Molekulare Karyotypisierung in der klinischen Diagnostik

The term “molecular karyotyping” refers to the genome-wide analysis of copy number variations using arrays that cover the genome with genomic markers with varying density. Currently the main application is the investigation of patients with otherwise unexplained mental retardation and multiple congenital anomalies. Studies of such patients who remained without etiological diagnosis after conventional karyotyping, subtelomeric screening, and targeted molecular–cytogenetic studies for well-known microdeletion syndromes revealed chromosomal microaberrations in about 10% of cases and allowed the delineation of several new microdeletion and microduplication syndromes. Nevertheless, because of the large number of copy number polymorphisms, interpretation of unique findings needs thorough consideration.     

Discovery of Potential New Gene Variants and Inflammatory Cytokine Associations with Fibromyalgia Syndrome by Whole Exome Sequencing

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (<2.5%) in the 1000 Genomes database, and presence in at least 2/19 probands sequenced. Two nonsense mutations, W32X in C11orf40 and Q100X in ZNF77 among 150 FMS trios had a significantly elevated frequency of transmission to affected probands (p = 0.026 and p = 0.032, respectively) and were present in a subset of 13% and 11% of FMS patients, respectively. Among 9 patients bearing more than one of the variants we have described, 4 had onset of symptoms between the ages of 10 and 18. The subset with the C11orf40 mutation had elevated plasma levels of the inflammatory cytokines, MCP-1 and IP-10, compared with unaffected controls or FMS patients with the wild-type allele. Similarly, patients with the ZNF77 mutation have elevated levels of the inflammatory cytokine, IL-12, compared with controls or patients with the wild type allele. Our results strongly implicate an inflammatory basis for FMS, as well as specific cytokine dysregulation, in at least 35% of our FMS cohort.     

Nichtinvasive molekulargenetische Methoden in der pränatalen Diagnostik

Work on the development of noninvasive prenatal tests to avoid risk to the fetus in traditional amniocentesis or chorion villus biopsy has been ongoing for many years. Until recently, most approaches were extremely expensive and limited only to selected applications, thus they failed to develop beyond a “proof-of-principle” status. This has changed radically as a result of the introduction of new sequencing methods, since initial studies have shown that fetal aneuploidies from maternal plasma DNA can be identified correctly. In addition, these techniques make it possible to establish even the mutation status of the fetus. While on the one hand this offers completely new options in prenatal diagnosis, progress of this kind is associated with significant ethical challenges on the other. This overview article presents the development of these new methods.     

An image analysis method for determination of spatial colonization patterns of bacteria in plant rhizosphere

A method that allows the rapid visualization of bacterial spatial colonization patterns on roots for the determination of general colonization trends was developed. This method, which analyzes images of roots, and bioluminescence-enhanced images of bacterial colonization patterns on these roots, was used to study the colonization patterns of seed-applied Enterobacter cloacae strain E6 on 3-day-old cucumber plants. Conventional dilution-plating methods indicated that E6 colonized cucumber tap roots in high populations and that these populations significantly decreased as the distance from the seed increased. In addition to confirming these observations, image analysis indicated that colonization by E6 significantly decreased on lateral roots as the distance increased horizontally away from the tap root, and that this bacterium did not evenly cover the most densely colonized regions of the cucumber root system. Results from these experiments indicate that the majority of E6 populations on cucumber roots after seed application are limited to the upper regions of the tap root and that E6 does not effectively colonize other regions of the root system. Received: 15 June 1988 / Received revision: 19 November 1998 / Accepted: 29 November 1998     

Die Genetik der bipolaren Störung

With a life-time prevalence of about 0.5–1.5%, bipolar (manic depressive) disorder represents a common psychiatric disease. Family, twin, and adoption studies have consistently shown that genetic factors contribute to disease development. Genome-wide linkage studies have detected chromosomal regions that are very likely to harbor predisposing genes. Meta-analyses suggest, however, that the genetic contribution of the individual loci must be relatively small which could be one reason for the difficulties in identifying the genes responsible. Very recently, genome-wide association analyses, investigating hundreds of thousands of single nucleotide polymorphisms (SNPs) in phenotypically well-characterized patient and control cohorts, promise a major breakthrough in search of disease-associated genes.     

Properties,morphogenesis, and effect of acidification on spines of the cidaroid sea urchin Phyllacanthus imperialis

Cidaroid sea urchins are the sister clade to all other extant echinoids and have numerous unique features, including unusual primary spines. These lack an epidermis when mature, exposing their high‐magnesium calcite skeleton to seawater and allowing the settlement of numerous epibionts. Cidaroid spines are made of an inner core of classical monocrystalline skeleton and an outer layer of polycrystalline magnesium calcite. Interestingly, cidaroids survived the Permian‐Triassic crisis, which was characterized by severe acidification of the ocean. Currently, numerous members of this group inhabit the deep ocean, below the saturation horizon for their magnesium calcite skeleton. This suggests that members of this taxon may have characteristics that may allow them to resist ongoing ocean acidification linked to global change. We compared the effect of acidified seawater (pH 7.2, 7.6, or 8.2) on mature spines with a fully developed cortex to that on young, growing spines, in which only the stereom core was developed. The cortex of mature spines was much more resistant to etching than the stereom of young spines. We then examined the properties of the cortex that might be responsible for its resistance compared to the underlying stereomic layers, namely morphology, intramineral organic material, magnesium concentration, intrinsic solubility of the mineral, and density. Our results indicate that the acidification resistance of the cortex is probably due to its lower magnesium concentration and higher density, the latter reducing the amount of surface area in contact with acidified seawater. The biofilm and epibionts covering the cortex of mature spines may also reduce its exposure to seawater.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.