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La neurofibromatose de type 1 (NF1) est une condition génétique entraînant des répercussions dans différents systèmes, y compris le système nerveux. Les enfants atteints de la NF1 sont à risque de rencontrer différents défis sur le plan cognitif et comportemental. Un chevauchement avec les symptômes associés à d’autres troubles neurodéveloppementaux, notamment le trouble déficitaire de l’attention avec ou sans hyperactivité (TDAH) et le trouble du spectre de l’autisme (TSA), est d’ailleurs présent dans cette condition génétique. Les études réalisées avec des modèles animaux de NF1 ont permis d’identifier des déséquilibres dans la neurotransmission qui pourraient sous-tendre les déficits cognitifs. Comment ces déséquilibres se traduisent sur le plan de l’activité cérébrale chez l’humain ayant la NF1 demeure peu compris à ce jour. L’objectif général de cette thèse est donc d’étudier, à l’aide de l’électroencéphalographie (EEG), les réponses neuronales lors du traitement sensoriel chez les enfants ayant la NF1 ainsi que les associations avec le fonctionnement cognitif et comportemental. Dans le premier article, nous avons étudié l’intégrité de la réponse de synchronisation neuronale lors de stimulations visuelles rythmiques à différentes fréquences. Considérant l’importance des déficits visuo-perceptifs ainsi que les indices de perturbations de la transmission GABAergique au niveau du cortex occipital dans la NF1, nous avons émis l’hypothèse d’une réduction de la synchronisation neuronale dans le groupe NF1 par rapport au groupe d’enfants neurotypiques. Une puissance réduite de la synchronisation neuronale a effectivement été trouvée dans la NF1, particulièrement en réponse à la plus haute fréquence de stimulation. Cette réponse neuronale affaiblie a été corrélée à une plus grande sévérité des symptômes comportementaux du TDAH. Cela soulève la possibilité d’une modulation de la réponse sensorielle par les réseaux attentionnels et soulève également des questions en lien avec l’impact du diagnostic de TDAH en comorbidité avec la NF1. L’impact de la médication psychostimulante, testé avec un sous-groupe de l’échantillon NF1, s’est d’ailleurs révélé par une amélioration de la réponse de synchronisation neuronale à la plus faible fréquence de stimulation. La réponse de synchronisation neuronale apparait donc comme un marqueur à investiguer davantage pour comprendre les mécanismes qui sous-tendent les déficits cognitifs dans la NF1 et pour évaluer l’impact de traitements. Dans le deuxième article, nous avons étudié les oscillations cérébrales dans la réponse de suppression neuronale et de détection du changement, toujours en comparant un groupe d’enfants ayant la NF1 à un groupe d’enfants neurotypiques. Une modulation atypique des oscillations cérébrales était attendue dans la NF1 en réponse à la répétition et au changement dans la séquence de stimuli audio-visuels. Les résultats ont démontré une réponse de suppression neuronale préservée dans le groupe NF1, mais une puissance accrue des oscillation thêta dans la condition de détection du changement. Les symptômes comportementaux du TDAH ont été associés à une réduction de la puissance des oscillations dans la réponse de détection du changement et ne contribueraient donc pas à la réponse accrue identifiée dans le groupe NF1. En somme, les résultats de la thèse ont permis d’identifier des particularités dans les réponses électrophysiologiques du traitement sensoriel chez les enfants ayant la NF1 et de mettre de l’avant des mesures dont le potentiel translationnel pourrait faciliter le transfert des connaissances accumulées dans les modèles animaux de NF1. Les résultats soulignent la complexité d’établir les bases neuronales des déficits cognitifs dans la NF1 compte tenu de l’hétérogénéité du profil cognitif. Cela dit, les associations établies entre les réponses neuronales et les symptômes comportementaux du TDAH suggèrent la possibilité de considérer le profil électrophysiologique pour guider le développement de traitements et interventions visant à améliorer le fonctionnement cognitif chez les enfants ayant la NF1. / Neurofibromatosis type 1 (NF1) is a genetic condition that has implications across different systems, including the nervous system. Children with NF1 are at risk of facing various cognitive and behavioral challenges. An overlap with symptoms associated with other neurodevelopmental disorders, including attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD), is also present in this genetic condition. Studies conducted using animal models of NF1 have identified imbalances in neurotransmission that may underlie cognitive deficits. However, how these imbalances translate into human brain activity in individuals with NF1 remains poorly understood to date. The overall objective of this thesis is to investigate, using electroencephalography (EEG), neural responses during sensory processing in children with NF1 and their associations with cognitive and behavioral functioning. In the first article, we studied the integrity of the neural synchronization response during rhythmic visual stimulation at different frequencies. Given the significance of visuo-perceptual deficits as well as indications of disrupted GABAergic transmission in the occipital cortex of individuals with NF1, we hypothesized a reduction in neural synchronization in the NF1 group compared to neurotypical children. Reduced power of neural synchronization was indeed found in NF1, particularly in response to the highest stimulation frequency. This weakened neural response was correlated with greater severity of ADHD behavioral symptoms. These results suggest a possible modulation of this sensory response by attentional networks and raise questions regarding the impact of comorbid ADHD diagnosis on neural activity in NF1. The intake of psychostimulant medication, in a subgroup of the NF1 sample, resulted in an increased neural synchronization at the lowest stimulation frequency. Neural synchronization should be further investigated as a potential biomarker in NF1 and could help understand the underlying mechanisms of cognitive deficits and assess the impact of treatments. In the second article, we studied brain oscillations in the repetition suppression and change detection response, again comparing a group of children with NF1 to a group of neurotypical children. Atypical modulation of brain oscillations was expected in NF1 in response to repetition and change in the audio-visual stimuli sequence. Results showed preserved repetition suppression response in the NF1 group, but increased power of theta oscillations in the change detection condition. Behavioral symptoms of ADHD were associated with reduced oscillatory power in the change detection response, which suggest that the ADHD comorbidity did not contribute to the increased response identified in the NF1 group. In summary, results of this thesis have demonstrated distinct electrophysiological responses during sensory processing in children with NF1 and have highlighted measures with translational potential that could facilitate the transfer of knowledge gained from animal models of NF1. The results emphasize the complexity of establishing the neural basis of cognitive deficits in NF1, given the heterogeneity of the cognitive profile. However, the associations established between neural responses and ADHD behavioral symptoms suggest the possibility of considering the electrophysiological profile to guide the development of treatments and interventions aimed at improving cognitive functioning in children with NF1.

Indiana University-Purdue University Indianapolis (IUPUI) / Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin is the protein product of NF1 and functions as a negative regulator of Ras activity in both hematopoietic and vascular wall cells, which are critical for maintaining blood vessel homeostasis. NF1 patients are predisposed to chronic inflammation and premature cardiovascular disease, including development of large arterial aneurysms, which may result in sudden death secondary to their rupture. However, the molecular pathogenesis of NF1 aneurysm formation is completely unknown. Utilizing a novel model of Nf1 murine aneurysm formation, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/-) results in enhanced aneurysm formation with myeloid cell infiltration and increased reactive oxygen species in the vessel wall. Using cell lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/- aneurysm phenotype in vivo. Additionally, oral administration of simvastatin, a statin with antioxidant and anti-inflammatory effects, significantly reduced aneurysm formation in Nf1+/- mice. Finally, the antioxidant apocynin was administered orally and also resulted in a significant reduction of Nf1+/- aneurysms. These data provide genetic and pharmacologic evidence that neurofibromin-deficient myeloid cells are the central cellular triggers for aneurysm formation in a novel model of NF1 vascular disease, implicated oxidative stress as the key biochemical mechanisms of NF1 aneurysm formation and provide a potential therapeutic target for NF1 vasculopathy.

Einleitung: Die Entdeckung somatischer homoplasmischer Mutationen der mitochondrialen DNA (mtDNA) in Tumoren gab Anlass zu der Frage, ob Mutationen der mtDNA einen Einfluss auf Entstehung und Wachstum von Tumoren haben könnten. Die Neurofibromatose Typ 1 (NF1, von Recklinghausen) ist eine der häufigsten erblichen Tumorerkrankungen mit einer Penetranz von 100%, aber hoher phänotypischer Variabilität. Selbst eineiige Zwillinge können sich erheblich in ihrem Phänotyp unterscheiden. Durch die ungleiche Verteilung der Mitochondriengenome auf die Embryonen könnten heteroplasmische mtDNA-Polymorphismen den Phänotyp der Neurofibromatose Typ 1 unterschiedlich beeinflussen. Ziel dieser Arbeit war es herauszufinden, ob es interindividuelle Unterschiede in der mtDNA-Sequenz monozygoter Zwillinge gibt, die an Neurofibromatose Typ 1 erkrankt sind, sich jedoch im Phänotyp unterscheiden. Des Weiteren habe ich nach intraindividuellen Unterschieden der mtDNA-Sequenz zwischen Blut und Tumorgewebe gesucht. Die Frage war, ob es somatische mtDNA-Mutationen gibt, die einen Einfluss auf das Entstehen der Tumore haben könnten. Innerhalb der mtDNA gibt es hypervariable Regionen (HVR), von denen der oft in heteroplamischer Form vorkommende D310-Trakt im D-loop als Marker für klonales Wachstum in Tumoren empfohlen wurde. Ich habe versucht, durch Analyse des D-loops der mtDNA aus Neurofibromen klonales Wachstum nachzuweisen. Methoden: Ich habe die mitochondriale DNA vier monozygoter Zwillingspaare untersucht. Die DNA wurde sowohl aus Blutleukozyten als auch aus Neurofibromen extrahiert. Ich habe zunächst mit mtDNA-spezifischen Primern eine Long-range PCR durchgeführt. Mit dem Long-range PCR-Produkt als Matrize habe ich in 17 verschachtelten PCR Reaktionen Fragmente generiert und diese sequenziert. Den relativen Anteil heteroplasmischer Längenvarianten des D310-Traktes ermittelte ich mittels Genotypisierung. Ergebnisse: Beim Vergleich der mtDNA-Sequenzen mit der mtDNA Standardsequenz (Genbank, NC_001807) habe ich insgesamt 88 Abweichungen gefunden. Die meisten waren in der Datenbank Mitomap verzeichnet. Es fanden sich keine interindividuellen Unterschiede innerhalb der einzelnen Paare. Beim Vergleich der mtDNA-Sequenzen aus Blut- mit denen aus Tumorzellen eines Zwillingspaares fand ich keinen intraindividuellen Unterschied. Der D310-Trakt innerhalb der HVR2 kam bei allen Zwillingspaaren in heteroplasmischer Form vor. Bei den Zwillingen A1 und A2 sowie deren Mutter MA konnte ich annähernd die gleiche Verteilung der Löngenvarianten in Blutzellen sowie in Neurofibromen von A1 und A2 zeigen. Schlussfolgerungen: Ich konnte keinen Hinweis dafür finden, dass Veränderungen in der mtDNA die phänotypische Ausprägung der NF1 beeinflussen. In Neurofibromen konnte ich durch Untersuchung des D310-Traktes keinen Hinweis auf klonales Wachstum finden. / Introduction: The discovery of homoplasmic somatic mutations of the mitochondrial DNA (mtDNA) led to the question whether mutations of mtDNA could influence tumor development and growth. Neurofibromatosis Type 1 (NF1) is one of the most common inherited disorders. Penetrance of the disease is 100%, but phenotypic variability is high, even amongst identical twins. I wanted to test the hypothesis, whether the unequal distribution of heteroplasmic mtDNA variants between the embryos might influence NF1 phenotype. The aim of this study was to look for interindividual differences of the mtDNA sequence between identical twins. In order to detect somatic mutation that could possibly influence tumor development I searched for intraindividual differences between blood- and tumor-mtDNA. The hypervariable D310-tract within the D-loop is heteroplasmic in most individuals, but shows a tendency towards homoplasmy in tumors. Therefore, it has been proposed as marker for clonal tumor growth. I tried to identify clonal growth in cutaneous neurofibromas by examination of the D310-tract. Methods: I examined the mtDNA from four pairs of identical twins. MtDNA was extracted from blood-leucocytes as well as from neurofibromas. With DNA-specific primers I first performed a long-range PCR. The product was then reamplified as 17 nested PCR fragments and sequenced afterwards. The relative amount of heteroplasmic D310-tract length variants was analyzed by genotyping. Results: Taken together, I identified 88 deviations from the mtDNA standard sequence (Genbank NC_001807). Most of these variants were already known as polymorphisms in the database MITOMAP. I could neither find any interindividual differences between the individuals of a twin pair nor intraindividual differences between blood- and tumor-mtDNA. The D310-tract was heteroplasmic in all twin pairs. Twins A1 and A2 as well as their mother showed almost the same distribution of length variants in blood and tumor. Conclusion: I could not show that mtDNA polymorphisms play a role in phenotypic variability of NF1. Examination of the D310 tract in cutaneous neurofibromas did not show signs of clonal growth.

La Neurofibromatose 1 (NF1) est une maladie autosomique dominante dont l’évolutivité est inconnue. En effet, ni le type de mutation du gène, la gravité d’éventuels cas familiaux, ni une première complication ne permettent de prédire le pronostic de la maladie. L’objectif général de ce travail de thèse était de cibler les malades les plus à risque de morbi-mortalité au cours de la NF1. Méthode. Les différents travaux se sont appuyés sur les données phénotypiques de patients NF1 suivis dans le Réseau NF-France labellisé par le ministère de la Santé. Il s’agit d’une filière nationale monothématique ayant pour mission la prise en charge des malades atteints de NF1. Une cohorte d’environ 2500 malades est actuellement suivie dans ce réseau. Résultats. La mortalité des patients NF1 a tout d’abord été comparée à celle de la population générale française par l’estimation du rapport de mortalité standardisée (SMR). Entre 1980 et 2006, 1 895 patients NF1 ont été rétrospectivement inclus dans la cohorte. Un excès de mortalité était observé chez les [10-20[ ans (SMR=5.2, IC95% : 2.6 – 9.3, p<10-4) et les [20-40[ ans (SMR=4.1, IC95% : 2.8 – 5.8, p<10-4). Les principales causes de décès étaient la transformation de neurofibromes internes en tumeurs malignes des gaines nerveuses (TMGN). Une étude cas témoins portant sur 208 patients NF1 a permis d’expliquer le risque de mortalité accru chez les patients présentant des neurofibromes sous cutanés (SC-NF) en confirmant en IRM la présence chez ces patients de neurofibromes internes à fort risque de transformation en TMGN (OR=4.3, IC95% : 2.2 – 8.2). Cet effet était d’autant plus marqué que le nombre de SC-NF était important et notamment au-delà d’un seuil de 10 (OR=82, IC95% : 10.4 – 647.9) et que les neurofibromes internes étaient diffus (OR=14.7, IC95% : 3.8 – 57.3) et de taille ≥ 3 cm (OR=6.3, IC95% : 2.3 – 17.4). Les patients présentant des SC-NF représentent 20 à 30% de la population NF1. Afin d’identifier les patients à risque de développer une TMGN, nous avons élaboré un score prédictif de la présence des neurofibromes internes à partir des caractéristiques phénotypiques des patients. La présence de SC-NF (OR=4.7, IC95% : 2.1 – 10.5), l’absence de neurofibromes cutanés (OR=2.6, IC95% : 0.9 – 7.5), un âge inférieur ou égal à 30 ans (OR=3.1, IC95% : 1.4 – 6.8) et moins de 6 tâches café au lait (OR=2, IC95% : 0.9 – 4.6) étaient les variables qui constituaient le NF1Score. Le NF1Score = 10*(âge ≤ 30 ans) + 10*(absence de neurofibromes cutanés) + 5*(moins de 6 tâches café-au-lait) + 15*(plus de 2 neurofibrome sous cutanés) avait une excellente adéquation (test C de Hosmer-Lemeshow=4,53 avec 7ddl, p>0,50) et une capacité discriminante satisfaisante (aire sous la courbe ROC non paramétrique = 0,75 [0,68-0,82]). Enfin, l’expression phénotypique variant au cours du temps chez un même patient nous avons réalisé une étude spécifique chez l’enfant. Ainsi, l’âge (OR=1.1, IC95% : 1.0 – 1.2), la présence de xanthogranulomes (OR=4.5, IC95% : 0.9 – 21.7), celle de neurofibromes sous cutanés et plexiformes (OR=5.0, IC95% : 1.8 – 13.6) étaient indépendamment associés à celle des neurofibromes internes chez l’enfant NF1 de moins de 17 ans. Dans cette dernière étude, les neurofibromes internes se développaient de façon exponentielle pendant l’adolescence et plus précocement chez les femmes en accord avec les données de la littérature. Conclusion. La période à risque de développer des neurofibromes internes semblent donc sesituer entre l’adolescence et l’âge de 30 ans. Les recommandations de suivi pourraient prendre en compte le phénotype à risque, mais également la période de survenue de ces complications en réévaluant l’intérêt dans ce contexte d’investigations complémentaires / Neurofibromatosis-1 (NF1) is a common autosomal dominant condition which is a source of various multisystemic manifestations related either to the accumulation of neurofibromas or to specific developmental abnormalities. There are no obvious factors that predict disease progression. Thus, the aim of our project was to characterize the phenotype of NF1 patients with a severe prognosis. Patients were identified among adults with NF-1 followed up in the Réseau NF-France. The Réseau NF-France is a French medical network devoted to neurofibromatosis 1. It has elaborated recommendations for the management of the disease and recommended a coordinated follow-up in specialized multidisciplinary centres. About 2 500 patients were enrolled. We first evaluated the mortality in a large retrospective cohort of NF1 patients. The standardized mortality ratio (SMR) with its 95% confidence interval (CI) was calculated as the ratio of observed over expected numbers of deaths. Between 1980 and 2006, 1895 NF1 patients were seen. The excess mortality occurred among patients aged 10 to 20 years (SMR=5.2; CI, 2.6-9.3; P<10-4) and 20 to 40 years (SMR, 4.1; 2.8-5.8; P<10-4). The main cause of death was the malignant tumors of the nerve sheath (MPNSTs) developing from preexisting internal neurofibromas. Then, a case-control study including 208 patients with NF1 allowed us to explain the increased risk of mortality among NF1 patients harboring subcutaneous neurofibromas (SC-NF) by the presence of internal neurofibromas (NF) at risk of MPNSTs systematically investigated with imaging (MRI) (OR=4.3, IC95% : 2.2 – 8.2). The association with SC-NF was stronger for patients with ten or more SC-NFs (OR=82, IC95% : 10.4 – 647.9) and for diffuse (OR=14.7, IC95% : 3.8 – 57.3), and ≥ 3 cm (OR=6.3, IC95% : 2.3 – 17.4) internal neurofibromas. Patients with SC-NF constituted 20 to 30% of the NF1 population. So, to characterize patients at risk of developping MPNSTs, we developped and validated a clinical score for predicting internal neurofibromas in adults. Four variables were independently associated with internal neurofibromas: at least two subcutaneous neurofibromas (OR=4.7, IC95% : 2.1 – 10.5), age ≤30 years (OR=3.1, IC95% : 1.4 – 6.8), absence of cutaneous neurofibromas (OR=2.6, IC95% : 0.9 – 7.5), and fewer than six café-au-lait spots (OR=2, IC95% : 0.9 – 4.6). The NF1Score was computed as 10 . [age ≤30 years] + 10 • [absence of cutaneous neurofibromas] + 15 • [≥2 subcutaneous neurofibromas] + 5 • [<6 café-au-lait spots]). Calibration was excellent (Hosmer-Lemeshow statistic=4.53; degrees of freedom=7; P>0.5) and discrimination was good (AUC-ROC= 0.75; 95%CI, 0.7-0.8). Finally clinical expressivity is variable and manifestations of NF1 change at different times in an individual’s life. Consequently, a specific study was needed in pediatric patients. We identified easily recognizable clinical characteristics associated with internal neurofibromas in children with NF1. By multivariate analysis, age (OR=1.1, IC95% : 1.0 – 1.2), xanthogranulomas (OR=4.5, IC95% : 0.9 – 21.7), and presence of both subcutaneous and plexiform neurofibromas (OR=5.0, IC95% : 1.8 – 13.6) were independently associated with internal neurofibromas. Moreover internal neurofibromas increased during adolescence. Excess risk of developing internal neurofibromas seems to occur between the adolescence and the age of to 30 in NF1 patients. These clinical features in adults and children would define a new population at risk for complications that may need closer clinical and imaging follow-up

Made available in DSpace on 2016-01-26T12:51:47Z (GMT). No. of bitstreams: 1 fabianomoraisnogueira_dissert.pdf: 1824799 bytes, checksum: 73140945d3aa23f7080cfee361148ad6 (MD5) Previous issue date: 2013-07-11 / Introduction: Neurofibromatosis type 1 (NF1) is among the most common genetic disorders caused by mutations on chromosome 17 and characterized by a broad spectrum of clinical manifestations. Scoliosis is one of the most frequently musculoskeletal alterations and may be accompanied by dystrophic changes and tumor-related spine. Objective: To identify the prevalence of tumors and dystrophic changes in the spine of children and adolescents with neurofibromatosis type 1 assessed by magnetic resonance imaging and to analyze possible correlations between these findings and the presence of spinal deformities. Methods: Twenty-two patients with NF1, less than 21 years, underwent clinical and magnetic resonance imaging of the spine between September 2009 and July 2011. The group had 13 girls and 9 boys, with a mean age of 13.04 years (range 4-20 years). Only patients with clinical evidence of scoliosis were subjected to x-ray total spine for measuring the Cobb angle. Statistical analysis was performed using the Statistical Analysis Systems. The level of significance for all tests was 5%. Results: Scoliosis was diagnosed in 13 patients at the apex of the curve in the thoracic region predominantly represented by nine patients (69.23%). Excluding 3 patients did not undergo X-ray, 4 patients presented with dystrophic scoliosis mean Cobb angle of 57.75 degrees and 6 patients with non-dystrophic scoliosis and average Cobb angle of 15.33 degrees (p=0.0017). Neurofibromas associated to the spine were present in 9 patients (40.91%) and predominated in children older than 12 years (77.7%) but without statistical significance (p=0.2031). Among the patients with neurofibromas, 6 patients (66.7%) had associated with scoliosis (p=0.674). The dystrophic changes were found in 5 patients (22.72%), all with scoliosis. The vertebral erosion was the most frequent finding dystrophic, with 31 lesions in 25 vertebrae, mostly located in the posterior region of the vertebra and thoracic spine (54.84%). The vertebral erosions were associated with scoliotic curve in 96.7% of cases, dural ectasia in 87.5% of cases and patients with the greatest number of these lesions had a higher magnitude of their curves (Pearson=0.8275; p=0.0838). One patient had multiple meningoceles and one patient had two ribs intracanal, both associated with dystrophic curves. Conclusion: The evaluation with magnetic resonance imaging of the spine was able to identify the main tumor and dystrophic changes, correlate with the presence of vertebral deformities and analyze their distribution area of the curve. The vertebral erosion was the most common finding in dystrophic scoliosis curve and showed a tendency of correlation between the curves more severe and patients with higher number of vertebrae eroded. Neurofibromas that were associated with the spine were mainly found in the older children and tended to occur more frequently in the patients with scoliosis. In both cases, studies with larger samples are needed to assess whether these trends are evident. / Introdução: A neurofibromatose tipo 1 (NF1) está entre as desordens genéticas mais comuns causada por mutações no cromossomo 17 e caracterizada por um amplo espectro de manifestações clínicas. A escoliose é uma das alterações musculoesqueléticas mais frequentes, podendo estar acompanhada por lesões distróficas e tumorais associadas a coluna. Objetivo: Identificar a prevalência das alterações distróficas e tumorais presentes na coluna vertebral de crianças e adolescentes portadores de NF1 avaliados por imagens de ressonância magnética, bem como analisar possíveis correlações entre esses achados e a presença de deformidades espinhais. Casuística e Métodos: Vinte e dois pacientes portadores de NF1, menores de 21 anos, foram submetidos a exames clínicos e de ressonância magnética da coluna vertebral entre setembro de 2009 e julho de 2011. O grupo apresentava 13 meninas e 9 meninos, com idade média de 13,04 anos (variação de 4 a 20 anos). Apenas os pacientes com evidências clínicas de escoliose foram submetidos ao raio x de coluna total para medição do ângulo de Cobb. A análise estatística foi realizada no programa Statistical Analysis Systems. O nível de significância adotado foi de 5%. Resultados: A escoliose foi diagnosticada em 13 pacientes com ápice da curva predominando na região torácica (69,23%). Excluindo-se 3 pacientes não submetidos ao raio X de coluna total, 4 pacientes apresentavam escoliose distrófica com ângulo de Cobb médio de 57,75 graus e 6 pacientes com escoliose não distrófica e ângulo de Cobb médio de 15,33 graus (p=0,0017). Os neurofibromas associados à coluna vertebral estavam presentes em 9 pacientes e predominavam nas crianças maiores de 12 anos (77,7%) porém sem significância estatística (p=0,2031). Entre os portadores de neurofibromas, 6 pacientes (66,7%) apresentavam associação com escoliose (p=0,674). As alterações distróficas foram encontradas em 5 pacientes (22,72%), todos com escoliose. A erosão vertebral foi o achado distrófico mais frequente, sendo 31 lesões distribuídas em 25 vértebras, a maioria localizada concomitantemente na região posterior da vértebra e no segmento torácico da coluna (54,84%). As erosões vertebrais estavam associadas à curva escoliótica em 96,7% dos casos, a ectasias durais em 87,5 % dos casos e pacientes com maior número dessas lesões apresentavam maior magnitude de suas curvas (Pearson=0,8275; p=0,0838). Um paciente apresentou múltiplas meningoceles e um paciente apresentou duas costelas intracanal, ambos associados a curvas distróficas. Conclusão: A avaliação das imagens de ressonância magnética da coluna vertebral, foram capazes de identificar as principais alterações distróficas e tumorais, correlacionar com a presença de deformidades vertebrais e analisar sua distribuição em relação à área da curva. A erosão vertebral foi o achado mais frequente nas escolioses distróficas com uma tendência de correlação entre as curvas mais graves e maior número de vértebras erodidas. Neurofibromas associados a coluna predominaram nas crianças mais velhas e tenderam a ocorrer mais frequentemente em pacientes com escoliose. Em ambos os casos, estudos com maior casuística são necessários para que essas tendências sejam evidenciadas.

La neurofibromatose de type 2 (NF2) est une maladie autosomique causée soit par l'inactivation du gène NF2, soit par la perte de la protéine issue due ce gène, Merline. Cela entraîne à son tour la formation de plusieurs tumeurs nerveuse bénignes (non invasives) comme les schwannomes, méningiomes et les épendymomes. De plus, une diminution de l'expression de Merline est observée dans les cancers du sein invasifs, toutefois le rôle de Merline dans ces tumeurs invasives est peu étudié. Merline est la seule protéine ayant un rôle de suppresseur de tumeur dans la famille des ERM (Ezrin / Radixin / Moesin). Nous, ainsi que d'autres groupes, avons montré que la partie C-terminale de Merline est importante pour sa fonction inhibitrice de la croissance cellulaire. Par conséquent, j'ai cherché à mettre en évidence de nouveaux partenaires d'interaction non décrits à ce jour, ainsi que de nouveaux sites de phosphorylation sur l'extrémité C-terminale de Merline qui pourrait expliquer la fonction de suppresseur de tumeur de Merlin. L'utilisation d’expériences d'immunoprécipitation couplées à la spectrométrie de masse nous a permis d’identifier de nouveaux interacteurs ainsi que de nouveaux sites de phosphorylation sur ce domaine C-terminal de Merline. Nous avons analysé l'importance d'un nouvel interacteur, AmotL1, ainsi que d'un nouveau site de phosphorylation sur la threonine 581 (T581), dans la fonction suppresseur de tumeur de Merline. La protéine AmotL1 appartient à la famille des motines, qui sont connues pour être impliquées dans la régulation de la migration cellulaire. A cet égard, nous avons montré qu’AmotL1 est un nouveau partenaire d'interaction de Merline. Nous avons étudié l'importance de cette interaction entre Merline et AmotL1 dans la migration cellulaire et nos données suggèrent fortement que Merlin pourrait inhiber la migration cellulaire médiée par AmotL1 dans les cellules du cancer du sein, via notamment la régulation de son expression et de sa localisation. Enfin, nous avons également identifié plusieurs nouveaux interacteurs de Merline, qui pourraient expliquer comment Merlin pourrait agir comme une protéine d'échafaudage à la membrane plasmique, en interagissant avec des composants essentiels de la voie Hippo, comme AmotL1, Kibra, Lats et YAP, pour réguler la prolifération et la migration cellulaire. Dans la deuxième partie, nous avons identifié un nouveau site de phosphorylation spécifique à l'isoforme 1 de Merline, la T581, et nous avons démontré que la phosphorylation de cette threonine est importante pour la progression en mitose au moment approprié. De plus, dans cette étude, nous avons montré que Merlin est un substrat potentiel de la kinase Aurora A, un oncogène majeur, au cours de la mitose et de l'interphase, dans des lignées cellulaires de cancer du sein. Enfin, nous avons fourni des données préliminaires sur la façon dont Aurora A régule la signalisation Hippo et la fonction de DCAF1 en phosphorylant Merline. En résumé, cette thèse met en évidence deux fonctions importantes de Merline : premièrement comment Merline régule la migration/invasion cellulaire dans des tumeurs non-nerveuses telles que les cancers du sein et deuxièmement, comment Merline est régulé au cours de la mitose et de l'interphase dans des lignées de cancer du sein, en agissant comme un substrat pour la kinase Aurora A qui est surexprimée dans plusieurs cancers comme celui du sein, du côlon et l'HCC. Prise dans son ensemble, notre étude montre le rôle potentiel de Merline dans les tumeurs invasives telles que celles rencontrées dans les cancers du sein. / Neurofibromatosis type 2 (NF2) is an autosomal disorder caused by inactivation of NF2 gene or loss of the NF2 product, Merlin. This in turn results in formation of multiple benign (noninvasive) nerve tumors such as schwannomas, meningiomas and ependymomas. Additionally reduced expression of Merlin is observed in invasive breast cancers however the role of Merlin in these invasive tumors is poorly investigated. Merlin is the only tumor suppressor protein in Ezrin/Radixin/Moesin (ERM) family proteins. Previously we and others have shown that C-terminus of Merlin is important for its growth suppressive function. In this regard, I set out to investigate whether there were undiscovered interacting partners and novel phosphorylation sites on the C-terminus of Merlin that could account for tumor suppressor function of Merlin. Using immunoprecipitation coupled to mass spectrometry we have identified new interactors as well as novel phosphorylation on this C-terminus domain of Merlin. We analyzed importance of new interactor, AmotL1, as well as novel phosphorylation site on T581 in the tumor suppressor function of Merlin. AmotL1 belongs to AMOT family proteins which are known to involve in the regulation of cell migration. In this regard, we have shown that AmotL1 is novel interacting partner of Merlin. We have investigated the importance of Merlin and AmotL1 interactions in cell migration and our data strongly suggest that Merlin might inhibit AmotL1 mediated cell migration in breast cancer cells by regulating its expression and localization. Finally, we have also found several new interactors of Merlin and that could explain how Merlin might acts as scaffolding protein at the plasma membrane by interacting with Hippo core components such as AmotL1, Kibra, Lats and YAP to regulate cell proliferation and migration. In the second part, we have identified a novel phosphorylation site at T581 which is specific to Merlin isoform 1 and demonstrated that phosphorylation of Merlin on T581 is important for the timely mitotic progression. Further in this study, we have shown that Merlin is a potential substrate for major oncogene Aurora kinase A in mitosis as well as in interphasic breast cancer cell lines. Finally we have provided initial clues how Aurora A regulates Hippo signaling and DCAF1 function by phosphorylating Merlin. In the summary, this thesis highlights two important functions of Merlin: firstly how Merlin regulates the cell migration/invasion in non-nerve tumors such as breast cancers and secondly how Merlin is regulated in mitosis and interphasic breast cancer cells by acting as a substrate to Aurora Kinase A which is over expressed in several cancers such as breast, colon and HCC. All together our study indicates the potential role for Merlin in invasive tumors such as breast cancers.

<p>Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the <i>NF2 </i>gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the <i>NF2</i> gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the <i>NF2</i> locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the <i>NF2 </i>locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the <i>GSTT1/CABIN1</i> locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the <i>CABIN1</i> gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.</p>

<p>The array-based form of comparative genomic hybridization (array-CGH) is a new methodology that has shown to be of significant importance. This thesis focuses on the development of array-CGH with the aim to define candidate regions/genes on chromosome 22 in a wide spectrum of cancer-related conditions. In <b>paper I</b>, we developed and applied the first comprehensive genomic microarray, representing human chromosome 22, for analysis of DNA copy number. Using this array-based approach, we identified gene copy number alterations, including heterozygous/homozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of imbalanced translocation, in several 22q-associated disorders. In <b>paper II</b>, we applied the same array to perform DNA copy number profiling of a series of ovarian carcinoma. cDNA arrays were also used in this study to correlate gene expression levels with DNA-copy number. In the course of this analysis, we determined a small 3.5 Mb candidate 22q telomeric region and suggested a number of specific candidate genes. <b>Paper III</b> described the comprehensive and high-resolution analysis of chromosome 22 in a large set of various stage breast cancers. Multiple distinct patterns of genetic aberrations were observed. The smallest identified candidate locus was 220 kb in size and mapped to a gene-rich region in the vicinity of telomere of 22q. Intriguing result of this study was the detection of high frequency (26.6%) of intra-tumoral clonal variation in gene copy number profiles, which should be viewed as a high number, considering that we study in detail only a single human chromosome. In <b>paper IV</b>, we profiled a series of 28 Wilms tumor samples using 22q-array in order to assess specific regions affected with DNA dosage-alterations. The distribution of aberrations defined a complex amplifier genotype and delimited two tumor suppressor/oncogene candidate loci. These results open up for several avenues for continued research of these tumor forms. These findings also demonstrate the power of array-CGH in the precise determination of minute DNA copy number alterations and strengthen the notion that further studies, preferentially in the context of the entire human genome, are needed.</p>