Comparison of HIF1 Gene Polymorphism (Rs11549465) Among Elite and Amateur Power Athletes Versus Non-athletes

Background: One of the major energy systems for power performance in muscles is glycolysis that is regulated by the levels of Hypoxia-inducible factor-1α (Hif1) which in response to hypoxia act as a transcription factor. The goal of this survey was the comparison of HIF1 gene polymorphism (rs11549465) among elite and amateur power karate-kas versus non-athletes. Methods and Results: The C/T polymorphism allelic and genotypic's distribution of Hif1 rs11549465 gene was determined in this survey throughout 550 healthy Iranian subjects that divided in power karate-kas groups that sub-divided into elite (86 male and 86 female) and amateur karate-kas (100 male and 72 female) versus 206 individuals in non-athlete group (100 male and 106 female). After obtaining the written consent of all participants in the study, in seated mode, at a given time and in the diagnostic-laboratory, the amount of 5cc blood from brachiocephalic vein was taken. The Hif1 gene T/C polymorphism was assessed with PCR by using the forward and the overturn primers. Amplicons, the length were treated with the restriction endonuclease Hph I (Enzyme). Also RFLP were analyzed by electrophoresis separation on 1% agarose gel. Non–parametric Chi-Square and multinomial regression tests were used for assess the differences through the SPSS software and the P value less than 0.05 was considered as a signicant different. Chi-square test showed genotype frequencies of Hif1 TC rs11549465 (TC genotype: 25.6% vs. TT genotype: 11.4%; CC genotype: 63%) were signicantly different in these population (χ 2 = 99.889, P=0.000). Furthermore, the results of multiple regressions demonstrated that there isn’t general linear relationship between Hif1 gene expression and power ability of karate-kas. Conclusions: From the genetic talent aspect, there wasn't any correlation between Hif1 gene expressions with Iranian karate competitor’s levels.


Introduction
Genetic factors have major effects on elements of the athletic performance like psychological traits and phenotypes (1). Evidence suggested that genetic markers may explain variation of physical performance specs in reply to endurance or strength training (2). For example, there are valid roles for genetic research and the Australian Institute of Sport (AIS) valid genetic study which aims to increase comprehension of athlete talent to injury or illness (3). Nowadays many commercial companies being predict athletic ability throughout genetic testing. Polymorphisms identifying sport talent (genetic testing) could revolutionary to the eld of physical performance (4). The result of sport talent is identify athletes suitable to the specs of the sportive branch selected (5); so sport talent companies may explore the might of initial success for sportsmen partly on genetic database. Hence, genetic sport talent can bring many bene ts from economic e ciency and success, likely. For this reason, the SNPs were investigated in order to expedite the choice process for talented individuals who wish to be a huge champion. Although be notice that due to much number of SNPs related to sport performance and much cost of review them, we may not be able to test all of them, therefore it needs to provide exact survey to ensure the correct relation information for each famous sport that be rely about it via genetic sport talent.
Exercise condition increases oxygen demand which is required in muscle contraction (6). Glycolytic system is one of the most important energy supplies of skeletal muscles during highly intense exercise under anaerobic performance in sport. This imbalance condition between the amounts of oxygen demand and supply in body is named hypoxia (6). There is a hypothesis that continuous exercise training induced hypoxia, as a bene cial muscular adaptations, would result in induce included increased expression of genes that codes glycolysis enzymes (7). Hypoxia via the expression of peroxisome proliferator activated receptor-gamma coactivator 1α (PGC-1α) regulates the HIF-1α (8). Likely, HIF1α initiates the transcription of various hypoxia-adaptive genes like glycolysis, hence improves glucose metabolism (9). The Pro582Ser polymorphism (rs11549465) was detected in the HIF1A gene that encodes α subunit of HIF-1 protein, resulting in the replacement of proline (Pro) with serine (Ser) at amino acid 582 (10). Pro582Ser is present in exon 12 (C/T at bp 85; rs11549465) and T allele (Ser582) improves glucose metabolism (1). The HIF1A Ser allele can be associate with allele favoring the development of the speed/ power capacity; hence it subdivided in power/strength-related genetic markers. It is reported that increased ratio of fast-twitch muscle bers in Vastus Lateralis was associated by 582Ser allele (11).
Karate is one of the most competitive sports in Olympic. Base on the world karate federation rules, the rst qualify punch or kick of competitors; result in the point and the next punch or kick of his/her opponent will not get the score; even though they be correct. So, high speed of karate-ka among the other kind of tness factors would be important for each elite karate-ka, and hence, this kind of sport would appropriate in power/strength-related sport (Batavani et al. unpublished data).
To date, Ahmetov et al (2008) reported that the frequency of the HIF1A 582Ser allele in Russian weightlifters vs. controls was signi cantly higher and increase of its frequency had association with their competition levels (11). Also Cięszczyk et al (2011) reported that the incidence of the HIF1A Ser allele genotype polymorphism in power-orientated runners, swimmers and weightlifters was signi cantly higher than sedentary controls, too (12). These results were ingeminate by Gabbasov et al (2013)  upgraded their rankings to the black belt, but they hadn't get to the elite group's places. Subjects in the control group didn't have any regular exercise experience in their life.
The methods of sampling were explained to the subjects and they signed their written consent form to participate in the survey. The patient samples were excluded out from the survey. Besides, all people with familial relation were left out from survey; and bioethics items were considered relying on the standards described by advisory board of the faculty of sport sciences. In seated mode, at a given time and in the diagnostic-laboratory, the amount of 5cc blood from brachiocephalic vein was taken and according to the alkali treatment protocol genome was isolated (16).
In this study salting-out method was used for DNA extraction. All of samples were kept at 4 o C. Then, the amount of 0/5 ml of blood sample with cell lysis buffer add to micro tube (1.5 ml), 15minutes was incubated by ice that result to fragility on the membrane of red blood cells and centrifuged at 9000 rpm for 5 min, hence of the supernatant discarded and cell lysis buffer was added. After 10 minutes of incubation, 5 minutes centrifuged at 9000 rpm 5 times until the time of observation white pink pellet. The produced pellet by nuclease lysis buffer (to lysing the membrane of nucleus) was dissolved. Then sodium per chlorate was added and cloudy protein made. chloroform was added then at 12000 rpm for 10 minutes centrifuged. Ethanol (96%) was added to water phase of tube that was contained DNA, then it was shacked again. by observing the DNA cluster, tube at 12000 rpm was centrifuged for 10 minuted, then ethanol (70%) was added, again at 12000 rpm centrifuged for 10 minutes. After drying the tube at room temperature, sterile distilled water was added and extracted DNA was placed at -20 °C (17).
Quality checking of isolated DNA By the spectrophotometer and measured via the ratio of DNA optical density (A260) and protein optical density (A280) the concentration and purity of the extracted DNA were analyzed. also for determining of DNA degradation, electrophoresis was performed on 1% agarose gel, too.
Analysis of DNA ampli cation and restriction fragment length polymorphism (RFLP) Via PCR using a speci c primer pair (the forward primer, 5´_AGGACACAGATTTAGACTTGG_3´; the overturn primer, 5´_GGAATACTGTAACTGCGCTTTG_3) and the enzyme of Hph I the HIF-1α gene T/C Analyses of Data.
The graphs are drawn via Graph Pad in Stat and gure is drawn via Excel software, too. Non-parametric Chi-Square and multinomial regression tests were used for assess the differences through the SPSS software and the P value less than 0.05 was considered as a signi cant different. Con dence interval is 95% and for greater certainty the multinomial regression test was used.

Results
Quantitative analysis of DNA samples by spectrophotometer for 5 samples (typical) on agarose gel 1% has been shown (Fig. 1). Genotype and allelic frequencies analyses by the HIF1gene (T/C polymorphism) in karate-kas and nonathletes have showed that the CC genotype (63%) had the most common incidence, while the TT genotype (11.4%) and TC genotype (25.6%) were less common. Totally, Chi-Square tests demonstrated that there is a signi cant difference in frequencies of HIF1 gene allelic (χ2= 99.889, p = 0.000).  Fig. 3).  Result showed HIF1 gene T/C polymorphism is not signi cantly associated with the physical performance´s levels of karate-kas in Iranian population. The frequency of T/T genotype vs. C/C genotype decrease in professional karate-kas and amateur karate-kas versus to control non-athletes (odd ratio < 1), but it wasn't signi cant (P > 0/05). Also the frequency of T/C genotype vs. C/C genotype increase in professional karate-kas and amateur karate-kas versus to control non-athletes (odd ratio > 1), and however it was not signi cant, too (P > 0/05). Odd ratio of T/T and T/C genotype compare to C/C in professional group versus control were 0.

Discussion
This survey compared HIF1 gene polymorphism rs11549465 among elite/ amateur power athletes vs. non-athletes. Analyze showed that the HIF1 gene TC genotype occurrence in Iranian population (elite and amateur karate-kas and non-athletes, in both male and female) were signi cantly different among high incidence of CC, TC and TT; respectively. It's worth noting that HIF1 genotype frequency and T/C allele occurrence incidence between all groups (among elite, amateur and non-athlete) were not statistically differences signi cant and wasn't associated with the physical performance´s levels of karate-kas.
Our results were in con rm by the results that reported by Eynon et al. (2011) that did not nd signi cant differences in their survey in HIF1A genotype distribution and Ser582 allele frequency of endurance and sprinters athletes vs. controls (15). These results showed that HIF1A Ser allele carriers are not more predisposed to power-orientated karate-kas.
Our survey doesn't con rm previews studies by Ahmetov et al (2008) that reported a association between the HIF1A Pro582Ser polymorphism and increased levels of Russian sprint/strength weightlifters achievement and Zoll et al. (2006) that showed long exercise plan (i.e., 6 weeks) signi cantly (up to 104%) increased hif-1 gene expression in Quadriceps muscle, too (11,18).
Differences in the sports conditions, training design (frequency and duration), methods of DNA extraction, size of subjects, and experience and training sessions of subjects, etc…could explain these discrepancies, likely. Especially our study, had blood sampling protocol instead of muscle biopsies. Mounier et al (2009) found decrease in transcriptional regulations of the hif-1α gene when they used muscle biopsy; while it didn't change when they had blood sampling to extract DNA (7). In our survey DNA was extracted from peripheral blood samples.
In the state of hypoxia many factors such as the amount of ATP in muscles, high enzymatic performance for energy supply via glycolysis and phosphagenic systems, creatine phosphate, glycogen, etc... affecting the high anaerobic ability (11).
From the other hand, a complex interaction of psychological, socio-cultural, and etc factors result in variation of athletic performance (23). Mitchell et al (2005) reported that karate-Do is a low dynamic and high static sport; hence it need moderate total cardiovascular demands, too (24). Aligned with it Batavani et al (2017) had been proposed to be the association between the CK-MM gene A/G genotype and brilliant karate-kas performance (19). Aerobic energy supply and power ability of muscles among exercise are important, too. Indeed elite karate performance is a polygenic trait, with over one polymorphism like ACE and CKMM association (19). Also, elite performance in karate seems depends on the simultaneous presence of multiple tness factors such skill, speed and etc..., likely.
Indeed might be having a suggestion that the HIF1A Pro582Ser polymorphism is not important in karate athletes. Hence, for some branches of sport which are yielded by intensive training, suitable genetic is not enough to attach the optimal sport performances, basically. However, the effect of genes for adaptation and responding to exercise, unable to be ignored (25). Athletic performance that predicted by genetic pro les examination alone, is not reliably (26). The most branches of sports need the combination of sprint or power and endurance demands together such as many other factors, including a broad variety of genetic, physical, environmental and psychological elements to reach the success, too (3). Specially, it be suggested that between the HIF1 gene polymorphism and athletic status replicate additional large scale prospective studies in some worldwide branches of sports, separately. However, this conclusion

Conclusion
Our study investigated the relation among the HIF1A Pro582Ser polymorphism and karate performance. The investigated group included of elite and amateur karate-kas, and the results of our survey did not indicate the relation between HIF1A Ser 582 allele and karate level achievement. Indeed, the ndings showed no association between HIF1A gene Pro582Ser polymorphism and Iranian karate-kas performance. Therefore it suggested that the HIF1A gene can't be taken into consideration as a genetic marker in Iranian power-orientated karate-kas. Hence that genetic association studies must be interpreted with caution, likely and the accordingly use of Hif1 genetic experiments as an only predictor of karate talent is unscienti c and invalid.  It shows simultaneous bar plot of female and male HIF1 allelic in different conditions: elite, amateur and non-athlete.