Whole Transcriptome Profiling of Successful Immune Response to Vibrio Infections in the Oyster Crassostrea gigas by Digital Gene Expression Analysis

The cultivated Pacific oyster Crassostrea gigas has suffered for decades large scale summer mortality phenomenon resulting from the interaction between the environment parameters, the oyster physiological and/or genetic status and the presence of pathogenic microorganisms including Vibrio species. To obtain a general picture of the molecular mechanisms implicated in C. gigas immune responsiveness to circumvent Vibrio infections, we have developed the first deep sequencing study of the transcriptome of hemocytes, the immunocompetent cells. Using Digital Gene Expression (DGE), we generated a transcript catalog of up-regulated genes from oysters surviving infection with virulent Vibrio strains (Vibrio splendidus LGP32 and V. aestuarianus LPi 02/41) compared to an avirulent one, V. tasmaniensis LMG 20012T. For that an original experimental infection protocol was developed in which only animals that were able to survive infections were considered for the DGE approach. We report the identification of cellular and immune functions that characterize the oyster capability to survive pathogenic Vibrio infections. Functional annotations highlight genes related to signal transduction of immune response, cell adhesion and communication as well as cellular processes and defence mechanisms of phagocytosis, actin cytosqueleton reorganization, cell trafficking and autophagy, but also antioxidant and anti-apoptotic reactions. In addition, quantitative PCR analysis reveals the first identification of pathogen-specific signatures in oyster gene regulation, which opens the way for in depth molecular studies of oyster-pathogen interaction and pathogenesis. This work is a prerequisite for the identification of those physiological traits controlling oyster capacity to survive a Vibrio infection and, subsequently, for a better understanding of the phenomenon of summer mortality

Whole transcriptome profiling of successful defence response to Vibrio infections in Pacific oysters (Crassostrea gigas) using digital gene expression (DGE) analysis

L'huître creuse C. gigas a connu des épisodes récurrents de mortalités estivales. En 2009, l'ostréiculture a connu la plus grave crise écologique et économique jamais observée depuis l'introduction de cette espèce sur les côtes françaises. Les études réalisées précédemment dans le but de comprendre les causes de ces mortalités, attribuent une origine multifactorielle faisant intervenir, de manière concomitante, des paramètres environnementaux, des conditions physiologiques particulières de l'huître, en association à la présence de microorganismes pathogènes tels que les vibrions appartenant aux espèces V. aestuarianus, V. splendidus et le virus OsHV-1. Ainsi, l'ensemble des travaux menés au cours de cette thèse représentent une contribution à l'étude d'une réponse immunitaire efficace de l'huître creuse Crassostrea gigas aux infections vibrions pathogènes.Le travail de thèse qui vous a été présenté a été réalisé dans le but de progresser encore sur la compréhension de la réponse immunitaire de l'huître creuse. Ce travail nous a permis d'établir une cartographie des modifications transcriptionnelles par la technique DGE (Digital Gene Expression) et notamment par l'identification d'effecteurs ou mécanismes entrant en jeu dans une réponse efficace vis-à-vis des agents infectieux et conduisant à son élimination. Le modèle (Crassostrea gigas – V. aestuarianus/V. splendidus) s'est avéré adapté à cette étude. Les mécanismes identifiés au cours de cette thèse, devront être étudiés plus précisément, puisque la plupart des gènes surreprésentés chez les huîtres survivantes ont des fonctions putatives. Nous avons suggéré l'implication de ces gènes dans le processus de survie des huîtres au cours d'une réponse efficace aux infections bactériennes. Il est apparu nécessaire de développer des recherches autour des défenses de l'huître creuse C. gigas avec pour objectif le développement de stratégies destinées à limiter l'impact des maladies. A long terme, ces stratégies seront applicables soit en prophylaxie afin de détecter des déficiences et prévenir le développement de maladies, soit en sélection génétique pour le criblage de géniteurs présentant des capacités de défenses optimales.Aquatic organisms and particularly marine invertebrates, such as the oyster Crassostrea gigas, harbour an abundant and diverse microflora on their surface (epibiosis) or inside their tissues (endobiosis) where Vibrio splendidus is found as a dominant culturable Vibrio. With Evolution, oysters have developed effective systems for maintaining their homeostasis and discriminating the bacteria beneficial for their physiological fitness from the potentially harmful and pathogenic ones. However, for decades, the cultivated Pacific oyster C. gigas is suffering large scale summer mortality phenomenon that is reported in all areas of the world where this species is cultivated. Considerable effort has been invested in advanced genomic technologies to understand and characterize the major traits that govern the tolerance of oysters to stressful culture conditions and to pathogenic bacteria. In particular, immune-related genes have been characterized from C. giga. Briefly, a variety of antimicrobials have been fully characterized. Whereas most of these immune genes were shown to be modulated during infections, the molecular mechanisms by which the oyster can survive virulent Vibrio infections remained totally unknown. Here, our objective was to develop a better understanding of the genetic-level responses of oysters to pathogenic versus non pathogenic bacteria and to identify genes that are involved in physiological and immune responsiveness to circumvent the infections. In this attempt, we have performed a comprehensive analysis of the transcriptome of oyster immunity (hemocytes), using Digital Gene Expression (DGE). The study aimed to compare the expression profiles of the two libraries and, beyond gene identification and functional annotation, to explore the putative functions involved in the capability of oysters to circumvent and to survive infections. This is the first report on genome-wide transcriptional analysis of oyster survival-responsiveness

Évaluation des capacités de survie de l'huître creuse Crassostrea gigas suite à des infections bactériennes

L'huître creuse C. gigas a connu des épisodes récurrents de mortalités estivales. En 2009, l'ostréiculture a connu la plus grave crise écologique et économique jamais observée depuis l'introduction de cette espèce sur les côtes françaises. Les études réalisées précédemment dans le but de comprendre les causes de ces mortalités, attribuent une origine multifactorielle faisant intervenir, de manière concomitante, des paramètres environnementaux, des conditions physiologiques particulières de l'huître, en association à la présence de microorganismes pathogènes tels que les vibrions appartenant aux espèces V. aestuarianus, V. splendidus et le virus OsHV-1. Ainsi, l'ensemble des travaux menés au cours de cette thèse représentent une contribution à l'étude d'une réponse immunitaire efficace de l'huître creuse Crassostrea gigas aux infections vibrions pathogènes.Le travail de thèse qui vous a été présenté a été réalisé dans le but de progresser encore sur la compréhension de la réponse immunitaire de l'huître creuse. Ce travail nous a permis d'établir une cartographie des modifications transcriptionnelles par la technique DGE (Digital Gene Expression) et notamment par l'identification d'effecteurs ou mécanismes entrant en jeu dans une réponse efficace vis-à-vis des agents infectieux et conduisant à son élimination. Le modèle (Crassostrea gigas V. aestuarianus/V. splendidus) s'est avéré adapté à cette étude. Les mécanismes identifiés au cours de cette thèse, devront être étudiés plus précisément, puisque la plupart des gènes surreprésentés chez les huîtres survivantes ont des fonctions putatives. Nous avons suggéré l'implication de ces gènes dans le processus de survie des huîtres au cours d'une réponse efficace aux infections bactériennes. Il est apparu nécessaire de développer des recherches autour des défenses de l'huître creuse C. gigas avec pour objectif le développement de stratégies destinées à limiter l'impact des maladies. A long terme, ces stratégies seront applicables soit en prophylaxie afin de détecter des déficiences et prévenir le développement de maladies, soit en sélection génétique pour le criblage de géniteurs présentant des capacités de défenses optimales.Aquatic organisms and particularly marine invertebrates, such as the oyster Crassostrea gigas, harbour an abundant and diverse microflora on their surface (epibiosis) or inside their tissues (endobiosis) where Vibrio splendidus is found as a dominant culturable Vibrio. With Evolution, oysters have developed effective systems for maintaining their homeostasis and discriminating the bacteria beneficial for their physiological fitness from the potentially harmful and pathogenic ones. However, for decades, the cultivated Pacific oyster C. gigas is suffering large scale summer mortality phenomenon that is reported in all areas of the world where this species is cultivated. Considerable effort has been invested in advanced genomic technologies to understand and characterize the major traits that govern the tolerance of oysters to stressful culture conditions and to pathogenic bacteria. In particular, immune-related genes have been characterized from C. giga. Briefly, a variety of antimicrobials have been fully characterized. Whereas most of these immune genes were shown to be modulated during infections, the molecular mechanisms by which the oyster can survive virulent Vibrio infections remained totally unknown. Here, our objective was to develop a better understanding of the genetic-level responses of oysters to pathogenic versus non pathogenic bacteria and to identify genes that are involved in physiological and immune responsiveness to circumvent the infections. In this attempt, we have performed a comprehensive analysis of the transcriptome of oyster immunity (hemocytes), using Digital Gene Expression (DGE). The study aimed to compare the expression profiles of the two libraries and, beyond gene identification and functional annotation, to explore the putative functions involved in the capability of oysters to circumvent and to survive infections. This is the first report on genome-wide transcriptional analysis of oyster survival-responsiveness.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies

International audienceDiagnosis of dystrophinopathies needs to combine several techniques for detecting copy number variations (CNVs; two-thirds of mutations) and single nucleotide variations (SNVs). We participated in the design of an amplicon-based PCR kit (Multiplicom) for sequencing with a GS-Junior instrument (Roche) and later with a MiSeq instrument (Illumina). We compared two different software programs, MiSeq Reporter (Illumina) and SeqNext (JSI Medical Systems) for data analyses. Testing of six patient DNA samples carrying 72 SNVs in the DMD gene showed an experimental sensitivity of 91.7% with MiSeq Reporter, 98.6% with SeqNext, and >99.9% with both, demonstrating the need to use two different software programs. Analytical specificity was >98%. Fifty-eight additional patient DNAs were analyzed, and 25 deleterious mutations were identified, without false-negative results. We also tested the possibility for our protocol to identify CNVs. We performed additional next-generation sequencing experiments on 50 DNAs and identified 28 CNVs, all confirmed by multiple ligation probe amplification. Statistical analyses on amplicons without CNV (n = 3797), amplicons with heterozygous deletions (n = 51) or duplications (n = 191), and with hemizygous duplications (n = 63) showed a sensitivity and specificity of >99.9%. We implemented a strategy to simultaneously detect SNVs and CNVs in the DMD gene with one comprehensive technique, allowing considerable reduction of time and cost burden for diagnosis of dystrophinopathies

The importance of an integrated genotype-phenotype strategy to unravel the molecular bases of titinopathies

International audienc

A Reliable Targeted Next-Generation Sequencing Strategy for Diagnosis of Myopathies and Muscular Dystrophies, Especially for the Giant Titin and Nebulin Genes

International audienc

An Integrated Clinical-Biological Approach to Identify Interindividual Variability and Atypical Phenotype-Genotype Correlations in Myopathies: Experience on A Cohort of 156 Families

International audienceDiagnosis of myopathies is challenged by the high genetic heterogeneity and clinical overlap of the various etiologies. We previously reported a Next-Generation Sequencing strategy to identify genetic etiology in patients with undiagnosed Limb-Girdle Muscular Dystrophies, Congenital Myopathies, Congenital Muscular Dystrophies, Distal Myopathies, Myofibrillar Myopathies, and hyperCKemia or effort intolerance, using a large gene panel including genes classically associated with other entry diagnostic categories. In this study, we report the comprehensive clinical-biological strategy used to interpret NGS data in a cohort of 156 pediatric and adult patients, that included Copy Number Variants search, variants filtering and interpretation according to ACMG guidelines, segregation studies, deep phenotyping of patients and relatives, transcripts and protein studies, and multidisciplinary meetings. Genetic etiology was identified in 74 patients, a diagnostic yield (47.4%) similar to previous studies. We identified 18 patients (10%) with causative variants in different genes (ACTA1, RYR1, NEB, TTN, TRIP4, CACNA1S, FLNC, TNNT1, and PAPBN1) that resulted in milder and/or atypical phenotypes, with high intrafamilial variability in some cases. Mild phenotypes could mostly be explained by a less deleterious effect of variants on the protein. Detection of inter-individual variability and atypical phenotype-genotype associations is essential for precision medicine, patient care, and to progress in the understanding of the molecular mechanisms of myopathies