Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis

Arthrogryposis multiplex congenita (AMC) is characterized by the presence of multiple joint contractures resulting from reduced or absent fetal movement. Here, we report two unrelated families affected by lethal AMC. By genetic mapping and whole-exome sequencing in a multiplex family, a heterozygous truncating MAGEL2 mutation leading to frameshift and a premature stop codon (c.1996delC, p.Gln666Serfs∗36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfs∗7) and occurring de novo on the paternal allele of MAGEL2 was identified in the affected individual. In both families, RNA analysis identified the mutated paternal MAGEL2 transcripts only in affected individuals. MAGEL2 is one of the paternally expressed genes within the Prader-Willi syndrome (PWS) locus. PWS is associated with, to varying extents, reduced fetal mobility, severe infantile hypotonia, childhood-onset obesity, hypogonadism, and intellectual disability. MAGEL2 mutations have been recently reported in affected individuals with features resembling PWS and called Schaaf-Yang syndrome. Here, we show that paternal MAGEL2 mutations are also responsible for lethal AMC, recapitulating the clinical spectrum of PWS and suggesting that MAGEL2 is a PWS-determining gene

Développement de modèles in vitro et in vivo pour analyser la réponse aux radiations ionisantes du tissu thyroïdien normal humain

The only demonstrated etiologic factor for thyroid cancer is an exposure to ionizing radiation (IR) during childhood. Epidemiological studies can measure a significant risk to develop a cancer following thyroid doses above 50 mGy. This risk is observed after external exposure to high doses/dose rates (radiotherapy) and after contamination of iodine radioisotopes at lower doses.As the risk decreases with the dose, if radiation-induced cancers (R) develop after thyroid doses <50mGy, an excess of cancers cannot be measured by conventional epidemiology, especially because the incidence of sporadic cancers (S) increases. Consequently, it is not possible to answer the societal debates concerning the risk associated to low doses exposure, such as the risk for patients during medical diagnosis by imagery technics in the "head and neck" area especially for children, following Chernobyl and Fukushima accidents fallout, and following contamination after the atmospheric nuclear tests in French Polynesia.To answer the questionnning about the risk of low doses IR on human thyroid, a better knowledge of the exposure according to doses and dose rates is necessary,. Thyroid carcinogenesis presents specificities among species. Indeed, in mice, S or R thyroid cancers incidence is low, and the few R tumors developping are mainly follicular cancers whereas there are mainly papillary cancers in humans (S and R).In order to analyze the normal human thyroid tissue response to IR, we have developed several approaches using biopsies of non-pathological tissues from patients who have undergone thyroidectomy: primary thyrocyte cultures, in vitro 3D models (thyrospheres and organotypic cultures) and xenografts in mice.For 3D models, maintenance of polarity, cell differentiation, tissue complexity and physiological activity of the cultures were controlled. We obtained thyrospheres (6 donors) organized in follicles delimiting a lumen composed of thyroglobulin. We used the matrigel-based protocol described by Toda et al. (2002) for organotypic cultures of porcin tissue, and showed an hypoxic stress in the human tissue (7 donors). An optimal oxygenation of the tissue was obtained by air-liquid interface culture (3 donors). In ALI organotypic cultures, a secretion of free thyoid hormone T4 was observed (1 donor, at 1 week), and this protocol allowed the tissue maintenance over 4 weeks. Mouse xenografts permitted human tissue maintenance over a period of at least one year, we already succeed in maintaining the thyroid tissue over 5 weeks in SCID/beige mice (3 donors).In parallel, we compared the proliferation, the survival and the kinetics of DNA strand breaks induction/repair after exposure of thyrocytes from patients exposed (radiotherapy) during childhood (2 donors) or unexposed (3 donors), in primary cultures. We reproducibly observed that exposed thyrocytes are more radioresistant than unexposed thyrocytes. These results strongly suggest the existence of a long-term phenotypic signature of exposure to IR in normal thyroid tissue, consistent with the identification of molecular signatures discriminating exposed and unexposed normal thyroid tissue by C Ory and N Ugolin in the laboratory. As suggested by C Dupuy's team, this imprinting could be due to the development of a chronic oxidative stress following IR exposure in the thyroid.The models developed during this thesis will be essential to understand low and high doses IR effects and risks on the human thyroid. They will also be usefull to asses the effects of endocrine disruptors (ED) on human thyroid, alone or in cocktail and estimate if this ED exposure may modify the radiosensitivity of the thyroid. These models will be used to analyse the first steps on radiation-induced thyroid carcinogenesis as well as the origin of this persistent long-term exposure imprinting.Le seul facteur étiologique démontré pour le cancer de la thyroïde est une exposition aux radiations ionisantes (RI) durant l’enfance. Les études épidémiologiques mesurent un risque significatif de développer un cancer suite à l’exposition pour une dose reçue à la thyroïde d’au minimum 50 mGy, le risque étant proportionnel à la dose. Ce risque est observé après une exposition externe à fortes doses/débits de dose (radiothérapie) et après une contamination à de plus faibles doses de radioisotopes de l’iode. Si des cancers radio-induits (R) se développent à des doses <50mGy, l’excès de cancers ne peut pas être mesuré par l’épidémiologie conventionnelle, d’autant plus que l’incidence des cancers spontanés de la thyroïde (S) augmente. En conséquence, il n’est pas possible de répondre aux interrogations sociétales sur les risques pour la santé d’exposition aux faibles doses délivrées lors d’examens d’imagerie médicale dans la région « tête et cou » chez l’enfant, ou reçues suite aux retombées des accidents de Tchernobyl et Fukushima, et aux essais nucléaires atmosphériques en Polynésie française.Pour répondre à ces interrogations, une meilleure connaissance de l’effet des RI en fonction de la dose et du débit de dose sur la thyroïde humaine est nécessaire. La carcinogenèse thyroïdienne présente des spécificités d’espèces : chez la souris, l’incidence de cancers de la thyroïde S et R est faible, et les quelques tumeurs R qui se développent sont principalement des cancers folliculaires alors qu’ils sont majoritairement papillaires chez l’Homme (S et R).Pour analyser la réponse aux RI du tissu thyroïdien humain normal, nous avons développé plusieurs approches à partir de biopsies de tissu normal provenant de patients ayant subi une thyroïdectomie : des cultures primaires de thyrocytes, des modèles 3D in vitro (thyrosphères, cultures organotypiques) et des xénogreffes chez la souris.Pour les modèles 3D, le maintien de la polarité, de la différenciation cellulaire, de la complexité tissulaire et de l’activité physiologique des cultures ont été contrôlés. Nous avons obtenu des thyrosphères (6 donneurs) organisées en follicules délimitant un lumen composé de thyroglobuline. Nous avons développé des cultures organotypiques en matrigel selon le protocole publié par Toda et al. (thyroïde de porc, 2002) (7 donneurs), et montré un choc hypoxique dans les tissus. Une oxygénation optimale du tissu a été obtenue par une culture en interface air-liquide (ALI) (3 donneurs). En culture ALI, les biopsies sécrétent l’hormone thyroïdienne T4 en réponse à la TSH (1 donneur, 1 semaine). Ce protocole a permis un maintien du tissu sur 4 semaines. Les xénogreffes devraient permettre une analyse à des temps plus tardifs, et ont déjà permis le maintien du tissu sur 5 semaines sur des souris SCID/beige (3 donneurs).En parallèle, nous avons comparé la prolifération, la survie et la cinétique d’induction/réparation des cassures après exposition aux RI de cultures primaires de thyrocytes de patients exposés (radiothérapie) pendant l’enfance (2 donneurs) ou non exposés (3 donneurs). Nous observons de façon reproductible que les thyrocytes exposés sont plus radiorésistants que les thyrocytes non exposés. Ces résultats suggèrent fortement l’existence d’une empreinte à long terme d’exposition aux RI dans le tissu normal, en accord avec l’identification de signatures moléculaires discriminant le tissu normal exposé et non exposé par C Ory et N Ugolin au LCE. Comme suggéré par l’équipe de C Dupuy, cette empreinte pourrait être due à la mise en place d’un stress oxydatif chronique dans la thyroïde suite à l’exposition aux RI.Les modèles développés pendant cette thèse seront essentiels pour comprendre les effets et les risques des RI à faibles et fortes doses, associées ou non aux perturbateurs endocriniens, sur la thyroïde humaine, ainsi que les premières étapes de la carcinogenèse et l’origine de cette empreinte d’exposition persistante sur le long terme

Development of In Vitro and In Vivo Models To Analyze the Normal Human Thyroid Tissue Response to Ionizing Radiation

Le seul facteur étiologique démontré pour le cancer de la thyroïde est une exposition aux radiations ionisantes (RI) durant l’enfance. Les études épidémiologiques mesurent un risque significatif de développer un cancer suite à l’exposition pour une dose reçue à la thyroïde d’au minimum 50 mGy, le risque étant proportionnel à la dose. Ce risque est observé après une exposition externe à fortes doses/débits de dose (radiothérapie) et après une contamination à de plus faibles doses de radioisotopes de l’iode. Si des cancers radio-induits (R) se développent à des doses <50mGy, l’excès de cancers ne peut pas être mesuré par l’épidémiologie conventionnelle, d’autant plus que l’incidence des cancers spontanés de la thyroïde (S) augmente. En conséquence, il n’est pas possible de répondre aux interrogations sociétales sur les risques pour la santé d’exposition aux faibles doses délivrées lors d’examens d’imagerie médicale dans la région « tête et cou » chez l’enfant, ou reçues suite aux retombées des accidents de Tchernobyl et Fukushima, et aux essais nucléaires atmosphériques en Polynésie française.Pour répondre à ces interrogations, une meilleure connaissance de l’effet des RI en fonction de la dose et du débit de dose sur la thyroïde humaine est nécessaire. La carcinogenèse thyroïdienne présente des spécificités d’espèces : chez la souris, l’incidence de cancers de la thyroïde S et R est faible, et les quelques tumeurs R qui se développent sont principalement des cancers folliculaires alors qu’ils sont majoritairement papillaires chez l’Homme (S et R).Pour analyser la réponse aux RI du tissu thyroïdien humain normal, nous avons développé plusieurs approches à partir de biopsies de tissu normal provenant de patients ayant subi une thyroïdectomie : des cultures primaires de thyrocytes, des modèles 3D in vitro (thyrosphères, cultures organotypiques) et des xénogreffes chez la souris.Pour les modèles 3D, le maintien de la polarité, de la différenciation cellulaire, de la complexité tissulaire et de l’activité physiologique des cultures ont été contrôlés. Nous avons obtenu des thyrosphères (6 donneurs) organisées en follicules délimitant un lumen composé de thyroglobuline. Nous avons développé des cultures organotypiques en matrigel selon le protocole publié par Toda et al. (thyroïde de porc, 2002) (7 donneurs), et montré un choc hypoxique dans les tissus. Une oxygénation optimale du tissu a été obtenue par une culture en interface air-liquide (ALI) (3 donneurs). En culture ALI, les biopsies sécrétent l’hormone thyroïdienne T4 en réponse à la TSH (1 donneur, 1 semaine). Ce protocole a permis un maintien du tissu sur 4 semaines. Les xénogreffes devraient permettre une analyse à des temps plus tardifs, et ont déjà permis le maintien du tissu sur 5 semaines sur des souris SCID/beige (3 donneurs).En parallèle, nous avons comparé la prolifération, la survie et la cinétique d’induction/réparation des cassures après exposition aux RI de cultures primaires de thyrocytes de patients exposés (radiothérapie) pendant l’enfance (2 donneurs) ou non exposés (3 donneurs). Nous observons de façon reproductible que les thyrocytes exposés sont plus radiorésistants que les thyrocytes non exposés. Ces résultats suggèrent fortement l’existence d’une empreinte à long terme d’exposition aux RI dans le tissu normal, en accord avec l’identification de signatures moléculaires discriminant le tissu normal exposé et non exposé par C Ory et N Ugolin au LCE. Comme suggéré par l’équipe de C Dupuy, cette empreinte pourrait être due à la mise en place d’un stress oxydatif chronique dans la thyroïde suite à l’exposition aux RI.Les modèles développés pendant cette thèse seront essentiels pour comprendre les effets et les risques des RI à faibles et fortes doses, associées ou non aux perturbateurs endocriniens, sur la thyroïde humaine, ainsi que les premières étapes de la carcinogenèse et l’origine de cette empreinte d’exposition persistante sur le long terme.The only demonstrated etiologic factor for thyroid cancer is an exposure to ionizing radiation (IR) during childhood. Epidemiological studies can measure a significant risk to develop a cancer following thyroid doses above 50 mGy. This risk is observed after external exposure to high doses/dose rates (radiotherapy) and after contamination of iodine radioisotopes at lower doses.As the risk decreases with the dose, if radiation-induced cancers (R) develop after thyroid doses <50mGy, an excess of cancers cannot be measured by conventional epidemiology, especially because the incidence of sporadic cancers (S) increases. Consequently, it is not possible to answer the societal debates concerning the risk associated to low doses exposure, such as the risk for patients during medical diagnosis by imagery technics in the "head and neck" area especially for children, following Chernobyl and Fukushima accidents fallout, and following contamination after the atmospheric nuclear tests in French Polynesia.To answer the questionnning about the risk of low doses IR on human thyroid, a better knowledge of the exposure according to doses and dose rates is necessary,. Thyroid carcinogenesis presents specificities among species. Indeed, in mice, S or R thyroid cancers incidence is low, and the few R tumors developping are mainly follicular cancers whereas there are mainly papillary cancers in humans (S and R).In order to analyze the normal human thyroid tissue response to IR, we have developed several approaches using biopsies of non-pathological tissues from patients who have undergone thyroidectomy: primary thyrocyte cultures, in vitro 3D models (thyrospheres and organotypic cultures) and xenografts in mice.For 3D models, maintenance of polarity, cell differentiation, tissue complexity and physiological activity of the cultures were controlled. We obtained thyrospheres (6 donors) organized in follicles delimiting a lumen composed of thyroglobulin. We used the matrigel-based protocol described by Toda et al. (2002) for organotypic cultures of porcin tissue, and showed an hypoxic stress in the human tissue (7 donors). An optimal oxygenation of the tissue was obtained by air-liquid interface culture (3 donors). In ALI organotypic cultures, a secretion of free thyoid hormone T4 was observed (1 donor, at 1 week), and this protocol allowed the tissue maintenance over 4 weeks. Mouse xenografts permitted human tissue maintenance over a period of at least one year, we already succeed in maintaining the thyroid tissue over 5 weeks in SCID/beige mice (3 donors).In parallel, we compared the proliferation, the survival and the kinetics of DNA strand breaks induction/repair after exposure of thyrocytes from patients exposed (radiotherapy) during childhood (2 donors) or unexposed (3 donors), in primary cultures. We reproducibly observed that exposed thyrocytes are more radioresistant than unexposed thyrocytes. These results strongly suggest the existence of a long-term phenotypic signature of exposure to IR in normal thyroid tissue, consistent with the identification of molecular signatures discriminating exposed and unexposed normal thyroid tissue by C Ory and N Ugolin in the laboratory. As suggested by C Dupuy's team, this imprinting could be due to the development of a chronic oxidative stress following IR exposure in the thyroid.The models developed during this thesis will be essential to understand low and high doses IR effects and risks on the human thyroid. They will also be usefull to asses the effects of endocrine disruptors (ED) on human thyroid, alone or in cocktail and estimate if this ED exposure may modify the radiosensitivity of the thyroid. These models will be used to analyse the first steps on radiation-induced thyroid carcinogenesis as well as the origin of this persistent long-term exposure imprinting

Ocular Barriers and Their Influence on Gene Therapy Products Delivery

The eye is formed by tissues and cavities that contain liquids whose compositions are highly regulated to ensure their optical properties and their immune and metabolic functions. The integrity of the ocular barriers, composed of different elements that work in a coordinated fashion, is essential to maintain the ocular homeostasis. Specialized junctions between the cells of different tissues have specific features which guarantee sealing properties and selectively control the passage of drugs from the circulation or the outside into the tissues and within the different ocular compartments. Tissues structure also constitute selective obstacles and pathways for various molecules. Specific transporters control the passage of water, ions, and macromolecules, whilst efflux pumps reject and eliminate toxins, metabolites, or drugs. Ocular barriers, thus, limit the bioavailability of gene therapy products in ocular tissues and cells depending on the route chosen for their administration. On the other hand, ocular barriers allow a real local treatment, with limited systemic side-effects. Understanding the different barriers that limit the accessibility of different types of gene therapy products to the different target cells is a prerequisite for the development of efficient gene delivery systems. This review summarizes actual knowledge on the different ocular barriers that limit the penetration and distribution of gene therapy products using different routes of administration, and it provides a general overview of various methods used to bypass the ocular barriers

Chronic Systemic Dexamethasone Regulates the Mineralocorticoid/Glucocorticoid Pathways Balance in Rat Ocular Tissues

International audienceCentral serous chorioretinopathy (CSCR) is a retinal disease affecting the retinal pigment epithelium (RPE) and the choroid. This is a recognized side-effect of glucocorticoids (GCs), administered through nasal, articular, oral and dermal routes. However, CSCR does not occur after intraocular GCs administration, suggesting that a hypothalamic-pituitary-adrenal axis (HPA) brake could play a role in the mechanistic link between CSCR and GS. The aim of this study was to explore this hypothesis. To induce HPA brake, Lewis rats received a systemic injection of dexamethasone daily for five days. Control rats received saline injections. Baseline levels of corticosterone were measured by Elisa at baseline and at 5 days in the serum and the ocular media and dexamethasone levels were measured at 5 days in the serum and ocular media. The expression of genes encoding glucocorticoid receptor (GR), mineralocorticoid receptors (MR), and the 11 beta hydroxysteroid dehydrogenase (HSD) enzymes 1 and 2 were quantified in the neural retina and in RPE/ choroid. The expression of MR target genes was quantified in the retina (Scnn1A (encoding ENac-α, Kir4.1 and Aqp4) and in the RPE/choroid (Shroom 2, Ngal, Mmp9 and Omg, Ptx3, Plaur and Fosl-1). Only 10% of the corticosterone serum concentration was measured in the ocular media. Corticosterone levels in the serum and in the ocular media dropped after 5 days of dexamethasone systemic treatment, reflecting HPA axis brake. Whilst both GR and MR were downregulated in the retina without MR/GR imbalance, in the RPE/choroid, both MR/GR and 11β-hsd2/11β-hsd1 ratio increased, indicating MR pathway activation. MR-target genes were upregulated in the RPE/ choroid but not in the retina. The psychological stress induced by the repeated injection of saline also induced HPA axis brake with a trend towards MR pathway activation in RPE/ choroid. HPA axis brake causes an imbalance of corticoid receptors expression in the RPE/choroid towards overactivation of MR pathway, which could favor the occurrence of CSCR

Phenotypic spectrum and genomics of undiagnosed arthrogryposis multiplex congenita

Arthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families