The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans.

Expression of lactase in the intestine persists into adult life in some people and not others, and this is due to a cis-acting regulatory polymorphism. Previous data indicated that a mutation leading to lactase persistence had occurred on the background of a 60 kb 11-site LCT haplotype known as A (Hollox et al. 2001). Recent studies reported a 100% correlation of lactase persistence with the presence of the T allele at a CT SNP at -14 kb from LCT, in individuals of Finnish origin, suggesting that this SNP may be causal of the lactase persistence polymorphism, and also reported a very tight association with a second SNP (GA -22 kb) (Enattah et al. 2002). Here we report the existence of a one megabase stretch of linkage disequilibrium in the region of LCT and show that the -14 kb T allele and the -22 kb A allele both occur on the background of a very extended A haplotype. In a series of Finnish individuals we found a strong correlation (40/41 people) with lactose digestion and the presence of the T allele. The T allele was present in all 36 lactase persistent individuals from the UK (phenotyped by enzyme assay) studied, 31/36 of whom were of Northern European ancestry, but not in 11 non-persistent individuals who were mainly of non-UK ancestry. However, the CT heterozygotes did not show intermediate lactase enzyme activity, unlike those previously phenotyped by determining allelic transcript expression. Furthermore the one lactase persistent homozygote identified by having equally high expression of A and B haplotype transcripts, was heterozygous for CT at the -14 kb site. SNP analysis across the 1 megabase region in this person showed no evidence of recombination on either chromosome between the -14 kb SNP and LCT. The combined data shows that although the -14 kb CT SNP is an excellent candidate for the cause of the lactase persistence polymorphism, linkage disequilibrium extends far beyond the region searched so far. In addition, the CT SNP does not, on its own, explain all the variation in expression of LCT, suggesting the possibility of genetic heterogeneity.

Analysis of thyroid hormone responsive gene expression in osteoblastic cells.

Thyroid hormones regulate gene expression to influence the development and metabolism of many tissues including bone. The identification of genes that are regulated by thyroid hormones during skeletal development requires sensitive and quantitative techniques that are not limited by small amounts of available tissue and RNA. We have compared the efficiencies of differential display and poly A PCR subtraction hybridisation methods for the detection of thyroid hormone responsive genes expressed in osteoblastic cells. The utility of each technique was evaluated with respect to its sensitivity, specificity, cost and ability to identify novel genes. Subtraction hybridisation was rapid and more efficient in all categories. Poly A PCR facilitates quantitative and representative global amplification of cDNAs from low concentrations of RNA extracted from small tissue samples. The method, in combination with microarray analyses, may prove useful as an additional, complementary strategy to subtraction hybridisation for the analysis of differential gene expression in tissues where sample size is limiting.

Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus.

Thyroid hormone receptors are encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) loci. These genes are transcribed into multiple variants whose functions are unclear. Analysis by gene inactivation in mice has provided new insights into the functional complexity of these products. Different strategies designed to modify the TRalpha locus have led to strikingly different phenotypes. In order to analyze the molecular basis for these alterations, we generated mice devoid of all known isoforms produced from the TRalpha locus (TRalpha(0/0)). These mice are viable and exhibit reduced linear growth, bone maturation delay, moderate hypothermia, and reduced thickness of the intestinal mucosa. Compounding TRalpha(0) and TRbeta(-) mutations produces viable TRalpha(0/0)beta(-/-) mice, which display a more severe linear growth reduction and a more profound hypothermia as well as impaired hearing. A striking phenotypic difference is observed between TRalpha(0/0) and the previously described TRalpha(-/-) mice, which retain truncated TRDeltaalpha isoforms arising from a newly described promoter in intron 7. The lethality and severe impairment of the intestinal maturation in TRalpha(-/-) mice are rescued in TRalpha(0/0) animals. We demonstrate that the TRDeltaalpha protein isoforms, which are natural products of the TRalpha locus, are the key determinants of these phenotypical differences. These data reveal the functional importance of the non-T3-binding variants encoded by the TRalpha locus in vertebrate postnatal development and homeostasis.

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.

The genetically programmed down-regulation of lactase in children.

BACKGROUND & AIMS: Intestinal lactase activity is high in all healthy human babies, but in adults a genetic polymorphism, which acts in cis to the lactase gene, determines high or low messenger RNA (mRNA) expression and activity (lactase persistence and nonpersistence, respectively). Our aim was to investigate the onset of expression of this polymorphism in children. METHODS: Activities were analyzed in relation to age in normal biopsy specimens from a 20-year collection of diagnostic specimens. In a smaller set of 32 samples, aged 2-132 months, RNA was extracted for semiquantitative reverse-transcription polymerase chain reaction. Marker polymorphisms were used to determine the allelic origin of lactase mRNA transcripts. RESULTS: Analysis of 866 children showed evidence that the lactase persistence/nonpersistence polymorphism began before 5 years of age. The 32 children tested had high lactase mRNA and activity. Six children aged 2-16 months showed equal expression of two alleles, 2 children aged 7 and 14 months showed slightly asymmetric expression, and 7 children aged 22-132 months showed very asymmetric expression, the second allele being undetectable in the 11-year-old, as previously seen in lactase-persistent heterozygote adults. CONCLUSIONS: Genetically programmed down-regulation of the lactase gene is detectable in children from the second year of life, although the onset and extent are somewhat variable.

Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5' end of the lactase gene on chromosome 2q21.

Four exons of a human homologue of a yeast cell division cycle gene (MCM6/mis5, which is thought to encode a DNA replication licensing factor) have been identified 3.3 kb upstream from the start of transcription of the intestinal lactase gene on human chromosome 2q21, initially by similarity to a rat 'intestinal crypt-cell replication factor'. RT-PCR analysis shows, that unlike lactase, MCM6 is not restricted in its tissue distribution and does not show person-to-person variation in the level of expression in adult intestine.

The lactase persistence/non-persistence polymorphism is controlled by a cis-acting element.

Lactase activity is present at high levels in the small intestine of some human adults and not others. This is due to a genetically determined polymorphism which affects the developmental regulation of the expression of the lactase gene. This polymorphism is of considerable interest in relation to cultural differences in nutrition but despite exhaustive studies, the molecular basis has not yet been found. It has not even been shown whether the sequence differences reside within or adjacent to the lactase gene itself or in a trans-acting factor. We have therefore exploited known DNA 'marker' polymorphisms within the exons of the lactase gene to examine the expression of the individual lactase mRNA transcripts from persistent and non-persistent individuals in order to determine whether the regulation is in cis or trans. Our results show that in certain lactase persistent individuals one allele of the lactase gene is expressed at much lower levels than the other and these individuals tend to have intermediate lactase activities. It is proposed that these people are heterozygous for the lactase persistence and non-persistence alleles and that this means that the nucleotide substitutions responsible for the lactase persistence/non-persistence polymorphism are cis-acting. This narrows down considerably the area of the genome that needs to be searched for the relevant sequence differences.

Studies on the expression of intestinal lactase in different individuals.

Sixty one duodenal biopsy specimens were examined for the expression of lactase at the level of enzyme activity, protein, and messenger RNA. Of the 51 samples with normal villous architecture, 39 were lactase persistent, 11 were nonpersistent (adult type hypolactasia), and one was of indeterminate status. All the lactase persistent individuals showed high mRNA and a high level of the lactase protein as detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis. All the 11 non-persistent individuals tested showed a low level of lactase protein. Nine of the 10 samples tested showed low mRNA and one high mRNA. These results suggest that the lactase persistence polymorphism is controlled at the level of the expression of the lactase gene, though there may be some heterogeneity of the lactase non-persistence phenotype.

DNA polymorphisms in the lactase gene. Linkage disequilibrium across the 70-kb region.

The enzyme lactase, which is responsible for the digestion of dietary lactose, is present in the intestine of some adults but not others. As a means of providing a platform to explore the molecular basis of this nutritionally relevant genetic variation we have screened for polymorphism in several regions of the lactase gene. In each case simple polymerase chain reaction-based procedures (including single-strand conformation analysis and denaturing gradient gel electrophoresis) were used, combined with silver staining as a method of detection. Allelic variation was found at 6 different sites. One previously published polymorphism was also tested. The frequencies of the alleles were determined in more than 100 unrelated individuals of the Centre d'Etude du Polymorphisme Humain (CEPH) panel, and the haplotypes were deduced. A region of linkage disequilibrium was observed, which spans the whole coding region of the lactase gene (approximately 60-70 kb); there were only 3 common haplotypes in this population. When the CEPH sample was subdivided according to the population of origin (France or Utah) the haplotype frequencies were shown to be markedly different.

Regional localization of the lactase-phlorizin hydrolase gene, LCT, to chromosome 2q21.

The gene LCT which codes for the intestinal disaccharidase lactase-phlorizin hydrolase has previously been mapped, using somatic cell hybrids and linkage analysis, using the CEPH families, to chromosome 2. We describe here the regional localization of LCT to chromosome 2q21 by polymerase chain reaction (PCR) analysis of somatic cell hybrids and in situ hybridization. LCT is closely linked to D2S44, with a lod score of 30.6 at theta = 0.10.

Spatial conceptions in Chinese and Canadian children.

Four-, five-, and six-year-old Chinese and Canadian boys and girls were presented a series of 11 graphic models and were asked to draw them. Supporting Piaget's theory, the data indicated that drawing performance on the topological dimension was better than the performance on the Euclidean dimension, and that there were neither gender nor cultural differences in performance. The research offers strong support for a main effect of biological or maturational factors as the foundation for the developmental influences in the acquisition of spatial conceptions.

Velocities and stress levels of axisymmetric, azimuthal flow within the toroidal rotary seal of the IBM 2997 continuous flow cell separator and the implications.

Continuous flow blood fraction separators are used to facilitate the removal of specific blood components for donation or for certain medical procedures. Problems with one such device, the IBM 2997 Blood Processor, have been noted in a number of independent investigations. A key feature of this particular unit is a ceramic rotary seal that allows the continuous separation by centrifugation to take place. The equation of motion for flow inside a split toroid cavity within the rotary seal has been solved numerically; velocities and shear stresses found numerically compare favorably with limiting case, analytical solutions. Predicted torque values as a function of rotation rate and fluid viscosity also served as an experimental check on the validity of the mathematical findings. Comparison of calculated shear stress levels and exposure times with known thresholds for cell damage shows that platelet and leukocyte losses may indeed be caused by the seal. Suggestions are made to improve performance of the IBM Blood Processor.