Estudamos duas grandes famílias com manifestações de doenças neurodegenerativas. Uma delas é originária do alto oeste do estado do Rio Grande do Norte e a outra, da região de São José do Rio Preto, SP. A primeira, uma extensa família com tradição de casamentos consangüíneos, apresenta 68 indivíduos afetados pela síndrome a qual nomeamos Spoan (Spastic Paraplegia, Optic Atrophy, Neuropathy). A mesma é uma doença neurodegenerativa de herança autossômica recessiva, caracterizada por atrofia óptica congênita, espasticidade, polineuropatia periférica axonal sensitivo-motora, sobressaltos à estimulação sonora, deformidades articulares e da coluna e disartria. Estes resultados foram publicados em 2005 no Ann Neurol. 57(5):730-7. Dando continuidade ao estudo, selecionamos 23 genes que tiveram todos os exons seqüenciados. Nenhuma mutação foi observada. Amostras de 65 afetados e seus parentes foram estudados para seis marcadores de microsatélite, totalizando 149 indivíduos genotipados. Cinqüenta SNPs foram investigados, o que nos permitiu reduzir a região candidata de 4.8 para 2.3Mb em 11q13, entre o SNP rs1939212 e o microssatélite D11S987. Para o marcador D11S1889, com alelos em homozigose para todo os pacientes, foi obtido um lod score máximo de 27 em .=0.0. Os resultados deste estudo se encontram em fase de submissão. A segunda família foi estudada pela equipe da Dra. Mayana Zatz há alguns anos. Nela, investigamos 12 indivíduos afetados e 12 normais. Dentre estes, sete, com idades entre 30 e 60 anos, foram clinicamente avaliados. A idade de início foi a partir da terceira década de vida, sendo a paraplegia espástica o único sintoma. Para o marcador DXS8057 localizado em Xq25 foi obtido um lod score máximo de 4.13 em .=0.0. Com o estudo de marcadores moleculares, delimitamos uma região candidata entre os marcadores DXS1001 e DXS8033, de cerca de 14Mb, e demonstramos a existência de um novo loco gênico no cromossomo X, por nós denominado SPG34. Os resultados deste estudo estão publicados no Neurogenet on line em 08/05/200 / We studied two large families with expressions of neurodegenerative diseases. One is from the high west of the state of Rio Grande do Norte and the other from São José do Rio Preto region, in São Paulo. The first, an extended family with a tradition of consanguineous marriages, has 68 individuals affected by the syndrome named by us Spoan (Spastic Paraplegia, Optic Atrophy, Neuropathy). The Spoan syndrome is a neurodegenerative disease, autosomal recessive, characterized by congenital Optic Atrophy, spasticity, axonal polyNeuropathy peripheral sensory-motor, shocks to the sound stimuli, joint and spine deformities, and dysarthria. These results were published in 2005 in Ann Neurol. 57 (5):730-7. Latter we analyzed 23 genes that were entirely sequenced. No mutation was observed. Samples of 65 affected and their relatives were studied for six microsatellite markers, totaling 149 individuals genotiped. Fifty single nucleotide polymorphisms (SNPs), located in the critical region, were also investigated, which allowed us to reduce the region for the SPOAN gene from 4.8 to 2.3 Mb, between the SNP rs1939212 and microsatellite D11S987 in 11q13. All patients are homozygous only at D11S1889, which two-point lod score with a Zmax of 27 at .=0.0 was obtained. The results of this study are being submitted. The second family was studied by Dr. Mayana Zatz group a few years ago. We investigated 12 affected and 12 normal relatives. Among these, seven patients, aged between 30 and 60 years, were clinically evaluated. The age of onset was from the third decade of life and disease showed behaviour very uniform, all affected showed Spastic Paraplegia as the only symptom. For the marker DXS8057, in Xq25, was obtained a maximum lod score of 4.13 at .=0.0. The candidate region was maped between the markers DXS1001 and DXS8033, about 14Mb and demonstrate the existence of a new gene locus on chromosome X, named by us SPG34. The results of this study were published in Neurogenet on line on may.08.2008.

Estudamos duas grandes famílias com manifestações de doenças neurodegenerativas. Uma delas é originária do alto oeste do estado do Rio Grande do Norte e a outra, da região de São José do Rio Preto, SP. A primeira, uma extensa família com tradição de casamentos consangüíneos, apresenta 68 indivíduos afetados pela síndrome a qual nomeamos Spoan (Spastic Paraplegia, Optic Atrophy, Neuropathy). A mesma é uma doença neurodegenerativa de herança autossômica recessiva, caracterizada por atrofia óptica congênita, espasticidade, polineuropatia periférica axonal sensitivo-motora, sobressaltos à estimulação sonora, deformidades articulares e da coluna e disartria. Estes resultados foram publicados em 2005 no Ann Neurol. 57(5):730-7. Dando continuidade ao estudo, selecionamos 23 genes que tiveram todos os exons seqüenciados. Nenhuma mutação foi observada. Amostras de 65 afetados e seus parentes foram estudados para seis marcadores de microsatélite, totalizando 149 indivíduos genotipados. Cinqüenta SNPs foram investigados, o que nos permitiu reduzir a região candidata de 4.8 para 2.3Mb em 11q13, entre o SNP rs1939212 e o microssatélite D11S987. Para o marcador D11S1889, com alelos em homozigose para todo os pacientes, foi obtido um lod score máximo de 27 em .=0.0. Os resultados deste estudo se encontram em fase de submissão. A segunda família foi estudada pela equipe da Dra. Mayana Zatz há alguns anos. Nela, investigamos 12 indivíduos afetados e 12 normais. Dentre estes, sete, com idades entre 30 e 60 anos, foram clinicamente avaliados. A idade de início foi a partir da terceira década de vida, sendo a paraplegia espástica o único sintoma. Para o marcador DXS8057 localizado em Xq25 foi obtido um lod score máximo de 4.13 em .=0.0. Com o estudo de marcadores moleculares, delimitamos uma região candidata entre os marcadores DXS1001 e DXS8033, de cerca de 14Mb, e demonstramos a existência de um novo loco gênico no cromossomo X, por nós denominado SPG34. Os resultados deste estudo estão publicados no Neurogenet on line em 08/05/200 / We studied two large families with expressions of neurodegenerative diseases. One is from the high west of the state of Rio Grande do Norte and the other from São José do Rio Preto region, in São Paulo. The first, an extended family with a tradition of consanguineous marriages, has 68 individuals affected by the syndrome named by us Spoan (Spastic Paraplegia, Optic Atrophy, Neuropathy). The Spoan syndrome is a neurodegenerative disease, autosomal recessive, characterized by congenital Optic Atrophy, spasticity, axonal polyNeuropathy peripheral sensory-motor, shocks to the sound stimuli, joint and spine deformities, and dysarthria. These results were published in 2005 in Ann Neurol. 57 (5):730-7. Latter we analyzed 23 genes that were entirely sequenced. No mutation was observed. Samples of 65 affected and their relatives were studied for six microsatellite markers, totaling 149 individuals genotiped. Fifty single nucleotide polymorphisms (SNPs), located in the critical region, were also investigated, which allowed us to reduce the region for the SPOAN gene from 4.8 to 2.3 Mb, between the SNP rs1939212 and microsatellite D11S987 in 11q13. All patients are homozygous only at D11S1889, which two-point lod score with a Zmax of 27 at .=0.0 was obtained. The results of this study are being submitted. The second family was studied by Dr. Mayana Zatz group a few years ago. We investigated 12 affected and 12 normal relatives. Among these, seven patients, aged between 30 and 60 years, were clinically evaluated. The age of onset was from the third decade of life and disease showed behaviour very uniform, all affected showed Spastic Paraplegia as the only symptom. For the marker DXS8057, in Xq25, was obtained a maximum lod score of 4.13 at .=0.0. The candidate region was maped between the markers DXS1001 and DXS8033, about 14Mb and demonstrate the existence of a new gene locus on chromosome X, named by us SPG34. The results of this study were published in Neurogenet on line on may.08.2008.

Les mutations du gène SPG4 codant la spastin sont responsables de la forme la plus fréquente de Paraplégies Spastiques Héréditaires (PSH), des maladies neurologiques caractérisées par une dégénérescence des faisceaux cortico-spinaux. La spastin, ainsi que son homologue p60-katanin sont des enzymes de cassure des microtubules (MSE) essentielles à la croissance des neurones moteurs spinaux (NMS) chez l'embryon de poisson-zèbre mais dont le rôle dans les processus de guidage axonal également dépendant des microtubules (MTs) demeurent énigmatiques. Les principaux objectifs de ma thèse ont consisté à préciser le rôle et le degré de redondance fonctionnelle existant entre ces deux MSE lors de l'établissement des circuits moteurs chez ce téléoste et de clarifier les mécanismes pathogéniques à l¿origine des PSH liées au gène SPG4.J'ai tout d'abord contribué à montrer que la p60-Katanin contrôle la trajectoire des axones des NMS et la mobilité des larves de façon dose-dépendante et non redondante avec la spastin. De plus, notre étude identifie la polyglutamylation des MTs par TTLL6 comme un élément clé de l'activité de la p60-Katanin lors de ce processus. Sur le même modèle, j'ai révélé un rôle différentiel des isoformes majoritaires de la spastin (résultant d¿une traduction alternative, M1 et M61) au cours du développement des NMS en démontrant un rôle coopératif de M1 et d'autres protéines de PSH dans l'inhibition de la voie des BMPs et révélant un rôle pour M61 en aval de la signalisation Neuropilin-1. Ces données suggèrent que l'altération de ces deux grandes voies de signalisation essentielles au développement des NMS pourrait contribuer à la pathogénèse des formes SPG4. / Mutations in SPG4, encoding spastin cause the major form of Hereditary Spastic Paraplegias (HSP), a paralytic disorder characterised by the degeneration of the corticospinal tracts. Spastin and its close homologue p60-katanin are microtubule-severing enzymes (MSE) required for spinal motor neuron (SMN) axon extension during zebrafish development. However, their roles in SMN axon navigation which also rely on microtubules (MTs) remain elusive. My PhD work aimed at refining the functional specificity and redundancy of these MSE during motor circuit wiring and clarifying the physiopathology of SPG4-linked HSP. I have first contributed to show that p60-Katanin controls SMN axon targeting and larval locomotion in a dose-dependent manner. We also demonstrated that Spastin and p60-Katanin play differential roles in SMN navigation and identified TTLL6-mediated MT polyglutamylation as a key event in regulating p60-Katanin activity in this process. Concomitantly, I have conducted a functional analysis of spastin main isoforms (resulting from alternative translation, M1 and M61) during zebrafish development, which reveals their critical and specific involvement in two distinct signalling pathways that are both essential for motor circuit wiring and locomotor behaviours. This study has provided compelling evidences for a concerted role for M1 and other HSP proteins in the down-regulation of the BMP pathway and reveals a specific role for M87 as a downstream effector of Neuropilin-1 signalling. Altogether, our study emphasizes defective BMP signalling as a key pathogenic mechanism in HSP, and shows that dysregulation of the Neuropilin-1 pathway may equally contribute to SPG4-linked HSP.

Les paraplégies spastiques héréditaires (PSH) sont des maladies cliniquement et génétiquement hétérogènes, qui se manifestent par la présence de signes pyramidaux (spasticité, réflexes myotatiques vifs et diffusés) et d’un déficit moteur des membres inférieurs. On distingue des formes pures et complexes de PSH, ces dernières étant associées à la présence de signes additionnels tels que troubles cognitifs, neuropathie périphérique, signes cérébelleux, etc. Les mécanismes physiopathologiques des PSH sont également hétérogènes, incluant une anomalie du transport axonal (SPG3A, SPG4, SPG10, SPG20), du métabolisme mitochondrial (SPG7, SPG13), une anomalie de la formation de la myéline (SPG1) ou un dysfonctionnement du développement neuronal (SPG2). Elles peuvent se transmettre selon le mode autosomique dominant (AD), récessif (AR), ou récessif lié au chromosome X. Actuellement, 13 loci dont 9 gènes de PS-AD sont connus, mais seulement 5 gènes responsables de PS-AR ont été identifiés, alors que 14 loci sont connus. De par leur hétérogénéité clinique, génétique et physiopathologique, les PSH sont encore des maladies mal connues. Une meilleure connaissance du phénotype associé à chaque locus/gène permettrait aux cliniciens de mieux orienter les analyses moléculaires pour un diagnostic rapide. L’établissement de corrélations génotype-phénotypes et de la fréquence des gènes impliqués dans les PSH permettrait tant aux cliniciens qu’aux biologistes de cibler les gènes, les exons à analyser ou les mutations à rechercher en priorité. L’identification des mécanismes physiopathologiques des mutations est une première étape vers des études fonctionnelles et des traitements spécifiques. Nous avons montré que la forme de PS AD liée à des mutations dans le gène SPG3A était caractérisée par un début très précoce, avant l’âge de 10 114 ans. Cette forme en générale pure de PS peut se compliquer, notamment par une neuropathie périphérique ou un syndrome cérébelleux après une longue durée d’évolution de la maladie. Ces résultats permettent d’orienter les analyses moléculaires vers le gène SPG3A avant le gène SPG4, devant tout patient qui a débuté la maladie précocement, quelque soit le tableau clinique. Nous avons montré que les mutations dans le gène SPG3A, dont les mutations récurrentes p.R239C et p.R495W dans les exons 7 et 12 peuvent apparaître de-novo, justifiant l’analyse de ce gène chez des patients isolés. Nous avons étendu le phénotype des PS AD liées à des mutations dans le gène SPG4, qui doit être analysé chez les patients présentant une PS associée à un retard mental sans malformation cérébrale. De plus, nous avons montré que les délétions de ce gène ne sont pas rares, atteignant une fréquence de 20% chez les patients présentant une PS-AD sans mutation retrouvée par DHPLC. Ceci entraîne un changement des stratégies d’analyses moléculaires utilisées chez les patients atteints de PS, avec l’instauration systématique d’un MLPA chez chaque patient. Nous avons précisé le tableau clinique des paraplégies spastiques AR liées aux loci SPG26 et 27. Nous avons réduit l’intervalle génomique de ces loci. L’identification d’autres familles liées à ces loci permettra de réduire encore plus leurs intervalles génomiques, voire d’identifier les gènes responsables de ces maladies.

No description available.

Les paraplégies spastiques héréditaires (PSH) font partie d’un groupe plus large de pathologies neurodégénératives associant une spasticité. J’ai exploré la variabilité clinique et moléculaire de ces pathologies à l’aide d’une cohorte de familles soudanaises. Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J’ai extrait l’ADN génomique et constitué une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l’exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l’effet biologique des mutations. J’ai pu identifier la cause génétique dans 17 familles. Dans 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a été identifié. 5 gènes candidats restent à départager dans 2 familles. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles, dans la même branche ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l’analyse des données du SNG difficile. Une autre particularité a été l’hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature. Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents. / Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort. We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all-remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionally, functional studies were conducted when feasible and relevant. I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms.