Human Lactase Research Articles

Development of an Isothermal Point-of-care Genetic Rapid Test for the Detection of the HLA-B*57:01 Allele, a Predictor for Hypersensitivity Reaction Caused by Abacavir, for Stratifying Patients for Antiretroviral Abacavir HIV Therapy

Background: Abacavir is used in the treatment of HIV-infected patients. A hypersensitive reaction (HSR) occurs in about 5-8% of patients treated with Abacavir. The HLA-B*57:01 allele is a valuable predictor for HSR and its screening is mandatory prior to treatment with Abacavir. Objective: Current screening methods require considerable investments for equipment. In order to lower the required investments and enable physician practices to perform the screening in a point-of-care (PoC) setting, our objective was to develop a novel isothermal genetic rapid test that requires a minimal setup cost, does not require specific training and thus is suitable for a physician practice setting. Methods: We developed an isothermal recombinase polymerase amplification (RPA) for the specific amplification of the HLA-B*57:01 allele using allele-specific primers coupled to Biotin. Primers specific for human lactase gene, coupled to Digoxigenin, were used as an internal amplification control (IAC). Lateral flow dipstick provided rapid and accurate detection of HLA-B*57:01 allele and IAC via the respective antibodies sprayed on the strips surface. Results: The reference method identified the HLA-B*57:01 allele in the reference sample, in 2 out of 28 buccal swab samples and in 2 out of 13 blood samples. The initial isothermal RPA resulted in unspecific amplification of the HLA-B*57:01 allele. By further optimization steps the specific amplification of the allele and the detection on lateral flow dipstick was observed. The newly developed isothermal RPA was validated. Conclusion: The method developed fulfils the requirements for a genetically based PoC screening system for the HLA-B*57:01 variant, requiring a minimal investment for a heating block and a pipette.

The Relationship Between the Human Genome and Microbiome Comes into View.

The body's microbiome, composed of microbial cells that number in the trillions, is involved in human health and disease in ways that are just starting to emerge. The microbiome is assembled at birth, develops with its host, and is greatly influenced by environmental factors such as diet and other exposures. Recently, a role for human genetic variation has emerged as also influential in accounting for interpersonal differences in microbiomes. Thus, human genes may influence health directly or by promoting a beneficial microbiome. Studies of the heritability of gut microbiotas reveal a subset of microbes whose abundances are partly genetically determined by the host. However, the use of genome-wide association studies (GWASs) to identify human genetic variants associated with microbiome phenotypes has proven challenging. Studies to date are small by GWAS standards, and cross-study comparisons are hampered by differences in analytical approaches. Nevertheless, associations between microbes or microbial genes and human genes have emerged that are consistent between human populations. Most notably, higher levels of beneficial gut bacteria called Bifidobacteria are associated with the human lactase nonpersister genotype, which typically confers lactose intolerance, in several different human populations. It is time for the microbiome to be incorporated into studies that quantify interactions among genotype, environment, and the microbiome in order to predict human disease susceptibility.

Frequency of adult type-associated lactase persistence LCT-13910C/T genotypes in the Czech/Slav and Czech Roma/Gypsy populations

Lactase non-persistence (leading to primary lactose intolerance) is a genetically dependent inability to digest lactose in adulthood. As part of the human adaptation to dairying, the human lactase LCT-13910C/T mutation (which propagates adult expression of lactase) developed, spread and participated in the adaptation to dairying. This variant is associated with lactase activity persistence, and its carriers are able to digest lactose. We compared the frequencies of lactase 13910C/T (rs4988235) genotypes in Czechs/Slavs (N = 288) and Czech Gypsies/Roma (N = 300), two ethnically different groups where this polymorphism has not yet been analysed. Allelic frequencies significantly differed between the populations (p < 0.0001). In Czechs/Slavs, the lactase persistence T allele was present in 76% of the individuals, which is in agreement with frequencies among geographically neighbouring populations. In the Czech Gypsy/Roma population, only 27% of the adults were carriers of at least one lactase persistence allele, similar to the Indian population. In agreement with this result, dairy product consumption was reported by 70.5% of Czechs/Slavs and 39.0% of the Czech Gypsy/Roma population. Both in the Czech Gypsy/Roma and in the Czech/Slavs populations, the presence of carriers of the lactase persistence allele was similar in subjects self-reporting the consumption of unfermented/fresh milk, in comparison to the others.

Adult type associated lactase persistence genotypes in Czech and Roma/Gypsy populations

Background Dairy products are important source of calcium. Lactase non-persistence/lactose intolerance is a physiologically dependent nonability to digest lactose in adult age. Due to the human adaptation on dairying, new mutation influencing the activity of human lactase LCT C-13,910T occurs and spread out. This mutation is associated with lactase activity …

Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene.

Lactose intolerance in northern Europeans is strongly associated with a single-nucleotide polymorphism (SNP) located 14 kb upstream of the human lactase gene: -13,910 C/T. We examined whether SNPs in the 5' flanking region of the pig lactase gene are similar to those in the human gene and whether these polymorphisms play a functional role in regulating pig lactase gene expression. The 5' flanking region of the lactase gene from several different breeds of pigs was cloned and analyzed for gene regulatory activity of a luciferase reporter gene. One SNP was found in the enhancer region (-797 G/A) and two were found in the promoter region (-308G/C and -301 A/G). The promoter C-308,G-301(Pro-CG) strongly promotes the expression of the lactase gene, but the promoter G-308,A-301(Pro-GA) does not. The enhancer A-797(Enh-A) genotype for Pro-GA can significantly enhance promoter activity, but has an inhibitory effect on Pro-CG. The Enhancer G-797(Enh-G) has a significant inhibitory effect on both promoters. In conclusion, the order of effectiveness on the pig lactase gene is Enh-A+Pro-GA>Enh-A/G+Pro-CG>Enh-G+Pro-GA.

Latitude, sunshine, and human lactase phenotype distributions may contribute to geographic patterns of modern disease: the inflammatory bowel disease model.

Countries with high lactase nonpersistence (LNP) or low lactase persistence (LP) populations have lower rates of some “western” diseases, mimicking the effects of sunshine and latitude. Inflammatory bowel disease (IBD), ie, Crohn’s disease and ulcerative colitis, is putatively also influenced by sunshine. Recent availability of worldwide IBD rates and lactase distributions allows more extensive comparisons. The aim of this study was to evaluate the extent to which modern day lactase distributions interact with latitude, sunshine exposure, and IBD rates. National IBD rates, national distributions of LP/LNP, and population-weighted average national annual ultraviolet B exposure were obtained, estimated, or calculated from the literature. Negative binomial analysis was used to assess the relationship between the three parameters and IBD rates. Analyses for 55 countries were grouped in three geographic domains, ie, global, Europe, and non-Europe. In Europe, both latitude and ultraviolet B exposure correlate well with LP/LNP and IBD. In non-Europe, latitude and ultraviolet B exposure correlate weakly with LP/LNP, but the latter retains a more robust correlation with IBD. In univariate analysis, latitude, ultraviolet B exposure, and LP/LNP all had significant relationships with IBD. Multivariate analysis showed that lactase distributions provided the best model of fit for IBD. The model of IBD reveals the evolutionary effects of the human lactase divide, and suggests that latitude, ultraviolet B exposure, and LP/LNP mimic each other because LP/LNP follows latitudinal directions toward the equator. However, on a large scale, lactase patterns also follow lateral polarity. The effects of LP/LNP in disease are likely to involve complex interactions.

The Iceman’s Genome, New-Age Foot Care

The complete genome sequence of Otzi the Iceman was reported on Feb. 28 in Nature Communications (DOI: 10.1038/ncomms1701). Recall that Otzi lived about 5,300 years ago and was found mummified with an arrowhead stuck in his back in a snowy mountain pass in Italy in 1991. According to the genomic analysis, Otzi had brown eyes, type O+ blood, a predisposition to cardiovascular disease, possibly Lyme disease, and was lactose intolerant. That the iceman was lactose intolerant gives the Newscripts gang food for thought. Lactose intolerance arises from the inability to produce enough of the enzyme lactase to metabolize lactose, the disaccharide sugar found in milk. Symptoms might not be noticeable, but at the extreme they involve unpleasant digestive distress after consuming milk or milk products. Most mammals lose the ability to digest milk after weaning. But the gene coding for adult human lactase production arose in Europe about 7,000 years ...

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.

RFLP analysis and allelic discrimination with real-time PCR using the human lactase persistence trait

We describe here two open-ended laboratory investigations for an undergraduate laboratory course that uses students' DNA as templates for quantitative real-time PCR and for traditional PCR followed by RFLP analysis. Students are captivated by the immediacy of the application and the relevance of the genotypes and traits, lactase persistence or nonpersistence, under study. Concepts ranging from nucleotide polymorphisms to natural selection can be demonstrated and reinforced by these laboratories. Students have a high degree of success with the laboratories and both objective and self-assessments of student learning indicate that concepts and techniques are better understood following implementation of these laboratory investigations.

Human Adult-Onset Lactase Decline: An Update

Human adult-onset lactase decline is a biologic feature characteristic of the maturing intestine in the majority of the world's population. The digestion and absorption of lactose, the major carbohydrate in milk and also the main substrate for lactase, is often variable, a consequence of lactase levels, gastric emptying rate, and colonic salvage. Although commercially available "lactase" products alleviate symptoms in many lactose-intolerant people, a greater understanding of this variability in lactose tolerance could lead to interventions that reduce the rate of gastric emptying and/or increase the proliferation of lactose-metabolizing bacteria in the colon, leading to more efficient lactose utilization. Adult-onset lactase decline appears to be a risk factor for developing osteoporosis, owing to avoidance of dairy products or interference of undigested lactose with calcium absorption. Elderly with both adult-onset lactase decline and atrophic gastritis or those undergoing anti-ulcer treatment may have an increased risk of low calcium absorption owing to the lack of gastric acid that facilitates calcium uptake. Thus, lactose-intolerant elders should monitor their calcium nutrition status carefully.

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.

Transcriptional regulation of the lactase-phlorizin hydrolase promoter by PDX-1.

Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox-1 (PDX-1) protein is expressed maximally in the proximal duodenum. This study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were cotransfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100-bp lactase gene promoter. EMSAs demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence TAAT is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independently of DNA binding to its consensus binding site. Stable overexpression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.

Erratum: Gene-culture coevolution between cattle milk protein genes and human lactase genes

Erratum: Gene-culture coevolution between cattle milk protein genes and human lactase genes

Gene-culture coevolution between cattle milk protein genes and human lactase genes

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.

Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element.

Lactase persistence is a heritable, autosomal dominant, condition that results in a sustained ability to digest the milk sugar lactose throughout adulthood. The majority of the world's human population experiences a decline in production of the digestive enzyme lactase-phlorizin hydrolase during maturation. However, individuals with lactase persistence continue to express high levels of the lactase gene into adulthood. Lactase persistence has been strongly correlated with single nucleotide genetic variants, C/T_(13910) and G/A_(22018), located 13.9 and 22 kb upstream from the lactase structural gene. We aimed to characterize a functional role for the polymorphisms in regulating lactase gene transcription. DNA in the region of the C/T_(13910) or G/A_(22018) human lactase variants was cloned upstream of the 3.0 kb rat lactase gene promoter in a luciferase reporter construct. Human intestinal Caco-2 cells were transfected with the lactase variant/promoter-reporter constructs and assayed for promoter activity. A 200 bp region surrounding the C_(13910) variant, associated with lactase non-persistence, results in a 2.2-fold increase in lactase promoter activity. The T_(13910) variant, associated with lactase persistence, results in an even greater 2.8-fold increase. The DNA sequence of the C/T_(13910) variants differentially interacts with intestinal cell nuclear proteins on EMSAs. AP2 co-transfection results in a similar repression of the C/T_(13910) variant/promoter-reporter constructs. The DNA region of the C/T_(13910) lactase persistence/non-persistence variant functions in vitro as a cis element capable of enhancing differential transcriptional activation of the lactase promoter. Such differential regulation by the C and T variants is consistent with a causative role in the mechanism specifying the lactase persistence/non-persistence phenotypes in humans.

Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.

In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear.

Caco-2 cell disaccharidase activities are unaffected by gestational hormones.

We previously reported that lactose handling was significantly improved during late-phase pregnancy in women with a genetically determined adult-type hypolactasia. However, the adaptive mechanisms underlying the enhanced lactose digestion during pregnancy are not clear. Progesterone therapy has been associated in animals with increased intestinal lactase activity. To investigate the potential role of progesterone and estrogen as modulators of human lactase activity during pregnancy, we employed the human-derived intestinal epithelial Caco-2 cell line. Measurements of lactase and sucrase activities were performed in parallel with DNA content in progesterone- and estrogen-treated cells after 5, 10, and 30 days of confluency. Caco-2 monolayer DNA content was observed to increase with duration of culture to an equivalent extent in both hormone-treated and control cultures. Lactase and sucrase activities were similarly unaltered by incubation with either progesterone or estrogen, at any time point tested. These data demonstrate that gestational hormones do not influence intestinal cell number nor disaccharidase activity in Caco-2 cells, at the doses tested. Although these studies were carried out in a malignant cell line, our data suggest that the improved lactose handling observed during pregnancy is probably related to prolonged intestinal transit.

Human Lactase–Phlorizin Hydrolase: Evidence of Dimerization in the Endoplasmic Reticulum

Human lactase–phlorizin hydrolase [EC 3.2.1.23–3.2.1.62] is a disaccharidase located in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a precursor protein in the endoplasmic reticulum and in addition to being glycosylated is subsequently proteolytically processed to the mature microvillus membrane-bound form after passing thetrans-Golgi compartment. We studied the oligomerization of human lactase–phlorizin hydrolase in transfected polarized Madin Darby canine kidney cells using metabolic labeling and sucrose-density centrifugation analysis. We detected high mannose dimers of the lactase–phlorizin hydrolase precursor molecule after metabolic labeling with [35S]methionine at 37 and 15°C. In addition, both complex-glycosylated lactase–phlorizin hydrolase precursor molecule and the mature microvillus membrane-bound enzyme showed this oligomeric structure. Chemical crosslinking resulted in the detection of covalently crosslinked lactase–phlorizin hydrolase dimers after sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results provide evidence that oligomerization of lactase–phlorizin hydrolase is an early event and begins in the endoplasmic reticulum.

Trans-acting nuclear proteins in human lactase and sucrase-isomaltase regulation

Trans-acting nuclear proteins in human lactase and sucrase-isomaltase regulation

The polymorphic expression of lactase in adults is regulated at the messenger RNA level

Background/Aims: Lactase phlorizin hydrolase (LPH) activity is high in infants but declines 80%–90% before adulthood in most mammals, including humans. However, 95% of whites show autosomal dominant inheritance of a lifelong high lactose digesting capacity (LDC). This study attempted to clarify the molecular mechanism(s) of this phenomenon (posttranslational vs. pretranslational). Methods: A race- and sex-balanced cohort (n = 20) was studied, and lactose tolerance and levels of jejunal lactase protein, activity, and messenger RNA (mRNA) were measured. Results: These data confirm that black heritage predicts low LDC, and white heritage predicts high LDC. Lactase breath hydrogen and determination of lactase/sucrase ratio (L/S) from jejunal biopsy specimens divide the group by high and low LDC phenotypes concordantly. All subjects with an L/S ratio >0.5 had immunodetectable LPH protein and measurably higher LPH mRNA levels than the remaining subjects. LPH mRNA levels are highly correlated with lactase specific activity (r = 0.80) and L/S ratio (r = 0.88). Conclusions: The direct correlation between LPH mRNA levels and lactase expression argues that the gene responsible for the human lactase polymorphism regulates the level of LPH mRNA.