Diversity of Lactase Persistence Alleles in Ethiopia: Signature of a Soft Selective Sweep

Abstract

The persistent expression of lactase into adulthood in humans is a recent genetic adaptation that allows the consumption of milk from other mammals after weaning. In Europe, a single allele (−13910∗T, rs4988235) in an upstream region that acts as an enhancer to the expression of the lactase gene LCT is responsible for lactase persistence and appears to have been under strong directional selection in the last 5,000 years, evidenced by the widespread occurrence of this allele on an extended haplotype. In Africa and the Middle East, the situation is more complicated and at least three other alleles (−13907∗G, rs41525747; −13915∗G, rs41380347; −14010∗C, rs145946881) in the same LCT enhancer region can cause continued lactase expression. Here we examine the LCT enhancer sequence in a large lactose-tolerance-tested Ethiopian cohort of more than 350 individuals. We show that a further SNP, −14009T>G (ss 820486563), is significantly associated with lactose-digester status, and in vitro functional tests confirm that the −14009∗G allele also increases expression of an LCT promoter construct. The derived alleles in the LCT enhancer region are spread through several ethnic groups, and we report a greater genetic diversity in lactose digesters than in nondigesters. By examining flanking markers to control for the effects of mutation and demography, we further describe, from empirical evidence, the signature of a soft selective sweep. The persistent expression of lactase into adulthood in humans is a recent genetic adaptation that allows the consumption of milk from other mammals after weaning. In Europe, a single allele (−13910∗T, rs4988235) in an upstream region that acts as an enhancer to the expression of the lactase gene LCT is responsible for lactase persistence and appears to have been under strong directional selection in the last 5,000 years, evidenced by the widespread occurrence of this allele on an extended haplotype. In Africa and the Middle East, the situation is more complicated and at least three other alleles (−13907∗G, rs41525747; −13915∗G, rs41380347; −14010∗C, rs145946881) in the same LCT enhancer region can cause continued lactase expression. Here we examine the LCT enhancer sequence in a large lactose-tolerance-tested Ethiopian cohort of more than 350 individuals. We show that a further SNP, −14009T>G (ss 820486563), is significantly associated with lactose-digester status, and in vitro functional tests confirm that the −14009∗G allele also increases expression of an LCT promoter construct. The derived alleles in the LCT enhancer region are spread through several ethnic groups, and we report a greater genetic diversity in lactose digesters than in nondigesters. By examining flanking markers to control for the effects of mutation and demography, we further describe, from empirical evidence, the signature of a soft selective sweep. Lactase, the enzyme that digests the milk sugar lactose, persists into adult life in approximately 35% of the world’s population.1Ingram C.J. Mulcare C.A. Itan Y. Thomas M.G. Swallow D.M. Lactose digestion and the evolutionary genetics of lactase persistence.Hum. Genet. 2009; 124: 579-591Crossref PubMed Scopus (309) Google Scholar This genetic trait of adult lactase persistence (LP [MIM 223100]) is a recent human adaptation permitting those who carry it to use animal milk more readily as a source of nutrition. This LP phenotype is in contrast to the ancestral mammalian phenotype shared by most of the human population where lactase is downregulated before adulthood. LP is attributable to nucleotide changes in a regulatory region that acts as an enhancer of the expression of the gene encoding lactase, LCT (MIM 603202). This LCT enhancer is located in intron 13 of the neighboring gene, MCM6 (MIM 601806), immediately upstream of LCT (for review see Ingram et al.1Ingram C.J. Mulcare C.A. Itan Y. Thomas M.G. Swallow D.M. Lactose digestion and the evolutionary genetics of lactase persistence.Hum. Genet. 2009; 124: 579-591Crossref PubMed Scopus (309) Google Scholar). LP in Europe is generally attributable to a single allele (−13910∗T, rs4988235) that seems to have been under strong directional selection in the last 5,000–10,000 years.2Itan Y. Powell A. Beaumont M.A. Burger J. Thomas M.G. The origins of lactase persistence in Europe.PLoS Comput. Biol. 2009; 5: e1000491Crossref PubMed Scopus (326) Google Scholar The evidence for selection comes from the fact that the allele lies on an extended haplotype3Poulter M. Hollox E. Harvey C.B. Mulcare C. Peuhkuri K. Kajander K. Sarner M. Korpela R. Swallow D.M. The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans.Ann. Hum. Genet. 2003; 67: 298-311Crossref PubMed Scopus (108) Google Scholar, 4Bersaglieri T. Sabeti P.C. Patterson N. Vanderploeg T. Schaffner S.F. Drake J.A. Rhodes M. Reich D.E. Hirschhorn J.N. Genetic signatures of strong recent positive selection at the lactase gene.Am. J. Hum. Genet. 2004; 74: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (737) Google Scholar with low microsatellite diversity and is at a significantly higher frequency than expected for the age of the allele.1Ingram C.J. Mulcare C.A. Itan Y. Thomas M.G. Swallow D.M. Lactose digestion and the evolutionary genetics of lactase persistence.Hum. Genet. 2009; 124: 579-591Crossref PubMed Scopus (309) Google Scholar, 4Bersaglieri T. Sabeti P.C. Patterson N. Vanderploeg T. Schaffner S.F. Drake J.A. Rhodes M. Reich D.E. Hirschhorn J.N. Genetic signatures of strong recent positive selection at the lactase gene.Am. J. Hum. Genet. 2004; 74: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (737) Google Scholar, 5Anagnostou P. Battaggia C. Coia V. Capelli C. Fabbri C. Pettener D. Destro-Bisol G. Luiselli D. Tracing the distribution and evolution of lactase persistence in Southern Europe through the study of the T(-13910) variant.Am. J. Hum. Biol. 2009; 21: 217-219Crossref PubMed Scopus (19) Google Scholar, 6Coelho M. Luiselli D. Bertorelle G. Lopes A.I. Seixas S. Destro-Bisol G. Rocha J. Microsatellite variation and evolution of human lactase persistence.Hum. Genet. 2005; 117: 329-339Crossref PubMed Scopus (97) Google Scholar Tests of haplotype homozygosity and population differentiation in genome-wide studies that focus on European samples show that the region of chromosome 2 containing LCT has one of the highest “signatures” of selection.7Sabeti P.C. Schaffner S.F. Fry B. Lohmueller J. Varilly P. Shamovsky O. Palma A. Mikkelsen T.S. Altshuler D. Lander E.S. Positive natural selection in the human lineage.Science. 2006; 312: 1614-1620Crossref PubMed Scopus (808) Google Scholar In Africa and the Middle East, the situation is more complicated and three additional alleles (−13907∗G, rs41525747; −13915∗G, rs41380347; −14010∗C, rs145946881) in the same LCT enhancer region have been reported to cause lactase persistence.8Jensen T.G. Liebert A. Lewinsky R. Swallow D.M. Olsen J. Troelsen J.T. The -14010∗C variant associated with lactase persistence is located between an Oct-1 and HNF1α binding site and increases lactase promoter activity.Hum. Genet. 2011; 130: 483-493Crossref PubMed Scopus (36) Google Scholar, 9Olds L.C. Ahn J.K. Sibley E. 13915∗G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1.Hum. Genet. 2011; 129: 111-113Crossref PubMed Scopus (28) Google Scholar, 10Sibley E. Ahn J.K. Theodore E. Woodward Award: lactase persistence SNPs in African populations regulate promoter activity in intestinal cell culture.Trans. Am. Clin. Climatol. Assoc. 2011; 122: 155-165PubMed Google Scholar, 11Troelsen J.T. Olsen J. Møller J. Sjöström H. An upstream polymorphism associated with lactase persistence has increased enhancer activity.Gastroenterology. 2003; 125: 1686-1694Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Wang Z. Maravelias C. Sibley E. Lactase gene promoter fragments mediate differential spatial and temporal expression patterns in transgenic mice.DNA Cell Biol. 2006; 25: 215-222Crossref PubMed Scopus (7) Google Scholar, 13Enattah N.S. Jensen T.G. Nielsen M. Lewinski R. Kuokkanen M. Rasinpera H. El-Shanti H. Seo J.K. Alifrangis M. Khalil I.F. et al.Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture.Am. J. Hum. Genet. 2008; 82: 57-72Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 14Ingram C.J. Elamin M.F. Mulcare C.A. Weale M.E. Tarekegn A. Raga T.O. Bekele E. Elamin F.M. Thomas M.G. Bradman N. Swallow D.M. A novel polymorphism associated with lactose tolerance in Africa: multiple causes for lactase persistence?.Hum. Genet. 2007; 120: 779-788Crossref PubMed Scopus (180) Google Scholar, 15Ingram C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar, 16Tishkoff S.A. Reed F.A. Ranciaro A. Voight B.F. Babbitt C.C. Silverman J.S. Powell K. Mortensen H.M. Hirbo J.B. Osman M. et al.Convergent adaptation of human lactase persistence in Africa and Europe.Nat. Genet. 2007; 39: 31-40Crossref PubMed Scopus (987) Google Scholar In Tanzania and Kenya, one particular allele, −14010∗C, is at high frequency and gives a significant signal of positive selection in tests of haplotype homozygosity, with expansion of the −14010∗C allele dated to approximately 3,000–7,000 years ago.16Tishkoff S.A. Reed F.A. Ranciaro A. Voight B.F. Babbitt C.C. Silverman J.S. Powell K. Mortensen H.M. Hirbo J.B. Osman M. et al.Convergent adaptation of human lactase persistence in Africa and Europe.Nat. Genet. 2007; 39: 31-40Crossref PubMed Scopus (987) Google Scholar In some cases, however, including the Jaali from Sudan and the Somali camel herders from Ethiopia, several different LP alleles are associated with lactase persistence in the same ethnic group.15Ingram C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar Indeed, we observed that in these groups, the sequence of the enhancer region was much more diverse in lactose digesters than in nondigesters. Analysis of mitochondrial DNA, Y chromosome, and autosomal microsatellite variations demonstrated that this difference in diversity was not attributable to hidden population stratification.15Ingram C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar Although it is possible that not all of the described enhancer alleles are functional, we speculated that this difference in diversity had been influenced by what has been described as a soft selective sweep—the phenomenon by which several different variants of similar function rise in frequency simultaneously.17Hermisson J. Pennings P.S. Soft sweeps: molecular population genetics of adaptation from standing genetic variation.Genetics. 2005; 169: 2335-2352Crossref PubMed Scopus (658) Google Scholar, 18Pennings P.S. Hermisson J. Soft sweeps III: the signature of positive selection from recurrent mutation.PLoS Genet. 2006; 2: e186Crossref PubMed Scopus (199) Google Scholar, 19Pennings P.S. Hermisson J. Soft sweeps II—molecular population genetics of adaptation from recurrent mutation or migration.Mol. Biol. Evol. 2006; 23: 1076-1084Crossref PubMed Scopus (215) Google Scholar Such soft selective sweeps might not be detectable by published methods of genome-wide detection of selection.20Sabeti P.C. Reich D.E. Higgins J.M. Levine H.Z. Richter D.J. Schaffner S.F. Gabriel S.B. Platko J.V. Patterson N.J. McDonald G.J. et al.Detecting recent positive selection in the human genome from haplotype structure.Nature. 2002; 419: 832-837Crossref PubMed Scopus (1411) Google Scholar, 21Sabeti P.C. Varilly P. Fry B. Lohmueller J. Hostetter E. Cotsapas C. Xie X. Byrne E.H. McCarroll S.A. Gaudet R. et al.International HapMap ConsortiumGenome-wide detection and characterization of positive selection in human populations.Nature. 2007; 449: 913-918Crossref PubMed Scopus (1324) Google Scholar Here, we examine genetic diversity of the LCT enhancer in a larger lactose-tolerance-tested cohort (>350 individuals), which consists of volunteers from several ethnic groups in Ethiopia. We aim to determine whether any further genetic variants are likely to be functional, whether the different Ethiopian groups in our cohort carry private mutations, and whether the pattern of increased diversity of the enhancer region in lactose digesters that we detected previously is widespread in Ethiopia. We examine this diversity in relation to that of flanking markers to control for the effects of mutation and demography to describe this signal of selection. The Ethiopian participants who by a was from the of and for the and a to and of and and and samples from all by DNA B. N. C. J. DNA from by of effects on and for Genet. 2003; PubMed Scopus Google Scholar and from individuals. as lactose or a as described by Ingram and C.J. Elamin M.F. Mulcare C.A. Weale M.E. Tarekegn A. Raga T.O. Bekele E. Elamin F.M. Thomas M.G. Bradman N. Swallow D.M. A novel polymorphism associated with lactose tolerance in Africa: multiple causes for lactase persistence?.Hum. Genet. 2007; 120: 779-788Crossref PubMed Scopus (180) Google Scholar, 15Ingram C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar from from the study of recent use or between in a from individuals. A of of the volunteers from this Ethiopian cohort as lactose digesters and as nondigesters. A of and the The volunteers to with and from the same ethnic as or and those with other or as as as possible by We a DNA the LCT enhancer (for and see and a of 13 of the significant from not of the derived alleles private to any particular ethnic of the derived enhancer alleles for all lactose digesters are in The strong of and with is in this the other previously −14010∗C was in digesters at low a frequency to in this cohort and was in one who also a enhancer In this we show a significant of the −14009∗G variant with lactose-digester with a of for and more in than in which that these alleles not cause In this is of particular it is located to other functional other alleles rare to of in 13 of MCM6 and with allele reported are from a of volunteers of the alleles also after of all who carry two different derived alleles in are for the of digesters and nondigesters. significant after for tests that is in one the a also was from of The of immediately to the of in a The reported are from a of volunteers of the alleles also after of all who carry two different derived alleles in are for the of digesters and nondigesters. significant after for tests that is in one the a also was from of The of immediately to the of determine whether −14009∗G has a functional on the LCT enhancer the −14009∗G mutation was by into the LCT enhancer region to and into the promoter with as a as previously T.G. Liebert A. Lewinsky R. Swallow D.M. Olsen J. Troelsen J.T. The -14010∗C variant associated with lactase persistence is located between an Oct-1 and HNF1α binding site and increases lactase promoter activity.Hum. Genet. 2011; 130: 483-493Crossref PubMed Scopus (36) Google Scholar, 11Troelsen J.T. Olsen J. Møller J. Sjöström H. An upstream polymorphism associated with lactase persistence has increased enhancer activity.Gastroenterology. 2003; 125: 1686-1694Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar with and the are in A significant in activity was observed for the −14009∗G in with the ancestral This is with the observed for the −14010∗C variant as a positive and our studies in confirm of binding and The fact that as as who carry the allele as however, that this allele than the in one of the LP variants and as lactose other as had variant in the LCT least of this is attributable to one of the of this of control for of function in the from C.A. Weale M.E. B. D. Tarekegn A. Swallow D.M. Bradman N. Thomas M.G. The allele of a polymorphism upstream of the lactase gene not or cause the lactase-persistence phenotype in J. Hum. Genet. 2004; 74: Full Text Full Text PDF PubMed Scopus Google Scholar the lactose digesters with enhancer variation and are more to and might be that a different causal be at A mutation in a different region be responsible for LP in these or a variants are likely to be causal are more likely than variants to have a Swallow D.M. The of on the human J. 2011; 5: PubMed Scopus Google Scholar). also a in the persistence of lactase in some of these individuals. In an of the diversity of the LCT enhancer we greater sequence diversity in the digesters than in the for digesters for for digesters for which is with our in Ethiopian Somali camel C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar further the causes of this greater diversity in we to control for the possible effects of demography and mutation we two control of the LCT enhancer that in most to have been from the enhancer by the of the spread of these alleles (for and see We a upstream of the LCT enhancer in intron of MCM6 and a 13 of the approximately 1 upstream of LCT a of observed in for that region or of the are likely to have the last of MCM6 is likely to have a similar to that of intron 13 a similar to as a control region with similar mutation The on samples with sequence for all three of phenotype the previously cohort of Ethiopian Somali camel C.J. Raga T.O. Tarekegn A. Browning S.L. Elamin M.F. Bekele E. Thomas M.G. Weale M.E. Bradman N. Swallow D.M. Multiple rare variants as a cause of a common phenotype: several different lactase persistence associated alleles in a single ethnic group.J. Mol. Evol. 2009; 69: 579-588Crossref PubMed Scopus (73) Google Scholar for The of sequence variants in the two control for digesters and are in with the M. P. A of methods for haplotype from population J. Hum. Genet. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar and the by of the The that in this those with than three are in 2 in relation to our M. M. V. A. T. N. A.I. Swallow D.M. Lactase haplotype diversity in the J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar the of and and of show evidence of between the flanking and the frequency of detectable is for the the derived enhancer alleles previously is on a or a haplotype which of are all three and is on an extended or in of 2 and The −14009∗G allele in of on a haplotype that has a at to our haplotype be an or haplotype. of the LCT sequence of a of the with the −14009∗G that carry a that this is most an than the more common M. M. V. A. T. N. A.I. Swallow D.M. Lactase haplotype diversity in the J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar an additional that the enhancer region in intron 13 is not more than the other that we have we the of the three from by the from the We observed that the of the intron 13 region is in fact than that of the other two in with the the and for control region the enhancer and control region We haplotype diversity and nucleotide diversity of all three sequence in the digesters and from the and and the previously of or those to groups with than in our for the of this of as other for the 13 of the of genetic diversity with those samples with a of lactose-digester or that for the enhancer the of of and for and are in larger in the digesters than nondigesters. on samples for which was and a single additional was of all in intron and one in the upstream LCT are as SNPs in the of are in in a the of genetic diversity with those samples with a of lactose-digester or that for the enhancer the of of and for and are in larger in the digesters than nondigesters. on samples for which was and a single additional was of all in intron and one in the upstream LCT are as SNPs in the of are in the distribution of nucleotide diversity for the enhancer in all cases, was greater in the digesters than the nondigesters. We the of the difference in for ethnic by in which the and to digesters and in A was as the of with an difference to or larger than the observed difference between digesters and nondigesters. the was a significant difference in between digesters and for all ethnic groups of the control one ethnic with a significant difference for the control region in intron diversity of the enhancer region was also significantly greater in digesters than for all ethnic groups by the M.G. Weale M.E. M. A. N. A. R. Tarekegn A. et of groups by J. Hum. Genet. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar This distribution is in for the the significant difference in the control was the intron region in not it is that in most the diversity of the flanking is in the than digesters of the same This which is in the Somali is a of the high of a haplotype that not carry any derived alleles on and see the haplotype in we confirm our on the diversity of the LCT enhancer sequence region and show with a cohort of samples that is significantly greater nucleotide and haplotype diversity in the enhancer region in digesters than in nondigesters. By two flanking which have the same history as the enhancer the last control have high diversity in as as in Indeed, it is the of diversity in the enhancer sequence of that is and this seems to the of this sequence and the are approximately and and on of this this sequence region have been under evolutionary of function as a regulatory element for lactase expression in This appears to have been in some human of the of adult lactase expression. This study the of LP causal variants to −13915∗G, and all the most of the to have enhancer that the functional region is than the sequence that we have and this be further in The of all alleles in some to the greater nucleotide diversity the LCT enhancer in intron 13 of MCM6 in has been a of human in the last 5,000 years the LP alleles are likely to have under and studies on other African and Middle populations and show different with in Ethiopia, that origins are all where these and spread is likely to be The of large population and selection has been to be in this of pattern of selection of multiple alleles of similar a soft selective J. Pennings P.S. Soft sweeps: molecular population genetics of adaptation from standing genetic variation.Genetics. 2005; 169: 2335-2352Crossref PubMed Scopus (658) Google Scholar Here we confirm this pattern of diversity in the LCT enhancer region in Ethiopia, and by flanking we at a for possible in population and mutation between the digesters and nondigesters. increased genetic diversity in digesters is to the LCT we can that recent selection is on this functional sequence has the of the frequency of the on which the derived alleles the on diversity of the flanking is not This pattern with that observed from the selective for the LCT enhancer allele, where a single extended haplotype is in at least one in haplotype is significantly higher for the M. Hollox E. Harvey C.B. Mulcare C. Peuhkuri K. Kajander K. Sarner M. Korpela R. Swallow D.M. The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans.Ann. Hum. Genet. 2003; 67: 298-311Crossref PubMed Scopus (108) Google Scholar, 4Bersaglieri T. Sabeti P.C. Patterson N. Vanderploeg T. Schaffner S.F. Drake J.A. Rhodes M. Reich D.E. Hirschhorn J.N. Genetic signatures of strong recent positive selection at the lactase gene.Am. J. Hum. Genet. 2004; 74: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (737) Google Scholar, C.B. Hollox M. Y. M. S. R. Sarner M. et haplotype in with the lactase persistence/non-persistence Hum. Genet. PubMed Google Scholar and This the sequence of in other of the This was by the for the European to the of and and for is the and of the has any or other with to the of the We and for and for with and Hollox for with and The for are as in