Introdução: Dentre as neuropatias hereditárias, as neuropatias hereditárias motoras e sensitivas (HMSN), também conhecidas como Doença de Charcot-MarieTooth (CMT) são as mais comuns, podendo acometer 1:2500 pessoas. Elas podem ser classificadas com base nas características clínicas, eletrofisiológicas, padrão de herança e mutação em localização gênica/mutação. Atualmente, mais de 80 genes estão relacionados à CMT, dentre eles o GDAP1 que é responsável pelas formas CMT4A, AR-CMT2, CMTRIA e CMT2K. O gene codifica a proteína GDAP1 que é expressa pelos neurônios do sistema nervoso periférico e central e também pelas células de Schwann. Mutações no GDAP1 geralmente estão relacionados com CMT de herança recessiva, mas autossômica dominante também pode ocorrer. Geralmente, as neuropatias recessivas são de diagnóstico molecular mais difíceis, são mais graves e tem rápida progressão. É necessário que haja um maior número de casos de pacientes com CMT envolvendo mutações no GDAP1, juntamente com os dados clínicos, patológicos e de eletrofisiologia detalhados, para estabelecer uma relação de confiança entre o genótipo e o fenótipo das diferentes formas da doença. O objetivo do trabalho foi investigar mutações no gene GDAP1 em uma amostra da população brasileira com quadro clínico de CMT tanto axonal quanto desmielinizantes. Métodos: Screening mutacional do gene GDAP1 por sequenciamento direto em 100 pacientes com diagnóstico clínico sugestivo de CMT4, CMT2, CMTi e CMT esporádico, onde mutações nos genes MPZ, MFN2 e GJB1 foram previamente excluídos. Resultados: Foram encontradas alterações no GDAP1 em 6 pacientes índices não relacionados, sendo que 1 deles foi homozigoto para a alteração Q163*, 1 heterozigoto composto para as alterações N64S e R125*, dois pacientes não relacionados foram heterozigotos compostos para as alterações P119T e Q163*, 1 paciente em heterozigose para a alteração P119T e 1 paciente em heterozigose para alteração K207T. Dentre essas alterações, as variantes N64S, P119T, K207T ainda não foram descritas na literatura. Conclusão: Os resultados obtidos mostraram que mutações no gene GDAP1 estão presentes em pacientes da população brasileira com fenótipo de CMT2, AR-CMT2 e CMT esporádico. A frequência de ocorrência pode ser considerada alta (3,88%). Ademais, foram encontradas na população de estudo, duas mutações conhecidamente patogênicas (Gln163Ter e Arg125Ter) e três novas variantes (Asn64Ser, Pro119Thr, Lys207Thr) que ainda não haviam sido relacionadas ao fenótipo de CMT. / Introduction: Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth disease, are the most common ones and may affect 1:2500 people. They can be classified based on their clinical features, electrophysiology, inheritance pattern and mutation on a gene location/mutation. Today, more than 80 genes have been related to the CMT disease and among them the GDAP1 gene, responsible for the CMT4A, AR-CMT2, CMTRIA and CMT2K forms. This gene codes the GDAP1 protein, which is expressed by neurons from the peripheral and central nervous system and also by Schwann cells. GDAP1 mutations are usually related to recessive inheritance, although autosomal dominant cases can also occur. Most of the times, recessive neuropathies tend to have a more difficult molecular diagnosis, besides being more severe and presenting fast progression. A larger number of patients with CMT related to GDAP1 mutations, along with detailed clinical, pathological and electrophysiological data, is necessary in order to establish a trustful relation between genotype and phenotype in the different forms of this disease. The objective of this research was to investigate mutations in the GDAP1 gene in a brazilian sample of patients with clinical picture of both axonal and demyelinating CMT. Methods: Mutational screening of the GDAP1 gene by direct sequencing of 100 patients presenting suggestive clinical diagnosis of CMT4, CMT2, CMTi and sporadic CMT, where mutations in the MPZ, MFN2 and GJB1 genes have been previously excluded. Results: Alterations in the GDAP1 were found in 6 unrelated index patients, including 1 homozygous for the Q163* alteration, 1 compound heterozygous for the N64S and R125* alterations, 2 compound heterozygous for the P119T and Q163* alterations, 1 heterozygous for the P119T alteration alone and 1 heterozygous for the K207T alteration. Among these alterations, the variants N64S, P119T, K207T haven\'t been described in the literature yet. Conclusion: The results obtained showed that mutations in GDAP1 gene are present in patients of brazilian population with phenotype of CMT2, AR-CMT2 and sporadic CMT. The occurrence frequency can be considered high (3,88%). Furthermore, were found in the studied population two mutations known as pathogenic (Gln163Ter and Arg125Ter) and three news variants (Asn64Ser, Pro119Thr, Lys207Thr), that had not been related to the CMT phenotype.

Introdução: Dentre as neuropatias hereditárias, as neuropatias hereditárias motoras e sensitivas (HMSN), também conhecidas como Doença de Charcot-MarieTooth (CMT) são as mais comuns, podendo acometer 1:2500 pessoas. Elas podem ser classificadas com base nas características clínicas, eletrofisiológicas, padrão de herança e mutação em localização gênica/mutação. Atualmente, mais de 80 genes estão relacionados à CMT, dentre eles o GDAP1 que é responsável pelas formas CMT4A, AR-CMT2, CMTRIA e CMT2K. O gene codifica a proteína GDAP1 que é expressa pelos neurônios do sistema nervoso periférico e central e também pelas células de Schwann. Mutações no GDAP1 geralmente estão relacionados com CMT de herança recessiva, mas autossômica dominante também pode ocorrer. Geralmente, as neuropatias recessivas são de diagnóstico molecular mais difíceis, são mais graves e tem rápida progressão. É necessário que haja um maior número de casos de pacientes com CMT envolvendo mutações no GDAP1, juntamente com os dados clínicos, patológicos e de eletrofisiologia detalhados, para estabelecer uma relação de confiança entre o genótipo e o fenótipo das diferentes formas da doença. O objetivo do trabalho foi investigar mutações no gene GDAP1 em uma amostra da população brasileira com quadro clínico de CMT tanto axonal quanto desmielinizantes. Métodos: Screening mutacional do gene GDAP1 por sequenciamento direto em 100 pacientes com diagnóstico clínico sugestivo de CMT4, CMT2, CMTi e CMT esporádico, onde mutações nos genes MPZ, MFN2 e GJB1 foram previamente excluídos. Resultados: Foram encontradas alterações no GDAP1 em 6 pacientes índices não relacionados, sendo que 1 deles foi homozigoto para a alteração Q163*, 1 heterozigoto composto para as alterações N64S e R125*, dois pacientes não relacionados foram heterozigotos compostos para as alterações P119T e Q163*, 1 paciente em heterozigose para a alteração P119T e 1 paciente em heterozigose para alteração K207T. Dentre essas alterações, as variantes N64S, P119T, K207T ainda não foram descritas na literatura. Conclusão: Os resultados obtidos mostraram que mutações no gene GDAP1 estão presentes em pacientes da população brasileira com fenótipo de CMT2, AR-CMT2 e CMT esporádico. A frequência de ocorrência pode ser considerada alta (3,88%). Ademais, foram encontradas na população de estudo, duas mutações conhecidamente patogênicas (Gln163Ter e Arg125Ter) e três novas variantes (Asn64Ser, Pro119Thr, Lys207Thr) que ainda não haviam sido relacionadas ao fenótipo de CMT. / Introduction: Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth disease, are the most common ones and may affect 1:2500 people. They can be classified based on their clinical features, electrophysiology, inheritance pattern and mutation on a gene location/mutation. Today, more than 80 genes have been related to the CMT disease and among them the GDAP1 gene, responsible for the CMT4A, AR-CMT2, CMTRIA and CMT2K forms. This gene codes the GDAP1 protein, which is expressed by neurons from the peripheral and central nervous system and also by Schwann cells. GDAP1 mutations are usually related to recessive inheritance, although autosomal dominant cases can also occur. Most of the times, recessive neuropathies tend to have a more difficult molecular diagnosis, besides being more severe and presenting fast progression. A larger number of patients with CMT related to GDAP1 mutations, along with detailed clinical, pathological and electrophysiological data, is necessary in order to establish a trustful relation between genotype and phenotype in the different forms of this disease. The objective of this research was to investigate mutations in the GDAP1 gene in a brazilian sample of patients with clinical picture of both axonal and demyelinating CMT. Methods: Mutational screening of the GDAP1 gene by direct sequencing of 100 patients presenting suggestive clinical diagnosis of CMT4, CMT2, CMTi and sporadic CMT, where mutations in the MPZ, MFN2 and GJB1 genes have been previously excluded. Results: Alterations in the GDAP1 were found in 6 unrelated index patients, including 1 homozygous for the Q163* alteration, 1 compound heterozygous for the N64S and R125* alterations, 2 compound heterozygous for the P119T and Q163* alterations, 1 heterozygous for the P119T alteration alone and 1 heterozygous for the K207T alteration. Among these alterations, the variants N64S, P119T, K207T haven\'t been described in the literature yet. Conclusion: The results obtained showed that mutations in GDAP1 gene are present in patients of brazilian population with phenotype of CMT2, AR-CMT2 and sporadic CMT. The occurrence frequency can be considered high (3,88%). Furthermore, were found in the studied population two mutations known as pathogenic (Gln163Ter and Arg125Ter) and three news variants (Asn64Ser, Pro119Thr, Lys207Thr), that had not been related to the CMT phenotype.

Les cellules souches induites à la pluripotence (iPSc) apparaissent comme une solution très intéressante pour créer et observer le comportement de cellules spécifiques et inaccessibles d'un patient. Notre équipe travaille sur les pathologies génétiques des nerfs périphériques et en particulier la maladie de Charcot-Marie-Tooth (CMT). Un de nos objectifs est le développement de modèles de motoneurones de patients utilisant la stratégie des iPSc afin de mieux comprendre la physiopathologie des neuropathies liées au gène GDAP1. Ce gène a été décrit en 1998 pour être responsable d'une forme axonale de CMT ; il code une protéine de la membrane externe mitochondriale dont la fonction précise reste encore méconnue. Des fibroblastes dermiques (FD) ont été obtenus après une biopsie de peau d'une personne saine (témoin) et d'un patient homozygote porteur de la mutation non-sens p.Gln163* dans le gène GDAP1. Par la suite, les FDs ont été reprogrammés en cellules iPSc en utilisant le cocktail de Yamanaka (plasmides non intégratifs composés d’Oct4, Sox2, Klf4 et l-Myc). Après amplification, tous les contrôles ont été effectués pour conclure que nos iPSc avaient les mêmes propriétés et les mêmes capacités que les cellules souches embryonnaires ainsi qu’un caryotype normal. Enfin, nous avons optimisé le protocole de différenciation avec succès de manière à obtenir à partir des iPSc des rosettes (structures pleines de progéniteurs neuronaux), puis des neurones et finalement des motoneurones pour le contrôle et le patient. Les premières différences entre le contrôle et le patient ont été observées lors de l’obtention de rosettes. Les cellules du patient présentaient de nombreuses gouttelettes lipidiques et la proportion de rosettes obtenue était plus faible. Une fois les motoneurones obtenus, des tests de microscopie confocale et électroniques ont montré des différences du réseau mitochondrial entre le témoin et le patient, ainsi qu’une morphologie des mitochondries se rapprochant de celle observée lors de biopsie de nerf de patient (rondes / accumulées). De manière à réduire la durée de différenciation, une méthode de tri cellulaire a été utilisée la SdFFF. Cette méthode nous a permis de trier différents progéniteurs (neuraux / endothéliaux). La génération de motoneurones à partir de fibroblastes dermiques de patient atteint de CMT axonale via les iPSc était une première étape cruciale pour mieux comprendre le rôle de GDAP1 dans cette pathologie. Ce modèle cellulaire de CMT4A est un premier pas pour réaliser des tests précliniques de médicaments afin d'identifier de futurs candidats pharmacologiques. / Induced pluripotent stem cells (iPSc) are a highly interesting tool to create and observe the behavior of specific and unattainable cells from a patient. Our team is interested in genetic peripheral nerves disorders and especially in Charcot-Marie-Tooth disease (CMT). One of our objectives is the development of motor neurons models from patients using the iPSc strategy in order to better understand the pathophysiology of GDAP1-related neuropathies. This gene was found in 1998 to be mutated in an axonal form of CMT and encodes a mitochondrial outer membrane protein, which function remains unclear. We first obtained dermal fibroblasts (DF) from skin biopsies of a healthy person and of a homozygous patient carrying GDAP1 non-sense mutation (p.Gln163*). Then, we reprogrammed DFs into iPSc using non-integrative plasmids (Oct4, Sox2, Klf4 and l-Myc). After amplification, all quality controls were performed to conclude that our iPSc had the same properties and capacities than embryonic stem cells and a normal karyotype. Finally, we optimized protocols to successfully differentiate these iPSc into rosettes (structures full of neural progenitors), then into neurons and finally into motor neurons for control and GDAP1 patients. The first differences between control and patient cells were observed during the rosette formation, where a lot of patient cells were full of lipid droplets, and the rosette proportion was lower than the control cells. Mitochondria morphology was totally different in motor neurons between control and patient, where mitochondria had the same morphology than the mitochondria observed in patient nerve biopsies (round and accumulated). In order to reduce the time of differentiation, a cell sorting method was used (SdFFF). It allowed us to sort different progenitors (neural / endothelial). Generation of motor neurons using axonal CMT-patient-derived iPSc was a first crucial step to better understand the role of GDAP1 in this pathology. This cellular model of CMT4A should ultimately allow us to perform preclinical drug screening in order to identify candidate pharmacological treatments for CMT patients.

La maladie de Charcot-Marie-Tooth (CMT) représente un large groupe hétérogène de neuropathies périphériques héréditaires. Les mutations du gène GDAP1 (ganglioside-induced differentiation-associated protein 1), codant pour une protéine de la membrane externe mitochondriale, sont associées à des formes récessives (CMT4A) et à des formes dominantes de CMT (CMT2K). GDAP1 participerait au processus de fission des mitochondries sans que son rôle soit bien défini. L'objectif de ce travail a été d'étudier le métabolisme énergétique mitochondrial de cellules de peau issues de patients porteurs de mutations du gène GDAP1. Nous avons mis en évidence un déficit énergétique mitochondrial associé au complexe I sans altération majeure du réseau mitochondrial. Ce déficit fonctionnel du complexe I est associé à une production accrue de radicaux libres et un défaut de régulation de la protéine sirtuine 1, une désacétylase NAD-dépendante impliquée dans la biogenèse mitochondriale. Nos travaux ont ainsi montré que GDAP1 a un rôle important dans le métabolisme énergétique mitochondrial. Dans le but d'établir des corrélations génotype-phénotype, nous avons créé une base de données internationale permettant de répertorier les données cliniques et les variations de séquence de GDAP1.