The Telomere-Telomerase System Is Detrimental to Health at High-Altitude

Abstract

The hypobaric-hypoxia environment at high-altitude (HA, >2500 m) may influence DNA damage due to the production of reactive molecular species and high UV radiation. The telomere system, vital to chromosomal integrity and cellular viability, is prone to oxidative damages contributing to the severity of high-altitude disorders such as high-altitude pulmonary edema (HAPE). However, at the same time, it is suggested to sustain physical performance. This case-control study, comprising 210 HAPE-free (HAPE-f) sojourners, 183 HAPE-patients (HAPE-p) and 200 healthy highland natives (HLs) residing at ~3500 m, investigated telomere length, telomerase activity, and oxidative stress biomarkers. Fluidigm SNP genotyping screened 65 single nucleotide polymorphisms (SNPs) in 11 telomere-maintaining genes. Significance was attained at p ≤ 0.05 after adjusting for confounders and correction for multiple comparisons. Shorter telomere length, decreased telomerase activity and increased oxidative stress were observed in HAPE patients; contrarily, longer telomere length and elevated telomerase activity were observed in healthy HA natives compared to HAPE-f. Four SNPs and three haplotypes are associated with HAPE, whereas eight SNPs and nine haplotypes are associated with HA adaptation. Various gene-gene interactions and correlations between/among clinical parameters and biomarkers suggested the presence of a complex interplay underlining HAPE and HA adaptation physiology. A distinctive contribution of the telomere-telomerase system contributing to HA physiology is evident in this study. A normal telomere system may be advantageous in endurance training.

Keywords: adaptation; genetic predisposition; high-altitude; high-altitude pulmonary edema; telomerase; telomere.

Figure 1

Schematic presentation of the study. (A) Hypothesis of the study. The telomere-telomerase system is influenced by oxidative stress under the hypobaric hypoxic environment of high-altitude. In a sequential process, they play a crucial role in HAPE pathophysiology and HA adaptation. (B) Study design. Study subjects were recruited, and their sampling was conducted at high-altitude (~3500 m above sea level). Socio-demographic and clinical data were collected at the time of recruitment. Plasma biomarkers namely, 8-isoPGF2α and total antioxidants, telomere length and telomerase activity were estimated, followed by SNP genotyping, gene expression and in silico analyses to evaluate the site-specific protein structure variations. All the data were analyzed using relevant statistical and bioinformatic tools and are described in the Methods section. ROS, reactive oxygen species; HAPE, high-altitude pulmonary edema; HAPE-p, HAPE patients; HAPE-f, HAPE-free healthy controls; HLs, high-land natives; SNPs, single nucleotide polymorphisms.

Figure 2

The clinical parameters and circulating biomarkers differ in the three study groups HAPE-f, HAPE-p and HLs. (A) Arterial oxygen saturation level is lowest in patients. (B) Mean arterial pressure has increased in patients whereas SaO₂ and MAP present a reverse trend in both the control groups. (C) Relative Telomere Length is shorter in patients. (D) Telomerase Activity decreased in patients causing the shortening of the telomere length. (E) 8-isoprostaglandin F2α level is increased in patients reflecting stress. (F) Total Antioxidant Activity decreased in patients. The increased stressor and decreased antioxidant complement the two clinical parameters and the shortened telomere length and the telomerase activity in HAPE patients compared to the reverse trend in the two control groups suggesting their contribution to HAPE pathophysiology. The data are represented as mean ± SD. SPSS 16.0 was used to obtain p-values after adjusting with age, gender, and BMI by performing UNIANOVA. A p-value of ≤ 0.05 was considered statistically significant. * represents p ≤ 0.05, ** represents p ≤ 0.01, *** represents p ≤ 0.001 and **** represents p ≤ 0.0001. HAPE-f, HAPE-free healthy controls; HAPE-p, HAPE-patients; HLs, High-land natives; SaO₂, arterial oxygen saturation; MAP, mean arterial pressure; T/S, ratio between telomere repeat copy number (T) and the copy number of single copy gene (S); 8-iso PGF2α, 8-isoprostaglandin F2α; p, p-value.

Figure 3

MDR generated distribution of genotype interactions in observed best models. In the Figure, sub-figures (A–C) represent a comparison of the two to four loci genotype interactions between the HAPE patients and HAPE-free controls. In sub-figures (D,E), two to three loci genotype interactions are compared between the two control groups, i.e., HAPE-f (HAPE-free healthy controls) and HLs (high-land natives). (A) HAPE-associating shelterin genes in a 2-locus best model. The TT-TT (TPP1/ACD rs72556537A/T-RAP1/TERF2IP rs59297469G/T) interaction was observed as risk in which the protective allele TPP1 rs72556537A was absent. (B) HAPE-associating telomerase genes in a 2-locus best model. A risk-predicting TT-AG (TEP1 rs2228036G/T-TERC rs2293607A/G) interaction was observed in which risk allele TEP1 rs2228036T was present as homozygous genotype TEP1 rs2228036TT and risk allele TERC rs2293607G was present as heterozygous rs2293607AG genotype. (C) HAPE-associating genes of the telomere-telomerase system in a 4-locus best model. The TT-GT-AA-TT (RAP1 rs59297469G/T-TEP1 rs2228036G/T-TERC rs2293607A/G-TPP1 rs72556537A/T) risk-predicting interaction consisted of risk allele RAP1 rs59297469T as homozygous rs59297469TT genotype and risk allele TEP1 rs2228036T as heterozygous rs2228036GT genotype. The GG-GT-AG-TT interaction consisted of risk allele TEP1 rs2228036T as heterozygous rs2228036GT genotype and risk allele TERC rs2293607G as heterozygous rs2293607AG genotype. (D) HA adaptation-associating shelterin genes and HA adaptation-associating genes of the telomere-telomerase system in a 3-locus best model. Two interactions, GG-AA-TC (TERF1 rs10099824G/A-TPP1 rs6979G/A-TERF1 rs2975843T/C) and GG-AA-TT, predicted HA adaptation consisting of adaptive allele TERF1 rs10099824G as homozygous rs10099824GG genotype and adaptive allele TPP1 rs6979A as homozygous rs6979AA genotype. The adaptive allele TERF1 rs2975843T was present as heterozygous rs2975843TC genotype in GG-AA-TC and as homozygous rs2975843TT genotype in GG-AA-TT. (E) HA adaptation-associating telomerase genes in a 2-locus best model. Three adaptive interactions, i.e., AA-TT, AG-TT. GG-TT (TEP1 rs2184282A/G-TEP1 rs4246977T/C) were obtained. The adaptive allele TEP1 rs4246977T as homozygous rs4246977TT genotype is present in all the three adaptive interactions revealing a strong association with HA adaptation. In (A–C) presentation, the dark grey cells associate with HAPE risk, and the light grey cells associate with HAPE protection; the first bar in a cell represents the number of HAPE-p and the second bar represents the number of HAPE-f. In (D,E), the dark grey cells represent interactions enriched in HAPE-f and the light grey cells associate with HA adaptation; the first bar in a cell represents the number of HAPE-f and the second bar represents the number of HLs. * marked cells represent the significant genotype interactions, which are summarized in respective tables. The genotypes of the SNPs appear in alphabetical order. Chi-square test or Fisher’s exact test (when the number of samples was ≤5) was applied to calculate p-value, OR (odds ratio) and 95% CI (confidence interval) using SISA online tool.

Figure 4

The gene expression, allele specificity with TFs and the genes/proteins interactions differentiate the physiological variations. (A) Heatmap of log2 fold-change value of differently expressed genes in the three study groups. TERF2IP was down-regulated by 3.1 folds in HAPE-p and up-regulated by 1.4 folds in HLs. TERT was down-regulated by 1.8 folds in HAPE-p and 1.3 folds in HLs. Further HSP90AA1 was down-regulated 2.1 folds in HAPE-p. * represents p ≤ 0.05, ** represents p ≤ 0.01 and **** represents p ≤ 0.0001. (B) Docking image of TFs, USF1 and N-myc on RAP1 or TERF2IP with wild type allele, rs59297469G (WtRAP1). (C) Docking image of TFs, USF1 and N-myc, on RAP1 or TERF2IP with variant/risk type allele, rs59297469T (VtRAP1). Figure (B,C) differentiates the binding affinity for the same locus in the presence of risk and the protective allele that would influence the expression and function of the gene, which is depicted in the subsequent presentation. RMS, root mean square. (D) RAP1 expression is significantly down-regulated with a higher ΔCt in the presence of risk allele RAP1 rs59297469T compared to the protective allele rs59297469G. (E) Interactions among the telomere-maintaining proteins. (F) Telomere system shows stronger interactions among its proteins contributing to the regulation of other pathways such as the nitric oxide synthase (NOS) (marked red and blue) and cellular response to stress (marked green and yellow) which are depicted in subsequent presentation. (G) Enriched biological processes such as the nitric oxide synthase (NOS) and cellular response, which are important pathways in HAPE pathophysiology. (H) Interactions among the proteins of the telomere-maintaining and the hypoxia-maintaining systems in HA physiology influence several pathways. (I) Prominent biological processes as a result of the interaction of the proteins of the two major systems. In silico tools were applied to gain insight into the differential physiological system.

Figure 5

The proposed pathophysiology of HAPE. Hypobaric hypoxia is a stimulant to stress, which may in turn stimulate a normal physiological system to sustain a normal status which may not be possible in a few subjects. The same is depicted in the given graphical sketch. The expression of various proteins and genes, which are essential to regulate ROS production is dysregulated on exposure to HA. The telomere system is one of the major systems, whose genes/proteins are significantly disturbed. Down-regulation of HSP90AA1, TERT and the telomerase is observed and contributes to telomere attrition and may also enhance ROS production in a reversible process. Overall, in this milieu, the exaggerated ROS and the down-regulated candidate markers lead to the NOS3-mediated decrease in NO production and enhanced vasoconstriction leading to the disturbance of vascular homeostasis causing pulmonary hypertension, a hallmark of HAPE.