Lactase-phlorizin Hydrolase Gene Research Articles

A Genetic Lab-on-Phone Test for Point-of-Care Diagnostic of Lactose Intolerance near Patient and in less than 90 Minutes.

The -13910 C/T single nucleotide polymorphism located within the MCM6 gene, an enhancer region located upstream of the lactase-phlorizin hydrolase gene, is associated with lactase persistence/non-persistence traits among the Caucasian population. The performance of a new point-of-care CE-IVD (In Vitro Diagnostic) marked isothermal lab-on-phone lactose intolerance assay, using crude samples, was assessed in comparison with Sanger sequencing using purified DNA, as reference method. The study was conducted following a non-probability sampling using direct buccal swab (n = 63) and capillary blood (n = 43) clinical samples from a total of 63 volunteers. A 3 × 3 confusion matrix/contingency table was used to evaluate the performance of the isothermal lab-on-phone lactose intolerance assay. The isothermal lab-on-phone lactose intolerance assay successfully detected the -13910 C/T variant with a limit of detection of 5 cells/assay and demonstrated an overall accuracy of 98.41% (95% CI, 91.47%-99.96%) for buccal swab samples and 100% (95% CI, 91.19%-100%) for capillary blood, taking just 90 min from sample to result, with only 2 min hands-on. The lab-on-phone pocket-sized assay displayed good performance when using direct buccal swab and capillary blood samples, enabling a low-cost, real-time, and accurate genotyping of the -13910 C/T region for the rapid diagnosis of primary lactose intolerance at point-of-care, which enables a prompt implementation of appropriate diet habits and/or intolerance therapies. To our knowledge, this is the first point-of-care genetic test for lactose intolerance to be made available on the market.

Development of RFLP and qPCR Assays to Detect the Human C/T‐13910 SNP, which Is Associated with Lactase Persistence

Human infants, like those of other mammals, express the lactase enzyme to digest the lactose sugar in mother’s milk. After weaning, the lactase‐phlorizin hydrolase gene (LCT) ceases to produce lactase, resulting in the lactase non‐persistence (LNP) phenotype. However, in 35% of the world’s population of humans, expression of the lactase enzyme continues into adulthood, known as lactase persistence (LP). The LP phenotype occurs when an individual has one of more than 20 single nucleotide polymorphisms (SNPs) located near the lactase gene. The most commonly studied SNP is the “European” SNP, C/T‐13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct‐1 regulatory transcription factor and/or prevention of gene silencing. Several studies have been conducted in non‐European, isolated populations to determine the frequency of LP within the ethnic group. However, fewer studies have been conducted in ethnically diverse locations such as the US. To lay the groundwork for such a study, we have successfully implemented a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011, doi:10.1136/bmjopen‐2011‐000125) to detect the C/T‐13910 SNP in human cheek cell lysate, confirming the results by direct sequencing of the DNA. We used the TOPO cloning technique to generate control plasmids containing inserts with either the “C” or “T” genotype to use as positive controls in future assays. We confirmed that the sequence of the control plasmid inserts match the original target sequence. We are using the control plasmids to optimize a SYBR green quantitative polymerase chain reaction (qPCR) genotyping assay based on Weinlander et al. (2010, doi:10.1002/bmb.20357) as an alternate method of genotyping. Finally, we have curated a list of SNPs from the literature and public databases that are known to affect the LP phenotype in different world populations.Support or Funding InformationLoyola Marymount University Summer Undergraduate Research Program (S.M.C., A.N.W.), Loyola Marymount University Honors Summer Research Fellowship (S.M.C.)

Congenital Lactase Deficiency: Mutations, Functional and Biochemical Implications, and Future Perspectives.

Congenital lactase deficiency (CLD) is a severe autosomal recessive genetic disorder that affects the functional capacity of the intestinal protein lactase-phlorizin hydrolase (LPH). This disorder is diagnosed already during the first few days of the newborn’s life due to the inability to digest lactose, the main carbohydrate in mammalian milk. The symptoms are similar to those in other carbohydrate malabsorption disorders, such as congenital sucrase-isomaltase deficiency, and include severe osmotic watery diarrhea. CLD is associated with mutations in the translated region of the LPH gene that elicit loss-of-function of LPH. The mutations occur in a homozygote or compound heterozygote pattern of inheritance and comprise missense mutations as well as mutations that lead to complete or partial truncations of crucial domains in LPH, such as those linked to the folding and transport-competence of LPH and to the catalytic domains. Nevertheless, the identification of the mutations in CLD is not paralleled by detailed genotype/protein phenotype analyses that would help unravel potential pathomechanisms underlying this severe disease. Here, we review the current knowledge of CLD mutations and discuss their potential impact on the structural and biosynthetic features of LPH. We also address the question of whether heterozygote carriers can be symptomatic for CLD and whether genetic testing is needed in view of the severity of the disease.

Compound heterozygous mutations elicit congenital lactase deficiency in a Japanese infant

Congenital lactase deficiency (CLD) is a rare severe autosomal recessive disorder, with symptoms like watery diarrhea, meteorism and malnutrition, which start a few days after birth by the onset of nursing. The most common rationales identified for this disorder are missense mutations or premature stop codons in the coding region of the lactase‐phlorizin hydrolase (LPH) gene. Recently, two heterozygous mutations, c.4419C>G (p.Y1473X) in exon 10 and c.5387delA (p.D1796fs) in exon 16, have been identified within the coding region of LPH in a Japanese infant with CLD.Here, we investigate the influence of these mutations on the structure, biosynthesis and function of LPH. We show that both mutant proteins are mannose‐rich glycosylated proteins that are not capable of exiting the endoplasmic reticulum. These mutant proteins are misfolded and turnover studies show that they are ultimately degraded. The enzymatic activities of these mutant forms are not detectable, despite the presence of lactase and phlorizin active sites in the polypeptide backbone of LPH‐D1796fs and LPH‐Y1473X respectively. Interestingly, wild type LPH retains its complete enzymatic activity and intracellular transport competence in the presence of the pathogenic mutants suggesting that heterozygote carriers presumably do not show symptoms related to CLD.Our study strongly suggests that the onset of severe forms of CLD is elicited by mutations in the LPH gene that occur in either a compound heterozygous or homozygous pattern of inheritance.

Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene.

BackgroundCongenital lactase deficiency (CLD) is a rare severe autosomal recessive disorder, with symptoms like watery diarrhea, meteorism and malnutrition, which start a few days after birth by the onset of nursing. The most common rationales identified for this disorder are missense mutations or premature stop codons in the coding region of the lactase-phlorizin hydrolase (LPH) gene. Recently, two heterozygous mutations, c.4419C > G (p.Y1473X) in exon 10 and c.5387delA (p.D1796fs) in exon 16, have been identified within the coding region of LPH in a Japanese infant with CLD.MethodsHere, we investigate the influence of these mutations on the structure, biosynthesis and function of LPH. Therefore the mutant genes were transiently expressed in COS-1 cells.ResultsWe show that both mutant proteins are mannose-rich glycosylated proteins that are not capable of exiting the endoplasmic reticulum. These mutant proteins are misfolded and turnover studies show that they are ultimately degraded. The enzymatic activities of these mutant forms are not detectable, despite the presence of lactase and phlorizin active sites in the polypeptide backbone of LPH-D1796fs and LPH-Y1473X respectively. Interestingly, wild type LPH retains its complete enzymatic activity and intracellular transport competence in the presence of the pathogenic mutants suggesting that heterozygote carriers presumably do not show symptoms related to CLD.ConclusionsOur study strongly suggests that the onset of severe forms of CLD is elicited by mutations in the LPH gene that occur in either a compound heterozygous or homozygous pattern of inheritance.

Differential expression of Homeobox C11 protein in water buffalo Bubalus bubalis and its putative 3D structure.

BackgroundThe Homeobox (Hox) family complex contains 39 genes, clustered into four groups (A-D) all expressing in sequential manner. The HOX proteins are transcriptional factors involved in regulation of pattern formation of the anterio-posterior body axis across the species. Most of the Hox family genes have been studied with respect to their organization and expression during the embryonic stages. However, expression pattern of Homeobox C11 (Hoxc11) gene in the 5′ region, particularly in higher mammals remains largely unexplored.ResultsWe cloned and expressed Homeobox C11 (Hoxc11) gene from water buffalo Bubalus bubalis. The recombinant HOXC11 protein expressed as inclusion bodies was solubilized in Tris buffer (10 mM, pH-6.5) and purified using Ni-NTA affinity column. The purity and molecular weight of HOXC11 protein (~33 kDa) were confirmed by SDS-PAGE and western blot analysis. Employing immunohistochemistry approach, we localized HOXC11 protein in the nuclei across the tissues of buffalo. Western blot analysis showed highest expression of HOXC11 protein in kidney and lung although its possible renal and respiratory roles are not yet established. Electrophoretic mobility shift assay (EMSA) demonstrated the specific binding of HOXC11 protein with the promoter element, CE-LPH1 of lactase-phlorizin hydrolase (LPH) gene showing reduced mobility of the protein-DNA complex, corroborating with earlier report on the possible role of this protein in intestinal functions. In silico analysis of HOXC11 showed predominance of α helices and presence of six conserved domains. We deduced the putative 3D structure of HOXC11 protein and fifteen possible DNA interacting residues within the homeodomain.ConclusionsPresent study augments our understanding on the specific expression of HOXC11 protein in kidney and lung in water buffalo. The fifteen DNA interacting residues reported herein provide an opportunity to establish much broader structural and functional perspectives of HOXC11 protein in the context of genome analysis in general and animal biotechnology in particular.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-638) contains supplementary material, which is available to authorized users.

Thyroid and glucocorticoid hormones induce expression of lactase-phlorizin hydrolase gene in CDX-2/HNF-1α co-transfected IEC-6 cells.

Thyroid and glucocorticoid hormones and several transcriptional factors such as caudal type homeobox (CDX)-2 and hepatocyte nuclear factor (HNF)-1α are important for the differentiation of small intestinal absorptive cells and the consequent expression of genes related to the digestion/absorption of carbohydrates. In this study, we investigated whether thyroid and glucocorticoid hormones enhanced the expression of lactase-phlorizin hydrolase (LPH) gene, an intestine-specific gene that encodes an enzyme for lactose digestion, in small intestinal stem-like IEC-6 cells co-transfected with CDX-2 and HNF-1α using a retrovirus system. Changes in expression of intestine-specific genes caused by treatment with thyroid and/or glucocorticoid hormones were monitored in empty vector-transfected cells and in CDX-2/HNF-1α co-transfected cells by qRT-PCR. Stable co-transfection with CDX-2 and HNF-1α evoked the expression of the LPH gene in IEC-6 cells. Furthermore, treatment with a thyroid hormone, triiodothyronine, and a glucocorticoid receptor agonist, dexamethasone, significantly enhanced expression of the LPH, CDX-2 and HNF-1α genes in CDX-2/HNF-1α co-transfected IEC-6 cells. These results suggest that thyroid and glucocorticoid hormones synergistically enhance expression of the LPH gene in CDX-2/HNF-1α co-transfected IEC-6 cells.

The T-13910C polymorphism in the lactase phlorizin hydrolase gene is associated with differences in serum calcium levels and calcium intake

The C-variant of a T-13910C polymorphism (rs4988235; NT_022135.15:g.25316568G > A) upstream of the lactase phlorizin hydrolase (LPH) gene causes lactose intolerance. Association studies with differences in bone parameters and fracture risk have been inconclusive. The objective of this study was to examine the association of LPH rs4988235 with body height and bone parameters and calcium homeostasis in two elderly populations of Dutch Caucasians and assess interaction with vitamin D receptor (VDR) polymorphisms. Genotyping of LPH and VDR polymorphisms was performed in 6367 individuals from the Rotterdam Study and 844 from the Longitudinal Aging Study Amsterdam (LASA). Associations with age, height, weight, bone mineral density (BMD), skeletal morphometric parameters and serum vitamin D and calcium levels, and dietary calcium intake were assessed using ANOVA or analysis of covariance, and allele dose effect was assessed using linear regression analysis. Fracture risk was analyzed using Cox's proportional hazard regression analysis. Associations with body height (p = 2.7 × 10(-8)) and vertebral area (p = .048) found in the Rotterdam Study were explained by population stratification, as assessed by principal-component analyses, and disappeared after additional adjustments. No associations with femoral neck or lumbar spine BMD or with fracture risk were detected. Calcium intake and serum ionized serum calcium were significantly lower in C-homozygotes (p = 9.2 × 10(-7), p = .02, respectively). For none of the parameters studied was interaction between the T-13910C polymorphism and VDR block 5 haplotype 1 observed. We show that the C allele of the T-13910C polymorphism causing lactose intolerance is associated with lower dietary calcium intake and serum calcium levels but not with BMD or fractures. The associations observed with height and vertebral area were the result of population stratification. This demonstrates the impact of population stratification and urges researchers to carefully take this into account in genetic associations, in particular, in dietary intake-related phenotypes, of which LPH and lactose intolerance are a strong example.

Higher expression of jejunal LPH gene in rats fed the high-carbohydrate/low-fat diet compared with those fed the low-carbohydrate/high-fat diet is associated with in vitro binding of Cdx-2 in nuclear proteins to its promoter regions

It has been previously demonstrated that the expression of lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) genes are higher in rats fed a high-carbohydrate/low-fat (HCT) diet than in those fed a low-carbohydrate/high-fat (LCT) diet. In the present study, using a nuclear run-on assay we clearly show that higher expression of LPH and SI genes in jejunum of rats fed the HCT diet compared with those fed a LCT diet was regulated at the transcription levels. DNase I foot printing analysis of the 5′ flanking region of the rat LPH gene demonstrated that by incubating the jejunal nuclear extract the protected region was conserved as the same sequence as the homeodomain protein-binding element designated as CE-LPH1. UV-cross linking and electromobility shift assay in vitro clearly showed that Cdx-2 was including proteins bound to CE-LPH1. Moreover, in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1, a cis-element identified as the binding element of Cdx-2 on the SI gene, in jejunal nuclear extracts of rats fed a HCT diet were greater than those fed a LCT diet. These results suggest that in vitro binding of Cdx-2 to CE-LPH1 as well as SIF1 in jejunal nuclear extracts is associated with the higher expression of the LPH and SI genes in rats fed the HCT diet compared with those fed a LCT diet.

Correlation between lactose absorption and the C/T-13910 and G/A-22018 mutations of the lactase-phlorizin hydrolase (LCT) gene in adult-type hypolactasia

The C/T-13910 mutation is the major factor responsible for the persistence of the lactase-phlorizin hydrolase (LCT) gene expression. Mutation G/A-22018 appears to be only in co-segregation with C/T-13910. The objective of the present study was to assess the presence of these two mutations in Brazilian individuals with and without lactose malabsorption diagnosed by the hydrogen breath test (HBT). Ten milk-tolerant and 10 milk-intolerant individuals underwent the HBT after oral ingestion of 50 g lactose (equivalent to 1 L of milk). Analyses for C/T-13910 and G/A-22018 mutations were performed using a PCR-based method. Primers were designed for this study based on the GenBank sequence. The CT/GA, CT/AA, and TT/AA genotypes (lactase persistence) were found in 10 individuals with negative HBT. The CC/GG genotype (lactase non-persistence) was found in 10 individuals, 9 of them with positive HBT results. There was a significant agreement between the presence of mutations in the LCT gene promoter and HBT results (kappa = -0.9, P < 0.001). The CT/AA genotype has not been described previously and seems to be related to lactase persistence. The present study showed a significant agreement between the occurrence of mutations G/A-22018 and C/T-13910 and lactose absorption in Brazilian subjects, suggesting that the molecular test used here could be proposed for the laboratory diagnosis of adult-type primary hypolactasia.

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.

Lactase persistence/non-persistence variants, C/T_13910 and G/A_22018, as a diagnostic tool for lactose intolerance in IBS patients

Background Irritable bowel syndrome (IBS) is a symptom-based disorder characterized by abdominal pain related to altered bowel habit. We evaluated the predictive power of 2 genetic markers of hypolactasia, C/T_13910 and G/A_22018, in IBS patients with and without lactose intolerance in order to gain insight into the role of lactose intolerance in IBS. Methods Seventy five patients (59F/16M, mean age: 49.6 ± 14.2 years) with an IBS diagnosis based on Rome II criteria and 272 healthy individuals, where 74 (58F/16M, 54.1 ± 10.9 years) were matched-controls, were evaluated. IBS and healthy individuals were genotyped for the C/T_13910 and G/A_22018 polymorphisms nearby the lactase-phlorizin hydrolase gene. Hydrogen breath test (HBT) with gas chromatography was performed in IBS patients to assess for lactose intolerance. Results Of the 75 IBS patients, 28 (37%) were defined as lactose intolerants. The grade/severity of symptoms after an oral lactose load were positively correlated to the expiratory H2 excretion ( P < 0.001). Alleles and genotypes frequencies from C/T_13910 and G/A_22018 were not significantly different between IBS patients and control individuals ( P > 0.05;NS). Presence of the C and G allele were positively associated with a higher expiratory hydrogen excretion and more intense gastrointestinal symptoms ( P < 0.001). Considering these polymorphisms as a diagnostic test for lactose intolerance in IBS patients, presence of the CC and GG genotypes were estimated to have, a sensitivity of 100% and 96%, respectively; and a specificity of 83% and 79%, positive predictive value of 76% and 73%, and negative predictive value of 100% and 97%. Conclusions In IBS patients, genotyping of C/T_13910 and G/A_22018 polymorphisms predicts gastrointestinal symptoms after lactose ingestion and are a diagnostic tool for lactose intolerance.

Gata4andHnf1αare partially required for the expression of specific intestinal genes during development

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

The possible roles of homeobox protein, Cdx-2 for the expression of LPH gene during postnatal development

The expression of intestinal lactase-phlorizin hydrolase (LPH) gene normally decreases after completion of weaning in almost all mammals. To elucidate the mechanism whereby LPH gene expression is regulated during the suckling–weaning period, we studied the effects of the thyroid hormone (T 3) on LPH gene expression in the small intestine during postnatal development in the rat. Firstly, we measured LPH mRNA level in rat jejunum at 5, 13, 20 and 27 days after birth. The amount of LPH mRNA at 27 days was significantly lower than that at 5 days. The transcript level of Cdx-2, which is a putative transcriptional factor for regulation of LPH gene expression, was also significantly decreased after 21 days. The binding of nuclear protein to the cis element CE-LPH1 on the promoter region of the LPH gene was reduced at the end of the weaning period. Daily intraperitoneal (i.p.) injection of T 3 for 6 days during days 22–27 significantly reduced LPH mRNA level by day 27 (50%, P < 0.01), but injection of T 3 during days 8–13 did not. Moreover, i.p. T 3 injection during days 22–27 was accompanied by a reduction in the level of Cdx-2 mRNA. Our study suggests that the decrease in the LPH gene expression during the weaning period is associated with a reduction of Cdx-2 expression caused by thyroid hormone.

Dietary Sucrose Enhances Intestinal Lactase Gene Expression in Euthyroid Rats

It is postulated that dietary carbohydrates and thyroid hormones are major regulators for expression of the lactase/phlorizin hydrolase (LPH) gene in rat jejunum. In this study, we investigated the effects of thyroid hormones and dietary sucrose on LPH gene expression and lactase activity in starved rats. Firstly, animals at 8 wk of age were fed a low-starch diet (5.5% energy as cornstarch) or high-starch diet (71% energy as cornstarch) for 7 d (experiment 1). The mRNA level of LPH as well as lactase activity significantly decreased in rats fed the low-starch diet as compared to those fed the high-starch diet. To investigate the effects of thyroid hormone status, the animals previously fed the low-starch diet were starved for 3 d, and half of the animals were given intraperitoneal (i.p.) injections of 20 microg/ 100 g body weight triiodothyronine (T3) twice daily (experiment 2). The LPH mRNA level and lactase activity were elevated by starvation for 3 d, but they were repressed by the injection of T3 during starvation. To investigate the effects of dietary sucrose in starved rats, they were force-fed a sucrose diet for 6 h (experiment 3). The LPH gene expression and lactase activity were up-regulated by force-feeding a sucrose diet, only when the animals were kept in euthyroid status by daily T3 administrations. In contrast, the sucrase-isomaltase mRNA levels and sucrase activity were unaffected by force-feeding the sucrose diet for both T3-treated and untreated starved rats. Our work suggests that dietary sucrose is capable of enhancing lactase gene expression in starved rats when they have a sustainable thyroid hormone level.

Hepatocyte nuclear factor-1α is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo

Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.

Regulatory Regions in the Rat Lactase‐Phlorizin Hydrolase Gene that Control Cell‐Specific Expression

ABSTRACTObjectives:Lactase‐phlorizin hydrolase (LPH) is an enterocyte‐specific gene whose expression has been well‐characterized, not only developmentally but also along the crypt‐villus axis and along the length of the small bowel. Previous studies from the authors’ laboratory have demonstrated that 2 kb of the 5′‐flanking region of the rat LPH gene control the correct tissue, cell, and crypt‐villus expression in transgenic animals.Methods:To examine further the regulation conferred by this region, protein‐DNA interactions were studied using DNase I footprint analyses in LPH‐expressing and nonexpressing cell lines. Functional delineation of this 5′‐flanking sequence was performed using deletion analysis in transient transfection assays.Results:Studies revealed a generally positive activity between −74 and −37 bp, a cell‐specific negative region between −210 and −95 bp, and additional elements further toward the 5′ terminus that conferred a highly cell‐specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co‐transfection and electromobility shift assay experiments indicated binding of HNF3β at three sites relevant to LPH expression.Conclusions:The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5′‐flanking region.

Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression.

Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb of the 5'-flanking region of the rat LPH gene control the correct tissue, cell, and crypt-villus expression in transgenic animals. To examine further the regulation conferred by this region, protein-DNA interactions were studied using DNase I footprint analyses in LPH-expressing and nonexpressing cell lines. Functional delineation of this 5'-flanking sequence was performed using deletion analysis in transient transfection assays. Studies revealed a generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5'-terminus that conferred a highly cell-specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co-transfection and electromobility shift assay experiments indicated binding of HNF3beta at three sites relevant to LPH expression. The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5'-flanking region.

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4.

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.

P0066 PP GENETIC TESTING OF ADULT-TYPE HYPOLACTASIA

Introduction: Adult-type hypolactasia affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia has been difficult to establish due to unsatisfactory diagnostic methods. We have identified a DNA variant residing 13910 bp upstream of the lactase-phlorizin hydrolase (LPH) gene at chromosome 2q21–22 that shows complete association with lactase non-persistence/persistence trait (Enattah 2002, Kuokkanen 2003). The genotype C/C-13910 is associated with adult-type hypolactasia and genotypes C/T-13910 and T/T-13910 with lactase persistence. We have assessed the applicability of the C/T-13910 variant as a diagnostic test at the time of downregulation of the lactase activity in children of African, Finnish and other Caucasian origins. Methods: Intestinal biopsies were obtained from 333 children and adolescents aged from 0.1 to 20 years undergoing upper gastrointestinal endoscopy because of unspecified abdominal complaints. The biopsies were assayed for lactase, sucrase and maltase activity and genotyped for the C/T-13910 variant using PCR-minisequencing (Messer 1966, Syvänen 1994). Results: The prevalence of the genotype C/C-13910 defining adult-type hypolactasia was 14.5% among Finnish (n=256), 95.4% among African (n=65) and 75.0% among the other Caucasian children (n=12). The majority of Finnish children (92%) with C/C-13910 genotype had high lactase activity (>10 U/g protein) before the age of five years. A gradual decline took place between 5–12 years of age. The decline of the lactase activity in African children occurred somewhat earlier than in Finnish children as 30% of the children already had low levels (<10 U/g protein) at the age of five years. In Finnish children with C/C-13910 genotype the probability of low lactase activity (<10 U/g protein) was 8% before five years of age, 63% between 6–11 years of age and 100% after 12 years of age. The C/C-13910 genotype was associated with low lactase activity (<10 U/g protein) in all children >12 years of age in all ethnic groups studied giving a sensitivity of 94% and specificity of 100% for this genetic test defining adult-type hypolactasia. Conclusion: Genetic testing of adult-type hypolactasia is a clinical aid in diagnosis of abdominal complaints in populations like those of Northern Europeans where the prevalence of adult-type hypolactasia is relatively high and consumption of dairy products common. This test is easily performed with a semiautomated analysis from a drop of peripheral blood without fasting making this test suitable not just for diagnostic purposes at individual level but also as a screening method for population studies. Reference(S):