polymerase chain reaction using the following primers:

forward 5′-CTGTTGCCTGTGGTAAGTGGG-3′

reverse 5′-TGGTCACAGTATGCAGGAGGG-3′.

Polymerase chain reaction products were digested with DdeI enzyme. The 577R and 577X alleles (CGA and TGA codons, respectively) were distinguished by the presence (577X) or absence (577R) of a DdeI restriction site in exon 16. Allele 577X shows two fragments with 205 and 85 bp, while allele 557R presents three fragments with 108, 97, and 86 bp. The obtained fragments were separated by 10% polyacrylamide gel electrophoresis and stained with ethidium bromide [27]. To ensure proper internal control, for each genotype analysis, we used positive and negative controls from different DNA aliquots that were previously genotyped with the same method.

Chi-squared tests were used to test for the presence of Hardy-Weinberg equilibrium (HWE). Multinomial logistic regression analyses were conducted to assess the association between genotype and athletic status/competition level. In each case, analyses were made comparing 577XX (reference group) vs. 577RX, 577XX vs. 577RR (co-dominant effect), 577XX vs. 577RR and 577RX combined (dominant effect), and 577XX and 577RX combined (reference group) vs. 577RR (recessive effect). Significance was accepted when p ≤ 0.05. Chi-squared tests for HWE were conducted using Genepop (v. 9), while statistical analyses were conducted using STATISTICA (Statsoft v. 7)

Table 1 shows the genotype and allele frequency distributions in the non-athletes and athletes divided by group and subgroup. The genotype distributions met HWE (all p > 0.05) for all groups of participants.

We tested, for the first time, the association between ACTN3 R577X polymorphism and team sport athletic status in Italian athletes. Our results indicate that team sport athletes have a lower 577RR than 577XX genotype frequency, compared to sprint/power athletes (p = 0.044). No significant difference in the ACTN3 R577X polymorphism genotype distribution was found between the team sport athletes, endurance athletes, and control group. Furthermore, the ACTN3 R577X genotype distribution was similar between elite-level and national-level team sport athletes, suggesting that this polymorphism is not influential among team sport athletes.

Our findings agree with those of Eynon et al. [26], who recently analyzed elite team sport athletes from three European countries (i.e., Spain, Poland, and Russia) and showed that their performance was not significantly influenced by ACTN3 R577X polymorphism. Similarly, our results agree with theirs in that team sport athletes were less likely to possess the 577RR genotype than the 577XX genotype compared to power athletes (p = 0.045).

The ACTN3 R577X polymorphism was consistently associated with elite sprint/power status [2,3] and muscle power [6]. The functional consequence of the ACTN3 R577X polymorphisms on sport performance is supported by the Actn3 knock-out (KO) mouse model [28]. The authors demonstrated that the loss of alpha-actinin expression results in a shift in muscle metabolism toward the more efficient aerobic pathway, with a subsequent increase in running distance achieved before exhaustion during a treadmill test [28]. Moreover, the Actn3 KO mice (ACTN3 577XX), compared to wild-type (WT) counterparts, had lower muscle mass (smaller diameter fast-twitch muscle fibers), significantly lesser grip strength, significantly lower anaerobic enzyme activity, and higher oxidative/mitochondrial enzyme activity without a shift in fiber-type distribution [29].

In team sports, most of the decisive actions during competition are characterized by jumping and/or sprinting, and it is for that reason that we hypothesized that the 577RR genotype would be associated with team sport performance. However, team sports generally involve mixed aerobic-anaerobic demands and players may not need to have extraordinary aerobic/anaerobic capacity. During a soccer game, for example, aerobic metabolism dominates energy delivery [30], yet the most decisive actions, characterized by jumping and sprinting, are supported by anaerobic metabolism. In similar team sports, many factors contribute to success, including muscle power, aerobic endurance, ball skills, and psychological traits. Therefore, muscle power represents just one of several factors that determine a player’s ability but a substantial development of this factor is decisive during the crucial phases of a match.

When studying the possible role of genetics in team sports, it should be noted that not all players have similar characteristics in terms of physiological demand because it varies based on the position played. For example, during a rugby match, players perform various activities based on their position [31]. Forwards, rather than backs, are involved in significantly more physical collisions and tackles, experience a greater proportion of high-intensity activity than low-intensity activity, and travel greater distances during a match (9,929 vs. 8458 m) [31], demonstrating that a wide range of skills and physiological demands exist for the different playing positions.

Indeed, while ACTN3 R577X polymorphism has been previously associated with sprint/power or endurance performance, the results of this study suggest that it is not associated with team sport athletic status.

The most obvious limitation of this study is the sample size. However, the very large population samples required to reach a sufficient statistical power in the field of genetic studies do not harmonize with the limited number of elite athletes for each sport discipline. We believe that the small sample size could be justifiable by the number of top-level athletes and by the homogeneity of the sample in terms of geographical and ethnical origin, recruited for the present study.

In conclusion, our results confirm, for the Italian population, the recent data published by Eynon et al. [26] that ACTN3 R577X polymorphism is not significantly associated with team sport athletic status, when team sport athletes are compared to endurance athletes and non-athletic controls, and the 577RR genotype is overrepresented in power/sprint athletes compared to team sport athletes.