Low Prevalence of Lactase Persistence in Neolithic South-West Europe
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European Journal of Human Genetics (2012) 20, 778–782 & 2012 Macmillan Publishers Limited All rights reserved 1018-4813/12 www.nature.com/ejhg ARTICLE Low prevalence of lactase persistence in Neolithic South-West Europe This article has been corrected since Advance Online Publication and a corrigendum is also printed in this issue Theo S Plantinga1,2,SantosAlonso3,NeskutsIzagirre3,MontserratHervella3,RosaFregel4, Jos WM van der Meer1,2, Mihai G Netea1,2 and Concepcion de la Ru´a*,3 The ability of humans to digest the milk component lactose after weaning requires persistent production of the lactose- converting enzyme lactase. Genetic variation in the promoter of the lactase gene (LCT) is known to be associated with lactase production and is therefore a genetic determinant for either lactase deficiency or lactase persistence during adulthood. Large differences in this genetic trait exist between populations in Africa and the Middle-East on the one hand, and European populations on the other; this is thought to be due to evolutionary pressures exerted by consumption of dairy products in Neolithic populations in Europe. In this study, we have investigated lactase persistence of 26 out of 46 individuals from Late Neolithic through analysis of ancient South-West European DNA samples, obtained from two burials in the Basque Country originating from 5000 to 4500 YBP. This investigation revealed that these populations had an average frequency of lactase persistence of 27%, much lower than in the modern Basque population, which is compatible with the concept that Neolithic and post-Neolithic evolutionary pressures by cattle domestication and consumption of dairy products led to high lactase persistence in Southern European populations. Given the heterogeneity in the frequency of the lactase persistence allele in ancient Europe, we suggest that in Southern Europe the selective advantage of lactose assimilation in adulthood most likely took place from standing population variation, after cattle domestication, at a post-Neolithic time when fresh milk consumption was already fully adopted as a consequence of a cultural influence. European Journal of Human Genetics (2012) 20, 778–782; doi:10.1038/ejhg.2011.254; published online 11 January 2012 Keywords: lactase persistence; evolutionary pressure; ancient DNA; Neolithic INTRODUCTION as cattle domestication and dairy consumption was introduced in Throughout the world, large differences in the ability to digest lactose Europe. From that point a strong selective advantage of the ability after weaning, designated as lactase persistence, are observed between to digest milk would have become apparent, which has resulted in a populations. Although lactase persistence reaches up to 60–90% in survival benefit of the already existing rare lactase persistent Central-European and Northern European populations, it is much less individuals in the population. As a consequence, the frequency of frequently present in Southern European, Middle-Eastern, African and lactase persistence has increased rapidly in the generations to some South-Asian populations, while being completely absent in the follow. This hypothesis has been described in literature as the rest of the world population.1–3 The persistent activity of the lactase ‘culture–historical hypothesis’7,8 andhasbeensupportedbyrecent enzyme (ie, lactase phlorizin hydrolase, encoded by the lactase gene studies in the Neolithic samples from Central, Mediterranean and LCT) during adulthood, expressed especially in the intestine, has been Northern Europe9–11 and medieval samples from Central Europe.3 demonstrated to be linked with variations in the lactase promoter, Burger et al9 reported the absence of lactase persistence in a total such as a C/T transition in the promoter region 13910 basepairs number of nine early Neolithic Central Europeans (7500 YBP), upstream of the LCT gene (rs4988235), which is suggested to influence which argues that in this era Europeans were predominantly lactase LCT gene expression.4 The T allele, dominant over the C allele, has deficient in adulthood. The absence of lactase persistence is also been described to be the allele associated with lactase persistence reported by Lacan et al11 in 26 samples from a late Neolithic burial in European populations.5 In other populations, different polymor- located in Southern France. However, in Neolithic Scandinavians phisms in the LCT promoter have been also shown to be associated the prevalence of the À13910T allele was only 5%.10 The opposing with lactase persistence.6 ‘reverse cause hypothesis’, states that human populations were Several studies support the perspective that these differences in already differentiated with regard to lactase persistence frequency lactase persistence between populations can be ascribed to pro- before the development of dairying, and that the presence of lactase cesses of positive selection.1,2 Indeed, it has been suggested that the persistence determined the adoption of milk production and frequency of lactase persistence has increased in several populations consumption practices.12 1Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 2Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Bizkaia, Spain; 4Department of Genetics, University of La Laguna, Santa Cruz de Tenerife, Spain *Correspondence: Dr C de la Ru´ a, Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Barrio Sarriena s/n 48940 Leioa, Bizkaia, Spain. Tel +34 946 012 544, Fax +34-946013145; E-mail: [email protected] Received 21 July 2011; revised 2 November 2011; accepted 7 December 2011; published online 11 January 2012 Lactase persistence in Neolithic South-West Europe TS Plantinga et al 779 Table 1 Primer sequences and annealing temperatures used to amplify the promoter region of the LCT gene Primer pair Forward primer Reverse primer Anneal temperature (1C) 15¢-TACTAGTAGG 5¢-TGCAACCTAA 54 CCTCTGCGCT-3¢ GGAGGAGAGT-3¢ 25¢-AAATGTACTTAG 5¢-AACCTAAGGA 50 ACCCTACAATGTA-3¢ GGAGAGTTCC-3¢ 35¢-AAAATGTACTTA 5¢-ATACAAATGCA 50 GACCCTACAATG-3¢ ACCTAAGGAG-3¢ contamination of the endogenous DNA, we carefully selected those teeth that did not show any signs of caries or deep fissures that might extend into the pulp, our source of DNA. The samples were processed according to a series of previously detailed Figure 1 Late Neolithic burial locations in the Basque Country. criteria.24–26 Thus, the extraction of DNA and set-up of the PCR were done in a positive pressure, sterile chamber, which was physically separated from the In Northern Europe, the advantage of the calcium assimilation laboratory where the post-PCR processes are usually carried out. All of the working surfaces were regularly cleaned with sodium hypochlorite and were, in hypothesis is likely to be the cause of the high lactase persistence at addition, regularly irradiated with UV light. If possible, PCR reagents were this latitude,13 but in Southern Europe the presence of the À13910T 2 exposed to intense UV light before use. Suitable disposable clothing was worn allele displays a large variation between present populations. The (lab coat, mask, gloves and cap). Iberian Peninsula represents one of the end points of Neolithic In order to eliminate surface contamination, the teeth were washed with migration. The arrival or adoption of the Neolithic way of life in acids to depurinate possible contaminating external DNA. Besides, the entire the Northern part of the Iberian Peninsula, here referred to as the tooth surface was irradiated with UV light. Then, after cutting the root of the Basque Country, is believed to have taken place around 7000 YBP. tooth, it was incubated at 37 1C and with agitation overnight in a lysis buffer Several burials dating from this period have been identified and (5 ml; 0.5 M EDTA pH 8.0–8.5; 0.5% SDS; 50 mM Tris HCl pH 8.0; 0.01 mg/ml investigated archaeologically in the Basque Country, dating from proteinase K). Then, DNA was extracted using the conventional phenol– 7000 to 4000 YBP.14–16 There are various genetic arguments favouring chloroform procedure. After the extraction, the DNA was concentrated and purified by means of Centricon-30 Amicon spin columns (Millipore, Billerica, the Basques as the most homogeneous relict population of the pre- MA, USA). Each extraction session involved two contamination controls, Neolithic inhabitants of Europe. Therefore, the genetic make-up of the which were applied during both the extraction and amplification processes. Neolithic Basques is likely to mirror the general genetic signature of Neolithic populations in Europe.17–22 Although the prevalence of the 2 Amplification and sequencing of the LCT promoter region À13910T allele in the modern Basque population reaches 66%, and PCR amplification was performed with the incorporation of the extraction the selection coefficient for lactase persistence has been shown to be controls obtained during the DNA extraction process. Negative controls for the 23 relatively high in the Iberian Peninsula, the processes that shaped PCR reaction were included. Three different primer pairs were used to amplify this process are controversial. the promoter region of the LCT gene containing the À13910 position, which all In order to assess whether lactase persistence was a common