Abstract
Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30–50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.
Main
Infectious diseases have presented one of the strongest selective pressures in the evolution of humans and other animals1,2. Not surprisingly, many candidates for population-specific positive selection in humans involve immune response genes, consistent with the hypothesis that exposure to new and/or re-emerging pathogens has driven adaptation5,6. However, it is challenging to connect signatures of natural selection with their causative pathogens unless the underlying loci are still associated with susceptibility to the same pathogen in modern populations7,8. Clarifying the dynamics that have shaped the human immune system is key to understanding how historical diseases contributed to disease susceptibility today.
We sought to identify signatures of natural selection in Europeans imposed by Yersinia pestis, the bacterium responsible for bubonic plague3. The first recorded plague pandemic began with the Plague of Justinian in AD 541 (refs. 9,10). Nearly 800 years later, the Black Death (1346–1350) marked the beginning of the second pandemic of plague, which spread throughout Europe, the Middle East and Northern Africa, reducing the population by up to 30–50%4,11. With no recent exposure to plague, Europeans living through the Black Death probably represented immunologically naive populations with little to no prior adaptation to Y. pestis. The high mortality rate suggests that genetic variants that conferred protection against Y. pestis infection might have been under strong selection during this time. Indeed, the nearly decadal plague outbreaks over the subsequent four hundred years of the second pandemic in Europe often (but not always) were associated with reduced mortality rates11,12, which could have been due to pathogen evolution or changing cultural practices, but potentially also linked to human genetic adaptation to Y. pestis.
Positive selection on immune genes
Genomic targets of selection imposed by Y. pestis during the Black Death, if present, have remained elusive13,14,15. To better identify such loci, we characterized genetic variation from ancient DNA extracts derived from individuals who died shortly before, during or soon after the Black Death in London and across Denmark. This unique sampling design differentiates, to the greatest extent possible, signatures due to Y. pestis from those associated with other infectious diseases or other selective processes (although we cannot exclude these entirely). From London, individuals were sampled from three cemeteries close to one another, tightly dated by radiocarbon, stratigraphy and historical records to before, during and after the Black Death (Fig. 1, Supplementary Table 1 andSupplementary Methods). From Denmark, individuals were sampled from five localities, geographically spread across the country, which were dated via archaeological means (such as burial arm positions), stratigraphy and historical records. We grouped all individuals into those that lived pre-Black Death (London: around AD 1000–1250, Denmark: around AD 850 to around AD 1350) and post-Black Death (London: AD 1350–1539, Denmark: around AD 1350 to around AD 1800). Within London, we also included individuals buried in the plague cemetery, East Smithfield, all of whom died during a two-year window of the Black Death between 1348 and 1349 (ref.16). Analysis of the mitogenomic diversity from these individuals identifies solely European mitogenomic haplotypes, avoiding a possible confound between natural selection and population replacement from non-European sources17.
In total we screened 516 samples (n = 318 from London; n = 198 from across Denmark) for the presence of human DNA using a modified polymerase chain reaction (PCR) assay for the single copy nuclear c-myc gene17,18 and identified 360 with sufficient endogenous DNA content for downstream enrichment and sequencing of additional nuclear loci (Supplementary Methods). As many of our samples were poorly preserved and had low endogenous DNA content, we used hybridization capture to enrich for and sequence 356 immune-related genes, 496 genome-wide association study (GWAS) loci previously associated with immune disorders and 250 putatively neutral regions (1.5 kb each), as defined by Gronau and colleagues19 (Supplementary Table 2; Supplementary Methods). The targeted immune genes were manually curated on the basis of their role in immune-related processes, and include innate immune receptors, key immune transcription factors, cytokines and chemokines, and other effector molecules (Supplementary Table 3). To ensure that deamination and other forms of ancient DNA damage did not lead to spurious genotype calls, we trimmed 4 base pairs (bp) from the start and end of each sequencing read (Supplementary Fig. 1) and excluded all singleton variants (n = 106,757). Our final dataset contained 33,110 biallelic variants within the targeted regions (2,669 near GWAS loci, 19,972 in immune genes and 10,469 in putatively neutral regions), with a mean coverage of 4.6× reads per site per individual (see Supplementary Table 1 for per-individual coverage). We further filtered our results by excluding samples with missing genotype calls at more than 50% of those sites (retaining n = 206 individuals, Fig. 1c) and excluding variants with genotype calls for less than 10 individuals per time period and population. Using genotype likelihoods, we then calculated the minor allele frequency (MAF) per population at each time point. Finally, we retained only sites with a mean MAF (averaged across London and Denmark) greater than 5% (n = 22,868 sites), as our power to detect selection for variants below 5% is very low (Supplementary Fig. 2).
To detect alleles that may have conferred protection from, or susceptibility to, Y. pestis, we searched within candidate regions (immune genes and GWAS loci) for variants that exhibit unexpectedly large changes in allele frequency between pre- and post-Black Death samples. Specifically, we identified alleles for which the degree of differentiation (FST) was larger than expected by chance, when compared to variants in the putatively neutral genetic regions sampled from the same populations. We used the larger sample set from London as our discovery cohort. Burials in London were also more precisely dated and better geographically controlled than those from Denmark, improving our relative ability to detect selection in the cohort from London (Supplementary Methods). We found an enrichment of highly differentiated variants for all frequency bins with MAF greater than 10%, relative to a null expectation established using our neutral loci (Fig. 2a). Across these variants, differentiation at immune loci exceeded the 99th percentile of neutral variants at 2.4× the rate expected by chance (binomial test P = 7.89 × 10−12). For variants with an MAF greater than 30%, this enrichment was even more pronounced (3.9× the rate expected by chance; binomial test P = 1.16 × 10−14), probably due to increased power (Supplementary Fig. 2). Simulations show that differences in recombination rate and background selection between neutral and candidate loci are insufficient to explain the observed enrichments (Extended Data Fig. 1). To further validate the signatures of selection we observed among immune loci from our London sample, we performed the same analyses using the allele frequencies estimated from our Danish cohort. These samples were also enriched for highly differentiated sites relative to the expectation from neutral loci (1.6× the rate expected by chance, binomial test P = 9.21 × 10−4; Fig. 2b), further supporting evidence for plague-induced selection on immune genes.
Functional dissection of candidate loci
None of our top candidate variants overlaps with (nor is in strong linkage disequilibrium with) coding variants, although one, near the ERAP2 gene, is strongly linked to a variant that affects splicing21,22. Their selective advantage may stem from an impact on gene expression levels, particularly in immune cell types that participate in the host response to Y. pestis infection. Macrophages in particular are recruited to sites of infection, where they interact with bacteria and contribute to plague resistance23,24. Macrophages phagocytize Y. pestis, but some bacteria survive and spread to the lymph node, where they replicate uncontrollably25,26. …
