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Ancient European, Pathogen DNA Data Promises to Shed Light on Brain Disorders, Infectious Disease

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NEW YORK (GenomeWeb) – An international research team is reaching back in time — and into many ancient mouths — to watch human evolution in reverse, using ancient human and pathogen DNA to explore the possibility that selective pressure related to infectious diseases played a role in neuropsychiatric conditions and other brain diseases in European populations.

As part of a recently announced collaboration with Illumina, researchers at the University of Copenhagen, the University of California Berkeley, and elsewhere plan to sequence both human and pathogen DNA from around 5,000 ancient tooth or bone samples from Western Eurasia, stretching back up to 10,000 years. They will initially use the resulting sequences, along with available genome-wide association study data from contemporary human populations, to explore potential ties between neuropsychiatric disorders and infectious disease.

"We are particularly interested, but not only interested, in mental disorders," explained Eske Willerslev, director of the University of Copenhagen's Centre of Excellence GeoGenetics, who is also a professor of ecology and evolution at the University of Cambridge in the UK. "We'll look at other diseases as well, and I'm sure that many other people will also … as new results come from GWAS," he said.

The team expects to continue tapping into the ancient human and pathogen sequence datasets to explore the infectious potential of pathogens that plagued humans in the past, human adaptations to various dietary or environmental conditions, heritable disease risk in specific populations, and more.

"If we look at various quantitative phenotypes — disease-related phenotypes and other phenotypes — how do we best look at how they have changed over time and between different places in the world?" explained UC Berkeley computational and statistical genetics researcher Rasmus Nielsen, a principal investigator on the project. "A major aim of this project is to use ancient DNA to be able to track phenotypes in time and be able to address these questions using statistical methods."

The project is largely being financed by foundations, including the Lundbeck Foundation, and will involve a technical development-focused collaborative partnership with Illumina, Willerslev said. He noted that the project would not be possible without a high-throughput sequencing platform such as the NovaSeq 6000, given the low endogenous levels of human DNA in the ancient samples.

"To get [an ancient human] genome out, you have to sequence an enormous amount," he said. "For this project to be successful, we're relying on tremendous sequencing power."

Humans made some other pretty major lifestyle changes over the past 10,000 years, Willerslev explained — finding new ways to produce food, industrializing, and welcoming potentially germ-riddled domesticated animals into their environments.

"The last 10,000 years is the time in human history where we have undergone the biggest lifestyle changes. This is where we go from hunter-gatherers to farmers," he said. "We know that this has had a massive impact on human health, but also even our genome." 

For the moment, the team's focus is firmly on brain and neuropsychiatric disease, which has been well studied by GWAS and sequencing in modern-day populations but is still poorly understood from an evolutionary perspective.

In particular, the researchers will explore the hypothesis that infectious disease outbreaks deep in human history may have helped to shape the immune environment in ways that affected the brain. In other words, they want to know if neuropsychiatric disorders and other brain diseases might be a byproduct of selective pressure on human populations dealing with historical infectious diseases.

"I'm not saying I'm supporting [the hypothesis], but people have suggested that … because of pressure from pathogens on the immune system — and the part of the genome associated with the immune system — some of these variants are then kind of surfing with that," Willerslev said.

There are other theories about the evolution of brain conditions, from proposed tradeoffs that increased intelligence to indirect forms of selection that involve the immune system.

For example, some have speculated that individuals with neuropsychiatric conditions such as depression may have dodged historical infectious disease outbreaks because of characteristic disease-related behaviors, he explained. If being shut in with a bout of the blues could unwittingly ward off the plague and other transmissible maladies by preventing the human interactions needed for disease distribution, those individuals might theoretically have survived epidemics that others did not.

Some of these ideas might be a stretch, Willerslev said, but given the widespread prevalence of brain diseases and disorders —from migraine headaches to Alzheimer's to schizophrenia — combined with their frequent heritability, it doesn't hurt to keep an open mind about why these conditions have arisen and persisted in human populations.

"Why are we suffering from these disorders that clearly have very strong genetic associations?" Willerslev said. "It's been known for a long time that these disorders are running in families. And recent GWAS, of course, show that although there are a lot of variants with smaller effects, [the conditions] are clearly associated with genetics."

The researchers will use Illumina's NovaSeq to perform shotgun sequencing on ancient human and microbial DNA in parallel on the same samples, he noted, and expect to automate many of the DNA extraction, library preparation, and other steps to get through the large number of samples.

The team plans to give priority to the tooth samples since prior studies suggest the so-called cementum layer in those samples is apt to contain DNA from blood-borne pathogens that were circulating in an individual before death.

The petrous bone — which forms early in life and is known as the hardest bone in the human body — typically retains relatively high levels of endogenous DNA over time with good preservation, Willerslev explained, making it a favorite DNA source for ancient sequencing studies. On the other hand, those samples are expected to contain far less DNA from microbes that were associated with the individual.

"The chance of finding pathogens is way, way lower [in petrous bone samples], and therefore we prefer the teeth, if possible," he said.

After taking out the human DNA reads for their own genome analyses, the team will compare the sequences that remain to those from known pathogens, performing more targeted, in-depth analyses, phylogenetics, and genome assembly on suspected pathogens that do turn up.

Because many microbes make their way into human bodies from soil and other sources after we die, the researchers will rely on a combination of phylogenetic information and DNA degradation patterns to focus in on authentic bacterial, viral, or fungal pathogens in the ancient Europeans.

"The major challenge for the researchers is to distinguish which pathogens were really in the individual when he or she died and which ones came subsequently as a type of contamination," Nielsen noted. "That's also a bioinformatic challenge."

The decision to start with shotgun sequencing was likewise very deliberate: by maximizing the amount of human and microbial DNA profiled across the ancient samples, the investigators hope to get an unbiased look at as many genes and variants as possible.

"We are not doing capture — the genome-wide capture that other groups have been applying in recent years," Willerslev said, emphasizing the precious nature of many of the ancient samples. "When we're trying to get the full genome, it's also to make sure that we're trying to get the maximum information out of destructive sampling,"

"The beauty of doing the shotgun [sequencing] to start with is also that we have no pre-assumptions of what to expect to find," he added.

In addition, Nielsen noted that genes and variants that investigators focus on now may differ from those of interest in the future, making it appealing to get information across as much of the ancient genomes as possible.

As in the pathogen analysis, the shotgun sequences will form the foundation for any additional targeted or enrichment sequencing done on the human DNA.

The investigators are still debating the ideal coverage depth and number of reads to target for the ancient human genomes and corresponding microbial species. Willerslev noted that they may use a combination of deep genome sequencing — something on the order of 20X to 30X coverage — on a limited number of samples, with low-coverage sequencing to between 1X and 3X coverage on many more samples.

"There are certain types of analyses — modeling of population structure and other things — that demand at least the scaffold of a very deep genome," he noted, "so we will probably take a subset from each period" for deeper sequencing, "based on the preservation of DNA in the sample as well as the time and location."

Similar studies are expected to become somewhat more feasible in other populations with the growth of projects such as the National Institutes of Health's All of Us Research Program, combined with genetic susceptibility insights gained from related GWAS. Even so, the investigators cautioned that the ancient DNA analyses will inevitably be limited to locations with sufficient sample availability and endogenous DNA content.

"Hopefully it can provide an example of how things can develop in other parts of the world," Willerslev said. For now, though, the research is most feasible in European populations because the anticipated ancient DNA analyses will rely "very heavily" on information from GWAS, which have largely been done on individuals with European ancestry.

"Where are the disease variants coming from? When have they been under selection? When do they become highly frequent? And so forth," he said. Those questions "will be much more challenging for other populations than Europeans," at least for the time being.

Although the team plans to study samples from sites in Europe and from the area around Mongolia, many of the samples used in the current study will come from Denmark, Nielsen said, noting that the country has a long history of fossil collection, combined with climate and soil conditions that are favorable for fossil and DNA preservation.

From the samples that are already becoming available, it may even be possible to dig into the family and social structures for individuals from certain intersections of time and space.

"We actually don't know too much about how any of these traits have evolved through time," Nielsen said. "The more we can pull in information from studies done in other parts of the world, particularly other regions in Europe, the better we will be able to do predictions where you can transfer predictions from one pace to another."

He is already anticipating addition applications for the upcoming collections of ancient human and pathogen sequences — from exploring population adaptations and human evolution in general to exploring the history and prevalence of diseases in specific populations over time.

"It will really be the first time that we can look at one place in the world and ask 'How has allele frequency changed through time?'" Nielsen said. "As an evolutionary biologist, this is a tremendously exciting project for me, because we can do what we always wanted: take a time machine back and see what happened back in time in great detail."

The investigators expect to finish sequencing the samples in the next two to three years and will wrap up the project within around five years. They have committed to making the full collections of ancient human and pathogen DNA publicly available and hope that it will also serve as a resource for understanding other types of human disease risk, while providing a look at the versions of different pathogens that have posed a threat to humans in our past.

"Evolution, of course, creates kind of this natural experiment that you could never undertake in the lab: you can't infect people with plague and study what happens in the population," Willerslev said. "This has all played out in history. We've gone through bottlenecks because of pathogen outbreaks, we've had changes in the genome because of climate changes and adaptations to that, et cetera."

On the pathogen side, the work will also lead to a "catalog of the possible variants that will, at one point, return again, because that's how pathogens work," he added. "This gives you, then, an opportunity to test also through these resurrecting studies whether current vaccines are covering ancient pathogens that could be our future pathogens."

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