Prevalence of ACTN 3 R 577 X Polymorphism in Chinese University Students

The study on single-nucleotide polymorphism (SNP) of alpha-actinin (ACTN)3 gene was mainly focused on R577X codon (rs1815739), which leads to replacement of an arginine (577R) with a premature stop codon (577X), The ACTN3 R577X polymorphism has been reported to be related to the elite athlete performance, disease and even history of human evolution. The objective of the current study was to analysis genotype and allelic frequencies of ACTN3 R577X in Chinese University Students. We used TaqMan SNP assay for genotyping 638 Chinese Han Nationality University Students. Total of 638 Northwestern University students in this study, of whom 322 were female and 316 were male, The different genotypes were as follow; 32.13% RR (205/638), 51.25% RX(327/638), 16.61% XX (106/638), R allele 0.58(737/1276), X allele 0.42(539/1276). Respectively, 322 female 31.37% RR (101/322), 49.69% RX (160/322), 18.94% XX(61/322),R allele 0.56(362/644), X allele 0.44(282/644). 316 male 32.91% RR (104/316), 52.85% RX(167/316), 14.24% XX (45/316), R allele 0.59(375/632), X allele 0.41(257/632). 638 Northwestern University students in this study were confirmed to have representativeness, It is similar to Iranian population and Caucasian, which is also favors the hypothesis of an adaptive evolutionary history for ACTN3 R577X.


INTRODUCTION
Human athletic ability has a high level heritability, with the development of molecular biology techniques and detection methods, researchers are increasingly concerned with the influence of gene variation on the elite athlete performance and also come to realize the important predictive value of genes.Recently a large number of candidate gene polymorphisms have been studied to investigate potential relations with musclerelated phenotypes in different ethnic, geographic, occupational population (Appell Coriolano and Duarte, 2012).Family of α-actinin proteins consists of four forms protein (ACTN 1-4) which are the major structural elements of the Z-discs in the human skeletal muscle, ACTN1 and ACTN4 assist in cytoskeletal anchoring which belong to non-sarcomeric, while ACTN 2 and ACTN 3 are expressed in muscle fibers.ACTN 3 protein is encoded by ACTN 3 gene which is actin-binding protein and expressed only in type II skeletal muscle fibers (fast-twitch), maintain the integrity of the muscle contraction element and many of the signaling and metabolism of protein interactions (Mills et al., 2001).A common nonsense singlenucleotide polymorphism (R577X) in ACTN3 gene, which result in a premature stop codon Ter (X) replacing the Arg (R) at amino acid 577 (rs1815739) (North et al., 1999).There are two allele versions in humans, R(functional allele) and X(null allele).It is estimated the more than one billion people have R577X null allele in the world, XX genotype is prevalent in 16-18% of European Caucasian population and 25-29% of the Japanese population (Kikuchi et al., 2015), many researchers in the field of sports science are concerned about the relation between the lack of ACTN3 in the human fast muscle fiber and performance of sports (Papadimitriou et al., 2016).Although many studies have shown that ACTN3 gene polymorphism is associated with the performance of elite endurance and power athletes (Mikami et al., 2014), there are many studies reported the opposite results (Ruiz et al., 2013).In addition, ACTN3 gene polymorphism has been discovered in the cardiometabolic health (Deschamps et al., 2015), spinal cord injury (Broos et al., 2012) and idiopathic Inflammatory Myopathies (IIMs) (Sandoval-García et al., 2012), however the data of ACTN3 R577X polymorphism in Chinese University students remains unknown, so the aim of this study was to investigate the prevalence of the allele in Chinese University students.

MATERIALS AND METHODS
In this study, Subjects were 638 from Northwestern University students (322 female, 316 male; age 20.62±2.74years), all the subjects were excluded patients with acute or chronic liver, kidney disease, cardiovascular, endocrine or metabolic disorders patients.Physical activity is normal without any professional training, physical examination qualified and no family history of sports training.All the subjects were from Shaanxi provinces in Northwest China descent for ≥3 generations.The experimental procedures were been performed with the approval and Ethics Committee of Research Centre in Sports, Department of Physical Education of Northwestern University, approved all procedures.
Genotyping: During fall 2015, Peripheral venous blood samples were collected in 2 mL EDTA-K2 anticoagulant tube at -20°C until DNA extraction.Genomic DNA was extracted from blood samples during the years 2015-2016 and genotyping was performed in Clinical Laboratory of Xi'an Central Hospital, China.ACTN3 R577X (rs1815739) genotyping methods were carried out by using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, California, USA) (Schadock et al., 2015).PCR reaction system as follows: genomic DNA 2 uL, TaqMan Universal PCR amplified premixed reagent (Applied Biosystems, Foster City, California, USA) 13.75 uL, primers and probe (TakaRa, Dalian, China) per 0.5 uL.Finally, the complement of double distilled water to the total 25 uL reaction volume.Reaction parameters: 1 cycles of 50°C for 2 min, 95°C for 10 min, 40 cycles of 92°C for 15 s and 60°C for 1 min, the fluorescence detection period of 60°C 1 min.PCR reactions and genotype analysis were run on ABI 7300 instrument.

Statistical analysis:
The anthropometric measures are presented as mean and SEM.Genotypes and alleles frequencies were calculated by direct counting method, Hardy-Weinberg equilibrium was used chi-square test.Statistical significance was accepted at p value of less than 0.05.All statistical analyses were performed using the SPSS 13.0 software for windows.

RESULTS AND DISCUSSION
A total of 638 Northwestern University students in this study, of whom 322 (322/638, 50.5%) were female and (316/638, 49.5%) were male, All subjects were Han Nationality, The ACTN3 R577X polymorphism's distribution from 322 female University Students was met the condition of HWE (F = 0.028, df = 1, p>0.05) and 316 male University Students was met the condition of HWE (F = 2.86, df = 1, p>0.05).The total of 638 University students was also met the condition of HWE (F = 1.62, df = 1, p>0.05).Therefore, the polymorphism distribution of 322 female and 316 male University Students were confirmed to have representativeness.Which is shown in Table 1.
It is well-known that skeletal muscle was made up of individual muscle fibers, muscle fibers are classified into slow twitch fibers (Type I fibers) and fast twitch fibers (Type II fibers).Type I fibers are more efficient at using oxygen to generate ATP to participate in the need for endurance exercise, such as marathon and bicycle for hours.Whereas Type II fibers are mainly involved in the need of high force and low endurance, directly to create energy via anaerobic metabolism, such as sprint (Vincent et al., 2007).Fast fibers produce ACTN3, which reflects the body's ability to produce fast power.Researchers in many fields of science are very concerned about whether ACTN3 gene polymorphism affect movement ability and quality or not, especially the quality of the explosive force.A study by Eynon et al. (2009) showed that the proportion of group velocity athletes ACTN3 R577X polymorphism RR genotype were found to be significantly higher than that of the endurance athlete group and the control group and endurance athletes of group in the XX genotype proportion was even higher.In addition, it was found that the proportion of R allele was higher in top level athletes according to the level of achievement points to the top level and the national level athletes, So they think that the R allele of R577X ACTN3 polymorphism was associated with the velocity and quality of explosive force.These findings were also validated in others studies Mikami et al. (2014).In contrast, there were quite a number of studies which had reported the opposite result.A study by Ruiz et al. (2011) reported no significant influence of the ACTN3 R577X polymorphism on explosive leg muscle power  in elite volleyball player.Similar results had been obtained from the other study (Ruiz et al., 2013).Recently a many of studies have described association ACTN 3 R577X polymorphism with the diseases (Broos et al., 2012).A study by Amorim et al. (2015) suggested that the functional ACTN3 R577X polymorphism might have evolved due to selection in Eurasian populations, that The R577X ACTN3 allele frequency shows a general trend of increase with distance from Africa (577X allele frequencies lowest: 0.093), reaching its highest frequencies in the Americas continent (577 X allele frequencies highest: 0.764).Currently an ideal sample for studies of population genetics generally need more than 500 people, there are mostly about 150 people due to various reasons (which is the minimum).The results of the above study may be related to the sample size, population and ethnic differences.So it is very important to analysis genotype and allelic frequencies of ACTN3 R577X in Chinese University Students.Subjects were selected from Han nationality students of Northwestern University, age span less ideal reference population, Most important all the subjects were from Shaanxi provinces in Northwest China descent for ≥3 generations and without any professional sports training.

CONCLUSION
In summary, we investigated that 638 Northwestern University students in this study were confirmed to have representativeness under the conditions that the genotype and allele frequencies attained the Hard-Weinberg equilibrium, The R allelic distributions was 0.58 and X allelic distributions was 0.42.It is similar to Iranian population and Caucasian, which is also favors the hypothesis of an adaptive evolutionary history for ACTN3 R577X (Fattahi and Najmabadi, 2012).

Table 1 :
Genotypes of ACTN3 genotype and alleles in Chinese university students Genotype