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

Deep learning from phylogenies to uncover the transmission dynamics of epidemics

Widely applicable, accurate and fast inference methods in phylodynamics are needed to fully profit from the richness of genetic data in uncovering the dynamics of epidemics. Standard methods, including maximum-likelihood and Bayesian approaches, which are both model specific, often rely on complex mathematical formulae and approximations, and do not scale well with dataset size. We develop a likelihood-free, simulation-based approach, which combines deep learning with (1) a large set of summary statistics measured on phylogenies or (2) a complete and compact vectorial representation of trees, which avoids potential limitations of summary statistics and applies to any phylodynamic model. Our method enables both model selection and estimation of epidemiological parameters. We demonstrate its speed and accuracy on simulated data, where it performs better than the state-of-the-art methods. To illustrate its applicability, we assess the dynamics induced by superspreading individuals in an HIV dataset of men- having-sex-with-men in Zurich

CKD Increases Carbonylation of HDL and Is Associated with Impaired Antiaggregant Properties

International audienceBACKGROUND: CKD is associated with increased oxidative stress that correlates with occurrence of cardiovascular events. Modifications induced by increased oxidative stress particularly affect circulating lipoproteins such as HDL that exhibit antiatheromatous and antithrombotic properties in vitro. METHODS: To explore the specific role of oxidative modifications of HDL in CKD and their effect on the platelet-targeting antiaggregant properties of HDL, we used a CKD (5/6 nephrectomy) rabbit model. For ex vivo assessment of the antiaggregant properties of HDL, we collected blood samples from 15 healthy volunteers, 25 patients on hemodialysis, and 20 on peritoneal dialysis. We analyzed malondialdehyde, 4-hydroxynonenal (HNE), and 4-hydroxy-2-hexenal protein adduct levels. Platelet aggregation and activation were assessed by aggregometry, thromboxane B2 assay, or FACS. We modified HDL from controls by incubating it overnight at 37°C with 100 µM of HNE. RESULTS: HDL from CKD rabbits and patients on hemodialysis had HNE adducts. The percentage of platelet aggregation or activation induced by collagen was significantly higher when platelets were incubated with HDL from CKD rabbit and hemodialysis groups than with HDL from the control group. In both rabbits and humans, platelet aggregation and activation were significantly higher in the presence of HNE-modified HDL than with HDL from their respective controls. Incubation of platelets with a blocking antibody directed against CD36 or with a pharmacologic inhibitor of SRC kinases restored the antiaggregative phenotype in the presence of HDL from CKD rabbits, patients on hemodialysis and peritoneal dialysis, and HNE-modified HDL. CONCLUSIONS: HDL from CKD rabbits and patients on hemodialysis exhibited an impaired ability to inhibit platelet aggregation, suggesting that altered HDL properties may contribute to the increased cardiovascular risk in this population

Molecular analyses of juvenile granulosa cell tumors bearing AKT1 mutations provide insights into tumor biology and therapeutic leads.

International audienceJuvenile granulosa cell tumors (JGCTs) of the ovary are pediatric neoplasms representing 5% of all granulosa cell tumors (GCTs). Most GCTs are of adult type (AGCTs) and bear a mutation in the FOXL2 gene. The molecular basis of JGCTs is poorly understood, although mutations in the GNAS gene have been reported. We have detected in-frame duplications within the oncogene AKT1 in >60% of the JGCTs studied. Here, to evaluate the functional impact of these duplications and the existence of potential co-driver alterations, we have sequenced the transcriptome of four JGCTs and compared them with control transcriptomes. A search for gene variants detected only private alterations probably unrelated with tumorigenesis, suggesting that tandem duplications are the best candidates to underlie tumor formation in the absence of GNAS alterations. We previously showed that the duplications were specific to JGCTs. However, the screening of eight AGCTs samples without FOXL2 mutation showed the existence of an AKT1 duplication in one case, also having a stromal luteoma. The analysis of RNA-Seq data pinpointed a series of differentially expressed genes, involved in cytokine and hormone signaling and cell division-related processes. Further analyses pointed to the existence of a possible dedifferentiation process and suggested that most of the transcriptomic dysregulation might be mediated by a limited set of transcription factors perturbed by AKT1 activation. Finally, we show that commercially available AKT inhibitors can modulate the in vitro activity of various mutated forms. These results shed light on the pathogenesis of JGCTs and provide therapeutic leads for a targeted treatment

Real-Time Quantification of AFP mRNA to Assess Hematogenous Dissemination After Transarterial Chemoembolization of Hepatocellular Carcinoma

OBJECTIVE: To determine whether the number of hepatocytes containing AFP mRNA shed into the bloodstream during transarterial chemoembolization (TAE) affects the incidence and pattern of recurrence of hepatocellular carcinoma (HCC). PATIENTS AND METHODS: We developed a Taqman procedure to quantify AFP mRNA prospectively in 52 consecutive patients before and after TAE. Results are expressed in hepatocytes /mL. RESULTS: Thirteen of the patients (24.5%) were positive for AFP mRNA (42 ± 19 hepatocytes/mL) before TAE and 13 (24.5%) (80 ± 32 hepatocytes/mL) after TAE; the difference was not significant. The presence of AFP mRNA in the bloodstream before TAE was associated with larger nodules (85.2 ± 73.8 mm versus 34.8 ± 26.1 mm; P = 0.006). Six of the patients were excluded from the analysis because they underwent curative surgery or were lost to follow-up. The circulating levels of AFP mRNA released in the 46 remaining patients after TAE did not affect metastasis-free survival. A significant number of extrahepatic metastases were found in patients exhibiting at least 1 AFP mRNA-positive blood sample either before or after TAE. However, the TAE procedure did not increase the risk of extrahepatic recurrences. CONCLUSION: Cells containing AFP mRNA are inconsistently released into the circulation during TAE. The amount of these cells released does not affect the recurrence of HCC

Identification of novel recurrent ETV6-IgH fusions in primary central nervous system lymphoma

International audienceBackground: Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCNSL discovered by using RNA sequencing (RNA-seq).Methods: Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, 6 were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion.Results: We identified a novel recurrent gene fusion, E26 transformation-specific translocation variant 6-immunoglobulin heavy chain (ETV6-IgH). Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6, and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, P = 0.03, hazard ratio = 0.33, 95% CI = 0.12-0.88. The overall survival at 5 years was 69% for PCNSL harboring ETV6-IgH versus 29% for samples without this gene fusion.Conclusions: ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsufficiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies

Genomic Epidemiology of 2015–2016 Zika Virus Outbreak in Cape Verde

International audienceDuring 2015-2016, Cape Verde, an island nation off the coast of West Africa, experienced a Zika virus (ZIKV) outbreak involving 7,580 suspected Zika cases and 18 microcephaly cases. Analysis of the complete genomes of 3 ZIKV isolates from the outbreak indicated the strain was of the Asian (not African) lineage. The Cape Verde ZIKV sequences formed a distinct monophylogenetic group and possessed 1-2 (T659A, I756V) unique amino acid changes in the envelope protein. Phylogeographic and serologic evidence support earlier introduction of this lineage into Cape Verde, possibly from northeast Brazil, between June 2014 and August 2015, suggesting cryptic circulation of the virus before the initial wave of cases were detected in October 2015. These findings underscore the utility of genomic-scale epidemiology for outbreak investigations