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The genetic trait of lactase persistence (LP) is associated with at least five independent functional single nucleotide variants in a regulatory region about 14 kb upstream of the lactase gene [−13910*T (rs4988235), −13907*G (rs41525747), −13915*G (rs41380347), −14009*G (rs869051967) and −14010*C (rs145946881)]. These alleles have been inferred to have spread recently and present-day frequencies have been attributed to positive selection for the ability of adult humans to digest lactose without risk of symptoms of lactose intolerance. One of the inferential approaches used to estimate the level of past selection has been to determine the extent of haplotype homozygosity (EHH) of the sequence surrounding the SNP of interest. We report here new data on the frequencies of the known LP alleles in the ‘Old World’ and their haplotype lineages. We examine and confirm EHH of each of the LP alleles in relation to their distinct lineages, but also show marked EHH for one of the older haplotypes that does not carry any of the five LP alleles. The region of EHH of this (B) haplotype exactly coincides with a region of suppressed recombination that is detectable in families as well as in population data, and the results show how such suppression may have exaggerated haplotype-based measures of past selection. Electronic supplementary material The online version of this article (doi:10.1007/s00439-017-1847-y) contains supplementary material, which is available to authorized users.

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, -14009TG (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.

Cis-acting polymorphisms that affect gene expression are now known to be frequent, although the extent and mechanisms by which such variation affects the human phenotype are, as yet, only poorly understood. Key signatures of cis-acting variation are differences in gene expression that are tightly associated with regulatory SNPs or expression Quantitative Trait Loci (eQTL) and an imbalance of allelic expression (AEI) in heterozygous samples. Such cis-acting sequence differences appear often to have been under selection within and between populations and are also thought to be important in speciation. Here we describe the example of lactase persistence. In medical research, variants that affect regulation in cis have been implicated in both monogenic and polygenic disorders, and in the metabolism of drugs. In this review we suggest that by further understanding common regulatory variations and how they interact with other genetic and environmental variables it will be possible to gain insight into important mechanisms behind complex disease, with the potential to lead to new methods of diagnosis and treatments.

Genetic variants that regulate the expression level of a transcript are known as expression quantitative trait loci (eQTLs) or regulatory single nucleotide polymorphisms. Cis-variants are those that only act on a target gene (or genes) on the same chromosomal homologue. They cause additive differences in gene expression that are tightly associated with the three genotypes of the regulatory eQTL, and cause allelic expression imbalance in heterozygous samples. Unlike trans-variants, which can act on many genes located at a distance, cis-acting variants are less likely to have all-or-none effects if inactivated, and these may be tissue specific and thus less severe. Such cis-variants are found occasionally in Mendelian disease and now increasingly implicated in polygenic disorders. The interactions of these regulatory variations with other genetic and also environmental factors are likely to account for some of the complexity of multifactorial diseases. Key Concepts: Regulatory polymorphisms, otherwise known as eQTLs (expression quantitative trait loci), are DNA sequence variations that alter the level of expression of particular transcripts. Cis-eQTLs are cis-acting polymorphisms that cause variation in the expression of a nearby gene or genes on the same chromosomal homologue. In heterozygous individuals there is a difference in the level of expression from each of the pair of genes transcribed from the two chromosomal homologues. This difference in expression in heterozygous individuals can be observed as allelic expression imbalance (AEI) if there are also allelic sequence differences in the transcript. Such AEI is distinguishable from imprinting in that unlike the case of imprinting the higher expressing transcript can come from either the mother or the father. Cis-acting polymorphisms can affect expression in a tissue-specific and developmentally specific manner. They are strong candidates for positive selection and evolutionary adaptation but such variants might be maladaptive in different environments. Cis-acting regulatory sequence changes have been identified as a class of rare mutations in Mendelian disorders, and more common variants are increasingly implicated as causal polymorphisms involved in multifactorial diseases. Keywords: cis-acting polymorphism; allelic expression imbalance; AEI; eQTL; gene expression; regulation; allelic expression

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

Gene therapy for cystic fibrosis lung disease will likely require repeated delivery of gene transfer agents (GTA) to the terminally differentiated cells of the airway epithelium. We are currently testing several non-viral GTAs for efficiency of gene transfer following aerosol delivery to the lungs of mice and sheep. The most sensitive method available to quantify gene expression from plasmid vectors is quantitative (TaqMan) reverse transcriptase (RT)-PCR, which has been widely used to quantify gene expression in pre-clinical and clinical samples. TaqMan PCR can potentially detect a single copy of the target sequence per reaction. We designed TaqMan assays to quantify both vector-specific mRNA and endogenous mRNA of a normalising gene. Specifically, assays were developed for plasmids containing the CMV promoter, or the human polyubiquitin C (UbC) promoter, shown to have improved duration of expression in the lung. In addition, assays have been developed to detect endogenous murine and ovine Cftr mRNA. Assays were designed to be specific for mRNA by placing one of the primer sequences across an exon splice site. In order to determine the absolute copy number of mRNA in a given sample, RNA standard curves were produced. DNA templates for the assays were constructed and included the PCR amplicon plus surrounding sequences totalling 160bp. RNA standards were generated incorporating RNase resistant ribonucleotides into RNA transcripts, using the Ambion Competitor Construction Kit. The mimics were quantified using the Molecular Probes RiboGreen assay and dilutions were produced containing 1 to 510 copies/|[mu]|l. 1|[mu]|l of each dilution was used in paired RT reactions with sequence specific primers and MultiScribe RT. Quantitative PCR was then carried out using an Applied Biosystems TaqMan 7700 Sequence Detector and standard curves generated for the four assays. When paired (multiplexed), the CMV and sheep Cftr assays were linear between 100 and 106 starting copies per reaction (R2=0.984 and 0.981). The UbC and mouse Cftr assays were paired and the UbC assay was linear between 100 and 106 starting copies (R2=0.987) while the mouse Cftr assay was linear between 1500 and 106 starting copies per reaction (R2=0.890). Control samples lacking RT did not result in amplification, indicating that the standards were free of template DNA. These results show the high degree of sensitivity and linearity that can be obtained with TaqMan RT-PCR. These assays will now be used to quantify absolute levels of expression in our pre-clinical models to determine the optimal GTA formulation for future clinical studies.

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. Results We used surface interpolation of Old World lactase persistence genotype and phenotype frequency estimates obtained from all available literature and perform a comparison between predicted and observed trait frequencies in continuous space. By accommodating additional data on sample numbers and known false negative and false positive rates for the various lactase persistence phenotype tests (blood glucose and breath hydrogen), we also apply a Monte Carlo method to estimate the probability that known lactase persistence-associated allele frequencies can explain observed trait frequencies in different regions. Conclusion Lactase persistence genotype data is currently insufficient to explain lactase persistence phenotype frequency in much of western and southern Africa, southeastern Europe, the Middle East and parts of central and southern Asia. We suggest that further studies of genetic variation in these regions should reveal additional nucleotide variants that are associated with lactase persistence.