ГЕНЕТИКА, 2015, том 51, № 3, с. 389-392
CKM GENE POLYMORPHISM IN RUSSIAN AND POLISH ROWERS
© 2015 г. J. Eider1, I. I. Ahmetov2, 3 4, O. N. Fedotovskaya2, W. Moska5, P. Cieszczyk1, A. Zarebska5,
Z. Czubek5, T. Klocek6, M. Stepien-Slodkowska1, A. Maciejewska-Karlowska1, M. Sawczuk1
1University of Szczecin, Faculty of Physical Culture and Health Promotion, Szczecin 71-065, Poland
2St. Petersburg research institute of physical culture, sports genetics laboratory, St. Petersburg 191040, Russia 3Kazan state medical university, laboratory of molecular genetics, Kazan 420012, Russia 4Volga region state Academy of physical culture, sport and tourism, sport technology education research laboratory, Kazan 420138, Russia 5Academy ofphysical education and sport, faculty of tourism and recreation, Gdansk 80-336, Poland 6University school of physical education, faculty of physical education and sport, Krakow 31-571, Poland
Received April 29, 2014
Muscle-specific creatine kinase (CKMM) plays a vital role in the energy homeostasis of muscle cells. The A/G variation (rs8111989) located in the 3'-untranslated region of the CKM gene has been found to be the most relevant in terms of genetic testing in sport. The aim of the presented study was to test the hypothesis that the G allele might represent a genetic element that contributes to the improvement of endurance performance in Polish and Russian rowers. The distribution of the CKM genotypes was examined in a group of Polish and Russian athletes in comparison with non-athlete controls. There were no statistical differences between the rowers and the control groups across the CKM genotypes when Polish or Russian participants were analyzed. Based on the obtained results, it may be speculated that the CKM A/G polymorphism is not an important determinant of endurance performance level in Polish and Russian rowers. However, these results should be interpreted with caution as they can be limited by many factors.
Creatine kinase (CK) catalyzes the reversible transfer of a phosphate from phosphocreatine to adenosine diphosphate, generating adenosine triphosphate in tissues such as brain and muscle that require large amounts of energy. The muscle-specific creatine kinase (CKMM) enzyme is one of several tissue-specific isozymes of creatine kinase (CK) that play a vital role in energy homeostasis of muscle cells [1, 2]. It has been shown that CKMM activities differ with respect to skeletal muscle fiber types, and the type II (fast-twitch) fibers showed significantly higher CKMM activity than type I (slow-twitch) fibers [3, 4]. It has been suggested that CKMM reduced activity may be a characteristic feature of the skeletal muscle of endurance athletes [2, 3, 5]. In humans, the CKMM-encoding gene CKM has been mapped to chromosome 19q13.2—13.3 [6, 7]. Within the sequence of the CKMM gene, more than 260 single nucleotide polymorphisms (SNPs) exist. Several studies have tried to link CKM gene polymorphisms with physical performance related phenotypes [2, 5, 8, 9]. However, only the A/G variation (rs8111989) located in the 3'-untranslated region of the CKM gene, also known as the CKM-NcoI SNP, was found to be relevant in terms of genetic testing in sport. It was suggested that the CKM-NcoI polymorphism might be associated with differential CKMM activities in myocytes . Other authors have postulated that the CKM genotype may be associated with
the expression and stability of its mRNA . Moreover, it has been reported that blood levels of creatine kinase (CK) may be used as a marker to reflect the magnitude of skeletal muscle destruction in response to exercise . Such exertion-induced muscle injury causes an increase in blood CK and it was also revealed that some individuals exhibit extreme increases in blood CK after exercise — those people have been characterized as high responders (HR) . It has been shown that athletes or individuals with the CKM-NcoI AA genotype had a six-fold higher likelihood of being HR compared with GG and AG genotypes. Because the G allele is the rare allele for the CKM-NcoI polymorphism, some have speculated that this allele is associated with a protective mechanism against exer-tional muscle breakdown . Several articles have been published showing a positive association of A/G variation with endurance athlete status or endurance capacity [2, 5, 9], while others failed to show any significant association [13—15]. This discrepancy raises the question of whether A and G allelic variants of the CKM gene are indeed genetic factors that can influence physical performance. We have decided to test the hypothesis that the G allele might represent a genetic element that contributes to greater endurance performance in Polish and Russian rowers. With the objective of confirming or rejecting this hypothesis, we examined the distribution of CKM genotypes in a
CKM genotypes and allele frequencies in Polish and Russian participants
Group n CK-MM genotype
AA AG GG
Polish rowers Polish controls 127 57 (44.9%) 57 (44.9%) 13 (10.2%)
684 348 (50.9%) 293 (42.8%) 43 (6.3%)
Russian rowers Russian controls 93 40 (43.0%) 36 (38.7%) 17 (18.3%)
1170 497 (42.5%) 520 (44.4%) 153 (13.1%)
G allele frequency, %
group of Polish and Russian athletes in comparison with non-athlete controls.
All procedures followed in this study meet the ethical standards in Sport and Exercise Science Research approved by appropriate local Ethics Committees. 127 Polish male rowers (27.5 ± 5.3 yr; both former and current competitors) were recruited for this study. The whole group ofPolish rowers included 21 athletes classified as "top-elite" (gold medalists in the World and European Championships, World Cups or Olympic Games) and 23 athletes classified as "elite" (silver or bronze medallists in the World and European Championships, World Cups or Olympic Games). An additional 49 athletes were classified as "sub-elite" (participants in international competitions). The others (n = = 34) were classified as "non-elite" rowers, all regional competitors with no less than 4 years of experience participating in the sport. For Polish controls, the samples were prepared from 684 volunteers (students from the University of Szczecin, 20.6 ± 0.9 yr). The athletes and controls were all Caucasians to avoid racial gene skew and to overcome any potential problems of population stratification. For the second part of the study, 93 Russian rowers (21.9 ± 3.8 yr) of a regionally or nationally competitive standard were recruited. There were 4 athletes classified as "top-elite", 44 athletes classified as "elite", 45 athletes classified as "sub-elite". Russian controls were 1170 healthy unrelated citizens of St. Petersburg, Moscow and Kazan (18.5 ± 2.2 yr) without any competitive sport experience. The athletes and control groups were all Caucasians.
The DNA was extracted from the buccal cells donated by the Polish and Russian subjects using a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Germany) or by alkaline extraction  or by using DNK-sorb-A and Proba-GS sorbent kits (Central Research Institute of Epidemiology and DNA-Technology, Russia) according to the manufacturers' instructions. To discriminate CKM G and A alleles (rs8111989) of the Polish samples were genotyped using an allelic discrimination method with Taqman probes  (Applied Biosystems, USA) and StepOne Real-Time Polymerase Chain Reaction (PCR) instrument (Applied Biosystems, USA) were used. The Russian samples were genotyped for the rs8111989 poly-
morphism by PCR and restriction enzyme digestion method . PCR primers were forward 5'-GGG ATG CTC AGA CTC ACA GA-3'; reverse 5'-AAC TTG AAT TTA GCC CAA CG-3', generating a fragment of 359 bp. PCR products were digested with Bspl9I restriction endonuclease (SibEnzyme, Russia). A x2 two-sided test or x2 test with Yates correction for small groups were used to compare the CKM alleles and genotype frequencies between athletes and control subjects. The level of statistical significance was set atp < 0.05.
All CKM genotype distributions for both athletes and controls met Hardy—Weinberg expectations (p > > 0.05 in all groups tested separately). The distributions of the CKM genotypes and alleles in Polish and Russian athletes and controls are given in table. The study revealed no statistical differences in the observed allelic and genotypic frequencies of the CKM A/G polymorphism between cases and controls, indicating no significant associations between the CKM DNA sequence variants and athletic status among rowers.
Several lines of evidence suggest that the CKM-NcoI single-nucleotide polymorphism (rs8111989) in the 3'-untranslated region might contribute to individual differences in physical performance. Previously, a CKM-NcoI restriction fragment length polymorphism was shown to be associated with enhanced physical performance and to contribute to differences in VO2max responses following endurance training in the HERITAGE family study [5, 9]. This A/G DNA variation, in the 3' untranslated region has also shown an association with running economy responses to an 18-week 5000-m training program . However, four independent studies investigating the CKM-NcoI SNP, including the original investigation in the Genathlete study [14, 15, 19, 20], found no significant association between the SNP and endurance performance. The findings of the classical case-control study indicated that the SNP rs8111989 at CKM gene did not contribute to the proper classification of subjects into high or low VO2max status . Subsequently, the association between the same CKM SNP and performance status was examined in professional cyclists, sedentary controls, and Olympic class runners and revealed no statistical differences for the CKM SNP prevalence among the three grou
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