Dr Horia C. Stanescu
University College London, UK

Dr Catalin D. Voinescu
University College London, UK

A hundred thousand years ago, mammoths, woolly rhinoceroses, sabretooth tigers and cave bears roamed the icy expanses of Europe. They were hunted by small bands of large-brained, robust hominins, well-adapted to the difficult conditions of the last ice age: our cousins, the Neanderthals. They have recently been thrust back into the spotlight due to the 2022 Nobel Prize in Physiology and Medicine, which was awarded to Svante Pääbo “for his research in the field of genomes of extinct hominins and human evolution”. While the hominins in question may be extinct, namely Neanderthals and Denisovans, their legacy still lives on in us intertwined with our DNA.

Neanderthals

The first remains to be recognised as Homo sapiens neanderthalensis were found in 1856 in Germany, in a cave in the Neander Valley (formerly Neanderthal, now spelled Neandertal) on the river Dussel. It is ironic that the remains of the “ancient” man were discovered in “the valley of the new man”. The Neander Valley was named after a 17th century pastor, Joachim Neander, whose name comes from neo (Latin/Greek for “new”) + andros (Greek for “man”). Quarrying for limestone in this valley led to the discovery of the fossils, and subsequently to the unfortunate destruction of the cave (Schmitz and Thissen, 2010).

Neanderthals are the best-known subspecies of archaic humans. Archaic humans are usually defined as being the group of now extinct human species that successively branched out of Homo erectus in the last million years. The two populations that would become Neanderthals and, respectively, anatomically modern humans (Homo sapiens sapiens) diverged about 550,000 years ago (Prufer et al., 2017).

Neanderthals probably evolved locally in Eurasia and disappeared about 40,000 years ago. Compared to anatomically modern humans, Neanderthals had much longer to adapt morphologically, physiologically, and culturally to cold glacial climates.

Fossil records facilitate our understanding of Neanderthal morphology and can help us to infer information about their functional capacities. For the most part, Neanderthal morphology seems to follow the so called “ecogeographical rules”, which describe the underlying relationship between morphology and climate. Short sturdy limb bones and broad stocky bodies with barrel shaped chests are thought to be cold climate adaptations, which all served to increase their heat-conserving abilities via a high body volume to low surface area ratio (Ocobock, Lacy, and Niclou, 2021).

Before the sequencing era, scientists used to build their understanding of morphological and functional features on fossil records, and contextualised these with the information extracted from the archaeological settings in which they were found. Nowadays the availability of ancient genomic data has spectacularly enhanced our capabilities to infer physiological aspects of Neanderthal biology.

Ancient DNA and modern technology

For a long time, scientists believed it would be impossible to extract usable DNA from historical samples, but in 1984 Higuchi et al. made an important breakthrough and sequenced 129 base pairs extracted from a 200-year-old specimen of an extinct zebra-like animal (the Quagga)! Around the same year Svante Paabo also succeeded in extracting ancient DNA, this time from a millennia-old mummy (Jones, 2022). Extracting ancient human DNA is, indeed, difficult. In addition to the almost inevitable bacterial contamination and chemical degradation of the samples, DNA extraction and analysis of archaic and ancient human remains are made even more difficult by contamination with present-day human DNA from the researchers themselves.

The first complete Neanderthal genome to be sequenced was that of their mitochondria (Green et al., 2008). The process of extracting fragmentary Neanderthal DNA was streamlined, improved, and standardised, borrowing heavily from clean-room techniques used in the semiconductor industry, with stringent physical contamination prevention augmented by contamination filtering software. These improvements in technique led to the publication of the first complete nuclear Neanderthal genome a mere two years later (Green et al., 2010).

Thanks to these efforts, three good-quality nuclear Neanderthal genomes are currently available: one from the Vindija cave in present day Croatia (about 50,000 years old), one from the Denisova cave (about 110,000 years old) and another from the Chagyrskaya cave (about 80,000 years old), both in the Altai mountains of present-day Russia (Fig. 1). These Neanderthal sequences, along with a large number of ancient and present-day H. sapiens sapiens genomes, can be publicly accessed through the Allen Ancient DNA Resource (Reich, 2021).

The mixing of DNA: introgression signals

Introgression is defined as the incorporation of stretches of DNA from one species into the gene pool of a second one. The presence of genetic material from a relatively recent admixture with an evolutionary branch that diverged a long time ago can, in principle, be detected with statistical
methods, even in the absence of genetic information related to the donor population (in our case, the Neanderthals). Simply put, during the time they were separated from us, Neanderthals accumulated mutations (variants), not shared by modern humans. Introgressed material has a higher rate of variants than either the background or the subpopulations without introgression.

Another observation is that genetic material is more likely to undergo recombination at multiple points along its length over many generations, so only short contiguous fragments occur today. Comparatively, newer introgressed material has less time to get fragmented, so it occurs in longer stretches, which makes it identifiable.

The arrival of H.sapiens sapiens

About 45,000 years ago, modern humans started populating Eurasia, arriving from Africa via the Middle East. This allowed for an overlap between the two species of approximately 5,000 years (Hublin et al., 2020). We currently believe that during that time interaction between the two groups followed all possible avenues: avoidance, conflict, and mixing – the latter scenario leading to introgression.

As a result, 1%-3% of the H. sapiens sapiens genome is believed to be of Neanderthal origin, except for genomes of sub-Saharan Africans, which is consistent with introgression having occurred outside Africa. While the average DNA fraction of Neanderthal origin in a non-African individual is around 2%, the fragments of ancient DNA do not occur at the same positions for everyone. It is believed that between 12% and 20% of the Neanderthal genome survives in modern humans today. This archaic DNA is not uniformly distributed within our genome. Neanderthal genetic material is particularly rich in some areas, and conspicuously absent in others, suggesting that both positive and negative selection pressures have affected the present day distribution.

Although not the principal focus of this piece, it is worth noting that there is also evidence of another archaic human subspecies introgressing with previously Anatomically Modern Humans.
The highest levels are found in individuals from Oceania who have inherited ~5% of their genome from Denisovans (Browning, 2018), and those in mainland Asia and Americas sharing ~0.2% of their genome with Denisovans (Prufer, 2013).

Present-day consequences

The functional effect of Neandertal variants versus modern human ones can be assessed by computational analysis. For instance, the depletion of Neanderthal sequence in and around functional elements of the modern human genome suggests that a large proportion of the archaic variants were deleterious for H.sapiens sapiens (Dannemann and Kelso, 2017).

However, there are also some Neanderthal variants that have increased in frequency in modern humans. One possible explanation is that these variants have contributed to a better adaptation to new environments, having therefore been positively selected, and therefore retained in the recipient population. As such they are most likely the result of “adaptive introgression” (Reilly et al., 2022).

The computational/statistical assessment of the correlation between Neanderthal alleles and modern human phenotypes, derived from large populational databases (Dannemann and Kelso, 2017) and electronic health records (Simonti et al., 2016), has contributed to the definition of a set of traits that are influenced by archaic ancestry: hair and skin phenotypes (pigmentation, tanning, sunburn, skin lesions); psychological/behavioural traits (mood, depression, chronotype – morning or evening person, addictive behaviour – tobacco use); metabolic traits (obesity); height, blood disorders, heart rate, etc.

One of the main conclusions of the Neanderthal trait association studies is that a major influence
of the introgressed alleles is exerted through their effects on gene regulation (Dannemann and Kelso, 2017). This effect can also be computationally interrogated genome-wide, given the accessibility of datasets from high throughput studies of human gene expression (The Genotype-Tissue Expression – GTEx – Project; Geuvadis, etc).

Most spectacularly though, the ultimate biological significance of the Neanderthal-associated traits can be tested by in vitro functional assays in the wet laboratory. For instance, in Massively Parallel Reporter Assays (MPRAs), introgressed and non-introgressed alleles are barcoded and cloned into reporter vectors, in front of a minimal promoter whose efficiency they are going to influence. The vectors are then transfected into cells that will transcribe the reporter mRNA. The reporter transcripts can subsequently be sequenced and counted, allowing the comparison of the two variants’ efficiency when it comes to gene expression (Jagoda et al., 2022).

Genome engineering of induced Pluripotent Cell Stem (iPSC) lines can also be used to demonstrate causality of associated variants, by inserting them in model organisms or by organoid modelling. For example, a Neanderthal-specific nonsynonymous substitution in the NOVA1 gene was assessed by CRISPR editing of iPSCs to generate cortical organoids, showing neurodevelopmental differences between Neanderthal and modern human-derived organoids (Trujillo et al., 2021).

One of the first instances of comparing Neanderthal vs. modern variants, assessed the functionality of FOXP2, a gene previously involved in the evolution of language, in “humanised” mice. This historically important study showed that the mice bearing the modern human specific variant have altered vocalisations, modified behaviour and decreased brain dopamine concentrations (Enard et al., 2009).

(Auto)Immunity

One of the most important effects of introgressed variants is manifest in the immune function of present-day humans. This importance is highlighted by the recent identification of Neanderthal variants associated with susceptibility to and severity of COVID-19 infection. Several of these variants have also been associated with lung or autoimmune diseases, and inflammatory responses to infection (COVID-19 Host Genetics Initiative, 2021).

Due to the strong mortality burden brought about by infections, genes involved in the immune response are under the strongest selection pressure. It has been shown that instances of variants from archaic populations have introgressed in modern humans in both adaptive and innate immunity genes, with the innate immunity genes having an even higher average of Neanderthal ancestry than the rest of the genome (Deschamps et al., 2016).

Neanderthals were probably better adapted to the local regional pathogen spectrum when anatomically modern humans started their forays in Europe, hence introgression might have contributed to the adaptation of H. sapiens sapiens to the newly encountered pathogens (Kerner, Patin, and Quintana-Murci, 2021).

It is likely that the lag observed between the appearance of modern humans in the Middle East (~100,000 years ago) and their further dispersion into Eurasia (~45,000 years ago) was necessary for them to acquire, by introgression, the immune-related variants adapted to the local regional infectious conditions. This “epidemiologic transition” lag is observed in invasive species between the moment of their appearance in the new environment and the start of efficient population growth (Hawks, 2017).

Risk factors for autoimmune diseases probably originated as adaptations to the infectious diseases of the time, but what we see today might be an unfortunate rebalancing of the immune system. Many autoimmune disorders have been linked to variants inherited from Neanderthals. Some of these deleterious traits could have survived simply due to chance and insufficient negative selective pressure, especially those occurring later in life. It is also possible that they presented a heterozygote advantage with respect to a pathogen, even one no longer present (as is the case of thalassamia/sickle cell in the Haldane hypothesis: wherein homozygous individuals suffer from the respective disease, while heterozygous individuals are unaffected and have increased resistance to malaria) (Haldane, 1949).

Concluding remarks

Thus, the study of recent human evolution contributes not only to our understanding of population history and adaptive physiology, but also to the genetic architecture of diseases. In the case of autoimmune diseases, it is becoming clear that the infectious context is a major source of evolutionary pressure, with effects seen over comparatively short timescales. Investigation of Neanderthal and other archaic human subspecies DNA can advance both our understanding of the complex genetic makeup of disease risk factors and, more generally, the way we conceptualise the relationship between environmental conditions and evolution. Our archaic ancestors have much yet to teach us about ourselves.

References

Browning SR et al. (2018). Analysis of human sequence data reveals two pulses of archaic Denisovan admixture. Cell. 173 (1): 53–61.e9. doi:10.1016/j.cell.2018.02.031. ISSN 0092-8674. PMC 5866234. PMID 29551270

COVID-19 Host Genetics Initiative (2021). Mapping the human genetic architecture of COVID-19. Nature 600 (7889): 472–77. https://doi.org/10.1038/s41586-021-03767-x.

Dannemann M and Janet K. (2017. ‘The contribution of Neanderthals to phenotypic variation in modern humans’. American Journal of Human Genetics 101 (4): 578–89. https://doi.org/10.1016/j.ajhg.2017.09.010.

Deschamps M et al. (2016). Genomic Signatures of Selective Pressures and Introgression from Archaic Hominins at Human Innate Immunity Genes. The American Journal of Human Genetics 98 (1): 5–21. https://doi.org/10.1016/j.ajhg.2015.11.014.

Enard W et al. (2009). A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice’. Cell 137 (5):961–71. https://doi.org/10.1016/j.cell.2009.03.041.

Green RE et al. (2010). A Draft Sequence of the Neandertal Genome. Science 328 (5979): 710–22. https://doi.org/10.1126/science.1188021

Green RE et al. (2008). A complete Neandertal mitochondrial genome sequence determined by highthroughput sequencing. Cell 134 (3): 416–26. https://doi.org/10.1016/j.cell.2008.06.021.

Haldane JBS. (1949). Disease and Evolution. La Ricerca Scientifica – Supplement 19: 68–76.

Hawks J. (2017). Neanderthals and Denisovans as biological invaders. Proceedings of the National Academy of Sciences of the United States of America 114 (37): 9761–63. https://doi.org/10.1073/pnas.1713163114.

Higuchi RB et al. (1984). DNA sequences from the Quagga, an extinct member of the horse family. Nature 312 (5991): 282–84. https://doi.org/10.1038/312282a0.

Hublin J et al. (2020). Initial upper Palaeolithic Homo Sapiens from Bacho Kiro Cave, Bulgaria. Nature 581 (7808): 299–302. https://doi.org/10.1038/s41586-020-2259-z.

Jagoda E et al. (2022). Detection of Neanderthal adaptively introgressed genetic variants that modulate reporter gene expression in human immune cells. Molecular Biology and Evolution 39 (1): msab304. https://doi.org/10.1093/molbev/msab304.

Jones ED. (2022). Svante Paabo: The Dark Lord of ancient DNA. Yale University Press (blog). 4 November 2022. https://yalebooks.yale.edu/2022/11/04/svante-Paabo-the-dark-lord-of-ancient-dna/.

Kerner G et al. (2021). New insights into human immunity from ancient genomics. Current Opinion in Immunology 72 (October): 116–25. https://doi.org/10.1016/j.coi.2021.04.006.

Ocobock C et al. (2021). Between a rock and a cold place: Neanderthal biocultural cold adaptations. Evolutionary Anthropology 30 (4): 262–79. https://doi.org/10.1002/evan.21894.

Prufer K et al. (2013). The complete genome sequence of a Neanderthal from the Altai Mountains. Nature. 505 (7481): 43–49. Bibcode:2014Natur.505…43P.doi:10.1038/nature12886. PMC 4031459. PMID 24352235.

Prufer K et al. (2017). A high-coverage Neandertal genome from Vindija Cave in Croatia. Science (New York, N.Y.) 358 (6363): 655–58. https://doi.org/10.1126/science.aao1887.

Reich D. (2021). Allen ancient DNA Resource (AADR): Downloadable genotypes of present-day and ancient DNA data | David Reich Lab. 10 October 2021. https://reich.hms.harvard.edu/allen-ancient/dna-resource-aadrdownloadable-genotypes-present-day-and-ancientdna-data.

Reilly PF et al. 2022. The contribution of Neanderthal introgression to modern human traits. Current Biology: CB 32 (18): R970–83. https://doi.org/10.1016/j.cub.2022.08.027.

Schmitz RW and Thissen J. (2010). Neandertal: die Geschichte geht weiter. 1st ed. Heidelberg: Spektrum, Akad. Verl.

Simonti CN et al. (2016). The Phenotypic Legacy of Admixture between Modern Humans and Neandertals. Science (New York, N.Y.) 351 (6274): 737–41. https://doi.org/10.1126/science.aad2149.

Trujillo CA et al. (2021). Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment. Science (New York, N.Y.) 371 (6530):eaax2537. https://doi.org/10.1126/science.aax2537.