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Рецензент: д.мед.н., доц. В.А. Пастухова УДК 577.21: 796.015 Національний університет фізичного виховання і спорту України Национальный университет физического воспитания и спорта Украины ул. Физкультуры, 1, Киев, Украина, 03680 National University of Physical Education and Sports of Ukraine Str. Fizkultury, 1, Kyiv, Ukraine, 03680 sdrozdovska@gmail.com Introduction. Rapid progress in molecular genetics of muscle activity and the application of modern molecular techniques revealed that physical performance in sports depends on a set of gene polymorphisms whose protein products are involved in the processes of adaptation to muscular activity [1, 17]. The combination of favorable gene alleles leads to creation of optimal conditions for busy functioning of all body systems with intensive muscular activity. One of the candidate genes whose polymorphisms affect physical performance in various sports is the gene for peroxisome proliferator- activated receptor α (PPARA). This gene encodes the synthesis of α- receptor protein (PPARα), which is a transcription factor that activates the expression of dozens of genes involved in lipid, carbohydrate and energy metabolism; and, controls body weight and inflammation in blood vessels. The PPARα is expressed in cardiac and skeletal muscles, adipose tissues, and liver [4, 5, 11]. We have found that the expression level of PPARA is higher in slowly-contracting muscle fibers [15], while endurance training leads to increase in oxidative capacity of skeletal muscles by regulating by PPARA regulation of gene expression [12, 13]. The PPARA regulates expression of genes encoding important muscle enzymes involved in the oxidation of fatty acids [4, 16]. There is some data confirming the important role of the PPARA in the adaptive processes in response to training with preferred development of endurance [2, 7]. The PPARA gene localized in chromosome 22 (22q13.31) consists of 93.230 bases and contains, according to data from the NCBI (National Center for Biotechnology Information) database, 2493 SNP (single nucleotide polymorphisms). The most studied polymorphism in this gene is a substitution of G nucleotide to C nucleotide in position 2528 of the 7th intron (rs 4253778 G/C). The frequency of minor C allele in the European population ranges from 0.195 to 0.212 (according to NCBI), while being considerably higher in the African-American population (from 0.625 to 0.833), and considerably less in the Asian population (≈ 0%). The G/C polymorphism in 7-th intron of the PPARA gene is associated with predominant metabolism of fatty acids and glucose. In G-allele carriers the oxidation of fatty acids in the liver, myocardium, skeletal muscles and other organs is more intensive compared to the C- allele carriers [10, 11]. The insufficient oxidation of fatty acids in the C-allele carriers is counterbalanced by increase in glucose utilization. Therefore, the G-allele is related to the alleles of endurance, while the C-allele is related to the alleles of speed and power. The correlation analysis of G/C polymorphism of the PPARA gene with the data from echocardiographic examination of athletes has indicated an association of the PPARA C-allele with the risk of myocardial hypertrophy in left ventricle. Moreover, the PPARA G-allele was also associated with predominance of slow muscle fibers [2, 13]. Some authors have found that there are no probable differences in the distribution of genotypes for the A/C polymorphism of intron 1 of the PPARA gene among sprinters, athletes specializing in sport types requiring endurance, and representatives of control groups [8]. Therefore, the indicated polymorphism cannot be regarded as a marker of genetic predisposition to various types of sport. Relationship of this work with academic programs, plans and themes. The work is performed under the subject 2.22 “Development of comprehensive system for identification of individual-typological characteristics of athletes based on genome manifestations”, as well as under the consolidated plan of research work in the field of physical culture and sports for 2011 - 2015 (state registration no. 0111U001729). Purpose. The purpose is to examine the prevalence of polymorphisms of this gene among Ukrainian athletes; and, to establish its relationship with physical performance in sports. Methods and materials of research. The survey involved 287 persons, including 202 qualified athletes, and 85 persons comprising the control group while having no experience of regular sports. All tested athletes were divided into 3 groups depending on the nature of energy supply for muscular activity in selected sport type: 1) athletes specializing in sport types requiring physical endurance (n=80); 2) athletes specializing in sport types requiring strength and power (n=71); 3) athletes specializing in sport types requiring endurance and strength (n=51). The DNA was extracted from buccal epithelium using a set of reagents “DiatomTM DNA Prep” (by Biokom). Gene polymorphism was determined by polymerase chain reaction (PCR), followed by treatment with restrictases and subsequent analysis of restriction fragments’ length (PCR-RFLP). Genotyping of athletes was performed at the premises of the Laboratory of Molecular Genetics at the Department of General and Molecular Pathophysiology of the O.O. Bohomolets Institute of Physiology at the National Academy of Sciences of Ukraine. The G/C polymorphism of 7th intron of the PPARA gene (rs4253778) was determined by amplification of gene site involving the direct primer “5'-ACAATCACTCCTTAAATATGGTGG- 3'”, and the reverse primer “5'-AAGTAGGGACAGACAGGACCAGTA-3'” [10] (primers synthesized by «Metabion», Germany). The reaction composition included: 5 µl -PCR- buffer (“Amplisense”, Russia); 2.5 µl dNTP; 30 pmol/l of each primer; and 0.1 U Tag- polymerase (“Amplisense”, Russia); the volume adjusted to 25 µl with deionized water. The reaction mixture was added with 1 ml DNA, followed by use of the following PCR temperature mode in the “Applied Biosystems 2700” thermal cycler (USA). The products of this PCR amplification are DNA fragments 266 bp long. The presence of substitution nucleotide G with nucleotide C in position 2528 of intron 7 of the gene PPARA creates a restriction site (T ↓ CGA) for endonuclease Taq I. The presence of restriction site determines the distribution of amplicons into two fragments, 216 bp and 50 bp. The genotype G/G was matched by unrestricted fragments, 266 bp ; genotype G/C - by three fragments of 266 bp, 216 bp and 50 bp; and genotype C/C – by two fragments of 216 bp and 50 bp. Identification of single nucleotide substitution was achieved by joint incubation of amplicon and Taq I using restriction endonuclease (Taq I refSNP ID: rs4253778) (“Fermentas”, Lithuania). The composition of the restriction mixture included: deionized water - 0.8 µl; buffer - 0.8 ml; and Taq I restriction - 0.4 ml. Incubation of restrictive mixture (2 ml) with amplification products (6 ml) in a separate tube positioned in a thermostat at 65 ºC (overnight). Following restriction, the amplifiers were separated in 2.5% agarose gel containing 10 µg/ml ethidium bromide. Visualization of DNA after horizontal electrophoresis (160 V during 40 min) was performed using a transilluminator (“Biokom”, Russia) and the ViTran video system (Russia). The probability of differences in the distribution of samples was determined by the criterion c2. The value P < 0.05 was considered as probable. Results and discussion. Some researchers are of the opinion that the G/C polymorphism in intron 7 of the PPARA gene may be treated as a marker of disposition to muscular activity [1]. This analysis revealed that only the authors of one study claim that both alleles of this polymorphism are prognostic markers of predisposition to performance of muscular work, while others tend to think that only the G-allele may be a marker of probable endurance (Table 1). Table 1 Major trends of impact of G/C polymorphism of 7th intron of the PPARA gene on athletic performance [2, 6, 9, 14].
Since the frequency of occurrence of different genotypes under this polymorphism in the Ukrainian population have not been studied yet, we established the distribution of genotypes in the group of persons who are not involved in any sport types (Table 2). Table 2 Comparative analysis of the distribution of G/C polymorphism of 7th intron of the PPARA gene in Eastern European populations [2, 6]
As is seen from the above, our results coincide with those obtained from other Slavic populations, particularly among Russians. [2] The distribution of genotypes in our choice corresponds to the Hardy-Weinberg equilibrium (pχ2 = 0.64). Since the prognostic value of this marker for athletes of different sports has not been fully established, we conducted a study of the prevalence of allelic variants of this polymorphism among Ukrainian athletes of different sports (Table 3). A comparative analysis of the distribution of genotypes and alleles in the total group of athletes and in the control group failed to establish the likely differences between the samples (rχ2 gen = 0.37), (rχ2 al = 0.19). But in a group of athletes, the frequency of occurrence of the genotype G/G was by 7.2 % higher than the appropriate frequency in the control group; while the frequencies of occurrence of the genotypes G/C and C/C were by 5.8% and 1.3 %, and the G-allele frequency - by 4.2%, higher than the appropriate values in the control group (Fig. 1). Thus, G/G-genotype and G-allele of G/C polymorphism in 7th intron of the PPARA gene may contribute to high performance in sports. Table 3 Frequency of occurrence of allelic variants of G/C polymorphism of 7th intron of the PPARA gene among athletes of different sports (n=287)
P1 - statistical probability of differences in distribution of genotypes compared to the control group; P2 - statistical probability of differences in distribution of alleles compared to the control group; P3 - statistical probability of differences in distribution of genotypes compared to athletes in endurance; *- probable differences by c2-criterion.
Analysis of genotyping results in athletes in various sports shows that in all the groups of sports studied herein there is a predominance of frequency of G-allele and G/G genotype compared to the control group (Fig. 1). The highest frequency of G-allele is observed in athletes involved in sport types requiring a combination of strength and endurance, while the smallest frequency is observed in athletes of sport types requiring strength and alertness. No significant difference was established between the athletes engaged in endurance sports and the athletes engaged in power sports. However, we did observe a tendency for increase in the frequency of G-allele at increase in sportsmanship in types of sport primarily requiring manifestation of endurance: MSC (Master-of-Sports Candidates) (0.58) → MS (Master of Sports) (0.59) → WCMS (World-Class Master of Sports) (0.68). Similar results were observed in studies of Israeli athletes [9]. Analysis of sport types indicated a variation of results between different sports (Table 4). The highest frequency of G-allele among the groups herein surveyed is characteristic of the athletes specializing in throwing athletics and sprint. Figure 1: Frequency of G-allele in athlete groups and the control group, where 1 –mixed group; 2 - endurance sports; 3 - power sports; 4 - all athletes; 5 - control group. The results of our combined group, which includes athletes of different combat kind of sports, coincide with the results obtained from the Polish athletes specializing in combat sports [6]. We also detected a high frequency of G-allele (0.83). It is known that in G-allele carriers the oxidation of fatty acids in the liver, myocardium, skeletal muscles and other organs is more intensive than in C-allele carriers; obviously, because the G-allele is related to the alleles contributing to high performance in sports. Table 4. Distribution of allelic variants of G/C polymorphism of 7th intron of the PPARA gene among athletes of different sport types
P - statistical probability of differences in distribution of genotypes between the control group and athletes, p <0.05; * - probable differences in the frequency of alleles compared to the control group by c2-criterion;
Conclusions. The distribution of genotypes and alleles in the group of athletes differs from the control group. The frequency of occurrence of genotype G/G and G-allele in G/C polymorphism of 7th intron of the PPARA gene is higher in athletes than in the control group. The G/G-genotype and G-allele in G/C polymorphism of 7th intron of the PPARA gene contribute to high physical ability. This polymorphism can be used as a marker of predisposition to high physical performance in all sports. Література
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