Lorna G. Moore

Identifying signatures of natural selection in Tibetan and Andean populations using dense genome scan data

High-altitude hypoxia (reduced inspired oxygen tension due to decreased barometric pressure) exerts severe physiological stress on the human body. Two high-altitude regions where humans have lived for millennia are the Andean Altiplano and the Tibetan Plateau. Populations living in these regions exhibit unique circulatory, respiratory, and hematological adaptations to life at high altitude. Although these responses have been well characterized physiologically, their underlying genetic basis remains unknown. We performed a genome scan to identify genes showing evidence of adaptation to hypoxia. We looked across each chromosome to identify genomic regions with previously unknown function with respect to altitude phenotypes. In addition, groups of genes functioning in oxygen metabolism and sensing were examined to test the hypothesis that particular pathways have been involved in genetic adaptation to altitude. Applying four population genetic statistics commonly used for detecting signatures of natural selection, we identified selection-nominated candidate genes and gene regions in these two populations (Andeans and Tibetans) separately. The Tibetan and Andean patterns of genetic adaptation are largely distinct from one another, with both populations showing evidence of positive natural selection in different genes or gene regions. Interestingly, one gene previously known to be important in cellular oxygen sensing, EGLN1 (also known as PHD2), shows evidence of positive selection in both Tibetans and Andeans. However, the pattern of variation for this gene differs between the two populations. Our results indicate that several key HIF-regulatory and targeted genes are responsible for adaptation to high altitude in Andeans and Tibetans, and several different chromosomal regions are implicated in the putative response to selection. These data suggest a genetic role in high-altitude adaption and provide a basis for future genotype/phenotype association studies necessary to confirm the role of selection-nominated candidate genes and gene regions in adaptation to altitude.

Acute Mountain Sickness in a General Tourist Population at Moderate Altitudes

Rapid ascent from low to high altitude is often followed by headache, fatigue, shortness of breath, sleeplessness, and anorexia, a symptom complex called acute mountain sickness. Although some of these symptoms may occur as a result of travel not associated with altitude, only 5% of adults traveling at sea level report similar symptoms [1]. A long-standing interest has existed in the study of acute mountain sickness because it affects a large number of mountain visitors [2-4] and can progress to the life-threatening conditions of high-altitude pulmonary edema or high-altitude cerebral edema [5]. Previous estimates of the incidence of acute mountain sickness have been obtained primarily from small groups of physically fit young men going to altitudes above 12 000 feet [2-4, 7-9]. Little information exists on the frequency and severity of the disorder in the general population at moderate altitudes, yet the population at risk is large. For example, more than 13 million persons visited the Colorado mountains in 1990 for business, conferences, or recreational activities including skiing, climbing, hiking, hunting, and fishing [10]. More needs to be learned about the incidence of acute mountain sickness at moderate altitudes in the general population and about the characteristics of those most likely to be at risk for symptom development. We therefore surveyed groups of persons visiting resorts in the Colorado mountains for conferences and seminars. Specifically, we sought to determine 1) the incidence of acute mountain sickness in visitors exposed to moderate elevations; 2) the effect of acute mountain sickness on physical activity; and 3) the visitor characteristics associated with the development of acute mountain sickness. This information would be useful for developing strategies to minimize symptoms in travelers to moderate altitudes. Methods The study cohort consisted of 4212 adults attending 45 conferences at resorts located at elevations of 6300 to 9700 feet in the Rocky Mountains of Colorado from July 1989 to May 1991. Resorts were chosen on the basis of the willingness of conference organizers to participate. Conferences whose schedules required all participants to attend a meeting within 48 hours of arrival when the study questionnaire could be distributed were included. Study personnel attended these meetings, briefly introduced the study, and distributed the questionnaires. Questions by participants concerning acute mountain sickness or the effects of altitude on health were not answered until all questionnaires were collected. Completion of the survey usually took less than 10 minutes. The participants in each meeting were counted to calculate the response rate. The questionnaire was completed by 3158 (75%) of the persons registered for these conferences, and information satisfactory for analysis was obtained from 99% of those completed. Visitors ranged in age from 16 to 87 years (mean age [SD], 657e43.8 11.8 years) (Figure 1). Of the completed surveys, 2023 (64%) were conducted at resorts located at elevations over 9000 feet and 2603 (82%) were completed in the winter season (November through April). The study was approved by the Human Research Committee of the University of Colorado Health Sciences Center. Figure 1. Distribution of visitors by age and physical condition. n n The questionnaire was designed to obtain demographic information concerning age, gender, height, weight, and permanent residence for each visitor; previous or current medications; and the duration of stops, if any, made en route. Questions asked regarding underlying health conditions included whether the participant had ever been treated for lung disease (asthma, bronchitis, or emphysema); heart conditions (angina or heart attacks); diabetes; high blood pressure; or pregnancy. Participants were asked How do you rate your physical condition? Responses were rated as great, good, average, or poor. To determine whether participants had acute mountain sickness, they were asked if they had experienced any of the following symptoms while at the resort: loss of appetite, vomiting, shortness of breath, dizziness or lightheadedness, unusual fatigue, sleep disturbance (other than related to normal travel), and headache. If the response to headache was yes, they were asked to describe it as mild or severe. Acute mountain sickness was defined as the presence of three or more of these symptoms in the setting of recent altitude exposure. This case definition was similar to that used by previous investigators [1, 2, 5, 6, 9, 12, 13] and is in accordance with the case definition recently developed and codified for international use by the International Hypoxia Symposium [14]. If participants had any of these symptoms of acute mountain sickness, they were asked to determine how the symptoms affected their activity. Response options included no limitation, reduced activity, and required to stay in bed or room. A determination of symptom onset was assessed by asking participants How long after arrival at the resort did symptoms begin? Response options included less than 12 hours, 12 to 24 hours, 25 to 48 hours, 49 to 72 hours, or 72 hours. Alcohol use was measured as the number of beer, wine, or hard-liquor drinks consumed within the first 24 hours of arrival at the resort. Season was determined by the date of questionnaire administration, with winter defined as November through April in each of the study years. Body mass index was calculated as weight kg/in2 and was used to identify obese persons (women with a body mass index >27.3 and men with a body mass index >27.8) [11]. Persons with or without acute mountain sickness were compared using the Wilcoxon rank-sum test for ordinal variables and using the chi-square test for categoric variables. The Student t-test was used for normally distributed variables. The Fisher exact test was used for small sample sizes. Associations were considered significant at P < 0.05. A forward, stepwise, multiple logistic regression analysis was used to examine the independent effects of participant characteristics on the occurrence of acute mountain sickness. All variables associated with the occurrence of acute mountain sickness at P < 0.25 were initially included in the regression analysis. Data acquired in year 2 (June 1990 to May 1991), comprising 1241 cases after the revision of the questions concerning underlying health conditions and habitual activity level before travel, were used for the regression analysis. Variables were dichotomized for ease in presentation. The adjusted odds ratios were computed with 95% confidence intervals (CIs). All calculations were done using the Statistical Analysis Systems statistical package (Cary, North Carolina) [15]. Results Most of the visitors were middle-aged men whose permanent addresses were at sea level, who did not smoke, and who considered themselves to be in good physical condition (Table 1; Figure 1). Approximately one third (28%) stopped overnight at an intermediate altitude (5280 feet) enroute to their destination. Most (64%) had consumed one or more alcoholic beverages in the first 24 hours after arrival. Small proportions of the visitors were obese, pregnant, or had chronic illnesses (Table 1). Table 1. Characteristics and Incidence of Acute Mountain Sickness in Visitors to Areas of Moderate Altitude* Twenty-five percent (CI, 24.98% to 25.01%) of the visitors reported having three or more symptoms and thus met the case definition for acute mountain sickness, whereas 73% had at least one reported symptom. The most common symptom was headache, and the least common was vomiting (Figure 2). For most participants (65%), the onset of symptoms occurred within the first 12 hours after arrival at altitude; symptom onset occurred between 12 and 36 hours in 34% and after more than 36 hours in 1%. Most (58%) of those with symptoms took analgesics (for example, aspirin, acetaminophen, or ibuprofen). Although 44% of persons with acute mountain sickness had no reduction in activity, 51% had moderate activity reduction, and a small proportion (5%) stayed in bed. Figure 2. Distribution of symptoms of acute mountain sickness in 3072 visitors. Visitors whose permanent residence was at an elevation below 3000 feet were more likely to develop acute mountain sickness (see Table 1). The frequency with which it developed was inversely related to age and physical condition (Figure 3). Altitude visited and a previous history of acute mountain sickness were associated with an increased occurrence, whereas development was inversely related to alcohol consumption. Visitors who stopped over at lower elevations for more than 38 hours were less likely to develop acute mountain sickness than were those who did not. Obesity, female gender, and chronic lung disease were also associated with the development of acute mountain sickness. Figure 3. Percentage of acute mountain sickness in visitors to moderate altitudes according to age, physical condition, and altitude visited. n P n P n P The following nine variables were entered into the regression analysis as dichotomous variables: age (younger than 60 years), sex, altitude of permanent residence (below 3000 feet), obesity, lung disease, diabetes, overnight stops before arrival at the resort, previous symptoms during past altitude travel, and self-reported physical condition (poor or average). Although alcohol was statistically associated with acute mountain sickness, it was not included in the model because of the inability to exclude a temporal effect (that is, participants may have become sick and subsequently decided not to drink). The five independent predictors of acute mountain sickness, based on the logistic regression, were residence at an altitude less than 3000 feet; symptoms of acute mountain sickness during previous altitude travel; age younger than 60 years; physical condition self-assessed as poor or average; and the presence of lun

HUMAN ADAPTATION TO HIGH ALTITUDE : REGIONAL AND LIFE-CYCLE PERSPECTIVES

Studies of the ways in which persons respond to the adaptive challenges of life at high altitude have occupied an important place in anthropology. There are three major regions of the world where high-altitude studies have recently been performed: the Himalayas of Asia, the Andes of South America, and the Rocky Mountains of North America. Of these, the Himalayan region is larger, more geographically remote, and likely to have been occupied by humans for a longer period of time and to have been subject to less admixture or constriction of its gene pool. Recent studies of the physiological responses to hypoxia across the life cycle in these groups reveal several differences in adaptive success. Compared with acclimatized newcomers, lifelong residents of the Andes and/or Himalayas have less intrauterine growth retardation, better neonatal oxygenation, and more complete neonatal cardiopulmonary transition, enlarged lung volumes, decreased alveolar-arterial oxygen diffusion gradients, and higher maximal exercise capacity. In addition, Tibetans demonstrate a more sustained increase in cerebral blood flow during exercise, lower hemoglobin concentration, and less susceptibility to chronic mountain sickness (CMS) than acclimatized newcomers. Compared to Andean or Rocky Mountain high-altitude residents, Tibetans demonstrate less intrauterine growth retardation, greater reliance on redistribution of blood flow than elevated arterial oxygen content to increase uteroplacental oxygen delivery during pregnancy, higher levels of resting ventilation and hypoxic ventilatory responsiveness, less hypoxic pulmonary vasoconstriction, lower hemoglobin concentration, and less susceptibility to CMS. Several of the distinctions demonstrated by Tibetans parallel the differences between natives and newcomers, suggesting that the degree of protection or adaptive benefit relative to newcomers is enhanced for the Tibetans. We thus conclude that Tibetans have several physiological distinctions that confer adaptive benefit consistent with their probable greater generational length of high-altitude residence. Future progress is anticipated in achieving a more integrated view of high-altitude adaptation, incorporating a sophisticated understanding of the ways in which levels of biological organization are articulated and a recognition of the specific genetic variants contributing to differences among high-altitude groups.

Intrauterine growth restriction, preeclampsia, and intrauterine mortality at high altitude in Bolivia

Infant mortality and stillbirth rates in Bolivia are high and birth weights are low compared with other South American countries. Most Bolivians live at altitudes of 2500 m or higher. We sought to determine the impact of high altitude on the frequency of preeclampsia, gestational hypertension, and other pregnancy-related complications in Bolivia. We then asked whether increased preeclampsia and gestational hypertension at high altitude contributed to low birth weight and increased stillbirths. We performed a retrospective cohort study of women receiving prenatal care at low (300 m, Santa Cruz, n = 813) and high altitude (3600 m, La Paz, n = 1607) in Bolivia from 1996 to 1999. Compared with babies born at low altitude, high-altitude babies weighed less (3084 ± 12 g versus 3366 ± 18 g, p < 0.01) and had a greater occurrence of intrauterine growth restriction [16.8%; 95% confidence interval (CI): 14.9-18.6 versus 5.9%; 95% CI: 4.2-7.5; p < 0.01]. Preeclampsia and gestational hypertension were 1.7 times (95% CI: 1.3-2.3) more frequent at high altitude and 2.2 times (95% CI: 1.4-3.5) more frequent among primiparous women. Both high altitude and hypertensive complications independently reduced birth weight. All maternal, fetal, and neonatal complications surveyed were more frequent at high than low altitude, including fetal distress (odds ratio, 7.3; 95% CI: 3.9-13.6) and newborn respiratory distress (odds ratio, 7.3; 95% CI: 3.9-13.6; p < 0.01). Hypertensive complications of pregnancy raised the risk of stillbirth at high (odds ratio, 6.0; 95% CI: 2.2-16.2) but not at low altitude (odds ratio, 1.9; 95% CI: 0.2-17.5). These findings suggest that high altitude is an important factor worsening intrauterine mortality and maternal and infant health in Bolivia.

Arterial oxygen saturation in Tibetan and Han infants born in Lhasa, Tibet.

BACKGROUND Reduced oxygen availability at high altitude is associated with increased neonatal and infant mortality. We hypothesized that native Tibetan infants, whose ancestors have inhabited the Himalayan Plateau for approximately 25,000 years, are better able to maintain adequate oxygenation at high altitude than Han infants, whose ancestors moved to Tibet from lowland areas of China after the Chinese military entered Tibet in 1951. METHODS We compared arterial oxygen saturation, signs of hypoxemia, and other indexes of neonatal wellbeing at birth and during the first four months of life in 15 Tibetan infants and 15 Han infants at 3658 m above sea level in Lhasa, Tibet. The Han mothers had migrated from lowland China about two years previously. A pulse oximeter was placed on each infants foot to provide measurements of arterial oxygen saturation distal to the ductus arteriosus. RESULTS The two groups had similar gestational ages (about 38.9 weeks) and Apgar scores. The Han infants had lower birth weights (2773 +/- 92 g) than the Tibetan infants (3067 +/- 107 g), higher concentrations of cord-blood hemoglobin (18.6 +/- 0.8 g per deciliter, vs. 16.7 +/- 0.4 in the Tibetans), and higher hematocrit values (58.5 +/- 2.4 percent, vs. 51.4 +/- 1.2 percent in the Tibetans). In both groups, arterial oxygen saturation was highest in the first two days after birth and was lower when the infants were asleep than when they were awake. Oxygen saturation values were lower in the Han than in the Tibetan infants at all times and under all conditions during all activities. The values declined in the Han infants from 92 +/- 3 percent while they were awake and 90 +/- 5 percent during quiet sleep at birth to 85 +/- 4 percent while awake and 76 +/- 5 percent during quiet sleep at four months of age. In the Tibetan infants, oxygen saturation values averaged 94 +/- 2 percent while they were awake and 94 +/- 3 percent during quiet sleep at birth and 88 +/- 2 percent while awake and 86 +/- 5 percent during quiet sleep at four months. Han infants had clinical signs of hypoxemia--such as cyanosis during sleep and while feeding--more frequently than Tibetans. CONCLUSIONS In Lhasa, Tibet, we found that Tibetan newborns had higher arterial oxygen saturation at birth and during the first four months of life than Han newborns. Genetic adaptations may permit adequate oxygenation and confer resistance to the syndrome of pulmonary hypertension and right-heart failure (subacute infantile mountain sickness).

Development of a panel of genome-wide ancestry informative markers to study admixture throughout the americas

Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2>0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.

Placental contribution to the origins of sexual dimorphism in health and diseases: sex chromosomes and epigenetics

Sex differences occur in most non-communicable diseases, including metabolic diseases, hypertension, cardiovascular disease, psychiatric and neurological disorders and cancer. In many cases, the susceptibility to these diseases begins early in development. The observed differences between the sexes may result from genetic and hormonal differences and from differences in responses to and interactions with environmental factors, including infection, diet, drugs and stress. The placenta plays a key role in fetal growth and development and, as such, affects the fetal programming underlying subsequent adult health and accounts, in part for the developmental origin of health and disease (DOHaD). There is accumulating evidence to demonstrate the sex-specific relationships between diverse environmental influences on placental functions and the risk of disease later in life. As one of the few tissues easily collectable in humans, this organ may therefore be seen as an ideal system for studying how male and female placenta sense nutritional and other stresses, such as endocrine disruptors. Sex-specific regulatory pathways controlling sexually dimorphic characteristics in the various organs and the consequences of lifelong differences in sex hormone expression largely account for such responses. However, sex-specific changes in epigenetic marks are generated early after fertilization, thus before adrenal and gonad differentiation in the absence of sex hormones and in response to environmental conditions. Given the abundance of X-linked genes involved in placentogenesis, and the early unequal gene expression by the sex chromosomes between males and females, the role of X- and Y-chromosome-linked genes, and especially those involved in the peculiar placenta-specific epigenetics processes, giving rise to the unusual placenta epigenetic landscapes deserve particular attention. However, even with recent developments in this field, we still know little about the mechanisms underlying the early sex-specific epigenetic marks resulting in sex-biased gene expression of pathways and networks. As a critical messenger between the maternal environment and the fetus, the placenta may play a key role not only in buffering environmental effects transmitted by the mother but also in expressing and modulating effects due to preconceptional exposure of both the mother and the father to stressful conditions.

Fetal growth restriction and maternal oxygen transport during high altitude pregnancy.

High altitude reduces birth weights, averaging a 100-g fall per 1000 m elevation gain, as the result of restriction of third trimester fetal growth. Intrauterine growth restriction (IUGR) raises neonatal or infant mortality at low as well as at high altitude, but existing studies are unclear as to whether IUGR-specific mortality at high altitude is similar to, less than, or greater than at low altitude. Pregnancy increases maternal ventilation and raises arterial O(2) saturation at high altitude, which helps to protect against altitude-associated IUGR. Chronic hypoxia interferes with the maternal circulatory adjustments to pregnancy such that blood volume is lower and the rise in cardiac output diminished compared with sea level. The growth and remodeling of the uterine artery and other uteroplacental vessels is incomplete at high compared with low altitude, with the result that there is less redistribution of common iliac flow from the external iliac to the uterine arteries and lower uterine artery blood flow near term. Adaptations in multigenerational high altitude populations (e.g., Andeans and Tibetans) permit higher uterine artery blood flows and protect against altitude-associated IUGR.

Humans at high altitude: hypoxia and fetal growth.

High-altitude studies offer insight into the evolutionary processes and physiological mechanisms affecting the early phases of the human lifespan. Chronic hypoxia slows fetal growth and reduces the pregnancy-associated rise in uterine artery (UA) blood flow. Multigenerational vs. shorter-term high-altitude residents are protected from the altitude-associated reductions in UA flow and fetal growth. Presently unknown is whether this fetal-growth protection is due to the greater delivery or metabolism of oxygen, glucose or other substrates or to other considerations such as mechanical factors protecting fragile fetal villi, the creation of a reserve protecting against ischemia/reperfusion injury, or improved placental O(2) transfer as the result of narrowing the A-V O(2) difference and raising uterine P(v)O₂. Placental growth and development appear to be normal or modified at high altitude in ways likely to benefit diffusion. Much remains to be learned concerning the effects of chronic hypoxia on embryonic development. Further research is required for identifying the fetoplacental and maternal mechanisms responsible for transforming the maternal vasculature and regulating UA blood flow and fetal growth. Genomic as well as epigenetic studies are opening new avenues of investigation that can yield insights into the basic pathways and evolutionary processes involved.

Oxygen transport in tibetan women during pregnancy at 3,658 m.

High-altitude reduces infant birth weight as a result of intrauterine growth restriction (IUGR) and is associated with increased neonatal mortality. We hypothesized that babies born to Tibetan compared to Han (Chinese) high-altitude residents were protected from IUGR as the result of increased maternal O(2) transport due, in turn, to increased uterine artery (UA) blood flow. We studied 68 nonpregnant or pregnant Tibetan or Han residents of Lhasa, Tibet Autonomous Region, China (3,658 m). The pregnant women had higher hypoxic ventilatory responses (HVR A) and resting ventilations (V(E)) than their nonpregnant counterparts (Tib HVR = 134 +/- 16 (SEM) vs. 30 +/- 8, Han HVR = 134 +/- 16 vs. 66 +/- 18 A units; Tib V(E) = 11.8 +/- 0.3 vs. 10.1 +/- 0.5, Han V(E) = 10.7 +/- 0.5 vs. 9.4 +/- 0.5 l BTPS/min; all P < 0.05). Pregnancy did not change hemoglobin concentration in the Han but lowered values more than 2 g/dl in the Tibetans, serving to reduce arterial O(2) content below Han values (15.4 +/- 0.3 vs. 17.4 +/- 0.5 ml O(2)/100 ml whole blood, P < 0.05). Compared with the Han, the pregnant Tibetans had higher UA blood flow velocity (58.5 +/- 2.9 vs. 49.1 +/- 3.2, P < 0. 05) and distributed a higher portion of common iliac (CI) blood flow to the UA (4.8 +/- 0.4 vs. 3.3 +/- 0.3, P < 0.05). Birth weights averaged 635 g greater in the Tibetan than Han high-altitude residents (3,280 +/- 78 vs. 2,645 +/- 96 g, P < 0.01), or 694 g more when adjusted for maternal age, parity, height, and near-term body weight. Heavier birth weight babies were born to women with higher V(E) (r = 0.62, P < 0.01) and greater distribution of CI blood flow to the UA (r = 0.42, P < 0.05). We conclude that increased UA blood flow, and not higher arterial O(2) content, permits Tibetan women to increase uteroplacental O(2) delivery and protect their infants from altitude-associated IUGR.