A doença de Charcot-Marie-Tooth tipo 1A (CMT1A), associada à duplicação do gene da proteína da mielina periférica 22(PMP22), é a neuropatia hereditária mais comum. Administração diária de progesterona a modelos animais desta doença resultou em progressão mais rápida da neuropatia. Algumas mulheres por ela afetadas desenvolveram piora neurológica durante suas gravidezes. O objetivo deste estudo foi avaliar a influência de gestações sobre a neuropatia de pacientes com CMT1A. Mulheres afetadas responderam questões sobre sinais e sintomas apresentados durante suas gravidezes presentes e passadas. Pontuações nas escalas CMTNS (Charcot-Marie-Tooth Neuropathy Score) e SF-36 (Short Form Health Survey - 36) e dados coletados de avaliações clínicas e eletrofisiológicas dessas pacientes, de mulheres que nunca tiveram filhos e de homens foram comparados. Seis pacientes foram prospectivamente avaliadas durante suas gestações. Cinquenta e uma mulheres responderam questões sobre 130 gravidezes. Vinte e nove delas relataram piora de seus sintomas neurológicos em 61 gravidezes: vinte e cinco, tiveram cãibras dolorosas, três, fraqueza progressiva, duas, ataxia sensitiva, oito referiram sintomas sensitivos positivos. Algumas dessas pacientes apresentaram mais de um tipo de sintomas. As comparações entre mulheres e homens mostraram diferenças significativas nos seguintes aspectos, com pior desempenho pelas mulheres: pontuação no item do CMTNS relacionado a sintomas de membros superiores, força de abdução dos primeiros e segundos quirodáctilos, força da flexão do quadril bilateralmente, pelo MRC (Medical Research Council), amplitude do CMAP (potencial de ação muscular composto) do nervo ulnar direito em millivolts. O SF-36 teve diferença significativa nos seguintes itens, também com piores pontuações pelas mulheres: limitações devido a problemas emocionais, estado geral de saúde, saúde mental, funcionamento social, dor e vitalidade. Quando realizado cotejo entre homens, mulheres com filhos e mulheres sem filhos, dados relativos a força dos abdutores curtos dos polegares direito e esquerdo, força da flexão do quadril bilateralmente, amplitude do CMAP do nervo ulnar direito e os mesmo itens do SF-36, exceto dor e estado geral de saúde, continuaram a ter diferença significativa entre homens e mulheres, mas não entre mulheres com filhos e mulheres sem filhos. Também não houve diferença entre os dados relativos a mulheres que tiveram piora na gravidez e mulheres que não referiram piora. Das seis pacientes avaliadas prospectivamente, cinco, com a duplicação do PMP22, apresentaram piora de algum aspecto de sua neuropatia, porem retornaram à sua condição clínica prévia à gravidez em períodos variáveis de tempo. Uma, que apresentava duas muatçoes, a duplicação 17p11.2-p12 e a mutação de ponto Ser72Leu, não teve qualquer alteração. Nesta casuística, embora tenham sido encontradas diferenças significativas entre os dois gêneros, e até entre homens e mulheres com filhos, não foi observada nenhuma diferença significativa entre mulheres que referiram piora na gravidez e as que não apresentaram. A gestação teve diferentes consequências sobre a neuropatia de pacientes avaliadas, prospectivamente, porem houve retorno às condições clínica anteriores à gravidez. / The Charcot-Marie-Tooth type 1A (CMT1A) associated with PMP22 duplication is the most common hereditary neuropathy. Daily administration of progesterone resulted in more progressive clinical neuropathy of transgenic rats over-expressing PMP22. There are reports of women with CMT1A who had worsening of their neurological status during pregnancy. The aim of this study was to investigate the influence of pregnancy on CMT1A neuropathy and its natural history. Women with CMT1A answered questions about neurological signs and symptoms during their past and present pregnancies. CMT NS (Charcot-Marie-Tooth Neuropathy Score) and SF 36 (Short Form Survey - 36) scores and data collected from clinical and electrophysiological evaluations of these patients, of women who didn\'t have child and of men were compared. Six patients were prospectively evaluated during pregnancies. Fifty-one patients answered questions about 130 pregnancies. Twenty-nine patients (56%) reported worsening of their neurological symptoms during 61 pregnancies: twenty-five women had painful cramps, three, reported progressive weakness, two patients had sensitive ataxia, six patients had positive sensory symptoms. Some of these patients had more than one kind of symptoms. Comparison between men and women showed some significant differences: CMT NS item about upper limbs symptoms; strength of right and left first dorsal interosseos muscle(ID), strength of right and left abductor pollicis brevis(APB), strength of right and left flexors of the hip, CMAP of right ulnar nerve in millivolts; SF-36 had significant difference in the following items: limitations due to emotional problems, general health, mental health, social functioning, pain and vitality, in which female had worst scores. When comparison between men, women who had child and women who didn\'t have child was made, data regarding strength of right and left APB, strength of right and left flexors of the hip, amplitude of right CMAP and the same items of SF-36, except pain and general health, continued to have significant differences between men and women, but not between women who had child and women who didn\'t had child. There was no difference between data of women who got worst during their pregnancy and women who didn\'t. CMT worsening during pregnancy has previously been described. Some of these patients recovered their previous status after delivery, while others did not. Several explanations have been proposed for this deterioration. In our series, five patients had deterioration during pregnancy. It seems that pregnancy may affect CMT patients in different ways. There were also some differences between men and women, but no significant worsening was found in the neurological status of women that had been pregnant. A larger prospective study should be conducted to better understand the effects of pregnancy on CMT.

A doença de Charcot-Marie-Tooth tipo 1A (CMT1A), associada à duplicação do gene da proteína da mielina periférica 22(PMP22), é a neuropatia hereditária mais comum. Administração diária de progesterona a modelos animais desta doença resultou em progressão mais rápida da neuropatia. Algumas mulheres por ela afetadas desenvolveram piora neurológica durante suas gravidezes. O objetivo deste estudo foi avaliar a influência de gestações sobre a neuropatia de pacientes com CMT1A. Mulheres afetadas responderam questões sobre sinais e sintomas apresentados durante suas gravidezes presentes e passadas. Pontuações nas escalas CMTNS (Charcot-Marie-Tooth Neuropathy Score) e SF-36 (Short Form Health Survey - 36) e dados coletados de avaliações clínicas e eletrofisiológicas dessas pacientes, de mulheres que nunca tiveram filhos e de homens foram comparados. Seis pacientes foram prospectivamente avaliadas durante suas gestações. Cinquenta e uma mulheres responderam questões sobre 130 gravidezes. Vinte e nove delas relataram piora de seus sintomas neurológicos em 61 gravidezes: vinte e cinco, tiveram cãibras dolorosas, três, fraqueza progressiva, duas, ataxia sensitiva, oito referiram sintomas sensitivos positivos. Algumas dessas pacientes apresentaram mais de um tipo de sintomas. As comparações entre mulheres e homens mostraram diferenças significativas nos seguintes aspectos, com pior desempenho pelas mulheres: pontuação no item do CMTNS relacionado a sintomas de membros superiores, força de abdução dos primeiros e segundos quirodáctilos, força da flexão do quadril bilateralmente, pelo MRC (Medical Research Council), amplitude do CMAP (potencial de ação muscular composto) do nervo ulnar direito em millivolts. O SF-36 teve diferença significativa nos seguintes itens, também com piores pontuações pelas mulheres: limitações devido a problemas emocionais, estado geral de saúde, saúde mental, funcionamento social, dor e vitalidade. Quando realizado cotejo entre homens, mulheres com filhos e mulheres sem filhos, dados relativos a força dos abdutores curtos dos polegares direito e esquerdo, força da flexão do quadril bilateralmente, amplitude do CMAP do nervo ulnar direito e os mesmo itens do SF-36, exceto dor e estado geral de saúde, continuaram a ter diferença significativa entre homens e mulheres, mas não entre mulheres com filhos e mulheres sem filhos. Também não houve diferença entre os dados relativos a mulheres que tiveram piora na gravidez e mulheres que não referiram piora. Das seis pacientes avaliadas prospectivamente, cinco, com a duplicação do PMP22, apresentaram piora de algum aspecto de sua neuropatia, porem retornaram à sua condição clínica prévia à gravidez em períodos variáveis de tempo. Uma, que apresentava duas muatçoes, a duplicação 17p11.2-p12 e a mutação de ponto Ser72Leu, não teve qualquer alteração. Nesta casuística, embora tenham sido encontradas diferenças significativas entre os dois gêneros, e até entre homens e mulheres com filhos, não foi observada nenhuma diferença significativa entre mulheres que referiram piora na gravidez e as que não apresentaram. A gestação teve diferentes consequências sobre a neuropatia de pacientes avaliadas, prospectivamente, porem houve retorno às condições clínica anteriores à gravidez. / The Charcot-Marie-Tooth type 1A (CMT1A) associated with PMP22 duplication is the most common hereditary neuropathy. Daily administration of progesterone resulted in more progressive clinical neuropathy of transgenic rats over-expressing PMP22. There are reports of women with CMT1A who had worsening of their neurological status during pregnancy. The aim of this study was to investigate the influence of pregnancy on CMT1A neuropathy and its natural history. Women with CMT1A answered questions about neurological signs and symptoms during their past and present pregnancies. CMT NS (Charcot-Marie-Tooth Neuropathy Score) and SF 36 (Short Form Survey - 36) scores and data collected from clinical and electrophysiological evaluations of these patients, of women who didn\'t have child and of men were compared. Six patients were prospectively evaluated during pregnancies. Fifty-one patients answered questions about 130 pregnancies. Twenty-nine patients (56%) reported worsening of their neurological symptoms during 61 pregnancies: twenty-five women had painful cramps, three, reported progressive weakness, two patients had sensitive ataxia, six patients had positive sensory symptoms. Some of these patients had more than one kind of symptoms. Comparison between men and women showed some significant differences: CMT NS item about upper limbs symptoms; strength of right and left first dorsal interosseos muscle(ID), strength of right and left abductor pollicis brevis(APB), strength of right and left flexors of the hip, CMAP of right ulnar nerve in millivolts; SF-36 had significant difference in the following items: limitations due to emotional problems, general health, mental health, social functioning, pain and vitality, in which female had worst scores. When comparison between men, women who had child and women who didn\'t have child was made, data regarding strength of right and left APB, strength of right and left flexors of the hip, amplitude of right CMAP and the same items of SF-36, except pain and general health, continued to have significant differences between men and women, but not between women who had child and women who didn\'t had child. There was no difference between data of women who got worst during their pregnancy and women who didn\'t. CMT worsening during pregnancy has previously been described. Some of these patients recovered their previous status after delivery, while others did not. Several explanations have been proposed for this deterioration. In our series, five patients had deterioration during pregnancy. It seems that pregnancy may affect CMT patients in different ways. There were also some differences between men and women, but no significant worsening was found in the neurological status of women that had been pregnant. A larger prospective study should be conducted to better understand the effects of pregnancy on CMT.

No description available.

X-linked Charcot-Marie-Tooth disease (CMTX1), a peripheral neuropathy, is clinically characterised by slow progressive weakness and wasting of the distal muscles with associated sensory loss. The CMTX1 locus had previously been localised to the pericentromeric region of the X chromosome. Our initial linkage analysis utilising Restriction Fragment Length Polymorphisms (RFLPs) confirmed that CMTX1 mapped proximally to the DXYS1X locus (Xq21.31). Subsequent linkage analysis, carried out as part of an international consortium, utilising microsatellite polymorphisms further delineated the CMTX1 locus to a 2cM region around DXS453 (Xq31.1). To help with this analysis new microsatellites are generated at the loci DXS106 and DXS227. In parallel with the linkage analysis a physical map of the region was under construction simultaneously with candidate gene evaluation. The physical map was constructed by Pulsed Field Gel Electrophoresis (PFGE) used in conjunction with partial digestion of Yeast Artificial Chromosomes (YACs). The physical map of nine YACs had been obtained in which seven potential CpG islands were identified. Candidate genes were investigated by sequencing Polymerase Chain Reaction (PCR) amplified gene fragments from DNA isolated from patients with CMTX1. This led to the identification of missense, nonsense and base pair deletion mutations within the previously described GJβ1 gene. This gene encodes, connexin 32, a gap junction subunit. Gap junctions are channels which allow direct transfer of cellular components, which are below 900D in diameter, between coupled cells. It is proposed that mutations affecting the GJβ1 gene are the underlying biological defect which results in the CMTX1 phenotype.

The present study is focused on understanding the aetiology of the human clubfoot deformity. Although this pathology has been studied since Classical Antiquity, the mechanisms that lead to this abnormality in new-born patients remain elusive. Clubfoot is a deformity of one or both feet present at birth, in which the foot is abnormally positioned in a hand-like position, that is, the foot is turned and rotated inwards while pointing down; and is resistant to any further movements. Very little is known about the aetiology and genetics of clubfoot in the human population. Only recently, mutations in the PITX1 gene have been associated with a small number of patients. Because the genetic basis is not understood and the phenotypic observations are complex and variable in human patients, many mechanisms have been proposed to explain clubfoot. In this study, these pressing questions were addressed using the peroneal muscular atrophy (pma) mouse, a spontaneous mutant that has been shown to be a surprisingly good model for clubfoot, recapitulating the key features of the human phenotype. In order to confirm that the pma mouse is in fact an idiopathic model of clubfoot, it was important to understand if the pma clubfoot-like phenotype occurs in isolation or within a syndromic pathology. In addition to clubfoot, it was found that these animals show a retinal degeneration phenotype. However, this phenotype was associated with the Pde6brd1 mutation, suggesting that clubfoot occurs independently of the retinal phenotype and thus the pma is a good model for human idiopathic clubfoot. Clubfoot in the pma mouse has been associated with the observed failure of the foot rotation during embryonic development. This defect is thought to result from the extensive regional muscular atrophy that occurs at earlier stages. The peroneal nerve is also absent in the adult pma mouse, a defect that has remained unexplored. As such, this neuronal defect was studied to understand the reason for the peroneal nerve absence in the adult animals. The results indicate that the nerve fails to branch from the developing sciatic nerve during embryogenesis and is unable to innervate its target muscles. This abnormal branching process is associated with a neural growth delay. In respect to the genetics of the pma, it was not possible to identify the exact mutation that is responsible for the inheritance of the clubfoot phenotype. However, strong evidence was found in favour of a regulatory mutation resulting in over-expression of the gene Limk1, which encodes for a kinase involved in neuronal guidance and growth. Further work was performed on chicken embryos to understand the foot rotation process. By removing defined regions of muscle tissue from the developing limb zeugopod, it was possible to conclude that lack of function of the anterior and lateral hindlimb tissue is associated with abnormal foot rotation, resulting in a similar phenotype to clubfoot. By examining the affected muscles, it was possible to identify the tibialis cranialis and the peroneus longus muscles as relevant candidates involved in clubfoot aetiology. In summary, the evidence presented here suggests that the pma clubfoot results from a regulatory mutation that induces Limk1 over-expression and nerve growth delay. This in turn prevents the proper development of the peroneal nerve, resulting in the degeneration of its target muscle tibialis anterior and peroneus longus muscles. This degeneration will interfere with foot rotation and result in clubfoot. Thus, the results described by this work are of utmost importance for the understanding of the clubfoot pathology, as it supports a neuro-muscular aetiology dependent on a physical dynamic equilibrium of muscular forces. This is of scientific interest as it expands the current understanding of the foot rotation and the integrative interactions during the limb organogenesis; poorly described developmental processes, and of clinical relevance as it establishes important ideas and concepts for study in human patients.

Thesis (Ph.D.)--University of Western Australia, 2005.

Proefschrift Universiteit van Amsterdam. / Met samenvatting in het Nederlands.

Charcot-Marie-Tooth disease (CMT) is the commonest inherited neuromuscular disorder, which affects the peripheral nervous system leading to nerve degeneration. CMT is categorised into two forms, ‘axonal’ and ‘demyelinating’, which reflects the main site of pathology as the axon or Schwann cells respectively. Over 90 genes have been identified associated with the disease. Among the genes associated with demyelinating CMT, the focus of my thesis is LITAF, mutations in which lead to an autosomal dominant demyelinating CMT known as CMT type 1C. LITAF is a 17 kDa protein likely to be involved in endocytic degradation and trafficking of specific cargo proteins. It contains an N-terminal proline-rich region mediating protein-protein interactions and a C-terminal LITAF domain consisting of a zinc-ribbon structure with a hydrophobic region incorporated. Most of the CMT1C mutations are clustered in this highly conserved LITAF domain. LITAF was predicted to play roles in recruiting ESCRT components and exosome formation, but the precise function remains unclear. Furthermore, why mutations in LITAF lead to CMT is not known. My work therefore focused on characterising the function of the LITAF protein both in health and disease. In my thesis, I first tried to determine the subcellular localisation of LITAF proteins and also investigated the function of the highly conserved C-terminal LITAF domain in targeting protein to the membrane. With regards to the function of LITAF, potential binding partners were screened using the traditional GST pull-down assay and the in-situ proximity labelling assay, BioID. A number of novel potential binding partners were identified in both assays. Among the list of potential binding partners, BAG3 was captured in both pull-down assays and was chosen for further studies. The interaction with LITAF was characterised and the potential role of this interaction in autophagy was investigated. Integrin, which was also captured in the BioID assay, was another protein chosen for further studies. Internalisation and recycling assay were developed to investigate the function of LITAF in integrin trafficking. The potential of CMT mutations in impairing the internalisation of integrin in A431 cells and the difficulty in performing the assays were discussed. Lastly, with patient fibroblasts available in our lab, disease phenotypes were analysed using two types of imaging technique: transmission electron microscopy (TEM) (in collaboration with J. Edgar) and immunofluorescence microscopy. Swollen vacuoles were observed in the TEM images of the patient fibroblasts only, and various uptake assays were performed to identify these enlarged compartments. In summary, this work offers an insight into the function of LITAF both in health and disease as well as identifying potential intracellular binding partners that might shed light on pathogenesis. Furthermore, the modified trafficking assays described in this thesis can be applied to Schwann cell and patient fibroblasts, providing further tools to probe the underlying membrane trafficking pathways that are dysfunctional in CMT.

A doença de Charcot-Marie-Tooth (CMT) caracteriza-se por comprometimento dos nervos periféricos de predomínio distal, tendo curso clínico variável. Observa-se quadro de evolução lenta de atrofia e fraqueza distal em membros inferiores, seguidos por diminuição da sensibilidade. Os reflexos estão em geral abolidos, mas podem estar exaltados e acompanhados de sinal de Babinski. É frequente o encontro de atrofia do terço distal das pernas, de pes cavos e de deformidades em artelhos. A doença de CMT pode ser classificada, com o auxílio da eletroneuromiografia, em desmielinizante (CMT1) ou axonal (CMT2). A CMT1 possui velocidade de condução motora do nervo mediano < 38 m/s e a CMT2 > 38m/s. A CMT1 é de herança autossômica dominante, e a CMT2 pode ser de herança dominante ou recessiva. A CMT2 é geneticamente heterogênea e conhecem-se até o momento 13 loci associados a essa condição, com nove genes identificados. O objetivo deste estudo é investigar do ponto de vista clínico, neurofisiológico e genético uma família com muitos portadores de CMT2. A família multigeneracional que apresenta CMT2 é procedente de Tobias Barreto, SE. Foi feita avaliação neurológica de 50 indivíduos e eletroneuromiografia em 22 pacientes. Com dados da avaliação clínica e eletroneuromiográfica foi aplicado o escore que avalia a gravidade da doença, o CMTNS. Para o estudo genético, foram coletadas 42 amostras de sangue de indivíduos afetados e de familiares não afetados. Entre os 50 indivíduos avaliados, 30 tinham sinais clínicos de neuropatia sensitivo-motora de predomínio distal. Paresia dos músculos distais foram os sinais clínicos mais precoces. Redução da sensibilidade superficial e profunda foi detectada nos segmentos distais. O sinal de Babinski estava presente em 14 indivíduos. A eletroneuromiografia demonstrou alterações compatíveis com polineuropatia axonal sensitiva e motora. O estudo genético demonstrou que, nesta família, CMT2 não está ligada a nenhum dos loci já conhecidos para esta condição, más o lócus do gene responsável não foi identificado até o momento. Em conclusão, as características clínicas e neurofisiológicas dessa família não diferem significativamente das observadas em outras formas de CMT, exceto pela alta prevalência de sinal de Babinski, e nossos resultados indicam a existência de um novo locus para CMT2 / Charcot-Marie-Tooth (CMT) disease is characterized by predominantly distal peripheral neuropathy with variable clinical course. Initial presentation is of a slowly progressive distal atrophy and weakness in lower limbs, followed by sensory compromise. Reflexes are usually abolished, but might be brisk and accompanied by Babinski sign. It is frequent to find distal atrophy of lower limbs, pes cavus and toe deformities. Electromyography can recognize two patterns of CMT: demyelinating (CMT1), which has a conduction median nerve velocity < 38 m/s and axonal (CMT2), with velocity > 38m/s. CMT1 is inherited as an autosomal dominant trait, and CMT2 might be transmitted as an autosomal dominant or recessive. CMT is genetically heterogeneous, and, up to now, 13 loci have been recognized and nine genes identified. The aim of this study was to conduct an investigation of clinical, genetics and neurophysiological investigation of a multigenerational family with several individuals with CMT2 and to characterize phenotype, neurophysiological pattern and genetic basis of this condition. Fifty individuals were clinically evaluated and nerve conduction velocity studies and distal muscular activity in lower limbs using concentric needle were performed in 22 patients. A blood sample was collect from 42 individuals, in order to perform linkage analysis. Thirty, among the 50 evaluated individuals, had clinical signs of predominantely distal sensory motor neuropathy. Distal muscle paresis was an early clinical sign. Reduction of superficial and deep sensory was detected distally. Babinski sign was present in 14 affected individuals. Neurophysiological study was characteristic of axonal sensory-motor neuropathy. Linkage analysis demonstrated that in this family, CMT2 was not linked to any already known loci for this condition, but the responsible gene locus was not identified so far. In conclusion, clinical and neurophysiological characteristics of this family did not differ substantially from other forms of CMT, except by the high prevalence of Babinski sign. Our study also suggests the presence of a new locus for CMT2

A doença de Charcot-Marie-Tooth (CMT) é desordem hereditária do sistema nervoso periférico, caracterizada por fraqueza dos membros inferiores, atrofia muscular e perda sensitiva. CMTX é doença ligada ao cromossomo X a qual corresponde aproximadamente 10% das famílias com CMT, sendo a segunda causa mais frequente da doença, após a duplicação 17p11.2-p12. Neste estudo analisamos 66 pacientes com CMT, negativos para duplicação 17p, por método de DHPLC, confirmado por sequenciamento direto. Foram identificadas seis mutações, em que quatro destas não foram descritas. Desta maneira confirmou uma frequência de mutações em torno de 9% no gene da Cx32. Portanto, nesta pesquisa do gene da Cx32 com CMT demonstrou que em uma população brasileira teve um comportamento mutacional semelhante ao das demais populações estudadas. / The Charcot-Marie-Tooth (CMT) is inherited disorder of the peripheral nervous system characterized by weakness of the lower limbs, muscular atrophy and sensory loss. CMTX is X-linked disease which accounts for approximately 10% of families with CMT, the second most frequent cause of disease after duplication 17p11.2-p12. This study analyzed 66 patients with CMT, negative for duplication 17p, by method of DHPLC, confirmed by direct sequencing. We identified six mutations, four of which were not described. In this way confirmed a mutation frequency of around 9% in the Cx32 gene. Therefore, this research Cx32 gene with CMT showed that in a Brazilian population had a mutational behavior similar to that of other populations studied.