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Minier, Lisa. „Evaluation de la personnalité, du coping et de la régulation émotionnelle de patients atteints de Dystrophie Myotonique de type 1 (DM1)“. Thesis, Paris 10, 2019. http://faraway.parisnanterre.fr/login?URL=http://bdr.parisnanterre.fr/theses/intranet/2019/2019PA100112/2019PA100112.pdf.
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Le Dystrophie Myotonique de type 1 (DM1) est une maladie neuromusculaire aux atteintes multiples qui induisent notamment de l’émoussement affectif, de l’apathie, de l’hypersomnolence, de la fatigue, ainsi qu’un déficit de cognition sociale et de théorie de l’esprit. Nous avons évalué les traits de personnalité, le coping et la régulation émotionnelle de 60 patients atteints de DM1. In fine, il s’agissait de proposer, à partir de ces éléments, une prise en charge psychothérapeutique adaptée à leurs besoins. Concernant la personnalité, le résultat le plus frappant concerne la dimension N. Contrairement à ce que nous attendions (des scores élevés du fait de la maladie et de ses répercussions), les patients inclus dans notre échantillon obtiennent des scores similaires à ceux de notre groupe contrôle tout-venant. Nos résultats relatifs au coping témoignent d’une utilisation variée des 10 stratégies que nous avons évaluées. Toutefois, l’apathie et la motivation réduite ressortent comme des obstacles qui limiteraient leur mise en place pour faire face à la DM1. Enfin, l’apathie et la fatigue ne semblent pas influencer la régulation émotionnelle dans notre échantillon. De plus, la stratégie Réévaluation cognitive ne semble pas être impactée par la maladie, ce qui pourrait se révéler un atout important dans la préservation de la qualité de vie des patients malgré la progression de leurs atteintes. En termes de psychothérapie, une Thérapie Comportementale et Cognitive a été développée spécifiquement pour ces patients et apporte des résultats prometteurs. D’autres pistes de psychothérapies pourraient être intéressantes à explorer, notamment la thérapie d’acceptation et d’engagementMyotonic Dystrophy type 1 (DM1) is a neuromuscular disease with multiple impairments leading to blunted affect, apathy, hypersomnia, fatigue, social cognition deficit and theory of mind deficit. In this research, personality traits, coping, and emotion regulation of 60 DM1 patients were assessed. All this information will help us design DM1 adapted psychological care.Regarding personality, our main result is that patients show similar N scores to the healthy control group despite our expectations (high scores in relation with the severity of the disease and its complications). In the light of our coping results, it seems that DM1 patients are using a large variety of coping strategies. However, apathy and reduced motivation constitute obstacles for coping strategies. Finally, apathy and fatigue do not influence emotion regulation in our sample DM1. Furthermore, Cognitive reevaluation strategy seems preserved from the disease’s consequences. This strategy might be an important advantage in the preservation of quality of life in DM1, despite the disease progression. A DM1 specific Cognitive Behavioral Therapy showed promising results. Other psychotherapeutic approaches could be explored, namely Acceptance and Commitment Therapy
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De, Dea Diniz Damily. „The study of the consequences of serca1’s missplicing on muscle function in myotonic dystrophy type 1“. Electronic Thesis or Diss., Sorbonne université, 2018. http://www.theses.fr/2018SORUS569.
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La Dystrophie myotonique de type 1 (DM1) est une maladie neuromusculaire affectant notamment le muscle squelettique avec la présence d’une myotonie et d’une atrophie progressive et causée par une expansion anormale de triplets CTG dans le 3’UTR du gène DMPK. L’expression des ARN mutés induit la perte de fonction du facteur d’épissage MBNL1 et conduit à la réexpression de formes fœtales de certains transcrits dans les tissus adultes de patients DM1. J’ai développé un modèle de cellules musculaires exprimant de manière conditionnelle de 960 répétitions CTG interrompues, afin d’identifier des nouveaux mécanismes impliqués dans la dysfonction musculaire. Suite à l’expression ciblée d’ARN-960CUG dans des myotubes, une analyse du transcriptome montre la présence d’un certain nombre de fonction/processus biologiques typiques de la DM1. En revanche, l’induction de voies non associées à la DM1 et l’absence de phénotype suggèrent que notre modèle n’est pas approprié pour l’étude des mécanismes moléculaires. J’ai fait aussi une étude de l’impact du défaut d’épissage d’ATP2A1 (SERCA1), présent chez les patients DM1, sur la fonction musculaire. J’ai utilisé une approche antisens afin de favoriser l’exclusion de l’exon 22 d’Atp2a1 dans un muscle contrôle, conduisant à la réexpression de l’isoforme fœtal Serca1b. Chez la souris sauvage adulte, ce défaut provoque un ralentissement de la contraction et une perte de masse musculaire. Chez le poisson-zèbre, cette modification d’épissage provoque une altération de la locomotion. L’ensemble de ces résultats indique que la réexpression de Serca1b affecte la fonction musculaire et pourrait contribuer aux symptômes musculaires dans la DM1Myotonic Dystrophy Type 1 (DM1) is a neuromuscular disease that affects mainly the skeletal muscle with the presence of myotonia and progressive atrophy and is caused by abnormal CTG expansion in the 3'UTR of the DMPK gene. The expression of the mutated RNA induces the loss of function of the MBNL1 splicing factor and leads to the re-expression of fetal isoforms of certain transcripts in the adult tissues of DM1 patients. In order to identify new mechanisms involved in muscle dysfunction, I developed a model of muscle cells conditionally expressing 960 interrupted CTG repeats. Following the targeted expression of RNA-960CUG in myotubes, transcriptome analysis shows that despite the presence of functions/biological processes typical of DM1, the induction of non-DM1 associated pathways and the absence of phenotype suggest that this model is not appropriate for this study of molecular mechanisms. I also did a study of the impact of the ATP2A1 (SERCA1) misplicing, present in DM1 patients, on the muscular function. I used an antisense approach to promote the exclusion of exon 22 from Atp2a1 in the muscle of two animal models, leading to the reexpression of the Serca1b fetal isoform. The re-expression of Serca1b in the muscle of adult wild-type mice leads to a slowing contraction and a loss of muscle mass. In zebrafish, this modification on Atp2a1 splicing causes an alteration on the locomotion. All of these results indicate that reexpression of Serca1b affects muscle function and may contribute to muscle symptoms in DM1
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Lallemant, Louison. „Pathologie neuronale et gliale en lien avec les atteintes neurologiques de la dystrophie myotonique de type 1 (DM1)“. Electronic Thesis or Diss., Sorbonne université, 2023. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2023SORUS404.pdf.
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La dystrophie myotonique de type 1 (DM1) est une maladie neuromusculaire grave affectant de nombreux tissus et organes. Les manifestations neurologiques varient du dysfonctionnement exécutif chez les adultes aux déficits d'attention chez les enfants, en passant par une déficience intellectuelle sévère dans les cas congénitaux. Les manifestations neurologiques de la DM1 ont un impact important sur la vie quotidienne des patients et de leurs familles, et il n’existe actuellement aucun traitement pour cette maladie. La DM1 est causée par l'expansion anormale d'une répétition CTG dans le gène DMPK. Les transcrits DMPK mutés sont toxiques car ils s’accumulent dans le noyau de la cellule, perturbant l’activité d’importantes protéines de liaison à l’ARN. En conséquence, les cellules DM1 présentent un métabolisme anormal de l’ARN et une régulation anormale de nombreux transcrits en aval. Malgré les progrès dans la compréhension de la physiopathologie musculaire, les mécanismes de la maladie restent flous et méconnus dans le SNC. Nous ne savons toujours pas quels types de cellules et voies moléculaires sont principalement affectés dans le cerveau, ni comment ils contribuent aux symptômes neurologiques de la DM1. Afin d'étudier ce problème, notre laboratoire a développé un modèle murin transgénique de la DM1 : les souris DMSXL expriment des transcrits DMPK humain contenant plus de 1000 répétitions CUG dans plusieurs tissus, notamment dans le cerveau. Ces souris présentent des phénotypes comportementaux, électrophysiologiques et neurochimiques pertinents de la DM1. A l'aide de ce modèle murin, l'objectif de ma thèse était de mieux comprendre les mécanismes cellulaires et moléculaires impliqués dans les anomalies neuronales, mais aussi non neuronales liées aux atteintes neurologiques de la DM1. Je me suis d'abord concentrée sur la caractérisation des différents types cellulaires du cerveau des souris DMSXL. Une étude multi-omique a été réalisée sur les neurones, les astrocytes et les oligodendrocytes ces souris. Nos résultats, qui montrent que les cellules gliales sont davantage impactées par les répétitions CTG, ont permis de mieux comprendre les mécanismes cellulaires et moléculaires de la DM1 dans le SNC, mais surtout de souligner l'importance d'étudier non seulement les neurones, mais aussi les astrocytes et oligodendrocytes dans le contexte pathologique de la DM1. Je me suis ensuite impliquée dans l'étude de la pathologie des astrocytes dans la DM1. Nous avons ainsi démontré que les astrocytes DMSXL présentaient une ramification réduite et une adhésion cellulaire altérée, et avaient un fort impact négatif sur la neuritogenèse. Parallèlement, j'ai également participé à l'étude de l'altération oligodendrogliale dans la DM1. Nous avons constaté que l'ARN CUG toxique perturbe le programme moléculaire de différenciation des oligodendrocytes (OL), en association avec des modifications du transcriptome se produisant au cours de la transition des cellules progénitrices des oligodendrocytes (OPC) en OL et conduisant à une hypomyélinisation transitoire chez la souris. J'ai également étudié la pathologie neuronale chez la souris DMSXL. Nos résultats ont démontré que l’accumulation de foci d’ARN toxiques dans les neurones perturbe principalement la phosphorylation des protéines, ce qui semble conduire à des défauts morphologiques neuronaux associés à des troubles de la dynamique des vésicules et à des défauts de transport axonal.Les 3 types cellulaires du cerveau présentent donc des dommages importants dans le cadre de la DM1, qui pourraient avoir un impact sur des processus cruciaux du fonctionnement cérébral. En effet, nous avons démontré une altération de la neurotransmission et de la plasticité synaptique chez la souris DMSXL. Dans l'ensemble, mes travaux ont permis de mieux comprendre les mécanismes cellulaires et moléculaires spécifiques des astrocytes, des oligodendrocytes et des neurones dans la pathologie cérébrale de la DM1Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease affecting many tissues and organs. The debilitating neurological manifestations vary from executive dysfunction in adults, to attention deficits and low processing speed in pediatric patients, to severe intellectual disability in congenital cases. DM1 neurological manifestations have a profound impact on the daily life of patients and their families, and there is currently no treatment for this disease. DM1 is caused by the abnormal expansion of a CTG repeat in DMPK gene. Expanded DMPK transcripts are toxic because they accumulate in the cell nucleus, disrupting the activity of important RNA-binding proteins. As a consequence, DM1 cells show abnormal RNA metabolism and processing of many downstream transcripts. Despite progress in the understanding of the muscle pathophysiology, the disease mechanisms remain unclear in the CNS. We still do not know which cell types and molecular pathways are primarily affected in the brain and how they contribute to DM1 neurological symptoms. In order to investigate this problem, our laboratory has developed a transgenic mouse model of DM1: DMSXL mice express expanded human DMPK transcripts in multiple tissues, notably in the brain, and display relevant behavioral, electrophysiological and neurochemical phenotypes. Using this mouse model, the objective of my thesis was to better understand the cellular and molecular mechanisms involved in the neuronal and non-neuronal impairment linked to the neurological damages of DM1. I first focused on the characterization of the different cell types in the DMSXL brain. A multi-omics study was carried out on DMSXL neurons, astrocytes and oligodendrocytes. Our results, which show that glial cells are more impacted by CTG repeats, have allowed us to better understand the cellular and molecular mechanisms of DM1 in the CNS, but above all to emphasize the importance of studying not only the neurons, but also astrocytes and oligodendrocytes in the pathological context of DM1. I then got involved in the study of astrocyte pathology in DM1. We thus demonstrated that DMSXL astrocytes exhibited reduced ramification and impaired cell adhesion, and had a strong negative impact on neuritogenesis. In the same time, I also participated in the study of oligodendroglia impairment in DM1. We found that the toxic CUG RNA disrupts the molecular program of oligodendrocyte (OL) differentiation, impairing the transcriptome changes occurring during the oligodendrocyte precursor cells (OPC)-OL transition and leading to transient hypomyelination in mice. I also studied the neuronal pathology in DMSXL mice. Our results demonstrated that the accumulation of toxic RNA foci in neurons perturbs mainly protein phosphorylation, which seems to lead to neuronal morphological defects associated with vesicle dynamics impairment and axonal transport defects. The three main cell types of the brain therefore present significant damage in the context of DM1, which could have an impact on crucial processes of cerebral functioning. Indeed, we have demonstrated an alteration in neurotransmission and synaptic plasticity in DMSXL mice. All together my work has provided novel insight into the cell-specific mechanisms operating in DM1, demonstrating the implication of astrocyte, oligodendrocyte and neuron defects in a DM1mouse model, and contributing towards an integrative understanding of brain pathology
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Vergnol, Amélie. „Les isoformes CaVβ1 : rôle dans la formation de la jonction neuromusculaire et implication dans la physiopathologie de la Dystrophie Myotonique de type 1“. Electronic Thesis or Diss., Sorbonne université, 2024. http://www.theses.fr/2024SORUS305.
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Quatre protéines CaVβ (CaVβ1 à CaVβ4) sont connues comme des sous-unités régulatrices des canaux Ca2+ dépendants du voltage (Voltage-gated Ca2+ Channels, VGCC), chacune ayant des profils d'expression spécifiques selon leur fonction. Bien qu'elles soient principalement reconnues pour leur rôle dans la régulation des VGCC, les protéines CaVβ peuvent également agir indépendamment de ces canaux, en tant que régulateurs de l'expression génique. Parmi ces protéines, CaVβ1 est exprimée dans le muscle squelettique sous différentes isoformes. L'isoforme adulte constitutive, CaVβ1D, est localisée au niveau du sarcolemme et plus précisément à la triade, où elle joue un rôle crucial dans la régulation de CaV1 et ainsi du mécanisme de Couplage Excitation-Contraction (CEC), essentiel à la contraction musculaire. Dans cette thèse, nous nous sommes concentrés sur les isoformes embryonnaires/périnatales de CaVβ1, moins étudiées, y compris la CaVβ1E précédemment décrite. Nous avons étudié leurs rôles dans les systèmes neuromusculaire et musculaire. En effet, la protéine CaVβ1 s'est révélée essentielle au développement correct de la Jonction NeuroMusculaire (JNM), mais l'implication spécifique de ses isoformes y reste inconnue. Notre étude a donc évalué le rôle des isoformes CaVβ1 à différents stades de la formation et de la maturation/maintien de la JNM. Parallèlement, étant donné la dérégulation de CaVβ1 dans la dystrophie myotonique de type 1 (DM1), nous avons exploré son rôle fonctionnel dans ce contexte pathologique. Nous avons tout d'abord identifié CaVβ1A comme une isoforme exprimée pendant l'embryogenèse et les stades périnataux. Nos résultats ont révélé que l'expression des isoformes CaVβ1 est régulée par l'activation différentielle des promoteurs au cours du développement : un promoteur1 dans l'exon 1 contrôle l'expression de CaVβ1A/E, tandis qu'un promoteur2 dans l'exon 2B contrôle l'expression de CaVβ1D. Il est intéressant de noter qu'un endommagement du nerf déclenche une réactivation du promoteur1, conduisant à la réexpression des transcrits de CaVβ1A/E.De plus, nous avons découvert que les isoformes embryonnaires/périnatales de CaVβ1 sont essentielles pour la pré-empreinte in vitro des myotubes et que leur expression postnatale influence la maturation/maintien de la JNM. Dans le contexte pathologique de la DM1, nous avons observé une augmentation de l'expression de CaVβ1A/E, qui semble atténuer la myotonie, un symptôme caractéristique de cette pathologie. De plus, nous avons trouvé que la modulation de leur expression est liée aux protéines MBNL, centrales dans la physiopathologie de la DM1. En conclusion, ce travail de thèse a permis de clarifier les connaissances sur les différentes isoformes de CaVβ1 dans le muscle squelettique et d'apporter de nouveaux éléments sur leur rôle dans deux contextes indépendants : le développement de la JNM et de la physiopathologie de la DM1. Comprendre la régulation des isoformes protéiques de CaVβ1 dans le muscle squelettique est essentiel pour déchiffrer les mécanismes de l'homéostasie musculaire et potentiellement identifier de nouvelles approches thérapeutiques pour faire face aux pathologies musculairesFour CaVβ proteins (CaVβ1 to CaVβ4) are described as regulatory subunit of Voltage-gated Ca2+ channel (VGCC), each exhibiting specific expression pattern in excitable cells based on their function. While primarily recognized for their role in VGCC regulation, CaVβ proteins also function independently of channels, acting as regulators of gene expression. Among these, CaVβ1 is expressed in skeletal muscle as different isoforms. The adult constitutive isoform, CaVβ1D, is located at the sarcolemma and more specifically at the triad, where it plays a crucial role in regulating CaV1 to control Excitation-Contraction Coupling (ECC) mechanism, essential for muscle contraction.In this thesis, we further explored the less studied CaVβ1 isoforms, with a particular focus on embryonic/perinatal variants, including the previously described CaVβ1E. We investigated their roles in the neuromuscular and muscular systems. Indeed, CaVβ1 proteins have been showed as essential for NeuroMuscular Junction (NMJ) development, though the involvement of specific isoform remains unclear. Our investigation assessed the role of CaVβ1 isoforms at different stages of NMJ formation and maturation/maintenance. Additionally, given the deregulation of CaVβ1 in Myotonic Dystrophy Type 1 (DM1), we explored its functional role in this muscular pathological context.First, we identified CaVβ1A as another isoform expressed during embryogenesis and perinatal stages. Our findings revealed that CaVβ1 isoforms expressions are regulated by the differential activation of promoters during development: a promoter1 in exon 1 drives CaVβ1A/E expressions, while a promoter2 in exon 2B controls CaVβ1D expression. Interestingly, nerve damage in adult muscle triggers a shift toward the promoter1 activation and leading to the re-expression of CaVβ1A/E transcripts. Furthermore, we found that CaVβ1 embryonic/perinatal isoforms are critical for proper in vitro pre-patterning of myotubes and that their postnatal expressions influences NMJ maturation/maintenance. In the pathological context of DM1, we observed the increased expression of CaVβ1A/E, which appears to mitigate myotonia symptoms. In addition, we found that the modulation of their expression is linked with MBNL proteins, which are central in the pathophysiology of DM1. In conclusion, this thesis work has clarified knowledge of the various CaVβ1 isoforms in skeletal muscle and provides new insights into their role in two independent contexts of NMJ development and DM1 pathophysiology. Understanding CaVβ1 protein regulation in skeletal muscle is essential to decipher muscle homeostasis mechanisms and potentially identify new therapeutic targets to face muscular disorders
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Ney, Michel. „Rôle de l'inclusion de l'exon 7 de BIN1 dans la faiblesse musculaire des patients atteints de dystrophie myotonique“. Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ077/document.
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La dystrophie myotonique de type 1 (DM1), est une maladie génétique héréditaire affectant environ 1/8000 personnes. Les patients souffrant de DM1 développent essentiellement des troubles musculaires tels qu’une faiblesse et une atrophie musculaire. La cause de la DM1 est expliquée par la mutation du gène "DMPK". Lors de ma thèse, j’ai pu démontrer que l’épissage de l’ARNm BIN1 était altéré dans le muscle DM1. En effet, l’exon 7 de BIN1, qui est absent du muscle normal, est exprimé de façon aberrante chez les patients DM1. En utilisant un modèle murin, j’ai prouvé que l’expression forcée de l’exon 7 de BIN1 altérait simultanément la structure et la fonction du muscle. Nous avons notamment observés une diminution de la taille des fibres musculaires et une augmentation de la faiblesse musculaire, comparé à des souris normales. Par conséquent, ce travail aidera à la compréhension du mécanisme de la maladie et pourrait expliquer les causes de la faiblesse musculaire et de l’atrophieMyotonic dystrophy of type 1 (DM1), is an inherited genetic disease affecting around 1 in 8000 person. Patients suffering from DM1 develop essentially muscle disorders such as muscle weakness, muscle loss and atrophy. The cause of DM1 is explained by the mutation of a gene called “DMPK“.During my thesis, I discovered that the alternative splicing of BIN1 mRNA was altered in the muscle of DM1 patients. Indeed, the BIN1 exon 7, which is normally absent in healthy muscle, is aberrantly expressed in DM1 muscle. By using a mouse model, I found that the forced expression of BIN1 exon 7 was responsible of the alteration of both muscle structure and function. Notably, we found a decrease in muscle fibers area (atrophy) and an increase of muscle weakness, compared to wild-type mice. Therefore, this work will help in the understanding of the disease mechanism and could explain the causes of muscle weakness and atrophy, which have never been elucidated to this date
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Winblad, Stefan. „Myotonic dystrophy type 1 : cognition, personality and emotion /“. Göteborg : Göteborg University, Dept. of Psychology, 2006. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=015464022&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Haworth, Christine. „Understanding the pathogenesis of myotonic dystrophy type 1“. Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/478/.
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To identify the full range of targets and the pathogenic consequences, we sought to mimic the pathogenesis of myotonic dystrophy type 1 with temporal and spatial control: temporal to reproduce the developmental pathogenesis of the congenital form, and spatial to isolate tissue specific pathology. To do this, we attempted to use the Cre-lox system for the conditional expression of an EGFP reporter-linked expanded CUG repeat RNA in the mouse. Expression of the transgene was controlled by Cre excision of a transcriptional stop, placed upstream of the EGFP-expanded repeat open reading frame. The transgenes were constructed and tested successfully, and a normal length repeat transgenic line was established. Unfortunately generation of the expanded repeat line was not successful. The constructs were used to generate cell-culture models of DM1, in both human and murine cells, which mimicked the nuclear foci formation and MBNL1 co-localisation seen in patient cells. Expression of exogenous MBNL1/GFP fusion protein in this model resulted in an increase in the size of foci, indicating that MBNL1 protein is limiting within the cell, and may possibly play a protective role. The murine DM1 cell-culture model was used to investigate the effects of expanded CUG repeat expression on splicing within the transcriptome. The differential effect between 5 and 250 repeat RNA expression using Affymetrix whole transcript and exon arrays was compared. Using whole genome arrays, 6 genes were down-regulated and 128 upregulated. With exon arrays, 58 genes showed alternative exon usage. Six genes were selected for further bioinformatics analysis: MtmR4, which has possible neuromuscular involvement; Kcnk4, Narg1, Ttyh1 and Bptf, potentially related to brain development; and Cacna1c, a promising candidate for heart conductance defects and sudden death.
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Osborne, Robert J. „Caenorhabditis elegans models of myotonic dystrophy type 1“. Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408632.
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Langlois, Marc-André. „RNA-based gene therapies for myotonic dystrophy type 1“. Thesis, Université Laval, 2003. http://www.theses.ulaval.ca/2003/21404/21404.pdf.
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La dystrophie myotonique de type 1 (DM1) est une maladie neuromusculaire grave qui engendre une perte d’autonomie des patients et diminue leur espérance de vie. Cette maladie est la plus fréquente des dystrophies musculaires chez l’adulte avec une incidence mondiale d’une personne atteinte sur 15 000. Au Québec, cette maladie est d’une importance particulière, car elle touche une personne sur 500 dans les régions du Saguenay et de Charlevoix. La DM1 est causée par l’expansion du triplet CTG situé dans la région 3’ non-codante de la myotonine protéine kinase (DMPK). Toutefois, il a été démontré que la grande part des symptômes de la maladie seraient liés à l’accumulation nucléaire de l’ARNm de DMPK portant l’expansion. Ces ARNm mutés se lient à des facteurs nucléaires formant des foci dans les noyaux des cellules DM1, engendrant des effets toxiques sur le métabolisme cellulaire et sur l’épissage alternatif de certains ARNm.
Nos travaux avaient comme but premier d’évaluer si la destruction de l’ARNm mutant de DMPK dans des myoblastes provenant de muscle squelettique DM1 permettrait de rétablir certaines fonctions et caractéristiques normales dans ces cellules. Trois technologies à base d’ARNs: les antisens, les ribozymes et les shRNAs ont diminué avec succès ces niveaux d’ARN mutés. Les ARNs antisens et les ribozymes, contrairement aux shRNA, ont permis un ciblage préférentiel des ARNs mutés de DMPK dans le noyau de myoblastes DM1. Ceci permet donc de maintenir un niveau basal de la protéine DMPK dans les myoblastes, un détail important advenant l’utilisation de ces molécules en thérapie génique chez l’humain. En utilisant les ribozymes, nous avons diminué la quantité et l’intensité des foci ce qui a permis de libérer les facteurs cellulaires se liant aux expansions de CUG. Ceci a eu comme effet de corriger un défaut d’épissage alternatif dans le récepteur à l’insuline. En exprimant de longs antisenses à l’ARNm de DMPK par un oncorétrovirus, nous avons constaté une restauration de la fusion cellulaire, de la capture de glucose ainsi qu’une diminution de CUGBP, un facteur d’épissage alternatif. Nous avons également démontré que la surexpression de hnRNP H, un facteur d’épissage liant les expansions de CUG, permettait aussi de diminuer les niveaux de CUGBP et ainsi corriger le défaut d’épissage alternatif du récepteur à l’insuline. Ces résultats démontrent donc pour la première fois le lien direct entre la rétention des ARNs mutés, la déplétion nucléaire d’un facteur d’épissage s’y liant et l’exacerbation de certaines caractéristiques du phénotype DM1. La somme de nos observations a permis deux choses importantes: en premier lieu, d’établir un nouveau modèle détaillé expliquant la pathogenèse de la DM1. En second lieu, nos résultats ont permi de valider la pertinence de détruire spécifiquement les transcrits mutés de DMPK afin de développer une thérapie génique efficace pour la DM1.Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease that ultimately causes loss of mobility and premature death. DM1 is the most common muscular dystrophy in adults with a world wide incidence of 1 affected individual in every 15 000. This disease is of special relevance in the Saguenay and Charlevoix regions in Quebec, where 1 in every 500 individuals is a carrier of the mutation. DM1 is caused by the expansion of an unstable CTG trinucleotide repeat located in 3’UTR of the DMPK (DM protein kinase) gene. However, it has been shown that most DM1 symptoms are related to the nuclear retention of mutant DMPK mRNA. These mutant transcripts bind to nuclear proteins and form foci in DM1 cell nuclei. This is though to be the leading cause of metabolical disruptions and defective alternative splicing of several mRNAs observed in DM1 cells. Our main project objective was to evaluate whether destruction of mutant DMPK mRNA could restore normal phenotype features in DM1 human skeletal myoblasts. The use of three RNA-based approaches: antisense RNAs, ribozymes and shRNAs, all displayed significant reductions in mutant DMPK mRNA. Antisense RNAs and ribozymes, as opposed to shRNAs, allowed specific targeting and destruction of mutant DMPK mRNAs in the nucleus of DM1 myoblasts. This feature thus allows a basal level of DMPK protein expression which is of particular relevance in the advent of developing a gene therapy for DM1. Ribozymes were effective in reducing the number and intensity of foci present in the nucleus of the myoblasts, thus allowing the release of certain CUG-binding proteins. This resulted in restoration of the defective splicing of the insulin receptor mRNA. Antisense RNAs to the DMPK mRNA expressed by an oncoretrovirus restored myoblast fusion, glucose uptake and lowered nuclear levels of CUGBP, an alternative splicing factor. Over expression of hnRNP-H, an alternative splicing factor that we showed could bind to CUG repeats, also reduces expression of CUGBP and restores defective splicing of the insulin receptor. These results reveal for the first time the intricate link between mutant DMPK mRNA nuclear retention, depletion of a CUG-binding protein that is also a splicing factor and exacerbation of related DM1 features. In conclusion, our work has allowed to better define the mechanisms involved in DM1 pathogenesis and has validated the relevance of developing a gene therapy that specifically targets mutant DMPK mRNAs.
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Nasser, Khalidah K. „Genetic and symptomatic variations in Myotonic Dystrophy Type 1“. Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7874/.
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Myotonic dystrophy type 1 (DM1) is an extremely variable genetic disorder showing an autosomal dominant inheritance that is characterised by myotonia, insulin resistance, cardiac conduction defects and cataracts. It is caused by a trinucleotide repeat expansion of CTG sequence located in the 3’-untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19 at q13.3. The severity of symptoms ranges from mild adult onset to severe congenital form. A characteristic clinical feature of DM1 is anticipation phenomenon where disease severity increases and age of onset decreases over successive generations. The DM1 mutation is highly unstable in both the germline and soma, and showed to be an age-dependent, tissue-specific (skeletal muscles comprised the largest allele length of approximately thousand units) and expansion biased. The unaffected level of the repeat sequence falls between ~5-37 repeats whereas the disease associated range starts from ~50 repeats, reaching several thousand units. These properties account for the observed anticipation and contribute toward the tissue-specificity and progressive nature of the symptoms. The manifested phenotypes, symptoms severity and age at onset are extremely variable within and between families. This is mostly accounted for by the progenitor allele length (PAL) passed on from affected parents in addition to the level of somatic instability over time. Though, recent data have shown that additional sequence variations (CCG, CGG variant repeats) within the repeat and immediate flanking DNA are associated with additional symptomatic variation, modified stability and delayed age of onset. Furthermore, individual specific genetic factors have shown to be clustered within and between families as a heritable trait. Therefore, it has been verified that PAL, in addition to individual specific genetic variations are the main modifier of disease onset. More recently, it has been observed that mismatch repair (MMR) genes play a key role in modulating the dynamic of DM1 mutation, and subsequently impact on the age at onset. Therefore, these genes serve as powerful trans-acting modifiers of repeat instability and subsequent severity. Also, sequestration and up-regulation of RNA binding proteins (MBNL1, CELF1 respectively) against the trapped mutant transcripts are the hall mark of DM1 pathogenicity associated with alternative splicing defects that account for the variability of symptoms. Thus, sequence variations within these genes may underlie the genetic and phenotypic variability among DM1 patients. The current diagnostic test for DM1 only provides a qualitative value, and takes no account of the somatic instability and/or the presence of variations within or elsewhere in the genome. Thus, limited prognostic information is delivered to patients and their families. Although more elaborate genotyping approaches that measure the DM1 degree of instability was developed, they remain labour intensive, time consuming and are not suited to routine clinical diagnostics. In this project, we have evaluated the utility of more rapid and higher throughput next generation sequencing (NGS) technologies (Ion PGM and PacBio platforms) to simultaneously sequence the DM1 alleles of the Scottish patients, characterise the immediate flanking variants (5’-extra AAT and 5’-CCG variant repeats), elucidating the possible role of these variants on the DM1 instability, and finally sequencing the potential trans-acting modifiers in a massive customised panel (Ion AmpliSeq). Though, the accurate genotyping of the DM1 allele using NGS method remains challenging and cannot be used at the moment for accurate measurement of allele length. This is due to the sequencing biased nature towards shorter fragments resulting in differences of modal allele length measurement between PacBio and traditional SP-PCR methods. Additionally, Ion PGM platform was not successful at sequencing >20 CTG repeats. To correct for the sequencing biased distribution towards shorter alleles and distinguish between possible somatic variants from sequencing errors, safe sequencing (SafeSeq) method was conducted by tagging each original parental molecule with unique identifier (UID) sequences via PCR followed by sequencing using MiSeq platform. As the UID assignment was successful in tagging different population of repeats lengths, unfortunately we were not able to confidently differentiate between true somatic mutants from possible repeat slippage events in earlier cycles of PCR. Thus, it was decided to modify the incorporation of UID sequences using ligation based approach instead of PCR, and better optimise the method for more accurate results in the future. The identification of the immediate 5’-extra AAT flanking variant of the DM1 allele in a subset of the Scottish DM1 patients with and without CCG variant repeats has led us to speculate the possible presence of a new sub derived DM1 haplotype shared by a recent common ancestor in the Scottish population. In order to address this question, we were able to discriminate the normal allele haplotype of 11-13 repeats from >20 CTG haplotype among 18 DM1 patients whom were previously sequenced by Dr. Saeed Al ghamdi. These data have illustrated the most conserved haplotype around the DM1 allele. Therefore, the corresponding region was included in a customised Ion AmpliSeq sequencing panel for future larger scale haplotype analysis, in order to provide insights about future DM1 prevalence among the Scottish population. The data of this project highlighted the importance of using NGS technologies to characterise the structural pattern of the DM1 allele containing variants that may impact on symptom severity. It also showed the successful sequencing of trans-acting genetic modifiers in massive parallel fashion. Over larger scale analysis, these data could be used for better genotype-phenotype correlation and stratify patients in future clinical trials.
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Harrison, Eleanor G. „Redesign of trans-splicing molecules for the correction of dystrophia myotonica type 1 toxic RNA transcripts“. Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/honors/248.
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Dystrophia myotonica (DM1), one of the most common forms of muscular dystrophy, is caused by a repeated trinucleotide expansion in the DMPK gene. This mutation results in the accumulation of toxic cellular RNA transcripts. Spliceosome-mediated RNA trans-splicing (SMaRT) technology is a form of gene therapy that possesses the potential to correct these toxic RNA transcripts and thus cure the disease. Despite its promise, prior research applications of SMaRT technology to DM1 have been hampered by poor efficiency and have not been validated in a relevant model of the disease. In order to improve the efficiency of trans-splicing, this study examined the use of novel SMaRT molecules containing altered binding domains. These SMaRT molecules were tested in a clinically relevant cell model of DM1 and their corrective ability compared with that of a standard SMaRT molecule. The results were quantified by RT-PCR. The outcome of this study indicated the need to utilize more specific methods for measuring efficiency and for understanding the specific interactions of SMaRT molecules with target transcripts.
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Coldefy, Maurin Anne-Sophie. „Implication des voies de signalisation des MAPK, ERK1/2 et p38, dans la dystrophie myotonique de type 1 (DM1)“. Nice, 2006. http://www.theses.fr/2006NICE4059.
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Au cours de nos travaux de recherche, nous nous sommes principalement intéressés aux implications des voies de signalisation ERK1/2 et p38 dans la dystrophie myotonique de type ou DM1. La DM1 est la dystrophie musculaire la plus fréquente chez l’adulte. Cette pathologie multi-systémique atteint particulièrement les muscles squelettiques (myotonie, fonte musculaire progressive, altération de la différenciation musculaire). La DM1 est une maladie génétique à transmission autosomale dominante. Le défaut génétique est une insertion de triplets nucléotidiques CTG dans la région 3' non traduite du gène DMPK. Ce gène code pour une sérine/thréonine kinase, dont la fonction reste inconnue à ce jour. Les voies de signalisation des MAPK ERK1/2 et p38 jouent un rôle essentiel dans la transduction du signal et régulent de nombreuses fonctions cellulaires, notamment la différenciation musculaire. Nous avons montré que l'activation de ERK1/2 et p38 est significativement diminuée dans des biopsies de patients DM1. D’après nos études réalisées sur des souris transgéniques sur-exprimant ou invalidées pour le gène Dmpk, la diminution de l'activation de ERK1/2 et p38 ne semble pas corrélée à une diminution de l’expression de DMPK dans la DM1. Cependant, dans des cellules C2C12 exprimant des répétitions CUG en aval du gène de la GFP, l'activation de ERK1/2 et p38 est également diminuée. Ainsi, l’expression de transcrits contenant des triplets CUG répétés pourrait altérer l’activation des MAPK, ERK1/2 et p38, que nous avons observée dans la DM1. A ce jour, nous n’avons pu conclure sur un mécanisme précis d’altération des activations de ERK1/2 et p38 dans la DM1. Néanmoins, les outils que nous avons développés récemment et l’ensemble de nos résultats ouvrent de nouvelles perspectives pour une meilleure compréhension de l’implication des MAPK ERK1/2 et p38 dans la DM1, mais également de la régulation de Dmpk au cours de la différenciation musculaireThe aim of this work was to characterize a putative role of ERK1/2 and p38 MAPK in the myotonic dystrophy 1, called DM1. DM1, the most frequent dystrophy in adults, is a multi-systemic disorder which mainly affects skeletal muscles (myotonia, progressive wasting and weakness, delay in muscular differentiation). DM1 is an autosomal dominant inherited disease. The genetic mutation is an expansion of CTG trinucleotide repeats tract in the DMPK 3’ untranslated region. DMPK encodes a serine/threonine kinase but its function is still unknown. ERK1/2 and p38 MAPK signalling pathways play central and essential roles in cells physiology and are implicated in various cellular processes including muscular differentiation. We show that ERK1/2 and p38 activation is significantly diminished in muscular biopsies from DM1 patients. This diminished activation is not correlated with a diminution of DMPK expression in DM1, as we observed in transgenic mice, Dmpk knock-out or DMPK over-expressing mice. However, in C2C12 cells expressing CUG repeats in 3’ UTR of GFP, ERK1/2 and p38 activation is altered. In DM1, the diminution of ERK1/2 and p38 activation could be due to the expression of CUG repeats tract rather than to the decrease of DMPK expression. Our results and our recently developed molecular tools will enable us to further understand the implication of ERK1/2 and p38 MAPK in DM1 as well as Dmpk function in muscular differentiation
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Jimenez, Moreno Aura Cecilia. „Exploring outcome measures for adults with myotonic dystrophy type 1“. Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3933.
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Myotonic Dystrophy type 1 (DM1) is a multisystem progressive disorder with high heterogeneity. Novel emerging therapies require assessment tools that can effectively assess the effects of an intervention. The Outcome Measures in 5 Myotonic Dystrophy (OMMYD) Consortium has proposed a battery of functional outcome measures (FOM) identified as relevant for clinical trials in DM1. However, due to the variable nature of the disease and a scarcity of resources, there is a lack of systematic research that properly explores the use of these FOM. The current study examined three of these FOM and one extra related to 10 patients’ daily life performance. These are: (1) the ten-meters walk test; (2) the ten-meters walk/run test; (3) the 30-seconds sit and stand test; and, (4) a tri-axial accelerometer. By exploring the reliability, validity and responsiveness of these outcomes, we aimed to establish reference values and standard methodologies that could serve as guidance for clinical trials in DM1. A cohort of DM1 adults 15 screened for the two largest-to-date trials in DM1 (OPTIMSITIC and PHENO-DM1) were examined in relation to a set of pre-specified assessments and disease-burden scores. The results of this thesis supply disease-specific evidence of their validity, reliability and feasibility. The FOM, have shown to be psychometrically robust measures of functionality in DM1 and to be feasible for 20 clinical trials; they can provide a picture of patients’ muscle strength and perceived mobility and participation in life. The accelerometer can objectively quantify joints accelerations when walking at different speeds and summarise a DM1 patient’s habitual physical activity. The final choice of an outcome measure for a clinical trial in DM1 should be guided by disease domain that an intervention 25 is likely to impact on; but, a disease-specific study like this one will reduce the burden of protocol design whilst providing evidence supporting the decision-making process.
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Montero, Fernando Alberto Morales. „Somatic mosaicism and genotype-phenotype correlations in myotonic dystrophy type 1“. Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433224.
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Azimi, Mehrdad. „Analysis and Modulation of PACT, DICER and MBNL1 in the Context of Myotonic Dystrophy Type I“. Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35310.
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Myotonic Dystrophy Type I (DM1) is a multi-systemic genetic neuromuscular degenerative disease, has a prevalence in most populations of about 1:8000 and is caused by the nuclear retention of pathogenically expanded DMPK mRNA. A previous DM1 RNAi-kinome screen in our lab has identified kinases that reduced both count and area of DMPK mRNA foci in vitro.
One such discovered kinase is PACT, which has showed to decrease foci count and area in DM1 fibroblasts by 30-50%. This study explored PACT as well as binding partner DICER involved in cellular RNA processing machinery, to highlight potential therapeutic targets in DM1. DM1 fibroblasts treated with PACT siRNA showed a non-significant trend of upregulation in MBNL1 mRNA and protein expression. PACT knockdown also showed trend of missplicing normalization in SERCA-1, more prominently seen in DM1-2000 human fibroblasts, whereas IR (insulin receptor) splicing remained unaffected. On the other hand, DICER knockdown did not
have profound affect on foci integrity as well as MBNL1 RNA and protein xpressions in DM1 fibroblasts. SERCA-1 splicing in DICER siRNA treated samples also remained unchanged. We report here our findings in pursuit of potential therapeutic targets for the treatment of DM1.
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Loro, Emanuele Loro. „Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells“. Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3423200.
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Myotonic dystrophy type 1 (DM1) is caused by (CTG)n expansion in the 3’-untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating their functions (i.e. splicing regulation). Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy.
Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG)n range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and Western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG)n expansions were confirmed by long PCR and RNA fluorescence in-situ hybridization. Moreover, the DM1 myotubes displayed the splicing alteration of insulin receptor and MBNL1 genes associated to the DM1 phenotype.
Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Treatment with the pancaspase inhibitor Z-VAD significantly reduced the decrease in myonuclei number and in average width in15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenance-regeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis.La distrofia miotonica di tipo 1 (DM1) è causata dall'espansione (CTG)n nella regione trascritta ma non tradotta al 3' del gene DMPK. I trascritti mutati sono trattenuti in foci nucleari, i quali sequestrano diverse proteine leganti RNA spesso alterandone le funzioni (i.e. regolazione dello splicing). A livello del muscolo, i meccanismi patogenetici che portano a miotonia, debolezza e perdita di massa dei muscoli distali, non sono ad oggi chiari. Otto linee di mioblasti primari umani, ottenuti da biopsie di pazienti affetti da DM1 nelle forme adulta e congenita (range di espansione tra 90 e 1800 CTG), sono state differenziate ed innervate con successo, ottenendo miotubi in grado i contrarre. L'analisi morfologica e la quantificazione di diversi marker di miogenesi mediante RT-PCR e Western blotting, hanno indicato che il diferenziamento in vitro dei mioblasti primari DM1 è indistinguibile da quello ottenuto con mioblasti di controllo. In ciascuna linea DM1 è stata confermata l'espansione (CTG)n mediante long-PCR ed ibridizzazione in situ. Inoltre, nei miotubi DM1 è stato rilevata l'alterazione dello splicing del recettore per l'insulina e di MBNL1, caratteristica del fenotipo DM1. A 15 giorni di differenziamento, una considerevole perdita di miotubi DM1 ha suggerito l'attivazione di pathways catabolici, come confermato dalla presenza di marker di apoptosi (taglio proteolitico della caspasi 3, rilascio di citocromo c dai mitocondri, frammentazione della cromatina) e di autofagia (aumento dei livelli di LC3 lipidato e di P62). Il trattamento con l'inibitore delle caspasi Z-VAD si è dimostrato efficace nell'attenuare la riduzione del numero di mionuclei e del calibro medio dei miotubi a 15 giorni di differenziamento. Proponiamo quindi che la compromissione muscolare tipica della DM1 sia dovuta, più che ad un'alterata miogenesi, a problemi nei meccanismi di mantenimento/rigenerazione, che si esplicano attraverso la prematura attivazione di apoptosi e/o autofagia
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Mérola, Claudia Braida. „Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis“. Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/359/.
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Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterised by multiple tissue involvement and caused by an expansion of a (CTG)n repeat within the 3’-UTR of the DMPK gene (19q13.3). Normal individuals contain between 5 and 35 CTG repeats, whereas the repeats in DM1 patients expand in the range of 50 to several thousands. Longer alleles are very unstable and generally always increase in size when transmitted from parent to child, explaining the phenomenon of anticipation defined by earlier age of onset and an increase in the severity of the symptoms. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous, hereditary motor and sensory neuropathy of the peripheral nervous system. To date, 30 different loci have been mapped and mutations have been identified in more than 20 different genes. The DM1+CMT++ family is a very unusual three generation family in which all patients co-segregate both DM1 and CMT (LOD score = 7.03). It was postulated that either a single or two closely linked mutations near the APOC2 marker must be the cause of DM1 and CMT. Southern blot analysis of restriction digested genomic DNA revealed a fragment equivalent to a small CTG expansion (~200-400) at the DM1 locus, but an expanded allele could not be amplified by PCR. We postulated that the expanded repeats may have predisposed the repeat tract and the flanking regions to further DNA instability, leading to a secondary deletion, insertion and/or rearrangement. These novel mutations might modify the expression of DMPK and/or nearby genes explaining the unusual clinical presentation. To identify the lesion in the DM1+CMT++ family, a variety of molecular approaches was performed. The molecular lesion identified was an insertion of a GC rich region within the CTG repeats. The allele was comprised of a variable number of CTGs at the 5'-end followed by (GGC)3 G (CCG)20 (CCGCTG)14 (CTG)35. Analysis of single molecule separated alleles revealed 3 that the interrupted 3'-end of the array was stable, while the CTG repeats at the 5'-end were unstable. Postulated mechanisms to explain the DM1 and CMT symptoms in the family were: a novel RNA gain-of-function, and/or a novel effect on the downstream genes. Finding an imperfect CTG repeat allele in the DM1+CMT++ family led us to suggest that imperfect CTG repeat alleles may not be unique events and other DM1 patients may also contain similar alleles. To investigate this DNA samples from 14 DM1 patients with an unusual molecular diagnosis were analysed. The majority of these patients presented with an imperfect CTG repeat allele containing CCGCTG hexamers and/or CCG repeats. Five patients contained two or three higher order repeats containing between 18 and 30 bp such as ((CTG)5 (CCG)5), ((CTG)2 (CCGCTG)4) and ((CTG)5 (CCG)2 (CCGCTG)). These findings further suggest that imperfect CTG repeat alleles might not be as rare as was previously believed. The results of this project point out the importance of performing a more detailed molecular characterisation of the DM1 patients, which could lead to the provision of more accurate prognoses and the development of effective therapies.
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Braida, Claudia. „Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis“. Thesis restricted. Connect to e-thesis to view abstract, 2008. http://theses.gla.ac.uk/359/.
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Thesis (Ph.D.) - University of Glasgow, 2008.Ph.D. thesis submitted to the Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.
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Ueki, Junko. „Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability“. Kyoto University, 2018. http://hdl.handle.net/2433/230991.
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O'Reilly, Sean W. P. „RNAi Screening of the Kinome Identifies PACT as a Novel Genetic Modifier of Foci Integrity in Myotonic Dystrophy type 1“. Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30639.
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Myotonic Dystrophy type 1 (DM1), the most common form of adult muscular dystrophy (~1:8000) currently has no effective treatment. In DM1, expansion of a tri-nucleotide repeat in the 3' UTR of the DMPK gene results in DMPK mRNA hairpin structures, aggregating as insoluble ribonuclear foci. The resulting mis-regulation of important splicing factors, causes the inclusion of fetal exons in dozens of transcripts that contribute to the disease phenotype. In order to identify novel gene targets and kinase signalling pathways for potential therapeutics we have performed a high-throughput RNAi. RNA foci were visualized and quantified by in-situ hybridization. From our screen, we have identified a novel gene, PACT, as a modulator of foci integrity and that PACT knockdown can induce MBNL1 protein levels. The identified signalling complex represents a valid target for DM1 therapeutics. Our data further emphasizes the utility of RNAi screens in identifying disease-associated genes.
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Ballester, López Alfonsina. „Myotonic Dystrophy Type 1: the heterogeneity of a complex disease in a global research approach“. Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671860.
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Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion.
In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor Resumen de la Tesis:
Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion.
In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor CTG expansion found in muscle correlates with the age of disease onset of these patients. We studied as well the presence of interruptions in the CTG expansion and how these variant repeats can affect the genetic transmission in between generations and the phenotype of the patients. After analyzing a cohort of 49 DM1 patients we have seen that 10% of the patients carried CCG interruptions. Moreover, the presence of interruptions resulted, in one case, in a contraction of the CTG expansion in between generations, but also resulted in an expansion of the CTG track in the other case, linked to anticipation. As it has been previously described, we have seen that the presence of interruptions can be linked to non-typical phenotypic traits of DM1, such as proximal weakness. However, contrary of what is believed, we have seen that patients carrying interruptions can develop a clinical phenotype with a high severity.
We also studied the pathomolecular mechanism of RNA gain-of function, trough 3D imaging. We studied myoblasts cells derived from DM1 patients, analyzing the presence of RNA toxic aggregates. 3D imaging allowed us to have a full characterization of the cell, quantifying the number of the RNA toxic aggregates and studying the relationship between the main molecular players. We have seen that the CTG expansion leads the number of RNA foci that is accumulated in the cell and the number of the RNA foci correlates with the DMPK expression. We have seen as well that the RNA foci is not only present in the nucleus, but also in the cytoplasm, and the area of these toxic aggregates is related with its presence in the cytoplasm.
Finally, we studied the efficacy (reduction of RNA foci number) and toxicity (cell mortality) of a promising therapeutic approach for DM1, using antisense oligonucleotides with BNANC modifications, in cells derived from DM1 patients. We have studied the treatment response in three different subtypes, since due to the multisystemic nature of DM1, it is necessary to know how the different cells would react to a specific treatment. We have seen that the RNA foci reduction and the cell mortality is different in fibroblasts, lymphoblasts and myoblasts of Dm1 patients. Myoblasts, derived from one of the most affected tissues in DM1 patients, are the cells responding less to the treatment, which highlights the importance to perform experiments in order to improve the delivery in these cells. Moreover, we have seen that the treatment response seems to not be related with the CTG expansion size.
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Fortune, Maria Teresa. „Developmental timing and the role of cis and trans acting modifiers on CTG repeat instability in murine models“. Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341709.
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Wei, Christina. „The Role of GSK3ß-CUGBP1 Pathway in the Correction of Myotonic Dystrophy Type 1 Muscle Pathology“. University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1490350842490709.
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Malik, Naveed Altaf. „Development and analysis of CTG repeat expansion cell lines to understand molecular events in myotonic dystrophy type 1“. Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/53025/.
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Myotonic dystrophy type 1 (DM1) is a dominant human neuromuscular disorder caused by a CTG repeat expansion at the 3' end of the DMPK gene. Pathogenesis of DM1 is linked to a toxic gain of function due to mutant RNA and is manifested by nuclear retention of expanded CUG repeats and aberrant splicing. The development of an inducible model for DM1 with uninterrupted CTG repeats could help us to better understand early pathogenic changes in DM1 due to CUG repeat expansion. In the first part of this thesis, I report an inducible C2C12 mouse myoblast cell line in which the pTetOne inducible system was used to express 174 CTG repeats. This resulted in the production of RNA foci in 26% of cells. Efforts to make larger un-interrupted CTG repeats were unsuccessful due to their instability in the E.coli plasmid. In the second part of the thesis, I used the CRISPR/Cas9-induced genome-editing technique to knock-in an inducible promoter into the endogenous DMPK gene in a DM1 fibroblast cell line. For this, I employed two different CRISPR/Cas9 based strategies which exploit homology-directed repair (HDR) and non-homologous end joining (NHEJ). Our results suggest that CRISPR/Cas9 induced knock-in is enabled by non-homologous end joining more efficiently as compared to homology-directed repair. In the last part of the thesis, TruSeq RNA Sequencing was used to quantify the number of mutant DMPK transcripts and other molecular markers of DM1 pathogenesis that could be a valuable tool for the evaluation of the efficacy of therapeutic compounds. The sequencing results reveal the significant low abundance of mutant DMPK transcripts in the cytoplasmic fraction of DM1 lines and confirm the previously reported nuclear retention of mutant DMPK transcripts. We identify six potential genes which are dysregulated in DM1 fibroblasts and the absolute quantification of mutant DMPK transcripts along with these six reported dysregulated genes can be suitable biomarkers for disease severity and therapeutic response in DM1 fibroblasts. Additionally, these assays could be further refined to provide valuable tools to assess therapeutic compounds.
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Higham, Catherine F. „Dynamic DNA and human disease : mathematical modelling and statistical inference for myotonic dystrophy type 1 and Huntington disease“. Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4228/.
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Several human genetic diseases, including myotonic dystrophy type 1 (DM1) and Huntington disease (HD), are associated with inheriting an abnormally large unstable DNA simple sequence tandem repeat. These sequences mutate, by changing the number of repeats, many times during the lifetime of those affected, with a bias towards expansion. High repeat numbers are associated with early onset and disease severity. The presence of somatic instability compromises attempts to measure intergenerational repeat dynamics and infer genotype-phenotype relationships. Modelling the progression of repeat length throughout the lifetime of individuals has potential for improving prognostic information as well as providing a deeper understanding of the underlying biological process. Dr Fernando Morales, Dr Anneli Cooper and others from the Monckton lab have characterised more than 25,000 de novo somatic mutations from a large cohort of DM1 patients using single-molecule polymerase chain reaction (SM-PCR). This rich dataset enables us to fully quantify levels of somatic instability across a representative DM1 population for the first time. We establish the relationship between inherited or progenitor allele length, age at sampling and levels of somatic instability using linear regression analysis. We show that the estimated progenitor allele length genotype is significantly better than modal repeat length (the current clinical standard) at predicting age of onset and this novel genotype is the major modifier of the age of onset phenotype. Further we show that somatic variation (adjusted for estimated progenitor allele length and age at sampling) is also a modifier of the age of onset phenotype. Several families form the large cohort, and we find that the level of somatic instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. We develop new mathematical models, the main focus of this thesis, by modifying a previously proposed stochastic birth process to incorporate possible contraction. A Bayesian likelihood approach is used as the basis for inference and parameter estimation. We use model comparison analysis to reveal, for the first time, that the expansion bias observed in the distributions of repeat lengths is likely to be the cumulative effect of many expansion and contraction events. We predict that mutation events can occur as frequently as every other day, which matches the timing of regular cell activities such as DNA repair and transcription, but not DNA replication. Mutation rates estimated under the models described above are lower than expected among individuals with inherited repeat lengths less than 100 CTGs, suggesting that these rates may be suppressed at the lower end of the disease causing range. We propose that a length-specific effect may be operating within this range and test this hypothesis by introducing such an effect into the model. To calibrate this extended model, we use blood DNA data from DM1 individuals with small alleles (inherited repeat lengths less than 100 CTGs) and buccal DNA from HD individuals who almost always have inherited repeat lengths less than 100 CAGs. These datasets comprise single DNA molecules sized using SM-PCR. We find statistical support for a general length-specific effect which suppresses mutational rates among the smaller alleles and gives rise to a distinctive pattern in the repeat length distributions. In a novel application of this new model, fitted to a large cohort of DM1 individuals, we also show that this distinctive pattern may help identify individuals whose effective repeat length, with regards to somatic instability, is less than their actual repeat length. A plausible explanation for this distinction is that the expanded repeat tract is compromised by interruptions or other unusual features. For these individuals, we estimate the effective repeat length of their expanded repeat tracts and contribute to the on-going discussion about the effect of interruptions on phenotype. The interpretation of the levels of somatic instability in many of the affected tissues in the triplet repeat diseases is hindered by complex cell compositions. We extend our model to two cell populations whose repeat lengths have different rates of mutation (fast and slow). Swami et al. have recently characterised repeat length distributions in end stage HD brain. Applying our model, we infer for each frontal cortex HD dataset the likely relative weight of these cell populations and their corresponding contribution towards somatic variation. By comparison with data from laser captured single cells we conclude that the neuronal repeat lengths most likely mutate at a higher rate than glial repeat lengths, explaining the characteristic skewed distributions observed in mixed cell tissue from the brain. We confirm that individual-specific mutation rates in neurons are, in addition to the inherited repeat length, a modifier of age of onset. Our results support a model of disease progression where individuals with the same inherited repeat length may reach age of onset, as much as 30 years earlier, because of greater somatic expansions underpinned by higher mutational rates. Therapies aimed at reducing somatic expansions would therefore have considerable benefits with regard to extending the age of onset. Currently clinical diagnosis of DM1 is based on a measure of repeat length from blood cells, but variance in modal length only accounts for between 20 - 40% of the variance in age of onset and, therefore, is not a an accurate predictive tool. We show that in principle progenitor allele length improves the inverse correlation with age of onset over the traditional model length measure. We make use of second blood samples that are now available from 40 DM1 individuals. We show that inherited repeat length and the mutation rates underlying repeat length instability in blood, inferred from samples at two time points rather than one, are better predictors of age of onset than the traditional modal length measure. Our results are a step towards providing better prognostic information for DM1 individuals and their families. They should also lead to better predictions for drug/therapy response, which is emerging as key to successful clinical trials. Microsatellites are another type of tandem repeat found in the genome with high levels of intergenerational and somatic mutation. Differences between individuals make microsatellites very useful biomarkers and they have many applications in forensics and medicine. As well as a general application to other expanded repeat diseases, the mathematical models developed here could be used to better understand instability at other mutational hotspots such as microsatellites.
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Dincã, Diana Mihaela. „Mechanisms of brain dysfunction in myotonic dystrophy type 1 : impact of the CTG expansion on neuronal and astroglial physiology“. Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB054/document.
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La dystrophie myotonique de type 1 (DM1), ou maladie de Steinert, est une maladie qui touche plusieurs tissus, dont le système nerveux central (SNC). L’atteinte neurologique est variable et inclut des troubles de la fonction exécutive, des changements de comportement et une hypersomnolence dans la forme adulte, ainsi qu’une déficience intellectuelle marquée dans la forme congénitale. Dans leur ensemble, les symptômes neurologiques ont un fort impact sur le parcours académique, professionnel et les interactions sociales. Aujourd’hui aucune thérapie n’existe pour cette maladie. La DM1 est due à une expansion anormale d’un triplet CTG non-codant dans le gène DMPK. Les ARN messagers DMPK, porteurs de l’expansion, s’accumulent dans le noyau des cellules (sous forme de foci) et perturbent la localisation et la fonction de protéines de liaison à l’ARN, notamment des familles MBNL et CELF, ce qui entraîne des défauts d’épissage alternatif, d’expression, de polyadenylation et de localisation d’autres ARN cibles. Malgré le progrès récent dans la compréhension des mécanismes de la maladie, les aspects cellulaires et moléculaires de l’atteinte neurologique restent méconnus: nous ne connaissons ni la contribution de chaque type cellulaire du cerveau, ni les voies moléculaires spécifiquement dérégulées dans chaque type cellulaire. L’objectif de ma thèse a été de répondre à ces deux questions importantes en utilisant un modèle de souris transgéniques et des cellules primaires dérivées de celui-ci. Pour mon projet, j’ai utilisé les souris DMSXL générées par mon laboratoire. Ces souris reproduisent des caractéristiques importantes de la DM1, notamment l’accumulation des ARN toxiques et la dérégulation de l’épissage alternatif dans plusieurs tissus. L’impacte fonctionnel des transcrits DMPK toxiques dans le SNC des souris DMSXL se traduit par des problèmes comportementaux et cognitifs et par des défauts de la plasticité synaptique. Afin d’identifier les mécanismes moléculaires associés à ces anomalies, une étude protéomique globale a montré une dérégulation de protéines neuronales et astrocytaires dans le cerveau des souris DMSXL. De plus, l’étude de la distribution des foci d’ARN dans les cerveaux des souris et des patients a montré un contenu plus élevé dans les astrocytes par rapport aux neurones. Ensemble, ces résultats suggèrent une contribution à la fois neuronale et gliale dans la neuropathogenèse de la DM1. L’étude protéomique globale des cerveaux des souris DMSXL, a aussi montré des défauts de protéines synaptiques spécifiques des neurones, que nous avons par la suite validés dans le cerveau des patients. SYN1 est hyperphosphorylée d’une façon CELF-dépendante et RAB3A est surexprimé en réponse à l’inactivation de MBNL1. Les protéines MBNL et CELF régulent l’épissage alternatif d’un groupe de transcrits au cours du développement, et leur dérégulation dans la DM1 entraîne l’expression anormale d’isoformes d’épissage embryonnaires dans le tissu adulte. Dans ce contexte, j’ai étudié si les défauts des protéines RAB3A et SYN1 sont associés à une dérégulation d’épissage, et si les anomalies des protéines synaptiques identifiées dans la DM1 reproduisent des évènements embryonnaires de la régulation de RAB3A et SYN1. Mes résultats indiquent que les défauts de ces protéines dans les cerveaux adultes ne sont pas dus à une altération de l’épissage alternatif des transcrits et ne recréent pas des évènements embryonnaires. La neuropathogenèse de la DM1 va, donc, au delà de la dérégulation de l’épissage et d’autres voies moléculaires restent à explorer dans les cerveaux DM1. Afin d’identifier des sous-populations cellulaires susceptibles à l’accumulation des ARN toxiques, nous avons étudié la distribution des foci dans plusieurs régions cérébrales. (...)Myotonic dystrophy type 1 (DM1) is a severe disorder that affects many tissues, including the central nervous system (CNS). The degree of brain impairment ranges from executive dysfunction, attention deficits, low processing speed, behavioural changes and hypersomnia in the adult form, to pronounced intellectual disability in the congenital cases. The neurological manifestations have a tremendous impact on the academic, professional, social and emotional aspects of daily life. Today there is no cure for this devastating condition. DM1 is caused by the abnormal expansion of a CTG trinucleotide repeat in the 3’UTR of the DMPK gene. Expanded DMPK transcripts accumulate in RNA aggregates (or foci) in the nucleus of DM1 cells, disrupting the activity of important RNA-binding proteins, like the MBNL and CELF families, and leading to abnormalities in alternative splicing, gene expression, RNA polyadenylation, localisation and translation. In spite of recent progress, fundamental gaps in our understanding of the molecular and cellular mechanisms behind the neurological manifestations still exist: we do not know the contribution of each cell type of the CNS to brain dysfunction, or the molecular pathways specifically deregulated in response to the CTG expansion. The aim of my PhD project has been to gain insight into these two important questions using a relevant transgenic mouse model of DM1 and cell cultures derived thereof. In my studies I used the DMSXL mice, previously generated in my host laboratory. The DMSXL mice express expanded DMPK mRNA with more than 1,000 CTG repeats. They recreate relevant DM1 features, such as RNA foci and missplicing in multiple tissues. The functional impact of expanded DMPK transcripts in the CNS of DMSXL mice translates into behavioural and cognitive abnormalities and defective synaptic plasticity. To identify the molecular mechanisms behind these abnormalities, a global proteomics analysis revealed changes in both neuron-specific and glial-specific proteins in DMSXL brain. We also investigated RNA foci in DMSXL and human DM1 brains and found non-homogenous distribution between cell types, with a higher foci content in astrocytes relative to neurons. Together these results suggest that both neuronal and glial defects contribute to DM1 neuropathogenesis. The global proteomics analysis of DMSXL brains also identified abnormalities in neuronal synaptic proteins that we have validated in human brain samples. SYN1 is hyperphosphorilated in a CELF-dependent manner while RAB3A is upregulated in association with MBNL1 depletion. CELF and MBNL proteins regulate the alternative splicing of a subset of transcripts throughout development, and their deregulation in DM1 leads to abnormal expression of fetal splicing isoforms in adult DM1 brains. In this context, I have studied if RAB3A and SYN1 deregulations observed in adult brains are associated with splicing abnormalities or if they recreated embryonic expression and phosphorylation events. My results indicate that the synaptic proteins abnormalities observed in adult DMSXL brains are not caused by defective alternative splicing and do not recreate embryonic events. Thus, DM1 neuropathogenesis goes beyond missplicing and other molecular pathways must be explored in DM1 brains. To better understand the cellular sub-populations susceptible of accumulating toxic RNA foci we have studied foci distribution in different brain regions. We identified pronounced accumulation of toxic RNAs in Bergman astrocytes of DMSXL mice cerebellum and DM1 patients, associated with neuronal hyperactivity of Purkinje cells. A quantitative proteomics analysis revealed a significant downregulation of GLT1 – a glial glutamate transporter expressed by the Bergmann cell in the cerebellum. I have confirmed the GLT1 downregulation in other brain regions of mouse and human brain. (...)
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Ramon, Duaso Carla 1987. „Neurobiological correlates of behavioural alterations in mouse models of MBNL2 deficiency and pharmacological interventions : relevance for myotonic dystrophy type 1“. Doctoral thesis, Universitat Pompeu Fabra, 2019. http://hdl.handle.net/10803/668245.
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Myotonic dystrophy type 1 (DM1) is a rare disease characterized by muscular defects, as well as, cognitive and mood alterations. Although these neurophysiological disturbances are very debilitating, there is lack of data on the neurobiological mechanisms involved, and no specific treatment is available. Here, we investigated the neurobiological correlates underlying MBNL2 loss-of-function using two transgenic mouse lines: (i) constitutive Mbnl2 knockout (KO) mice and (ii) tissue-specific KO mice, where Mbnl2 expression is selectively deleted in the glutamatergic neurons of the forebrain. We demonstrated that both models exhibit long-term memory recognition deficits and a depressive-like state associated with increased microglia and altered neurochemical levels. Moreover, chronic treatments with the psychostimulant methylphenidate and the atypical antidepressant mirtazapine attenuated these behavioural alterations through a reduction of pro-inflammatory microglial overexpression and restoring the levels of several neurotransmitters and its receptors. Together, these data reveal new insights into the neurobiology of DM1 and provide evidence that methylphenidate and mirtazapine could be novel potential candidates to alleviate the debilitating symptoms related to the central nervous system in patients with DM1.La distrofia miotónica tipo 1 (DM1) es una enfermedad rara caracterizada por defectos musculares, así como por alteraciones cognitivas y afectivas. Aunque estas alteraciones neurofisiológicas son muy debilitantes, faltan datos sobre los mecanismos neurobiológicos involucrados, y no hay un tratamiento específico disponible. Aquí, investigamos los correlatos neurobiológicos subyacentes a la pérdida de función de MBNL2 utilizando dos líneas de ratones transgénicos: (i) ratones knockout (KO) constitutivos del gen Mbnl2 y (ii) ratones KO condicionales con una deleción específia del gen Mbnl2 en las neuronas glutamatérgicas del cerebro anterior. Los resultados demostraron que ambos modelos exhiben déficits de reconocimiento de memoria a largo plazo y un estado depresivo, asociado con un aumento de la microglia y niveles neuroquímicos alterados. Además, los tratamientos crónicos con el psicoestimulante metilfenidato y el antidepresivo atípico mirtazapina, atenuaron estas alteraciones del comportamiento mediante una reducción de la sobreexpresión microglial proinflamatoria y el restablecimiento de los niveles de varios neurotransmisores y sus receptores. Juntos, estos datos aportan nuevos conocimientos sobre la neurobiología de DM1 y proporcionan evidencia de que el metilfenidato y la mirtazapina podrían ser nuevos candidatos potenciales para aliviar los síntomas debilitantes relacionados con el sistema nervioso central en pacientes con DM1.
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Tran-Ladam, Hélène. „Mécanismes moléculaires associés aux changements d'épissage de Tau dans une Tauopathie, la dystrophie myotonique de type 1“. Thesis, Lille 2, 2010. http://www.theses.fr/2010LIL2S037/document.
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La pathologie Tau est une lésion neuronale commune à plus d’une vingtaine de maladies neurodégénératives. Elle correspond à l’agrégation des protéines Tau anormalement modifiées. Les mécanismes moléculaires impliqués dans l’agrégation de Tau demeurent encore mal compris. Toutefois, parmi les différentes hypothèses étiologiques, celle d’une dérégulation de l’épissage alternatif de Tau nous intéresse tout particulièrement. Ici, nous considérons la dystrophie myotonique de type 1 (DM1) comme maladie « modèle » pour étudier cette relation, puisqu’elle présente à la fois une dérégulation de l’épissage alternatif de Tau et des agrégats Tau. La DM1 est la forme adulte la plus fréquente de dystrophie musculaire. Il s’agit d’une maladie héréditaire à transmission autosomale dominante caractérisée par des répétitions CTGn>50 instables localisées dans la région 3’UTR du gène DMPK. Les mécanismes impliqués supposent un gain de fonction toxique des ARN mutés conduisant à une modification de l’épissage alternatif de nombreux transcrits parmi lesquels Tau. Dans ce contexte, nos objectifs étaient 1) de caractériser le défaut d’épissage de Tau dans le cerveau de plusieurs cas DM1 2) de modéliser ce défaut d’épissage afin d'identifier les facteurs trans-régulateurs impliqués et 3) de proposer une approche visant à restaurer un épissage normal. Le défaut d’épissage de Tau a été observé dans tous les cas analysés. Celui de l’exon 10, en revanche, n’a été rapporté que chez deux cas, qui, de façon intéressante, présentaient également une augmentation de l’expression des protéines CELF, décrites comme protéines régulatrices de l’épissage de Tau. Outre les protéines CELF, nous nous sommes également intéressés à MBNL1. MBNL1 est un facteur d’épissage jouant un rôle essentiel dans la physiopathologie de la DM1 où il a été décrit comme séquestré dans les foci. Peu de choses sont connues sur MBNL1 dans le cerveau et sur son rôle sur l’épissage alternatif des transcrits neuronaux. Ici, nous montrons que le niveau d’expression cérébrale de MBNL1 ne varie pas entre les cas DM1 et contrôles. En revanche, nous montrons que son épissage alternatif est dérégulé dans le cerveau. Notre étude de relation entre la structure et la fonction de la protéine suggère que ce changement d’épissage favorise sa séquestration dans les foci en modifiant sa localisation nucléaire, son activité de facteur d’épissage et ses propriétés d’oligomérisation. Le changement d’épissage de MBNL1 n’influence pas celui de Tau. Cependant, sa perte de fonction reproduit un profil d’épissage similaire à celui observé dans les cerveaux DM1. De plus, nous montrons que la surexpression de MBNL1, en présence des répétitions CTG suffit à restaurer un épissage normal de Tau et de plusieurs autres transcrits dérégulés dans la DM1. Enfin, des expériences complémentaires réalisées avec des protéines tronquées non fonctionnelles en tant que facteur d’épissage suggèrent que la restauration d’un profil d’épissage normal dans la DM1 serait due à la saturation des sites de liaisons CUG, ce qui permettrait de libérer les protéines MBNL1 séquestrées. Ces constructions semblent donc présenter un potentiel intérêt pour inverser les changements d’épissage observés dans la DM1 et sont actuellement en cours d’étudesTau pathology is a brain lesion common to more than twenty neurodegenerative disorders. It consists of the abnormal aggregation of the microtubule-associated protein Tau into neurofibrillary tangles. Mechanisms underlying Tau aggregation are not fully understood yet. However, among the different etiological hypothesises, the one of a relationship between Tau mis-splicing and Tau aggregates particularly interests us. Here, we proposed a disease model, being myotonic dystrophy type I (DMI), in which Tau mis-splicing and Tau aggregate occur. DM1 is the most common adult form of muscular dystrophy. It is an inherited autosomal disorder characterised by a dynamic instable CTG repeats (over 50) in the 3’UTR of DMPK gene. DM1 pathogenesis is suggested to result from a RNA toxic gain of function whereby mutant transcripts modify the splicing machinery activity leading thus to a mis-splicing of several pre-mRNA targets including Tau. In this context, our objectives were to 1) characterize Tau mis-splicing in several DM1 brain patients 2) Model it and identify the trans-regulating splicing factors likely involved and 3) Propose a therapeutic approach to reverse it. Tau mis-splicing was always observed for both exons 2 and 3 in human adult DM1 brain and consisted of a reduced inclusion. Tau exon 10 splicing was seldom mis-regulated and associated with an increase of the CELF proteins family. CELF proteins are splicing factors previously described to regulate alternative splicing of Tau exons 2, 3 and 10. In addition to the CELF proteins, we also investigated the potential role of the splicing factor MBNL1, which was shown to play an essential role in DM1 physiopathology through its sequestration by the CUG repeats. MBNL1’s brain expression was ill-defined. Here, we report that MBNL1’s expression level was not altered but its splicing modified in adult DM1 brain. In addition, we provide evidences by a relationship study between the structure and the function of MBNL1 that this mis-splicing event favored its sequestration to the foci by modifying its cell-localization, splicing activity and oligomerization properties. MBNL1 mis-splicing does not influence Tau mis-splicing. However its loss of expression reproduced the mis-splicing of Tau exons 2/3 as observed in DM1 brain. Interestingly, the overexpression of MBNL1 in the presence of the CTG repeats partially restored a normal splicing of Tau as well as several other mis-regulated pre-mRNA targets. Further experiments performed with different molecular constructs lead us to hypothezied that the reversal of the abnormal splicing events observed in DM1 was mediated by a saturation of the CUG binding sites that lead to the release of a free pool of MBNL1, recovering thus its splicing function. This work leads us to design a new molecular tool that might be of interest to reverse the pathological events observed in DM1
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Coleman, Stewart M. „Changes in localisation and dynamics of splicing and alternative splicing factors in human lens epithelial cells of myotonic dystrophy type 1 patients“. Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/3647.
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Lens growth and development is a life-long process in which epithelial cells from the perimeter of the lens migrate towards the centre of the lens and follow a dynamic programme of differentiation and structured DNA and organelle degradation resulting in the optical clarity of the lens. Myotonic dystrophy type 1 (DM1) is a genetic disease resulting in multiple symptoms including skeletal muscle wasting, cardiac conduction defects, myotonia and endocrine system malfunction. DM1 is caused by an expanded CTG repeat in the 3' untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Mutant DMPK RNA has been demonstrated to form abnormal foci within the cell nucleus in muscle cells from DM1 patients. The link between these foci and the multiple symptoms of DM1 is not fully understood. The alternative pre-mRNA splicing factor muscle blind-like 1 (MBNL1) is found in the foci and sequestration of MBNL1 is a leading hypothesis for pathogenesis. Results shown in this thesis demonstrate that only a small percentage of nuclear MBNL1 is sequestrated into foci. Furthermore MBNL1 is shown to co-localise with splicing speckles in a transcriptionally dependent manner. Interestingly eye lens histological samples suggest a change in MBNL1 distribution during lens growth. The data presented in this thesis shows a strong relationship between MBNL1 and CUGexp pre-mRNA foci and presents data which highlights the sensitivity of lens epitheial cells to changes in MBNL1.
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Laurent, François-Xavier. „Une nouvelle fonction pour la DEAD-box ARN hélicase p68/DDX5 dans la Dystrophie Myotonique de type 1“. Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00662593.
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La Dystrophie Myotonique de type 1 (DM1) est cause par l'expansion anormale d'un triplet CTG dans la partie 3'UTR du gène DMPK, entrainant l'agrégation du transcrit mutant dans des inclusions ribonucléoprotéiques appelées foci. D'après plusieurs études structurales sur des courtes répétitions CUG, il a été proposé que les expansions CUG se replient en une structure en tige-boucle qui interfère avec l'activité de plusieurs facteurs lié au métabolisme de l'ARN et altère leur fonction cellulaire. Le facteur d'épissage muscleblind-like 1 (MBNL1) a été identifié par sa capacité à interagir avec les répétitions CUG. In vivo, ces répétitions entrainent la séquestration de cette protéine aboutissant en une déplétion nucléaire. Un autre facteur d'épissage, la CUG Binding protein (CUGBP1), est également impliqué dans la pathologie. Au lieu d'être séquestré par les répétitions, la stabilité protéique de CUGBP1 est augmentée dans les tissus DM1 entrainant un gain d'activité pour ce facteur. La séquestration de MBNL1 et la stabilisation de CUGBP1 résultent en la dérégulation de l'épissage alternatif de plusieurs transcrits musculaires et du cerveau et la réexpression d'isoformes protéiques fœtales dans les tissus adultes. Cependant, de récentes études suggèrent que d'autres facteurs ou voies de signalisation que celles faisant intervenir MBNL1 et CUGBP1 pourraient être impliquées dans la pathologie DM1.Le but de mon travail de thèse a été d'identifier de nouveaux facteurs ayant la capacité d'interagir avec les répétitions CUG. A l'aide d'une purification sur chromatographie d'affinité utilisant un ARN contenant 95 répétitions CUG comme appât, nous avons identifié l'ARN hélicase p68/DDX5. p68 fait partie de la famille des protéines DEAD-box, caractérisée par un core protéique conservé constitué de neufs domaines hautement conservés, dont le motif DEAD, à l'origine du nom de ces protéines. p68 est impliquée dans de nombreux aspects du métabolisme de l'ARN, dont la transcription, l'épissage, l'export, la traduction et la dégradation des ARN. Nous avons montré, que p68 colocalise avec les foci CUG dans un modèle cellulaire exprimant la partie 3'UTR du gène DMPK contenant de longues répétitions CTG. Nous avons identifié que p68 augmente l'interaction de MBNL1 sur les répétitions CUG et une structure secondaire particulière d'un élément régulateur de l'ARN pré-messager cardiac Troponin T (TNNT2), dont l'épissage est dérégulé dans la pathologie. L'insertion de mutations dans le core de l'hélicase de p68 abolit l'effet de p68 sur la fixation de MBNL1 ainsi que la colocalisation de p68 avec les expansions CUG in vivo, suggérant que le remodelage des structures secondaires ARN de manière ATP-dépendante par p68 facilite l'interaction de MBNL1. Nous trouvons également que la compétence de p68 pour réguler l'inclusion de l'exon alternatif 5 de TNNT2 dépend de l'intégrité des sites de fixation de MBNL1.Nous proposons que p68 agit comme un modificateur de l'activité de MBNL1 sur ces cibles d'épissage ainsi que sur les expansions CUG à l'origine de la pathologie.
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Merien, Antoine. „Étude de la fonction des protéines MBNL au cours du développement à l’aide de cellules souches humaines induites à la pluripotence“. Thesis, université Paris-Saclay, 2021. https://www.biblio.univ-evry.fr/theses/2021/interne/2021UPASQ015.pdf.
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L'épissage alternatif est apparu comme un mécanisme fondamental non seulement pour la diversification des isoformes des protéines mais aussi pour la régulation spatio-temporelle du développement. Par conséquent, une meilleure compréhension de la manière dont ce mécanisme est régulé permettrait non seulement d'élucider les principes biologiques fondamentaux, mais aussi de déchiffrer les mécanismes pathologiques impliqués dans les maladies dans lesquelles ces processus moléculaires sont dérégulés. Dans le cadre de cette thèse, nous avons utilisé des cellules souches pluripotentes humaines pour déchiffrer, au cours de la myogenèse humaine, le rôle des protéines MBNL, une famille de régulateurs d'épissage spécifiques aux tissus dont la perte de fonction est associée à la dystrophie myotonique de type 1 (DM1), une maladie neuromusculaire héréditaire. Grâce à la technologie CRISPR/Cas9, nous avons généré des cellules souches pluripotentes d'origine humaine (hiPSC) déplétées en protéines MBNL et évalué les conséquences moléculaires et fonctionnelles de cette perte sur la génération de cellules musculaires squelettiques. Nos résultats ont indiqué que les protéines MBNL sont spécifiquement requises pour la maturation myogénique tardive mais pas pour l'engagement myogénique précoce. Par une analyse transcriptomique, nous avons pu mettre en évidence les voies moléculaires régulées par ces protéines durant la myogenèse, ainsi que les phénomènes de compensation entre les paralogues MBNL. Cette étude nous a également permis d’identifier un nouveau défaut d’épissage alternatif dans la DM1, régulé par les protéines MBNL, et qui aboutit à des anomalies structurelles du compartiment post-synaptique musculaire. Ensemble, nos résultats révèlent à la fois la temporalité de l’action des protéines MBNL dans la myogenèse humaine et permettent également d’identifier de nouvelles voies moléculaires régulées par ces protéines et pouvant être impliquées dans le développement de la DM1. A plus long terme, les outils développés dans cette étude devraient également faciliter l'identification de nouvelles stratégies thérapeutiques capables de faire face à la perte de fonction de ces protéinesAlternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of development. Therefore, a better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms involved in diseases where normal splicing networks are mis-regulated. As part of this thesis, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of MBNL proteins, a family of tissue-specific splicing regulators whose loss of function is associated with Myotonic Dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the molecular and functional consequences of this loss on the generation of skeletal muscle cells. Our results indicated that MBNL proteins are specifically required for the late myogenic maturation but not for early myogenic commitment. By a transcriptomic analysis, we were able to highlight the molecular pathways regulated by these proteins during myogenesis, as well as the compensatory effects between MBNL paralogs. This study also allowed us to identify a new alternative splicing defect in DM1, regulated by MBNL proteins, which leads to structural abnormalities of the muscular post-synaptic compartment. Together, our results reveal the temporal requirement of MBNL proteins in human myogenesis and allow the identification of new molecular pathways regulated by these proteins that could be involved in the development of DM1. In the longer term, the tools developed in this study should also facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these proteins
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Maury, Yves. „Utilisation de cellules souches pluripotentes humaines pour le développement de criblages phénotypiques dans le cadre de la dystrophie myotonique de type 1 et l'amyotrophie spinale infantile“. Thesis, Evry-Val d'Essonne, 2013. http://www.theses.fr/2013EVRY0019/document.
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Les cellules souches pluripotentes (CSP) humaines sont devenues en quelques années des modèles de choix pour étudier les mécanismes cellulaires et moléculaires qui gouvernent l'apparition de maladies monogéniques, mais également pour le développement de criblages à haut débits afin d'identifier parmi plusieurs milliers de molécules chimiques celles qui ont un potentiel thérapeutique. C'est dans ce contexte de criblage que mes travaux de thèse s'inscrivent, alliant automatisation et miniaturisation de la biologie des CSP dans le cadre de deux maladies monogéniques, l'amyotrophie spinale infantile (SMA) et la dystrophie myotonique de type I (DM1). De manière générale, la mise en place d'une telle stratégie repose sur trois étapes essentielles qui sont l'obtention de CSP porteuses d'une mutation donnée, l'identification d'un modèle d'étude pertinent et la réalisation du criblage à proprement parlé. L'obtention de CSP humaines repose sur deux approches principales. La première consiste en la dérivation de cellules embryonnaires humaine (hES) issues de diagnostiques préimplantatoires et la seconde repose sur la reprogrammation de cellules somatiques par l'induction de pluripotence (iPS). Une partie de mon travail a consisté en la création de cellules iPS modèles de la SMA et leur caractérisation par une approche à haut débit. Par la suite un travail d'optimisation du protocole de génération de motoneurones à partir de CSP humaines a permis d'accélérer et augmenter les rendements de production de ces cellules qui sont principalement affectées dans la SMA. Enfin, l'utilisation de cellules hES porteuses de la mutation causale de la DM1 a permis le criblage de 12000 molécules et a conduit à l'identification d'une famille chimique capable de restaurer plusieurs défauts typiques de cette maladie tels que des défauts d'épissage et de fusion moléculaireFor only few years, Human pluripotent stem cells (PSC) have become wide spread models in order to study and decipher cellular or molecular mechanims involved in monogenic diseases, but also for the development of large scale screening strategies allowing the identification of new therapeutics among thousands of chemicals. Mythesis research aimed at the development of such strategies, miniaturizing and automating PSC biology within the framework of two monogenic diseases, namely spinal muscular atrophy (SMA) and myotonic dystrophy type 1 (DM1).Basically, PSC based screening programs are generally built around three main steps which are the access to a stem cell model, the identification of a relevant cell type and lastly the screening campaign. There is actually two main ways to generate human PSC. Firstly, human embryonic stem cells (hES) can be derived from the inner cell mass of blastocyte through a pre-implantation diagnosis and secondly, induced pluripotent stem cells (iPS) can be generated after somatic cell reprogramming in vitro. A part of my work has consisted in the generation of hiPS cellular models for SMA by reprogramming fibroplasts that carried SMN1 gene deletion, followed bay the characterization of several dozen of independant clones with high throughput. Then an optimization process of the protocol for the generation of Motoneuron from PSC has been done multiplying experimental conditions. This finally allowed the description of a fast and efficient protocol to generate the most affected cell type in SMA. Finally, DM1 mutated hES were uded for the screening of 12.000 compounds among which a chemical family has been identified to rescue DM1 typical splicing and myogenesis defects
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Légaré, Cecilia. „Déterminants génétiques et épigénétiques de la variabilité phénotypique de la dystrophie myotonique de type 1“. Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/11602.
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La dystrophie myotonique de type 1 (DM1) est une maladie à transmission autosomale dominante causée par une répétition trinucléotidique CTG située dans la région 3’ non-traduite du gène dystrophia myotonica protein kinase (DMPK). La prévalence mondiale de la DM1 est de 8,26 personnes atteintes par 100 000 habitants : celle-ci est presque 20 fois plus importante au Saguenay-Lac-St-Jean en raison d’un effet fondateur. La présentation clinique de la DM1 peut comprendre divers symptômes dont de la faiblesse musculaire, de la myotonie, des cataractes, de l’insuffisance respiratoire, de l’arythmie cardiaque, de l’hypersomnolence et des troubles cognitifs et endocriniens. Par ailleurs, une grande variation dans la présence et la sévérité de ces symptômes est observée chez les patients et celle-ci n’est qu’en partie expliquée par la longueur des répétitions CTG. Plusieurs mécanismes pourraient expliquer la variabilité inexpliquée dont les défauts d’épissage, la mauvaise régulation des facteurs de transcription, la traduction non-ATG associée aux répétitions et les modifications épigénétiques, en particulier la méthylation de l’ADN. L’objectif de ce projet était donc d’évaluer l’impact de la méthylation de l’ADN au locus DMPK sur la variabilité phénotypique des patients atteints de DM1. Nous rapportons que la méthylation de l’ADN mesurée en amont et en aval de la répétition CTG est respectivement corrélée négativement et positivement avec la longueur de la répétition CTG. La présence d’une interruption de la répétition est associée à un niveau plus élevé de méthylation de l’ADN. À l’aide de modèles de régression linéaire multiple, nous démontrons que la méthylation de l’ADN contribue significativement et indépendamment de la longueur des répétitions CTG, à expliquer la variabilité́ de la force des dorsifléchisseurs de la cheville, de la force de préhension, de la force des pinces, de la capacité́ vitale forcée, du débit expiratoire de pointe, de la pression expiratoire et inspiratoire maximale. La méthylation de l’ADN explique une fraction de la variabilité phénotypique en DM1 et en association avec la longueur de la répétition CTG pourrait aider à améliorer la prédiction de la progression de la maladie chez ces patients.Abstract : Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by a CTG repeat extension in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Worldwide, the prevalence of DM1 is 8.26 affected persons per 100 000 persons, but it goes up to 158 affected persons per 100 000 in the Saguenay-Lac-St-Jean region of the province of Quebec (Canada) due to a founder effect. Clinical presentation includes muscular weakness, myotonia, cataracts, respiratory insufficiency, cardiac arrhythmia, hypersomnolence and endocrine and cognitive problems. There is a large variability in the presence and severity of these symptoms that is only partially explained by the CTG repeat length. Many mechanisms such as splicing defects, impaired regulation of transcription factors, repeat-associated non-ATG translation and epigenetic modifications, including DNA methylation, may explain this variability. The objective of this study was to assess the impacts of DNA methylation measured at the DMPK gene locus on phenotypic variability in DM1. We report that DNA methylation upstream of the repeat was negatively correlated with CTG repeat length whereas downstream DNA methylation was positively correlated. The presence of a variant repeat within the CTG repeat was associated with a higher level of DNA methylation. Linear multiple regression models support that DNA methylation contributes significantly and independently of the CTG repeat length to the variability of the ankle dorsiflexor, grip and pinch strengths, as well as forced vital capacity, peak expiratory flow and maximal inspiratory and expiratory pressures. DNA methylation could thus explain part of the phenotypic variability in DM1 and, together with CTG repeat length, could help improve the prediction of the progression of the disease.
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Picchio, Lucie. „Mise en place, caractérisation phénotypique et transcriptomique d'un modèle de Drosophilie de la Dystrophie Myotonique de type 1“. Thesis, Clermont-Ferrand 1, 2013. http://www.theses.fr/2013CLF1MM15/document.
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La dystrophie myotonique de type 1 (DM1) ou maladie de Steinert est la maladie génétique neuromusculaire la plus commune avec une incidence de 1/8000 à travers le monde. Cette maladie multisystémique touche particulièrement les muscles squelettiques (myotonie, faiblesse et perte musculaires) et le coeur qui présente des symptômes variés comme des troubles de la conduction et des arythmies. La DM1 est causée par une expansion instable de répétitions CTG dans la région 3’ non traduite du gène DMPK. Les individus sains possèdent entre 5 et 37 répétitions CTG tandis que les patients DM1 portent entre 50 et plusieurs milliers de répétitions. Il est bien établi que les expansions de répétitions non codantes forment des foci dans les noyaux musculaires où elles séquestrent le facteur d'épissage MBNL1. Toutefois, l'implication de la stabilisation et l'accumulation de CUGBP1 hyperphosphorylé par la PKC dans la maladie est un sujet controversé dans la communauté DM1. Dernièrement, en plus de la rupture de l'équilibre entre MBNL1/CUGBP1, plusieurs mécanismes ont été mis en cause dans la pathogenèse de la DM1. Parmi eux, l'expression perturbée de facteurs de transcription, la maturation altérée de miARNs, l'activation de kinases... chacune de ces altérations menant au final à une perturbation du transcriptome. Afin d'étudier l'effet de la toxicité des répétitions sur les phénotypes et lestranscriptomes, nous avons généré trois lignées de Drosophile inductibles et site-spécifiques exprimant 240, 600 et 960 répétitions de triplets. Nous avons travaillé en parallèle sur une lignée atténuée pour mbl (orthologue de MBNL1) et deux lignées gain de fonction bru -3 (orthologue de CUGBP1). Exprimées dans les muscles somatiques, les répétitions CTG conduisent à une mobilité réduite, le fractionnement des fibres musculaires, une réduction de leur taille et une altération du processus de fusion des myoblastes de manière dépendante de Mbl et Bru-3. En outre, l'expression des répétitions cause une hypercontraction musculaire dépendante de Mbl et due à un mauvais épissage de dSERCA. L'analyse transcriptionnelle comparative réalisée sur les muscles larvaires des différentes conditions pathologiques montre que l'atténuation de mbl reproduit 70-82% des dérégulations transcriptomiques des larves DM1 alors que le gain de fonction bru-3 représente 32-53% des altérations transcriptomiques des lignées DM1. Ainsi Mbl est un facteur clé des dérégulations observées dans les muscles somatiques des lignées DM1. Au contraire, les analyses physiologiques effectuées sur les coeurs adultes suggèrent que Bru-3 est un facteur clé dans la mise en place des phénotypes cardiaques. En effet, d'une part, l'atténuation de mbl dans le coeur cause une cardiomyopathie dilatée, un symptôme rarement diagnostiqué chez les patients. D'autre part, les lignées gain de fonction bru-3 et DM1 présentent de la fibrillation qui évolue avec l'âge ou la taille des répétitions vers un phénotype qui rappelle l'insuffisance cardiaque chez les patientsMyotonic Dystrophy Type 1 (DM1) or Steinert's disease is the most common genetic neuromuscular disorder affecting 1 out of 8000 people worldwide. This multisystemic disease affects particularly the skeletal muscles (myotonia, muscle weakness and wasting) and the heart, which can exhibit various symptoms like conduction disturbances and arrhythmia (auricular fibrillation and flutter). DM1 is caused by an unstable CTG repeat expansion in the 3' non-translated region of the DMPK gene. In healthy individuals, the number of CTG repeats ranges from 5 to 37 whereas DM1 patients carry from 50 to thousands repeats. It is well established that when expanded non-coding repeats aggregate into foci within muscle nuclei and sequester the MBNL1 splicing factor. However, the involvement of the stabilization and accumulation of CUGBP1 following PKC hyper-phosphorylation in the disease is a controversial matter in the DM1 community. Lately, in addition to the disruption of the balance between MBNL1/CUGBP1, several mechanisms were identified as part of the DM1 pathogenesis. Among them, transcription factors perturbations, altered maturation of miRNA, kinases activation… each of them leading eventually to transcriptomic alterations. In order to investigate the effect of toxic repeat expression on phenotypic and transcriptomic alterations, we generated three inducible site-specific Drosophila lines expressing 240, 600 and 960 triplet repeats. We worked in parallel on a mbl (MBNL1 orthologue) knocked-down line and two bru-3 (CUGBP1 orthologue) gain of function lines. When expressed in somatic muscles, CTG repeats lead to altered motility, fiber splitting, reduced fiber size and affected myoblast fusion process in a Mbl and Bru-3 dependent manner. In addition, toxic repeats cause fiber hyper-contraction in a Mbldependentmanner due to dSERCA mis-splicing. Comparative transcriptional profiling performed on larval muscles of different conditions show that mbl attenuation reproduces 70-82% of DM1 transcriptomic deregulations whereas bru-3 gain of function represents 32-53% of transcritomic alterations. Thus Mbl appears as a key factor of transcripts deregulations observed in DM1 muscles. On the contrary, physiologic analyses performed on adult hearts suggest that Bru-3 is a key factor for cardiac phenotypes. Indeed, on one hand, mbl attenuated flies display dilated cardiomyopathy, a symptom barely diagnosed in patients. On the other hand, bru-3 gain of function line and DM1 lines display fibrillation, which evolves withage or repeat size into a phenotype reminiscent of heart insufficiency in patients
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Pontual, Laure de. „Identification de nouveaux facteurs chimiques capables de moduler l'instabilité des répétitions CTG dans la dystrophie myotonique de type 1“. Electronic Thesis or Diss., Sorbonne université, 2024. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2024SORUS198.pdf.
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La dystrophie myotonique de type 1 (DM1) est la dystrophie la plus fréquente chez l'adulte avec une prévalence estimée à 1 : 8000 individus. C'est une maladie multi-systémique caractérisée par des atteintes musculaires, cardiaques, cognitives et digestives responsables d'une réduction de l'espérance et de la qualité de vie des patients. Elle est causée par une expansion anormale de répétitions CTG en 3'UTR du gène DMPK. Dans la population générale, le nombre de répétitions est inférieur à 35 CTG tandis qu'il dépasse 50 CTG et peut atteindre jusqu'à plusieurs milliers de répétitions chez les patients. Comme dans d'autres maladies causées par une expansions de triplets répétés, l'expansion CTG est instable chez les patients DM1. La taille des répétitions CTG augmente entre les générations (instabilité intergénérationnelle) et au sein des tissus au cours de la vie des patients (instabilité somatique). Or, le nombre de répétitions héritées ainsi que le niveau d'instabilité somatique corrèlent avec l'âge d'apparition des symptômes ainsi qu'avec leur sévérité. S'attaquer à la mutation elle-même pour stabiliser ou réduire le nombre de répétitions CTG est la voie thérapeutique la plus prometteuse puisque cela permettrait d'agir sur l'ensemble des mécanismes physiopathologiques qui découlent de la présence de la mutation.Dans un premier temps, mes travaux de thèse ont porté sur l'identification de molécules chimiques de repositionnement capables de moduler l'instabilité des répétitions. Le criblage des 1280 molécules de la chimiothèque Prestwick m'a permis d'identifier 39 molécules candidates qui modifient l'expression d'un gène rapporteur suggérant qu'elles pourraient moduler l'instabilité des répétitions. Après étude directe de leur effet sur l'instabilité, j'ai pu exclure quatre de ces molécules qui ne modulent pas l'expression des répétitions. J'ai montré qu'une cinquième molécule, la clomipramine, était capable de moduler l'instabilité des répétitions dans le modèle cellulaire de criblage mais pas dans des modèles cellulaires DM1 murins et humains.Parallèlement, j'ai démontré que RGFP966, un inhibiteur sélectif d'HDAC3, induisait des contractions des répétitions CTG dans des fibroblastes murins DM1 avec environ 650 répétitions CTG. Cet effet semble dépendre de la dose de RGFP966 ou de la taille de la répétition CTG car il n'a pas été reproduit dans des fibroblastes humains DM1 avec 350 CTG. Une approche RNA-seq dans les cellules murines traitées avec RGFP966 a permis d'identifier plusieurs gènes candidats impliqués dans la réplication de l'ADN, comme modificateurs possibles de l'instabilité. J'ai également montré une diminution de la transcription bidirectionnelle de DMPK associée à une probable hyperméthylation en aval des répétitions dans les cellules murines DM1. En conclusion, mes données suggèrent que RGFP966 module l'instabilité des répétitions CTG dans la DM1 par de multiples mécanismes, incluant potentiellement une modification de la structure chromatinienne au locus DM1 et des altérations de la réplication de l'ADN.L'ensemble de mon projet de thèse a permis de progresser dans la compréhension des mécanismes de l'instabilité et dans l'identification de molécules chimiques modulant la dynamique de l'instabilité. Mes travaux de thèse ont également permis de relever les limites de chacun des modèles utilisés et la complexité d'identifier de petites molécules modifiant la dynamique des triplets CTG en utilisant des modèles cellulaires rapporteurs. Par ailleurs, j'ai participé au développement pour la DM1 du séquençage à longues lectures (avec et sans amplification), un nouvel outil rapide et très informatif pour analyser la mosaïque somatiqueMyotonic dystrophy type 1 (DM1) is the most common dystrophy in adults, with an estimated prevalence of 1:8000 individuals. It is a multisystemic disease characterized by muscle, cardiac, cognitive, and digestive impairments, which contribute to a reduction in both life expectancy and quality of life for patients. DM1 is caused by an abnormal expansion of CTG repeats in the 3'UTR of the DMPK gene. In the general population, the number of repeats is under 35 CTG, whereas in patients, it exceeds 50 CTG and can reach several thousand repeats. As in other diseases caused by repeat expansions, the CTG expansion in DM1 is unstable. The repeat size increases across generations (intergenerational instability) and within tissues during a patient's lifetime (somatic instability). The number of inherited repeats and the level of somatic instability correlate with the age of onset and severity of symptoms. Thus, targeting the mutation itself to stabilize or reduce CTG repeat length is the most promising therapeutic strategy, as it would address all the pathophysiological mechanisms resulting from the mutation.Initially, my thesis work focused on identifying repositioned chemical molecules capable of modulating repeat instability. Screening the 1280 molecules from the Prestwick Chemical Library allowed me to identify 39 candidate molecules that alter the expression of a reporter gene, suggesting they could modulate repeat instability. After directly studying their effect on instability, I excluded four of these molecules that do not modulate repeat expression. I demonstrated that a fifth molecule, clomipramine, can modulate repeat instability in the screening cell model but not in murine and human DM1 fibroblasts.Concurrently, I showed that RGFP966, a selective HDAC3 inhibitor, induced contractions of CTG repeats in murine DM1 fibroblasts with approximately 650 repeats. This effect appears to depend on the dose of RGFP966 or the size of the CTG repeat, as it was not replicated in human DM1 fibroblasts with 350 CTG repeats. An RNA-seq approach in murine cells treated with RGFP966 identified several candidate genes involved in DNA replication as possible modifiers of instability. I also showed a decrease in bidirectional DMPK transcription associated with a probable hypermethylation downstream of the repeats in murine DM1 cells. In conclusion, my data suggest that RGFP966 modulates CTG repeat instability in DM1 through multiple mechanisms, potentially including chromatin structure modification at the DM1 locus and alterations in DNA replication.Overall, my thesis project contributed to the understanding of repeat instability mechanisms and the identification of chemical compounds that modulate instability dynamics. My work also highlighted the limitations of each model used and the complexity of identifying small molecules that alter CTG triplet dynamics in reporter cell models. Additionally, I participated in developing long-read sequencing (with and without amplification) for DM1, providing a rapid and highly informative new tool for the analysis of somatic mosaicism
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Rodrigues, Luiza Paulsen. „Identificação e avaliação da distribuição alélica de repetições do trinucleotídeo CTG no gene DMPK em indivíduos saudáveis e em pacientes com distrofia miotônica tipo 1“. reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/158225.
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O gene DMPK (Dystrophia Myotonica-Protein Kinase) humano está localizado no locus 19q13.3, sendo dividido em 15 éxons, com uma região polimórfica de repetições CTG em sua região 3’ não traduzida. Indivíduos normais apresentam de 5 a 34 repetições CTG. Indivíduos com alelos com mais de 50 repetições CTG apresentam distrofia miotônica tipo 1 (DM1), uma doença multissistêmica de herança autossômica dominante. Os sintomas incluem miotonia, fraqueza muscular progressiva, hipogonadismo, entre outros. Neste trabalho, a distribuição dos alelos do gene DMPK em indivíduos controles foi estabelecida em duas populações (brasileira e peruana), por meio de PCR convencional utilizando iniciadores fluorescentes e repeat-primed PCR. O protocolo confirmou 93 casos não relacionados de DM1 (76 brasileiros e 17 peruanos) após a análise de 224 amostras com suspeita clínica. A distribuição e as frequências dos alelos normais foram estabelecidas em ambas as populações e os alelos mais frequentes foram 5 (frequência = 0,326) e 13 (frequência = 0,545) repetições de CTG em brasileiros e peruanos, respectivamente. A frequência de alelos normais grandes (aqueles com mais de 45 repetições CTGs) foi de 9% e 4% em brasileiros e peruanos, respectivamente. Neste trabalho é descrita a análise molecular de DM1 na maior coorte brasileira até o momento e é o primeiro trabalho em que foi analisada a população peruana. A distribuição e a frequência de alelos normais também foram estabelecidas e alelos mutáveis foram detectados entre os indivíduos controles.The human DMPK (Dystrophia Myotonica-Protein Kinase) gene is located at 19q13.3 locus, being organized into 15 exons, with a polymorphic tract of CTG repeats in its 3' untranslated region. Normal individuals have 5-34 CTG repeats. Individuals carrying alleles with more than 50 CTG repeats have myotonic dystrophy type 1 (DM1), a multisystemic disease of autosomal dominant inheritance. Symptoms include myotonia, progressive muscle weakness, hypogonadism, among others. Disease prevalence is variable among populations and may be related to the frequency of large normal alleles (those with more than 18 CTG repeats). Here we determined here the distribution of alleles of DMPK gene in healthy and DM1 patients in Brazilian and Peruvian populations, through conventional PCR using fluorescent primers and repeat-primed PCR. This protocol confirmed 93 unrelated cases of DM1 (76 Brazilians and 17 Peruvians) following the analysis of 224 samples with clinical suspicion. Distribution and frequencies of normal alleles were also established in both populations and the most frequent alleles were 5 (frequency of 0.326) and 13 (frequency of 0.545) CTG repeats in Brazilians and Peruvians, respectively. Frequency of large normal alleles (those with more than 45 CTG repeats) was established to be 9% and 4% in Brazilians and Peruvians, respectively. This report describes molecular analysis of DM1 in the largest Brazilian cohort so far, and is the first to report any data in the Peruvian population. Distribution and frequency of normal alleles were also established and mutable alleles were detected among controls.
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Arandel, Ludovic. „Développement d'une thérapie génique pour la Dystrophie Myotonique de type 1“. Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS229.
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Les Dystrophies Myotoniques de type 1 (DM1) et (DM2) sont des maladies multisystémiques autosomiques dominantes avec une forte composante neuromusculaire. Ces pathologies se caractérisent essentiellement par une myotonie, une faiblesse musculaire progressives, des déficiences cognitives, et des défauts de conduction cardiaque. Ces maladies sont causées par une amplification anormale de séquences répétées C(C)TG situées respectivement dans la région 3’UTR du gène de la DMPK et dans l’intron du gène CNBP. Ces séquences contenant les expansions sont transcrites et retenues dans le noyau des cellules sous forme d’agrégats riboprotéiques. La présence de ces ARN-C(C)UG toxiques induisent la séquestration des protéines de liaisons à l’ARN de la famille MBNL conduisant à leur perte de fonction et à la dérégulation d’évènements d'épissages alternatifs dont plusieurs ont été associés à des symptômes cliniques chez les patients. A jour ce jour, il n’existe aucun traitement pour la DM. Dans ce travail de thèse, j’ai développé un outil de thérapie génique basé sur une modification de la protéine MBNL1. Ce dérivé de MBNL tronqué dans sa partie C-terminale (MBNLΔ), agit comme leurre pour libérer les protéines MBNL endogènes séquestrées par les ARN mutés. Notre approche été validée dans des cellules musculaires issue de patient DM1 et dans un modèle murin de la maladie après injection de virus AAV. Le traitement par le MBNLΔ permet la délocalisation des protéines MBNL endogènes des foci, modifie la dynamique des foci, corrige le transcriptome ainsi que la myotonie, et ce 1 an après injectionMyotonie dystrophy types 1(DM1) and 2 (DM2) are autosomal dominant multisystem diseases with a strong neuromuscular component. They are characterized by progressive myotonia, muscle weakness, cognitive impairment, and cardiac conduction defects. These diseases are caused by abnormal amplification of C(C)TG repeat sequences located in the 3'UTR region of the DMPK gene and in the intron of the CNBP gene, respectively. These expansion-containing sequences are transcribed and retained in the nucleus as riboprotein aggregates. The presence of these toxic C(C)UG RNAs induces sequestration of the MBNL family of RNA-binding proteins, leading to their loss of function and deregulation of alternative splicing events, many ofv/hich are associated with clinical symptoms in patients. There is currently no1reatment for DM. In this thesis, I have developed a gene therapy tool based on a modification of the MBNL1 protein. This C- terminal truncated MBNL derivative (MBNLΔ) acts as a decoy to release endogenous MBNL proteins sequestered by mutant RNAs. Our approach was validated in muscle cells from DM1 patients and in a mouse model of the disease after AAV virus injection. Treatment with MBNLΔ allows the delocalisation of endogenous MBNL proteins from the foci, modifies the foci dynamics, corrects the transcriptome and myotonia, which is maintained 1 year after injection
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Petitclerc, Émilie. „Association entre le profil de force musculaire et les capacités fonctionnelles aux membres inférieurs chez les personnes atteintes des phénotypes adulte classique et adulte tardif de dystrophie myotonique de type 1“. Mémoire, Université de Sherbrooke, 2015. http://hdl.handle.net/11143/8031.
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Résumé: But : Les objectifs étaient de 1) décrire les profils de force musculaires aux membres inférieurs (MIs) et les capacités aux déplacements des personnes présentant les phénotypes adulte classique (DM1-AC) et adulte tardif (DM1-AT) de la dystrophie myotonique de type 1 (DM1), et 2) d’explorer l’influence de la faiblesse des MIs sur les capacités aux déplacements dans cette population. Méthode : Cette étude consiste en une analyse secondaire de données issues d’une plus large recherche qui visait à identifier les déterminants de la participation sociale et de la qualité de vie de personnes atteintes de DM1 (n = 158 DM1-AC et n = 42 DM1-AT). La force de quatre groupes musculaires des MIs a été mesurée à l’aide du bilan musculaire manuel (BMM) et du bilan musculaire quantitatif (BMQ) par dynamométrie manuelle. Les capacités aux déplacements ont été évaluées à l’aide de tests standardisés (échelle d’équilibre de Berg, vitesse de marche et Timed Up & Go). Résultats : Le phénotype DM1-AT présente moins de faiblesse et d’incapacités que le phénotype DM1-AC (p < 0,001 – 0,020). Le BMM ne détecte pas de faiblesse chez le phénotype DM1-AT mais des pertes de force au BMQ de 12 % à 20 % ont été identifiées chez ce phénotype, excepté pour les fléchisseurs du genou, entrainant des limitations aux déplacements chez 22 % à 48 % de ces individus. Dans le phénotype DM1-AC, l’atteinte musculaire était légèrement plus importante en distal qu’en proximal. Selon ces résultats, les phénotypes DM1-AC et DM1-AT présentent des portraits distincts et les données relatives à chacun devraient être analysées séparément. Une progression générale de la faiblesse au BMQ et des scores aux tests fonctionnels a été observée en fonction des cotes de l’échelle Muscular Impairment Rating Scale (MIRS). Un déficit de force au BMQ (excepté pour les fléchisseurs du genou) et des incapacités fonctionnelles ont aussi été observés dès les premières cotes de la MIRS. Finalement, les dorsifléchisseurs de la cheville et les extenseurs du genou semblent être de bons indicateurs de la fonction des membres inférieurs en DM1. Conclusion : Cette étude a permis de dresser un portrait des atteintes de la force musculaire aux MIs et des capacités fonctionnelles liées aux déplacements pour chacun des phénotypes DM1-AC et DM1-AT de la DM1, ainsi que d’explorer la contribution de la faiblesse des groupes musculaires évaluées sur les capacités aux déplacements dans cette population. Ces résultats contribueront à mieux déterminer les cibles d’évaluation et d’interventions en réadaptation et à mieux définir le processus d’évaluation dans le contexte des essais thérapeutiques à venir.Abstract: Purpose: The purposes of this study were 1) to describe lower limbs muscle strength and mobility capacities, and 2) to explore the respective contribution of lower limb muscle weaknesses on mobility in the adult and late-onset phenotypes of myotonic dystrophy type 1 (DM1). Methods: This study is a secondary analysis of part of the results of a larger study, whose purpose was to identify social participation and quality-of-life determinants in 200 DM1 patients (158 adult and 42 late-onset). The strength of four lower limb muscle groups was assessed using manual muscle testing (MMT) and handheld dynamometry quantitative muscle testing (QMT). Mobility capacities were assessed using standardized tests (Berg balance scale, 10 Meter Walk Test and Timed Up & Go). Results: Although the late-onset phenotype showed less weaknesses and mobility limitations than the adult phenotype (p <0.001-0.020), and although MMT showed no weakness in the late-onset phenotype, quantitative strength losses of 12-20% were measured in this phenotype, with the exception of the knee flexors. These weaknesses led to mobility limitations in 22-48% of participants with the late-onset phenotype. In the adult phenotype, muscle strength impairment was slightly more important distally than proximally (2-2.5/10 and 5.8-8.2% for MMT and QMT, respectively) (p <0.001-0.002). According to those results, the adult and late-onset phenotypes show different profiles of lower limb impairment, and should not be pooled for data analysis. A general progression of quantitative muscle weakness and of mobility scores was observed according to the Muscular Impairment Rating Scale (MIRS) classification. Quantitative weaknesses, with the exception of the knee flexors, and mobility limitations were observed from the first MIRS grades. QMT is therefore definitely a more effective tool for measuring weakness in DM1. Finally, ankle dorsiflexors and knee extensors seem to be good indicators of lower limb function in DM1. Conclusion: This study allowed a better characterization of lower limb weaknesses and mobility limitations in the adult and late-onset phenotypes of DM1, and explored the contribution of lower limb weaknesses on mobility capacities in this population. These results will be useful for developing more specific rehabilitation programs and for optimizing the evaluation of these impairments in the context of the upcoming therapeutic trials. Keywords: Myotonic dystrophy type 1, phenotypes, muscle strength, mobility capacities, lower limbs, explanatory variables, physiotherapy.
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Antonio, Marie de. „Statistiques et modèles de survie pour améliorer la connaissance d’une maladie rare, la dystrophie myotonique The DM-Scope registry: a rare disease innovative framework bridging the gap between research and medical care Unraveling the myotonic dystrophy type 1 clinical spectrum: a systematic registry-based study - Implications for disease classification“. Thesis, Sorbonne université, 2020. http://www.theses.fr/2020SORUS096.
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La dystrophie myotonique (DM) est considérée comme l'une des maladies neuromusculaires les plus complexes. Bien que les travaux de recherche de ces 30 dernières années aient permis de mieux comprendre les mécanismes moléculaires sous-jacents, la nature de l'anomalie génétique hors norme, son expression multisystémique et son large spectre clinique ne permettent pas, à l'heure actuelle, une prise en charge optimale des patients. Mon travail a eu pour but d'approfondir les connaissances et de préciser l'histoire naturelle de cette maladie rare. La première partie du manuscrit est consacrée à la présentation de l'observatoire DM-Scope, sur lequel s'appuie tout mon travail de thèse. Après la description du concept, du fonctionnement et de la plateforme de recueil, les caractéristiques de la cohorte DM1, à partir de laquelle les analyses ont été réalisées, sont présentées : spectre clinique couvert, atteinte multisystémique, corrélations génotype/phénotype, interrelations entre les symptômes et comparaison à la dystrophie myotonique de type II (DM2). Ensuite, dans une deuxième partie, nous abordons les avancées majeures obtenues dans les DM grâce à DM-Scope et aux analyses réalisées pendant ma thèse : (i) précision de l'histoire naturelle de la maladie, notamment avec la proposition d'une nouvelle classification ; (ii) mise en exergue de facteurs déterminants du phénotype comme le genre, la taille de la mutation ou les interrelations entre les symptômes. Ces travaux ont conduit à des recommandations de soins, notamment pour la transition enfants-adultes mais aussi la validation de critères d'inclusion importants pour les essais cliniques comme le genre. DM-Scope permet d'accéder à des échantillons biologiques pour des études de recherche fondamentale et valider de nouvelles approches thérapeutiques. Il est aujourd'hui un leader à l'international et un outil incontournable dans la recherche translationnelle dans la DM. Ce concept transférable à n'importe quelle autre population, peut être utilisé pour la prise en charge d’autres maladies rares. Enfin, le développement d'un modèle de survie construit à partir de la cohorte DM de l'observatoire est présenté. Ce modèle a trois spécificités : (i) il est applicable en grande dimension, à des cas comme DM-Scope, où l'on a un nombre important de variables; (ii) il prend en compte les risques compétitifs, lorsque les patients sont exposés simultanément à plusieurs évènements. Dans notre observatoire, l'étude des décès de cause respiratoire est biaisée sans la prise en compte des évènements concurrents tels que le décès de cause cardiaque; (iii) il modélise l'hétérogénéité entre les groupes de patients (effets centres), potentiellement due à une prise en charge différente. L'analyse des données de DM-Scope nécessite cette spécificité issue des modèles à fragilité car l'observatoire est multicentrique (55 centres). Le modèle est transférable et applicable à d'autres données car de plus en plus de bases sont de grandes dimensions, la majorité des analyses de survie ont une censure liée à la survenue de l'événement d'intérêt et les études multicentriques sont de plus en plus communesMyotonic dystrophy (DM) is considered one of the most complex neuromuscular diseases. Although research work over the past 30 years has permitted a better understanding of its underlying molecular mechanisms, the unusual nature of its genetic anomalies, its multisystemic expression and its broad clinical spectrum do not allow, at the moment, optimal patient management. The purpose of my work was to deepen our knowledge of this rare disease and to clarify its natural history. The first part of my manuscript is dedicated to the presentation of the DM-Scope Registry, on which all my thesis work is based. After the description of the concept, the functioning and the data collection platform, the manuscript features the characteristics of the DM1 cohort, from which our analyses were conducted : the clinical spectrum covered, multisystemic impairment, genotype/phenotype correlations, interrelations between symptoms and comparison to myotonic dystrophy type II (DM2). In the second part, we focus on the major progress achieved through the existence of DM-Scope and the analyses conducted during my thesis: (i) detailing the natural history of the disease, in particular proposing a new classification; (ii) highlighting the phenotype’s determining factors such as gender, mutation size, interrelations between symptoms. This work has led to recommendations for care, in particular for the transition from child to adult, but also the validation of important inclusion criteria for clinical trials such as gender. DM-Scope provides access to available biological samples for basic research studies and validates new therapeutic approaches. DM-Scope is now a worldwide leader and an essential tool in translational research in DM. The DM-Scope concept can be transferred to any other population and can be used for care management in other rare diseases. Finally, we present the development of a survival model built from the DM-Scope cohort. This model has three specificities: (i) it is applicable to high dimensional data, in such cases as DM-Scope, where there is a large number of measurements; (ii) it takes into account competitive risks, when patients are simultaneously exposed to several events. In our registry, the study of respiratory-related deaths is biased if competing events such as heart disease deaths are not taken into account ; (iii) it models the heterogeneity between patient groups probably due to divergent care, called \og centres effects \fg{}. DM-Scope data analysis requires such specificity of frailty models due to its multicentric coverage (55 centres). This model can be transferred and applied to other data, considering the following : more and more large-scaled registries are being used ; a majority of survival analyses includes censorship caused by the occurrence of the event of interest ; multicentre studies have become increasingly common
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Poggi, Lucie. „Gene editing approaches of microsatellite disorders : shortening expanded repeats“. Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS412.
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Les maladies à triplet sont dues à des expansions de trinucléotides dans l’ADN. Aucun traitement n’existe pour les soigner. Le but de cette thèse est de mettre au point de nouvelles approches de thérapie génique pour supprimer les expansions pathologiques dans le génome humain. Dans une première partie, un système expérimental dans la levure a été construit afin d’évaluer l’efficacité de différentes nucléases associées au système CRISPR sur des microsatellites. La seconde partie est concentrée sur une maladie à triplet en particulier ; la dystrophie myotonique de type 1 (DM1), qui est due à une expansion d’une répétition de triplets CTG dans la région 3’UTR du gène DMPK. Une nucléase, TALENCTG , construite pour induire une cassure double-brin dans les répétitions CTG en 3’UTR du gène DMPK, induit de manière très efficace des contractions de triplets CTG dans la levure. Des événements de contraction ont été observés lorsque cette nucléase est exprimée. Des expériences in vivo dans un modèle de souris contenant un fragment d’ADN génomique humain de patient contenant 1000 CTG ont été menées. Des particules virales AAV recombinantes portant le gène de la TALEN ont été produites. Après injection intramusculaire, les cellules musculaires expriment la nucléase, mais dû à une toxicité ou immunogénicité de la protéine, l’expression est perdue. Enfin, le système mis au point dans la levure a été transposé dans une lignée cellulaire humaine établie, les HEK293FS. Ce système pourra servir à sélectionner des nucléases actives dans les cellules humainesMicrosatellite disorders are a specific class of human diseases that are due to the expansion of repeated sequences above pathological thresholds. These disorders have varying symptoms and pathogenic mechanisms, caused by the expanded repeat. No cure exists for any of these dramatic conditions. This thesis is investigating new gene editing approaches to remove pathological expansions in the human genome. In a first part, a yeast-based screen was constructed to identify potent CRISPR-associated nucleases that can cut these microsatellites. The second part focuses on myotonic dystrophy type 1 (DM1), which is due to and expanded CTG repeat tract located at the 3’UTR of the DMKP gene. A nuclease, TALENCTG was designed to induce a double strand break into the CTG repeats. It was previously shown to be active in yeast cells, inducing contractions of CTG repeats from a DM1 patient integrated into the yeast genome. The TALEN was tested in DM1 patient cells. The nuclease was found to trigger some contraction events in patient cells. In vivo experiments were carried out in a mouse model of myotonic dystrophy type 1 containing a human genomic fragment from a patient and 1000 CTG. Intramuscular injections of recombinant AAV encoding the TALENCTG revealed that the nuclease is toxic and/or immunogenic in muscle cells in the tested experimental conditions. Finally, the reporter assay integrated in yeast to screen nucleases was transposed in HEK293FS cell line. The integrated cassette contains a CTG expansion from a myotonic dystrophy type 1 patient flanked by two halves of GFP genes. This system would enable to find nucleases active in human cells
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Nguyen, Xuan-Tam. „Approches globales afin d’élucider les mécanismes pathogéniques de la dystrophie myotonique de type 1“. Thèse, 2016. http://hdl.handle.net/1866/18667.
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La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites.Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions.
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Cleary, John. „DNA Replication and Trinucleotide Repeat Instability in Myotonic Dystrophy Type 1“. Thesis, 2010. http://hdl.handle.net/1807/24723.
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The expansion of gene-specific trinucleotide repeats is responsible for a growing list of human disorders, including myotonic dystrophy type 1 (DM1). Repeat instability for most of these disorders, including DM1, is characterized by complex patterns of inherited and ongoing tissue-specific instability and pathogenesis. While the mechanistic basis behind the unique locus-specific instability of trinucleotide repeats is currently unknown, DNA metabolic processes are likely to play a role. My thesis involves investigating the contribution of DNA replication to the trinucleotide instability of myotonic dystrophy type 1.
Herein I have designed an in vivo primate model system, based on the SV40 replication system, to assess the contribution of DNA replication to DM1 repeat instability. This system allows the assessment, under controlled conditions, and manipulation of variables that may affect replication-associated repeat instability, under a primate cellular system. Using the SV40 model system, I not only confirmed previous observations that repeat length and replication direction affect repeat instability, but also for the first time determined that the location of the replication origin relative to the repeat tract plays an important role in repeat instability. This novel observation allowed for the development of a fork-shift model of repeat instability, in which cis-elements adjacent to the repeat tract affect replication, in turn altering the propensity for repeat instability.
To further my study of DNA replication in DM1 repeat instability, I have mapped the origin of replication adjacent to the DM1 locus in human patient cells and the tissues of DM1 transgenic mice actively undergoing repeat instability. The position of the replication origins adjacent to the repeat tract at the DM1 locus places several known cis-elements, including CTCF binding sites, in a position to alter replication as predicted by the fork-shift model. My analysis of the CTCF sites showed them capable of altering replication and repeat instability at the DM1 locus. Taken together these results suggest that the placement of replication origins, repeat tracts and cis-elements, may mark trinucleotide repeat tracts, such as the DM1, for locus-, tissue- and development-specific replication-associated repeat instability.
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Basílio, Ana Cláudia Miquelino. „Characterization of the nuclear envelope alterations in Myotonic Dystrophy Type 1“. Master's thesis, 2019. http://hdl.handle.net/10773/28409.
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The myotonic dystrophy type 1 (DM1) is a multisystem genetic disorder mainly associated with degeneration of skeletal muscle and caused by a CTG repeat expansion in the 3′ untranslated region of the DMPK gene, accumulating as nuclear foci and compromising the nuclear function. In the last few years, some studies associate nuclear envelope proteins as a significant component in the response of cells to mechanical stress, helping to maintain muscle cell integrity, demonstrating a clear association to DM1 pathology. In fact, it has been demonstrated that DM1 patient’s fibroblasts presents an impaired nuclear structure and an altered localization of some NE proteins. However, the contribution of the NE for DM1 was not fully elucidated. In this work, we performed an ATR-FTIR spectroscopy study of human control and DM1 fibroblasts to identify spectral differences. The human patients’ fibroblasts included in this study have 1000 CTG and 2000 CTG repeats, representing the adult and congenital DM1 types, respectively. DM1 fibroblasts present differences at the molecular level, mainly in protein and lipids structures: DM1 donors can be distinguished from controls by a larger lipidic stretching, more saturated lipidic stretching and presence of protein aggregates. These differences can be explained by the dysregulation of lipins in DM1, affecting the lipid metabolism, and RNA and polyglutamine expansion proteins accumulation as nuclear aggregates, respectively. Moreover, the nuclear profile in human fibroblasts derived from DM1 patients was evaluated, in order to characterize some particular nuclear features, namely the nuclear area, circularity, deformations (blebs and misshaped nuclei) and micronucleus presence. Our results evidenced a tendency to increase the nuclear area, the number of cells with micronuclei and the % of deformed nuclei in DM1 donors, comparing to the control. Further, in order to unravel some nuclear envelope proteins alterations in DM1, namely in lamin A/C, emerin, SUN1 and LAP1, we performed immunocytochemistry analysis in human fibroblasts. Regarding lamin A/C, emerin and LAP1, these proteins showed an altered localization in nuclear envelope, in both DM1 fibroblasts. Also, both lamin A/C and emerin are highly present in protein positive-nuclear inclusions whereas SUN1 and LAP1 are highly present in protein positive-nuclear invaginations. To conclude, spectral differences have been identified between controls and DM1 fibroblasts. Moreover, the nuclear architecture and nuclear envelope proteins seems to be altered in DM1 donors. Therefore, further studies should be performed in order to elucidate nuclear envelope proteins contribution to the disease.A distrofia miotónica tipo 1 (DM1) é uma disfunção genética multissistémica associada principalmente à degeneração do músculo esquelético e causada por uma expansão repetida de trinucleótidos CTG na região 3' não traduzida do gene DMPK, acumulando-se na forma de inclusões nucleares e comprometendo a função nuclear. Nos últimos anos, alguns estudos associaram algumas proteínas do envelope nuclear (NE) como sendo componentes importantes na resposta das células ao stress mecânico, ajudando a manter a integridade das células musculares, o que demonstra uma associação com a DM1. De facto, já foi demonstrado que os fibroblastos de pacientes com DM1 apresentavam uma estrutura nuclear comprometida e uma localização alterada de algumas proteínas do NE. No entanto, a contribuição que o envelope nuclear possa ter para a DM1 ainda não foi totalmente elucidada. Neste trabalho, realizámos um estudo espectroscópico ATR-FTIR usando fibroblastos humanos de controlos e de pacientes com DM1 de forma a identificar diferenças espectrais. Os fibroblastos dos pacientes incluídos neste estudo têm 1000 repetições de CTG e 2000 repetições de CTG, representando os tipos DM1 adulto e congénito, respectivamente desta doença. Os modelos de DM1 usados neste estudo apresentaram diferenças a nível molecular, principalmente nas estruturas das proteínas e dos lipídos: os modelos de DM1 podem ser diferenciados dos controlos através de cadeias lipídicas maiores, cadeias lipídicas mais saturadas e presença de agregados proteicos. Estas diferenças podem ser explicadas por um metabolismo lipídico alterado e pela acumulação de agregados tóxicos sob a forma de RNA e de proteínas de poliglutaminas expandidas. Além disso, foi avaliado o perfil nuclear em fibroblastos humanos de controlos e de pacientes com DM1, de forma a caracterizar algumas características nucleares específicas, nomeadamente: área nuclear, circularidade, deformações (blebs e núcleos irregulares) e presença de micronúcleos. Os nossos resultados evidenciaram uma tendência para a área nuclear, o número de células com micronúcleos e os núcleos deformados aumentarem nos modelos de DM1, em comparação aos controlos. Além disso, para observar a localização de algumas proteínas do envelope nuclear, nomeadamente, lamina A/C, emerina, SUN1 e LAP1, foi realizada uma análise imunocitoquímica nos fibroblastos humanos de controlos e de pacientes com DM1. Em relação à lamina A/C, emerina e LAP1, estas proteínas apresentaram uma localização alterada no envelope nuclear em ambos os modelos de DM1 (DM1 1000 e DM1 2000). Além disso, a lamina A/C e a emerina mostraram estar acumuladas nas inclusões nucleares marcadas por estas proteínas, enquanto que a SUN1 e a LAP1 mostraram estar acumuladas nas invaginações nucleares marcadas por estas proteínas. Para concluir, foram identificadas diferenças espectrais entre os controlos e os modelos de DM1. Além disso, a arquitetura nuclear e as proteínas do envelope nuclear mostraram estar alteradas nos modelos de DM1. Portanto, mais estudos devem ser realizados para elucidar a contribuição que as proteínas do envelope nuclear possam ter para esta doença.
Mestrado em Biomedicina Molecular
44
Viegas, Diana Ferreira. „Nuclear envelope dysfunctions observed in patients with myotonic dystrophy type 1“. Master's thesis, 2021. http://hdl.handle.net/10773/30799.
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Muscular dystrophies are a very heterogeneous group of inherited diseases that
are characterized by muscle weakness, loss of muscle mass and, in some cases,
alterations in nervous system. To date, more than 30 different forms of muscular
dystrophies are known, including myotonic dystrophy (DM). Myotonic dystrophy
has two distinct variants, myotonic dystrophy type 1 (DM1) and myotonic
dystrophy type 2 (DM2), which result from an abnormal expansion of the CTG
trinucleotide and CCTG tetranucleotide, respectively. DM1 results from an
abnormal expansion of CTG in the 3 'untranslated region of the DMPK gene. The
mutant DMPK mRNA accumulates in the nucleus as ribonuclear foci
compromising the normal nuclear function. In recent years, nuclear envelope
(NE) proteins have been associated with muscular dystrophies and specifically
with DM1, as being an essential component in the response of cells to external
mechanical stresses and in maintaining the integrity of muscle cells. To date,
only few studies have shown that fibroblasts and myoblasts/myotubes from
patients with DM1 have a compromised nuclear structure, together with altered
location and intracellular levels of some NE proteins. Despite this, the role of NE
proteins in DM1 is remain unclear and further studies are needed. Therefore, in
present work, we analysed fibroblasts from an apparently healthy control and
from patients with DM1 with approximately 1000 and 2000 CTG repeat length
(representing the adult and congenital phenotypes, respectively) using the
techniques western blotting and immunocytochemistry to evaluate NE protein
levels and subcellular localization, respectively. Concerning intracellular levels of
NE proteins, we observed an increase in LAP1, SUN1, lamin A/C and lamin B1
intracellular levels, in patients with DM1 when compared with controls. In the
case of nesprin-1, a decrease in the intracellular was observed. The intracellular
levels of nesprin-2 were decreased only with DM1_2000 fibroblasts when
compared with controls. Finally, the emerin proteins levels were similar between
the DM1-derived fibroblasts and controls. In the study of immunocytochemistry,
we found that the nuclei of DM1-derived fibroblasts, when marked only with
DAPI, presented an increase in deformed nuclei, accompanied by a significant
increase in nuclear area (DM1_2000). In addition, we found that the nuclei of
DM1-derived fibroblasts, positive for lamin A/C proteins. emerin, LAP1 and
nesprin-1 showed also an increase in the number of deformed nuclei and nuclear
inclusions. In the case of the lamin A/C, emerin and LAP1, there was also an
increase in the immunolabelling of the NE and the nucleoplasm. In summary, we
demonstrated that DM1 patients tend to have changes in the intracellular levels
of NE proteins and that these are accompanied by changes in the nuclear
architecture. Thus, further studies are needed to elucidate the contribution of this
structure to the pathological mechanisms of DM1.As distrofias musculares são um grupo muito heterogêneo de doenças hereditárias que se caracterizam por fraqueza muscular, perda de massa muscular e, em alguns casos, alterações do sistema nervoso. Até o momento, são conhecidas mais de 30 formas diferentes de distrofias musculares, incluindo distrofia miotónica (DM). A distrofia miotónica possui duas variantes distintas, distrofia miotónica tipo 1 (DM1) e distrofia miotónica tipo 2 (DM2), que resultam de uma expansão anormal do trinucleótido CTG e do tetranucleótido CCTG, respetivamente. O DM1 resulta de uma expansão anormal de CTG na região 3 'não traduzida do gene DMPK. O mRNA DMPK mutante acumula-se no núcleo como focos ribonucleares comprometendo a função nuclear normal. Nos últimos anos, as proteínas do envelope nuclear (NE) têm sido associadas às distrofias musculares e, especificamente, á DM1, sendo um componente essencial na resposta das células a stresses mecânicos externos e na manutenção da integridade das células musculares. Até ao momento, poucos estudos demonstraram que fibroblastos e mioblastos / miotubos de pacientes com DM1 têm estrutura nuclear comprometida, juntamente com localização e níveis intracelulares alterados de algumas proteínas NE. Apesar disso, o papel das proteínas NE no DM1 ainda não está claro e mais estudos são necessários. Portanto, no presente trabalho, analisamos fibroblastos de um controle aparentemente saudável e de pacientes com DM1 com aproximadamente 1000 e 2000 repetições de CTG (representando os fenótipos adulto e congénito, respetivamente) usando as técnicas de western blotting e imunocitoquímica para avaliar os níveis de proteína NE e localização subcelular, respetivamente. Em relação aos níveis intracelulares das proteínas NE, observamos aumento nos níveis intracelulares de LAP1, SUN1, lamina A / C e lamina B1, em pacientes com DM1 quando comparados aos controles. No caso da nesprina-1, foi observada uma diminuição dos níveis intracelulares da proteína. Os níveis intracelulares de nesprina-2 diminuíram apenas em fibroblastos de DM1_2000 quando comparados aos controles. Finalmente, os níveis da proteína emerina foram semelhantes entre os fibroblastos derivados de DM1 e controles. No estudo da imunocitoquímica, constatamos que os núcleos dos fibroblastos derivados do DM1, quando marcados apenas com DAPI, apresentavam aumento de núcleos deformados, acompanhado de aumento significativo da área nuclear (em DM1_2000). Além disso, descobrimos que os núcleos de fibroblastos derivados de DM1 são positivos para proteínas da lâmina A/C. emerina, LAP1 e nesprina-1 também apresentaram aumento no número de núcleos deformados e inclusões nucleares. No caso da lâmina A/C, emerin e LAP1, também houve aumento da imunomarcação do NE e do nucleoplasma. Em resumo, demonstramos que pacientes com DM1 tendem a apresentar alterações nos níveis intracelulares das proteínas NE e que estas são acompanhadas por alterações na arquitetura nuclear. Assim, mais estudos são necessários para elucidar a contribuição dessa estrutura para os mecanismos patológicos do DM1.
Mestrado em Biomedicina Molecular
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Wang, Pei-Ying, und 汪佩瑩. „Investigation of Pathogenesis of Myotonic Dystrophy Type 1 in Central Nervous System“. Thesis, 2018. http://ndltd.ncl.edu.tw/handle/82g8ad.
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博士國立陽明大學
分子醫學博士學位學程
106
Myotonic dystrophy (DM) is the most common cause of adult onset muscular dystrophy. Brain involvement in DM type 1 (DM1) includes mental retardation, psychiatric disorders and neurodegeneration. Cognitive impairment associated with structural change in the brain is prevalent in DM1. The occurrence of cognitive impairment in individuals with DM1 is high, however, the mechanism of causing the deficits remains unknown. The genetic basis of DM1 is caused by an expansion of CTG repeats in the 3’ untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. DMPK mRNA containing expanded CUG repeats accumulates in nuclear foci and affect nuclear and cytoplasmic activities of RNA binding protein muscleblind like (MBNL) family. Dysfunction of MBNL has been implicated in DM1 neural pathogenesis. How expanded CUG RNA and MBNL dysfunction affect cognitive function and brain structure remains elusive. To study the neural pathogenesis of DM1, we established a brain-specific DM1 mouse model, EpA960/ CaMKII-Cre, expressing the expanded CUG RNA in the postnatal brain. The EpA960/ CaMKII-Cre model recapitulates several features of DM1 brain including nuclear RNA and MBNL foci formation, misregulated alternative splicing, learning disability and neuro-degeneration. The pathological abnormality characterized by a time-course study showed that hippocampus-related learning and synaptic potentiation were impaired before structural changes, followed by progressive reduction of axon and dendrite integrity and aberrant MBNL2-regulated alternative splicing. In addition, cytoplasmic MBNL1 on dendrites decreased before dendrite degeneration suggesting reduced cytoplasmic MBNL1 as an early event response to the pathogenic RNA. We further investigated the causal mechanism of cytoplasmic MBNL1 reduction and the function of cytoplasmic MBNL1. We found that neurons expressing expanded CUG RNA and MBNL1-depleted neurons exhibited similar morphological impairment. MBNL1 cytoplasmic, but not nuclear, isoform promoted neurite outgrowth and reversed the morphological defects caused by expanded CUG RNA. The reduced cytoplasmic MBNL1 caused by expanded CUG RNA was due to MBNL1 cytoplasm-to-nucleus translocation. The cytoplasmic localization of MBNL1 was regulated by lysine 63 (K63)-linked polyubiquitination. Reduced cytoplasmic MBNL1 in the EpA960/CaMKII-Cre brain was consistent with the reduced extent of K63 ubiquitination. Expanded CUG RNA induced the deubiqutination of cytoplasmic MBNL1, which resulted in nuclear translocation andmorphological defects that could be ameliorated by inhibiting K63-linked polyubiquitin chain degradation. These results suggest that K63-linked ubiquitination of MBNL1 is required for its cytoplasmic localization and that deubiquitination of cytoplasmic MBNL1 is pathogenic in DM1 brain.
46
Mateus, Tiago Duarte Cordeiro. „Study of the metabolome and muscle strength measures for the characterization of patients with myotonic dystrophy type 1“. Master's thesis, 2021. http://hdl.handle.net/10773/30819.
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Myotonic dystrophy type 1 (DM1) is an autosomal dominant hereditary disease caused by an
alteration leading to an abnormal expansion of unstable repetitions of CTG in the 3’
untranslated region of Myotonic Dystrophy Protein Kinase (DMPK) gene. DM1 is
characterized by myotonia, progressive distal muscle weakness and by multisystemic
involvement namely cataracts, muscle pain, cardiac and respiratory dysfunctions, endocrine
dysfunctions (insulin resistance, metabolic syndrome, dyslipidemia), cancer and alterations in
the central nervous system (CNS). Patients with DM1 have a frequency of metabolic
syndrome higher than in the general population. Thus, the study of the metabolome is of a
great importance since it can give new insight regarding the molecular pathways affected in
DM1 diseases as well as to discriminate between the different degrees of severities in
patients with DM1 and may also, lead to the development of new metabolic therapeutics.
Given the previously reported metabolic alterations observed in patients DM1, we considered
that the evaluation of the metabolic profile of those patients of paramount importance.
Therefore, we started with the literature review for summarizing the metabolic alterations
previously reported in patients with DM1 and the relationship of Lipin with the metabolic
alterations in DM1 (Chapter I). Essentially, the previous studies showed a clear metabolic
alteration between patients with DM1 and control groups, namely, increased total cholestrol,
Low-density lipoprotein, triacylglycerol, insulin and HOMA-Insulin resistance levels, increased
glucose levels and low levels of high-density lipoprotein. This review also showed a potential
relationship between Lipin and its association with metabolic abnormalities found in patients
with DM1, namely, the metabolic roles in adipose tissue, skeletal-muscle, liver and its
association with dyslipidemia and insulin resistance, which is a characteristic feature in
patients with DM1.
The metabolic profile of patients with DM1 then was evaluated using the ATR-FTIR
spectroscopy technique, together with multivariate analysis, which is suitable for providing a
(bio)chemical profile of patients with DM1 and controls. Essentially, DM1-derived fibroblast
and controls were used, and the results showed a clear discrimination within DM1-derived
fibroblast with different CTG repeat length and age at onset, meaning that they may have a
distinct metabolic profile. This discrimination can be attributed mainly to the altered lipid
metabolism in 1800-1500 region cm-1
. It was also possible to discriminate between the
control groups and both DM1-derived fibroblast from Coriell institute and Centro Hospitalar do
Tâmega e Sousa in 3000-2800 cm-1
region (Chapter II).
Additionally, a systematic review was made to gather information of all outcome and
measurements used to assess muscle strength in adult patients with DM1 (Chapter IV). The
cardiac, skeletal and respiratory muscle strength was evaluated. Briefly, the systematic
review showed a consistent use of echocardiography, quantitative muscle test, manual
muscle test and manometry to assess cardiac, skeletal and respiratory muscle strength. The
measures of choice to assess muscle strength were: (1) ejection fraction in cardiac muscle;
(2) muscle isometric torque, grip strength and medical research council (0-5 points and 0-60
points) in skeletal-muscle; (3) maximal inspiratory pressure and maximal expiratory pressure
in respiratory muscles. In conclusion, our results suggest that there is a need to further
research the lipid metabolism of patients with DM1, not only to better characterize these
patients but also to understander the underlying mechanism of lipid abnormalities and to
have new insights of Lipin in DM1. FTIR spectroscopy is a valuable tool to characterize
patients with DM1 severities, which is crucial for a proper diagnosis and further studies. We
successfully gather the more consensual and important measures to evaluate muscle
strength. The results obtained were important and useful given that they will be valuable for
muscle strength evaluation in future clinical trials and observational studies, particularly to
test if a drug is improving muscle strength in patients with DM1.A distrofia miotônica tipo 1 (DM1) é uma doença hereditária autossómica dominante causada por uma alteração que leva a uma expansão anormal de repetições instáveis de CTG na região 3' não traduzida do gene da proteína quinase da distrofia miotônica (DMPK). DM1 é caracterizado por miotonia, fraqueza muscular distal progressiva e por envolvimento multissistémica, nomeadamente cataratas, dores musculares, disfunções cardíacas e respiratórias, disfunções endócrinas (resistência à insulina, síndrome metabólica, dislipidemia), cancro e alterações no sistema nervoso central (SNC). Doentes com DM1 apresentam frequência de síndrome metabólica maior do que na população geral. Assim, o estudo do metaboloma é de grande importância, pois pode fornecer novos ideias sobre as vias moleculares afetadas nas doenças DM1, bem como discriminar entre os diferentes graus de gravidade em doentes com DM1 e também pode levar ao desenvolvimento de novas terapêuticas metabólicas. Dadas as alterações metabólicas previamente descritas e observadas em doentes com DM1, consideramos que a avaliação do perfil metabólico destes doentes é de grande importância. Portanto, elaborou-se uma revisão da literatura para resumir as alterações metabólicas previamente descritas em doentes com DM1 e a relação da Lipina com as alterações metabólicas na DM1 (Capítulo I). Essencialmente, os estudos anteriores mostraram uma clara alteração metabólica entre os doentes com DM1 e os grupos controlo, nomeadamente o aumento dos níveis de colesterol total, lipoproteína de baixa densidade, triacilglicerol, insulina e resistência HOMA-insulina, o aumento dos níveis de glicose, assim como a diminuição dos níveis de lipoproteína de alta densidade. Esta revisão também demonstrou uma potencial relação entre a Lipina e a sua associação com as anormalidades metabólicas encontradas em doentes com DM1, nomeadamente os papéis metabólicos no tecido adiposo, músculo esquelético, fígado e a sua associação com a dislipidemia e a resistência à insulina, que é uma das características em doentes com DM1. O perfil metabólico dos doentes com DM1 foi então avaliado pela técnica de espectroscopia ATR FTIR, em conjunto com a análise multivariada, sendo que é adequada para fornecer um perfil (bio) químico dos doentes com DM1 e controlos. Essencialmente, fibroblastos derivados de DM1 e controlos foram utilizados, e os resultados demonstraram uma clara discriminação dentro de fibroblastos derivados de DM1 com diferentes repetições de CTG e idades de início da doença, o que significa que estes podem ter um perfil metabólico distinto. Esta discriminação pode ser atribuída principalmente ao metabolismo lipídico alterado na região 1800-1500 cm-1 . Também foi possível discriminar entre os grupos controlo e fibroblastos derivados de DM1 do Instituto Coriell e Centro Hospitalar do Tâmega e Sousa na região de 3000-2800 cm-1 (Capítulo II). Além disso, foi feita uma revisão sistemática para reunir informações de todos os resultados e medidas utilizadas para avaliar a força muscular em doentes adultos com DM1 (Capítulo IV). Foi avaliada a força muscular cardíaca, esquelética e respiratória. Resumidamente, a revisão sistemática demonstrou uma utilização consistente da ecocardiografia, teste muscular quantitativo, teste muscular manual e manometria para avaliar a força muscular cardíaca, esquelética e respiratória. As medidas escolhidas para avaliar a força muscular foram: (1) fração de ejeção para a força do musculo cardíaco; (2) torque isométrico muscular, força de preensão e conselho de pesquisa médica (0-5 pontos e 0-60 pontos) para a força do músculo esquelético; (3) pressão inspiratória máxima e pressão expiratória máxima para a força dos músculos respiratórios. Em conclusão, os resultados sugerem que há uma necessidade de estudos adicionais relativamente ao metabolismo lipídico em doentes com DM1, não apenas para caracterizar melhor estes doentes, como também para compreender o mecanismo subjacente das anormalidades lipídicas e ter novas noções sobre a Lipina na DM1. A espectroscopia FTIR é uma ferramenta valiosa para caracterizar doentes com diferentes severidades da DM1, o que é crucial para um diagnóstico adequado e para estudos futuros. Reunimos com sucesso as medidas mais consensuais e importantes para avaliar a força muscular. Os resultados obtidos foram importantes e úteis, pois serão valiosos para avaliação da força muscular em futuros ensaios clínicos e estudos observacionais, principalm
Mestrado em Biologia Molecular e Celular
47
Costa, Adriana Emília Amaral. „Establishment of skin-derived fibroblast cell lines for the study of protein phosphorylation in Myotonic Dystrophy type 1“. Master's thesis, 2021. http://hdl.handle.net/10773/31035.
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Muscular dystrophies are a molecularly, genetically, and clinically
heterogeneous group of disorders characterized mainly by progressive muscle
weakness and degeneration. Within this group, the most common muscular
dystrophy in adults is Myotonic Dystrophy type 1 (DM1), an inherited autosomal
dominant disorder caused by an expansion of the (CTG)n trinucleotide repeats
on the 3’ untranslated region of the DMPK gene. Patients with DM1 not only
present muscular symptoms such as myotonia and muscle wasting, but also
extramuscular symptoms such as cataracts, cardiac conduction abnormalities
and insulin resistance. In DM1, the increased length of triplet expansions leads
to the accumulation of (CUG)n mRNA, that forms hairpin-like structures in the
nucleus, leading to a toxic “gain-of-function” that deregulates RNA binding
proteins such as MBNL1 and CUGBP1. This consequently affects alternative
splicing of different mRNAs, which damages the normal function of different
signalling pathways regulated through phosphorylation, an important regulatory
mechanism. To understand the different impaired phosphorylation signalling
pathways affected in DM1 we firstly conducted a systematic review about protein
phosphorylation in DM1. The results provided a compilation of the altered protein
phosphorylation events, namely the signalling pathways which regulate key
cellular events. Some main findings are the underactivation of signalling
pathways, such as AKT/mTOR and AMPK upon insulin signalling and starvation
conditions, respectively. Also, myoblast differentiation was impaired since,
during differentiation, there was an increase of activity of signalling pathways that
stimulate cell proliferation (e.g. MEK/ERK, PKR/PERK) and a decrease of
important proteins for muscle development, such as DMPK. In order to be able
to study the mechanisms underlying the impaired signalling pathways described
in the systematic review, it is necessary to establish DM1 cell models. For that
reason, fibroblasts have been widely used for the study of this disorder due to its
versatility and easy manipulation. We then successfully established skin-derived
human fibroblast cell lines from patients with DM1 through a skin punch biopsy
explant. These cell lines were subsequently characterized through indirect
immunocytochemistry using a fibroblast-specific marker TE-7. The intracellular
levels and localization of DMPK were also evaluated. It was possible to detect
differences, although not statistically significant, of a reduced expression of
DMPK in DM1-derived fibroblasts from late and juvenile onset with controls. To
conclude, these fibroblasts can be an important cell model for the study of
phosphorylation pathways and other mechanisms, such as nuclear envelope
alterations and being an important research tool for DM1. As future perspectives,
these cell models can be used to study protein phosphatases, such as PP1 and
PP2, since there is not enough evidence of how these are altered in DM1 and
could highly contribute to unravel new molecular mechanisms.As distrofias musculares são um grupo de patologias clínica e geneticamente heterogéneas, caracterizadas por fraqueza e degeneração muscular progressivas. Dentro deste grupo, a distrofia muscular mais comum em adultos é a distrofia miotónica tipo 1 (DM1), uma doença hereditária autossómica dominante causada por uma expansão das repetições de tripletos (CTG)n na região 3' não traduzida do gene DMPK. Os pacientes com DM1 apresentam não só sintomas musculares, como miotonia e perda de massa muscular, mas também extramusculares, como cataratas, problemas na condução cardíaca e resistência à insulina. Na DM1, o aumento das expansões CTG levam ao acúmulo de mRNA (CUG)n, que forma estruturas em hairpin no núcleo, levando a um "ganho de função" tóxico que desregula proteínas de ligação ao RNA, como a MBNL1 e CUGBP1. Isso, consequentemente, afeta o splicing alternativo de diferentes mRNAs, o que prejudica a função normal de diferentes vias de sinalização reguladas por fosforilação, um importante mecanismo regulatório. Para perceber as diferentes vias de sinalização de fosforilação afetadas na DM1, executamos uma revisão sistemática sobre a fosforilação de proteínas em DM1. Os resultados forneceram uma compilação das vias de sinalização alteradas e que regulam eventos celulares chave. Alguns dos principais resultados são a reduzida ativação das vias da AKT/mTOR e da AMPK quando estimuladas com insulina ou com condições de privação de nutrientes, respetivamente. Adicionalmente, a miogénese estava também alterada devido ao aumento de vias estimuladoras de proliferação celular (e.g. MEK/ERK, PKR/PERK) e uma diminuição de proteínas importantes para o desenvolvimento muscular, como a DMPK. Para poder estudar os mecanismos subjacentes às vias de sinalização prejudicadas descritas na revisão sistemática, é necessário estabelecer modelos de células DM1. Por esse motivo, os fibroblastos têm sido amplamente utilizados para o estudo dessa doença devido à sua versatilidade e fácil manipulação. Em seguida, estabelecemos com sucesso linhas de células de fibroblastos humanos derivadas da pele de pacientes com DM1 através de um explante de biópsia cutânea. Essas linhas celulares foram posteriormente caracterizadas por imunocitoquímica indireta usando um marcador específico para fibroblastos TE-7. Os níveis intracelulares e a localização de DMPK também foram avaliados. Foi possível detetar diferenças, embora não estatisticamente significativas, de uma expressão reduzida de DMPK em fibroblastos derivados de DM1 com fenótipos de início tardio e juvenil comparando com controlos. Concluindo, esses fibroblastos podem ser um importante modelo celular para o estudo das vias de fosforilação e outros mecanismos, como alterações do envelope nuclear, sendo uma importante ferramenta de pesquisa para DM1. Como perspetivas futuras, estas linhas celulares podem ser usadas para estudar as fosfatases, como a PP1 e PP2, uma vez que não há evidências suficientes de como estas se alteram no DM1 e podem contribuir fortemente para desvendar novos mecanismos moleculares.
Mestrado em Biomedicina Molecular
48
Burgoci, Vasile. „Étude du rôle des ARN non codants du cluster Dlk1-Dio3 dans la dystrophie myotonique de type 1“. Thèse, 2019. http://hdl.handle.net/1866/22793.
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