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HIV-1 protecting CCR5-Δ32 allele in medieval Poland
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Infection, Genetics and Evolution 8 (2008) 146–151 www.elsevier.com/locate/meegid
HIV-1 protecting CCR5-D32 allele in medieval Poland Przemysław Zawicki *, Henryk W. Witas Department of Molecular Biology, Chair of Oncology, Medical University of Ło´dz´, Sporna 36/50, PL-91738 Ło´dz´, Poland Received 23 August 2007; received in revised form 24 October 2007; accepted 13 November 2007 Available online 19 November 2007
Abstract CCR5-D32 is the mutation in the chemokine receptor CCR5 that gives its homozygous carriers nearly complete protections from HIV-1 infection. Restricted almost exclusively to Europe, the mutation is thought to have originated in the continent and risen in frequency to the presentday value of approximately 10% due to a selective advantage it gave its carriers. The mutation bearing allele was initially calculated to be 1000 years old and pandemic diseases, such as Bubonic Plague or smallpox were postulated to have selected it. However, new reports appear, that question these hypotheses. Data from ancient DNA (aDNA) studies prove the mutation to be much older, as suggested by calculations based on newer genetic maps. In order to investigate if the plagues of the last millennium selected the allele, and add to the discussion on CCR5-D32 origin and age, we searched for the mutation in aDNA isolated from individuals whose skeletal remains were collected at archaeological sites in Poland, dated back to 11–14th centuries. The calculated mean frequency of the allele in medieval Poland (5.06% as compared to contemporary 10.26%), implies its longer than previously believed presence in European populations, and suggests that historic pandemics had little effect on its presentday frequency. # 2007 Elsevier B.V. All rights reserved. Keywords: AIDS; Ancient DNA (aDNA); Black death; CCR5-D32; C–C chemokine receptor 5 (CCR5); HIV
1. Introduction Among many receptor molecules, C–C chemokine receptor 5 (CCR5) arouse particular interest, as it was proven that, when defective, it confers resistance to most of HIV-1 strains (Liu et al., 1996; Samson et al., 1996; Dean et al., 1996). Most of the many mutations in CCR5 gene, located in chromosome 3, region p21.3, are function altering, which may suggest their adaptive role in response to past selective pressures (AnsariLari et al., 1997; Carrington et al., 1997). A 32 base pair deletion within the second extracellular loop makes the protein shorter and prevents it from fusing with the cell membrane, which results in lack of the receptor expression on the surface of leukocytes (Blanpain et al., 2002). Homozygous carriers of CCR5-D32 exhibit no CCR5, whereas in heterozygotes, the amount of the receptor is on average lower. Because CCR5 is required for HIV-1 entry into leukocytes, this makes the first group nearly completely resistant to HIV-1 infection and in the latter, progression towards AIDS is slower, CD4+ T cells counts
* Corresponding author. Tel.: +48 42 6177773. E-mail address: [email protected] (P. Zawicki). 1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2007.11.003
higher and mortality rate lower (Dean et al., 1996; Rappaport et al., 1997; Zimmerman et al., 1997; Philpott et al., 2003). There is no known medical condition or an apparent impairment of body function that results from absence or reduced number of CCR5 (Liu et al., 1996; Zimmerman et al., 1997), which may be explained by the fact that there are a lot of other chemokine receptors of the kind (Premack and Schall, 1996; Carrington et al., 1997). The mutation is present almost exclusively in Europe and neighbouring areas of Asia and Mediterranean. Its mean frequency in the continent is around 10% and the distribution of the allele forms a north to south cline, with lowest frequency in Sardinia (4%) and highest in Finland (15.8%) (Libert et al., 1998). Its trace presence in other parts of the world results most probably from migrations and settlement of European descent. For instance, CCR5-D32 has not been found in indigenous Mexican populations but its frequency in Hispanic Mexicans is approximately 4.4% (in Spain it is present in 8% of the population) (Salas-Alanis et al., 1999). The mutation is absent in indigenous populations of Asian and Pacific islands (Lu et al., 1999), South America (Leboute et al., 1999) and Africa (Samson et al., 1996). Although carriers of the mutation have been found in nonCaucasian populations, e.g. Indian (Husain et al., 1998) and
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Chinese (Zhang et al., 2002), it might result from gene inflow to ancestral populations (Martinson et al., 1997). Because of the characteristic distribution of the mutation carrying allele in Europe and the whole world, there are two main theories concerning its place of origin. Libert et al. (1998) suggested, it first appeared in North-Eastern Europe and Balanovsky et al. (2005) seek its roots in a Uralic population. Alternatively, the mutation event may have taken place in a Scandinavian population and was disseminated in 8–10th centuries by Vikings (Lucotte and Mercier, 1998; Lucotte and Dieterlen, 2003). The age of the mutation was first estimated (using haplotype analysis and coalescence theory) by Stephens et al. (1998) to be approximately 700 years. The authors also suggested that to reach the present-day frequency, the allele must have been subjected to strong selective pressure imposed by a factor that operated only in Europe. It would have given an advantage of some kind to its carriers and thus quickly rose in frequency. The most obvious candidate seemed to be the Black Death, the disease that claimed millions during the last centuries in the continent. The bubonic plague of 14th century appeared as an especially suitable candidate for a selective factor since mortality rate was the highest among all epidemics (McEvedy, 1988), the pandemic route was similar to the contemporary gradient of CCR5-D32 in Europe, Yersinia pestis (the plague agent) infects CCR5 carrying macrophages and, above all, because of the time of the outbreak, which matched the calculations (Stephens et al., 1998). However, a different set of chromosome markers used by Libert et al. (1998) allowed the group to estimate the time of the allele appearance to be between 3400 and 1400 years before present. Still, they argue, the allele had a single origin and it was under a selective pressure. Plenty of pathogens were speculated to be involved in selecting CCR5-D32. These include: Shigella, Salmonella, Mycobacterium (which all target macrophages) (Stephens et al., 1998) and Variola major (the smallpox virus)—which infects cells in a similar manner as HIV-1 does and claimed a huge number of deaths cumulatively throughout the last millennium (Klitz et al., 2001; Galvani and Slatkin, 2003). Other suspected diseases were: viral haemorrhagic fever, which may have been responsible for the deadliest epidemics (Duncan and Scott, 2005) and anthrax (Winkler et al., 2004). Alternatively, the frequency of CCR5-D32 may have been influenced by some climatic or geographical factors (Limborska et al., 2002; Balanovsky et al., 2005). However, new reports appear that question the initial assumption regarding the allele’s age and discuss the need of any selection to account for the contemporary frequency and distribution of CCR5-D32. In order to add to the data and discussion on origin, dispersal and possible impact of the historically most deadly infectious diseases on the frequency of the allele, we decided to take advantage of ancient DNA (aDNA) methodology and search for the mutation in a medieval population that predates the biggest epidemics of 14th and 17th centuries. Comparison of medieval frequency of the allele with present-day values would shed light on the putative influence of pandemics on CCR5-D32 presence in Europe. Had the mutation been selected by any of the pathogens plaguing the
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European population during the last millennium, its frequency would have been much lower in the middle ages. Poland appears to be a very suitable region for such comparison as the population has always been uniform with very little ethnic minorities and no major migration or immigrant influx, which is reflected for example in the language, that is deprived of any major variability throughout the whole country. This ensures relative continuity between the medieval and contemporary populations. 2. Materials and methods 2.1. Materials Ancient DNA was isolated from skeletal remains of individuals excavated at Polish archaeological sites: Stary Brzes´c´ Kujawski 4 (central Poland)—dated to 12–14th century (121 individuals), Dziekanowice (central Poland)—dated to 11– 12th century (102 individuals), Daniłowo (eastern Poland)— dated to 11–13th century (23 individuals), Cedynia (western Poland)—dated to 12–14th century (21 individuals) and Gdan´sk (northern Poland)—dated to 14th century (9 individuals). Only teeth without any visible damage were chosen for the analysis. Specimens found in distinct areas of each site were sampled, to avoid possible relatedness, which would affect the final results. The material was excavated and handled with due caution, in order to avoid contamination with contemporary DNA. Contemporary reference group consisted of 190 unrelated individuals from central Poland. 2.2. Methods As true with aDNA work in general, the crucial requirement to achieve reliable results was to provide clear and contamination free environment for handling and analysing the historical samples. All staff involved in the procedures wore disposable clothing and all work was divided between separate rooms, where no contemporary DNA had ever been analysed. Disposables were used whenever possible, specially selected from those that had been found to be most reliable. All appliances were frequently cleaned with bleach and UV irradiated, similarly to all surfaces used. All work was done in a class II biosafety cabinet (Heraeus). Each tooth was first mechanically cleaned (the outer layer removed with a Dremel1 instrument) and rinsed in sodium hypochlorite, then in alcohol, washed in double-distilled water and finally exposed to UV light. Indirect assessment of biomolecules preservation was performed by means of collagen quantification (according to Collins and Galley, 1998). The material was subsequently powdered in an agate mortar and subjected to DNA purification and isolation procedure. The powder was incubated in an EDTA solution for 48 h, to which proteinase K and PTB were added for another 24 h. PTB (N-phenacylthiazolium bromide) – first applied to aDNA isolation procedure by Poinar et al. (1998) – is the chemical, which breaks intra- and intermolecular crosslinks formed during Mailard’s reaction (Vasan et al., 1996). The solution was
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Fig. 1. Fragment of CCR5 sequence (A), amplified with the primers used. Sequencing chromatogram (B) shows a fragment of medieval CCR5-D32 allele. Electrophoregram (C) presents genotypes determined within the study. Individuals from Stary Brzes´c´ Kujawski 4 (SBK-4) are assigned as: 113, 114 and 87 (wild-type homozygotes), 82 (heterozygote) and 85 (D32/D32 homozygote).
then subjected to DNA isolation in MagNA Pure1 Compact Nucleic Acid Purification System (Roche). Subsequent procedure of PCR amplification was carried out within 1 day, following the purification step, using AmpliTaq Gold1 polymerase (Applied Biosystems) and uracil N-glycosylase (UNG). Wild-type and D32 alleles were amplified with primers described by Hummel (2003), which yielded products of 130 and 98 bp, respectively. PCR products were then visualised on polyacrylamide gel and additionally sequenced for product identification (Fig. 1). Mock samples accompanied each step of the isolation and amplification procedures. As a further contamination control, we attempted to amplify longer sequences (977/788 bp fragments of AMELX/Y) from all of the aDNA solutions, to exclude the possibility of modern contaminating DNA presence (aDNA is unlikely to be isolated in fragments longer than 250 bp). Each result was confirmed in at least two separate analyses of different samples from the same individual typed. No member of personnel involved in sample collection and aDNA analysis is a carrier of CCR5-D32 allele. The contemporary reference group was typed in a different location than the aDNA analysis. DNA from blood samples was isolated following a phenol–chloroform extraction protocol.
four out of five locations and its frequency at the two most numerously represented burial sites (Stary Brzes´c´ Kujawski 4 and Dziekanowice) was similar (5.42% and 4.40%), which suggest the results are consistent and do not result from local accumulation of the mutated allele. The number of individuals sampled at the remaining three sites (Daniłowo, Cedynia and Gdan´sk) was not sufficient to draw any conclusions regarding CCR5-D32 frequency at those areas. The results, however, were used to calculate the mean frequency of the allele in the whole population of medieval Poland (Table 1). We also typed 190 individuals from contemporary control group and obtained a result similar to the previous published data for the country— Table 1 CCR5/CCR5-D32 alleles’ frequencies and distribution of genotypes in medieval and modern populations of Poland (genotypes, Fisher–Freeman–Halton exact test, P = 0.0171; alleles, Fisher’s exact test, one sided, upper tail P = 0.0263) Site
n
w/w
w/D32
D32/D32
D32 frequency
3. Results
Stary Brzes´c´ Kujawski Dziekanowice Daniłowo Cedynia Gdan´sk All sites Contemporary Poland
37 34 3 9 6 89 190
34 31 3 8 5 81 152
2 3 – 1 1 7 37
1 – – – – 1 1
5.41% 4.40% – – – 5.06% 10.26%
We typed 89 individuals from 5 sites and found the frequency of CCR5-D32 to be 5.06%. The allele was found in
D32 frequency was calculated as a per cent ratio of D32 alleles number to all alleles in the studied group. Frequency of the allele in Daniłowo, Cedynia and Gdan´sk was not calculated due to insufficient number of individuals typed.
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10.26% versus 10.9% (Jagodzin´ski et al., 2000). Table 1 shows genotypes distribution and CCR5-D32 frequency at each site, as well as mean values for medieval and contemporary populations of Poland. The frequencies do not deviate from Hardy– Weinberg equilibrium. 4. Discussion Similarly to our previous preliminary results (Witas and Zawicki, 2006), we found the frequency of CCR5-D32 in medieval Poland to be roughly half of today’s value (5.06% versus 10.26%) and within the range characteristic of contemporary European populations. The limited number of samples, from which aDNA could be retrieved, allowed us to calculate frequency of CCR5-D32 at only two medieval sites. However, the fact that we found the mutated allele in four locations (with similar frequencies at the two most represented sites: Stary Brzes´c´ Kujawski 4 and Dziakanowice) eliminates the possibility of familiar or local accumulation of the allele bearing chromosomes, resulting, e.g. from inbreeding. The slight difference between our data for the contemporary populations and the previously published figure may result from the fact that we sampled inhabitants of central region of the country (which corresponds to the location of the medieval sites), where the frequency is lower than in the east (the frequency is generally higher in the eastern countries) and the west (where people were relocated after the Second World War from the east) (Jagodzin´ski et al., 2000). Presence of CCR5-D32 allele in Poland at the beginning of the second millennium questions the initial calculations regarding its age. The allele must have been present in Europe much earlier than 700 years ago. To allow the mutation to spread to the country from the north or the east and rise to such substantial frequency would require at least a few centuries and a strong selection factor. Our results remain in accordance with other reports on CCR5-D32 presence in historical populations. The mutation was found in other medieval populations (Kremeyer et al., 2005) and even in specimens from bronze age excavated at sites in Italy and Germany (Hummel et al., 2005). These findings favour the calculations of Libert et al. (1998) who argue that the mutation event took place a few millennia ago and those of Sabeti et al. (2005), who used more advanced genetic maps to conclude that the allele arose most probably more than 5000 year ago. The mean frequency of CCR5-D32 in the population inhabiting medieval Poland suggests that 14th century’s Black Death did not select the allele, as it was already widespread at the time of the disease outbreak and its frequency did not rise considerably during the next centuries. Weakness of the theory that the Plague selected the allele was further proven by another aDNA study, in which frequency of CCR5-D32 was analysed and compared between the 14th century’s Plague victims and a control group from the same burial site but dated to the time before the pandemic; no significant difference was found between the two groups (Kremeyer et al., 2005). Smoljanovic´ et al. (2006), however, do not exclude the possibility of CCR5D32 selection by Y. pestis. The group analysed two isolated
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contemporary populations from Croatian islands with different history of the plague exposure and found that frequency of CCR5-D32 was higher in the community that was decimated by the Black Death. There is also not enough evidence confirming the role of CCR5 receptor in Y. pestis infection. Mecsas et al. (2004) studied growth of the pathogen in CCR5 deficient and normal mice and found no difference between the knockout and control breeds. Another group, however, extended the study to phagocytosis experiments and suggested that lack of the receptor may influence the disease progression in a more complex way (Elvin et al., 2004). Our results prove also, indirectly, that there was little or possibly no effect of other pandemics of the last millennium on the frequency of CCR5-D32. Unless, that is, we assume that there was also negative selection of the allele’s carriers throughout the history. Although both homo- and heterozygotes do not suffer from any apparent impairment nowadays, we cannot exclude the possibility of a disadvantage at times of long forgotten ailments, different bacterial strains, or perhaps different living conditions. Moreover, new reports have been published recently that suggest an important role of the CCR5 receptor, in immunological response to Toxoplasmosa gondii (Khan et al., 2006), to West Nile virus (Glass et al., 2006), to Mycobacterium (Kurashima et al., 1997; Floto et al., 2006), as well as in onset of schizophrenia (Rasmussen et al., 2006). It seems plausible that the receptor may be advantageous in immunological responses that involve inflammatory process. It may not have been proven yet, due to the fact that pathogens, which elicit an inflammatory response, such as parasites or the abovementioned, are very rare in European, North-American and Asian populations, where CCR5-D32 is studied (Galvani and Novembre, 2005). On the other hand, the fact that CCR5-D32 frequency figure characterising contemporary Polish population is approximately twofold higher than the medieval may not simply result from migrations or gene flow between past populations. There are plethora of microorganisms that target macrophages, which use or require the receptor during infection. In situation when there was no balancing selection, i.e. the CCR5-D32 allele was not eliminated (as its carriers did not suffer from any illness or body impairment) or the purifying selection was weaker, it is possible that the frequency of the allele was growing constantly as it was selected by more than one pathogen (and possibly environmental factors). Such situation may have taken place in European and Asian populations throughout history—just as it is taking place now, in the whole world, due to widespread HIV/ AIDS epidemic (Schliekelman et al., 2001; Hedrick and Verrelli, 2006). Perhaps in the future, the mutation bearing allele will reach much higher frequency, as it most probably happened in red-capped mangabeys—a 24 bp deletion (conferring resistance to simian deficiency virus in homozygotes) in their CCR5 gene reached 86.6% frequency (98% of them carry at least one copy of the allele) (Chen et al., 1998). We therefore conclude that although the presence of CCR5D32 in Eurasia only and its characteristic frequency gradient reflect most probably a selection of some kind at the loci, it cannot be attributed solely to the medieval plagues. New reports
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appearing recently suggest its long presence in Europe, and before any hypotheses are put forward, more data on CCR5 involvement in immunological response, as well as the allele’s frequency in different areas at different times are required. This can be achieved by analysing historic populations (as well as pathogens) by means of constantly improving methodologies of ancient DNA retrieval. Acknowledgements The authors cordially thank Prof. E. Z˙a˛dzin´ska, Prof. J. Gładykowska-Rzeczycka, Prof. B. Jerszyn´ska, J. Wrzesin´ski and A. Wrzesin´ska for the dated and carefully described archaeological material used in the study. Project (no. 502-18556) supported by Medical University of Ło´dz´. References Ansari-Lari, M.A., Liu, X.M., Metzker, M.L., Rut, A.R., Gibbs, R.A., 1997. The extent of genetic variation in the CCR5 gene. Nat. Genet. 16, 221–222. Balanovsky, O., Pocheshkhova, E., Pshenichnov, A., Solovieva, D., Kuznetsova, M., Voronko, O., Churnosov, M., Tegako, O., Atramentova, L., Lavryashina, M., Evseeva, I., Borinska, S., Boldyreva, M., Dubova, N., Balanovska, E., 2005. Is spatial distribution of the HIV-1-resistant CCR5Delta32 allele formed by ecological factors? J. Physiol. Anthropol. Appl. Hum. Sci. 24, 375–382. Blanpain, C., Libert, F., Vassart, G., Parmentier, M., 2002. CCR5 and HIV infection. Recept. Channels 8, 19–31. Carrington, M., Kissner, T., Gerrard, B., Ivanov, S., O’Brien, S.J., Dean, M., 1997. Novel alleles of the chemokine-receptor gene CCR5. Am. J. Hum. Genet. 61, 1261–1267. Chen, Z., Kwon, D., Jin, Z., Monard, S., Telfer, P., Jones, M.S., Lu, C.Y., Aguilar, R.F., Ho, D.D., Marx, P.A., 1998. Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus. J. Exp. Med. 188 (11), 2057–2065. Collins, M.J., Galley, P., 1998. Towards an optimal method of archaeological collagen extraction; the influence of pH and grinding. Anc. Biomol. 2, 209– 222. Dean, M., Carrington, M., Winkler, C., Huttley, G.A., Smith, M.W., Allikmets, R., Goedert, J.J., Buchbinder, S.P., Vittinghoff, E., Gomperts, E., Donfield, S., Vlahov, D., Kaslow, R., Saah, A., Rinaldo, C., Detels, R., O’Brien, S.J., 1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273, 1856–1862. Duncan, C.J., Scott, S., 2005. What caused Black Death? Postgrad. Med. J. 81, 315–320. Elvin, S.J., Williamson, E.D., Scott, J.C., Smith, J.N., Perez De Lema, G., Chilla, S., Clapham, P., Pfeffer, K., Schlondorff, D., Luckow, B., 2004. Evolutionary genetics: ambiguous role of CCR5 in Y. pestis infection. Nature 430, 417. Floto, R.A., MacAry, P.A., Boname, J.M., Mien, T.S., Kampmann, B., Hair, J.R., Huey, O.S., Houben, E.N., Pieters, J., Day, C., Oehlmann, W., Singh, M., Smith, K.G., Lehner, P.J., 2006. Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5. Science 314, 454–458. Galvani, A.P., Slatkin, M., 2003. Evaluating plague and smallpox as historical selective pressures for the CCR5-D32 HIV-resistance allele. PNAS 100, 15276–15279. Galvani, A.P., Novembre, J., 2005. The evolutionary history of the CCR5Delta32 HIV-resistance mutation. Microbes Infect. 7 (2), 302–309. Glass, W.G., McDermott, D.H., Lim, J.K., Lekhong, S., Yu, S.F., Frank, W.A., Pape, J., Cheshier, R.C., Murphy, P.M., 2006. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 203, 35–40. Hedrick, P.W., Verrelli, B.C., 2006. ‘‘Ground truth’’ for selection on CCR5Delta32. Trends Genet. 22 (6), 293–296.
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Infection, Genetics and Evolution 8 (2008) 146–151 www.elsevier.com/locate/meegid
HIV-1 protecting CCR5-D32 allele in medieval Poland Przemysław Zawicki *, Henryk W. Witas Department of Molecular Biology, Chair of Oncology, Medical University of Ło´dz´, Sporna 36/50, PL-91738 Ło´dz´, Poland Received 23 August 2007; received in revised form 24 October 2007; accepted 13 November 2007 Available online 19 November 2007
Abstract CCR5-D32 is the mutation in the chemokine receptor CCR5 that gives its homozygous carriers nearly complete protections from HIV-1 infection. Restricted almost exclusively to Europe, the mutation is thought to have originated in the continent and risen in frequency to the presentday value of approximately 10% due to a selective advantage it gave its carriers. The mutation bearing allele was initially calculated to be 1000 years old and pandemic diseases, such as Bubonic Plague or smallpox were postulated to have selected it. However, new reports appear, that question these hypotheses. Data from ancient DNA (aDNA) studies prove the mutation to be much older, as suggested by calculations based on newer genetic maps. In order to investigate if the plagues of the last millennium selected the allele, and add to the discussion on CCR5-D32 origin and age, we searched for the mutation in aDNA isolated from individuals whose skeletal remains were collected at archaeological sites in Poland, dated back to 11–14th centuries. The calculated mean frequency of the allele in medieval Poland (5.06% as compared to contemporary 10.26%), implies its longer than previously believed presence in European populations, and suggests that historic pandemics had little effect on its presentday frequency. # 2007 Elsevier B.V. All rights reserved. Keywords: AIDS; Ancient DNA (aDNA); Black death; CCR5-D32; C–C chemokine receptor 5 (CCR5); HIV
1. Introduction Among many receptor molecules, C–C chemokine receptor 5 (CCR5) arouse particular interest, as it was proven that, when defective, it confers resistance to most of HIV-1 strains (Liu et al., 1996; Samson et al., 1996; Dean et al., 1996). Most of the many mutations in CCR5 gene, located in chromosome 3, region p21.3, are function altering, which may suggest their adaptive role in response to past selective pressures (AnsariLari et al., 1997; Carrington et al., 1997). A 32 base pair deletion within the second extracellular loop makes the protein shorter and prevents it from fusing with the cell membrane, which results in lack of the receptor expression on the surface of leukocytes (Blanpain et al., 2002). Homozygous carriers of CCR5-D32 exhibit no CCR5, whereas in heterozygotes, the amount of the receptor is on average lower. Because CCR5 is required for HIV-1 entry into leukocytes, this makes the first group nearly completely resistant to HIV-1 infection and in the latter, progression towards AIDS is slower, CD4+ T cells counts
* Corresponding author. Tel.: +48 42 6177773. E-mail address: [email protected] (P. Zawicki). 1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2007.11.003
higher and mortality rate lower (Dean et al., 1996; Rappaport et al., 1997; Zimmerman et al., 1997; Philpott et al., 2003). There is no known medical condition or an apparent impairment of body function that results from absence or reduced number of CCR5 (Liu et al., 1996; Zimmerman et al., 1997), which may be explained by the fact that there are a lot of other chemokine receptors of the kind (Premack and Schall, 1996; Carrington et al., 1997). The mutation is present almost exclusively in Europe and neighbouring areas of Asia and Mediterranean. Its mean frequency in the continent is around 10% and the distribution of the allele forms a north to south cline, with lowest frequency in Sardinia (4%) and highest in Finland (15.8%) (Libert et al., 1998). Its trace presence in other parts of the world results most probably from migrations and settlement of European descent. For instance, CCR5-D32 has not been found in indigenous Mexican populations but its frequency in Hispanic Mexicans is approximately 4.4% (in Spain it is present in 8% of the population) (Salas-Alanis et al., 1999). The mutation is absent in indigenous populations of Asian and Pacific islands (Lu et al., 1999), South America (Leboute et al., 1999) and Africa (Samson et al., 1996). Although carriers of the mutation have been found in nonCaucasian populations, e.g. Indian (Husain et al., 1998) and
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Chinese (Zhang et al., 2002), it might result from gene inflow to ancestral populations (Martinson et al., 1997). Because of the characteristic distribution of the mutation carrying allele in Europe and the whole world, there are two main theories concerning its place of origin. Libert et al. (1998) suggested, it first appeared in North-Eastern Europe and Balanovsky et al. (2005) seek its roots in a Uralic population. Alternatively, the mutation event may have taken place in a Scandinavian population and was disseminated in 8–10th centuries by Vikings (Lucotte and Mercier, 1998; Lucotte and Dieterlen, 2003). The age of the mutation was first estimated (using haplotype analysis and coalescence theory) by Stephens et al. (1998) to be approximately 700 years. The authors also suggested that to reach the present-day frequency, the allele must have been subjected to strong selective pressure imposed by a factor that operated only in Europe. It would have given an advantage of some kind to its carriers and thus quickly rose in frequency. The most obvious candidate seemed to be the Black Death, the disease that claimed millions during the last centuries in the continent. The bubonic plague of 14th century appeared as an especially suitable candidate for a selective factor since mortality rate was the highest among all epidemics (McEvedy, 1988), the pandemic route was similar to the contemporary gradient of CCR5-D32 in Europe, Yersinia pestis (the plague agent) infects CCR5 carrying macrophages and, above all, because of the time of the outbreak, which matched the calculations (Stephens et al., 1998). However, a different set of chromosome markers used by Libert et al. (1998) allowed the group to estimate the time of the allele appearance to be between 3400 and 1400 years before present. Still, they argue, the allele had a single origin and it was under a selective pressure. Plenty of pathogens were speculated to be involved in selecting CCR5-D32. These include: Shigella, Salmonella, Mycobacterium (which all target macrophages) (Stephens et al., 1998) and Variola major (the smallpox virus)—which infects cells in a similar manner as HIV-1 does and claimed a huge number of deaths cumulatively throughout the last millennium (Klitz et al., 2001; Galvani and Slatkin, 2003). Other suspected diseases were: viral haemorrhagic fever, which may have been responsible for the deadliest epidemics (Duncan and Scott, 2005) and anthrax (Winkler et al., 2004). Alternatively, the frequency of CCR5-D32 may have been influenced by some climatic or geographical factors (Limborska et al., 2002; Balanovsky et al., 2005). However, new reports appear that question the initial assumption regarding the allele’s age and discuss the need of any selection to account for the contemporary frequency and distribution of CCR5-D32. In order to add to the data and discussion on origin, dispersal and possible impact of the historically most deadly infectious diseases on the frequency of the allele, we decided to take advantage of ancient DNA (aDNA) methodology and search for the mutation in a medieval population that predates the biggest epidemics of 14th and 17th centuries. Comparison of medieval frequency of the allele with present-day values would shed light on the putative influence of pandemics on CCR5-D32 presence in Europe. Had the mutation been selected by any of the pathogens plaguing the
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European population during the last millennium, its frequency would have been much lower in the middle ages. Poland appears to be a very suitable region for such comparison as the population has always been uniform with very little ethnic minorities and no major migration or immigrant influx, which is reflected for example in the language, that is deprived of any major variability throughout the whole country. This ensures relative continuity between the medieval and contemporary populations. 2. Materials and methods 2.1. Materials Ancient DNA was isolated from skeletal remains of individuals excavated at Polish archaeological sites: Stary Brzes´c´ Kujawski 4 (central Poland)—dated to 12–14th century (121 individuals), Dziekanowice (central Poland)—dated to 11– 12th century (102 individuals), Daniłowo (eastern Poland)— dated to 11–13th century (23 individuals), Cedynia (western Poland)—dated to 12–14th century (21 individuals) and Gdan´sk (northern Poland)—dated to 14th century (9 individuals). Only teeth without any visible damage were chosen for the analysis. Specimens found in distinct areas of each site were sampled, to avoid possible relatedness, which would affect the final results. The material was excavated and handled with due caution, in order to avoid contamination with contemporary DNA. Contemporary reference group consisted of 190 unrelated individuals from central Poland. 2.2. Methods As true with aDNA work in general, the crucial requirement to achieve reliable results was to provide clear and contamination free environment for handling and analysing the historical samples. All staff involved in the procedures wore disposable clothing and all work was divided between separate rooms, where no contemporary DNA had ever been analysed. Disposables were used whenever possible, specially selected from those that had been found to be most reliable. All appliances were frequently cleaned with bleach and UV irradiated, similarly to all surfaces used. All work was done in a class II biosafety cabinet (Heraeus). Each tooth was first mechanically cleaned (the outer layer removed with a Dremel1 instrument) and rinsed in sodium hypochlorite, then in alcohol, washed in double-distilled water and finally exposed to UV light. Indirect assessment of biomolecules preservation was performed by means of collagen quantification (according to Collins and Galley, 1998). The material was subsequently powdered in an agate mortar and subjected to DNA purification and isolation procedure. The powder was incubated in an EDTA solution for 48 h, to which proteinase K and PTB were added for another 24 h. PTB (N-phenacylthiazolium bromide) – first applied to aDNA isolation procedure by Poinar et al. (1998) – is the chemical, which breaks intra- and intermolecular crosslinks formed during Mailard’s reaction (Vasan et al., 1996). The solution was
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Fig. 1. Fragment of CCR5 sequence (A), amplified with the primers used. Sequencing chromatogram (B) shows a fragment of medieval CCR5-D32 allele. Electrophoregram (C) presents genotypes determined within the study. Individuals from Stary Brzes´c´ Kujawski 4 (SBK-4) are assigned as: 113, 114 and 87 (wild-type homozygotes), 82 (heterozygote) and 85 (D32/D32 homozygote).
then subjected to DNA isolation in MagNA Pure1 Compact Nucleic Acid Purification System (Roche). Subsequent procedure of PCR amplification was carried out within 1 day, following the purification step, using AmpliTaq Gold1 polymerase (Applied Biosystems) and uracil N-glycosylase (UNG). Wild-type and D32 alleles were amplified with primers described by Hummel (2003), which yielded products of 130 and 98 bp, respectively. PCR products were then visualised on polyacrylamide gel and additionally sequenced for product identification (Fig. 1). Mock samples accompanied each step of the isolation and amplification procedures. As a further contamination control, we attempted to amplify longer sequences (977/788 bp fragments of AMELX/Y) from all of the aDNA solutions, to exclude the possibility of modern contaminating DNA presence (aDNA is unlikely to be isolated in fragments longer than 250 bp). Each result was confirmed in at least two separate analyses of different samples from the same individual typed. No member of personnel involved in sample collection and aDNA analysis is a carrier of CCR5-D32 allele. The contemporary reference group was typed in a different location than the aDNA analysis. DNA from blood samples was isolated following a phenol–chloroform extraction protocol.
four out of five locations and its frequency at the two most numerously represented burial sites (Stary Brzes´c´ Kujawski 4 and Dziekanowice) was similar (5.42% and 4.40%), which suggest the results are consistent and do not result from local accumulation of the mutated allele. The number of individuals sampled at the remaining three sites (Daniłowo, Cedynia and Gdan´sk) was not sufficient to draw any conclusions regarding CCR5-D32 frequency at those areas. The results, however, were used to calculate the mean frequency of the allele in the whole population of medieval Poland (Table 1). We also typed 190 individuals from contemporary control group and obtained a result similar to the previous published data for the country— Table 1 CCR5/CCR5-D32 alleles’ frequencies and distribution of genotypes in medieval and modern populations of Poland (genotypes, Fisher–Freeman–Halton exact test, P = 0.0171; alleles, Fisher’s exact test, one sided, upper tail P = 0.0263) Site
n
w/w
w/D32
D32/D32
D32 frequency
3. Results
Stary Brzes´c´ Kujawski Dziekanowice Daniłowo Cedynia Gdan´sk All sites Contemporary Poland
37 34 3 9 6 89 190
34 31 3 8 5 81 152
2 3 – 1 1 7 37
1 – – – – 1 1
5.41% 4.40% – – – 5.06% 10.26%
We typed 89 individuals from 5 sites and found the frequency of CCR5-D32 to be 5.06%. The allele was found in
D32 frequency was calculated as a per cent ratio of D32 alleles number to all alleles in the studied group. Frequency of the allele in Daniłowo, Cedynia and Gdan´sk was not calculated due to insufficient number of individuals typed.
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10.26% versus 10.9% (Jagodzin´ski et al., 2000). Table 1 shows genotypes distribution and CCR5-D32 frequency at each site, as well as mean values for medieval and contemporary populations of Poland. The frequencies do not deviate from Hardy– Weinberg equilibrium. 4. Discussion Similarly to our previous preliminary results (Witas and Zawicki, 2006), we found the frequency of CCR5-D32 in medieval Poland to be roughly half of today’s value (5.06% versus 10.26%) and within the range characteristic of contemporary European populations. The limited number of samples, from which aDNA could be retrieved, allowed us to calculate frequency of CCR5-D32 at only two medieval sites. However, the fact that we found the mutated allele in four locations (with similar frequencies at the two most represented sites: Stary Brzes´c´ Kujawski 4 and Dziakanowice) eliminates the possibility of familiar or local accumulation of the allele bearing chromosomes, resulting, e.g. from inbreeding. The slight difference between our data for the contemporary populations and the previously published figure may result from the fact that we sampled inhabitants of central region of the country (which corresponds to the location of the medieval sites), where the frequency is lower than in the east (the frequency is generally higher in the eastern countries) and the west (where people were relocated after the Second World War from the east) (Jagodzin´ski et al., 2000). Presence of CCR5-D32 allele in Poland at the beginning of the second millennium questions the initial calculations regarding its age. The allele must have been present in Europe much earlier than 700 years ago. To allow the mutation to spread to the country from the north or the east and rise to such substantial frequency would require at least a few centuries and a strong selection factor. Our results remain in accordance with other reports on CCR5-D32 presence in historical populations. The mutation was found in other medieval populations (Kremeyer et al., 2005) and even in specimens from bronze age excavated at sites in Italy and Germany (Hummel et al., 2005). These findings favour the calculations of Libert et al. (1998) who argue that the mutation event took place a few millennia ago and those of Sabeti et al. (2005), who used more advanced genetic maps to conclude that the allele arose most probably more than 5000 year ago. The mean frequency of CCR5-D32 in the population inhabiting medieval Poland suggests that 14th century’s Black Death did not select the allele, as it was already widespread at the time of the disease outbreak and its frequency did not rise considerably during the next centuries. Weakness of the theory that the Plague selected the allele was further proven by another aDNA study, in which frequency of CCR5-D32 was analysed and compared between the 14th century’s Plague victims and a control group from the same burial site but dated to the time before the pandemic; no significant difference was found between the two groups (Kremeyer et al., 2005). Smoljanovic´ et al. (2006), however, do not exclude the possibility of CCR5D32 selection by Y. pestis. The group analysed two isolated
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contemporary populations from Croatian islands with different history of the plague exposure and found that frequency of CCR5-D32 was higher in the community that was decimated by the Black Death. There is also not enough evidence confirming the role of CCR5 receptor in Y. pestis infection. Mecsas et al. (2004) studied growth of the pathogen in CCR5 deficient and normal mice and found no difference between the knockout and control breeds. Another group, however, extended the study to phagocytosis experiments and suggested that lack of the receptor may influence the disease progression in a more complex way (Elvin et al., 2004). Our results prove also, indirectly, that there was little or possibly no effect of other pandemics of the last millennium on the frequency of CCR5-D32. Unless, that is, we assume that there was also negative selection of the allele’s carriers throughout the history. Although both homo- and heterozygotes do not suffer from any apparent impairment nowadays, we cannot exclude the possibility of a disadvantage at times of long forgotten ailments, different bacterial strains, or perhaps different living conditions. Moreover, new reports have been published recently that suggest an important role of the CCR5 receptor, in immunological response to Toxoplasmosa gondii (Khan et al., 2006), to West Nile virus (Glass et al., 2006), to Mycobacterium (Kurashima et al., 1997; Floto et al., 2006), as well as in onset of schizophrenia (Rasmussen et al., 2006). It seems plausible that the receptor may be advantageous in immunological responses that involve inflammatory process. It may not have been proven yet, due to the fact that pathogens, which elicit an inflammatory response, such as parasites or the abovementioned, are very rare in European, North-American and Asian populations, where CCR5-D32 is studied (Galvani and Novembre, 2005). On the other hand, the fact that CCR5-D32 frequency figure characterising contemporary Polish population is approximately twofold higher than the medieval may not simply result from migrations or gene flow between past populations. There are plethora of microorganisms that target macrophages, which use or require the receptor during infection. In situation when there was no balancing selection, i.e. the CCR5-D32 allele was not eliminated (as its carriers did not suffer from any illness or body impairment) or the purifying selection was weaker, it is possible that the frequency of the allele was growing constantly as it was selected by more than one pathogen (and possibly environmental factors). Such situation may have taken place in European and Asian populations throughout history—just as it is taking place now, in the whole world, due to widespread HIV/ AIDS epidemic (Schliekelman et al., 2001; Hedrick and Verrelli, 2006). Perhaps in the future, the mutation bearing allele will reach much higher frequency, as it most probably happened in red-capped mangabeys—a 24 bp deletion (conferring resistance to simian deficiency virus in homozygotes) in their CCR5 gene reached 86.6% frequency (98% of them carry at least one copy of the allele) (Chen et al., 1998). We therefore conclude that although the presence of CCR5D32 in Eurasia only and its characteristic frequency gradient reflect most probably a selection of some kind at the loci, it cannot be attributed solely to the medieval plagues. New reports
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