Molecular genetic determination of functional performance of combatants of different skill levels

Фотографии: 

ˑ: 

PhD M.S. Terzi1
PhD E.V. Lekontsev2
PhD D.A. Saraykin1
Dr.Biol. V.I. Pavlova1
Dr.Med. J.G. Kamskova1
1Chelyabinsk State Pedagogical University, Chelyabinsk
2Ural State University of Physical Culture, Chelyabinsk

Keywords: sport genetics, functional state, martial arts, taekwondo

Introduction. The relevance of the issue of molecular-genetic determinants of functional performance of combatants of different skill levels is accounted for by the fact that its solution will make it possible to provide a scientific foundation for the technology of their sport training for a more efficient implementation of the training and competition processes, to adjust the sport training based on the genetic profile as well as professionally carry out the system of individual selection.  

Modern achievements in Olympic martial arts (taekwondo, boxing, judo, etc.) were made possible due to a significant increase in the volume and intensity of training and competition loads. Insufficient development of anaerobic and aerobic energy supply mechanisms [3] serves as a limiting factor of combatants’ activity, and it is aerobic performance that creates the foundation for the optimal development of anaerobic abilities. The effectiveness of aerobic and anaerobic mechanisms that create the basis for a high level of functional performance of athletes in martial arts is determined by the physiological state of the cardiorespiratory system as well as anaerobic energy supply systems (phosphagen and glycolytic). The optimal operation of these systems is ensured by some polymorphic variants of genes, for example, ACE I/D, BDKRB2 -9/+9 [1].

Objective of the research was to identify and analyse polymorphic loci of molecular-genetic markers ACE I/D, BDKRB2 -9/+9 that determine the integral functional performance of taekwondokas of different skill levels. 

Research method and organization. The study was conducted in the laboratory of Chelyabinsk State Pedagogical University: “Adaptation of athletes to physical loads of varying intensity” as well as at the premises of the Research Institute of Olympic sports of Ural State University of Physical Culture, in the laboratory of molecular-genetic research. It involved combatants from the Specialized Children and Youth Sports School of the Olympic Reserve (SCYSSOR) “Korio” (taekwondo) of the city of Chelyabinsk. All the athletes had been practicing taekwondo for a period of 5-12 years and were divided into three qualification groups (according to their categories and athletic titles): low-skilled athletes (I, II, III junior categories) – 46 persons (reference group); athletes with average skills (I, II, III senior categories) – 54 persons (study group-1) and highly skilled athletes (Candidates for Master of Sports and Masters of Sport) – 51 persons (study group-2).    

DNA was isolated from buccal cells of the oral cavity. Amplification of genomic DNA fragments containing polymorphic sites was carried out using the polymerase chain reaction (PCR) in iQ5 (Bio-Rad, USA) and GeneAmp PCR System 9700 (Applied Biosystems, USA) thermocyclers. Primers were synthesized by the Evrogen Company (Russia). PCR fragments containing single nucleotide polymorphisms were treated with matching restriction enzymes in accordance with conventional techniques. Restriction output as well as PCR products of polymorphisms containing insertions/deletions were separated using polyacrylamide gel electrophoresis 8% followed by staining with ethidium bromide and a visualization on a transilluminator by the UVP Company (USA). Separation profiles were photographed using a digital camera. Polymorphic variants of genes regulating the cardiovascular function of combatants (ACE I/D, BDKRB2 -9/+9) [1] were studied in the course of the research.  

Functional performance level of the taekwondokas of different skill levels was determined using the modified technique of A.N. Polikarpochkin [4] which involves the quantitative informative assessment of the integral readiness of athletes for training and successful competitive activity.   

Student’s test (t) was used to evaluate the significance of differences of mean values. Statistical interconnections were studied by means of Spearman’s nonparametric correlation analysis. Significance of the differences in the frequency of alleles, genotypes and genotype combinations of compared samples as well as compliance of the genotypes distribution with the Hardy-Weinberg equilibrium were determined using chi-square test (for large samples) or Fisher’s exact test (for small samples). In terms of the quantitative characteristic (physiological and other criteria) the groups were compared using the unpaired t-test (when comparing two groups, for example, carriers of two different genotypes) or analysis of variance (ANOVA) (when comparing three or more groups). Regression analysis was used to assess the genetic component contribution to phenotypic variance. Differences were considered significant at р<0.05.

Research results and discussion. Table 1 presents the results of the study of the functional performance level of taekwondokas of low, average and high skill levels. As seen from Table 1, tests to determine the functional performance of the athletes were meant primarily to assess the functional state of the cardiorespiratory system.

Table 1. Functional performance of taekwondokas of different fitness levels, M±m

 

Functional performance indicators

Groups of subjects

р 1-2

р 1-3

р 2-3

RG, n=46

 

SG-1, n=54

 

SG-2, n=51

 

TEC, s

28.2±2.37

100%

34.7±2.54

123.1%

38.3±3.89

135.8%

˃0.05

˂0.05

˃0.05

FI, c.u.

52.4±3.43

100%

60.1±4.25

114.7%

68.7±4.31

131.1%

˃0.05

˂0.05

˃0.05

HR, bpm

64.2±1.89

100%

57.6±1.11

89.7%

52.6±1.79

81.9%

˂0.05

˂0.05

˂0.05

SHEC, ms

221.3±3.84

100%

210.8±4.23

95.3%

205.5±3.16

92.8%

˃0.05

˂0.05

˃0.05

VC, ml

3579.4±123.5

100%

4015.6±168.4

112.2%

4433.7±125.5

123.9%

˂0.05

˂0.05

˂0.05

IFP, %

44.76±2.13

52.01±2.57

58.79±3.12

˂0.05

˂0.05

˃0.05

Note. RG – reference group (low-skilled taekwondokas), SG-1 – study group (taekwondokas of average skill level), SG-2 – study group (highly-skilled taekwondokas); M±m – arithmetic mean of the indicator in the sample of subjects of the group in question ± standard error of the arithmetic mean; p 1-2 – degree of significance of differences between the first and second groups, etc.

 

ACE gene (angiotensin I converting enzyme; АСЕ) is a zinc-containing protease that catalyzes the conversion of angiotensin-I into angiotensin-II. In addition, the enzyme carries out inactivation of bradykinin (vasolidator) into inactive metabolites. Bradykinin is one of the stimulators of nitrogen oxide (NO) – primary endothelial relaxation factor - production by endothelium [2].

The АСЕ gene is localized in locus q23.3 of chromosome 17 and contains 26 exons. An insertion/deletion polymorphism (indel) accounted for by the presence (insertion – allele I) or absence (deletion – allele D) of a fragment in 287 base pairs – Alu repeat – was detected in intron 16 of the gene during ACE gene cloning. This polymorphism is associated with the enzyme levels in the blood: protease enzyme content increases in individuals homozygous for the D allele [1]. Polymorphism I/D of the ACE gene is linked with the cardiovascular system and the musculoskeletal function, training impact on the muscle efficiency and hypertrophy. There are 3 ACE genotypes of I/D polymorphism that are relevant in descending order for “endurance”: I/I, I/D and D/D. Relevance for “speed/force” in descending order is as follows: D/D, I/D and I/I  [1;  2].

Table 2. Frequencies of alleles and genotypes of the polymorphic marker of angiotensin-converting enzyme gene (ACE) in the sample of taekwondokas

Allele

(genotype)

Groups of subjects

р 1-2

р 1-3

р 2-3

1st (RG)

n=46

2nd (SG-1)

n=54

3rd (SG-2)

n=51

ni

pi±sp %

(CI)

ni

pi±sp %

(CI)

ni

pi±sp %

(CI)

ACE

I/I

8

17.4±0.05

(7.8-31.4)

15

27.8±0.06

(16.5-41.6)

19

37.2±0.07

(24.1-51.9)

0.32

0.05

0.41

I/D

20

43.5±0.07

(28.9-58.9)

24

44.4±0.07

(30.9-58.6)

24

47.1±0.07

(32.9-61.5)

1.00

0.88

0.94

D/D

18

39.1±0.07

(25.1-54.6)

15

27.8±0.06

(16.5-41.6)

8

15.7±0.05

(7.02-28.6)

0.32

0.02

0.21

χ2 (p.value 1-2) 2.141 = (0.3429)

χ2 (p.value 1-3) = 8.456 (0.0146)

χ2 (p.value 2-3) = 2.517 (0.284)

ACE

I

36

39.1±0.05

(29.1-49.9)

54

50±0.048

(40.2-59.8)

67

62.6±0.047

(52.7-71.8)

0.16

0.002

0.08

D

56

60.9±0.05

(50.1-70.9)

54

50±0.048

(40.2-59.8)

40

37.4±0.047

(28.2-47.3)

0,16

0.002

0.08

χ2 (p.value 1-2) = 2.372 (0.1628)

χ2 (p.value 1-3) = 10.928 (0.0024)

χ2 (p.value 2-3) = 3.477 (0.0841)

Note. Here and in Table 3: CI – confidence interval; P 1-2 - degree of significance of differences between the first and second groups, etc.; χ2 (p.value 1-3) – chi-square (significance level achieved).

 

Table 2 presents the results of the study of polymorphic variants of ACE I/D genes in taekwondokas of different skill levels. 

Three genotypes and two alleles were detected while typing I/D polymorphism of the ACE gene. In the RG of low-skilled taekwondokas the ACE*I/*I genotype is presented with the frequency of 17.4%, the ACE*I/*D genotype – with the frequency of 43.5% and ACE*D/*D – with the frequency of 39.1% (see Table 2). The frequency of the ACE*I allele in this sample was 39.1%, the ACE*D allele is found with the frequency of 60.9%.

In the group of athletes of average skill level (SG-1) the ACE*I/*I genotype is presented with the frequency of 27.8%, the ACE*I/*D genotype – with the frequency of 44.4% and ACE*D/*D - with the frequency of 27.8%. Frequencies of the alleles ACE*I and ACE*D are found in equal proportions of 50±0.048% in this sample (see Table 2).

In the group of highly-skilled taekwondokas (SG-2) the ACE*I/*I genotype is presented with the frequency of 37.2%, the ACE*I/*D genotype – with the frequency of 47.1% and ACE*D/*D – with the frequency of 15.7%. The frequency of the ACE*I allele in this sample was 62.6%, the ACE*D allele is found with the frequency of 37.4%. The observed distribution of genotype frequencies corresponds to the theoretically expected Hardy-Weinberg equilibrium (χ2 = 10.928, р = 0.0024).

A statistically significant increase in the frequency of the ACE*I/*I genotype in the sample of highly-skilled taekwondokas with the highest level of integral functional performance compared to the group of low-skilled athletes and with the lowest level of integral functional performance (37.2% in SG-2 vs 17.4% in RG, р≤0.05) was revealed during pair-wise comparison of the frequencies of genotypes and alleles in groups of athletes with different levels of functional performance.

The frequency of the heterozygous ACE*I/*D genotype did not change significantly in any of the groups of taekwondokas.  

In the group of low-skilled taekwondokas with the lowest level of integral functional performance (IFP=44.76) the frequency of the ACE*D/*D genotype proved to be significantly higher (39.1% in RG vs 15.7 % in SG-2, р=0.02) only compared to the group of highly-skilled taekwondokas and with the highest (IFP=58.79) level of integral functional fitness among the three groups.

Calculation of the determination coefficient between the indicators of the integral functional performance of taekwondokas of different skill levels and the frequencies of the ACE*I allele showed a direct functional interrelation (R2=1.000, р<0.05), while calculation of the determination coefficient between the indicators of the integral functional performance of taekwondokas of different skill levels and the frequencies of the ACE*D allele (the significance of developing the strength/speed characteristic) showed an inverse functional interrelation (R2=1.000, р<0.05).

BDKRB2 gene is one of the major mediators of the bradykinin effect. Bradykinin is a polypeptide of the kinin group produced during activation of the kallikrein-kinin blood system. Bradykinin lowers the vascular tone (stimulates production of nitric oxide by endothelial cells, which causes blood pressure to fall), increases capillary permeability, promotes contraction of smooth muscles in the bronchi and other organs. It also improves ventricular stroke volume, is involved in the repair processes, protects cardiac muscle cells from ischemia and has insulin-like properties stimulating glucose uptake by peripheral tissues, modulates transmission of nerve impulses in the central and peripheral nervous systems [1].

A functional insertion/deletion polymorphism (insertion or deletion of 9 nucleotides; +9/-9) was found in the 1st exon of the BDKRB2 gene (localization - 14q32.1-q32.2). High-level expression of the gene is associated with the deletion of the insert (-9) and therefore a more pronounced vasolidating effect [1]. There are 3 genotypes of the BDKRB2 (-9/+9) polymorphism – «-9/-9», «-9/+9» and «+9/+9». Relevance for the characteristic of “endurance” is as follows in the descending order: «-9/-9», «-9/+9» and «+9/+9».  Allele BDKRB2*-9/-9 is associated with a high level of performance of athletes of various sports [1].

Table 3 presents the results of the study of polymorphic variants of BDKRB2 genes in taekwondokas of low, average and high skill levels.

Three genotypes and two alleles were detected while typing (-9/+9) polymorphism of the BDKRB2 gene. In the RG of low-skilled taekwondokas the BDKRB2*-9/-9 genotype is presented with the frequency of 13.04%, the BDKRB2*-9/+9 genotype – with the frequency of 47.83% and BDKRB2*+9/+9 – with the frequency of 39.13% (see Table 3). The frequency of the BDKRB2*+9 allele in this sample was 63.04%, the BDKRB2*-9 allele is found with the frequency of 36.96%.

Table 3. Frequencies of alleles and genotypes of the polymorphic marker of bradykinin receptor gene β2 (BDKRB2) in the sample of taekwondokas

Allele

(genotype)

Groups of subjects

р 1-2

р 1-3

р 2-3

1st (RG)

n=46

2nd (SG-1)

n=54

3rd (SG-2)

n=51

ni

pi±sp %

(CI)

ni

pi±sp %

(CI)

ni

pi±sp %

(CI)

BDKRB2

+9/+9

18

39.13±0.07

(25.1-54.6)

15

27.78±0.06

(16.5-41.6)

8

15.7±0.05

(7.02-28.6)

0.32

0.01

0.21

-9/+9

22

47.83±0.07

(32.9-63.1)

26

48.15±0.07

(34.3-62.2)

21

41.2±0.07

(27.6-55.8)

1.00

0.65

0.60

-9/-9

6

13.04±0.05

(4.94-26.3)

13

24.07±0.05

(13.5-37.6)

22

43.1±0.07

(29.3-57.8)

0.25

0.003

0.06

χ2 (p.value 1-2) = 2.685 (0.2612)

χ2 (p.value 1-3) = 12.887 (0.001)

χ2 (p.value 2-3) = 4.992 (0.0824)

BDKRB2

+9

58

63.04±0.05

(52.3-72.9)

56

51.85±0.048

(42.3-61.6)

37

36.3±0.05

(26.9-46.4)

0.15

0.004

0.12

-9

34

36.96±0.05

(27.1-47.7)

52

48.15±0.048

(38.4-57.9)

65

63.7±0.05

(53.6-73.0)

0.15

0.004

0.12

χ2 (p.value 1-2) = 2.103 (0.1474)

χ2 (p.value 1-3) = 9.106 (0.0035)

χ2 (p.value 2-3) = 2.462 (0.1168)

 

In the group of athletes of the average skill level (EG-1) the BDKRB2*+9/+9 genotype is presented with the frequency of 27.78%, the BDKRB2*-9/+9 genotype – with the frequency of 48.15% and BDKRB2*-9/-9 - with the frequency of 24.07%. Frequency of the allele BDKRB2*+9 was 51.85%, the allele BDKRB2*-9 is found with the frequency of 48.15% (see Table 3).

In the group of highly-skilled taekwondokas (SG-2) the BDKRB2*+9/+9 genotype is presented with the frequency of 15.7%, the BDKRB2*-9/+9 genotype – with the frequency of 41.2% and BDKRB2*-9/-9 – with the frequency of 43.1%. The frequency of the BDKRB2*+9 allele in this sample was 36.3%, the BDKRB2*-9 allele is found with the frequency of 63.7%. The observed distribution of genotype frequencies corresponds to the theoretically expected Hardy-Weinberg equilibrium (χ2 = 9.106, р = 0.0035).

A statistically significant increase in the frequency of the BDKRB2*-9/-9 genotype in the sample of highly-skilled taekwondokas with the highest level of integral functional performance compared to the group of low-skilled athletes with the lowest level of integral functional performance (43.1% in SG-2 vs 13.4% in RG, р=0.003) was revealed during the pair-wise comparison of the frequencies of genotypes and alleles in the groups of athletes with different levels of functional performance.

The frequency of the heterozygous BDKRB2*-9/+9 genotype did not change significantly in any of the groups of taekwondokas.  

In the group of low-skilled taekwondokas with the lowest level of integral functional performance (IFP=44.76) the frequency of the BDKRB2*+9/+9 genotype proved to be significantly higher (39.13% in RG vs 15.7 % in SG-2, р=0.01) only compared to the group of highly-skilled taekwondokas with the highest (IFP=58.79) level of integral functional performance among three groups.

Calculation of the determination coefficient between the indicators of the integral functional performance of taekwondokas of different skill levels and the frequencies of the BDKRB2*+9 allele showed an inverse functional interrelation (R2=1.000, р<0.05), while the calculation of the determination coefficient between the indicators of the integral functional performance of taekwondokas of different skill levels and the frequencies of the BDKRB2*-9 allele showed a direct functional interrelation (R2=1.000, р<0.05).

Conclusion. The following patterns were found by us while analyzing polymorphic loci of molecular-genetic markers ACE I/D, BDKRB2 -9/+9 that determine performance of the cardiorespiratory and anaerobic glycolytic systems of combatants: a direct correlation (r = 1.000) was observed between the high level of integral functional performance of taekwondokas and the frequencies of the alleles of   ACE*I, BDKRB2*-9 genes (determination coefficient was R2=1.000, р<0.05 in all the cases). The optimal number of alleles of these markers for achieving success in taekwondo is from 4 to 6. Thus, these alleles of the studied genes can be used as markers of the objective assessment of predisposition of taekwondokas for efficient training and competition activity. This phenomenon, in accordance with the genetic concept of sport qualification, reflects accumulation of variants of genes favoring certain motor activity in a sample of highly-qualified taekwondokas.

In terms of practical recommendations for optimizing the training process of taekwondokas we can suggest professional training adjustment based on the unique features of their particular genomes: for example, in case of the alleles of the ACE*D gene in place tools and techniques developing the functional state of the cardiorespiratory system should be additionally included into the training process of taekwondokas; in case of the alleles of the BDKRB2*+9 gene the share of tools and techniques stimulating aerobic performance enhancement in the training process should be increased.  

References

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Corresponding author: terzims@yandex.ru

Abstract
Polymorphic variants of genes associated with the integral functional performance in a sample of combatants of different skill levels were studied. To determine the influence of polymorphic variants of genes on the state of the cardiorespiratory and anaerobic glycolytic systems of combatants in their sport competency growth dynamics polymorphic variants of genes regulating these systems were studied (ACE I/D, BDKRB2 -9/+9). Prospects and feasibility of using the analysis of polymorphic loci of genetic mark-ers that influence aerobic and anaerobic performance of combatants as a physiological substantiation of the sport training technology to improve reliability and efficiency of the system of individual selection and optimization of functional performance of elite taekwondokas are shown.