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April 4, 2017

How Tibetans can function at high altitudes

Filed under: Altitude Adaptation,Evolution,Genetics,Genomics,Human Evolution,Tibetans — Razib Khan @ 11:10 am


About seven years ago I wrote two posts about how Tibetans manage to function at very high altitudes. And it’s not just physiological functioning, that is, fitness straightforwardly understood. High altitudes can cause a sharp reduction in reproductive fitness because women can not carry pregnancies to term. In other words, high altitude is a very strong selection pressure. You adapt, or you die off.

For me there have been two things of note since those original papers came out. First, one of those loci seem to have been introgressed from a Denisovan genetic background. I want to be careful here, because the initial admixture event may not have been into the Tibetans proper, but earlier hunter-gatherers who descend from Out of Africa groups, who were assimilated into the Tibetans as they expanded 5-10,000 years ago. Second, it turns out that dogs have been targeted for selection on EPAS1 as well (the “Denisovan” introgression) for altitude adaptation as well.

This shows that in mammals at least there’s a few genes which show up again and again. The fact that EPAS1 and EGLN1 were hits on relatively small sample sizes also reinforces their powerful effect. When the EPAS1 results initially came out they were highlighted as the strongest and fastest instance of natural selection in human evolutionary history. One can quibble about the details about whether this was literally true, but that it was a powerful selective event no one could deny.

A new paper in PNAS, Genetic signatures of high-altitude adaptation in Tibetans, revisits the earlier results with a much larger sample size (the research group is in China) comparing Han Chinese and Tibetans. They confirm the earlier results, but, they also find other loci which seem likely targets of selection in Tibetans. Below is the list:

SNP A1 A2 Frequency of A1 P value FST Nearest gene
Tibetan EAS (Han)
rs1801133 A G 0.238 0.333 6.30E-09 0.021 MTHFR
rs71673426 C T 0.102 0.013 1.50E-08 0.1 RAP1A
rs78720557 A T 0.498 0.201 4.70E-08 0.191 NEK7
rs78561501 A G 0.599 0.135 6.10E-15 0.414 EGLN1
rs116611511 G A 0.447 0.003 3.60E-19 0.57 EPAS1
rs2584462 G A 0.211 0.549 3.90E-09 0.203 ADH7
rs4498258 T A 0.586 0.287 1.70E-08 0.171 FGF10
rs9275281 G A 0.095 0.365 1.10E-10 0.162 HLA-DQB1
rs139129572 GA G 0.316 0.449 5.80E-09 0.036 HCAR2
P value indicates the P value from the MLMA-LOCO analysis. FST is the FST value between Tibetans and EASs. Nearest gene indicates the nearest annotated gene to the top differentiated SNP at each locus except EGLN1, which is known to be associated with high-altitude adaptation; rs139129572 is an insertion SNP with two alleles: GA and G. A1, allele 1; A2, allele 2.

Many of these genes are familiar. Observe the allele frequency differences between the Tibetans and other East Asians (mostly Han). The sample sizes are on the order of thousands, and the SNP-chip had nearly 300,000 markers. What they found was that the between population Fst of Han to Tibetan was ~0.01. So only 1% of the SNP variance in their data was partitioned between the two groups. These alleles are huge outliers.

The authors used some sophisticated statistical methods to correct for exigencies of population structure, drift, admixture, etc., to converge upon these hits, but even through inspection the deviation on these alleles is clear. And as they note in the paper it isn’t clear all of these genes are selected simply for hypoxia adaptation. MTFHR, which is quite often a signal of selection, may have something to due to folate production (higher altitudes have more UV). ADH7 is part of a set of genes which always seem to be under selection, and HLA is never a surprise.

Rather than get caught up in the details it is important to note here that expansion into novel habitats results in lots of changes in populations, so that two groups can diverge quite fast on functional characteristics.  The PCA makes it clear that Tibetans and Hans have very little West Eurasian admixture, and the Fst based analysis puts their divergence on the order of 5,000 years before the present. The authors admit honestly that this is probably a lower bound value, but I also think it is quite likely that Tibetans, and probably Han too, are compound populations, and a simple bifurcation model from a common ancestral population is probably shaving away too many realistic edges. In plainer language, there has been gene flow between Han and Tibetans probably <5,000 years ago, and Tibetans themselves probably assimilated more deeply diverged populations in the highlands as they expanded as agriculturalists. An estimate of a single divergence fits a complex history to too simple of a model quite often.

The take home: understanding population history is probably important to get a better sense of the dynamics of adaptation.

Citation: Jian Yang, Zi-Bing Jin, Jie Chen, Xiu-Feng Huang, Xiao-Man Li, Yuan-Bo Liang, Jian-Yang Mao, Xin Chen, Zhili Zheng, Andrew Bakshi, Dong-Dong Zheng, Mei-Qin Zheng, Naomi R. Wray, Peter M. Visscher, Fan Lu, and Jia Qu, Genetic signatures of high-altitude adaptation in Tibetans, PNAS 2017 ; published ahead of print April 3, 2017, doi:10.1073/pnas.1617042114

September 14, 2010

The silver age of altitude adaptation

tib1With all the justified concern about “missing heritability”, the age of human genomics hasn’t been a total bust. As I have observed before in 2005’s excellent book Mutants the evolutionary geneticist Armand M. Leroi asserted that we really didn’t have a good understanding of normal variation of human pigmentation. At the time I think it was a defensible claim, but within three years I’d say that most of the mystery had been cleared up. Though there are still some holes to be plugged, and details to be elucidated, the genetic architecture of pigmentation is now understood more or less. By the fall of 2006 Richard Sturm penned a review titled A golden age of human pigmentation genetics, an age I think which in some ways probably was closed with his 2009 review Molecular genetics of human pigmentation diversity. It’s not surprising that many of the traits that 23andMe tells you about have to do with your pigmentation. Of course there’s some limited utility in this, one assumes that most individuals don’t gain much benefit from the knowledge that they have an “85% change of having brown eyes,” though it may be useful in terms of offspring prediction (I would say I have an 85% chance of having brown eyes, but since I’m not European the genetic background isn’t right to make that probability assertion).

ResearchBlogging.orgBut as the golden age of pigmentation genetics comes to a close and the low hanging fruit is stripped bare, where next? I wonder if it may be altitude adaptations. Like pigmentation altitude genetics has been around for a while, but it seems there’s a recent cresting of papers in the area, focusing in particular on the three canonical high altitude peoples, the Tibetans, Andeans, and the Ethiopians. Last spring two major groups came out with papers on the genetics of Tibetan altitude adaptation, and its evolutionary history, using somewhat different techniques. A new paper in PLoS Genetics builds upon that work (verifying two of the loci as targets of selection in Tibetans implicated in the previous papers), and, adds Andean populations to the mix to assess the possibilities of convergent adaptations. Identifying Signatures of Natural Selection in Tibetan and Andean Populations Using Dense Genome Scan Data:

High-altitude hypoxia is caused by decreased barometric pressure at high altitude, and results in severe physiological stress to the human body. Three human populations have resided at high altitude for millennia including Andeans on the Andean Altiplano, Tibetans on the Himalayan plateau, and Ethiopian highlanders on the Semian Plateau. Each of these populations exhibits a unique suite of physiological changes to the decreased oxygen available at altitude. However, we are just beginning to understand the genetic changes responsible for the observed physiology. The aim of the current study was to identify gene regions that may be involved in adaptation to high altitude in both Andeans and Tibetans. Genomic regions showing evidence of recent positive selection were identified in these two high-altitude human groups separately. We found compelling evidence of positive selection in HIF pathway genes, in the globin cluster located on chromosome 11, and in several chromosomal regions for Andeans and Tibetans. Our results suggest that key HIF regulatory and targeted genes are responsible for adaptation to altitude and implicate several distinct chromosomal regions. The candidate genes and gene regions identified in Andeans and Tibetans are largely distinct from one another. However, one HIF pathway gene, EGLN1, shows evidence of directional selection in both high-altitude populations.

In this paper the authors looked at around 50 Andeans (Quechua and Aymara speakers) and 50 Tibetans, and compared them to various outgroups. In addition to the European and Asian HapMap populations they also looked at some Amerindian populations. The map below shows the geographical scope of their sampling (the right inset are the Amerindian lowland groups):

journal.pgen.1001116.g001

The ancestral relationships of the two highland groups sampled in relation to the lowlanders was relatively straightforward. Panel A and B show PCA plots for the Andeans and Tibetans, while C and D show frappe bar plots. The only thing notable for me is that the Quechua speakers seem to show residual European ancestry which the Aymara do not, and the Colombian indigenous groups seems to have more affinity with Mesoamerican populations than with the other South American samples. I can give no insight as to the latter, but if it is not just a quirk of non-representativeness one may be seeing the higher number of Spanish men who married into the nobility of the Quechua speaking highlands than further south in lands of the Aymara (though Potosi was in Bolivia, so this may not be plausible).

We already have some evolutionary expectations of how these groups came to have these adaptations to their high altitude environments. It seems that the physiological processes for the three groups are somewhat different, and this has been a source of curiosity for geneticists for a long time. It stands to reason if the physiology is somewhat varied, the genetics should be too, and that seems to be a broadly correct assumption. In this paper they took two general approaches, looking at the total genome, and focusing on specific candidate regions. From what I can tell they did not find much novel using the first technique, but they did clarify the relationship between Tibetans and Andeans in terms of their genetic adaptations a bit by looking at specific genes. As noted in the author summary it looks as if the two populations do have somewhat different genetic architectures. Many of the genes which seem to have been targets of selection do not overlap, and of those that do there seem different localized selection events so that the haplotypes being driven by positive selection differ.

They used a compound of techniques to detect possible regions of natural selection:

- locus specific branch length (LSBL)

- the log of ratio of heterozygosities (lnRH)

- a modified Tajima’s D statistic

- whole genome long range haplotype (WGRLH)

LSBL is an elaboration on Fst, so it is finding between population differences in allele frequency. Recall that at any given locus you don’t expect much between population difference, so if there is a great deal of ecological adaptation you may see a lot of variance as a function of geography. Heterozygosity is simply a measure of the fraction of loci where the two gene copies are in different states. It’s just a way to measure genetic variation (though there are others). The Tajima’s D statistic is a test for whether the locus seems deviated from neutral expectations. This means that there may have been a bottleneck, selective sweep, or, balancing selection. Finally, the last test looks for sets of correlated markers within the genome. If there is a haplotype, a sequence of markers, at high frequency then it may be that you’re witnessing a genomic region which is in, or just after, the occurrence of a selective sweep.

Why four different tests? Because one given test is not dispositive of natural selection. As noted with Tajima’s D, there are demographic processes of a stochastic nature which can produce false positives, so it is best not to live or die by one technique alone.

Here is figure 4, which shows the differences in allele frequencies on the EGLN1 gene:

journal.pgen.1001116.g004

We’ve seen EGLN1 before. In the figure above the left panels show the Andean derived SNPs, and the right panels the Tibetan ones. Note the differences in frequency in A and B. The red denotes statistically significant values for a statistic in panels C & D. Both Andeans and Tibetans show indications of selection, but the details in the patterns vary when you zoom in on the gene. The very last panel has an arrow which points to the SNPs in each population where the between population variance is maximized. Interestingly the ancestral allele seems to have risen in frequency here in the high altitude populations, as black denotes ancestral and red derived in the first and last panels.

Let me jump to their conclusion:

In summary, we performed a genome scan on high- and low-altitude human populations to identify selection-nominated candidate genes and gene regions in two long-resident high-altitude populations, Andeans and Tibetans. Several chromosomal regions show evidence of positive directional selection. These regions are unique to either Andeans or Tibetans, suggesting a lack of evolutionary convergence between these two highland populations. However, evidence of convergent evolution between Andeans and Tibetans is suggested based on the signal detected for the HIF regulatory gene EGLN1. In addition to EGLN1, a second HIF regulatory gene, EPAS1, as well as two HIF targeted genes, PRKAA1 and NOS2A, have been indentified as selection-nominated candidate genes in Tibetans (EPAS1) or Andeans (PRKAA1NOS2A). PRKAA1 and NOS2A play major roles in physiological processes essential to human reproductive success…Thus, in addition to demonstrating the likely targets of natural selection and the operation of evolutionary processes, genome studies also have the clear potential for elucidating key pathways responsible for major causes of human morbidity and mortality. Based on the findings of this study, it will be important to confirm the results with genotype-phenotype association studies that link genotype to a specific high-altitude phenotype.

I wanted to show the alphabet soup of genes in case you’re a geneticist with an interest in any of these loci. I’ve seen these before in previous papers, I assume the key that got this published in PLoS Genetics is the deep comparative dimension, as the researchers explored the lack or existence of evolutionary convergence between these two populations. Should the finding be surprising? I don’t think so. High altitudes are extreme environments, and the literature is filled with references to problems which emerge even in these populations because of the nature of their adaptations. There are likely deleterious side effects, especially if one of last spring’s papers on Tibetans is correct and that they’re relatively recent settlers of the highlands. But you never know until you play the game, so it is good to confirm.

A further exploration of the genetic architecture and nature of adaptations, especially when the research is extended to Ethiopians, may give us a further window into contingency in evolutionary history. These three occurrences are basically three independent experiments. In this paper they indicate that some of the variants being subject to natural selection may have been in the ancestral population, so standing variation. Others are new mutations, unique and novel. Though there are different pathways to the final expression of the phenotype, which in the details of implementation (physiology) still differ across the groups, there are also genes which in this comparison seem to be implicated in both Tibetans and Andeans as having been subject to selection. How constrained is the sample space subject to possible selection and the implied G-matrix? How contingent are the evolutionary pathways that different populations take to attain the state of adaptive fitness in similar ecologies? These are the sort of long term questions which I think will be possibly answered as the tentative silver age of altitude adaptation gives way to the golden age.

Citation: Bigham A, Bauchet M, Pinto D, Mao X, & Akey JM (2010). Identifying Signatures of Natural Selection in Tibetan and Andean Populations Using Dense Genome Scan Data PLoS Genetics

Image Credit: Micah MacAllen

Note: I am aware that classically the silver age follows the golden age, instead of precedes it. But we live in Whiggish times indeed!

May 14, 2010

Breathing like Buddha: altitude & Tibet

443px-PaldenLhamoYou probably are aware that different populations have different tolerances for high altitudes. Himalayan sherpas aren’t useful just because they have skills derived from their culture, they’re actually rather well adapted to high altitudes because of their biology. Additionally, different groups seem to have adapted to higher altitudes independently, exhibiting convergent evolution. But in terms of physiological function they aren’t all created equal, at least in relation to the solutions which they’ve come to to make functioning at high altitudes bearable. In particular, it seems that the adaptations of the peoples of Tibet are superior than those of the peoples of the Andes. Superior in that the Andean solution is more brute force than the Tibetan one, producing greater side effects, such as lower birth weight in infants (and so higher mortality and lower fitness).

The Andean region today is dominated by indigenous people, and Spanish is not the lingua franca of the highlands as it is everyone in in the former colonial domains of Spain in the New World. This is largely a function of biology; as in the lowlands of South America the Andean peoples were decimated by disease upon first contact (plague was spreading across the Inca Empire when Pizzaro arrived with his soldiers). But unlike the lowland societies the Andeans had nature on their side: people of mixed or European ancestry are less well adapted to high altitudes and women without tolerance of the environment still have higher miscarriage rates.

So despite the suboptimal nature of the Andean adaptations vis-a-vis the Tibetan ones, they are certainly better than nothing, and in a relative sense have been very conducive to higher reproductive fitness. And yet why might the Andeans have kludgier adaptations than Tibetans? One variable to consider is time. The probability is that the New World was populated by humans only for the past ~10,000-15,000 years or so, with an outside chance of ~20,000 years (if you trust a particular interpretation of the genetic data, which you probably shouldn’t). By contrast, modern humans have had a presence in the center of Eurasia for ~30,000 years. Generally when populations are exposed to new selective regime the initial adaptations are drastic and exhibit major functional downsides, but they’re much better than the status quo (remember, fitness is relative). Over time genetic modifications mask the deleterious byproducts of the genetic change which emerged initially to deal with the new environment. In other words, selection perfects design over time in a classic Fisherian sense as the genetic architecture converges upon the fitness optimum.*

Another parameter may be the variation available within the population, as the power of selection is proportional to the amount of genetic variation, all things equal. The peoples of the New World tend to be genetically somewhat homogeneous, probably due to the fact that they went through a bottleneck across Berengia, and that they’re already sampled from the terminus of the Old World. A physical anthropologist once told me that the tribes of the Amazon still resemble Siberians in their build. It may be that it takes a homogeneous population with little extant variation a long time indeed to shift trait value toward a local ecological optimum (tropical Amerindians are leaner and less stocky than closely related northern populations, just not particularly in relation to other tropical populations). In contrast, populations in the center of Eurasia have access to a great deal of genetic variation because they’re in proximity to many distinctive groups (the Uyghurs for example are a recent hybrid population with European, South Asian and East Asian ancestry).

So that’s the theoretical backdrop for the differences in adaptations. Shifting to the how the adaptations play out concretely, some aspects of the physiology of Tibetan tolerance of high altitudes are mysterious, but one curious trait is that they actually have lower levels of hemoglobin than one would expect. Andean groups have elevated hemoglobin levels, which is the expected “brute force” response. Interestingly it seems that evolution given less time or stabilizing at a physiologically less optimal equilibrium is more comprehensible to humans! Nature is often more creative than us. In contrast the Tibetan adaptations are more subtle, though interestingly their elevated nitric acid levels may facilitate better blood flow. Though the inheritance patterns of the trait had been observed, the genetic mechanism underpinning it has not been elucidated. Now a new paper in Science identifies some candidate genes for the various physiological quirks of Tibetans by comparing them with their neighbors, and looking at the phenotype in different genotypes with the Tibetan population. Genetic Evidence for High-Altitude Adaptation in Tibet:

Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. We report genome-wide scans that reveal positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia response pathways in humans.

Here’s what they did. First, Tibetans are adapted to higher altitudes, Chinese and Japanese are not. The three groups are relatively close genetically in terms of ancestry, so the key is to look for signatures of positive selection in regions of the genome which have been identified as possible candidates in terms of functional significance in relation to pathways which may modulate the traits of interest. After finding potential regions of the genome possibly under selection in Tibetans but not the lowland groups, they fixed upon variants which are at moderate frequencies in Tibetans and noted how the genes track changes in the trait.

This figure from the supplements shows how the populations are related genetically:

tib1

In a worldwide context the three groups are pretty close, but they also don’t overlap. The main issue I would have with this presentation is that the Chinese data is from the HapMap, and they’re from Beijing. This has then a northeast Chinese genetic skew (I know that people who live in Beijing may come from elsewhere, but recent work which examines Chinese phylogeography indicates that the Beijing sample is not geographically diversified), while ethnic Tibetans overlap a great deal with Han populations in the west of China proper. In other words, I wouldn’t be surprised if the separation between Han and Tibetan was far less if you took the Chinese samples from Sichuan or Gansu, where Han and Tibetans have lived near each other for thousands of years.

tib2But these issues of phylogenetic difference apart, we know for a fact that lowland groups do not have the adaptations which are distinctive to the Tibetans. To look for genetic differences they focused on 247 loci, some from the HIF pathway, which is important for oxygen homeostasis, as well genes from Gene Ontology categories which might be relevant to altitude adaptations. Table 1 has the breakdown by category.

Across these regions of the genome they performed two haplotype based tests which detect natural selection, EHH and iHS. Both of these tests basically find regions of the genome which have reduced variation because of a selective sweep, whereby selection at a specific region of the genome has the effect of dragging along large neutral segments adjacent to the original copy of the favored variant. EHH is geared toward detection of sweeps which have nearly reached fixation, in other words the derived variant has nearly replaced the ancestral after a bout of natural selection. iHS is better at picking up sweeps which have not resulted in the fixation of the derived variant. The paper A Map of Recent Positive Selection in the Human Genome outlines the differences between EHH and iHS in more detail. They looked at the three populations and wanted to find regions of the genome where Tibetans, but not the other two groups, were subject to natural selection as defined by positive signatures with EHH and iHS. They scanned over 200 kb windows of the genome, and found that 10 of their candidate genes were in regions where Tibetans came up positive for EHH and iHS, but the other groups did not. Since these tests do produce false positives they ran the same procedure on 240 random candidate genes (7 genes were in regions where Chinese and Japanese came up positive, so these were removed from the set of candidates), and came up with average EHH and iHS positive hits of ~2.7 and ~1.4 genes after one million resamplings (specifically, these are genes where Tibetans were positive, the other groups negative). Their candidate genes focused on altitude related physiological pathways yielded 6 for EHH and 5 for iHS (one gene came up positive for both tests, so 10 total). This indicates to them these are not false positives, something made more plausible by the fact that we know that Tibetans are biologically adapted to higher altitudes and we have an expectation that these genes are more likely than random expectation to have a relationship to altitude adaptations.

Finally, they decided to look at two genes with allelic variants which exist at moderate frequencies in Tibetans, EGLN1 and PPARA. The procedure is simple, you have three genotypes, and you see if there are differences across the 31 individuals by genotype in terms of phenotype. In this case you want to look at hemoglobin concentration, where those who are well adapted have lower concentrations. Figure 3 is rather striking:

tib3

Even with the small sample sizes the genotypic effect jumps out at you. This isn’t too surprising, previous work has shown that these traits are highly heritable, and that they vary within the Tibetan population. There’s apparently a sex difference in terms of hemoglobin levels, so they did a regression analysis, and it illustrates how strong the genetic effect from these alleles are:

tib4

My main question: why do Tibetans still have variation on these genes after all this time? Shouldn’t they be well adapted to high altitudes by now? A prosaic answer may be that the Tibetans have mixed with other populations recently, and so have added heterozygosity through admixture. But there are several loci here which are fixed in Tibetans, and not the HapMap Chinese and Japanese. For admixture to be a good explanation one presumes that the groups with which the Tibetans mixed would have been fixed for those genes as well, but not the ones at moderate frequencies. This may be true, but it seems more likely that admixture alone can not explain this pattern. As the Andean example suggests adaptation to high altitudes is not easy or simple. Until better options arrive on the scene, kludges will suffice. It may be that the Tibetans are still going through the sieve of selection, and will continue to do so for the near future. Or, there may be balancing dynamics on the genes which exhibit heterozygosity, so that fixation is prevented.

No matter what the truth turns out to be, this is surely just the beginning. A deeper investigation of the genetic architecture of Andeans and Ethiopians, both of which have their own independent adaptations, will no doubt tell us more. Finally, I wonder if these high altitude adaptations have fitness costs which we’re not cognizant of, but which Tibetans living in India may have some sense of.

Citation: Tatum S. Simonson, Yingzhong Yang, Chad D. Huff, Haixia Yun, Ga Qin, David J. Witherspoon, Zhenzhong Bai, Felipe R. Lorenzo, Jinchuan Xing, Lynn B. Jorde, Josef T. Prchal, & RiLi Ge (2010). Genetic Evidence for High-Altitude Adaptation in Tibet Science : 10.1126/science.1189406

* Additionally, it may be that archaic hominin groups were resident in the Himalaya for nearly one million years. Neandertal admixture evidence in Eurasians should change our priors when evaluating the possibility for adaptive introgression on locally beneficial alleles.

Image Credit: Wikimedia Commons

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