Le complexe P-gp/mdr de la drosophile (un bio-marqueur potentiel de la pollution atmosphérique due aux hydrocarbures aromatiques polycycliques)

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants, émis lors de combustion incomplètes, qui peuvent engendrer des effets cytotoxiques, génotoxiques ou cancérigène. Dans le milieu atmosphérique la teneur de ces composés a été récemment réglementée au niveau européen. La surveillance de leurs concentrations et de leur impacts sur la santé, doit être effectuée par des mesures physico-chimiques et par le suivi de bio-indicateurs. Cette bio-surveillance est encore peu développée et nécessite la recherche de bio-marqueurs répondant spécifiquement à ces polluants. L'utilisation du complexe P-gp/mdr bien connu chez les mammifères pour son implication dans des phénomènes de " multidrug resistance ", a été évaluée pour le développement d'un bio-marqueur chez le modèle drosophile. Chez cet insecte nous avons trouvé que la P-gp était exprimée dans chaque stade de développement et qu'elle participait très activement à la protection des cellules en rejetant dans le milieu extracellulaire les HAP. De plus, l'exposition des drosophiles, en laboratoire ou sur le terrain, à différentes concentrations en HAP a très clairement été à l'origine d'une augmentation dose dépendante du taux d'expression de la P-gp. Nous avons montré que les transcrits des 3 gènes de type mdr répertoriés chez la mouche, étaient présents chez l'adulte d'une manière ubiquitaire et étaient exprimés au cours des différents stades de développement de cet insecte. L'analyse plus approfondie du gène mdr49 nous a permis de mettre en évidence une augmentation rapide et dose dépendante du taux de ses transcrits en réponse à une exposition des drosophiles à différents HAP. Les résultats obtenus ont permis de démontrer le rôle du complexe Pgp/mdr dans le système de protection de la drosophile vis-à-vis de HAP et sont en faveur de son utilisation potentielle en tant que bio-marqueur.Polycyclic aromatic hydrocarbons (PAHs) are cytotoxic, genotoxic and carcinogenic contaminants resulting from incomplete combustion. In the European community today, the release of PAHs in the atmosphere is tightly controlled. The monitoring of their concentrations and their impacts on health must be carried out by physicochemical measurements and the surveillance of biological indicators. Bio-monitoring is still poorly developed and requires the search for bio-markers responding specifically to these pollutants. We wished to develop a biomarker using Drosophila model. Therefore we evaluated the use of the P-gp/mdr complex known to be involved multidrug resistance in mammals. In Drosophila we found that P-gp is expressed at every developmental stage and is actively implicated in cell protection through the efflux of PAHs, either in the laboratory or in a natural environment, P-gp expression clearly increased in a dose dependant way. We also demonstrated that 3 identified mdr genes transcripts in Drosophila homologous of mammalian mdr, are ubiquitously expressed in the adult fly and are expressed during various stages of fly development. A thorough analysis of the mdr49 gene showed a rapid and dose-dependent increase of its expression after drosophila exposure to various PAHs. The results demonstrate the role of the Pgp/mdr complex in drosophila's defense system against PAHs and are in favour of its use as a bio-marker.CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

A potential genomic biomarker for the detection of polycyclic aromatic hydrocarbon pollutants: multidrug resistance gene 49 in Drosophila melanogaster.

International audiencePolycyclic aromatic hydrocarbons (PAHs) are a major source of air, water, and soil pollution. The multidrug resistance (mdr)/permeability glycoprotein (P-gp) complex is implicated in the multidrug resistance pattern developed against various drugs and xenobiotics, including polycyclic aromatic hydrocarbons. In order to develop a genomic biomarker, we investigated the response of the mdr49 gene (mdr49) of Drosophila melanogaster to PAHs. Structural analysis of mdr49-PA, which is the putative protein expressed from Drosophila mdr49 gene, demonstrated that this transmembrane protein indeed belongs to the adenosine triphosphate-binding cassette transporter superfamily. Polymerase chain reaction (PCR) and real-time PCR analysis revealed that the mdr49 gene is expressed continuously at all the stages of fly development, including embryos, pupae, larvae, and adults, as well as in embryonic Drosophila S12 cells. In the adult fly, the mdr49 gene was expressed in all the analyzed segments (head, thorax, and abdomen) and organs (olfactory and sexual organs). The quantification of mdr49 transcripts by real-time PCR in adult flies exposed to benzo[a]pyrene over time or in presence of increasing concentrations of this pollutant showed a clear dose-dependent response. Similarly, mdr49 gene expression increased after adult flies were exposed to structurally varied PAHs. The detection of tested PAHs by Drosophila P-gp efflux pump was checked by flow cytometry

Drosophila melanogaster P-glycoprotein : A membrane detoxification system toward polycyclic aromatic hydrocarbon pollutants

International audiencePolycyclic aromatic hydrocarbons (PAHs) are well-known ubiquitous environmental contaminants. Permeability glycoprotein (P-gp) is a transmembrane detoxification efflux pump transporting various lipophilic xenobiotics, such as PAHs, out of the cells. The existence of a P-gp detoxification system inducible by PAHs was investigated in Drosophila melanogaster. Western blot experiments showed that D. melanogaster expressed a 140-kDa P-gp in S12 cells, embryos, and adult flies. Permeability glycoprotein was expressed in adult flies in the head, abdomen, and thorax and sublocalized in the sexual and olfactory organs. Flow cytometry experiments using Drosophila S12 cells in the presence of PAHs and target P-gp drug compounds revealed that Drosophila P-gp acted as an efflux detoxification pump. In Drosophila exposed to benzo[a]pyrene or to ambient air polluted by higher or lower PAH concentrations, P-gp expression was clearly showed a dose-dependent increase response. The P-gp induction was detected both in adult flies and in different fly parts, such as the head, thorax, and antennae. Drosophila P-gp acts as a membrane barrier against PAH pollutants

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

International audienceMolecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes

Usher syndrome type 2 caused by activation of an USH2A pseudoexon: Implications for diagnosis and therapy

International audienceUSH2A sequencing in three affected members of a large family, referred for the recessive USH2 syndrome, identified a single pathogenic alteration in one of them and a different mutation in the two affected nieces. As the patients carried a common USH2A haplotype, they likely shared a mutation not found by standard sequencing techniques. Analysis of RNA from nasal cells in one affected individual identified an additional pseudoexon (PE) resulting from a deep intronic mutation. This was confirmed by minigene assay. This is the first example in Usher syndrome (USH) with a mutation causing activation of a PE. The finding of this alteration in eight other individuals of mixed European origin emphasizes the importance of including RNA analysis in a comprehensive diagnostic service. Finally, this mutation, which would not have been found by whole-exome sequencing, could offer, for the first time in USH, the possibility of therapeutic correction by antisense oligonucleotides (AONs)

Four-Year Follow-up of Diagnostic Service in USH1 Patients

International audiencePURPOSE: The purpose of this study was to establish the mutation spectrum of an Usher type I cohort of 61 patients from France and to describe a diagnostic strategy, including a strategy for estimating the pathogenicity of sequence changes.METHODS: To optimize the identification of Usher (USH)-causative mutations, taking into account the genetic heterogeneity, preliminary haplotyping at the five USH1 loci was performed to prioritize the gene to be sequenced, as previously described. Coding exons and flanking intronic sequences were sequenced and, where necessary, semiquantitative PCR and multiplex ligation-dependent probe amplification (MLPA) were performed to detect large genomic rearrangements.RESULTS: Four years ' experience confirms that the chosen approach provides an efficient diagnostic service. Sixty-one patients showed an abnormal genotype in one of the five USH1 genes. Genetic heterogeneity was confirmed, and, although MYO7A remains the major gene, involvement of other genes is considerable. Distribution of missense, splicing, premature termination codons (PTCs; due to point substitution and small deletions/ or insertions), and large genomic alterations was determined among the USH genes and clearly highlights the need to pay special attention to the diagnostic approach and interpretation, depending on the mutated gene.CONCLUSIONS: Over the 4 years of a diagnostic service offering USH1 patient testing, pathogenic genotypes were identified in most cases (>90%). The complexity and heterogeneity of mutations reinforces the need for a comprehensive approach. Because 32% of the mutations are newly described, the results show that a screening strategy based on known mutations would have solved less than 55% of the cases