Introdução: A Neurofibromatose tipo 1 (NF1) é uma doença genética autossômica dominante causada por mutação no gene NF1 (17q11.2; proteína neurofibromina). A incidência é de aproximadamente 1:3500 recém-nascidos e o diagnóstico clínico é possível na maioria dos casos. Alguns estudos sugeriram que pacientes com NF1 são mais suscetíveis a apresentar deficiência de vitamina D quando comparados à população geral. Objetivo: Determinar os níveis de 25(OH)D em indivíduos com NF1 e em controles saudáveis. Nos pacientes com NF1 nos também avaliamos o fenótipo clínico e analisamos polimorfismos comuns do gene VDR (BsmI e FokI) para avaliar se estavam correlacionados com os níveis de 25(OH)D. Métodos: Estudo de caso-controle que incluiu 45 pacientes com NF1 do Sul do Brasil e 45 controles saudáveis pareados por idade, sexo e tipo de pele de acordo com a classificação de Fitzpatrick. Resultados e Conclusões: Setenta e sete vírgula oito por cento dos 90 sujeitos de pesquisa apresentaram níveis de 25(OH)D abaixo de 30ng/ml. Deficiência de 25(OH)D (<20ng/mL) foi observada em 28 (31,1%) e insuficiência (de 20 a 30ng/mL) em 42 (46,6%). Deficiência ou insuficiência de 25(OH)D não foi mais frequente em pacientes com NF1 do que em controles (p=0,074). Nos não observamos qualquer associação entre os polimorfismos do gene VDR e os níveis de vitamina D sugerindo que o fenótipo da insuficiência ou deficiência bioquímica de 25(OH)D nos pacientes estudados não está associado com essas variantes genéticas. / Introduction: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene (17q11.2, neurofibromin). The estimated incidence is approximately 1:3500 newborns and clinical diagnosis is possible in the majority cases. A few studies suggest that patients with NF1 are more likely to have vitamin D deficiency when compared to the general population. Objectives: The goal of the study was to determine the levels of 25-OH-vitamin D [25(OH)D] in individuals with NF1 and disease-unaffected controls. In NF1 patients we also assessed clinical phenotype and analyzed common VDR gene polymorphisms (BsmI and FokI) to verify whether they were associated with lower vitamin D levels. Methods: Case-control study that included 45 NF1 patients from Southern Brazil and 45 healthy controls matched by sex, skin type and age to the cases. Results and conclusions: Overall, 70 (77.8%) of the individuals studied had levels of 25(OH)D below 30ng/ml: vitamin D deficiency was observed in 28 (31.1%) and vitamin D insufficiency in 42 (46.6%) subjects. 25(OH)D deficiency or insufficiency were not more frequent in NF1 patients than in controls (p=0,074). We did not observe an association of VDR gene polymorphisms and vitamin D levels suggesting that the insufficient or deficient biochemical phenotypes in the patients studied here are not associated with these genetic variants.

The aim of the study was to evaluate the importance of brain MRI's findings, and modify the criteria for brain MR imaging in NF1 patients according to this data, to improve the quality of life with early detection of important NF1 complications. Description of the whole cohort, with emphasise to possible cause of high range of sporadic NF1 cases. Evaluation the possibility diagnosis or follow up of brain gliomas by plasmatic values of neuron specific enolase (NSE) and S100B protein. Subjects and methods: I analysed data from 285 NF1 children followed up on our department from 1990 to 2010 by the same examination battery. I evaluated the incidence of brain MRI findings, clinical development, age at gliomas manifestation and necessity of treatment. I also described the whole cohort and made statistic analysis of plasmatic values of NSE and S100B protein in NF1 patients, with and without brain gliomas. Results: OPGs were found in 77/285 (27 %) children and GOOPs in 29/285 (10.2 %) of NF1 children, of who 19 had OPG and GOOP together, so the total number of brain glioma was 87/285 (30.5 %). Totally, 43/87 (49.4 %), respectively 43/285 (15.1 %) children with brain glioma were treated, and 4/285 (1.4 %) of this children died. Obstructive hydrocephalus was found in 22/285 (7.7 %) patients and was caused...

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder that affects roughly 1 in 3500 individuals. In addition to physical features (e.g., neurofibromas), developmental disorders are also common that can affect cognition, learning, attention and social function. The NF1 gene encodes neurofibromin, a GTPase activating protein (GAP)-like protein that negatively regulates Ras GTPase activation. Mutation at the NF1 locus increases the output of MAPK and PI3K signal transduction from the cellular membrane to the nucleus. Similar to humans, Nf1+/- mice show spatial learning abnormalities that are potentially correlated with increases in GABA-mediated inhibition and deficits in long-term potentiation in the hippocampus. Here, we demonstrate for the first time that Nf1+/- mice exhibit a selective loss of long-term social learning / memory and increased GABAergic inhibition in the basolateral amygdala, a critical brain region for regulating social behaviors. Next, utilizing a genetic intercross, we show that the co-deletion of p21-activated kinase type 1 (Pak1-/-), which positively regulates MAPK activation, restores Nf1+/--dependent MAPK hyperactivation in neurons cultured from the frontal cortex. We found that the co-deletion of Pak1 in Nf1+/- mice (Nf1+/- / Pak1-/-) also restores the deficits in long-term social learning / memory seen in Nf1+/- mice and normalizes the increases in GABA-mediated inhibition in the BLA, as compared to Nf1+/- mice. Together, these findings establish a role for Nf1 and Pak1 genes in the regulation of social learning in Nf1-deficient mice. Furthermore, proteomic studies identify dysregulation of F-actin and microtubule dynamics in the prefrontal cortex, and implicate proteins associated with vesicular release as well as neurite formation and outgrowth (e.g., LSAMP, STXBP1, DREB). In the BLA, disintegrin and metalloproteinase domain-containing protein 22 (ADAM22) was identified, and ADAM22 may play a role in the regulation of AMPA receptors. Finally, due to the increased co-occurrence of NF1 and autism, these findings may also have important implications for the pathology and treatment of NF1-related social deficits and some forms of autism.

NF1 ist eine autosomal dominante Erbkrankheit, die durch inaktivierende Mutationen im Neurofibromin-Gen verursacht wird. NF1 manifestiert sich durch eine erhöhte Tumor-Inzidenz des neuralen Gewebes in der Haut (Neurofibroma). Neben diesen häufigeren klinischen Manifestationen haben rund 50% der NF1-Patienten Skelett-Anomalien. Häufiger sind Röhrenknochen betroffen, die klinischen Symptome reichen von Tibia-Krümmung über Spontanfrakturen bis hin zu Nonunions. Diese Studie analysiert den Heilungsverlauf von Femurfrakturen in Nf1Prx1- Mäusen. Der Frakturkallus von Mäusen wurde an den Tagen 7, 10, 14 und 21 durch µCT, Histologie und molekulare Analysen evaluiert. µCT und histologische Analysen haben eine beeinträchtigte Knochenheilung in Nf1Prx1-Mäusen gezeigt. Eine erhöhte periostale Knochenbildung in den frühen Stadien der Heilung war zu beobachten, sowie eine reduzierte, aber anhaltende Knorpelbildung und Bindegewebs-Akkumulation innerhalb der Fraktur. Wir konnten zeigen, dass der normalen Heilungsprozess durch dieses Bindegewebe behindert wird, welches durch alpha smooth muscle actin-positive Myofibroblasten gebildet wird, die ihrerseits aus einer bisher noch nicht identifizierten Muskelfaszie abgeleitet sind. Dieser Zusammenhang wird durch eine Microarray-Analyse der Kallus-Gewebe bestätigt, die ergab, dass durch den Knock-Out Gene reguliert wurden, die in Physiologie, Proliferation und Differenzierung von Muskelzellen involviert sind. Darüber hinaus waren extrazelluläre-Matrix-Gene in den Mutanten hoch regeuliert. Zusammenfassend konnten wir zeigen, dass eine Ähnlichkeit des Heilungsverlauf zwischen dem Nf1Prx1-Mausmodell und NF1-Patienten besteht. Folglich kann an diesem Mausmodell untersucht werden, durch welche Mechanismen die Mutationen im NF1 zu Knochenheilungsstörungen führen. Außerdem konnte in einer Pilotstudie der Effekt des Neurofibromin-Mangels auf die Knochenheilung durch Behandlung mit MEK-Inhibitoren in vitro und in vivo weitestgehend behoben werden / Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 patients – around 50% – have abnormalities of the skeleton. Long bones are often affected, and the clinical signs range from tibial bowing to spontaneous fractures and even non-unions. Moreover, NF1 mice models could provide the understanding of the cell types involved in the resulting non-union and their behavior. This study analyzed the healing progress of femur fractures in a model of NF1 long bone dysplasia. Fracture callus was assessed at days 7, 10, 14, and 21 by µCT, histology, biomechanics, and molecular analyses. Bone healing was impaired in Nf1Prx1 mice femoral fracture. Results revealed increased periosteal bone deposition at the early stages of healing, decreased but persistent cartilage formation concomitant with fibrous tissue accumulation within the fracture site, decreased torsional stiffness, decreased bone mineral density, and increased fibrous tissue infiltration in the callus of mutant mice. This fibrous tissue accumulation hindered bone fracture healing, and was deposited by alpha smooth muscle actin-positive myofibroblasts, which were derived from a yet unidentified muscle fascia. This is further supported by the microarray analysis of callus tissues showing that genes crucial to muscle cells physiology, proliferation and differentiation were affected. In addition, extracellular matrix related genes were up-regulated in the mutants. In summary, this study shows a resemblance in the healing progression to the Nf1Prx1 mice model and NF1 patients, thereby, confirming the suitability of this mice model to explore the mechanism by which mutations in NF1 lead to non-unions. Moreover, in vitro and in vivo pilot assessments of MEK inhibitor treatment demonstrated a potential remedy for the lack of neurofibromin in bone healing.

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Made available in DSpace on 2016-01-26T12:51:31Z (GMT). No. of bitstreams: 1 joserobertolopesferrazfilho_tese.pdf: 1409103 bytes, checksum: de6715f2f3059b0c5617f571b89bfdbd (MD5) Previous issue date: 2011-10-21 / Neurofibromatosis type 1 (NF1) in children and adolescents is frequently associated with the appearance of focal lesion hyperintensities on T2-weighted images seen in the brain which are called Unidentified Bright Objects (UBOs). These lesions are not an accepted criterion in the diagnosis of NF1 and the exact nature of UBOs remains unclear. Objectives: article 1: This study employed diffusion tensor imaging (DTI) to evaluate the relation among fractional anisotropy (FA) patterns with the findings of T2 sequences in individuals with NF1; article 2: To evaluate the evolution of UBOs in individuals with NF1 by serial MRI, and to relate this to regional fractional anisotropy (FA); article 3: To evaluate the metabolic patterns by magnetic resonance spectroscopy (MRS) of the brain, in the presence of UBOS in patients with NF1. Methods: article 1: Forty-four individuals with NF1 and 20 control subjects were evaluated. The comparative analysis of FA between NF1 and control groups was based on four pre-determined anatomic regions of the brain and related to the presence or absence of UBOs; article 2: The signal pattern of the T2-weighted sequences in the basal ganglia, thalamus, brain stem, and cerebellum for 27 NF1 individuals and a control group were analyzed by DTI. The presence or absence of UBOs in 2 consecutive MRI examinations were related to FA. article 3: Forty-two individuals with NF1 and 25 control subjects were evaluated by examination of ERM univoxel placed in the region of the globus pallidus. Automated quantitative analysis was made of the relationship of the metabolites choline/creatine (Co / Cr), N-acetyl aspartate/creatine (NAA / Cr) and myoinositol/creatine (MI / Cr) and related to the occurrence of UBOS in region of the globus pallidus. Results: article 1: The FA values between the groups demonstrated statistically significant differences (p &#8804; 0.05) for the cerebellum and thalamus in NF1 patients, independent of the occurrence of UBOs; article 2: We demonstrated statistically significant differences in FA for the basal ganglia, cerebellum, and thalamus between NF1 patients and controls (P &#8804; 0.05), even with a reduction or disappearance of UBOs; article 3: We demonstrated statistically significant differences between of patients with NF1 and control groups as the average values Mi/Cr and Co/Cr (P<0.05) in the region of the globus pallidus. Conclusions: article 1: MR imaging using DTI technique suggests that UBOs are due to microstructural defect of the brain tissue in NF1 patients. article 2: MRI allows for adequate monitoring of the temporal and spatial distribution of UBOs in patients with NF1. DTI confirmed changes in FA despite the disappearance or reduction of UBOs. article 3: MR spectroscopy allows the characterization of the tissue abnormalities not demonstrable in the conventional MR sequences of patients with NF1 by analysis of metabolites Co and Mi. / A neurofibromatose tipo 1 (NF1) em crianças e adolescentes está frequentemente associada com o aparecimento ou desaparecimento de lesões focais de hipersinal no encéfalo na ponderação T2 (FHE-T2). Estas lesões não são aceitas como critério diagnóstico para NF1 e sua natureza exata ainda não está clara. Objetivos: artigo 1: Avaliar a relação entre a presença dos FHE-T2 e padrões de anisotropia fracionada (FA) em uma série de pacientes com NF1; artigo 2: Demonstrar o padrão de evolução dos FHE-T2 por exame de Ressonância Magnética (RM) em indivíduos com NF1 e relacionar com o valor regional de FA; artigo 3: Avaliar os padrões metabólicos por meio da espectroscopia por ressonância magnética (ERM) do encéfalo na presença dos FHE-T2 em pacientes com NF1. Métodos: artigo 1: Analisou-se uma série de 44 indivíduos com NF1, e 20 controles. A análise quantitativa do FA foi definida em quatro regiões anatômicas pré-determinadas e relacionada à presença de FHE-T2; artigo 2: Analisou-se com imagem de tensor de difusão (DTI) a evolução dos FHE-T2 nas regiões de núcleos da base, tálamos, cerebelo e tronco encefalico de um grupo de 27 pacientes com NF1 e 20 controles. A presença de FHE-T2 em dois exames de RM encefálica consecutivos foram relacionados com o valor de FA; artigo 3: Analisou-se 42 indivíduos com NF1, e 25 controles saudáveis por exame de ERM univoxel na região do globo pálido. Foi feita análise automatizada quantitativa da relação dos metabólitos colina/creatina (Co/Cr), N-acetil aspartato/creatina (Naa/Cr) e Mioinositol/creatina (Mi/Cr) e relacionada à ocorrência de FHE-T2 na região do globo pálido. Resultados: artigo 1: Os FHE-T2 foram diagnosticados em 50% dos pacientes com NF1. Observou-se redução do valor de FA nas regiões do cerebelo e tálamo de aparência normal ou com FHE-T2 de pacientes com NF1 em relação ao controle (P &#8804;.05); artigo 2: Houve redução significativa no valor de FA nas regiões de núcleos da base, cerebelo e tálamos em pacientes com NF1 em relação ao grupo controle (P &#8804;.05) mesmo com redução ou desaparecimento dos FHE-T2; artigo 3: Houve diferença estatisticamente significante entre os grupos de pacientes com NF1 e o controle quanto aos valores médios ( ) de Mi/Cr e Co/Cr (P<0,05) na região do globo pálido. Conclusões: artigo 1: A técnica de DTI confirma que os FHE-T2 estejam relacionados às alterações da microestrutura do tecido cerebral em pacientes NF1. artigo 2: A RM possibilita o adequado monitoramento da distribuição no tempo e espaço dos FHE-T2 em pacientes com NF1. DTI evidencia alterações no valor de FA mesmo com o desaparecimento ou redução dos FHE-T2; artigo 3: A ERM permite a caracterização de anormalidades teciduais não demonstráveis nas sequências convencionais de RM de pacientes com NF1 por meio da análise dos metabólitos Co e Mi.

Neurofibromatosis Type 1 (NF1) is a monogenetic autosomal-dominant disorder with a broad spectrum of clinical symptoms and is commonly associated with cognitive deficits. Patients with NF1 frequently exhibit cognitive impairments like attention problems, working memory deficits and dysfunctional inhibitory control. The latter is also relevant for the resolution of cognitive conflicts. However, it is unclear how conflict monitoring processes are modulated in NF1. To examine this question in more detail, we used a system neurophysiological approach combining high-density ERP recordings with source localisation analyses in juvenile patients with NF1 and controls during a flanker task. Behaviourally, patients with NF1 perform significantly slower than controls. Specifically on trials with incompatible flanker-target pairings, however, the patients with NF1 made significantly fewer errors than healthy controls. Yet, importantly, this overall successful conflict resolution was reached via two different routes in the two groups. The healthy controls seem to arrive at a successful conflict monitoring performance through a developing conflict recognition via the N2 accompanied by a selectively enhanced N450 activation in the case of perceived flanker-target conflicts. The presumed dopamine deficiency in the patients with NF1 seems to result in a reduced ability to process conflicts via the N2. However, NF1 patients show an increased N450 irrespective of cognitive conflict. Activation differences in the orbitofrontal cortex (BA11) and anterior cingulate cortex (BA24) underlie these modulations. Taken together, juvenile patients with NF1 and juvenile healthy controls seem to accomplish conflict monitoring via two different cognitive neurophysiological pathways.

Neurofibromatosis Type 1 (NF1) is a monogenetic autosomal-dominant disorder with a broad spectrum of clinical symptoms and is commonly associated with cognitive deficits. Patients with NF1 frequently exhibit cognitive impairments like attention problems, working memory deficits and dysfunctional inhibitory control. The latter is also relevant for the resolution of cognitive conflicts. However, it is unclear how conflict monitoring processes are modulated in NF1. To examine this question in more detail, we used a system neurophysiological approach combining high-density ERP recordings with source localisation analyses in juvenile patients with NF1 and controls during a flanker task. Behaviourally, patients with NF1 perform significantly slower than controls. Specifically on trials with incompatible flanker-target pairings, however, the patients with NF1 made significantly fewer errors than healthy controls. Yet, importantly, this overall successful conflict resolution was reached via two different routes in the two groups. The healthy controls seem to arrive at a successful conflict monitoring performance through a developing conflict recognition via the N2 accompanied by a selectively enhanced N450 activation in the case of perceived flanker-target conflicts. The presumed dopamine deficiency in the patients with NF1 seems to result in a reduced ability to process conflicts via the N2. However, NF1 patients show an increased N450 irrespective of cognitive conflict. Activation differences in the orbitofrontal cortex (BA11) and anterior cingulate cortex (BA24) underlie these modulations. Taken together, juvenile patients with NF1 and juvenile healthy controls seem to accomplish conflict monitoring via two different cognitive neurophysiological pathways.

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.