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At least 19 recordsLinked to original sources
PubMed2003

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.

Base Sequence↗
PubMed1990

Age dependency of the lactase persistence and lactase restriction phenotypes among children in Sri Lanka and Britain.

All neonates are born with intestinal lactase activity. In most of them the intestinal lactase activity is lost during childhood (lactase restriction phenotype). In a minority of children normal intestinal lactase activity is retained (lactase persistence phenotype). In this study the progression of the lactase restriction phenotypes has been studied in 94 Sri Lankan children by oral lactose loads and 162 British children by intestinal lactase estimation (adult Sri Lankans and British predominantly belong to the lactase restriction and lactase persistence phenotypes, respectively). Lactase was present in infancy at birth in all Sri Lankan children and declined around the age of eight years, the majority (59 per cent) of the 10-15-year-olds belonging to the lactase restriction phenotype. In contrast the majority of the British children (95 per cent of all the British children studied) demonstrated the lactase persistence phenotype. The low prevalence rate of the restriction phenotype found among British children was largely contributed by children of African and Asian origin.

Age Factors↗
PubMed2004

PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene.

The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.

Adult↗
PubMed2005

T-13910 DNA variant associated with lactase persistence interacts with Oct-1 and stimulates lactase promoter activity in vitro.

Two phenotypes exist in the human population with regard to expression of lactase in adults. Lactase non-persistence (adult-type hypolactasia and lactose intolerance) is characterized by a decline in the expression of lactase-phlorizin hydrolase (LPH) after weaning. In contrast, lactase-persistent individuals have a high LPH throughout their lifespan. Lactase persistence and non-persistence are associated with a T/C polymorphism at position -13,910 upstream the lactase gene. A nuclear factor binds more strongly to the T-13,910 variant associated with lactase persistence than the C-13,910 variant associated with lactase non-persistence. Oct-1 and glyceraldehyde-3-phosphate dehydrogenase were co-purified by DNA affinity purification using the sequence of the T-13,910 variant. Supershift analyses show that Oct-1 binds directly to the T-13,910 variant, and we suggest that GAPDH is co-purified due to interactions with Oct-1. Expression of Oct-1 stimulates reporter gene expression from the T and the C-13,910 variant/LPH promoter constructs only when it is co-expressed with HNF1alpha. Binding sites for other intestinal transcription factors (GATA-6, HNF4alpha, Fox and Cdx-2) were identified in the region of the -13,910 T/C polymorphism. Three of these sites are required for the enhancer activity of the -13,910 region. The data suggest that the binding of Oct-1 to the T-13,910 variant directs increased lactase promoter activity and this might provide an explanation for the lactase persistence phenotype in the human population.

Binding Sites↗
PubMed1989

New insights into lactase and glycosylceramidase activities of rat lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase, a small intestinal disaccharidase, has been considered mainly an enzyme important only for the hydrolysis of lactose. After weaning in most mammals lactase-specific activity falls markedly, and, functionally, adult mammals are considered to be lactase deficient. However, the persistence of low levels of lactase activity in adulthood has never been explained. In addition, it has been suggested that lactase-phlorizin hydrolase is associated with glycosylceramidase activity when the enzyme is prepared by column chromatography, but it is unclear whether this represents copurified activities or two catalytic sites on one peptide. The developmental patterns of lactase-phlorizin hydrolase and other disaccharidases were investigated in homogenates of total rat small intestine; lactase and several glycosylceramidases were measured in immunoprecipitates from these homogenates using a monoclonal antibody. The developmental pattern of total lactase activity showed a steady 2.3-fold increase to adult levels (specific activity decreased eightfold), whereas total phlorizin-hydrolase activity increased 10.7-fold (specific activity decreased threefold). As expected, levels of both total and specific sucrase and maltase activities increased during development. In lactating rats total lactase activity showed a significant increase compared with adult males. The developmental pattern of the enzyme activities for the glycolipid substrates was similar to that found for lactase, and the immunoprecipitated enzyme showed a 40- to 55-fold higher affinity for the glycolipids than for lactose. Galactosyl- and lactosylceramide inhibited lactose hydrolysis by 38%, without a competitive pattern, suggesting two different active sites for lactose and glycolipid hydrolysis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2002

Effect of lactase preparations in asymptomatic individuals with lactase deficiency--gastric digestion of lactose and breath hydrogen analysis.

We compared two lactase preparations derived from Aspergillus orizae (AOL) and Penicillinase multicolor (PML) for stability in the stomach and overall enzymatic activity in 10 asymptomatic subjects with lactase deficiency. The subjects were given 10,000 FCC units of either AOL or PML 30 min prior to or simultaneously with 300 ml of milk. Gastric juice was withdrawn through a nasogastric tube immediately after and every 15 min for 60 min, and breath was sampled before and every 15 min for 6 h after the milk ingestion. When lactase was given simultaneously with the milk, gastric juice lactase activity and galactose concentration were significantly higher than the control levels. When lactase preparations were given 30 min prior to the milk, neither lactase activity nor galactose was detected in the gastric juice. The pH of the gastric juice was about 6.0 after the milk ingestion. Breath hydrogen did not increase when milk was ingested simultaneously with enzymes, but did increase if enzymes were given 30 min prior to milk ingestion. There were no significant differences in lactase activity, galactose concentration in gastric juice, or breath hydrogen when AOL and PML were compared. In conclusion, with exogenous lactase, digestion of lactose begins in the stomach when pH is raised to 6.0 by the buffering action of milk. Lactase preparations are effective assessed by breath hydrogen analysis in asymptomatic individuals with lactase deficiency if the enzymes are given simultaneously with milk.

Adult↗
PubMed1998

Lactase haplotype frequencies in Caucasians: association with the lactase persistence/non-persistence polymorphism.

A genetic polymorphism is responsible for determining that some humans express lactase at high levels throughout their lives and are thus lactose tolerant, while others lose lactase expression during childhood and are lactose intolerant. We have previously shown that this polymorphism is controlled by an element or elements which act in cis to the lactase gene. We have also reported that 7 polymorphisms in the lactase gene are highly associated and lead to only 3 common haplotypes (A, B and C) in individuals of European extraction. Here we report the frequencies of these polymorphisms in Caucasians from north and south Europe and also from the Indian sub-continent, and show that the alleles differ in frequency, the B and C haplotypes being much more common in southern Europe and India. Allelic association studies with lactase persistence and non-persistence phenotypes show suggestive evidence of association of lactase persistence with certain alleles. This association was rather more clear in the analysis of small families, where haplotypes could be determined. Furthermore haplotype and RNA transcript analysis of 11 unrelated lactase persistent individuals shows that the persistence (highly expressed) allele is almost always on the A haplotype background. Non-persistence is found on a variety of haplotypes including A. Thus it appears that lactase persistence arose more recently than the DNA marker polymorphisms used here to define the main Caucasian haplotypes, possibly as a single mutation on the A haplotype background. The high frequency of the A haplotype in northern Europeans is consistent with the high frequency of lactase persistence.

Alleles↗
PubMed1993

Immunocytochemical heterogeneity of lactase-phlorizin hydrolase in adult lactase deficiency.

BACKGROUND: Adult lactase deficiency may result either from diminished synthesis of precursor lactase-phlorizin hydrolase (phenotype I deficiency) or from altered posttranslational processing of the precursor protein (phenotype II). The aim of this study was to compare the location of lactase-phlorizin hydrolase along the crypt-villus axis in control with adult lactase-deficient subjects. METHODS: The immunocytochemical distribution of lactase-phlorizin hydrolase was studied in subjects with adult hypolactasia and in controls with persistent high lactase expression. RESULTS: Duodenal biopsy samples from controls showed increasing intensity of brush border staining from upper crypt to midvillus. Subjects with phenotype I deficiency showed reduced and more patchy reaction product over brush borders. In some, maximal staining was localized over the upper half of the villi. The subject with phenotype II deficiency showed patchy staining but also intracellular accumulations of immunoreactivity within the apex of enterocytes mainly on the upper half of villi. Subsequent immunoelectronmicroscopy showed nearly a fourfold increase in label over the endoplasmic reticulum. CONCLUSIONS: These findings support prior observations of diminished enzyme synthesis in phenotype I lactase deficiency and suggest that the alterations in posttranslational processing in phenotype II deficiency involve a partial block in transport from endoplasmic reticulum to Golgi apparatus.

Biopsy↗
PubMed1993

In vitro biosynthesis of lactase in suckling and adult rabbits. Regulatory mechanisms involved in the decline of the lactase activity.

Steady state forms, levels and the in vitro biosynthesis of lactase-phlorizin hydrolase (LPH) proteins have been studied in proximal and middle intestine of suckling and adult rabbits. In most adult tissues the lactase activity and the LPH protein content were low and the synthesis rate of the 200 kDa lactase precursor was reduced in comparison to suckling tissues. In a few tissues with low enzymatic activity the LPH protein content was relatively high, and high lactase synthesis occurred. In addition, the ratio (labeled lactase)/(lactase protein) was lower in the middle jejunum of the adult rabbit than in the proximal region. Both decreased synthesis of LPH precursor and increased turnover or inactivation of the enzyme may cause the decline of the lactase activity.

Animals↗
PubMed2004

The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans.

The ability to digest the milk sugar lactose as an adult (lactase persistence) is a variable genetic trait in human populations. The lactase-persistence phenotype is found at low frequencies in the majority of populations in sub-Saharan Africa that have been tested, but, in some populations, particularly pastoral groups, it is significantly more frequent. Recently, a CT polymorphism located 13.9 kb upstream of exon 1 of the lactase gene (LCT) was shown in a Finnish population to be closely associated with the lactase-persistence phenotype (Enattah et al. 2002). We typed this polymorphism in 1,671 individuals from 20 distinct cultural groups in seven African countries. It was possible to match seven of the groups tested with groups from the literature for whom phenotypic information is available. In five of these groups, the published frequencies of lactase persistence are >/=25%. We found the T allele to be so rare that it cannot explain the frequency of the lactase-persistence phenotype throughout Africa. By use of a statistical procedure to take phenotyping and sampling errors into account, the T-allele frequency was shown to be significantly different from that predicted in five of the African groups. Only the Fulbe and Hausa from Cameroon possessed the T allele at a level consistent with phenotypic observations (as well as an Irish sample used for comparison). We conclude that the C-13.9kbT polymorphism is not a predictor of lactase persistence in sub-Saharan Africans. We also present Y-chromosome data that are consistent with previously reported evidence for a back-migration event into Cameroon, and we comment on the implications for the introgression of the -13.9kb*T allele.

Alleles↗
PubMed1985

Human lactase and the molecular basis of lactase persistence.

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in Mr (160K). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl-beta-galactoside and beta-glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with lactase under all conditions investigated; coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.

Antibodies, Monoclonal↗
PubMed1986

Calcium absorption from milk in lactase-deficient and lactase-sufficient adults.

To determine whether lactose influences the absorption of calcium, the uptake of calcium from lactose-hydrolyzed milk and from unhydrolyzed milk was measured in 20 adults: 10 were lactase-deficient and 10 were lactase-sufficient as defined by breath hydrogen test, plasma glucose determination after oral lactose dose, and presence or absence of symptoms after lactose ingestion. On different days, each subject received either lactose-hydrolyzed or unhydrolyzed milk. Calcium absorption was measured by a double-isotope technique. In the lactase-deficient group, the mean absorptions were 33.5% from hydrolyzed milk and 36.2% from the same volume of unhydrolyzed milk (P greater than 0.30). In the lactase-sufficient group, mean absorptions were 24.2% from hydrolyzed milk and 25.7% from unhydrolyzed milk. The mean calcium absorption from both lactose-hydrolyzed milk and unhydrolyzed milk was significantly greater (P less than 0.01) in the lactase-deficient group compared to the lactase-sufficient group, presumably reflecting lower dietary calcium intake in the former. These data indicate that, in lactase-deficient subjects, malabsorption of lactose does not affect calcium absorption.

Adult↗
PubMed1993

Surface staining on the villus of lactase protein and lactase activity in adult-type hypolactasia.

BACKGROUND: We have shown in previous studies the presence of a patchy pattern of lactase protein expression in the proximal jejunum of hypolactasic humans, adult rabbits, and rats. The present study investigated the mechanisms underlying the heterogeneous expression of lactase on the villus. METHODS: Proximal jejunal tissue from 18 adult humans and 12 adult rabbits was examined using enzymocytochemical and surface-staining techniques for lactase protein and activity. RESULTS: In the proximal jejunum of hypolactasic humans and adult rabbits, lactase activity is patchily distributed on the villus enterocytes. In humans, the patches of lactase-positive enterocytes are randomly distributed on the villus, whereas in rabbits, vertical, continuous sheets of positive enterocytes arise from the base of the villus. CONCLUSIONS: The presence of enterocytes without lactase activity is one of the mechanisms causing adult-type hypolactasia in the proximal jejunum of humans and mammals. The patchy pattern of lactase in rabbits suggests a clonal origin with heterogeneity of the cells arising from the crypts. In hypolactasic humans, the enterocyte heterogeneity occurs as a consequence of mechanisms that do not have a clonal origin.

Age Factors↗
PubMed1996

Heterogeneity of intestinal lactase activity in children: relationship to lactase-phlorizin hydrolase messenger RNA abundance.

Despite extensive study in both humans and nonhuman mammals the mechanisms which regulate intestinal lactase activity, particularly during development, are incompletely understood. Our previous studies of human adults are consistent with an important role of lactase-phlorizin hydrolase (LPH) mRNA abundance in determining the lactase persistence/nonpersistence phenotypes. Our intent in the present study was to determine the role of LPH mRNA in the regulation of lactase in children. We therefore studied duodenal mucosal biopsies from 39 children undergoing diagnostic upper endoscopy in whom significant small intestinal and nutritional disease was excluded. We found no relationship between the level of LPH mRNA and lactase enzymatic activity. Our observations suggest the importance of posttranscriptional mechanisms in lactase regulation in human children.

Adolescent↗
PubMed2005

[Analysis of lactase activities of small intestine mucous membrane by double labeled stable isotope technique in subjects with lactase deficiency].

OBJECTIVE: Low lactase activity in small intestine mucosa is the main reason for the occurrence of lactose malabsorption (LM) and lactose intolerance (LI). It would be the basis for the research on LM and LI to find an accurate method to analyze the activity of lactase. METHODS: In this study, 43 volunteers were selected and divided into LM and LI group according to the results of H2 breath test and symptoms record. Twenty-five grams of 13C-lactose and 0.5 g 2H-glucose in 250ml solution were consumed by all the volunteers. The concentration of total plasma glucose, 13C- glucose and 2H-glucose were measured, the ratio of [13C-glucose]/[2H-glucose] and lactose digestion index (LDI)were calculated which could reflect the lactase activities in the mucous membrane of small intestine. RESULTS: It was found that there was no significant difference in the concentration of total glucose and 13C-glucose, while the LDI in LM group (0.47 +/- 0.15) was significantly higher than LI group (0.34 +/- 0.14). There was no significant relationship between LDI and 6h cumulative breath H2 amount (r = 0.12, P = 0.46). The 13 C-lactose/2 H-glucose challenged test showed there was still residual lactase activity in small intestine. CONCLUSION: It was concluded that 13C-lactose/2H-glucosetest can accurately and sensitively determine the lactase activity on small intestinal mucous membrane and digestible lactose amount.

Adult↗
PubMed1989

Yogurt and fermented-then-pasteurized milk: effects of short-term and long-term ingestion on lactose absorption and mucosal lactase activity in lactase-deficient subjects.

Lactase-deficient subjects absorb lactose in yogurt more effectively than lactose in other dairy products. However, as all previous studies were performed without a double-blind design and only after a single ingestion of the test product, the mechanism of this enhanced absorption remains unclear. The aims of this double-blind study were 1) to evaluate lactose absorption after prolonged ingestion of yogurt and fermented-then-pasteurized milk (FPM) and 2) to assess the modification of the lactase activity of the duodenal mucosa. In 16 lactase-deficient subjects we confirmed that yogurt enhances lactose digestion, this beneficial effect being destroyed by pasteurization. Moreover, the long-term (8 d) ingestion of either yogurt or FPM does not modify the results of hydrogen breath tests in comparison with a 24-h ingestion. The mucosal lactase (Dahlquist method) and beta-galactosidase (ONPG method) activities were not significantly modified by yogurt or FPM ingestion. These results suggest that in lactase-deficient subjects no adaptation occurs after eating yogurt or FPM and that the increased lactose absorption in yogurt must be mainly related to an intraluminal process.

Adolescent↗
PubMed1995

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

Amino Acid Sequence↗
PubMed1998

Assignment of the locus for congenital lactase deficiency to 2q21, in the vicinity of but separate from the lactase-phlorizin hydrolase gene.

Congenital lactase deficiency (CLD) is an autosomal recessive, gastrointestinal disorder characterized by watery diarrhea starting during the first 1-10 d of life, in infants fed lactose-containing milks. Since 1966, 42 patients have been diagnosed in Finland. CLD is the most severe form of lactase deficiency, with an almost total lack of lactase-phlorizin hydrolase (LPH) activity on jejunal biopsy. In adult-type hypolactasia, the most common genetic enzyme deficiency in humans, this enzyme activity is reduced to 5%-10%. Although the activity of intestinal LPH has been found to be greatly reduced in both forms, the molecular pathogenesis of lactase deficiencies is unknown. On the basis of the initial candidate-gene approach, we assigned the CLD locus to an 8-cM interval on chromosome 2q21 in 19 Finnish families. At the closest marker locus, a specific allele 2 was present in 92% of disease alleles. On the basis of a genealogical study, the CLD mutation was found to be enriched in sparsely populated eastern and northern Finland, because of a founder effect. The results of both the genealogical study and the haplotype analysis indicate that one major mutation in a novel gene causes CLD in the Finnish population. Consequently, the critical region could be restricted further, to an approximately 350-kb interval, by ancient-haplotype and linkage-disequilibrium analyses. Surprisingly, the LPH gene was shown to lie outside the critical CLD region, excluding it as a causative gene for CLD. The LPH locus was found to reside >2 Mb from the critical CLD region.

Chromosome Mapping↗