R577X OF THE ACTN3 GENE AS PREDICTOR OF PHYSICAL PERFORMANCE IN ULTRAMARATHON RUNNERS

ABSTRACT Introduction: Genetic factors appear to explain why some athletes perform better in competition and training than their peers. Objective: To determine the occurrence of R577X polymorphism of the ACTN3 gene in mountain runners. Methods: The sample consisted of 19 female mountain runners with a mean age of 41.2 ± 6.1 years. Genotyping of R577X polymorphism of the ACTN3 gene was performed by the polymerase chain reaction (PCR) method with DNA extracted from saliva. The genotypic and allelic frequencies of the athletes were evaluated and compared with data from the literature. Hardy-Weinberg equilibrium and Chi-square with Yates correction were used, with a significance level of p<0.05. Results: The genotypic distributions did not show any significant differences between the athletes and the control group, with RR = 15.8%, RX = 57.9%, XX = 26.3%. In regard to allelic distribution, the nonfunctional allele was higher in the study group than in the control group, with R = 44.7%, X = 55.3% for p = 0.0350. Conclusion: The data revealed a possible relationship between the ACTN3 X allele and athletic performance in Brazilian female mountain runners. Level of evidence II; Development of diagnostic criteria in consecutive patients (with “gold” reference standard applied).


INTRODUCTION
The improvement of physical activity in high-performance sports may be linked to genetic predisposition. 1 For more than a decade, studies have shown that genetics may explain the different responses and better performance of some athletes in specific modalities when comparing them with their peers. 2 Moreover, genetic factors can influence up to 50% the phenotypic characteristics related to performance, training and physical fitness of high-performance athletes. 3 As many genes and gene areas have already been related to phenotypes of human physical performance, the association between genetics and phenotypic profile can help athletes developing ideal morphophysiological features for certain sports, making them less susceptible to injuries and more prepared for training and competitions. 1 Thus, the study of genetic polymorphisms can be a prognosis of physical performance. 4 However, when dealing with studies that consider ethnicity and race of the population studied, we must remember that the phenotypic effects of some polymorphisms may be expressed in multiple ways in different communities. 3 For Ahmetov et al. 1 and Yang et al. 5 , genetic polymorphisms associated with sportive success are those that bring benefits, especially related to endurance, sprint and muscle power. In this sense, we highlight R577X polymorphism of the alpha-actinin 3 gene (ACTN3), investigated here because of its association with activities that require muscle strength, sprint, and endurance. 6,7 Located on chromosome 11q13-q14, this polymorphism is the product of the switch from cytosine to thymine at position 1747 of exon 16, which results in the substitution of arginine (R allele) for a premature stop codon (X allele) at amino acid 577, and thus allowing three genotypes: RR, RX and XX. 8 The switch of arginine or R allele represents the natural transcription of the α-actinin-3 protein in the skeletal muscle, while the stop codon/X allele prevents homozygous individuals (genotype XX) from producing α-actinin-3, decreasing the transverse sections area in muscles composed of type 2 fiber predominance, and thus reducing muscle mass, when compared to RR and RX genotypes. 8,9 However, the non-expression of α-actinin-3 seems to improve aerobic metabolism, increasing the muscle and cardiorespiratory endurance. 9,10 The relation of ACTN3 with high performance in sports is described by studies that report a greater presence of the X allele in athletes with muscular endurance 11,12 , while those with muscle power and sprint have the R allele. 7,12 Considering this, our research aims at better understanding the interaction of ACTN3 R577X polymorphism with the physical performance of female mountain runners in Brazil, since such genetic variation can improve their physical fitness. Moreover, this is an unprecedented analysis of these type of athletes, with the objective of determining the occurrence of R577X polymorphism in mountain runners.

MATERIALS AND METHODS
This is a descriptive transversal study carried out according to Resolution 466/12, during the Ultramaratona dos Perdidos SkyMarathon®, a 45-km race with 2,900 m of ascent, relative height of 5,800 m, in the second week of July 2017, in Tijucas do Sul, Paraná, Brazil. The study was also approved by a Research Ethics Committee (CEP) under opinion 1,572,571.
The inclusion criteria were runners with experience of three to four years in mountain races, and in at least two races above 21 km and one above 42 km, between 2015 and 2016, who trained five to six times a week, one to two hours a day and more than three hours at weekends, and have not reported musculoskeletal diseases. Runners who did not complete the race within a limit time of 11 hours and who did not signed the Informed Consent Form (IFC) were excluded.

Sample
Of the 34 participants, 15 did not participate in the study because they did not meet the inclusion criteria; thus, the sample consisted of 19 mountain runners, with mean age of 41.2 ± 6.1 years.

Procedures
The runners selected were evaluated in relation to the presence of ACTN3 R577X polymorphism according to the following procedures:

Saliva collection
Saliva collection was performed in the field, in a specific area to support athletes, and the participants selected for the study sat in a chair with their feet supported. Initially, a 3% glucose solution was introduced in the participants' mouths, and they swished the product for two minutes. After this, they spat the liquid in a plastic cup. Then the researcher gently rubbed their jugal mucosa with a wooden spatula and washed the spatula in the cup where they spat the solution, and transferred the entire content to a 15ml Falcon tube.
After collection, the samples were taken in a polystyrene foam box with ice plates to the Laboratory of Genetics and Molecular Biology of a Private Higher Education Institution, in Curitiba/PR, where they were centrifuged at 3000 rpm for 10 minutes. After centrifugation, the supernatant liquid was discarded. We kept the precipitate, added 1300 µL of cell extraction buffer (TRIS 10mM, EDTA 5mM, SDS 0.5%, pH=8) and frozen it at −20 °C 13 .

DNA extraction
After defrosting the saliva samples, we added 10 μL of Proteinase K (BioLabs, New England) in each extraction tube and kept them at 65 °C in water bath overnight.
After taking the samples from the water bath, they were gently agitated and transferred to an 2 mL Eppendorf (microtube). The researchers added 500 μL of ammonium acetate (8mM acetate, 1mM EDTA) and mixed the solution in a vortex for five minutes. They were then centrifuged at 13,000 rpm for 16 minutes and separated into two 1.5 mL microtubes (900 μL in each one), disregarding the pellet deposited at the bottom of the tube and adding 540 μL of isopropanol. We gently reversed the tube 20 times to see the DNA.
The content with DNA was centrifuged at 13,000 rpm for seven minutes, the isopropanol was discarded, and we added 1 mL of 70% ethanol. The solution was centrifuged again at 13,000rpm for seven minutes (the supernatant was discarded), and kept at room temperature for drying. The DNA was resuspended at approximately 50 μL of TE (TRIS 10 mM, 1 mM EDTA, pH=7.76) and kept at room temperature for 24 hours. After this period, the samples were stored for two days in the refrigerator and stored at −20 °C.

ACTN3 R577X genotyping
The genotyping of ACTN3 alleles (RR, RX and XX) was performed by the RFLP-PCR technique (polymerase chain reaction through restriction fragment length polymorphism analysis). After amplification, 10 μL of the PCR product was submitted to digestion with 10 units of DdeI restriction enzyme (SIGMA) and incubated for 4 hours in water bath at 37 °C.

Electrophoresis in agarose gel
For analyzing the ACTN3 genotypes, we conducted agarose gel (3%) electrophoresis, colored with ethidium bromide and visualized with an UV transilluminator.

Statistical Analysis
The Pearson's Chi-square test was used to compare the frequencies of genotypes with other studies; when the incidence number of some genotype was less than five, we used the Fisher' s exact test. The associations between the alleles frequencies were analyzed by 2×2 contingency tables, and by the Chi-square test with Yates correction. The Hardy-Weinberg equilibrium was used to verify the distribution of ACTN3 genotypes. The support tool for tests was IBM SPSS Statistics 20 (New York, USA), except for the Hardy-Weinberg Law, which was verified with BioEstat version 5.3 (Belém, Pará, Brazil). All analyses had significance level of p<0.05.

RESULTS
The study included 19 ultramarathoners, with mean age of 41.2 ± 6.1 years, total body mass of 56.9 ± 7.4 kg, height of 163 ± 7.3 cm and fat percentage of 13.73 ± 1.72%. The distribution of the ACTN3 genotype followed the Hardy-Weinberg equilibrium (p=0.45), which shows that the genotypic frequencies found were not different from the values observed in the general population. Therefore, it had no genetic influence on the sample of our study. There were no significant differences in the distribution of ACTN3 genotypes (p>0.05) when compared to athletes from other studies. (Tabela 1) Regarding the allelic and absolute distribution of ACTN3, no significant differences were found (p=0.51), when compared with the studies by Shang et al. 15 (p=0.64), Belli et al. 16 (p=0.25), Herbert et al. 17 (p=0.23), Ben-Zaken etal. 12 (p=0.79). However, when comparing the results with the research by Coelho et al. 18 , there was a significant difference (p=0.03), showing that the X allele is higher for the study sample. (Table 2)

DISCUSSION
Regarding the ACTN3 R577X polymorphism in Brazilian athletes, we verified that, until now, there are no studies focused on this interaction in female mountain ultramarathoners, which reveals the unprecedented character of our study.
The results obtained in terms of absolute and relative ACTN3 genotypic frequency showed that, when compared with studies conducted with endurance athletes from China 15 and Israel, 12 ultra-endurance runners from Brazil, 16 European marathoners 17 and with the Brazilian population in general, 18 there is no statistically significant difference.
Although the literature 12,19,20,15 evidences the genotypic association of ACTN3 with athletic performance in high-performance modalities, this study did not find any connection of genotype with athletic performance, corroborating the studies conducted by Ahmetov et al., 4 Lucia et al. 21 , and Muniesa, 22 who researched the ACTN3 association and the athletic performance of elite Russian rowing athletes, elite European cyclists and European runners, respectively.
On the other hand, studies conducted by Ben-Zaken et al., 12 Druzhevskaya et al., 19 Gómez-Gallego et al. 20 showed a relationship between genotype and performance. Ben-Zaken et al. 12 showed evidence of the relationship between genotype XX and the fitness of endurance athletes. Druzhevskaya et al. 19 found that 3.4% of elite athletes focused on muscle strength expressed genotype XX, and Gómez-Gallego et al. 20 observed that RR and RX genotypes are related to the peak power of endurance cyclists.
When comparing the allelic distribution with the control group, 18 we noticed that our sample has a frequency of the higher X allele (55.3% vs 37%), which is in agreement with Shang et al. 15 According to these authors, women have higher frequency of genotype XX (21.2% vs 15.8%), as well as of the X allele (51.3% vs 41.1%), when compared to the control group; but the same is not valid for men, which suggests that the non-functional allele of the gene may provide advantages for women in endurance tests.
Most long-distance athletes have a higher distribution of the X allele, 12 which influences muscle function in fast fibers, directing muscle metabolism to an aerobic pathway, which results in improved endurance performance. 9,10 Although Yvert et al. 23 point out the R allele presence in elite endurance athletes, and other inconclusive studies have not linked physical performance with ACTN3 R577X polymorphism in athletes with high strength and power 20 and muscle endurance, 6 some studies 11,12 suggest   that the non-functional allele (X) is present in individuals focused on sprint/power modalities, while the mutant allele (R) is more present in muscle endurance athletes. 11,24

CONCLUSION
This study was the first to analyze ACTN3 in Brazilian female ultramarathoners and found that 84.2% of the sample had the non-functional X allele, distributed in heterozygous and/or homozygous pattern, which supports the hypothesis that the non-functional allele of ACTN3 is associated with a good performance in high-level sports in Brazilian women who are mountain ultramarathoners.
All authors declare no potential conflict of interest related to this article