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.     

In vitro propagation of <Emphasis Type="Italic">Helleborus</Emphasis> species

A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l⁻¹) and 1-naphthaleneacetic acid (NAA-1 mg l⁻¹) at 5°C.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.     

Calcium-dependent binding of EDTA-extractable proteins to calf lens fiber membrane structures

Calf lens fiber membranes and fractions enriched in junction-like structures have been isolated in the absence and presence of EDTA. Their biochemical features have been studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting experiments have provided evidence that a distinct group of EDTA-extractable proteins, being one of the main protein components of calf lens fiber membranes and very likely also of junction-like structures, is bound to these membranes via calcium ions. In addition to these proteins, four polypeptides with apparent molecular weights between 14 000 and 17 000 are characteristic for detergent-insoluble lens fiber structures prepared in calcium-rich medium. The absence of EDTA-extractable proteins in the urea-soluble calcium-containing fraction implies that they are not components of the cytoskeleton and that the calcium-dependent binding of these proteins to the membrane is urea-resistant. The use of EDTA throughout the whole membrane isolation procedure results in their complete removal from the membranes which already starts during buffer washing. This indicates that EDTA-extractable proteins exclusively consist of extrinsic membrane proteins which probably are not involved in cytoskeleton binding.     

Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.