Microdeletion and microduplication syndromes

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted "genomic disorders" or "contiguous gene syndromes" is given.     

Regulation of ecto-5'-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors

Ecto-5'-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several in vivo models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H(2)O(2)) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H(2)O(2) decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

The human genome puzzle - the role of copy number variation in somatic mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.     

Somatic mosaicism in cases with small supernumerary marker chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that.     

Epoxy resin as fixative during freeze-substitution

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO(4)) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381-386.] using OsO(4) as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.     

Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C)

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.     

Variation in Chromatin Structure of the rDNA Transcribed Region in Human Peripheral Blood Lymphocytes

The rDNA transcribed region (TR) was tested for its accessibility for RsaI recognizing 15 TR sites, DNase I, and photoinducible arylazide (N-(4-azido-2-hydroxybenzoyl)-N,N"-diaminoheptane acetate) in isolated nuclei, and for the arylazide in intact cells. Arylazide readily entered the cells and did not appreciably affect the chromatin structure. Its photolysis products efficiently modified DNA in accessible sites. Single-strand breaks made by DNase I were not transformed into two-strand ones in rDNA TR, suggesting the necessity of denaturing electrophoresis for such an analysis. About 70% of all rDNA copies proved poorly accessible for endonucleases and arylazide, the accessibility being higher in their 18S and 5.8S rRNA gene regions than in the regions of the external transcribed spacers (ETSs) and the 28S rRNA gene. Proteinase K disrupted this structure, and the corresponding copies were extracted from nuclei. This explained whyin situ hybridization occasionally fails to reveal rDNA in the nucleolar fibrillar center (FC) on electron microscopic preparations. In other rDNA copies, TR (excluding 5"-ETS) was accessible for nucleases and arylazide. These copies were not extracted from nuclei treated with proteinase K. Some of their RsaI sites were protected by tightly bound proteins. Seven such regions were identified in TR. Possible association of the molecular structure, nucleolar location, and functional state of rDNA is discussed.