Comparison of lactase persistence polymorphism in ancient and present-day Hungarian populations

The prevalence of adult-type hypolactasia varies ethnically and geographically among populations. A C/T-13910 single nucleotide polymorphism (SNP) upstream of the lactase gene is known to be associated with lactase non-persistence in Europeans. The aim of this study was to determine the prevalence of lactase persistent and non-persistent genotypes in current Hungarian-speaking populations and in ancient bone samples of classical conquerors and commoners from the 10th-11th centuries from the Carpathian basin; 181 present-day Hungarian, 65 present-day Sekler, and 23 ancient samples were successfully genotyped for the C/T-13910 SNP by the dCAPS PCR-RFLP method. Additional mitochondrial DNA testing was also carried out. In ancient Hungarians, the T-13910 allele was present only in 11% of the population, and exclusively in commoners of European mitochondrial haplogroups who may have been of pre-Hungarian indigenous ancestry. This is despite animal domestication and dairy products having been introduced into the Carpathian basin early in the Neolithic Age. This anomaly may be explained by the Hungarian use of fermented milk products, their greater consumption of ruminant meat than milk, cultural differences, or by their having other lactase-regulating genetic polymorphisms than C/T-13910. The low prevalence of lactase persistence provides additional information on the Asian origin of Hungarians. Present-day Hungarians have been assimilated with the surrounding European populations, since they do not differ significantly from the neighboring populations in their possession of mtDNA and C/T-13910 variants.     

Frequency of lactase persistence genotype in a healthy Polish population

The majority of mammals are unable to digest lactose due to post-weaning deactivation of the LCT gene, which is responsible for encoding the enzyme lactase (i.e., adult-type hypolactasia). A substitution of C with T at position −13910 bp upstream of the LCT gene has been linked to the lactase persistence phenotype in European populations. We investigated the frequency of LCT-13910C>T polymorphism in 223 blood donors from central Poland. All samples were genotyped by polymerase chain reaction and direct sequencing. The LCT-13910 T allele (lactase persistence) was present in 51% of individuals sampled from the Polish population. We did not find any non-European variants associated with lactase persistence (LCT-13907C>G, LCT-13913T>C, LCT-13915T>G), or any new polymorphisms within the sequenced region. Allele frequencies obtained are in agreement with results from other countries and confirm the unique pattern of distribution of the LCT-13910C>T genotype in Europe.     

The human lactase persistence-associated SNP ?13910*T enables in vivo functional persistence of lactase promoter–reporter transgene expression

Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.     

Lactose digestion and the evolutionary genetics of lactase persistence

It has been known for some 40 years that lactase production persists into adult life in some people but not in others. However, the mechanism and evolutionary significance of this variation have proved more elusive, and continue to excite the interest of investigators from different disciplines. This genetically determined trait differs in frequency worldwide and is due to cis-acting polymorphism of regulation of lactase gene expression. A single nucleotide polymorphism located 13.9 kb upstream from the lactase gene (C-13910 > T) was proposed to be the cause, and the −13910*T allele, which is widespread in Europe was found to be located on a very extended haplotype of 500 kb or more. The long region of haplotype conservation reflects a recent origin, and this, together with high frequencies, is evidence of positive selection, but also means that −13910*T might be an associated marker, rather than being causal of lactase persistence itself. Doubt about function was increased when it was shown that the original SNP did not account for lactase persistence in most African populations. However, the recent discovery that there are several other SNPs associated with lactase persistence in close proximity (within 100 bp), and that they all reside in a piece of sequence that has enhancer function in vitro, does suggest that they may each be functional, and their occurrence on different haplotype backgrounds shows that several independent mutations led to lactase persistence. Here we provide access to a database of worldwide distributions of lactase persistence and of the C-13910*T allele, as well as reviewing lactase molecular and population genetics and the role of selection in determining present day distributions of the lactase persistence phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microsatellite variation and evolution of human lactase persistence

The levels of haplotype diversity within the lineages defined by two single-nucleotide polymorphisms (SNPs) (–13910 C/T and –22018 G/A) associated with human lactase persistence were assessed with four fast-evolving microsatellite loci in 794 chromosomes from Portugal, Italy, Fulbe from Cameroon, São Tomé and Mozambique. Age estimates based on the intraallelic microsatellite variation indicate that the –13910*T allele, which is more tightly associated with lactase persistence, originated in Eurasia before the Neolithic and after the emergence of modern humans outside Africa. We detected significant departures from neutrality for the –13910*T variant in geographically and evolutionary distant populations from southern Europe (Portuguese and Italians) and Africa (Fulbe) by using a neutrality test based on the congruence between the frequency of the allele and the levels of intraallelic variability measured by the number of mutations in adjacent microsatellites. This result supports the role of selection in the evolution of lactase persistence, ruling out possible confounding effects from recombination suppression and population history. Reevaluation of the available evidence on variation of the –13910 and –22018 loci indicates that lactase persistence probably originated from different mutations in Europe and most of Africa, even if 13910*T is not the causal allele, suggesting that selective pressure could have promoted the convergent evolution of the trait. Our study shows that a limited number of microsatellite loci may provide sufficient resolution to reconstruct key aspects of the evolutionary history of lactase persistence, providing an alternative to approaches based on large numbers of SNPs.Electronic supplementary material Supplementary material is available for this article at     

Molecular diagnosis and frequencies of primary hypolactasia in populations of RUSSIA and neighboring countries

The digestion of the milk sugar (lactose) is observed every normal child but not in every adult. The decreased lactase synthesis in some adults results in problems with digestion of the whole milk (primary hypolactasy). An association of lactase activity in adults with carrying of the allele T within the polymorphism C/T-13910 located upstream of the lactase gene and 100% association of hypolactasy with the genotype C/C has recently been shown for a Finnish sample. In the present work we determined the LCT* C/T_13910 genotypes and allele frequencies in populations from Russia. The genotype C/C frequencies varied from 36.6% for Russians to 88.2% for Chukchi and were close to the published medical and epidemiological data on hypolactasy frequencies in respective populations. Genotyping was performed by three different methods to identify the optimal one. Our results have shown that the studied locus is the key determinant for the primary hypolactasy development in various human populations. Consequently, the DNA diagnostics of the C/C genotype carrying is a promising predictive test to detect the primary hypolactasy long before its clinical development. Practical application of this type of diagnostics would be a step towards the individual-oriented medicine.     

Molecular diagnosis and frequencies of primary hypolactasia in populations of Russia and neighboring countries

Normally, the ability to digest milk sugar (lactose) is present in every child, but not in every adult. The decrease in lactase synthesis (hypolactasia) results in the inability to digest whole milk. Recent studies of the Finnish population have associated lactase persistence in adults with allele T of the C/T_?13910 polymorphism located upstream of the lactase gene; a 100% correlation of primary hypolactasia with genotype C/C has been proved. In this study, the allele and genotype frequencies of C/T_?13910 were determined in populations of Russia. The frequencies of genotype C/C, varying from 36.6% in Russians to 88.2% in Chukchi, were close to the published medical and epidemiological data on the hypolactasia frequencies in these populations. Genotyping was carried out by three different methods to determine the optimal one. Genotype C/C proved to be the key determinant of primary hypolactasia. It was assumed that DNA diagnosis of genotype C/C provides a predictive test to detect primary hypolactasia long before its clinical manifestation.     

Correlation of G/A -22018 single-nucleotide polymorphism with lactase activity and its usefulness in improving the diagnosis of adult-type hypolactasia among North Indian children

Adult-type hypolactasia (AtH or lactase non-persistence) is the physiological decline in lactase activity that manifests in majority of the world’s population after weaning. Recently, various single-nucleotide polymorphisms (SNPs) upstream of lactase gene (LCT) have been suggested to be associated with AtH or the lactase persistent trait in different human populations. C/T -13910 SNP was found be completely associated with AtH in Finnish population, and G/A -22018 SNP was found to be strongly, but not completely, associated with AtH. The aim of this study was to correlate G/A -22018 SNP with intestinal lactase activity in North Indian children. These children were also genotyped for C/T -13910 SNP. We also examined the differences in milk consumption and milk-related clinical symptoms in children with different genotypes of G/A -22018 and C/T -13910 SNPs. Intestinal biopsies were obtained from 231 children aged 2–16 years undergoing routine endoscopy for various abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activities and genotyped for G/A -22018 and C/T -13910 SNPs using restriction fragment length polymorphism and DNA sequencing analysis. There was a significant correlation between lactase activity and different genotypes of G/A -22018 SNP. Children with G/G -22018 genotype had low lactase activity. With a reference value of <10 U/g protein (lactase activity) to be indicative of AtH, the sensitivity and specificity of genetic test based on G/A -22018 SNP was 94.4 and 94.1 %, respectively. Furthermore, the consumption of milk was lower in children with G/G -22018 genotype. Flatulence was the only symptom significantly more frequent among the children with G/G -22018 genotype compared to those with G/A and A/A -22018 genotypes. However, most of the children with G/G -22018 genotype seem to tolerate small amounts of milk without any significant difference in gastrointestinal symptoms from those with G/A and A/A -22018 genotypes.     

Evolution of lactase persistence: an example of human niche construction

Niche construction is the process by which organisms construct important components of their local environment in ways that introduce novel selection pressures. Lactase persistence is one of the clearest examples of niche construction in humans. Lactase is the enzyme responsible for the digestion of the milk sugar lactose and its production decreases after the weaning phase in most mammals, including most humans. Some humans, however, continue to produce lactase throughout adulthood, a trait known as lactase persistence. In European populations, a single mutation (-13910*T) explains the distribution of the phenotype, whereas several mutations are associated with it in Africa and the Middle East. Current estimates for the age of lactase persistence-associated alleles bracket those for the origins of animal domestication and the culturally transmitted practice of dairying. We report new data on the distribution of -13910*T and summarize genetic studies on the diversity of lactase persistence worldwide. We review relevant archaeological data and describe three simulation studies that have shed light on the evolution of this trait in Europe. These studies illustrate how genetic and archaeological information can be integrated to bring new insights to the origins and spread of lactase persistence. Finally, we discuss possible improvements to these models.     

Herders of Indian and European cattle share their predominant allele for lactase persistence

Milk consumption and lactose digestion after weaning are exclusively human traits made possible by the continued production of the enzyme lactase in adulthood. Multiple independent mutations in a 100-bp region--part of an enhancer--approximately 14-kb upstream of the LCT gene are associated with this trait in Europeans and pastoralists from Saudi Arabia and Africa. However, a single mutation of purported western Eurasian origin accounts for much of observed lactase persistence outside Africa. Given the high levels of present-day milk consumption in India, together with archaeological and genetic evidence for the independent domestication of cattle in the Indus valley roughly 7,000 years ago, we sought to determine whether lactase persistence has evolved independently in the subcontinent. Here, we present the results of the first comprehensive survey of the LCT enhancer region in south Asia. Having genotyped 2,284 DNA samples from across the Indian subcontinent, we find that the previously described west Eurasian -13910 C>T mutation accounts for nearly all the genetic variation we observed in the 400- to 700-bp LCT regulatory region that we sequenced. Geography is a significant predictor of -13910*T allele frequency, and consistent with other genomic loci, its distribution in India follows a general northwest to southeast declining pattern, although frequencies among certain neighboring populations vary substantially. We confirm that the mutation is identical by descent to the European allele and is associated with the same>1 Mb extended haplotype in both populations.     

Several Different Lactase Persistence Associated Alleles and High Diversity of the Lactase Gene in the Admixed Brazilian Population

Adult-type hypolactasia is a common phenotype caused by the lactase enzyme deficiency. The −13910 C>T polymorphism, located 14 Kb upstream of the lactase gene (LCT) in the MCM6 gene was associated with lactase persistence (LP) in Europeans. This polymorphism is rare in Africa but several other variants associated with lactase persistence were observed in Africans. The aims of this study were to identify polymorphisms in the MCM6 region associated with the lactase persistence phenotype and to determine the distribution of LCT gene haplotypes in 981 individuals from North, Northeast and South Brazil. These polymorphisms were genotyped by PCR based methods and sequencing. The −13779*C,−13910*T, −13937*A, −14010*C, −14011*T LP alleles previously described in the MCM6 gene region that acts as an enhancer for the LCT gene were identified in Brazilians. The most common LP allele was −13910*T. Its frequency was highly correlated with European ancestry in the Brazilian populations investigated. The −13910*T was higher (0.295) in southern Brazilians of European ancestry and lower (0.175) in the Northern admixed population. LCT haplotypes were derived from the 10 LCT SNPs genotyped. Overall twenty six haplotypes previously described were identified in the four Brazilian populations studied. The Multidimensional Scaling analysis showed that Belém, in the north, was closer to Amerindians. Northeastern and southern Afro-descendants were more related with Bantu-speaking South Africans whereas the Southern population with European ancestry grouped with Southern and Northern Europeans. This study shows a high variability considering the number of LCT haplotypes observed. Due to the highly admixed nature of the Brazilian populations, the diagnosis of hypolactasia in Brazil, based only in the investigation of the −13910*T allele is an oversimplification.     

菌群失调腹泻造模及七味白术散治疗对小鼠乳糖酶基因13910多态性的影响

目的探讨抗生素及七味白术散对菌群失调腹泻小鼠乳糖酶基因13910位点多态性的影响。方法将36只SPF级小鼠随机分为正常组(12只)、模型组(12只)、七味白术散组(6只)和乳糖酶组(6只),除正常组外的其余小鼠采用混合抗生素造成菌群失调腹泻模型,然后分别灌胃七味白术散和乳糖酶治疗。造模成功和治疗后分别提取小鼠肠道内容物和肠道黏膜宏基因组,对包括小鼠乳糖酶基因13910位点在内的1 036bp长度的DNA片段进行单核苷酸多态性(SNP)测序分析。结果造模和治疗后,小鼠乳糖酶基因13910位点没有发生碱基的变异,均为A/A型,13910位点在内的1 036bp范围内也没有新的SNP位点出现。结论菌群失调腹泻造模与七味白术散治疗对小鼠乳糖酶活性的干预均与小鼠乳糖酶基因13910位点多态性没有相关性,可能存在其他多态性位点或其他方面的调控机制。     

−13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1

Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (−13907, −13910 and −13915) of the lactase gene in different ethnic populations. In contrast to the −13907*G and −13910*T SNPs, the −13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the −13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter–reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the −13907 to −13915 SNP region therefore remains a candidate interaction involved in lactase persistence.     

Association of the European Lactase Persistence Variant (LCT-13910 C>T Polymorphism) with Obesity in the Canary Islands

Background

European lactose tolerance genotype (LCT -13910 C>T, rs4988234) has been positively associated to body mass indexes (BMI) in a meta-analysis of 31,720 individuals of northern and central European descent. A strong association of lactase persistence (LP) with BMI and obesity has also been traced in a Spanish Mediterranean population. The aim of this study was to analyze a potential association of LP compared to lactase non-persistence (LNP) with BMI in inhabitants of the Canary Islands of Spain using Mendelian randomization.

Methods

A representative, randomly sampled population of adults belonging to the Canary Islands Nutrition Survey (ENCA) in Spain, aged 18–75 years (n = 551), was genotyped for the LCT – 13910 C>T polymorphism. Milk consumption was assessed by a validated questionnaire. Anthropometric variables were directly measured. WHO classification of BMI was used.

Results

LP individuals were significantly more obese than LNP subjects (χ² = 10.59; p<0.005). LP showed in a multivariate linear regression analysis showed a positive association of LP with BMI compared to LNP, (β = 0.96; 95% CI: 0.08–1.85, p = 0.033). In a multinomial logistic regression analysis normal range weight LP subjects showed an odds ratio for obesity of 2.41; 95%CI 1.39–418, (p = 0.002) compared to LNP.

Conclusions

The T-13910 of the allele LCT-13910 C>T polymorphism is positively associated with BMI. LP increases significantly the risk to develop obesity in the studied population. The LCT-13910 C>T polymorphism stands proxy for the lifetime exposure pattern, milk intake, that may increase susceptibility to obesity and to obesity related pathologies.     

A worldwide correlation of lactase persistence phenotype and genotypes

Background  

The ability of adult humans to digest the milk sugar lactose - lactase persistence - is a dominant Mendelian trait that has been a subject of extensive genetic, medical and evolutionary research. Lactase persistence is common in people of European ancestry as well as some African, Middle Eastern and Southern Asian groups, but is rare or absent elsewhere in the world. The recent identification of independent nucleotide changes that are strongly associated with lactase persistence in different populations worldwide has led to the possibility of genetic tests for the trait. However, it is highly unlikely that all lactase persistence-associated variants are known. Using an extensive database of lactase persistence phenotype frequencies, together with information on how those data were collected and data on the frequencies of lactase persistence variants, we present a global summary of the extent to which current genetic knowledge can explain lactase persistence phenotype frequency.     

Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture

The T₋₁₃₉₁₀ variant located in the enhancer element of the lactase (LCT) gene correlates perfectly with lactase persistence (LP) in Eurasian populations whereas the variant is almost nonexistent among Sub-Saharan African populations, showing high prevalence of LP. Here, we report identification of two new mutations among Saudis, also known for the high prevalence of LP. We confirmed the absence of the European T₋₁₃₉₁₀ and established two new mutations found as a compound allele: T/G₋₁₃₉₁₅ within the −13910 enhancer region and a synonymous SNP in the exon 17 of the MCM6 gene T/C₋₃₇₁₂, −3712 bp from the LCT gene. The compound allele is driven to a high prevalence among Middle East population(s). Our functional analyses in vitro showed that both SNPs of the compound allele, located 10 kb apart, are required for the enhancer effect, most probably mediated through the binding of the hepatic nuclear factor 1 α (HNF1α). High selection coefficient (s) ~0.04 for LP phenotype was found for both T₋₁₃₉₁₀ and the compound allele. The European T₋₁₃₉₁₀ and the earlier identified East African G₋₁₃₉₀₇ LP allele share the same ancestral background and most likely the same history, probably related to the same cattle domestication event. In contrast, the compound Arab allele shows a different, highly divergent ancestral haplotype, suggesting that these two major global LP alleles have arisen independently, the latter perhaps in response to camel milk consumption. These results support the convergent evolution of the LP in diverse populations, most probably reflecting different histories of adaptation to milk culture.     

Lactase haplotype diversity in the Old World

Lactase persistence, the genetic trait in which intestinal lactase activity persists at childhood levels into adulthood, varies in frequency in different human populations, being most frequent in northern Europeans and certain African and Arabian nomadic tribes, who have a history of drinking fresh milk. Selection is likely to have played an important role in establishing these different frequencies since the development of agricultural pastoralism approximately 9,000 years ago. We have previously shown that the element responsible for the lactase persistence/nonpersistence polymorphism in humans is cis-acting to the lactase gene and that lactase persistence is associated, in Europeans, with the most common 70-kb lactase haplotype, A. We report here a study of the 11-site haplotype in 1,338 chromosomes from 11 populations that differ in lactase persistence frequency. Our data show that haplotype diversity was generated both by point mutations and recombinations. The four globally common haplotypes (A, B, C, and U) are not closely related and have different distributions; the A haplotype is at high frequencies only in northern Europeans, where lactase persistence is common; and the U haplotype is virtually absent from Indo-European populations. Much more diversity is seen in sub-Saharan Africans than in non-Africans, consistent with an "Out of Africa" model for peopling of the Old World. Analysis of recent recombinant haplotypes by allele-specific PCR, along with deduction of the root haplotype from chimpanzee sequence, allowed construction of a haplotype network that assisted in evaluation of the relative roles of drift and selection in establishing the haplotype frequencies in the different populations. We suggest that genetic drift was important in shaping the general pattern of non-African haplotype diversity, with recent directional selection in northern Europeans for the haplotype associated with lactase persistence.     

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.     

The Origins of Lactase Persistence in Europe

Lactase persistence (LP) is common among people of European ancestry, but with the exception of some African, Middle Eastern and southern Asian groups, is rare or absent elsewhere in the world. Lactase gene haplotype conservation around a polymorphism strongly associated with LP in Europeans (−13,910 C/T) indicates that the derived allele is recent in origin and has been subject to strong positive selection. Furthermore, ancient DNA work has shown that the −13,910*T (derived) allele was very rare or absent in early Neolithic central Europeans. It is unlikely that LP would provide a selective advantage without a supply of fresh milk, and this has lead to a gene-culture coevolutionary model where lactase persistence is only favoured in cultures practicing dairying, and dairying is more favoured in lactase persistent populations. We have developed a flexible demic computer simulation model to explore the spread of lactase persistence, dairying, other subsistence practices and unlinked genetic markers in Europe and western Asia''s geographic space. Using data on −13,910*T allele frequency and farming arrival dates across Europe, and approximate Bayesian computation to estimate parameters of interest, we infer that the −13,910*T allele first underwent selection among dairying farmers around 7,500 years ago in a region between the central Balkans and central Europe, possibly in association with the dissemination of the Neolithic Linearbandkeramik culture over Central Europe. Furthermore, our results suggest that natural selection favouring a lactase persistence allele was not higher in northern latitudes through an increased requirement for dietary vitamin D. Our results provide a coherent and spatially explicit picture of the coevolution of lactase persistence and dairying in Europe.