10 research outputs found

    Bioinformatics Tools and Databases to Assess the Pathogenicity of Mitochondrial DNA Variants in the Field of Next Generation Sequencing

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    The development of next generation sequencing (NGS) has greatly enhanced the diagnosis of mitochondrial disorders, with a systematic analysis of the whole mitochondrial DNA (mtDNA) sequence and better detection sensitivity. However, the exponential growth of sequencing data renders complex the interpretation of the identified variants, thereby posing new challenges for the molecular diagnosis of mitochondrial diseases. Indeed, mtDNA sequencing by NGS requires specific bioinformatics tools and the adaptation of those developed for nuclear DNA, for the detection and quantification of mtDNA variants from sequence alignment to the calling steps, in order to manage the specific features of the mitochondrial genome including heteroplasmy, i.e., coexistence of mutant and wildtype mtDNA copies. The prioritization of mtDNA variants remains difficult, relying on a limited number of specific resources: population and clinical databases, and in silico tools providing a prediction of the variant pathogenicity. An evaluation of the most prominent bioinformatics tools showed that their ability to predict the pathogenicity was highly variable indicating that special efforts should be directed at developing new bioinformatics tools dedicated to the mitochondrial genome. In addition, massive parallel sequencing raised several issues related to the interpretation of very low mtDNA mutational loads, discovery of variants of unknown significance, and mutations unrelated to patient phenotype or the co-occurrence of mtDNA variants. This review provides an overview of the current strategies and bioinformatics tools for accurate annotation, prioritization and reporting of mtDNA variations from NGS data, in order to carry out accurate genetic counseling in individuals with primary mitochondrial diseases

    Genome sequence of Vibrio diabolicus and identification of the exopolysaccharide HE800 biosynthesis locus

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    Vibrio diabolicus, a marine bacterium originating from deep-sea hydrothermal vents, produces the HE800 exopolysaccharide with high value for biotechnological purposes, especially for human health. Its genome was sequenced and analyzed; phylogenetic analysis using the core genome revealed V. diabolicus is close to another deep-sea Vibrio sp. (Ex25) within the Harveyi clade and Alginolyticus group. A genetic locus homologous to the syp cluster from Vibrio fischeri was demonstrated to be involved in the HE800 production. However, few genetic particularities suggest that the regulation of syp expression may be different in V. diabolicus. The presence of several types of glycosyltransferases within the locus indicates a capacity to generate diversity in the glycosidic structure, which may confer an adaptability to environmental conditions. These results contribute to better understanding exopolysaccharide biosynthesis and for developing new efficient processes to produce this molecule for biotechnological applications

    Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells

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    Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides

    IntĂ©rĂȘt du sĂ©quençage combinĂ© du gĂ©nome mitochondrial et d’un panel ciblĂ© de gĂšnes nuclĂ©aires impliquĂ©s dans les maladies mitochondriales

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    International audienceThe molecular study of mitochondrial diseases, essential for diagnosis, is special due to the dual genetic origin of these pathologies: mitochondrial DNA and nuclear DNA. Complete mtDNA sequencing still remains the first line diagnostic test followed if negative, by resequencing panels of several hundred mitochondrially-encoded nuclear genes. This strategy, with an initial entire mtDNA sequencing, is currently justified by the presence of nuclear mitochondrial DNA sequences (NUMTs) in the nuclear genome. We designed a resequencing panel combining the mtDNA and 135 nuclear genes which was evaluated compared to the performances of the standard mtDNA sequencing. Method validation was performed on the reading depth and reproducibility of the results. Thirty patients were analyzed by both methods. We were able to demonstrate that NUMTs did not impact the mtDNA sequencing quality, as the identified variants and mutant loads were identical with the reference mtDNA sequencing method. Reading depths were higher than the recommendations of the MitoDiag French diagnostic network, for the entire mtDNA for muscle and for 70% of the mtDNA for blood. These results highlight the usefulness of combining both mtDNA and mitochondrially nuclear-encoded genes and thus obtain more complete results and faster turnaround time for mitochondrial disease patients.Le diagnostic molĂ©culaire des maladies mitochondriales est complexe du fait de la double origine gĂ©nĂ©tique de ces pathologies : ADN mitochondrial (ADNmt) et ADN nuclĂ©aire (ADNn). Le sĂ©quençage complet de l’ADNmt reste l’analyse de premiĂšre intention complĂ©tĂ© si besoin par l’étude de l’ADNn. Cette stratĂ©gie avec un sĂ©quençage isolĂ© de l’ADNmt se justifie par l’existence de pseudogĂšnes mitochondriaux au niveau nuclĂ©aire. Nous avons Ă©laborĂ© un panel comprenant l’ADNmt et 135 gĂšnes nuclĂ©aires que nous avons comparĂ©s au sĂ©quençage isolĂ© de l’ADNmt. Trente patients ont Ă©tĂ© analysĂ©s par les deux mĂ©thodes. La validation de mĂ©thode a Ă©tĂ© faite sur la profondeur de lecture et la reproductibilitĂ© des rĂ©sultats. Nous avons mis en Ă©vidence l’absence d’impact des pseudogĂšnes sur la dĂ©tection et la quantification de l’hĂ©tĂ©roplasmie des variants de l’ADNmt. Les profondeurs sont supĂ©rieures aux recommandations du rĂ©seau pour l’intĂ©gralitĂ© de l’ADNmt pour le muscle et pour 70 % pour le sang. Ces rĂ©sultats mettent en avant l’intĂ©rĂȘt du sĂ©quençage commun de l’ADNmt et ADNn permettant l’obtention de rĂ©sultats complets, dans un dĂ©lai plus court

    A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters

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    Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains has lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species we show here that VarS is a key factor of pathogenicit

    Human iPSC Models to Study Orphan Diseases: Muscular Dystrophies

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    Stem Cell-Based and Tissue Engineering Approaches for Skeletal Muscle Repair

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    Skeletal muscle tissue exhibits signiïŹcant regeneration capacity upon injury or disease. This intrinsic regeneration potential is orchestrated by stem cells termed satellite cells, which undergo activation and differentiation in response to muscle insult, giving rise to fusion-competent myogenic progenitors responsible for tissue rejuvenation. Skeletal muscle diseases such as Duchenne muscular dystro-phy are characterized by progressive loss of muscle mass which precipitates reduced motility, paralysis, and in some occurrences untimely death. A manifold of muscle pathologies involve a failure to efïŹciently regenerate the muscle tissue, rendering stem cell-based approaches an attractive therapeutic strategy. Here we will present past and contemporary methods to treat skeletal muscle degeneration by stem cell therapy, covering prominent challenges facing this technology and potential means to overcome current hurdles. A primary focus of this chapter is directed toward illustrating innovative ways to utilize stem cells alone or in conjunction with biomaterials and tissue engineering techniques to remedy Duchenne muscular dystrophy or volumetric muscle loss
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