UDK 902(485)''633":577-2 Documenta Praehistorica XXXIII (2006) Pushing it back. Dating the CCR5-A32 bp deletion to the Mesolithic in Sweden and its implications for the Meso\Neo transition Kerstin Liden1, Anna Linderholm1, Anders Götherström2 1 Archaeological Research laboratory, Stockholm University, 106 91 Stockholm, Sweden kerstin.liden@arklab.su.se, anna.linderholm@arklab.su.se 2 Department of Evolutionary Biology, Norbyvägen 18D, 751 05 Uppsala, Sweden Anders.Gotherstrom@ebc.uu.se ABSTRACT - Genetic variation in the chemokine receptor gene CCR5 has received considerable scien- tific interest during the last few years. Protection against HIV-infection and AIDS, together with spe- cific geographic distribution are the major reasons for the great interest in CCR5 32bp deletion. The event for the occurrence of this mutation has been postulated by coalescence dating to the 14th cen- tury, or 5000 BP. In our prehistoric Swedish samples we show that the frequency of 32pb deletion in CCR5 in the Neolithic population does not deviate from the frequency in a modern Swedish popula- tion, and that the deletion existed in Sweden already during the Mesolithic period. IZVLEČEK - Med znanstveniki je bilo v zadnjih nekaj letih precej zanimanja za genetsko variacijo na kemokinskem receptornem genu CCR5. Glavna razloga velikega zanimanja za zdrs 32bp na CCR5 sta zaščita proti okužbi z virusoma HIV in AIDS ter specifična geografska porazdelitev. S pomočjo 'datiranja zlitja'je bilo predpostavljeno, da se je ta mutacija pojavila v 14. stoletju AD ali pa 5000 BP. Na švedskih prazgodovinskih vzorcih pokažemo, da ni bistvenega odklona v frekvenci zdrsa 32pb na CCR5 med neolitsko in moderno švedsko populacijo in da je mutacija na Švedskem obstajala že v mezolitiku. KEY WORDS - CCR5-A32; Bubonic plague; Smallpox; Mesolithic; Neolithic Introduction The product of the CCR5 gene is a member of a se- ven-transmembrane G-protein-coupled receptor fa- mily, and is important for the cell entry of the hu- man immunodeficiency virus HIV-1 (Alkhatib et al. 1996; Choe et al. 1996; Deng et al. 1996; Doranz et al. 1996; Trkola et al. 1996). In 1996 a 32bp de- letion in the CCR5 gene was reported which trunca- tes the protein and appears to provide almost com- plete protection against infection by HIV-1 (Liu et al. 1996; Samson et al. 1996; Dean et al. 1996; Omet- to et al. 1999). In homozygotes for 32bp deletion the protection is well documented, but there are dif- ferent opinions about the protective effect of hetero- zygosity against HIV-1, although it seems to delay the outbreak of AIDS (Samson et al. 1996; Dean et al. 1996; Huang et al. 1996; Michael et al. 1997). The CCR5 32bp deletion seems to be restricted to Caucasian populations. However, the frequency of the mutation varies widely among European popula- tions. Whereas the frequency is quite high in St. Pe- tersburg, Russia (O.166), and in Sweden (0.143), it is considerably lower in the southern parts of Europe, e.g., in Spain (0.050) and Greece (0.041) (Lucotte 2001). However heterozygosity for the deletion was found at a frequency of 0.23 in a Swedish HIV positive group (Bratt et al. 1998). Since that study was cross- sectional and not prospective, this frequency may represent a population subjected to a different selec- tion pressure than an ordinary Scandinavian popu- lation. In cases where the deletion has been found in non-Caucasian populations, there has usually been continuous contact and a likely admixture with Cau- casians (Liu et al. 1996; Samson et al. 1996; Dean et al. 1996). The geographic distribution of 32bp de- letion in Europe has been explained in several ways, as e.g., genetic drift (Martinson et al. 1997) or posi- tive selection due to resistance to a major disease (Dean et al. 1996). Based on coalescence, 32bp dele- tion has been estimated to have appeared approxi- mately 700 years ago (Stephens et al. 1998). Conse- quently, it has been speculated that the Black Death (Yersinia pestis) caused a positive selection (Ste- phens et al. 1998; O'Brien 1998). The basis for this hypothesis is that CCR5 may be involved in the me- chanism of Yersinia-induced macrophage apoptosis (Stephens et al. 1998). If the Black Death, believed to be caused by Yersenina pestis, resulted in a posi- tive selection, this has to be a fairly recent process, since the outbreak of this disease was in the mid 14th century. However, according to Galvani and Slatkin (2003), the bubonic plague, although severe, could never have driven the frequencies to over 10% in the short time span of 700 years. In another study, it was postulated that the mutation originated about 2000 years ago, somewhere in north-eastern Europe (Libert et al. 1998). In later publications, smallpox is thought to be a more likely candidate, where the selective pressure from this virus would have pushed the frequency of 32bp deletion across Europe into today's figures (Klitz et al. 2001; Galvani and Slat- kin 2003). In a recent publication Sabeti et al. (2005), has pushed back the estimate of the occurrence of the CCR5-A32 allele to over 5000 years ago. There are also several articles suggesting that the deletion was dispersed by the Vikings along its present gradient across Europe (Lucotte 2001 and Lucotte and Die- terlen 2003). In 2005 the CCR5-A32 was detected in human Bronze Age skeletons from Germany by Hum- mel et al., and this is the first evidence that the mu- tation existed before thel4th century plague outburst. Although there are disagreements about the time for the first occurrence of 32bp deletion, the cause of the event, and the distribution of the deletion all seem to agree on the fact that the deletion originated from a single historical mutation event. Here we want to address the question regarding the deletion's first occurrence and distribution by ex- tracting DNA from well-known and well-dated prehi- storic human and animal bones and teeth. Of speci- fic interest is a date for the occurrence of the dele- tion that precedes 5000 BP, i.e. approximately the Mesolithic-Neolithic transition in Scandinavia. In Scandinavia the Mesolithic stretches from around 8300 to 4000 BC. It is a period connected to the end of the last glaciation, and marks a shift in climate to- wards a warmer period. During this period people began to colonise southern Sweden, both along the coasts and inland. In this study we have included samples dating to the Mesolithic from Skateholm in the south, and Huseby Klev from the west coast. The Neolithic period began in Sweden with the in- troduction of farming around 4000 BC, and it ended around 1800 BC with the introduction of the Bronze Age. During the middle Neolithic in Sweden, there were three main cultures: the Funnel Beaker (TRB), the Pitted Ware (GRK) and the Battle Axe (STY) cul- tures. We have analysed human and animal bones and teeth from two of these cultures, the Funnel Beaker culture complex that is believed to be connec- ted with the introduction of farming and cereal cul- tivation, and the GRK-complex that consists mainly of a set of hunter/gatherer dwelling sites and ceme- teries along the Scandinavian Baltic coast and the major Baltic Islands. The GRK are represented by samples from the islands of Gotland and Aland, and the TRB samples are represented by two passage graves in central Sweden (Fig. 1). The individuals from Dragby represent the last phase of the Neoli- thic in Sweden, thus this case study encompasses more than 5000 years. Materials and methods Bones and teeth from two Mesolithic and six Neoli- thic sites were chosen for this study. The two Mesolithic sites used in this study are Hu- seby Klev, radiocarbon dated to 7000-6500 BC, and Skateholm, radiocarbon dated to 5250-4900 BC (Fig. 1). Four individuals were analysed from Huseby Klev (Hkl, Hk2, Hk3 and Hk4), a Preboreal/Boreal site that was subjected to a rescue excavation in 1993- 94 by Bengt Nordqvist (Nordqvist 2000). Next we analysed six individuals from the Skateholm site (burials 4, 5, 7, 12, 63a and 63b) that was excavated in the early 1980s by Lars Larsson (Larsson 1988). The first Neolithic site, Ire on the island of Gotland (Fig. 1), is a cemetery belonging to the Pitted Ware culture complex (GRK) which was excavated in diffe- Fig. 1. Southern Scandinavia with Mesolithic and Neolithic sites 1. Skateholm, 2. Huseby Klev, 3. Visby, 4. Ire, 5. Jettböle, 6. Rössberga, 7. Hjelmars Rör and 8. Dragby. The A32 mutation of CCR5 was found in human material from Skateholm, Rössberga, Visby, Ire, and Dragby, indicating that the mutation is at least as old as these sites. We added a bone from a cow depo- sited in this passage grave, for the same reason as stated above. The last samples are from Dragby, a passage grave excavated by Märten Stenberger in 1958/59 (Gejvall 1963). The dating of this tomb is complicated, since it was superim- posed by a Bronze Age mound. How- ever, the 14C dates, 2290-1690 cal BC (Roumelis 2002), imply that this site was in use during the late Neoli- thic. Four individuals were analysed (H4/A, F15/44, F8/44 and F2f2/45). rent periods after its discovery in 1914 (Janzon 1974). Five individuals (burials 6b, 6c, 7a, 7b and 7c) were analysed from this site, which has been 14C dated to 3000-2100 cal BC (Janzon 1974). We also extracted DNA from a horse deposited in the settlement layers connected to the burials, in order to control for contamination and bone preservation. The other GRK site on Gotland, Visby (Fig 1.), is a ce- metery that was last excavated in 1960-62 by Erik Nylen (Janzon 1974). Here 12 individuals were se- lected for analysis (burials 2/09, 2/24, 2/39, 3b, 13, 19, 19/37, 23, 27, 30b, 31 and 33). They have been 14C dated to 3000-2500 cal BC (Janzon 1974). The next site to be analysed is Jettböle (Fig 1.), also a GRK settlement on an island in the archipelago of Aland, excavated by Björn Cederhvarf from 1905 until 1911 (Cederhvarf 1912). From this site, 3 in- dividuals were analysed (J1, J2 and J3). They have been radiocarbon dated to 3370-2910 cal BC (Liden et al. 1995). The other samples are from two passage graves, Rös- sberga and Hjelmars Rör (Fig. 1), both from Väster- götland in central Sweden and belonging to the Fun- nel Beaker culture complex (TRB). Five different in- dividuals from each passage grave were analysed. Fourteen out of sixteen 14C samples date Rössberga to 3506-2143 BC; the other two samples date Rös- sberga to the late Bronze Age (Persson & Sjögren 1995). Rössberga, which was excavated in 1962 by Carl Cullberg, will be treated here as a middle Neo- lithic passage grave (Cullberg 1963). Eight 14C sam- ples date Hjelmars Rör to 3350-2700 BC. The pas- sage grave was first excavated in 1868 by Bror Emil Hildebrand, but was revisited in the 1990s by Tony Axelsson and Per Persson (Persson & Sjögren 1995). Most publications on DNA from pre- historic material concern mitochondrial DNA (e.g. Hagelberg & Clegg 1991; Krings et al. 1997; Krings et al. 1999; Handt et al. 1994, Torroni et al. 2000, Hofreiter et al. 2002, Forster 2004, Starikovskaya et al. 2005). However, a number of studies have also been performed on nuclear, single copy markers from ancient tissue (e.g. Beraud-Colomb et al. 1995; Zierdt et al. 1996; Ovchinnikov et al. 1998; Göther- ström et al. 1997; Greenwood et al. 1999; Noonan et al. 2005; Poinar et al. 2005). We designed a suitable (<150bp) primer system for the part of CCR5 that carries the 32 bp deletion, i.e. in this study we use a single copy nuclear marker ap- plied to ancient DNA. In all cases, except for the cow, teeth were used as the source material for DNA extraction. Samples were extracted and prepared for PCR according to Liden et al. (1997) and Anderung et al. (2005). This study was conducted in two stages, and because of this the DNA extraction methods varied. Part one was done using a guanidium thiocyanate and silica extraction (Liden et al. 1997), and the second part was done using an extraction method called fishing (Anderung et al. 2005). A set of three primers was designed to give an easily detectable indication of the presence or absence of the 32bp deletion (Tab. 1), i.e., a system that ampli- fies fragments of different length depending on whe- ther the 32bp deletion is present or not, and to am- plify highly degraded DNA. This primer system was used in both set-ups. DNA was amplified with Amplitaq gold™ (Perkin El- mer)/HotStarTaq (Qiagen) to receive the hot start and time-release effect {Götherström et al. 1997). The 25 |l reactions contained 50 mM KCl, 10 mM Tri-HCl pH 8.3, 2.25 mM MgCl2, 0.2 mM of each dNTP, 0.5 |M of each primer, 1 U Taq-polymerase (Amplitaq gold™, Perkin Elmer, HotStarTaq, Qiagen), 10/9 |l template DNA and 2% glycerol/none. Hot start was performed automatically, due to the en- zyme used. The amplification cycles were initiated by a 45 sec/5 min. denaturation step at 95° C to acti- vate part of the enzyme and save the rest for later cycles; this was then followed by 45/30 sec. at 94° C, 1i/2/1min. at 50° C and 1 min/30 sec. at 72° C. This cycle was repeated 55/45 times. Finally, an ex- tension step at 72° C for 7/5 min. followed. The re- sult was detected on a 3%/1.5% ethidium bromide stained agarose gel exposed to 40 V for 1h. One pri- mer (CCR5:1) anneals upstream the sequence, one (CCR5:2) anneals downstream on the other side of the 32bp deletion, and the third primer {CCR5:3) anneals within the 32bp deletion. Thus, an indivi- dual homozygous for the deletion will have one fragment (91bp) amplified, an individual homozy- gous for the wild type will have two fragments (105bp and 123bp) amplified, and a heterozygous will have three fragments (91bp, 105bp and 123bp) amplified. The result was confirmed by sequencing on a Pharmacia ALF express™ with an Amersham Pharmacia Biotech Cycle Sequencing kit. A short mtDNA fragment, 16131-16303, according to the reference sequence from Anderson et al. (.1981), was amplified and sequenced from a part of the material to confirm that the DNA was from Primer name Primer sequence CCR5>1 5'TCCTTAGTAGAAATGGTCTAG3' CCR5>2 5'GTCGGGGTTCTACTGATAG3' CCR5>3 5'GAAATTACAGACCTTTAAGAAG3' L16131 5'CACCATGAATATTGTACGGT3' H16303 5'TGGCTTTATGTACTATGTAC3' HBB>1 5'GATATAAAAAAGAAGACCCAGTAG3' HBB>2 5'TACCTGAGTCATATGTAATATTCC3' HTG10>1 5'GAATTCCCGCCCCACCCCCGGCA3' HTG10>2 5'TTTTTATTCTGATCTGTCACATTT3' Tab. 1. Primer name and primer sequences used in the study. The system used to detect presence of the 32bp deletion of the CCR5 gene is based on three primers CCR5:1, CCR5:2, CCR5:3. The D-loop system is a simple two-primer system L16131 & H16303 (Anderson et al. 1981). The primers used for the horse were HTG10:1 and HTG10:2 (Mark- lund et al. 1994) and for the cow HBB:1 and HBB:2 (Steffen et al. 1993). different individuals in the first set-up. If authentic DNA was extracted, there should among all samples be several haplotypes present based on this short HVR1 sequence, but only one haplotype in each sam- ple. The same protocols, with minor changes, used for CCR5 amplification and sequencing were used for the amplification and sequencing of the mtDNA fragment. However, only 45 cycles were used, and L16131 and H16303 {Tab. 1) replaced CCR5:1, CCR5:2 and CCR5:3. The primers used for the horse and cow were HBB:1 and HBB:2 {Marklund et al. 1994) and HTG10:1 and HTG10:2 {Steffen et al. 1993) respecti- vely. In the second part of the study, all replications were performed in an independent laboratory, Centro Mixto UCM-ISCIII de Evoulcion y Comportamiento Humanos {Madrid) in order to detect contaminations and prove the authenticity of the ancient DNA {Hof- reiter et al. 2001). One tooth permits one extraction, which gave enough material for ten PCRs. Whenever possible, at least two samples were taken from each individual, but only one extraction could be carried out on each in- dividual from the passage graves due to the problem of separating individuals in a passage grave. How- ever, thanks to high reproducibility within the ex- traction, we could trust the results from the ten in- dividuals from the passage graves. Moreover, a ro- bust protocol including extraction blanks, carrier ef- fect blanks, PCR blanks, UV irradiation of reagents, fragment size control etc was applied {Götherström & Liden 1998). To further test the state of preserva- tion, we extracted another large bone protein, colla- gen, according to Brown et al. {1988) on both hu- man and animal samples. Here the extracted amount of collagen, the absolute amounts of carbon and ni- trogen, as well as their ratio provide information on the state of preservation {DeNiro 1985). We also tried to amplify DNA extracted from the horse and the cow with the primers used for the humans, to test for contamination. Results and discussion The collagen data were all in accordance with bones that are well preserved. In bones positive for DNA, no samples contained less than 0.66% of collagen, calculated on total bone, and the C/N ratios were all within the accepted limit of 2.9 - 3.4 {except for 3 samples), as well as the absolute values of carbon and nitrogen {Tab. 2) {De Niro 1985). It is therefore reasonable to believe that DNA was also well pre- served in the samples (Götherström et al. 2002). There is also a correlation with previously published quantitative data (Malmström et al. 2005), where good preservation has been indicated on the Baltic island of Gotland. That the extracted DNA is authen- tic was further proved by the result of the mtDNA, where we have four different haplotypes distributed over two cemeteries, and that no products were ob- tained from the extracted animal DNA amplified with the human primers (Tab 2.). We were able to extract DNA from 19 out of 50 sam- ples, representing in total 46 individuals, i.e. a 38% success rate (Tab. 2). We were also able to extract and sequence DNA from two different teeth from the same individual in two of the burials from Ire. Of the 19 samples, now representing 17 individuals, one individual was dated to the Mesolithic and came from Skateholm, and 16 individuals were dated to the Neolithic. Of the Neolithic samples, six individu- als can be attributed to the pitted ware culture, nine to the funnel beaker culture, and one to the late Neo- lithic. The complete sample is in Hardy-Weinberg equilib- rium (p = 0.661), and does not differ in frequencies (17.1%) of the CCR-A32 mutation from a modern Swedish population (14.3%). It could be argued that the small sample size, 17 individuals or 34 alleles, is not statistically significant. A further argument could be that the five individuals from one megalith tomb could be related i.e., providing a smaller num- ber of alleles than we calculate. This is also obvious in the case of Ire, where the individuals were sam- pled from one burial, and one individual proved to be a heterozygous, while the others proved to be ho- mozygous for the mutant allele. Two alternative explanations for the specific distribu- tion of the CCR5 32bp allele have been suggested, where one is a selection process related to a specific disease within a population; alternatively, the appea- rance of a higher gene frequency could be related to the migration of individuals already having the de- letion. One such migration that has been discussed and that is of interest here is the transition from the Mesolithic to the Neolithic. Traditionally, this is con- nected to a change in economy from a hunter-gathe- rer way of life to pastoralism-farming. This change in economy is most often explained by three major themes, of which immigration is one. Subsistence during the Mesolithic was based on hun- ting and gathering, as we can see in the individuals analysed from Skateholm and Huseby Klev (Liden et al. 2005). This lifestyle continued during the Neo- lithic for the Pitted Ware culture. Their main eco- nomy seems to have been based on maritime hunt- ing and fishing, as seen in the sites studied here: Ire, Visby and Jettböle (Liden 1995). The Funnel Beaker culture is partly differentiated from the GRK by the change in subsistence towards an economy based on agro-pastoralism, and this pattern is clearly visible in the passage graves analysed here: Rös- sberga and Hjelmars Rör (Liden 1995). In the late Neolithic, agro-pastoralism continued, as seen in the Dragby samples (Roumelis 2002). However, since we only have one individual from whom it was pos- sible to extract DNA dating to the Mesolithic, we can- not draw any conclusions relating to the migration hypothesis. However, the sample allows for testing the frequencies between the two Neolithic cultures, GRK and TRB, and when using Fishers's exact test (p = 0.005), we do find a significant difference. This difference is in itself interesting, in that these cul- tures represent different economies, and where the "farming culture" has the lower frequency of the deletion. Does this mean that the hunting GRK cul- ture could be regarded as original, and that the TRB were the newcomers? We cannot say. The second explanation for the distribution of the CCR5-A32 gene was connected to a selection pro- cess due to a specific disease. It is, however, obvious that the 32bp deletion in the CCR5 gene had spread and reached a relatively high frequency in Scandi- navia at least during the Neolithic, and that selective advantage due to the plague is evidently not a likely cause of the present day allele distribution. Previous studies also suggest that this is an unlikely hypothe- sis, as the short period during which the plague ra- vaged Europe would not have caused a selection pressure strong enough to alter frequency to any greater extent (Galvani and Slatkin 2003). Conse- quently, we will have to seek other explanations for the spread of the 32bp deletion in the Mesolithic and thereafter in Scandinavia. Galvani and Slatkin (2003) proposed that the more continuous smallpox mor- tality that afflicted European children since the ori- gin of the allele could have provided the necessary selective pressure to generate the rise of CCR5-A32 deletion to current frequencies of 10%. Our evidence pushes the dating of the CCR5 32 bp deletion back to around 5000 BC, which supports the suggested date for the deletions first occurrence to more than 5000 years ago (Sabeti et al. 2005). Kerstin Liden, Anna Linderholm, Anders Götherström Site # extract. Success. ampl. WT\WT WT\A32 A32\A32 14C age BC Collagen (%) C\N C (%) N (%) mtDNA haplotype Skateholm 4 2 - - - - 5250-4900 n.a. n.a. n.a. n.a. n.a Skateholm 5 2 - - - - 5250-4900 n.a. n.a. n.a. n.a. n.a Skateholm 7 2 - - - - 5250-4900 4.° 3.2 41,1 15,0 n.a Skateholm 12 2 - - - - 5250-4900 n.a. n.a. n.a. n.a. n.a Skateholm 63a 2 - - - - 5250-4900 n.a. n.a. n.a. n.a. n.a Skateholm 63b 2 2/2 X 5250-4900 n.a. n.a. n.a. n.a. n.a Huseby Klev 1 2 - - - - 7000-6500 0.2 3.8 15.2 3.7 n.a Huseby Klev 2 2 - - - - 7000-6500 4.3 3.5 42.0 14.1 n.a Huseby Klev 3 2 - - - - 7000-6500 2.0 3.3 37.7 13.3 n.a Huseby Klev 4 2 - - - - 7000-6500 1.7 3.3 39.7 13.6 n.a Visby 2/09 2 2/2 X 3000-2500 3.5 3.1 39.5 14.7 n.a Visby 2/24 2 - - - - 3000-2500 4.4 3.2 39.2 14.5 n.a Visby 2/39 2 - - - - 3000-2500 6.0 3.2 42.2 15.4 n.a Visby 3b 2 - - - - 3000-2500 2.8 3.5 42.4 14.2 n.a Visby 13 2 - - - - 3000-2500 1.2 3.1 31.6 11.9 n.a Visby 19 2 - - - - 3000-2500 6.2 3.1 37.5 14.1 n.a Visby 19/37 2 - - - - 3000-2500 3.6 3.1 44.6 15.3 n.a Visby 23 2 2/2 X 3000-2500 2.2 3.4 41.9 14.2 n.a Visby 27 2 2/2 X 3000-2500 1.3 3.5 38.2 12.6 n.a Visby 30b 2 - - - - 3000-2500 2.3 3.4 39.7 13.4 n.a Visby 31 2 - - - - 3000-2500 1.7 3.5 42.1 14.0 n.a Visby 33 2 - - - - 3000-2500 1.9 3.4 37.6 12.6 n.a Ire 6b m2 2 - - - - 3028-2141 1.9 3.1 40.1 13.2 n.a. Ire 6c m2 2 - - - - 3028-2141 1.1 3.6 33.2 11.1 n.a. Ire 7a i1 2 2/2 X 3028-2141 1.2 3.2 31.2 11.5 n.a. Ire 7a p3 2 - - - - 3028-2141 1.8 3.1 34.7 11.3 n.a. Ire 7a m2 2 2/2 X 3028-2141 1.7 3.2 29.8 10.9 n.a. Ire 7b dm2 2 - - - - 3028-2141 2.1 3.3 27.0 9.4 n.a. Ire 7c i2 2 - - - - 3028-2141 4.3 3.0 37.9 12.7 n.a. Ire 7c m, 2 2/2 X 3028-2141 3.6 3.0 39.0 13.2 n.a. Ire 7c m2 2 2/2 X 3028-2141 4.2 3.5 28.3 9.5 n.a. Jettböle 1 2 - - - - 3370-2910 2.9 n.a. n.a. n.a Jettböle 2 2 - - - - 3370-2910 3.2 n.a. n.a. n.a Jettböle 3 2 - - - - 3370-2910 3.1 n.a. n.a. n.a Rössberga 1 4 3/4 X 3506-2143 2.2 3.2 37.7 135 CRF Rössberga 2 4 3/4 X 3506-2143 5.8 3.3 40.5 14.5 16224C Rössberga 3 4 3/4 X 3506-2143 4.0 3.4 33.0 11.3 CRF Rössberga 4 4 3/4 X 3506-2143 3.1 3.3 37.8 13.2 CRF Rössberga 5 4 3/4 X 3506-2143 3.8 3.8 33.8 10.3 CRF Rössberga 6 4 - - - - 3506-2143 n.a. n.a. n.a. n.a. n.a Hjelmars rör 17 4 - - - - 3350-2700 n.a n.a n.a n.a n.a Hjelmars rör 18 4 3/4 X 3350-2700 5.5 3.3 41.6 14.7 CRF Hjelmars rör 19 4 3/4 X 3350-2700 5.2 3.3 42.0 14.8 16189C Hjelmars rör 20 4 3/4 X 3350-2700 5.6 3.2 42.2 15.2 CRF Hjelmars rör 21 4 3/4 X 3350-2700 2.9 3.2 40.1 14.4 16145A, 16231C 16261T Hjelmars rör 22 4 3/4 X 3350-2700 3.3 3.2 40.3 14.5 16189C Dragby H4/a 2 - - - 2290-1690 1.0 3.3 36.3 13.0 n.a Dragby F15 p1 2 2/2 X 2290-1690 0.6 3.2 32.5 12.3 n.a Dragby F8 p1 2 - - - - 2290-1690 1.0 3.3 30.7 11,4 n.a Dragby F2 p2 2 - - - 2290-1690 0.7 3.1 34.4 12.7 n.a Site and animal # extract. Success. ampl. SCN 14C age BC Collagen (%) C\N C (%) N (%) Ire horse 2 2/2 X 3028-2141 6.4 3.2 39.7 14.5 Hjelmars rör cow 2 2/2 X 3350-2700 5.2 3.2 39.5 14.3 Tab. 2. Presence of the 32bp deletion of the (A32) gene in the prehistoric samples compared to the wild type (WT), (-) = no result (X) = positive indication, when the number of extractions are 2 it indicates that a reproduction has taken place in a laboratory in Madrid, i = incisor, m = molar, p = premolar, n.a. = Not analysed, SCN=Single Copy Nuclear. Collagen % is given as compared to bone weight. Mitochondrial DNA huplotypes are given as compared to Anderson et al. (1981). Samples from Ire represents in total 5 indi- viduals 6b, 6c, 7a, 7b and 7c. Conclusions The CCR5-A32 results indicate that the frequency of 17.1% in our samples corresponds to the frequen- cies of CCR5-A32 mutation in present-day Sweden. Thus there seems to be no difference in the occur- rence of the deletion from the Neolithic and onwards in Sweden. The mutation occurs in two different Neolithic cul- tures, both the GRK and the TRB, despite differences in subsistence and lifestyle. This indicates that the selective pressure that caused this deletion to evolve had nothing to do with subsistence or way of life. There is also evidence for the mutation having oc- cured in one of the Mesolithic populations, although the sample size is small and no definite conclusions can be drawn from this. 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