| BackgroundHypoxic adaptation is a hot point in the biological evolution. Also, diseases caused by hypoxia are global public health concern. Acute mountain sickness(AMS) refers to the pathological responses exposed to acute hypoxia. It is a unique disease in the environment of high altitude hypoxia. The initiation and development of AMS will harm people’s health, reduce the working capacity and even hinder human migration. More importantly, the plateau is huge in China, and it is of great practical and historical importance in the military and politics. Therefore, to screen for the people at high risk and give them prompt prevent is crucial to furtherly reduce the incidence of AMS and even severe AMS.Early studies showed that under the same conditions, some people are susceptible to AMS and there are differences in their extent and rate. Although AMS is not a typical hereditary disease, AMS is an environment-induced complicated condition and lots of genes are involved. Thus, the studies focusing on the hereditay information will to some extent help understand the molecular mechanisms underlying the pathophysilogical appearance of AMS. Recent studies revealed that hypoxia-inducible factor(HIF) pathway plays an important role in people on different altitudes, and the mechanisms of adaptation to different conditions varied. Recent genome-wide scan results found a close correlation between the single nucleotide polymorphism(SNP) in the HIF pathway and the hypoxic adaptation of plateau Tibetan population and the Andes population. Therefore, to start the investigation of AMS-related genetic information from some important genes in the HIF pathway will provide rapid and effective genetic technique and indicator to score and evaluate the AMS risk, and furtherly, provide a potent rule for cancer, cardiovascular diseases, extremely hypoxic vocational exposure and sports medicine.This study is aimed to seek and find the SNP in the HIF pathway related with AMS susceptibility. Firstly, we carried out a prospective and observational study in the enolled low altitude native population, conduct the Lake Louise acute mountain sickness coring system(LLss)and detect some indexes. Secondly, we selected candidated genes and found some important gene sites in the HIF pathway, combined with genome-wide scan results and literatures. Finally, we correlated the gene sites with the development and extent of AMS as well as physilogical process, in order to find the crucial SNP and haplotype. These will provide evidence for the AMS scoring.Materials and Methods1. Subjects. We enrolled 603 low altitude native heathy men. They ascended from 500 m to 3700 m by plane. The subjects were divided into groups according to the LLS. We also collected some physilogical indexes and plasma samples to extract DNA. Inclusion criteria were(1) 18 to 45 years in age;(2) the subject and his parents are all Chinese Han population;(3) residence at or below 500 m for a long time;(4) no high altitude exposure(>2500 m) in the past 3 months.2. LLS system. On the premise of headache, AMS was diagnosed by the LLS System. AMS with a score of 3-4 is defined as mild AMS, while severe AMS has a score of 5 or more.3. Selection of gene and SNP sites. We screened all the introns and extrons of EPAS1(HIF2A)、HIF1A、PPARA and VEGFA genes and transcription initiation site upstream and downstream terminator of 5 kbp tag combined with recent genome-wide scan breakthrough results. Genotyping data derived from Chinese Han population in Beijing in the the human haplotype map project Ⅲ. The Haploview 4.2 software was used to construct linkage disequilibrium mapping and haploid domain. The Tagger software was used to select label SNP.4. Genetyping. We used Primer Premier 5.0 software to select candidated SNP sites and primer PCR amplification and sequencing primers. We applied the time of flight mass spectrum methods to detect the DNA samples in the EPAS1, HIF1 A, PPARA and VEGFA genes. We also used the TYPER software and furtherly obtained the SNP data classification. The results of genotyping were randomly selected as much as 10% of the second repeat parting confirmation.5. Using the SHEsis software to analysis haplotypes of VEGFA and HIF1 A gene and to calculate the frequency distribution.6. Statistical analyses. Continuous variables between groups were compared by Student’s two-sample t test; classification variables were compared by Chi-square test. Each SNP allelic frequency measurement by counting gene acquisition and each SNP genotypes distribution whether in accordance with the hardy weinberg equilibrium(HWE) were all compared by Chi-square test. The following models including allele dose model, generalized genotype genetic model, and the dominant model, the implicit model were applied to evaluate the SNP and AMS risk. Genetypes and AMS incidence were analyzed by Chi-square test. The age and body mass idex were adjusted. The logistic regression was used to calculate 95% confidence interval(CI) and estimate the relative risk of AMS(OR). All the analyses were performed using SPSS statistical software(version 18.0, SPSS Inc, Chicago, IL, USA).Results1. The Chinese Han population in this study ascended from 500 m to 3700 m and the AMS incidence was 61.19%. Among the people with AMS, 53% suffered severe AMS, 47% suffered mild AMS. Within the first 24 hours upon high altitude exposure, AMS group had higher heart rate(HR) and lower Oxygen saturation(SaO2). The incidences of headache, dizziness, gastrointestinal symptoms, sleep difficulty and fatigue were more frequent in the AMS group than in the Non-AMS group.2. There was a strong correlation between the rs6756667 of the EPAS1 gene and the AMS incidence. Compared with the A-allele carriers, the incidence of AMS of the G-allele carriers was increased by 1.701 fold(OR, 1.701, 95% CI, 1.261-2.545, p = 0.001). In the dominant model, there was a significant difference in the AMS incidence between the rs6756667 GG and(AA+AG) genetypes. In addition, GG genetypes increased the AMS risk(OR, 1.815, 95% CI, 1.233-2.666, p = 0.002). The G-allele carriers are more likely to develop mild AMS and severe AMS than the A-allele carriers.3. The association analyses between rs6756667 of the EPAS1 gene and the red blood cells(RBCs), hematocrit(HCT), hemoglobin concentration(Hb), SaO2, HR and blood pressure(BP) showed that there was no correlation between rs6756667 and SaO2, HR and BP, however, there was a significant correlation between rs6756667 and Hb, RBCs and HCT. The GG genetype carriers had higher Hb, RBCs and HCT than the AG genetype carriers.carriers.4. There was a significant association between the rs3025040 of the VEGFA gene and the AMS incidence. There were stastistically significant differences in the frequencies of CC, TT and CT genetypes as well as C/T allele between the AMS group and Non-AMS group. Compared with the C-allele carriers, the incidence of T-allele carriers was increased by 1.669 fold(OR, 1.669, 95% CI, 1.190-2.341, p = 0.003). In the dominant model, there was a significant difference in the AMS incidence between the rs3025040 CC carriers and(TT+CT) carriers. Besides, the CC genetype reduced the AMS risk(OR, 0.605, 95% CI, 0.416-0.880, p = 0.008). The T-allele carriers are more likely to develop mild AMS and severe AMS than the C-allele carriers. 5. There were no significant correlations between the rs2301104, rs2301112, rs12434438, rs966824, rs2301113 and rs11549467 of the HIF1 A gene and AMS incidence of Chinese Han population. Similarly, there were no significant correlations between the rs4253747, rs4253623, rs4253681 and rs135538 of the PPARA gene and AMS incidence of Chinese Han population.6. According to haplotype analysis, We found TG haplotype of rs3025040 and rs10434 sites in VEFGA genes was significantly associated with the risk of AMS and mild AMS, but not associated with severe AMS. There was no association between AC, GC and GT haplotypes of HIF1 A gene and AMS, mild AMS and severe AMS. Conclusions Based on this nested case-control study, we found out some associations between several SNP sites of the HIF pathway and AMS onset. For the first time, we demonstrated that rs6756667 of the EPAS1 gene strongly correlated with the physilogical indexes in the AMS group, as well as AMS onset. Again, we validated that there was no correlation between the rs3025040 of VEGFA gene and AMS onset. We also found TG haplotype of VEGFA gene significant associated with the risk of AMS.The EPAS1 gene and VEGFA gene may play certain genetic effects in the hypoxic adaptation among the low altitude Chinese Han men. Our study offers a novel idea for the prevention, treatment and scoring of AMS and also provides a new angle for the treatment of the hypoxia-related diseases. |
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