Fast approach for clarification of chromosomal aberrations by using LM-PCR and FT-CGH in leukaemic sample

Chromosomal abnormalities, like deletions, amplifications, inversions or translocations, are recurrent features in haematological malignancies. However, the precise molecular breakpoints are frequently not determined. Here we describe a rapid analysis of genetic imbalances combining fine tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR). We clarified an inv(14)(q11q32) in a case of T cell acute lymphoblastic leukaemia with a breakpoint in the TRA/D in 68% of cells detected by fluorescence in situ hybridization. FT-CGH showed several mono- and biallelic losses within TRA/D. LM-PCR disclosed a TRA/D rearrangement on one allele. The other allele revealed an inv(14)(q11q32), joining TRDD2 at 21,977,000 of 14q11 together with the IGH locus at 105,948,000 and 3'-sequence of TRAC at 22,092,000 joined together with IGHV4-61 at 106,166,000. This sensitive approach can unravel complex chromosomal abnormalities in patient samples with a limited amount of aberrant cells and may lead to better diagnostic and therapeutic options

Identifizierung von an chromosomalen Translokationen beteiligten Genen

Maligne Erkrankungen zeigen oft charakteristische genetische Veränderungen. Das Auffinden derartiger Veränderungen wurde in den letzten Jahren durch verfeinerte molekulare Techniken erleichtert. Viele genetische Ereignisse in den maligne transformierten Zellen sind jedoch noch ungeklärt. Die präzise Bestimmung der Bruchpunktregionen chromosomaler Veränderungen bei T-Zell akuten lymphatischen Leukämien ist Inhalt dieser Arbeit. Hierzu wurde die „Fine Tiling-Comparative Genomhybridisierung“ (FT-CGH) mit der „Ligation mediated-PCR“ (LM-PCR) kombiniert. Diese Methoden wurden zunächst an Zelllinien etabliert und anschließend in verschiedenen Leukämieproben eingesetzt. Chromosomale Aberrationen gehen häufig mit Verlust oder Gewinn von genetischem Material einher. Diese unbalancierten Anomalien lassen sich durch die Comparative Genomhybridisierung (CGH) ermitteln. Dieses Verfahren ermöglicht Differenzen der DNA-Menge einer zu untersuchenden Probe bezogen auf eine interne Kontrollprobe zu detektieren. Bei der Fine Tiling-CGH werden gezielt chromosomale Abschnitte hochauflösend auf eventuelle Abweichungen des DNA-Gehaltes analysiert. Anschließend werden die detektierten Bruchpunktregionen der DNA Schwankungen mittels der LM-PCR untersucht. Ein Abgleich mit einer internen Kontrollzelllinie HEK 293-T lässt atypische PCR-Fragmente bei der untersuchten Probe aufspüren. Der anschließende Sequenzabgleich unter der Verwendung des BLASTn Suchprogramms (National Center for Biotechnology Information) führte in den untersuchten Zelllinien, wie auch in den T-Zell akuten lymphatischen Leukämieproben zur Identifizierung verschiedener genomischer Veränderungen. Neben einfachen Deletionen wurden auch bisher ungeklärte komplexere chromosomale Translokationen nachgewiesen. So konnte unter anderem bei einer lymphoblastischen T-Zell-Leukämie die Translokation t(12;14)(q23;q11.2) auf genomischer Ebene geklärt werden. Hierbei fand im Abschnitt 14q11 innerhalb des TRA/D Locus eine Deletion von 89 Kilobasen statt. Die Bruchenden wurden mit der Sequenz des open reading frames C12orf42, welches im 12q23 Chromosomenabschnitt lokalisiert ist, zusammengelagert. Bei dieser chromosomalen Aberration wurde die C12orf42 Sequenz zerstört und 1,3 Kilobasen deletiert. Des Weiteren konnte bei einer akuten lymphoblastischen T-Zell-Leukämie die Inversion inv(14)(q11q32) mit involvierten TRA/D und IGH Locus auf Sequenzebene geklärt werden. Der Bruch des 14q11 Bereiches fand zwischen dem Genabschnitt der konstanten Region (TRAC) des TRA/D Locus und dem DAD1 (defender against cell death 1) Gens statt, wobei im beteiligten genetischen Abschnitt keine Rekombinasesignalsequenz (RSS) zu finden ist. Dieses belegt, dass fehlerhafte Umlagerungen innerhalb des Genoms nicht ausschließlich auf die Rekombinase zurückzuführen sind. Die vorliegende Arbeit zeigt, dass die Kombination aus FT-CGH und LM-PCR eine präzise Bruchpunktanalyse unbekannter chromosomaler Aberrationen, welche mit Imbalancen einhergehen, ermöglicht. Diese genaue Analyse dient der Identifizierung von Genen, welche direkt und indirekt durch diese genomischen Umlagerungen betroffen sind. Das Wissen über diese Veränderungen kann für das Verständnis der Pathogenese, für diagnostische Zwecke und zum Nachweis der minimalen Resterkrankung eingesetzt werden. Eine Klärung beteiligter Gene und Signalwege wird es erlauben, zielgerichtete und individualisierte Therapiestrategien zu entwickeln.Malignant diseases show frequently characteristic genetic alterations. The detections of such alterations were facilitating by improved molecular approaches in recent years. The focus of this work is the precise characterisation of breakpoint regions of chromosomal alterations of T-cell acute lymphatic leukaemia. For this purpose the fine-tiling comparative genomic hybridization (FT-CGH) were combined with the ligation mediated-PCR (LM-PCR). These both approaches were established in cell lines and subsequently used in different leukaemia samples. Chromosomal aberrations are frequently accompanied by genetic losses or gains. Such unbalanced alterations can be detected by the comparative genomic hybridization, which allows the detection of DNA copy number differences between a test sample and a reference sample. The fine-tiling-CGH detects with high resolution the DNA amount differences in specific genomic areas. The detected breakpoint regions can be further analysed by the LM-PCR. A comparison with the internal control cell line HEK 293-T revealed atypical PCR fragments in the analysed leukaemia sample. Using the BLASTn search database resulted in the identification of different genomic alterations in cell lines and T-cell acute lymphatic leukaemia samples. The combination of the FT-CGH and the LM-PCR allowed the clarification of simple deletions as well as complex chromosomal translocations. Among other things it were possible to clarify the translocation t(12;14)(q23;q11.2) in a lymphoblastic T-cell leukaemia on genomic level. The data of this translocation revealed a deletion of 89 kb within the TRA/D locus in the chromosomal area 14q11.2. The ending of this deletion were agminate with the open reading frame C12orf42, which is localized on chromosome 12q23. This chromosomal event leads to the destruction of the C12orf42 sequence and a deletion of 1.3 kilobase within this gene. Furthermore it was possible to clarify the inversion inv(14)(q11q32) in a acute lymphoblastic T-cell leukaemia with involved TRA/D and IGH locus on genomic level. The 14q11 breakpoint took place within the sequence of the constant region (TRAC) and the DAD1 (defender against cell death 1) gene. No recombination signal sequence (RSS) was found in the involved genomic area. This result showed that incorrect rearrangements in the genome are not only due to recombination errors. The present work shows that the combination of FT-CGH and LM-PCR allows a precise breakpoint analysis of unknown chromosomal aberrations, which are accompanied by genetic imbalances. This exact analysis leads to the identification of genes, which are affected direct or indirect by this genomic alteration. The knowledge of this chromosomal alteration can lead to understanding of the pathogenesis and can be used for diagnostics and for detection of minimal residual disease (MRD). The clarification of involved genes and metabolic pathways will allow the development of target-oriented and individualized therapy concepts

Costs of outpatient and inpatient MRSA screening and treatment strategies for patients at elective hospital admission - a decision tree analysis

Abstract Background Nosocomial infections are among the most common complications in hospitals. A major part is caused by multidrug-resistant organisms (MDRO). MRSA is still the most prominent and frequent MDRO. The early detection of carriers of multidrug-resistant bacteria is an effective measure to reduce nosocomial infections caused by MDRO. For patients who are planning to go to the hospital, an outpatient screening for MDRO and pre-hospital decolonization is recommended. However, the effectiveness of such pre-admission MDRO management in preparation for a planned hospital stay has not yet been sufficiently scientifically examined from an economic perspective. Methods A decision tree will be used to develop scenarios for MDRO screening and treatment in the context of the outpatient and inpatient sectors using MRSA-positive patients as an example. Subsequently, the expected costs for the respective strategy are presented. Results The decision tree analysis shows that the expected costs of outpatient MRSA management are €8.24 and that of inpatient MRSA management are €672.51. Conclusion The forward displacement of the MRSA screening to the ambulatory sector and any subsequent outpatient decolonization for patients with a planned hospitalization is the most cost-effective strategy and should become a standard benefit. Excluding opportunity costs, the expected costs of inpatient MRSA management are €54.94

Occurrence of ESBL-Producing Escherichia coli in Livestock and Farm Workers in Mecklenburg-Western Pomerania, Germany.

In recent years, extended-spectrum β-lactamases (ESBL) producing bacteria have been found in livestock, mainly as asymptomatic colonizers. The zoonotic risk for people working in close contact to animal husbandry has still not been completely assessed. Therefore, we investigated the prevalence of ESBL-producing Escherichia spp. in livestock animals and workers to determine the potential risk for an animal-human cross-transmission.In Mecklenburg-Western Pomerania, northeast Germany, inguinal swabs of 73 individuals with livestock contact from 23 different farms were tested for ESBL-producing Escherichia spp. Two pooled fecal samples per farm of animal origin from 34 different farms (17 pig farms, 11 cattle farms, 6 poultry farms) as well as cloacal swabs of 10 randomly selected broilers or turkeys were taken at each poultry farm. For identification, selective chromogenic agar was used after an enrichment step. Phenotypically ESBL-producing isolates (n = 99) were tested for CTX-M, OXA, SHV and TEM using PCR, and isolates were further characterized using multilocus sequence typing (MLST). In total, 61 diverse isolates from different sources and/or different MLST/PCR results were acquired. Five farm workers (three from cattle farms and two from pig farms) harbored ESBL-producing E. coli. All human isolates harbored the CTX-M β-lactamase; TEM and OXA β-lactamases were additionally detected in two, resp. one, isolates. ESBL-producing Escherichia spp. were found in fecal samples at pig (15/17), cattle (6/11) and poultry farms (3/6). In total, 70.6% (24/36) of the tested farms were ESBL positive. Furthermore, 9 out of 60 cloacal swabs turned out to be ESBL positive. All isolated ESBL-producing bacteria from animal sources were E. coli, except for one E. hermanii isolate. CTX-M was the most prevalent β-lactamase at cattle and pig farms, while SHV predominated in poultry. One human isolate shared an identical MLST sequence type (ST) 3891 and CTX-M allele to the isolate found in the cattle fecal sample from the same farm, indicating a zoonotic transfer. Two other pairs of human-pig and human-cattle E. coli isolates encoded the same ESBL genes but did not share the same MLST ST, which may indicate horizontal resistance gene transfer. In summary, the study shows the high prevalence of ESBL-producing E.coli in livestock in Mecklenburg- Western Pomerania and provides the risk of transfer between livestock and farm workers

Macrofaunal Patterns in and around du Couedic and Bonney Submarine Canyons, South Australia.

Two South Australian canyons, one shelf-incising (du Couedic) and one slope-limited (Bonney) were compared for macrofaunal patterns on the shelf and slope that spanned three water masses. It was hypothesized that community structure would (H1) significantly differ by water mass, (H2) show significant regional differences and (H3) differ significantly between interior and exterior of each canyon. Five hundred and thirty-one species of macrofauna ≥ 1 mm were captured at 27 stations situated in depth stratified transects inside and outside the canyons from 100 to 1500 m depth. The macrofauna showed a positive relationship to depth in abundance, biomass, species richness and community composition while taxonomic distinctness and evenness remained high at all depths. Biotic variation on the shelf was best defined by variation in bottom water primary production while sediment characteristics and bottom water oxygen, temperature and nutrients defined biotic variation at greater depth. Community structure differed significantly (p<0.01) among the three water masses (shelf-flowing South Australian current, upper slope Flinders current and lower slope Antarctic Intermediate Water) (H1). Although community differences between the du Couedic and Bonney regions were marginally above significance at p = 0.05 (H2), over half of the species captured were unique to each region. This supports the evidence from fish and megafaunal distributions that the du Couedic and Bonney areas are in different bioregions. Overall, the canyon interiors were not significantly different in community composition from the exterior (H3). However, both canyons had higher abundance and/or biomass, increased species dominance, different species composition and coarser sediments near the canyon heads compared to outside the canyons at the same depth (500 m), suggestive of heightened currents within the canyons that influence community composition there. At 1000-1500 m, the canyon interiors were depauperate, typical of V-shaped canyons elsewhere. The large number of species captured, given the relatively low sampling effort and focus on the larger macrofauna, support previous studies that identify the South Australian coast as a high biodiversity area

Association between serum insulin-like growth factor I or IGF-binding protein 3 and estimated glomerular filtration rate: results of a population-based sample

Abstract Background Insulin-like growth factor I (IGF-I), which is mostly carried in blood by IGF-binding protein 3 (IGFBP-3), was associated to the glomerular filtration rate and chronic kidney disease in a multiethnic study among US adults. The aim of the present study was to investigate whether serum IGF-I or IGFBP-3 are associated with estimated glomerular filtration rate (eGFR) in a population-based study of Caucasian adults. Methods Data from 4028 subjects (2048 women) aged 20 to 81 years from the Study of Health in Pomerania (SHIP) were analyzed. Total serum IGF-I and IGFBP-3 concentrations were determined by chemiluminescence immunoassays and categorized into sex- and age-specific quartiles. Results After adjusting for age, waist circumference and type 2 diabetes mellitus, analysis of variance (ANOVA) revealed inverse associations between serum IGF-I concentrations and eGFR in men as well as between serum IGFBP-3 concentrations and eGFR in men and women. Logistic regression analyses confirmed these findings and showed that high IGF-I or IGFBP-3 concentrations were associated with an increased risk of decreased eGFR (2) in men or women. These relations became stronger when lower eGFR cut-offs were used for the analyses. Conclusion Our data revealed associations of increased serum IGF-I concentrations and decreased eGFR in men but not in women and an association of increased serum IGFBP-3 concentrations and decreased eGFR in both sexes.</p