Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma

International audienceThe mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomi-tant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra-and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation

The heterochromatic chromosome caps in great apes impact telomere metabolism.

In contrast with the limited sequence divergence accumulated after separation of higher primate lineages, marked cytogenetic variation has been associated with the genome evolution in these species. Studying the impact of such structural variations on defined molecular processes can provide valuable insights on how genome structural organization contributes to organismal evolution. Here, we show that telomeres on chromosome arms carrying subtelomeric heterochromatic caps in the chimpanzee, which are completely absent in humans, replicate later than telomeres on chromosome arms without caps. In gorilla, on the other hand, a proportion of the subtelomeric heterochromatic caps present in most chromosome arms are associated with large blocks of telomere-like sequences that follow a replication program different from that of bona fide telomeres. Strikingly, telomere-containing RNA accumulates extrachromosomally in gorilla mitotic cells, suggesting that at least some aspects of telomere-containing RNA biogenesis have diverged in gorilla, perhaps in concert with the evolution of heterochromatic caps in this species

Relationships Linking Amplification Level to Gene Over-Expression in Gliomas

Background: Gene amplification is thought to promote over-expression of genes favouring tumour development. Because amplified regions are usually megabase-long, amplification often concerns numerous syntenic or non-syntenic genes, among which only a subset is over-expressed. The rationale for these differences remains poorly understood. Methodology/Principal Finding: To address this question, we used quantitative RT-PCR to determine the expression level of a series of co-amplified genes in five xenografted and one fresh human gliomas. These gliomas were chosen because we have previously characterised in detail the genetic content of their amplicons. In all the cases, the amplified sequences lie on extra-chromosomal DNA molecules, as commonly observed in gliomas. We show here that genes transcribed in nonamplified gliomas are over-expressed when amplified, roughly in proportion to their copy number, while non-expressed genes remain inactive. When specific antibodies were available, we also compared protein expression in amplified and nonamplified tumours. We found that protein accumulation barely correlates with the level of mRNA expression in some of these tumours. Conclusions/Significance: Here we show that the tissue-specific pattern of gene expression is maintained upon amplification in gliomas. Our study relies on a single type of tumour and a limited number of cases. However, it strongly suggests that, even when amplified, genes that are normally silent in a given cell type play no role in tumour progression

Maîtrise de l'antibiothérapie à l'hôpital (étude de la situation dans un service de réanimation polyvalente adulte et impact de la mise en place de mesures d'amélioration)

NANCY1-SCD Pharmacie-Odontologie (543952101) / SudocNANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines

International audienceThe mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridiza-tion on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homolo-gous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/ or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution

OntoNeuroBase: a multi-layered application ontology in neuroimaging

OntoNeuroBase is an application ontology that is being developed within the NeuroBase project, which seeks to create a federated system for the management of distributed and heterogeneous information sources in neuroimaging. Having adopted a specific, multi-layered, modular approach to ontology design, we used DOLCE as a foundational ontology together with three core ontologies: I&DA for modelling documents (texts and images), COPS for modelling programs and software and OntoKADS for modelling problem solving activities. Here, we report on how we built OntoNeuroBase by refining the concepts present in the existing modules. Neuroimaging is a very active and rapidly changing field. It is essential to ensure that a newly developed ontology is compatible with other available ontologies and to enable extension of the new ontology to a variety of neuroscience applications. The work reported here is in line with these ambitious objectives

A methodology for generating normal and pathological brain perfusion SPECT images for evaluation of MRI/SPECT fusion methods: application in epilepsy.

International audienceQuantitative evaluation of brain MRI/SPECT fusion methods for normal and in particular pathological datasets is difficult, due to the frequent lack of relevant ground truth. We propose a methodology to generate MRI and SPECT datasets dedicated to the evaluation of MRI/SPECT fusion methods and illustrate the method when dealing with ictal SPECT. The method consists in generating normal or pathological SPECT data perfectly aligned with a high-resolution 3D T1-weighted MRI using realistic Monte Carlo simulations that closely reproduce the response of a SPECT imaging system. Anatomical input data for the SPECT simulations are obtained from this 3D T1-weighted MRI, while functional input data result from an inter-individual analysis of anatomically standardized SPECT data. The method makes it possible to control the 'brain perfusion' function by proposing a theoretical model of brain perfusion from measurements performed on real SPECT images. Our method provides an absolute gold standard for assessing MRI/SPECT registration method accuracy since, by construction, the SPECT data are perfectly registered with the MRI data. The proposed methodology has been applied to create a theoretical model of normal brain perfusion and ictal brain perfusion characteristic of mesial temporal lobe epilepsy. To approach realistic and unbiased perfusion models, real SPECT data were corrected for uniform attenuation, scatter and partial volume effect. An anatomic standardization was used to account for anatomic variability between subjects. Realistic simulations of normal and ictal SPECT deduced from these perfusion models are presented. The comparison of real and simulated SPECT images showed relative differences in regional activity concentration of less than 20% in most anatomical structures, for both normal and ictal data, suggesting realistic models of perfusion distributions for evaluation purposes. Inter-hemispheric asymmetry coefficients measured on simulated data were found within the range of asymmetry coefficients measured on corresponding real data. The features of the proposed approach are compared with those of other methods previously described to obtain datasets appropriate for the assessment of fusion methods

Evaluation of methods to detect interhemispheric asymmetry on cerebral perfusion SPECT: application to epilepsy.

International audienceDetecting perfusion interhemispheric asymmetry in neurologic nuclear medicine imaging is an interesting approach to epilepsy. METHODS: This study compared 4 methods that detect interhemispheric asymmetries of brain perfusion in SPECT. The first (M1) was conventional side-by-side expert-based visual interpretation of SPECT. The second (M2) was visual interpretation assisted by an interhemispheric difference (IHD) volume. The last 2 were automatic methods: unsupervised analysis using volumes of interest (M3) and unsupervised analysis of the IHD volume (M4). Use of these methods to detect possible perfusion asymmetry was compared on 60 simulated SPECT datasets by controlling the presence and location of asymmetries. From the detection results, localization receiver operating characteristic curves were generated and areas under curves were estimated and compared. Finally, the methods were applied to analyze interictal SPECT datasets to localize the epileptogenic focus in temporal lobe epilepsies. RESULTS: This study showed an improvement in asymmetry detection on SPECT images with the methods using IHD volume (M2 and M4), in comparison with the other methods (M1 and M3). However, the most useful method for analyzing clinical SPECT datasets appeared to be visual inspection assisted by the IHD volume, since the automatic method using the IHD volume was less specific. CONCLUSION: The use of quantitative methods can improve performance in detection of perfusion asymmetry over visual inspection alone

Evaluation of methods to detect interhemispheric asymmetry on cerebral perfusion SPECT: application to epilepsy.

International audienceDetecting perfusion interhemispheric asymmetry in neurologic nuclear medicine imaging is an interesting approach to epilepsy. METHODS: This study compared 4 methods that detect interhemispheric asymmetries of brain perfusion in SPECT. The first (M1) was conventional side-by-side expert-based visual interpretation of SPECT. The second (M2) was visual interpretation assisted by an interhemispheric difference (IHD) volume. The last 2 were automatic methods: unsupervised analysis using volumes of interest (M3) and unsupervised analysis of the IHD volume (M4). Use of these methods to detect possible perfusion asymmetry was compared on 60 simulated SPECT datasets by controlling the presence and location of asymmetries. From the detection results, localization receiver operating characteristic curves were generated and areas under curves were estimated and compared. Finally, the methods were applied to analyze interictal SPECT datasets to localize the epileptogenic focus in temporal lobe epilepsies. RESULTS: This study showed an improvement in asymmetry detection on SPECT images with the methods using IHD volume (M2 and M4), in comparison with the other methods (M1 and M3). However, the most useful method for analyzing clinical SPECT datasets appeared to be visual inspection assisted by the IHD volume, since the automatic method using the IHD volume was less specific. CONCLUSION: The use of quantitative methods can improve performance in detection of perfusion asymmetry over visual inspection alone