© 2004 Nature Publishing Group http://www.nature.com/naturegenetics B R I E F C O M M U N I C AT I O N S Gene-culture coevolution between cattle milk protein genes and human lactase genes a N E W S Albano Beja-Pereira1,2, Gordon Luikart1, Phillip R England1, Daniel G Bradley3, Oliver C Jann4, Giorgio Bertorelle5, Andrew T Chamberlain6, Telmo P Nunes7, Stoitcho Metodiev8, Nuno Ferrand2,9 & Georg Erhardt4 Milk from domestic cows has been a valuable food source for over 8,000 years, especially in lactose-tolerant human societies that exploit dairy breeds. We studied geographic patterns of variation in genes encoding the six most important milk proteins in 70 native European cattle breeds. We found substantial geographic coincidence between high diversity in cattle milk genes, locations of the European Neolithic cattle farming sites (>5,000 years ago) and present-day lactose tolerance in Europeans. This suggests a gene-culture coevolution between cattle and humans. Some, but not all, human populations have the genetically determined ability to digest milk lactose in adulthood, thereby benefiting from the rich food resources in cow’s milk1. These societies (e.g., Northern Europe) are lactose-tolerant and highly dependent on milk products. Lactose tolerance is an example of selection-based evolutionary change in humans from milk-drinking cultures2. Has there also been a detectable evolutionary change in the gene pool of domestic cattle from these cultures? Figure 1 Geographic coincidence between milk gene diversity in cattle, lactose tolerance in humans and locations of Neolithic cattle farming sites in NCE. (a) Geographic distribution of the 70 cattle breeds (blue dots) sampled across Europe and Turkey. (b) Synthetic map showing the first principal component resulting from the allele frequencies at the cattle genes. The dark orange color shows that the greatest milk gene uniqueness and allelic diversity occurs in cattle from NCE. (c) Geographic distribution of the lactase persistence allele in contemporary Europeans. The darker the orange color, the higher is the frequency of the lactase persistence allele. The dashed black line indicates the limits of the geographic distribution of early Neolithic cattle pastoralist (Funnel Beaker Culture) inferred from archaeological data15. b c 1,000 0 1,000 2,000 Km 1Laboratoire d'Ecologie Alpine, Génomique des Populations et Biodiversité, CNRS UMR 5553, Université Joseph Fourier, B.P. 53, 38041 Grenoble, Cedex 9, France. de Investigação em Biodiversidade e Recursos Genéticos (CIBIO-UP) and Secção Autónoma de Ciências Agrárias, Faculdade de Ciências, Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão (VCD), Portugal. 3Department of Genetics, Smurfit Institute, Trinity College, Dublin 2, Ireland. 4Institut für Tierzucht und Haustiergenetik, Justus-Liebig-Universität Gieβen, Ludwigstr. 21b, 35390 Gieβen, Germany. 5Department of Biology, University of Ferrara, 44100 Ferrara, Italy. 6Department of Archaeology and Prehistory, University of Sheffield, Sheffield S1 4ET, UK. 7CIISA/UISEE/DETSA, Faculdade de Medicina Veterinária, Polo Universitário da Ajuda, Rua Prof. Cid dos Santos, 1300-477 Lisboa, Portugal. 8Department of Genetics and Animal Breeding, Thracian University, Agricultural Faculty, 6000 Stara Zagora, Bulgaria. 9Departamento de Zoologia/Antropologia da Faculdade de Ciências Praça Gomes Teixeira, 4099-002 Porto, Portugal. Correspondence should be addressed to A.B.-P. (albano.beja-pereira@ujf-grenoble.fr). 2Centro Published online 23 November 2003; doi:10.1038/ng1263 NATURE GENETICS VOLUME 35 | NUMBER 4 | DECEMBER 2003 311 B R I E F C O M M U N I C AT I O N S Table 1 Spearman correlation coefficient values between the first principal component from the milk protein gene frequencies, the lactase persistence allele frequency and the presence or absence of archeological evidence for Neolithic cattle pastoralists © 2004 Nature Publishing Group http://www.nature.com/naturegenetics Spearman correlation Degrees of freedom P First principal component versus neolithic cattle pastoralists –0.750 21.2–27.3 <0.0005 First principal component versus lactase persistence allele frequency –0.593 17.7–24.6 <0.01 Neolithic cattle pastoralists versus lactase persistence allele frequency 0.730 19.3–24.8 <0.0005 Our study of nonsynonymous mutations in six milk protein genes in ∼20,000 cattle from 70 breeds across Europe (Fig. 1a) found high allelic richness and genetic distinctiveness in the native cattle from North Central Europe (NCE), as illustrated by the synthetic map of cattle milk protein genes (Fig. 1b and Supplementary Tables 1 and 2, Supplementary Fig. 1 and Supplementary Methods online). Notably, this synthetic map (inner contour) closely matches the European distribution of the allele for human lactase persistence that is most frequent in NCE (P < 0.0005; Table 1). This is in stark contrast to the lower levels of lactose tolerance found in people of Southern Europe and the Near East. There was also strong concordance (P < 0.001) of the geographic distribution of cattle milk gene diversity with the early Neolithic distribution of a European cattle pastoralist society3 (Fig. 1c). How can we explain the strong geographic concordance between cattle milk gene diversity, human lactose tolerance and the distribution of the earliest European cattle pastoralists? We propose that since Neolithic times, there has been gene-culture coevolution between the domestic cattle and human culture driven by the advantages conferred by milk consumption. This led to the maintenance of larger herds and selection for increased milk yield and altered milk protein composition. This coevolution seemingly influenced the frequencies of the important milk protein genes in cattle and the gene encoding lactase in humans. In fact, a recent study suggested that the relatively old variant for lactose tolerance was only recently driven to high frequencies in North Central Europeans after the introduction of dairy culture in this region4. This scenario is also supported by evidence for selection at milk protein loci in bovids5. For example, directional selection can explain high intraspecific divergence and low intraspecific polymorphism in k-casein sequences across bovids5. Our data also show patterns consistent with selection: 19 NCE breeds deviated significantly from neutrality (Ewens-Watterson test, 32% of 114 tests with P < 0.01 versus 2% of 306 tests with P < 0.01 in the 51 non-NCE breeds; Fu test and Tajima test, all NCE breeds showed P < 0.05 versus 4 of 51 nonNCE cattle). Our genetic data corroborate recent archaeological evidence suggesting that the early European cattle pastoralists in NCE were dependent on milk6,7, as early Neolithic sites in NCE are rich in cattle remains4. Based on the analysis of intratooth change in nitrogen isotope ratios from archaeological cattle teeth, it seems that cattle herds were managed for early weaning of calves, making cow’s milk more available for human consumption. Meat production, practiced outside NCE, necessitates later weaning to optimize weight gain7. Among several phenomena that might have shaped our data, selection seems the most probable explanation. Recent studies have shown that high diversity in human genes can evolve rapidly due to selection8. In addition, analysis of bovine myostatin alleles showed signals of balancing selection in a number of independently occurring mutations that cause double-muscling in beef breeds9. Given that population surveys of mtDNA sequence, microsatellite markers and protein polymorphisms in European cattle breeds show no evidence of elevated diversity in NCE10-12, it is likely that selection pressure imposed by early pastoralists and their successors in different regions of NCE has left the legacy of high allelic diversity at these specific milk genes. It is also possible that some of the diversity represents relatively recent mutation (<10,000 years), although, under a neutral model, mutation rates are too low (10–6–10–9; ref. 5) for this to be a primary factor. Selection may have maintained many favorable new mutations by protecting them from the normal process of attrition due to drift. Another possible source of the unique diversity found in cattle in NCE is historical gene flow from an as yet unidentified origin. Two candidates for this source are local wild aurochs (Bos primigenius), which persisted in NCE until the sixteenth century, and domestic cattle other than those that gave rise to present day European cattle (outside NCE). Extensive wild auroch introgression seems unlikely, and no mtDNA sequences have been detected in European cattle which match aurochs sequences identified using ancient DNA sequencing13. Notably, our findings contradict the results of previous surveys of genetic variation in European cattle10–12, which suggested that diversity declines with distance from the Fertile Crescent region. This discrepancy could be explained by selection on the milk genes, and it may also reflect different sampling strategies. Our analysis is based on a sample set that is unprecedented in size, geographic coverage and breed diversity. Furthermore, unlike previous studies, we analyzed only nonsynonymous polymorphisms in strong candidate genes most likely to yield unusual geographic patterns in milk gene diversity. Our study provides evolutionary insights and identifies high diversity in cattle genes that are economically important, suggesting that cattle in NCE are a potentially precious genetic resource for future agricultural productivity. Farming practices since the Neolithic seem to have left reciprocal genetic signatures in cattle and human populations from NCE. This may represent a rare example of cultural-genetic coevolution between humans and another species. Other examples of coevolution have been documented for human genes and genes of parasites, such as Plasmodium14. But our study represents the first nondisease-related example of genetic coevolution between humans and domestic animals, reflecting the extent to which domestication has shaped human societies and the genomes of both humans and cattle. 312 Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank M. Zvelebil for ideas and discussion. A.B.-P. is supported by a grant from Fundação para a Ciência e Tecnologia through the Graduate Programme in Areas of Basic and Applied Biology, and the work was partially supported by a Praxis project grant. G.L. and P.R.E. were funded by the European Union (Econogene). D.G.B. is a Science Foundation Ireland Investigator. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 19 June; accepted 29 October 2003 Published online at http://www.nature.com/naturegenetics/ VOLUME 35 | NUMBER 4 | DECEMBER 2003 NATURE GENETICS B R I E F C O M M U N I C AT I O N S © 2004 Nature Publishing Group http://www.nature.com/naturegenetics 1. Schrimshaw, N.S. & Murray, E.B. Am. J. Clin. Nutr. 48, 1059–1179 (1988). 2. Feldman, M.W. & Cavalli-Sforza, L.L. in Mathematical evolutionary theory (ed. Feldman, M.W.) 145–173 (Princeton University Press, Princeton, New Jersey, 1989). 3. Midgley, M.S. TRB Culture: The First Farmers of the North European Plain (Edinburgh University Press, Edinburgh, 1992). 4. Enattah, N.S. et al. Nat. Genet. 30, 233–237 (2002). 5. Ward, R., Honeycutt, L. & Derr, J.N. Genetics 147, 1863–1872 (1997). 6. Dudd, S. & Evershed, P. Science 282, 1478–1480 (1998). 7. Balasse, M. & Tresset, A. J. Archaeol. Sci. 29, 853–859 (2002). 8. Tishkoff, S.A. et al. Science 293, 455–462 (2001). Mutations in the polyglutamine binding protein 1 gene cause Xlinked mental retardation Vera M Kalscheuer1, Kristine Freude1, Luciana Musante1,9, Lars R Jensen1,9, Helger G Yntema2, Jozef Gécz3, Abdelaziz Sefiani4, Kirsten Hoffmann1, Bettina Moser1, Stefan Haas1, Ulf Gurok1, Sebastian Haesler1, Beatriz Aranda1, Arpik Nshedjan1, Andreas Tzschach1, Nils Hartmann1, Tim-Christoph Roloff1, Sarah Shoichet1, Olivier Hagens1, Jiong Tao1, Hans van Bokhoven2, Gillian Turner5, Jamel Chelly6, Claude Moraine7, Jean-Pierre Fryns8, Ulrike Nuber1, Maria Hoeltzenbein1, Constance Scharff1, Harry Scherthan1, Steffen Lenzner1, Ben C J Hamel2, Susann Schweiger1 & Hans-Hilger Ropers1 We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of Xlinked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder. XLMR is a prominent unsolved problem in clinical genetics. Based on the distribution of linkage intervals in 125 unrelated families, we recently showed that roughly one-third of all mutations underlying MRX are clustered on proximal Xp1. This observation prompted us to search for mutations in families with linkage intervals overlapping this region. In 5 of 29 families studied, we detected mutations in PQBP1 that cause frameshifts in the fourth coding exon (Supplementary Methods online), which contains a stretch of six AG dinucleotides in the DR/ER repeat (Fig. 1 and Supplementary Fig. 1 online). In two families (family N9 (not previously reported) and family SHS with Sutherland-Haan syndrome (MRXS3; ref. 2)), all affected males carry an extra AG dinucleotide (3898_3899dupAG), whereas in two others (family N45 (not previously reported) and family MRX55; ref. 3), two AG dinucleotides are deleted (3896_3899delAGAG). A single AG unit (3898_3899delAG) is 9. Dunner, S. et al. Genet. Sel. Evol. 35, 103–118 (2003). 10. Loftus, R.T. et al. Mol. Ecol. 8, 2015–2022 (1999). 11. MacHugh, D.E., Loftus, R.T., Cunningham, P. & Bradley, D.G. Anim. Genet. 29, 333–340 (1998). 12. Medjugorac, I., Kustermann, W., Lazar, P., Russ, I. & Pirchner, F. Anim. Genet. 25, 19–27 (1994). 13. Troy, C. et al. Nature 410, 1088–1091 (2001). 14. Hill, A.V., Jepson, A., Plebanski, M. & Gilbert, S.C. Philos. Trans. R. Soc. Lond. B Biol. Sci. 352, 1317–1325 (1997). 15. Zvelebil, M. in Archaeogenetics: DNA and the Population History of Europe (ed. Boyle, K.) 57–79 (MacDonald Institute Cambridge, Cambridge, 2000). deleted in affected males of family N40 (ref. 4). In all families, these mutations segregated with the disease and were present in all obligate heterozygotes that we tested. Except for one, all obligate heterozygotes that we examined have random X-chromosome inactivation (data not shown) and have IQs in the normal range. Apart from a single-nucleotide polymorphism (IVS2–3C→T), we found no sequence variation in control X chromosomes. The duplication observed in families N9 and SHS and the deletion found in families N45 and MRX55 give rise to almost the same frameshift (Fig. 1b). Still, there is considerable inter- and intrafamilial phenotypic variation (Supplementary Table 1 online). For example, males with SHS show mental retardation, short stature, microcephaly, brachycephaly, spastic diplegia, small testes and anal stenosis or atresia, whereas there is no spastic diplegia or small testes in family N9, with an identical mutation. In both families the disease is not progressive. In family MRX55, in whom the predicted mutant protein differs by only two amino acids, affected individuals are moderately retarded but have no other clinical signs, except for a somewhat smaller body size in one individual (height was 159 cm, ≥2 s.d. below normal at the age of 20 years). In contrast, in addition to mental retardation, all affected individuals in family N45 have microcephaly, one has anal atresia and another has complete situs inversus. Some of this clinical variability may be due to differences in the genetic background. Family MRX55 is from Morocco, families N9 and N45 are from the Netherlands and family SHS has English ancestry. In family N40, all affected males have congenital heart defects in addition to severe mental retardation, microcephaly, spasticity, short stature, cleft or highly arched palate and other craniofacial abnormalities4. The mother of two of the affected individuals has a corrected atrial septal defect. Facial features coarsened with age. Several PQBP1 splice variants have been described5. All but one very rare variant contain exon 4, which is mutated in the five families. The three different types of mutations cause frameshifts that lead to premature stop codons, resulting in truncated PQBP1 proteins that lack several important domains. The particularly severe clinical phenotype seen in family N40 may be due to the fact that the C-terminal end of the predicted truncated protein is entirely different from that of the mutant proteins in the other families (Fig. 1b) and may give rise to aberrant protein-protein interactions. 1Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany. 2Department of Human Genetics, University Medical Centre, Nijmegen, The Netherlands. 3Women’s and Children’s Hospital and The University of Adelaide, Adelaide, Australia. 4Département de Génétique et de Biologie Moléculaire INH, Rabat, Morocco. 5Hunter Genetics and University of Newcastle, P.O. Box 84, Waratah, New South Wales 2298, Australia. 6Institut Cochin de Génétique Moleculaire, CNRS/INSERM, CHU Cochin 75014 Paris, France. 7Services de Génétique-INSERM U316, CHU Bretonneau, Tours, France. 8Center for Human Genetics, Clinical Genetics Unit, Leuven, Belgium. 9These authors contributed equally to this work. Correspondence should be addressed to V.M.K. (kalscheu@molgen.mpg.de). Published online 23 November 2003; doi:10.1038/ng1264 NATURE GENETICS VOLUME 35 | NUMBER 4 | DECEMBER 2003 313 E R R ATA Erratum: Chipping away at the chip bias: RNA degradation in microarray analysis H Auer, S Lyianarachchi, D Newsom, M I Klisovic, G Marcucci & K Kornacker Nat. Genet. 35, 292–293 (2003). © 2004 Nature Publishing Group http://www.nature.com/naturegenetics The name of the fifth author was spelled incorrectly. The correct spelling is “Guido Marcucci”. Erratum: Gene-culture coevolution between cattle milk protein genes and human lactase genes A Beja-Pereira, G Luikart, P R England, D G Bradley, O C Jann, G Bertorelle, A T Chamberlain, T P Nunes, S Metodiev, N Ferrand & G Erhardt Nat. Genet. 35, 311–313 (2003). The paper mistakenly contained a reference to Supplementary Figure 1 online; there is no such figure. 106 VOLUME 35 | NUMBER 1 | SEPTEMBER 2003 NATURE GENETICS