Hypermethylation of BMPR2 and TGF-ß Promoter Regions in Tibetan Patients with High-Altitude Polycythemia at Extreme Altitude.

Although the expression of many genes is associated with adaptation to high-altitude hypoxic environments, the role of epigenetics in the response to this harsh environmental stress is currently unclear. We explored whether abnormal DNA promoter methylation levels of six genes, namely, ABCA1, SOD2, AKT1, VEGFR2, TGF-ß, and BMPR2, affect the occurrence and development of high-altitude polycythemia (HAPC) in Tibetans. The methylation levels of HAPC and the control group of 130 Tibetans from very high altitudes (> 4500 m) were examined using quantitative methylation-specific real-time PCR (QMSP). Depending on the type of data, the Pearson chi-square test, Wilcoxon rank-sum test, and Fisher exact test were used to assess the differences between the two groups. The correlation between the methylation levels of each gene and the hemoglobin content was explored using a linear mixed model. Our experiment revealed that the methylation levels of the TGF-ß and BMPR2 genes differed significantly in the two groups (p < 0.05) and linear mixed model analysis showed that the correlation between the hemoglobin and methylation of ABCA1, TGF-ß, and BMPR2 was statistically significant (p < 0.05). Our study suggests that levels of TGF-ß and BMPR2 methylation are associated with the occurrence of HAPC in extreme-altitude Tibetan populations among 6 selected genes. Epigenetics may be involved in the pathogenesis of HAPC, and future experiments could combine gene and protein levels to verify the diagnostic value of TGF-ß and BMPR2 methylation levels in HAPC.

Predictive model for estimating the risk of high-altitude pulmonary edema: a single-centre retrospective outcome-reporting study.

OBJECTIVE: To develop the first prediction model based on the common clinical symptoms of high-altitude pulmonary edema (HAPE), enabling early identification and an easy-to-execute self-risk prediction tool. METHODS: A total of 614 patients who consulted People's Hospital of Tibet Autonomous Region between January 2014 and April 2022 were enrolled. Out of those, 508 patients (416 males and 92 females) were diagnosed with HAPE and 106 were patients without HAPE (33 females and 72 males). They were randomly distributed into training (n=431) and validation (n=182) groups. Univariate and multivariate analysis were used to screen predictors of HAPE selected from the 36 predictors; nomograms were established based on the results of multivariate analysis. The receiver operating characteristic curve (ROC) was developed to obtain the area under the ROC curve (AUC) of the predictive model, and its predictive power was further evaluated by calibrating the curve, while the Decision Curve Analysis (DCA) was developed to evaluate the clinical applicability of the model, which was visualised by nomogram. RESULTS: All six predictors were significantly associated with the incidence of HAPE, and two models were classified according to whether the value of SpO2 (percentage of oxygen in the blood) was available in the target population. Both could accurately predict the risk of HAPE. In the validation cohort, the AUC of model 1 was 0.934 with 95% CI (0.848 to 1.000), and model 2 had an AUC of 0.889, 95% CI (0.779 to 0.999). Calibration plots showed that the predicted and actual HAPE probabilities fitted well with internal validation, and the clinical decision curve shows intervention in the risk range of 0.01-0.98, resulting in a net benefit of nearly 99%. CONCLUSION: The recommended prediction model (nomogram) could estimate the risk of HAPE with good precision, high discrimination and possible clinical applications for patients with HAPE. More importantly, it is an easy-to-execute scoring tool for individuals without medical professionals' support.

Neural network correlates of high-altitude adaptive genetic variants in Tibetans: A pilot, exploratory study.

Although substantial progress has been made in the identification of genetic substrates underlying physiology, neuropsychology, and brain organization, the genotype-phenotype associations remain largely unknown in the context of high-altitude (HA) adaptation. Here, we related HA adaptive genetic variants in three gene loci (EGLN1, EPAS1, and PPARA) to interindividual variance in a set of physiological characteristics, neuropsychological tests, and topological attributes of large-scale structural and functional brain networks in 135 indigenous Tibetan highlanders. Analyses of individual HA adaptive single-nucleotide polymorphisms (SNPs) revealed that specific SNPs selectively modulated physiological characteristics (erythrocyte level, ratio between forced expiratory volume in the first second to forced vital capacity, arterial oxygen saturation, and heart rate) and structural network centrality (the left anterior orbital gyrus) with no effects on neuropsychology or functional brain networks. Further analyses of genetic adaptive scores, which summarized the overall degree of genetic adaptation to HA, revealed significant correlations only with structural brain networks with respect to local interconnectivity of the whole networks, intermodule communication between the right frontal and parietal module and the left occipital module, nodal centrality in several frontal regions, and connectivity strength of a subnetwork predominantly involving in intramodule edges in the right temporal and occipital module. Moreover, the associations were dependent on gene loci, weight types, or topological scales. Together, these findings shed new light on genotype-phenotype interactions under HA hypoxia and have important implications for developing new strategies to optimize organism and tissue responses to chronic hypoxia induced by extreme environments or diseases.

Whole-Genome Sequencing Identifies the Egl Nine Homologue 3 (egln3/phd3) and Protein Phosphatase 1 Regulatory Inhibitor Subunit 2 (PPP1R2P1) Associated with High-Altitude Polycythemia in Tibetans at High Altitude.

BACKGROUND: The hypoxic conditions at high altitudes are great threats to survival, causing pressure for adaptation. More and more high-altitude denizens are not adapted with the condition known as high-altitude polycythemia (HAPC) that featured excessive erythrocytosis. As a high-altitude sickness, the etiology of HAPC is still unclear. METHODS: In this study, we reported the whole-genome sequencing-based study of 10 native Tibetans with HAPC and 10 control subjects followed by genotyping of selected 21 variants from discovered single nucleotide variants (SNVs) in an independent cohort (232 cases and 266 controls). RESULTS: We discovered the egl nine homologue 3 (egln3/phd3) (14q13.1, rs1346902, P = 1.91 × 10-5) and PPP1R2P1 (Protein Phosphatase 1 Regulatory Inhibitor Subunit 2) gene (6p21.32, rs521539, P = 0.012). Our results indicated an unbiased framework to identify etiological mechanisms of HAPC and showed that egln3/phd3 and PPP1R2P1 may be associated with the susceptibility to HAPC. Egln3/phd3b is associated with hypoxia-inducible factor subunit α (HIFα). Protein Phosphatase 1 Regulatory Inhibitor is associated with reactive oxygen species (ROS) and oxidative stress. CONCLUSIONS: Our genome sequencing conducted in Tibetan HAPC patients identified egln3/phd3 and PPP1R2P1 associated with HAPC.

Mechanism of apoptosis involved in gastric mucosal lesions in Tibetans with high-altitude polycythemia.

The Tibetan high plateau is a low-oxygen environment, which may cause the pathogenesis of high-altitude polycythemia (HAPC). Gastric mucosal lesions (GML) are a common complication of HAPC. The molecular mechanisms involved in HAPC-induced GML have remained to be fully elucidated and were therefore investigated in the present study. Gastric tissues of patients with heavy, HAPC-induced GML and healthy controls were assessed by ultrastructural and histopathological analysis. In addition, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect cell apoptosis in the gastric mucosa tissues. Moreover, the expression of genes associated with the phosphoinositide-3 kinase (PI3K) pathway was assessed by RT-qPCR to investigate the mechanism of cell apoptosis in HAPC-induced GML. The results revealed a significant increase in the number of red blood cells, gastric vessels and the diameter of gastric mucosal vessels in HAPC-induced GML patients compared with those in healthy controls. In addition, more red blood cells were distributed in gastric tissue not only at the vascular level but also in the tissue space. The number of vacuoles was increased in the gastric mucosal cells. Furthermore, a significant increase in apoptosis of the gastric mucosal cells was identified. The expression of phosphatase and tensin homolog was significantly higher in gastric mucosa from patients with HAPC-induced GML compared with that in the healthy controls. All of the pathologic changes suggested that significant cell apoptosis occurred in the HAPC-induced GML tissues, which may be associated with the PI3K pathway. These findings may provide novel insight for the treatment of gastric lesions caused by HAPC in the future.

Transcriptome reveals the overexpression of a kallikrein gene cluster (KLK1/3/7/8/12) in the Tibetans with high altitude-associated polycythemia.

High altitude-associated polycythemia (HAPC) is a very common disease. However, it the disease is still unmanageable and the related molecular mechanisms remain largely unclear. In the present study, we aimed to explore the molecular mechanisms responsible for the development of HAPC using transcriptome analysis. Transcriptome analysis was conducted in 3 pairs of gastric mucosa tissues from patients with HAPC and healthy residents at a similar altitude. Endoscopy and histopathological analyses were used to examine the injury to gastric tissues. Molecular remodeling was performed for the interaction between different KLK members and cholesterol. HAPC was found to lead to morphological changes and pathological damage to the gastric mucosa of patients. A total of 10,304 differentially expressed genes (DEGs) were identified. Among these genes, 4,941 DEGs were upregulated, while 5,363 DEGs were downregulated in the patients with HAPC (fold change ≥2, P<0.01 and FDR <0.01). In particular, the kallikrein gene cluster (KLK1/3/7/8/12) was upregulated >17-fold. All the members had high-score binding cholesterol, particularly for the polymers of KLK7. The kallikrein gene cluster (KLK1/3/7/8/12) is on chromosome 19q13.3-13.4. The elevated levels of KLK1, KLK3, KLK7, KLK8 and KLK12 may be closely associated with the hypertension, inflammation, obesity and other gastric injuries associated with polycythemia. The interaction of KLKs and cholesterol maybe play an important role in the development of hypertension. The findings of the present study revealed that HAPC induces gastric injury by upregulating the kallikrein gene cluster (KLK1/3/7/8/12), which can bind cholesterol and result in kallikrein hypertension. These findings provide some basic information for understanding the molecular mechanisms responsible for HAPC and HAPC-related diseases.

Genome-Wide Transcriptional Analysis Reveals the Protection against Hypoxia-Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways.

The molecular mechanisms for hypoxic environment causing the injury of intestinal mucosal barrier (IMB) are widely unknown. To address the issue, Han Chinese from 100 m altitude and Tibetans from high altitude (more than 3650 m) were recruited. Histological and transcriptome analyses were performed. The results showed intestinal villi were reduced and appeared irregular, and glandular epithelium was destroyed in the IMB of Tibetans when compared with Han Chinese. Transcriptome analysis revealed 2573 genes with altered expression. The levels of 1137 genes increased and 1436 genes decreased in Tibetans when compared with Han Chinese. Gene ontology (GO) analysis indicated most immunological responses were reduced in the IMB of Tibetans when compared with Han Chinese. Gene microarray showed that there were 25-, 22-, and 18-fold downregulation for growth factor receptor-bound protein 2 (GRB2), epidermal growth factor receptor (EGFR), and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) in the IMB of Tibetans when compared with Han Chinese. The downregulation of EGFR, GRB2, and PTPN11 will reduce the production of reactive oxygen species and protect against oxidative stress-induced injury for intestine. Thus, the transcriptome analysis showed the protecting functions of IMB patients against hypoxia-induced oxidative injury in the intestine of Tibetans via affecting GRB2/EGFR/PTPN11 pathways.

Reversible Brain Abnormalities in People Without Signs of Mountain Sickness During High-Altitude Exposure.

A large proportion of lowlanders ascending to high-altitude (HA) show no signs of mountain sickness. Whether their brains have indeed suffered from HA environment and the persistent sequelae after return to lowland remain unknown. Thirty-one sea-level college students, who had a 30-day teaching on Qinghai-Tibet plateau underwent MRI scans before, during, and two months after HA exposure. Brain volume, cortical structures, and white matter microstructure were measured. Besides, serum neuron-specific enolase (NSE), C-reactive protein, and interleukin-6 and neuropsychiatric behaviors were tested. After 30-day HA exposure, the gray and white matter volumes and cortical surface areas significantly increased, with cortical thicknesses and curvatures changed in a wide spread regions; Anisotropy decreased with diffusivities increased in multiple sites of white matter tracts. Two months after HA exposure, cortical measurements returned to basal level. However, increased anisotropy with decreased diffusivities was observed. Behaviors and serum inflammatory factor did not significant changed during three time-point tests. NSE significantly decreased during HA but increased after HA exposure. Results suggest brain swelling occurred in people without neurological signs at HA, but no negative sequelae in cortical structures and neuropsychiatric functions were left after the return to lowlands. Reoxygenation changed white matter microstructure.

Comparative Analysis of Gut Microbiota of Native Tibetan and Han Populations Living at Different Altitudes.

The factors driving the composition of gut microbiota are still only partly understood but appear to include environmental, cultural, and genetic factors. In order to obtain more insight into the relative importance of these factors, we analyzed the microbiome composition in subjects of Tibetan or Han descent living at different altitudes. DNA was isolated from stool samples. Using polymerase chain reaction methodology, the 16S rRNA V1-V3 regions were amplified and the sequence information was analyzed by principal coordinates analysis and Lefse analyses. Contrasting the Tibetan and Han populations both living at the 3600 m altitude, we found that the Tibetan microbiome is characterized by a relative abundance of Prevotella whereas the Han stool was enriched in Bacteroides. Comparing the microbiome of Han stool obtained from populations living at different altitudes revealed a more energy efficient flora in samples from those living at higher altitude relative to their lower-altitude counterparts. Comparison of the stool microbiome of Tibetan herders living at 4800 m to rural Tibetans living at 3600 m altitude shows that the former have a flora enriched in butyrate-producing bacteria, possibly in response to the harsher environment that these herders face. Thus, the study shows that both altitude and genetic/cultural background have a significant influence on microbiome composition, and it represents the first attempt to compare stool microbiota of Tibetan and Han populations in relation to altitude.

Transcriptome Reveals 1400-Fold Upregulation of APOA4-APOC3 and 1100-Fold Downregulation of GIF in the Patients with Polycythemia-Induced Gastric Injury.

High-altitude polycythemia (HAPC) inducing gastric mucosal lesion (GML) is still out of control and molecular mechanisms remain widely unknown. To address the issues, endoscopy and histopathological analyses were performed. Meanwhile, microarray-based transcriptome profiling was conducted in the gastric mucosa from 3 pairs of healthy subjects and HAPC-induced GML patients. HAPC caused morphological changes and pathological damages of the gastric mucosa of GML patients. A total of 10304 differentially expressed genes (DEGs) were identified, including 4941 up-regulated and 5363 down-regulated DEGs in gastric mucosa of GML patients compared with healthy controls (fold change ≥2, P<0.01 and FDR <0.01). Particularly, apolipoprotein genes APOA4 and APOC3 were 1473-fold and 1468-fold up-regulated in GML patients compared with the controls. In contrast, gastric intrinsic factor (GIF) was 1102-fold down-regulated in GML patients compared with the controls. APOA4 (chr11:116691770-116691711), APOC3 (chr11:116703530-116703589) and GIF (chr11:59603362-59603303) genes are all located on chromosome 11. APOA4 and APOC3 act as an inhibitor of gastric acid secretion while gastric acid promotes ulceration. GIF deficiency activates a program of acute anemia, which may antagonize polycythemia while polycythemia raises the risk of GML. Therefore, the present findings reveal that HAPC-induced GML inspires the protection responses by up-regulating APOA4 and APOC3, and down-regulating GIF. These results may offer the basic information for the treatment of HAPC-induced gastric lesion in the future.

CD14 knockdown reduces lipopolysaccharide-induced cell viability and expression of inflammation-associated genes in gastric cancer cells in vitro and in nude mouse xenografts.

The present study examined the role of CD14 in the regulation of lipopolysaccharide (LPS)-induced effects on gastric cancer cells. MGC­803 cells were stably transfected with CD14 short hairpin (sh)RNA and treated with LPS, followed by assessment of cell proliferation, apoptosis and gene expression using a cell counting kit­8 assay, flow cytometry, reverse transcription­polymerase chain reaction and western blot analysis, respectively. The cells subjected to CD14 knockdown were treated with 10 g/ml LPS and injected into nude mice to form tumor xenografts. CD14 shRNA­transfected MGC­803 cells did not exhibit any significant changes in cell viability compared with the control cells (P>0.05), but cell viability was markedly increased in the wild­type (WT) + LPS group (P<0.05). In contrast to the WT + LPS group, the cell viability of the sh­CD14 + LPS group was markedly decreased (P<0.05). In addition, compared with those in the controls, the level of sh­CD14 cell apoptosis did not change significantly; however, it was markedly reduced in the LPS group. Compared with that in the WT + LPS group, the rate of apoptosis in the sh­CD14 + LPS group increased to a certain extent, while it remained lower in the control group. In addition, compared with that in the control, the expression of tumor necrosis factor­α, interleukin (IL)­1, IL­6 and IL­12, and human ß­defensin 2 was significantly increased in the WT + LPS group, while, compared with that in the WT + LPS group, the expression of these genes was markedly reduced in the sh­CD14 + LPS group (P<0.05). The nude mouse experiments further confirmed the in vitro data, including the finding that LPS promoted the growth of xenografts, but knockdown of CD14 expression reduced the response of tumor cells to LPS treatment. In conclusion, LPS induced cell viability and the release of inflammatory cytokines, but inhibited gastric cancer cell apoptosis. Knockdown of CD14 expression had no significant effect on gastric cancer malignancy, but mediated LPS signal transduction.

Association of CD14/-260 polymorphism with gastric cancer risk in Highland Tibetans.

AIM: To investigate the relationship between CD14-260 and -651 polymorphisms and the risk of developing gastric cancer. METHODS: DNA was extracted from peripheral blood samples obtained from 225 Tibetans with gastric cancer and 237 healthy Tibetans, and analyzed using the polymerase chain reaction/ligase detection (PCR/LDR) method to determine the genotypes at -260 and -651 loci of the CD14 promoter. The allele frequencies, genotype frequencies, and haplotypes were analyzed for their association with gastric cancer risk using online SHEsis software. The luciferase reporter assay and point mutation analysis were used to construct in vitro plasmids expressing a C/T homozygote at the -260 locus of the CD14 promoter. RESULTS: The frequencies of CC, CT and TT genotypes in the CD14-260 C/T locus in gastric cancer patients were 19.1%, 38.7% and 42.2%, respectively, whereas they were 33.3%, 32.5% and 34.2%, respectively, in healthy control subjects. CT genotype carriers were more frequently found among gastric cancer patients than healthy controls (OR = 2.076; 95%CI: 1.282-3.360). Also, TT genotype carriers were more frequently found among gastric cancer patients (OR = 2.155; 95%CI: 1.340-3.466). Compared to the C allele of CD14/-260, the T allele was associated with an increased risk for gastric cancer (OR = 1.574; 95%CI: 1.121-2.045). Furthermore, the frequencies of CC, CT and TT in the CD14-651 C/T locus in gastric cancer patients were 64.4%, 29.3% and 6.2%, respectively, while they were 56.5%, 35.0% and 8.4%, respectively, in the healthy control subjects (P > 0.05). Data obtained using the luciferase reporter assay showed that the p260T homozygote was associated with greater CD14 promoter activity (P < 0.01). CONCLUSION: CD14/-260 polymorphism is associated with gastric cancer risk in Highland Tibetans and affects CD14 promoter activity, thereby regulating CD14 expression.

CD14 regulates gastric cancer cell epithelial­mesenchymal transition and invasion in vitro.

Cluster of differentiation 14 (CD14) protein functions as a co-receptor with either the Toll-like receptor TLR4 or MD-2 in the detection of bacterial lipopolysaccharide (LPS) and plays a role in the innate immune system. Recently, it was shown to have effects on the regulation of epithelial-mesenchymal transition (EMT). Thus, the present study investigated the effects of CD14 knockdown on the regulation of gastric cancer cell EMT and invasive capacity following treatment with or without LPS. Knockdown of CD14 expression using CD14 shRNA in MGC-803 cells significantly enhanced E-cadherin expression, but reduced N-cadherin and vimentin expression in both LPS-treated and untreated cells. Morphologically, the phenotype of LPS-treated CD14-knockdown cells was altered to a sporadic long spindle shape. Moreover, TNF-α-treated cells were further elongated, connections between cells were reduced, the gap between the cells was increased and the cells were transformed into mesenchymal cells. Furthermore, the invasive capacity of CD14-knockdown cells was significantly lower compared to that of the negative control shRNA-transfected MGC-803 cells. LPS-treated CD14-knockdown cells had significantly lower levels of tumor cell invasive ability when compared to the LPS-treated parental MGC-803 cells. However, addition of TNF-α to LPS-treated CD14-knockdown cells significantly increased tumor cell invasion. This study demonstrated that CD14 promoted tumor cell EMT and invasion through TNF-α, whereas knockdown of CD14 expression inhibited gastric cancer cell invasion and EMT.

A1166C polymorphism of the angiotensin II type 1 receptor gene and essential hypertension in Han, Tibetan and Yi populations.

Our aim was to clarify whether substitution of cytosine for adenine at position 1166 (A1166C) polymorphism of the angiotensin II type 1 receptor (AT1R) gene is associated with susceptibility to essential hypertension in Han, Tibetan and Yi populations in China. This study involved 302 normotensive and 446 hypertensive subjects. The polymorphism was detected by polymelase chain reaction of genomic DNA and restriction fragment length polymorphism (PCR-RFLP) in genomic DNA. The data were analyzed by analysis of covariance (ANCOVA), X2 test, and multiple logistic regression. In normotensive controls, the A1166 allele frequencies were 0.979, 0.939 and 0.965 in Han, Tibetan and Yi participants, respectively. There was no significant intergroup variation in frequency of the allele in normotensives (X2=4.166, p=0.125). The frequency of the A1166 allele was significantly higher in Tibetan male hypertensives than that in normotensives (X2=11.46, p=0.001). There was no significant difference in A1166C genotype distribution and allele frequency between normotensives and hypertensives either in the Han (p=0.465) or Yi (p=0.357) populations. Body mass index in the Han and Yi populations (p=0.0001), age in the Tibetan and Yi populations (p=0.0001), and AA genotype in the Tibetan male population (p=0.0034) all were independent risk factors for hypertension. Diastolic blood pressure levels were significantly higher in Tibetan male subjects with the AA genotype than in those with the AC+CC genotype (p=0.0040). We concluded that the A1166 allele is very common in Han, Tibetan and Yi populations, approximately 1.35-fold more common than in Caucasians. The A1166 allele of the AT1R gene may be a predisposing factor for essential hypertension in Tibetan males. A1166C polymorphism of the AT1R gene is probably not involved in the pathogenesis of essential hypertension in Han or Yi populations.

Angiotensin-converting enzyme gene polymorphism and its association with essential hypertension in a Tibetan population.

There is strong evidence to support the idea that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of essential hypertension (EH) and its complications. However, existing data about the association of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with blood pressure is conflicting, mainly due to racial differences and environmental exposure status. We therefore conducted a case control study to observe the relationship between ACE I/D polymorphism and EH in a Tibetan population who live in relatively isolated areas and are genetically homogeneous. The study was conducted at stable residential communities in the urban district of Lhasa, the capital of the Tibet autonomous region, China, and 106 unrelated EH patients and 135 normotensIve subjects were recruited. PCR, PCR/RFLP and PCR-SSCP were carried out to study the association between RAS genes and EH. Frequencies for the DD, ID and II genotypes were 27, 47 and 29 in hypertensive subjects, and 15, 60 and 48 in normotensive subjects, respectively. Derived allele frequencies for the I and D alleles were 0.51 and 0.49 in hypertensive subjects and 0.64 and 0.36 in normotensive subjects. There were significant differences in genotype distribution and derived allele frequency between these two groups. The genotype and allele frequencies of the ACE gene differed significantly between hypertensive and normotensive females (p>0.05), but there were no differences in males. In females, the DBP and MAP level were significantly higher for the DD than for the ID and II genotype, and SBP was significantly higher for the DD than for the II genotype. But in males, there were no significant differences in blood pressure among ACE genotypes. The results showed a significant association between the D allele of the ACE gene and hypertension in Tibetan women but not in Tibetan men.