Doctor of Philosophy

dissertationHumans have lived at high altitude for hundreds of generations despite unavoidable challenges imposed by hypobaric hypoxia. The lower barometric pressure at high altitude reduces the number of oxygen molecules available in each breath of air, yet oxygen-dependent physiological processes must be maintained for survival. Cellular and system responses to hypoxic stress can result in altitude illness and may prove fatal in a small proportion of maladapted individuals. Native high-altitude populations, however, exhibit a unique suite of heritable traits that afford tolerance to hypoxia. Compared to lowland visitors and Andean highlanders, Tibetans exhibit lower hemoglobin (Hb) levels at high altitude, which tend to be similar to those expected under sea-level conditions. Such differences suggest this population has unique adaptations to their native environment. It has been hypothesized that genes specifically involved in the hypoxia inducible factor (HIF) pathway could underlie adaptive changes in high-altitude populations. Genome-wide analyses provide the first lines of evidence in support of genetic adaptation to high altitude. Three regions of the genome that contain genes associated with the human response to hypoxia show evidence of selection and are associated with decreased Hb levels, and two of these are also associated with metabolite levels. These phenotypic associations provide corroborative evidence for adaptive roles of genomic regions targeted by strong positive selection in Tibetans. iv While many of the same selection candidate genes are reported by studies of different Tibetan populations, some signals of selection and association are unique to particular groups. The genetic makeup of Tibetan groups located throughout the plateau is therefore important to consider in studies of high-altitude adaptation. Taken together, the data presented in this dissertation demonstrate that multiple genes are involved in Tibetan adaptation to high altitude. Some of these genes have been linked to hematological and metabolic phenotypes characterized thus far, providing further support for roles in physiological adaptation to this extreme environment. Studies aimed to identify associations between specific genetic variants, mechanisms, and phenotypes will help bridge the gap between genetic variation and organismal responses to hypoxia, and will have important implications for understanding human health and disease

Cognitive function and mood at high altitude following acclimatization and use of supplemental oxygen and adaptive servoventilation sleep treatments.

Impairments in cognitive function, mood, and sleep quality occur following ascent to high altitude. Low oxygen (hypoxia) and poor sleep quality are both linked to impaired cognitive performance, but their independent contributions at high altitude remain unknown. Adaptive servoventilation (ASV) improves sleep quality by stabilizing breathing and preventing central apneas without supplemental oxygen. We compared the efficacy of ASV and supplemental oxygen sleep treatments for improving daytime cognitive function and mood in high-altitude visitors (N = 18) during acclimatization to 3,800 m. Each night, subjects were randomly provided with ASV, supplemental oxygen (SpO2 > 95%), or no treatment. Each morning subjects completed a series of cognitive function tests and questionnaires to assess mood and multiple aspects of cognitive performance. We found that both ASV and supplemental oxygen (O2) improved daytime feelings of confusion (ASV: p < 0.01; O2: p < 0.05) and fatigue (ASV: p < 0.01; O2: p < 0.01) but did not improve other measures of cognitive performance at high altitude. However, performance improved on the trail making tests (TMT) A and B (p < 0.001), the balloon analog risk test (p < 0.0001), and the psychomotor vigilance test (p < 0.01) over the course of three days at altitude after controlling for effects of sleep treatments. Compared to sea level, subjects reported higher levels of confusion (p < 0.01) and performed worse on the TMT A (p < 0.05) and the emotion recognition test (p < 0.05) on nights when they received no treatment at high altitude. These results suggest that stabilizing breathing (ASV) or increasing oxygenation (supplemental oxygen) during sleep can reduce feelings of fatigue and confusion, but that daytime hypoxia may play a larger role in other cognitive impairments reported at high altitude. Furthermore, this study provides evidence that some aspects of cognition (executive control, risk inhibition, sustained attention) improve with acclimatization

Relationships Between Chemoreflex Responses, Sleep Quality, and Hematocrit in Andean Men and Women.

Andean highlanders are challenged by chronic hypoxia and many exhibit elevated hematocrit (Hct) and blunted ventilation compared to other high-altitude populations. While many Andeans develop Chronic Mountain Sickness (CMS) and excessive erythrocytosis, Hct varies markedly within Andean men and women and may be driven by individual differences in ventilatory control and/or sleep events which exacerbate hypoxemia. To test this hypothesis, we quantified relationships between resting ventilation and ventilatory chemoreflexes, sleep desaturation, breathing disturbance, and Hct in Andean men and women. Ventilatory measures were made in 109 individuals (n = 63 men; n = 46 women), and sleep measures in 45 of these participants (n = 22 men; n = 23 women). In both men and women, high Hct was associated with low daytime SpO2 (p < 0.001 and p < 0.002, respectively) and decreased sleep SpO2 (mean, nadir, and time <80%; all p < 0.02). In men, high Hct was also associated with increased end-tidal PCO2 (p < 0.009). While ventilatory responses to hypoxia and hypercapnia did not predict Hct, decreased hypoxic ventilatory responses were associated with lower daytime SpO2 in men (p < 0.01) and women (p < 0.009) and with lower nadir sleep SpO2 in women (p < 0.02). Decreased ventilatory responses to CO2 were associated with more time below 80% SpO2 during sleep in men (p < 0.05). The obstructive apnea index and apnea-hypopnea index also predicted Hct and CMS scores in men after accounting for age, BMI, and SpO2 during sleep. Finally, heart rate response to hypoxia was lower in men with higher Hct (p < 0.0001). These data support the idea that hypoventilation and decreased ventilatory sensitivity to hypoxia are associated with decreased day time and nighttime SpO2 levels that may exacerbate the stimulus for erythropoiesis in Andean men and women. However, interventional and longitudinal studies are required to establish the causal relationships between these associations

Anthrax: Evolutionary approaches for genetic-based investigative tools

A TaqMan-minor groove binding assay designed around a nonsense mutation in the plcR gene was used to genotype Bacillus anthracis, B. cereus, and B. thuringiensis isolates. The assay differentiated B. anthracis from these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracis strains

Higher oxygen content and transport characterize high-altitude ethnic Tibetan women with the highest lifetime reproductive success

We chose the “natural laboratory” provided by high-altitude native ethnic Tibetan women who had completed childbearing to examine the hypothesis that multiple oxygen delivery traits were associated with lifetime reproductive success and had genomic associations. Four hundred seventeen (417) women aged 46 to 86 y residing at ≥3,500 m in Upper Mustang, Nepal, provided information on reproductive histories, sociocultural factors, physiological measurements, and DNA samples for this observational cohort study. Simultaneously assessing multiple traits identified combinations associated with lifetime reproductive success measured as the number of livebirths. Women with the most livebirths had distinctive hematological and cardiovascular traits. A hemoglobin concentration near the sample mode and a high percent of oxygen saturation of hemoglobin raised arterial oxygen concentration without risking elevated blood viscosity. We propose ongoing stabilizing selection on hemoglobin concentration because extreme values predicted fewer livebirths and directional selection favoring higher oxygen saturation because higher values had more predicted livebirths. EPAS1, an oxygen homeostasis locus with strong signals of positive natural selection and a high frequency of variants occurring only among populations indigenous to the Tibetan Plateau, associated with hemoglobin concentration. High blood flow into the lungs, wide left ventricles, and low hypoxic heart rate responses aided effective convective oxygen transport to tissues. Women with physiologies closer to unstressed, low altitude values had the highest lifetime reproductive success. This example of ethnic Tibetan women residing at high altitudes in Nepal links reproductive fitness with trait combinations increasing oxygen delivery under severe hypoxic stress and demonstrates ongoing natural selection

Bacillus anthracis in China and its relationship to worldwide lineages

<p>Abstract</p> <p>Background</p> <p>The global pattern of distribution of 1033 <it>B. anthracis </it>isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP). These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages) have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different <it>B. anthracis </it>isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP) and multiple locus VNTR analysis (MLVA) typing has been used to examine this archival collection of isolates.</p> <p>Results</p> <p>The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China.</p> <p>Conclusion</p> <p><it>B. anthracis </it>isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum) in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads for not only trade but the movement of diseases such as anthrax along the ancient "silk road". Phylogenetic inference also suggests that the A.Br.Ames sub-lineage, first identified in the original Ames strain isolated from Jim Hogg County, TX, is descended from the A.Br.001/002 sub-group that has a major presence in most of China. These results suggest a genetic discontinuity between the younger Ames sub-lineage in Texas and the large Western North American sub-lineage spread across central Canada and the Dakotas.</p

Inflammation biomarkers in OSA, chronic obstructive pulmonary disease, and chronic obstructive pulmonary disease/OSA overlap syndrome.

Study objectivesThe coexistence of obstructive sleep apnea (OSA) and chronic obstructive pulmonary disease (COPD) in a single individual, also known as overlap syndrome (OVS), is associated with higher cardiovascular risk and mortality than either OSA or COPD alone. However, the underlying mechanisms remain unclear. We hypothesized that patients with OVS have elevated systemic inflammatory biomarkers relative to patients with either disease alone, which could explain greater cardiovascular risk observed in OVS.MethodsWe included 255 participants in the study, 55 with COPD alone, 100 with OSA alone, 50 with OVS, and 50 healthy controls. All participants underwent a home sleep study, spirometry, and a blood draw for high-sensitivity C-reactive protein and total blood count analysis. In a randomly selected subset of 186 participants, inflammatory protein profiling was performed using Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assays. Biomarker level differences across groups were identified using a mixed linear model.ResultsLevels of interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and granulocyte colony stimulating factor (G-CSF) were higher in participants with OVS and COPD compared with healthy controls and participants with OSA. Furthermore, participants with OVS had higher circulating levels of leukocytes and neutrophils than those with COPD, OSA, and controls.ConclusionsCOPD and OVS are associated with higher systemic inflammation relative to OSA and healthy controls. This work proposes the potential utilization of interleukin 6, granulocyte colony stimulating factor, and high-sensitivity C-reactive protein as screening biomarkers for COPD in patients with OSA. Inflammatory pathways may not fully explain the higher cardiovascular risk observed in OVS, indicating the need for further investigation.CitationSanchez-Azofra A, Gu W, Masso-Silva JA, et&nbsp;al. Inflammation biomarkers in OSA, chronic obstructive pulmonary disease, and chronic obstructive pulmonary disease/OSA overlap syndrome. J Clin Sleep Med. 2023;19(8):1447-1456

Global Genetic Population Structure of Bacillus anthracis

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064–6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated

Functional EPAS1/ HIF2A Missense Variant Is Associated With Hematocrit in Andean Highlanders

Hypoxia-inducible factor pathway genes are linked to adaptation in both human and nonhuman highland species. EPAS1, a notable target of hypoxia adaptation, is associated with relatively lower hemoglobin concentration in Tibetans. We provide evidence for an association between an adaptive EPAS1 variant (rs570553380) and the same phenotype of relatively low hematocrit in Andean highlanders. This Andean-specific missense variant is present at a modest frequency in Andeans and absent in other human populations and vertebrate species except the coelacanth. CRISPR-base-edited human cells with this variant exhibit shifts in hypoxia-regulated gene expression, while metabolomic analyses reveal both genotype and phenotype associations and validation in a lowland population. Although this genocopy of relatively lower hematocrit in Andean highlanders parallels well-replicated findings in Tibetans, it likely involves distinct pathway responses based on a protein-coding versus noncoding variants, respectively. These findings illuminate how unique variants at EPAS1 contribute to the same phenotype in Tibetans and a subset of Andean highlanders despite distinct evolutionary trajectories

Ancestry of the Iban Is Predominantly Southeast Asian: Genetic Evidence from Autosomal, Mitochondrial, and Y Chromosomes

Humans reached present-day Island Southeast Asia (ISEA) in one of the first major human migrations out of Africa. Population movements in the millennia following this initial settlement are thought to have greatly influenced the genetic makeup of current inhabitants, yet the extent attributed to different events is not clear. Recent studies suggest that south-to-north gene flow largely influenced present-day patterns of genetic variation in Southeast Asian populations and that late Pleistocene and early Holocene migrations from Southeast Asia are responsible for a substantial proportion of ISEA ancestry. Archaeological and linguistic evidence suggests that the ancestors of present-day inhabitants came mainly from north-to-south migrations from Taiwan and throughout ISEA approximately 4,000 years ago. We report a large-scale genetic analysis of human variation in the Iban population from the Malaysian state of Sarawak in northwestern Borneo, located in the center of ISEA. Genome-wide single-nucleotide polymorphism (SNP) markers analyzed here suggest that the Iban exhibit greatest genetic similarity to Indonesian and mainland Southeast Asian populations. The most common non-recombining Y (NRY) and mitochondrial (mt) DNA haplogroups present in the Iban are associated with populations of Southeast Asia. We conclude that migrations from Southeast Asia made a large contribution to Iban ancestry, although evidence of potential gene flow from Taiwan is also seen in uniparentally inherited marker data