Molecular Reclassification of Crohn’s Disease: A Cautionary Note on Population Stratification

Complex human diseases commonly differ in their phenotypic characteristics, e.g., Crohn’s disease (CD) patients are heterogeneous with regard to disease location and disease extent. The genetic susceptibility to Crohn’s disease is widely acknowledged and has been demonstrated by identification of over 100 CD associated genetic loci. However, relating CD subphenotypes to disease susceptible loci has proven to be a difficult task. In this paper we discuss the use of cluster analysis on genetic markers to identify genetic-based subgroups while taking into account possible confounding by population stratification. We show that it is highly relevant to consider the confounding nature of population stratification in order to avoid that detected clusters are strongly related to population groups instead of disease-specific groups. Therefore, we explain the use of principal components to correct for population stratification while clustering affected individuals into genetic-based subgroups. The principal components are obtained using 30 ancestry informative markers (AIM), and the first two PCs are determined to discriminate between continental origins of the affected individuals. Genotypes on 51 CD associated single nucleotide polymorphisms (SNPs) are used to perform latent class analysis, hierarchical and Partitioning Around Medoids (PAM) cluster analysis within a sample of affected individuals with and without the use of principal components to adjust for population stratification. It is seen that without correction for population stratification clusters seem to be influenced by population stratification while with correction clusters are unrelated to continental origin of individuals.     

Introducing the Multivariate Dale Model in Population‐Based Genetic Association Studies

Until recently, the most common parametric approaches to study the combined effects of several genetic polymorphisms located within a gene or in a small genomic region are, at the genotype level, logistic regressions and at the haplotype level, haplotype analyses. An alternative modeling approach, based on the case/control principle, is to regard exposures (e.g., genetic data such as derived from Single Nucleotide Polymorphisms – SNPs) as random and disease status as fixed and to use a marginal multivariate model that accounts for inter‐relationships between exposures. One such model is the multivariate Dale model. This model is based on multiple logistic regressions. That is why the model, applied in a case/control setting, leads to straightforward interpretations that are similar to those drawn in a classical logistic modeling framework. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sequence specificity of DNA topoisomerase I in the presence and absence of camptothecin.

Previously, we have demonstrated that in Tetrahymena DNA topoisomerase I has a strong preference in situ for a hexadecameric sequence motif AAGACTTAGAAGAAAAAATTT present in the non-transcribed spacers of r-chromatin. Here we characterize more extensively the interaction of purified topoisomerase I with specific hexadecameric sequences in cloned DNA. Treatment of topoisomerase I-DNA complexes with strong protein denaturants results in single strand breaks and covalent linkage of DNA to the 3' end of the broken strand. By mapping the position of the resulting nicks, we have analysed the sequence-specific interaction of topoisomerase I with the DNA. The experiments demonstrate that: the enzyme cleaves specifically between the sixth and seventh bases in the hexadecameric sequence; a single base substitution in the recognition sequence may reduce the cleavage extent by 95%; the sequence specific cleavage is stimulated 8-fold by divalent cations; 30% of the DNA molecules are cleaved at the hexadecameric sequence while no other cleavages can be detected in the 1.6-kb fragment investigated; the sequence specific cleavage is increased 2- to 3-fold in the presence of the antitumor drug camptothecin; at high concentrations of topoisomerase I, the cleavage pattern is altered by camptothecin; the equilibrium dissociation constant for interaction of topoisomerase I and the hexadecameric sequence can be estimated as approximately 10(-10) M.     

Plateau distributions of DNA fragment lengths produced by extended light exposure of extranuclear photosensitizers in human cells.

We have exploited properties of photosensitizers to study an aspect of the packing of chromatin in the cell nucleus. The fluorescent photosensitizers mesotetra(3-hydroxyphenyl) porphyrin and Photofrin II were both localized in the nuclear membrane and other membrane structures, but could not be found inside the nuclei. Light exposure of cells at 1 degrees C in the presence of the sensitizers induced DNA double-strand breaks. The length distributions of DNA fragments were determined by pulsed field gel electrophoresis. Because DNA damage is produced mainly via singlet oxygen diffusing less than 0.1 microns from the sensitizer, DNA double-strand breaks were supposedly produced within this distance of the nuclear membrane. Consistent with this, with prolonged illumination and with increasing concentrations of sensitizer the distribution of DNA fragment lengths reached a plateau level. In contrast, with the hydrophilic, intranuclear sensitizer meso-tetra(4-sulphonatophenyl)porphyrin, no such plateau level was found. The plateau distributions of DNA fragment lengths of different cell types had the same general shape with average fragment lengths ranging from 174 to 194 kilobasepairs. Particular genes, c-myc, fos and p53, were found on broad distributions of photocleaved fragment lengths. The results indicate that on each side of the genes the locus of the chromatin fibre situated close to the nuclear membrane, varied randomly.     

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Nasal Tumor in a Fallow Deer (Dama Dama L)

A nasal, so called ethmoidal, tumor from a fallow deer is described. It appears to be the first reported case of that species. The etiology is discussed.     

Rat immunoglobulin E heavy chain locus

A 2100 base-pair long sequence has been established which covers all four constant domains of the rat epsilon-chain. An analysis of messenger RNA from an immunoglobulin E producing rat immunocytoma revealed two separate epsilon-chain mRNA species, 2.3 X 10(3) and 2.8 X 10(3) base-pairs long. The latter mRNA encodes the membrane binding form of the epsilon-chain. The membrane exons which are located approximately 2 X 10(3) base-pairs away from the 3'-side of the CH4 exon were also sequenced. A comparison between the rat and mouse epsilon-chains at the protein sequence level revealed an overall homology of 80% which, as expected, is considerably higher than the homology found between rat and human epsilon-chains. The fourth constant domain together with the two membrane exons exhibited the highest degree of homology, 81 to 89%. Only two differences were found when the epsilon-chains from LOU and Sprague Dawley rats were compared. The most striking difference at the nucleotide sequence level between the rat, mouse, and human epsilon genes was found within the first intron. The mouse genome contains a unique 366 base-pair long sequence in this region. The inserted sequence is repetitive and present in approximately 100 copies in the mouse genome. It is flanked by 22 base-pair long direct repeats and contains also 14 base-pair long inverted repeats, thus having properties in common with transposable elements.     

Water economy in bird nests

Summary Nest humidity ( ) under an incubating bantam hen was measured at ambient ranging from 1.3 to 25.0 mmHg. Weight loss of eggs was recorded in clutches of varying size. Nest and ambient were also measured in wild incubating willow ptarmigan nests in dry and wet habitats.Nest increased linearly with ambient in a way predictable on the assumption that the water vapour conductance ( ) of brood patch skin, plumage and eggs were constant and independent of ambient . Nest was also dependent of clutch size. Egg dehydration was quantitatively predicted from measured values and the laws of diffusion.Our findings confirm earlier conclusions that the adult bird does not actively regulate nest at varying ambient . Birds can presumably achieve appropriate egg dehydration by a strategy combining nest site, nest construction, egg shell conductance and incubation behaviour which meets the requirements of their breeding climate.Abbreviations water vapur pressure - water vapur conductance - water flux