8 research outputs found
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.Gln666Serfsâ36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfsâ7) 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 modÚles 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 Ă©tiologique dĂ©montrĂ© pour le cancer de la thyroĂŻde est une exposition aux radiations ionisantes (RI) durant lâenfance. Les Ă©tudes Ă©pidĂ©miologiques mesurent un risque significatif de dĂ©velopper un cancer suite Ă lâexposition pour une dose reçue Ă la thyroĂŻde dâau minimum 50 mGy, le risque Ă©tant proportionnel Ă la dose. Ce risque est observĂ© aprĂšs une exposition externe Ă fortes doses/dĂ©bits de dose (radiothĂ©rapie) et aprĂšs une contamination Ă de plus faibles doses de radioisotopes de lâiode. Si des cancers radio-induits (R) se dĂ©veloppent Ă des doses <50mGy, lâexcĂšs de cancers ne peut pas ĂȘtre mesurĂ© par lâĂ©pidĂ©miologie conventionnelle, dâautant plus que lâincidence des cancers spontanĂ©s de la thyroĂŻde (S) augmente. En consĂ©quence, il nâest pas possible de rĂ©pondre aux interrogations sociĂ©tales sur les risques pour la santĂ© dâexposition aux faibles doses dĂ©livrĂ©es lors dâexamens dâimagerie mĂ©dicale dans la rĂ©gion « tĂȘte et cou » chez lâenfant, ou reçues suite aux retombĂ©es des accidents de Tchernobyl et Fukushima, et aux essais nuclĂ©aires atmosphĂ©riques en PolynĂ©sie française.Pour rĂ©pondre Ă ces interrogations, une meilleure connaissance de lâeffet des RI en fonction de la dose et du dĂ©bit de dose sur la thyroĂŻde humaine est nĂ©cessaire. La carcinogenĂšse thyroĂŻdienne prĂ©sente des spĂ©cificitĂ©s dâespĂšces : chez la souris, lâincidence de cancers de la thyroĂŻde S et R est faible, et les quelques tumeurs R qui se dĂ©veloppent sont principalement des cancers folliculaires alors quâils sont majoritairement papillaires chez lâHomme (S et R).Pour analyser la rĂ©ponse aux RI du tissu thyroĂŻdien humain normal, nous avons dĂ©veloppĂ© plusieurs approches Ă partir de biopsies de tissu normal provenant de patients ayant subi une thyroĂŻdectomie : des cultures primaires de thyrocytes, des modĂšles 3D in vitro (thyrosphĂšres, cultures organotypiques) et des xĂ©nogreffes chez la souris.Pour les modĂšles 3D, le maintien de la polaritĂ©, de la diffĂ©renciation cellulaire, de la complexitĂ© tissulaire et de lâactivitĂ© physiologique des cultures ont Ă©tĂ© contrĂŽlĂ©s. Nous avons obtenu des thyrosphĂšres (6 donneurs) organisĂ©es en follicules dĂ©limitant un lumen composĂ© de thyroglobuline. Nous avons dĂ©veloppĂ© des cultures organotypiques en matrigel selon le protocole publiĂ© par Toda et al. (thyroĂŻde de porc, 2002) (7 donneurs), et montrĂ© un choc hypoxique dans les tissus. Une oxygĂ©nation optimale du tissu a Ă©tĂ© obtenue par une culture en interface air-liquide (ALI) (3 donneurs). En culture ALI, les biopsies sĂ©crĂ©tent lâhormone thyroĂŻdienne T4 en rĂ©ponse Ă la TSH (1 donneur, 1 semaine). Ce protocole a permis un maintien du tissu sur 4 semaines. Les xĂ©nogreffes devraient permettre une analyse Ă des temps plus tardifs, et ont dĂ©jĂ permis le maintien du tissu sur 5 semaines sur des souris SCID/beige (3 donneurs).En parallĂšle, nous avons comparĂ© la prolifĂ©ration, la survie et la cinĂ©tique dâinduction/rĂ©paration des cassures aprĂšs exposition aux RI de cultures primaires de thyrocytes de patients exposĂ©s (radiothĂ©rapie) pendant lâenfance (2 donneurs) ou non exposĂ©s (3 donneurs). Nous observons de façon reproductible que les thyrocytes exposĂ©s sont plus radiorĂ©sistants que les thyrocytes non exposĂ©s. Ces rĂ©sultats suggĂšrent fortement lâexistence dâune empreinte Ă long terme dâexposition aux RI dans le tissu normal, en accord avec lâidentification de signatures molĂ©culaires discriminant le tissu normal exposĂ© et non exposĂ© par C Ory et N Ugolin au LCE. Comme suggĂ©rĂ© par lâĂ©quipe de C Dupuy, cette empreinte pourrait ĂȘtre due Ă la mise en place dâun stress oxydatif chronique dans la thyroĂŻde suite Ă lâexposition aux RI.Les modĂšles dĂ©veloppĂ©s pendant cette thĂšse seront essentiels pour comprendre les effets et les risques des RI Ă faibles et fortes doses, associĂ©es ou non aux perturbateurs endocriniens, sur la thyroĂŻde humaine, ainsi que les premiĂšres Ă©tapes de la carcinogenĂšse et lâorigine de cette empreinte dâexposition 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 Ă©tiologique dĂ©montrĂ© pour le cancer de la thyroĂŻde est une exposition aux radiations ionisantes (RI) durant lâenfance. Les Ă©tudes Ă©pidĂ©miologiques mesurent un risque significatif de dĂ©velopper un cancer suite Ă lâexposition pour une dose reçue Ă la thyroĂŻde dâau minimum 50 mGy, le risque Ă©tant proportionnel Ă la dose. Ce risque est observĂ© aprĂšs une exposition externe Ă fortes doses/dĂ©bits de dose (radiothĂ©rapie) et aprĂšs une contamination Ă de plus faibles doses de radioisotopes de lâiode. Si des cancers radio-induits (R) se dĂ©veloppent Ă des doses <50mGy, lâexcĂšs de cancers ne peut pas ĂȘtre mesurĂ© par lâĂ©pidĂ©miologie conventionnelle, dâautant plus que lâincidence des cancers spontanĂ©s de la thyroĂŻde (S) augmente. En consĂ©quence, il nâest pas possible de rĂ©pondre aux interrogations sociĂ©tales sur les risques pour la santĂ© dâexposition aux faibles doses dĂ©livrĂ©es lors dâexamens dâimagerie mĂ©dicale dans la rĂ©gion « tĂȘte et cou » chez lâenfant, ou reçues suite aux retombĂ©es des accidents de Tchernobyl et Fukushima, et aux essais nuclĂ©aires atmosphĂ©riques en PolynĂ©sie française.Pour rĂ©pondre Ă ces interrogations, une meilleure connaissance de lâeffet des RI en fonction de la dose et du dĂ©bit de dose sur la thyroĂŻde humaine est nĂ©cessaire. La carcinogenĂšse thyroĂŻdienne prĂ©sente des spĂ©cificitĂ©s dâespĂšces : chez la souris, lâincidence de cancers de la thyroĂŻde S et R est faible, et les quelques tumeurs R qui se dĂ©veloppent sont principalement des cancers folliculaires alors quâils sont majoritairement papillaires chez lâHomme (S et R).Pour analyser la rĂ©ponse aux RI du tissu thyroĂŻdien humain normal, nous avons dĂ©veloppĂ© plusieurs approches Ă partir de biopsies de tissu normal provenant de patients ayant subi une thyroĂŻdectomie : des cultures primaires de thyrocytes, des modĂšles 3D in vitro (thyrosphĂšres, cultures organotypiques) et des xĂ©nogreffes chez la souris.Pour les modĂšles 3D, le maintien de la polaritĂ©, de la diffĂ©renciation cellulaire, de la complexitĂ© tissulaire et de lâactivitĂ© physiologique des cultures ont Ă©tĂ© contrĂŽlĂ©s. Nous avons obtenu des thyrosphĂšres (6 donneurs) organisĂ©es en follicules dĂ©limitant un lumen composĂ© de thyroglobuline. Nous avons dĂ©veloppĂ© des cultures organotypiques en matrigel selon le protocole publiĂ© par Toda et al. (thyroĂŻde de porc, 2002) (7 donneurs), et montrĂ© un choc hypoxique dans les tissus. Une oxygĂ©nation optimale du tissu a Ă©tĂ© obtenue par une culture en interface air-liquide (ALI) (3 donneurs). En culture ALI, les biopsies sĂ©crĂ©tent lâhormone thyroĂŻdienne T4 en rĂ©ponse Ă la TSH (1 donneur, 1 semaine). Ce protocole a permis un maintien du tissu sur 4 semaines. Les xĂ©nogreffes devraient permettre une analyse Ă des temps plus tardifs, et ont dĂ©jĂ permis le maintien du tissu sur 5 semaines sur des souris SCID/beige (3 donneurs).En parallĂšle, nous avons comparĂ© la prolifĂ©ration, la survie et la cinĂ©tique dâinduction/rĂ©paration des cassures aprĂšs exposition aux RI de cultures primaires de thyrocytes de patients exposĂ©s (radiothĂ©rapie) pendant lâenfance (2 donneurs) ou non exposĂ©s (3 donneurs). Nous observons de façon reproductible que les thyrocytes exposĂ©s sont plus radiorĂ©sistants que les thyrocytes non exposĂ©s. Ces rĂ©sultats suggĂšrent fortement lâexistence dâune empreinte Ă long terme dâexposition aux RI dans le tissu normal, en accord avec lâidentification de signatures molĂ©culaires discriminant le tissu normal exposĂ© et non exposĂ© par C Ory et N Ugolin au LCE. Comme suggĂ©rĂ© par lâĂ©quipe de C Dupuy, cette empreinte pourrait ĂȘtre due Ă la mise en place dâun stress oxydatif chronique dans la thyroĂŻde suite Ă lâexposition aux RI.Les modĂšles dĂ©veloppĂ©s pendant cette thĂšse seront essentiels pour comprendre les effets et les risques des RI Ă faibles et fortes doses, associĂ©es ou non aux perturbateurs endocriniens, sur la thyroĂŻde humaine, ainsi que les premiĂšres Ă©tapes de la carcinogenĂšse et lâorigine de cette empreinte dâexposition 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 11ÎČ-hsd2/11ÎČ-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
Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis
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