Genetic Signatures of Positive Selection in Human Populations Adapted to High Altitude in Papua New Guinea.

Papua New Guinea (PNG) hosts distinct environments mainly represented by the ecoregions of the Highlands and Lowlands that display increased altitude and a predominance of pathogens, respectively. Since its initial peopling approximately 50,000 years ago, inhabitants of these ecoregions might have differentially adapted to the environmental pressures exerted by each of them. However, the genetic basis of adaptation in populations from these areas remains understudied. Here, we investigated signals of positive selection in 62 highlanders and 43 lowlanders across 14 locations in the main island of PNG using whole-genome genotype data from the Oceanian Genome Variation Project (OGVP) and searched for signals of positive selection through population differentiation and haplotype-based selection scans. Additionally, we performed archaic ancestry estimation to detect selection signals in highlanders within introgressed regions of the genome. Among highland populations we identified candidate genes representing known biomarkers for mountain sickness (SAA4, SAA1, PRDX1, LDHA) as well as candidate genes of the Notch signaling pathway (PSEN1, NUMB, RBPJ, MAML3), a novel proposed pathway for high altitude adaptation in multiple organisms. We also identified candidate genes involved in oxidative stress, inflammation, and angiogenesis, processes inducible by hypoxia, as well as in components of the eye lens and the immune response. In contrast, candidate genes in the lowlands are mainly related to the immune response (HLA-DQB1, HLA-DQA2, TAAR6, TAAR9, TAAR8, RNASE4, RNASE6, ANG). Moreover, we find two candidate regions to be also enriched with archaic introgressed segments, suggesting that archaic admixture has played a role in the local adaptation of PNG populations.

Phospholipase D regulates ferroptosis signal transduction in mouse spleen hypoxia response.

High-altitude hypoxia exposure can lead to phospholipase D-mediated lipid metabolism disorder in spleen tissues and induce ferroptosis. Nonetheless, the key genes underlying hypoxia-induced splenic phospholipase D and the ferroptosis pathway remain unclear. This study aimed to establish a hypoxia animal model. Combined transcriptomic and proteomic analyses showed that 95 predicted target genes (proteins) were significantly differentially expressed under hypoxic conditions. Key genes in phospholipase D and ferroptosis pathways under hypoxic exposure were identified by combining Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis techniques. Gene set enrichment analysis (GSEA) showed that the differential gene sets of the phospholipase D and ferroptosis signaling pathways were upregulated in the high-altitude hypoxia group. The genes in the phospholipase D signalling pathway were verified, and the expression levels of KIT and DGKG were upregulated in spleen tissues under hypoxic exposure. Subsequently, the mRNA and protein expression levels of genes from the exogenous pathway such as TFRC, SLC40A1, SLC7A11, TRP53, and FTH1 and those from the endogenous pathway such as GPX4, HMOX1, and ALOX15 differentials in the ferroptosis signalling pathway were verified, and the results indicated significant differential expression. In summary, exposure to high-altitude hypoxia mediated phospholipid metabolism disturbance through the phospholipase D signalling pathway and further induced ferroptosis, leading to splenic injury.

"What We Know and What We Do Not Know about Evolutionary Genetic Adaptation to High Altitude Hypoxia in Andean Aymaras".

Three well-studied populations living at high altitudes are Tibetans, Andeans (Aymaras and Quechuas), and Ethiopians. Unlike Tibetans and Ethiopians who have similar hemoglobin (Hb) levels as individuals living at sea level, Aymara Hb levels increase when living at higher altitudes. Our previous whole genome study of Aymara people revealed several selected genes that are involved in cardiovascular functions, but their relationship with Hb levels was not elucidated. Here, we studied the frequencies of known evolutionary-selected variants in Tibetan and Aymara populations and their correlation with high Hb levels in Aymara. We genotyped 177 Aymaras at three different altitudes: 400 m (Santa Cruz), 4000 m (La Paz), and 5000 m (Chorolque), and correlated the results with the elevation of residence. Some of the Tibetan-selected variants also exist in Aymaras, but at a lower prevalence. Two of 10 Tibetan selected variants of EPAS1 were found (rs13005507 and rs142764723) and these variants did not correlate with Hb levels. Allele frequencies of 5 Aymara selected SNPs (heterozygous and homozygous) at 4000 m (rs11578671_BRINP3, rs34913965_NOS2, rs12448902_SH2B1, rs10744822_TBX5, and rs487105_PYGM) were higher compared to Europeans. The allelic frequencies of rs11578671_BRINP3, rs34913965_NOS2, and rs10744822_SH2B1 were significantly higher for Aymaras living at 5000 m than those at 400 m elevation. Variant rs11578671, close to the BRINP3 coding region, correlated with Hb levels in females. Variant rs34913965 (NOS2) correlated with leukocyte counts. Variants rs12448902 (SH2B1) and rs34913965 (NOS2) associated with higher platelet levels. The correlation of these SNPs with blood cell counts demonstrates that the selected genetic variants in Aymara influence hematopoiesis and cardiovascular effects.

The overlooked significance of plasma volume for successful adaptation to high altitude in Sherpa and Andean natives.

In contrast to Andean natives, high-altitude Tibetans present with a lower hemoglobin concentration that correlates with reproductive success and exercise capacity. Decades of physiological and genomic research have assumed that the lower hemoglobin concentration in Himalayan natives results from a blunted erythropoietic response to hypoxia (i.e., no increase in total hemoglobin mass). In contrast, herein we test the hypothesis that the lower hemoglobin concentration is the result of greater plasma volume, rather than an absence of increased hemoglobin production. We assessed hemoglobin mass, plasma volume and blood volume in lowlanders at sea level, lowlanders acclimatized to high altitude, Himalayan Sherpa, and Andean Quechua, and explored the functional relevance of volumetric hematological measures to exercise capacity. Hemoglobin mass was highest in Andeans, but also was elevated in Sherpa compared with lowlanders. Sherpa demonstrated a larger plasma volume than Andeans, resulting in a comparable total blood volume at a lower hemoglobin concentration. Hemoglobin mass was positively related to exercise capacity in lowlanders at sea level and in Sherpa at high altitude, but not in Andean natives. Collectively, our findings demonstrate a unique adaptation in Sherpa that reorientates attention away from hemoglobin concentration and toward a paradigm where hemoglobin mass and plasma volume may represent phenotypes with adaptive significance at high altitude.

New Insights into the Genetic Basis of Monge's Disease and Adaptation to High-Altitude.

Human high-altitude (HA) adaptation or mal-adaptation is explored to understand the physiology, pathophysiology, and molecular mechanisms that underlie long-term exposure to hypoxia. Here, we report the results of an analysis of the largest whole-genome-sequencing of Chronic Mountain Sickness (CMS) and nonCMS individuals, identified candidate genes and functionally validated these candidates in a genetic model system (Drosophila). We used PreCIOSS algorithm that uses Haplotype Allele Frequency score to separate haplotypes carrying the favored allele from the noncarriers and accordingly, prioritize genes associated with the CMS or nonCMS phenotype. Haplotypes in eleven candidate regions, with SNPs mostly in nonexonic regions, were significantly different between CMS and nonCMS subjects. Closer examination of individual genes in these regions revealed the involvement of previously identified candidates (e.g., SENP1) and also unreported ones SGK3, COPS5, PRDM1, and IFT122 in CMS. Remarkably, in addition to genes like SENP1, SGK3, and COPS5 which are HIF-dependent, our study reveals for the first time HIF-independent gene PRDM1, indicating an involvement of wider, nonHIF pathways in HA adaptation. Finally, we observed that down-regulating orthologs of these genes in Drosophila significantly enhanced their hypoxia tolerance. Taken together, the PreCIOSS algorithm, applied on a large number of genomes, identifies the involvement of both new and previously reported genes in selection sweeps, highlighting the involvement of multiple hypoxia response systems. Since the overwhelming majority of SNPs are in nonexonic (and possibly regulatory) regions, we speculate that adaptation to HA necessitates greater genetic flexibility allowing for transcript variability in response to graded levels of hypoxia.

New genetic and physiological factors for excessive erythrocytosis and Chronic Mountain Sickness.

In the last few years, genetic and functional studies have provided important insight on the pathophysiology of excessive erythrocytosis (EE), the main sign of Chronic Mountain Sickness (CMS). The recent finding of the association of the CMS phenotype with a single-nucleotide polymorphism (SNP) in the Sentrin-specific Protease 1 (SENP1) gene, and its differential expression pattern in Andean highlanders with and without CMS, has triggered large interest in high-altitude studies because of the potential role of its gene product in the control of erythropoiesis. The SENP1 gene encodes for a protease that regulates the function of hypoxia-relevant transcription factors such as Hypoxia-Inducible Factor (HIF) and GATA, and thus might have an erythropoietic regulatory role in CMS through the modulation of the expression of erythropoietin (Epo) or Epo receptors. The different physiological patterns in the Epo-EpoR system found among Andeans, even among highlanders with CMS, together with their different degrees of erythropoietic response, might indicate specific underlying genetic backgrounds, which in turn might reflect different levels of adaptation to lifelong high-altitude hypoxia. This minireview discusses recent genetic findings potentially underlying EE and CMS, and their possible physiological mechanisms in Andean highlanders.

Altitude Adaptation: A Glimpse Through Various Lenses.

Simonson, Tatum S. Altitude adaptation: A glimpse through various lenses. High Alt Med Biol 16:125-137, 2015.--Recent availability of genome-wide data from highland populations has enabled the identification of adaptive genomic signals. Some of the genomic signals reported thus far among Tibetan, Andean, and Ethiopian are the same, while others appear unique to each population. These genomic findings parallel observations conveyed by decades of physiological research: different continental populations, resident at high altitude for hundreds of generations, exhibit a distinct composite of traits at altitude. The most commonly reported signatures of selection emanate from genomic segments containing hypoxia-inducible factor (HIF) pathway genes. Corroborative evidence for adaptive significance stems from associations between putatively adaptive gene copies and sea-level ranges of hemoglobin concentration in Tibetan and Amhara Ethiopians, birth weights and metabolic factors in Andeans and Tibetans, maternal uterine artery diameter in Andeans, and protection from chronic mountain sickness in Andean males at altitude. While limited reports provide mechanistic insights thus far, efforts to identify and link precise genetic variants to molecular, physiological, and developmental functions are underway, and progress on the genomics front continues to provide unprecedented movement towards these goals. This combination of multiple perspectives is necessary to maximize our understanding of orchestrated biological and evolutionary processes in native highland populations, which will advance our understanding of both adaptive and non-adaptive responses to hypoxia.

Higher androgen bioactivity is associated with excessive erythrocytosis and chronic mountain sickness in Andean Highlanders: a review.

Populations living at high altitudes (HA), particularly in the Peruvian Central Andes, are characterised by presenting subjects with erythrocytosis and others with excessive erythrocytosis (EE)(Hb>21 g dl(-1) ). EE is associated with chronic mountain sickness (CMS), or lack of adaptation to HA. Testosterone is an erythropoietic hormone and it may play a role on EE at HA. The objective of the present review was to summarise findings on role of serum T levels on adaptation at HA and genes acting on this process. Men at HA without EE have higher androstenedione levels and low ratio androstenedione/testosterone than men with EE, suggesting low activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and this could be a mechanism of adaptation to HA. Higher conversion of dehydroepiandrosterone to testosterone in men with EE suggests nigher 17beta-HSD activity. Men with CMS at Peruvian Central Andes have two genes SENP1, and ANP32D with higher transcriptional response to hypoxia relative to those without. SUMO-specific protease 1 (SENP1) is an erythropoiesis regulator, which is essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia. SENP1 reverses the hormone-augmented SUMOylation of androgen receptor (AR) increasing the transcription activity of AR.In conclusion, increased androgen activity is related with CMS.

Genetic variation in SENP1 and ANP32D as predictors of chronic mountain sickness.

Chronic mountain sickness (CMS) is a serious illness that affects life-long high-altitude residents. A recent study analyzed whole genome sequence data from residents of Cerro de Pasco (Peru) in an effort to identify the genetic basis of CMS and reported SENP1 (rs7963934) and ANP32D (rs72644851) to show signatures consistent with natural selection and protective against CMS (Zhou et al. 2013 ). We set out to replicate these observations in two Andean cohorts from Cerro de Pasco, consisting of 84 CMS cases and 91 healthy controls in total. We report evidence of association for rs7963934 (SENP1) in the combined cohorts (meta-analysis p=8.8x10(-4) OR 2.91, CI 1.56-5.5, I=0). The direction of effect was the same as in the original publication. We did not observe any significant correlation between rs72644851 (ANP32D) and the CMS phenotype, within or across cohorts (meta-analysis p=0.204, OR 1.37, CI 0.84-2.241, I=0). Our results provide independent evidence in support of a role for SENP1 in CMS in individuals of Quechua ancestry and suggest the SENP1 and ANP32D signatures of selection are in tight linkage disequilibrium (LD).

Vascular endothelial growth factor-A is associated with chronic mountain sickness in the Andean population.

A study of chronic mountain sickness (CMS) with a candidate gene--vascular endothelial growth factor A (VEGFA)--was carried out in a Peruvian population living at high altitude in Cerro de Pasco (4380 m). The study was performed by genotyping of 11 tag SNPs encompassing 2.2 kb of region of VEGFA gene in patients with a diagnosis of CMS (n = 131; 49.1 ± 12.7 years old) and unrelated healthy controls (n = 84; 47.2 ± 13.4 years old). The VEGFA tag SNP rs3025033 was found associated with CMS (p < 0.05), individuals with AG genotype have 2.5 more risk of CMS compared to those with GG genotype (p < 0.02; OR, 2.54; 95% CI: 1.10-5.88). Pairwise Fst and Nei's distance indicate genetic differentiation between Cerro de Pasco population and HapMap3 population (Fst > 0.36, p < 0.01), suggesting selection is operating on the VEGF gene. Our results suggest that VEGFA is associated with CMS in long-term residents at high altitude in the Peruvian Andes.

Whole-genome sequencing uncovers the genetic basis of chronic mountain sickness in Andean highlanders.

The hypoxic conditions at high altitudes present a challenge for survival, causing pressure for adaptation. Interestingly, many high-altitude denizens (particularly in the Andes) are maladapted, with a condition known as chronic mountain sickness (CMS) or Monge disease. To decode the genetic basis of this disease, we sequenced and compared the whole genomes of 20 Andean subjects (10 with CMS and 10 without). We discovered 11 regions genome-wide with significant differences in haplotype frequencies consistent with selective sweeps. In these regions, two genes (an erythropoiesis regulator, SENP1, and an oncogene, ANP32D) had a higher transcriptional response to hypoxia in individuals with CMS relative to those without. We further found that downregulating the orthologs of these genes in flies dramatically enhanced survival rates under hypoxia, demonstrating that suppression of SENP1 and ANP32D plays an essential role in hypoxia tolerance. Our study provides an unbiased framework to identify and validate the genetic basis of adaptation to high altitudes and identifies potentially targetable mechanisms for CMS treatment.

Store-operated channels in the pulmonary circulation of high- and low-altitude neonatal lambs.

We determined whether store-operated channels (SOC) are involved in neonatal pulmonary artery function under conditions of acute and chronic hypoxia, using newborn sheep gestated and born either at high altitude (HA, 3,600 m) or low altitude (LA, 520 m). Cardiopulmonary variables were recorded in vivo, with and without SOC blockade by 2-aminoethyldiphenylborinate (2-APB), during basal or acute hypoxic conditions. 2-APB did not have effects on basal mean pulmonary arterial pressure (mPAP), cardiac output, systemic arterial blood pressure, or systemic vascular resistance in both groups of neonates. During acute hypoxia 2-APB reduced mPAP and pulmonary vascular resistance in LA and HA, but this reduction was greater in HA. In addition, isolated pulmonary arteries mounted in a wire myograph were assessed for vascular reactivity. HA arteries showed a greater relaxation and sensitivity to SOC blockers than LA arteries. The pulmonary expression of two SOC-forming subunits, TRPC4 and STIM1, was upregulated in HA. Taken together, our results show that SOC contribute to hypoxic pulmonary vasoconstriction in newborn sheep and that SOC are upregulated by chronic hypoxia. Therefore, SOC may contribute to the development of neonatal pulmonary hypertension. We propose SOC channels could be potential targets to treat neonatal pulmonary hypertension.

High-altitude medicine.

Medical problems occur at high altitude because of the low inspired Po(2), which is caused by the reduced barometric pressure. The classical physiological responses to high altitude include hyperventilation, polycythemia, hypoxic pulmonary vasoconstriction-increased intracellular oxidative enzymes, and increased capillary density in muscle. However, with the discovery of hypoxia-inducible factors (HIFs), it is apparent that there is a multitude of responses to cellular hypoxia. HIFs constitute a master switch determining the general response of the body to oxygen deprivation. The recent discovery of genetic changes in Tibetans has opened up an exciting area of research. The two major human populations that have adapted well to high altitude, the Tibetans and Andeans, have strikingly different phenotypes. Diseases of lowlanders going to high altitude include acute mountain sickness, high-altitude pulmonary edema, and high-altitude cerebral edema. Diseases affecting permanent residents or highlanders include chronic mountain sickness and high-altitude pulmonary hypertension. Important recent advances have been made on mitigation of the effects of the hypoxic environment. Oxygen enrichment of room air is very powerful. Every 1% increase in oxygen concentration reduces the equivalent altitude by about 300 m. This procedure is used in numerous facilities at high altitude and in a Chinese train to Lhasa. An alternative strategy is to increase the barometric pressure as in aircraft cabins. A hybrid approach combining both strategies shows promise but has never been used. Mines that are being developed at increasingly high altitudes pose great medical problems.

Mitochondrial haplogroup D4 confers resistance and haplogroup B is a genetic risk factor for high-altitude pulmonary edema among Han Chinese.

High-altitude pulmonary edema (HAPE) is a life-threatening condition caused by acute exposure to high altitude. Accumulating evidence suggests that genetic factors play an important role in the etiology of HAPE. However, conclusions from association studies have been hindered by limited sample size due to the rareness of this disease. It is known that mitochondria are critical for hypoxic adaptation, and mitochondrial malfunction can be an important factor in HAPE development. Therefore, we tested the hypothesis that mitochondrial DNA haplotypes and polymorphisms affect HAPE susceptibility. We recruited 204 HAPE patients and 174 healthy controls in Tibet (3658 m above sea level), all Han Chinese, constituting the largest sample size of all HAPE vulnerability studies. Among mtDNA haplogroups, we found that haplogroup D4 is associated with resistance to HAPE, while haplogroup B is a genetic risk factor for this condition. Haplogroup D4 (tagged by 3010A) may enhance the stability of 16S rRNA, resulting in reduced oxidative stress and protection against HAPE. Within haplogroup B, subhaplogroup B4c (tagged by 15436A and 1119C) was associated with increased risk for HAPE, while subhaplogroup B4b may protect against HAPE. We indicate that there are differences in HAPE susceptibility among mtDNA haplogroups. We conclude that mitochondria are involved in adverse reactions to acute hypoxic exposure; our finding of differences in susceptibility as a function of mitochondrial DNA haplotype may shed light on the pathogenesis of other disorders associated with hypoxia, such as chronic obstructive pulmonary disease.

High altitude renal syndrome (HARS).

More than 140 million people live permanently at high altitude (>2400 m) under hypoxic conditions that challenge basic physiology. Here we present a short historical review of the populating of these regions and of evidence for genetic adaptations and environmental factors (such as exposure to cobalt) that may influence the phenotypic responses. We also review some of the common renal physiologic responses focusing on clinical manifestations. The frequent presentation of systemic hypertension and microalbuminuria with relatively preserved GFR coupled with the presence of polycythemia and hyperuricemia suggests a new clinical syndrome we term high altitude renal syndrome (HARS). ACE inhibitors appear effective at reducing proteinuria and lowering hemoglobin levels in these patients.

Living the high life: high-altitude adaptation.

Genome-wide scans demonstrate that genetic variants associated with high-altitude adaptation in Tibetans and Andeans arose independently as a result of convergent adaptation.

A variant of the endothelial nitric oxide synthase gene (NOS3) associated with AMS susceptibility is less common in the Quechua, a high altitude Native population.

Endothelial nitric oxide synthase (eNOS) is a vascular enzyme that produces nitric oxide, a transient signaling molecule that by vasodilatation regulates blood flow and pressure. Nitric oxide is believed to play roles in both short-term acclimatization and long-term evolutionary adaptation to environmental hypoxia. Several laboratories, including ours, have shown that variants in NOS3 (the gene encoding eNOS) are overrepresented in individuals with altitude-related illnesses such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS), suggesting that NOS3 genotypes contribute to altitude tolerance. To further test our hypothesis that the G allele at the G894T polymorphism in NOS3 (dbSNP number: rs1799983; protein polymorphism Glu298Asp) is beneficial in hypoxic environments, we compared frequencies of this allele in an altitude-adapted Amerindian population, Quechua of the Andean altiplano, with those in a lowland Amerindian population, Maya of the Yucatan Peninsula. While common in both populations, the G allele was significantly more frequent in the highlanders. Taken together, our data suggest that this variant in NOS3, which has been previously associated with higher levels of nitric oxide, contributes to both acclimatization and adaptation to altitude.

Abnormal energy regulation in early life: childhood gene expression may predict subsequent chronic mountain sickness.

BACKGROUND: Life at altitude depends on adaptation to ambient hypoxia. In the Andes, susceptibility to chronic mountain sickness (CMS), a clinical condition that occurs to native highlanders or to sea level natives with prolonged residence at high altitude, remains poorly understood. We hypothesized that hypoxia-associated gene expression in children of men with CMS might identify markers that predict the development of CMS in adults. We assessed distinct patterns of gene expression of hypoxia-responsive genes in children of highland Andean men, with and without CMS. METHODS: We compared molecular signatures in children of highland (HA) men with CMS (n = 10), without CMS (n = 10) and in sea level (SL) children (n = 20). Haemoglobin, haematocrit, and oxygen saturation were measured. Gene expression in white cells was assessed at HA and then, in the same subjects, within one hour of arrival at sea level. RESULTS: HA children showed higher expression levels of genes regulated by HIF (hypoxia inducible factor) and lower levels of those involved in glycolysis and in the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase kinase 1(PDK1) and HIF prolyl hydroxylase 3 (HPH3) mRNA expressions were lowest in children of CMS fathers at altitude. At sea level the pattern of gene expression in the 3 children's groups was indistinguishable. CONCLUSION: The molecular signatures of children of CMS patients show impaired adaptation to hypoxia. At altitude children of CMS fathers had defective coupling between glycolysis and mitochondria TCA cycle, which may be a key mechanism/biomarker for adult CMS. Early biologic markers of disease susceptibility in Andeans might impact health services and social planning.