O objetivo deste estudo foi avaliar o efeito do uso noturno ou diurno da ankle-foot orthosis (AFO) na biomecânica da marcha de pacientes com DMD. Foram avaliados 20 pacientes deambuladores, do Ambulatório de Miopatias Infantis do CER do HCFMRP-USP, com diagnóstico de distrofia muscular de Duchenne (DMD), com idades entre 4 e 12 anos. Foi realizada a avaliação inicial (Av1) em todos os pacientes e, 7 pacientes foram reavaliados após 6 meses (Av2). Na Av1, os pacientes foram agrupados conforme o uso da órtese: grupo sem órtese (SO; n=7), grupo órtese noturna (ON; n=7), grupo órtese diurna (OD; n=6). Na Av1 e na Av2 foram obtidos dados de massa corporal, altura, composição corporal pela bioimpedância elétrica, escore funcional pela escala medida da função motora, amplitude passiva de movimento articular, força muscular isométrica pelo dinamômetro Handheld e avaliação biomecânica da marcha, na velocidade habitual do paciente. Os pacientes que faziam uso da órtese diurna foram avaliados sem e com órtese, sendo denominados grupos ODs e ODc, respectivamente. Os dados foram analisados de três formas: duas transversais e uma longitudinal. Nas análises transversais, foram realizados dois procedimentos: (i) comparando dados dos grupos SO x ON x ODs; (ii) comparando dados dos grupos SO x ON x ODc. Nestas, foi utilizado o teste ANOVA, considerando um nível de significância de 5%. Na análise longitudinal, foi realizada a análise descritiva comparando os dados obtidos na Av1 e Av2, individualmente para os 7 pacientes reavaliados. Transversalmente, o grupo ODc apresentou maiores picos do ângulo de dorsiflexão e do momento dorsiflexor, menor ângulo de flexão plantar e menor geração de potência de tornozelo (p<0,05) que o grupo SO. Porém, ao caminhar sem a AFO (grupo ODs) estes resultados não foram observados (p>0,05). Em relação ao grupo ON, o grupo ODc obteve menores picos do ângulo de flexão do quadril, de absorção de potência de quadril, do ângulo de flexão plantar e maior pico do momento dorsiflexor (p<0,05), sendo que ao retirar a AFO (ODs) essas diferenças não foram observadas (p>0,05). E ainda, o grupo ON obteve maior pico do ângulo de flexão do joelho e menor momento flexor de quadril (p<0,05) em relação ao grupo ON. Na comparação dos dados entre os grupos SO e ON, o grupo ON obteve maior pico do ângulo de flexão do joelho e maior absorção de potência de quadril (p<0,05). Na análise longitudinal individual foi observado que os 2 pacientes que iniciaram precocemente e mantiveram o uso noturno da AFO apresentaram na Av2 maior velocidade da marcha, maiores momentos extensor de quadril e flexor plantar e maior geração de potência de tornozelo, contrariamente aos paciente que interromperam o uso (noturno ou diurno) da AFO. Conclui-se que o uso diurno da AFO acarretou alterações positivas na biomecânica da marcha, minimizando compensações típicas da DMD na articulação do tornozelo. O uso noturno da AFO, quando iniciado precocemente, também afetou positivamente a marcha dos pacientes. Assim, sugere-se o início precoce e contínuo do uso diurno e noturno da AFO aos pacientes com DMD. / The aim of this study was to evaluate the effect of the ankle-foot orthosis (AFO) during nocturnal or daytime usage of the gait biomechanics in patients with Duchenne Muscular Dystrophy (DMD). Twenty ambulant patients from the Myopathies Infant Ambulatory of CER - HCFMRP-USP, were diagnosed with DMD between the ages of 4 and13 years and were evaluated. The initial evaluation (Ev1) was performed in all patients, and 7 patients were reevaluated after 6 months (Ev2). In Av1, patients were grouped according to orthosis use: group without orthosis (NoO, n = 7), group with nocturnal orthosis (NiO, n = 7), group with daytime orthosis (DO, n = 6). In Ev1 and Ev2 data were obtained according to the weight, height, body composition (bioelectrical impedance), functional score (Measure scale of motor function), passive joint range of motion, isometric muscle strength (dynamometer Handheld) and biomechanical gait analyses (usual velocity for the patient). Patients who used the daytime orthosis were evaluated with and without bracing, respectively. The data were analyzed in three ways; the first two were cross-sectional and the other one was longitudinal. In the cross-sectional analyzes, an exploratory analysis of the data from each evaluation was performed, and subsequently, the variables were compared between groups, considering the means and standard deviations. ANOVA test was used, and it was considered a significant level of 5%. In the longitudinal analysis, the description of the data obtained in the evaluation 1 compared to the data obtained in the evaluation 2 was individually performed in the 7 patients who were reevaluated. A cross-sectional analysis compared the data between NoO x NiO x DO groups considering the gait analysis data from the DO group without the orthosis (barefoot), being named DOno. The other cross-sectional analysis compared the data between NoO x NiO x DO groups considering the gait analysis data from the OD group with orthosis, being named DOwith. In individual longitudinal analysis, it was observed that patients who had started early and kept the nocturnal usage of AFO which has been already showed, in six months, an increment of gait velocity, hip extensor and plantar flexor moments and also the increment of ankle power generation, which is the opposite of the patient who has discontinued the AFO usage (daytime or nocturnal). In the cross-sectional analyzes it was observed that, compared to the NoO group, the DOwith group had a higher dorsiflexion angle peak and higher dorsiflexor moment peak (p<0.05). However, when they walked without the device these results were not maintained. There was no difference (p>0.05) between DOno and NoO groups for the kinematic parameters. And, the DOno group had lower plantar flexor moment maximum peak than the SO group (p>0.05). It was concluded that AFO daytime use cause positive changes in gait biomechanics, minimizing typical compensation of DMD in the ankle joint. The night use of AFO, when started early, also positively affected the gait of patients. Thus, it is suggested early prescription of daytime and nocturnal usage of AFO for DMD patients.

A Distrofia Muscular de Duchenne (DMD) é uma doença genética neuromuscular hereditária, ligada ao cromossomo X, sendo encontrada em humanos do sexo masculino. Esta doença muscular é descrita em outras espécies. O modelo de estudo pré-clínico GRMD (Golden Retrievier Muscular Dystrophy) apresenta sintomas clínicos fenotipicamente característicos da DMD em humanos e, por esta razão, tem sido amplamente utilizado como modelo de estudos pré-clínicos. O objetivo da presente pesquisa foi avaliar o tecido muscular, no modelo canino distrófico, ao longo da gestação. Quatro fêmeas, portadoras do gene distrófico, foram inseminadas com sêmen fresco de cães distróficos. No 25&ordm; dia, pós-inseminação, as fêmeas foram submetidas a exames de ultrassonografia para confirmar a gestação. As fêmeas gestantes passaram por uma ovariosalpingohisterectomia (OSH) para a retirada dos embriões e fetos nos seguintes períodos gestacionais: 28&ordm; , 33&ordm; , 38&ordm; e 42&ordm; dias. Em seguida fragmentos de tecido muscular foram analisados macroscopicamente e microscopicamente. Para verificar expressões proteicas, amostras de tecido foram submetidas a técnicas imunológicas, e PCR para distrofina, miostatina, e utrofina. Aos, 33&ordm; e 38&ordm; dias de gestação, no grupo distrófico, foram observadas características teciduais que corroboram com desenvolvimento tardio do tecido muscular. Os resultados para detecção proteica sugerem que, a distrofina, miostatina e utrofina foram expressas igualmente nos grupos controle e distrófico, durante todos os períodos do desenvolvimento gestacional analisado. Por fim, os dados sugerem que animais distróficos apresentam músculo sadio durante a fase gestacional, o que pode ser benéfico para testes farmacológicos em idade precoce. / Duchenne Muscular Dystrophy (DMD) is a hereditary neuromuscular genetic disease linked to the X chromosome, being found in male humans. This muscle disease is described in other species. The pre-clinical GRMD (Golden Retrievier Muscular Dystrophy) study model presents phenotypically characteristic clinical symptoms of DMD in humans and,for this reason, has been widely used as a model for preclinical studies. The aim of the present study was to evaluate the muscular tissue, in the dystrophic canine model, throughout the gestation. Four females, carriers of the dystrophic gene, were inseminated with fresh semen from dystrophic dogs. On the 25th day, post-insemination, the females were submitted to ultrasonography to confirm the pregnancy. The pregnant females underwent an ovariosalpingohisterectomy (OSH) for the removal of the embryos and fetuses in the following gestational periods: 28&ordm;, 33&ordm;, 38&ordm; and 42&ordm; days. Then fragments of muscle tissue were analyzed macroscopically and microscopically. To verify protein expression, tissue samples were submitted to immunological techniques, and PCR for dystrophin, myostatin, and utrophin. At the 33 and 38th days of gestation, tissue characteristics were observed in the dystrophic group, which corroborate the late development of muscle tissue. The results for protein detection suggest that dystrophin, myostatin and utrophin were also expressed in the control and affected groups, during all periods of the gestational development analyzed. Lastly, the data suggest that dystrophic animals present healthy muscle during the gestational phase, which may be beneficial for pharmacological tests at an early age.

No description available.

Schätzung der Segregation beim Keimzellmosaik in Familien mit DMD/BMD anhand ausgewählter Stammbäume zur Verbesserung der Situation in der genetischen Beratung / Estimation of the segregation in DMD/BMD-families with germ-line mosaicism with the help of selected pedigrees to improve the situation in genetic counseling

Thèse (Ph. D.)--Université Laval, 2007. / Titre de l'écran-titre (visionné le 5 mai 2008). Bibliogr.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. [95]-106. Publié aussi en version électronique.

Duchenne's muscular dystrophy (DMD) is a debilitating disease that affects approximately 1 in 3500 boys, with many DMD patients dying before the age of 20 due to cardio-respiratory complications. DMD is the result of defects in the gene that encodes dystrophin, an integral muscle membrane protein. Although the genetic defect has been identified, the relation between the absence of expressed dystrophin and the mechanisms leading to its onset are still unclear. One possibility is that disrupted calcium (Ca²⁺) handling by the sarcoplasmic reticulum (SR) leads to an increased cytosolic Ca²⁺ concentration that activates proteolytic and apoptotic pathways that initiate muscle fiber death. However, little is known about the role of disrupted SR function in the onset of DMD. The purpose of this study was to test the hypothesis that altered calcium cycling by the SR could contribute to elevated cytosolic Ca²⁺ levels in the early stages of DMD, and thereby account for the onset of disease pathogenesis. Rates of SR Ca²⁺ uptake and release were determined in quadriceps muscles obtained from maturing dystrophic and control mice prior to the overt signs of the disease at ages ~9 and 21 days. In addition, the content of several key Ca²⁺ handling proteins, including two isoforms of the sarco(endo)plasmic reticulum ATPase pump (SERCA 1 & 2), ryanodine receptor type 1 (RyR1), parvalbumin, and calsequestrin were determined by Western analysis. Two dystrophic mouse models were used, the mdx mouse which lacks dystrophin, and the mdx:utrophin-deficient (mdx:utrn^-/-) mouse which also lacks utrophin, a protein homolog of dystrophin. The rate of SR Ca²⁺ uptake in quadriceps muscles of mdx/utrn^-/- mice aged 21 days was 73.1% and 61.3% higher than age-matched control and mdx muscles, respectively (p < 0.05). There was no difference in SR Ca²⁺ release rates between the genotypes at either age. There were significant increases in the content of each of the calcium handling proteins with age (p < 0.05), but no significant differences were detected between genotypes at either age. These data demonstrate the Ca²⁺ release rates of dystrophic SR are not compromised, but suggest the increased uptake rates of mdx:utrn^-/- SR may be an adaptation to increased cytosolic calcium levels, and/or be due to changes in intrinsic SERCA function and/or regulation. The role of increased SR Ca²⁺ uptakes rates in onset of DMD pathogenesis can not be directly determined from the present data; therefore it is suggested that future studies directly assess cytosolic Ca²⁺ concentration and examine the role of SERCA regulatory proteins in intact fibers obtained from mdx:utrn^-/- muscles at age 21 days. / Master of Science

Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy caused by the loss of functional dystrophin. DMD is characterized by scoliosis, muscle wasting, loss of ambulation and a reduced life span. The first line of treatment for DMD is glucocorticoids (GCs). GCs are prescribed primarily for their anti-inflammatory and immunosuppressive effects; however, GC treatment is known to cause significant muscle atrophy. In DMD, GC treatment has been shown to improve muscle strength for the first 6 months and stabilization of the disease for up to 3 years. However, long term treatment reduces muscle function and accelerates disease progression. It is paradoxical that we use a medication that causes muscle wasting to treat a muscle wasting disease. The regeneration and function of muscle is dependent on the proper regulation and functioning of muscle satellite cells (MuSCs) to restore and repair muscle tissue. The impact GCs have on MuSCs from activation to proliferation and differentiation into muscle fibers is not well understood. GCs have many mechanisms of action by acting as a ligand to the glucocorticoid receptor (GR) to cause downstream effects by direct DNA binding or indirectly by regulating proteins. To study the role of GCs, we examined the effects of GC treatment on myoblast morphology, the cytoskeletal network, post-translational modifications (PTMs) of tubulin subunits, and the organization of microtubule organizing centers (MTOCs) in proliferating and differentiating myoblasts. This study shows that the GR is an essential regulator of myotube morphology and proper myonuclei placement. Furthermore, dexamethasone (DEX) treatment causes branching of the MT network, as well as an increase in the expression of the stabilizing MT markers, acetylated and detyrosinated tubulin during early differentiation. DEX treatment was also found to misposition the Golgi complex, a primary MTOC for the cytoskeletal network, from the periphery of the nucleus to the center of the nucleus during early differentiation. Finally, we found very few differentially expressed genes between WT and GRMuSC-/- myoblasts between early and late differentiation, indicating that these morphological defects we see are not due to GCs regulating gene expression. Thus, GCs act through the GR to modify the MT network during early differentiation, causing morphological changes in myoblasts that persist throughout differentiation.

Hamm et al., 2021 reported that voluntary wheel running (R) was complementary to micro-dystrophin gene therapy (GT) in mdx mice, a model of Duchenne muscular dystrophy (DMD). After 21 weeks of running, time to fatigue on a treadmill for the mdxRGT mice was increased 1.8-fold compared to mdxGT mice (no run) and ~5-fold compared to mdx mice (no micro-dystrophin, no run). Fatigue times for mdxRGT were similar to wild type runners (WTR), while mdxGT and WT (no run) were also similar. The diaphragm is an important muscle for endurance exercise. Remarkably, diaphragm power in mdxRGT was depressed compared to mdxGT, suggesting a negative impact of running on GT. To explore mechanisms to explain this decrease, transcriptome profiles for each of the study groups were assessed. RNASeq data revealed differentially expressed genes (DEGs) from groupwise comparisons. Transcripts identified using the Jackson Labs' Gene Expression Database and extensive literature review were organized into a master signaling pathway composed of two sub-pathways: muscle regeneration and fast-slow fiber type shift. Both sub-pathways were hypothesized to explain the improved treadmill performance despite decreased diaphragm power in mdxRGT as potential adaptive mechanisms. Analysis revealed that GT alone (mdxGT) rescued transcriptome expression to WT values in the mdx phenotype more than GT and running combined (mdxRGT). This outcome indicates that, at the 26-week timepoint of sacrifice, the signaling of the transcripts in the muscle regeneration and fast-slow fiber type shift sub-pathways was likely not responsible for the observed improved running performance of mdxRGT compared to mdx. / Master of Science / Muscular dystrophy is a group of diseases characterized by progressive muscle wasting and loss of function. Duchenne muscular dystrophy (DMD) is the most common of these conditions, with an occurrence of 18 per 100,000 live births. The muscles of people with DMD lack a protein called dystrophin, which provides structural integrity for muscle fibers during contraction. This lack of dystrophin leads to muscle deterioration over time, leading to people with DMD typically being wheelchair-bound by ten years of age. Animal models of DMD have been created over time to help study this condition. One such model, the mdx mouse, was used in the study that led to this thesis project. In this study, Hamm et al., 2021, some of these mdx mice were given a micro-dystrophin gene therapy (GT). This GT aimed to deliver a smaller, but still functional version of the missing dystrophin protein to the mice, which has been shown to be beneficial in other studies. This study aimed to measure the effect of this GT when combined with voluntary wheel running. As people with DMD cannot exercise under current clinical guidelines, measuring the response of mdx mice to GT and running combined is an important step in determining the safety of such a treatment in human patients. In the study conducted by Hamm et al., 2021, the mice that received the micro-dystrophin GT and access to running wheels (mdxRGT) performed almost twice as well on a treadmill running test than the mice that received GT alone (mdxGT). Despite this positive result, the mdxRGT mice showed decreased diaphragm power generation compared to mdxGT mice. As the diaphragm is the most important breathing muscle, it is also very important for running performance; therefore, the decreased diaphragm power generation seen in mdxRGT mice is apparently contrary to their improved running performance. To explain this discrepancy, this thesis project examined the diaphragm transcriptome of the different groups in Hamm et al., 2021. The transcriptome is the sum total of messenger ribonucleic acid (mRNA) expressed in a given tissue. Deoxyribonucleic acid, or DNA, is transcribed into mRNA, which is then translated into protein. As such, this thesis project looked at the mRNA expressed in the diaphragm of the mice in the various groups of Hamm et al., 2021, specifically comparing the mdxGT and mdxRGT groups. Important mRNA transcripts, or genes, were identified and assembled into signaling pathways, cascades that highlight how transcripts affect each other and ultimately lead to function once they are translated into their corresponding proteins. Two such signaling pathways were generated based on mechanisms that were thought to contribute to the improved running performance-decreased diaphragm power discrepancy in mdxRGT mice -slow fiber type shifting, and muscle fiber regeneration. The expression of many of the mRNA transcripts in these resulting pathways was closer to the control group in mdxGT compared to mdxRGT. The control group was made up of healthy mice, and as such, their transcript expression level is seen as normal. This outcome of mdxGT having more similar expression to the control group than mdxRGT suggests that the expression of the transcripts included in the two signaling pathways (fast-slow fiber type shift and muscle fiber regeneration) likely did not explain the improved running performance despite decreased diaphragm power in mdxRGT mice. As such, future studies are warranted.

La dystrophie musculaire de Duchenne (DMD) est une maladie génétique qui touche environ 1 garçon sur 3500. Cette pathologie liée au chromosome X est caractérisée par l’absence de dystrophine au niveau des muscles. Ce manque de dystrophine fragilise le sarcolemme des fibres musculaires menant à une faiblesse progressive du muscle. Les patients décèdent généralement dans la vingtaine et il n’y a pas à l’heure actuelle de traitement curatif pour cette maladie. Une approche pour restaurer la dystrophine chez le patient DMD est d’introduire un transgène codant pour cette protéine dans ses muscles. Cela peut être fait par thérapie génique et particulièrement par la thérapie génique ex vivo et l’électroporation. Bien que ces deux techniques aient fait leurs preuves dans différents modèles animaux, elles n’ont jamais été utilisées chez le chien dystrophique alors que c’est le modèle le plus proche de la DMD en termes de phénotype. Deux versions de la dystrophine de chien ont été utilisées dans nos expériences : une version pleine longueur et une autre plus petite afin qu’elle puisse être incluse dans un lentivirus. La transplantation de myoblastes génétiquement modifiés par ce lentivirus (thérapie génique ex vivo) nous a permis d’obtenir l’expression de micro-dystrophine dans les muscles des souris immunodéficientes greffés. Néanmoins, l’autotransplantation de myoblastes de chien génétiquement modifiés a mené à un rejet spécifique des cellules greffées. L’électroporation, c.-à-d. l’injection de plasmide suivie d’un choc électrique, a également été utilisée pour introduire ce transgène ainsi que celui de la dystrophine pleine longueur dans des muscles de souris et de chien. Ces deux transgènes furent retrouvés avec succès chez la souris et le chien. Cependant, des infiltrations de cellules de l’immunité spécifique furent retrouvées au niveau des fibres exprimant le transgène chez le chien (pour l’utilisation de micro-dystrophine) et chez le chien dystrophique (pour la dystrophine pleine longueur). Bien que les résultats obtenus avec la thérapie génique ex vivo et l’électroporation soient très bons chez la souris, ceux obtenus chez le chien sont plus modérés. Il reste donc encore beaucoup d’améliorations à apporter à ces deux méthodes avant qu’elles puissent être utilisées comme approche thérapeutique dans le cadre de la DMD. / Duchenne muscular dystrophy is a genetic disease affecting 1 out of every 3500 boys. This X-linked pathology is characterised by the absence of dystrophin in myofibers. This lack of dystrophin leads to a progressive muscular degeneration. DMD patients die between 17 and 30 years of age. There are currently no curative treatments for this disease. An approach to restore dystrophin in DMD patients is to introduce a transgene coding for this protein into their muscles. This can be done by gene therapy, particularly by ex vivo gene therapy or by electroporation. Even if these 2 techniques have shown good results in mouse models, they have not been used in the dystrophic dog. Two different isoforms of the dystrophin were used in our experiments: the full length dog dystrophin and a shorter version, the dog micro-dystrophin, introduced in a lentivirus backbone. Myoblasts were transduced with this lentivirus and transplanted successfully in immunodeficient mouse. However, the autotransplantation of genetically modified dog myoblasts led to a specific rejection of the grafted cells. A non viral gene therapy (electroporation, i.e., injection of a plasmid followed by a sequence of electric pulses) was used to introduce these two different isoforms of dystrophin in mouse and (normal and dystrophic) dog muscles. The two transgenes were electroporated with success in these muscles. However, a specific immune response was found in some myofibers expressing the transgene in the normal dog (using micro-dystrophin) and in the dystrophic dog (using full length dystrophin). Although the results obtained with the ex vivo gene therapy and with the electroporation were relatively effective in the mouse model, those obtained with the dog model were much lower. Thus, lots of improvements remain to be made in order to consider these two techniques as potential approaches to restore dystrophin in a large animal model and eventually in DMD patients.