A.A. Zolotareva¹
PhD O.S. Glotov^{1, 2}
PhD M.V. Aseev^{1, 2}
E.S. Vashkovova¹
Dr.Med., Professor S.G. Shcherbak^{1, 2
1}St. Petersburg State University, St. Petersburg
²City Hospital No. 40, St. Petersburg

Keywords: genetic polymorphism, acute altitude sickness (AAS), mountaineering, allele frequency analysis, hypoxia.

Introduction. At high altitudes, a person begins to develop hypoxia, or in other words - oxygen deficiency - reduction of oxygen supply to tissues. Hypoxia is stressful for the body and makes it adapt to the ambient conditions. Oxygen deficit is displaced by other methods of oxygen delivery. Tolerance to hypoxia in mountaineers represents an interesting model for studying in vivo the mechanisms of adaptation to the conditions of low oxygen level.

It has now been established that the key role in the cellular response to oxygen deficit (anoxia) is played by a number of closely related transcriptional factors called hypoxia-inducible factors (HIFs) that serve as transcriptional activators and key regulators of a variety of cellular and systemic responses to hypoxia (Wang et al., 1995). The most important role in the process of activation of adaptation processes belongs to HIF-1α. HIF-1α activates genes that allow the cells to respond to oxygen deficit. HIF-1α targets are the genes as follows: VEGFA, EPO, and NOS3 (eNOS) that exert a direct or indirect effect on the cellular tolerance to hypoxia. Changes in the nucleotide sequence of these genes may cause increased or decreased activity of their products, which as a result will affect the cellular tolerance to anoxia. One of the main mechanisms that ensure the body's tolerance to hypoxia is vascular regulation of blood pressure, and this process is associated with reduced nitric oxide level in the blood. Nitric oxide is a strong active agent that can cause relaxation of the smooth muscles in the vessel walls, prevent adhesion to the vessel walls, as well as increase endothelial permeability. When the nitric oxide level decreases, the artery opening narrows, which results in an increase in blood pressure, and enhances the likelihood of thrombosis. In addition, nitric oxide participates in the regulation of the respiratory function and gastrointestinal system, nerve impulse transmission, angiogenesis, as well as in the formation of olfactory memory. The nitric oxide synthesis is due to the work of nitric oxide synthetase, an enzyme that synthesizes nitric oxide from L-arginine, during which the final product L-citrulline is formed. The gene is located at the 7q36 locus, has a size of 21 kb., and consists of 26 exons (Miyahara et al., 1994). At hypoxia eNOS affects vascular tone, inhibits leukocyte adhesion, activation, platelet adhesion and aggregation. The mechanism of its interaction with HIF-1 is yet to be studied, however, a series of researches mention the activation of NOS3 by HIF-1 (Kravchenko N.A. et al., 2008). There is a number of SNP in the NOS3 gene that are associated with arterial hypertension, for instance, guanine (rs1800780) and thymine (rs1799983) are associated with hypertensive disease susceptibility, and cytosine (rs2070744) - with a decrease in the gene transcription activity, and better tolerance to aerobic exercise (Yang B., et al., 2012, Augeri A.L., et al., 2009).

Hypoxia is distinguished by the fact that a pathological condition can develop suddenly or gradually. If a person suffering from mountain sickness continues to climb regardless of feeling bad, the chances of acute altitude sickness (AAS) get higher, since the protective functions of the body can no longer provide sufficient compensation. There are no distinct objective clinical and neurological symptoms of this form of mountain sickness. The worst possible manifestation of severe AAS is the development of pulmonary or cerebral edema. These pathological conditions are the most common cause of death at high altitudes. Moreover, death occurs when the swollen cerebral cortex, constricted by the calvaria, and the cerebellum, wedged into the spinal cord, cause the brain to swell, while during pulmonary edema – when thick foaming results in asphyxia by blocking the air-passages (Harvey V., 2014). In this connection, any method of estimation of the risk of AAS is relevant and necessary to protect athletes’ health (Glotov O.S. et al., 2013).

Objective of the study was to determine the frequency of SNP markers of the NOS3 gene - the target of the transcription activator HIF-1α, associated with hypoxia, in mountaineers and population-based reference group depending on the altitude and presence/absence of AAS.

Methods and structure of the study. The study involved 80 mountaineers studying at St. Petersburg State University, aged 20 to 60 years, living in the territory of the Russian Federation, who have had an over 3,000 m climbing experience at least once in their life (study group) and a population-based reference group. This group was divided into several subgroups (see Table 1). All subjects provided their written informed consent. Physical condition, working capacity, well-being, as well as degree of tolerance to hypoxic exposure at altitude was estimated by means of a questionnaire survey, which included: individual information, anthropometric characteristics, physical activity, climbing (including personal achievements in this area), symptoms of mountain sickness, diet and habits, diseases and medicines (containing the data on the diseases of both the subject under study and his/her immediate family members). This questionnaire was developed by H. Montgomery and is being actively used in the hypoxia research (Montgomery H.E. et al., 1998).

Table 1. Quantitative characteristics of the studied groups.

Genomic DNA was extracted from the peripheral blood lymphocytes according to the protocol proposed in Sambrook’s manual (Sambrook et al., 1989).

SNP in NOS3 (eNOS) gene was analysed by choosing the TaqMan-probe-based reagents: TaqMan® genotyping assay mix primers ("Applied Biosystems", USA), and a special mix for real-time PCR TaqMan®GTXpress ™ Master Mix ("Applied Biosystems", USA). The 10 μl reaction mix (according to the manufacturer's protocol) included: 5 μl of TaqMan® GTXpress ™ Master Mix, 0.5 μl of TaqMan® genotyping assay mix, 2.5 μL of DNAase-free water and 2 μl of DNA nanomatrices. This mix was added to the basins of the MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode plate ("Applied Biosystems", USA). The reaction was carried out using the device Applied Biosystems 7500 Real Time PCR fast. The manufacturer recommended the amplification mode. ABI 7500 software ("Applied Biosystems", USA) was used to record the document. Alleles were identified both manually and in the automatic mode in the program. The genotype was determined based on the findings.

The allele and genotype frequencies were analyzed using the GraphPad InStat software (USA) by calculating the P value. Additionally, for the purpose of statistical significance the odds ratio (spot and interval coefficient) and positive predictive value (spot and interval) with their 95% confidence intervals (Rebrova, 2002). The data were compared with those obtained in the reference group. The critical level of significance, sufficient for rejection of the null hypothesis, was equal to 0.05 (p=0.05).

Results and discussion. A molecular genetic testing of 80 DNA samples of the mountaineers was conducted. The real-time PCR method was applied to study rs1799983 and rs2070744 (NOS3 gene) both in the study group and reference group, which included 70 DNA samples (40 men and 30 women) and formed a population sampling. A comparative analysis of the allele and genotype frequencies within the mountaineers' group was carried out with due regard to its division into several subgroups based on altitudes and presence/absence of AAS. The intra-group comparison of the allele frequencies at rs1799983 and rs2070744 of the NOS3 gene of the mountaineers with different altitude trekking experience (below 4,500 m, 4,500-6,000 m and above 6,000 m) and the reference group data, no statistically significant differences were detected. This result may indicate both the sampling specificity and the fact that these substitutions do not affect the mountaineers' tolerance to hypoxia. This may be due to the fact that the reference group is a mixed sampling - i.e. people in this group can be both resistant to hypoxic exposure at altitude and not.

For now it is known that protein products of these genes contribute mostly to the body's tolerance to hypoxia. Thus, it seems necessary to keep searching for SNP markers of these genes for this work to be continued. In addition, in order to make more accurate assessment of significance of the studied SNP markers, the sampling needs to be expanded; and it seems most perspective to expand it by means of DNA samples obtained in people with over 6,000 m climbing experience.

We also conducted a comparative inter-group study depending on the presence/absence of AAS. In our case, we examined the following groups:

• Mountaineers diagnosed with AAS;

• Mountaineers having no signs of AAS;

• Population-based reference group.

We found that thymine at rs2070744 of the NOS3 gene is associated with the development of AAS. Previously it had been shown that allele C of the rs2070744 polymorphism is associated with higher tolerance to aerobic exercise (Augeri AL et al., 2009). According to our study, it is T allele that is associated with the development of AAS (in T allele carriers the AAS risk is 4 times higher than in C allele carriers), which is not rejected by the literature data; what is more, PPV=0.61 (interval estimate - 0.45 to 0.75). This suggests that the AAS risk in the T allele carriers is 61%. However, since one of the PPV interval estimate levels is below 50%, it is not safe to say that T is a risk allele. The obtained data enable us to hypothesize that the T allele of the rs2070744 polymorphism affects the ability of the nitric oxide synthase to participate in vascular relaxation, thus increasing the AAS risk. Therefore, it is necessary to continue with research on this allele as a potential marker of the AAS risk.

Conclusion. Given that hypoxia is a complex disorder, further research is to be focused on the allelic associations of the studied genes not only with altitudes and development of AAS, being a virulent form of hypoxia, but also with the specific symptoms of these diseases. This task can be fulfilled by applying an in-depth statistical analysis.

The study was supported by Project Grant No 14-50-00069 of the Russian Research Foundation.

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Corresponding author: olglotov@mail.ru

Abstract

Hypoxic tolerance of mountaineers provides an interesting subject for in vivo studies of hypoxic adaptation mechanisms activated by oxygen deficit. We have studied rs2070744, rs1799983 SNP-markers in the NOS3 (eNOS) gene by Real-Time Polymerase Chain Reaction (PCR) method in 80 mountaineers who have had an over 3,000 m climbing experience at least once in their life versus the referance group. For the purposes of the statistical data analysis, we grouped the sampled mountaineers depending on the reportedly reached altitudes and diagnosed/ non-diagnosed acute altitude sickness (AAS).

No significant intergroup data differences were found for the frequencies of alleles in rs1799983 and rs2070744 positions in the altitude-specific groups (under 4500 m, 4500-6000 m and above 6000 m) versus the reference group. Furthermore, no statistically significant correlations were found in the SNP-markers in the NOS3 gene versus the maximal altitudes reported by the subjects. The AAS/ non-AAS group data showed correlation of the thiamine in rs2070744 position in the NOS3 gene versus the AAS degree. The study data give reasons to assume that the T-allele carriers are prone to AAS with a probability rate of 61%; and the data gives us the reasons to offer a hypothesis that the T-allele in rs2070744 position may be suppressing the nitrogen oxide synthase effects on the vessels relaxation mechanism thereby increasing the risks of AAS.