Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

Des difficultés de compréhension interpersonnelles sont régulièrement rapportées par les proches et les patients atteints de Dystrophie Myotonique de type I (DM1) ou de Dystrophie Facio-Scapulo-Humérale (FSHD). Nous nous sommes intéressés à différents facteurs qui peuvent éclairer la présence d’un contresens relationnel au sein des couples (le patient avec son conjoint). Les facteurs étudiés sont : les troubles cognitifs, l’atteinte des mimiques faciales et le style d’attachement de chacun des partenaires. La méthode utilisée est basée en partie sur l’observation de l’interaction (communication verbale et non verbale) au sein des couples lors la réalisation d’une tâche de résolution de problème. Un test de reconnaissance des émotions faciales avec des visages atteints et des visages contrôles a également été construit et proposé à 57 sujets naïfs. Les résultats observés permettent de confirmer que l’atteinte du visage et de la cognition sociale (théorie de l’esprit) ainsi que le style d’attachement des membres du couple jouent un rôle dans les difficultés à se comprendre entre partenaires de l’interaction. Il est important de sensibiliser chacun des partenaires au rôle de la communication non verbale (CNV) dans leur quotidien. La CNV transmet non seulement des informations sur l’état interne (notamment les émotions) de l’autre, mais également sur ses besoins d’attention, de soutien, de support émotionnel. / Interpersonal communication difficulties are regularly reported by individuals suffering from Myotonic Dystrophy type I (DMI) and Facioscapulohumeral muscular Dystrophy (FSHD), and their families. Our study focuses specifically on communication between patients and their spouses. In order to investigate the nature of communication difficulties, cognitive impairment, impaired facial expression and the attachment styles of subjects were examined. Our investigation procedure involved detailed observation of couples’ communication processes (verbal and non-verbal communication) while completing a problem-solving task. Furthermore, we construct a test of facial emotion recognition composed of images from the DM1 and control groups, which was administered to 57 naive subjects. Results enables us to confirm that impaired facial expression, social cognition difficulties (theory of mind) and attachment style play a major role, in the communication and comprehension difficulties reported by patients and their families. A phenomenon of relational misinterpretation seems to occur between patients and their partners. It is therefore important to raise couples’ awareness of the role of non-verbal communication in everyday life. Non-verbal communication not only provides information about an individual’s internal state (such as emotions), but also about his or her needs for attention and emotional support.

Interstitial Fluid is a complex sample, highly abundant in the human body that can give information regardingtissue secretion, intracellular signaling and tissue health status. The composition of the interstitial fluid can giveinformation regarding the processes occurring in muscles and alterations due to pathological changes occurringduring disease progression. Currently this sample has not yet been characterized within rare diseases like musculardystrophies. Facioscapulohumeral Muscular Dytrophy is an inherited progressive myopathy, characterized by thedegeneration and progressive muscular fiber necrosis of muscles from the face, upper arms and lower limbs. It canbe diagnosed; but in an advanced stage where weakness in the muscles have already occur. Meanwhile there is nocurrent understanding of the mechanisms happening in the muscle. In this project an immunoassay protocol wasdeveloped using suspension bead array technology to create an optimal method to analyze the protein content ofthese samples. The technological platform allows antibody-based capturing and detection of protein targets frombiotinylated biological samples. By modifying an existing protocol for analysis of serum and plasma samplesabundance of 63 protein targets was measured in muscle interstitial fluid from healthy individuals and patientsaffected by facioscapulohumeral dystrophy (FSHD), The optimized steps were the sample pre-treatment, the assaybuffer dilution ratio and the incubation time for capturing the protein targets. The findings of this project indicatethat using 1 μl of muscle interstitial fluid sample with minimized dilution factor and 60-fold molar excess biotinrelative to sample protein concentration enables detection of Interstitial fluid protein components. The proteinsdetected are ret finger protein-like 4B (RFPL4B) and albumin in from affected muscle and histone cluster(HIST1H3A) and albumin in non affected muscle.

Multi-User Virtual Environments (MUVEs) and Massively Multi-player On- line Games (MMOGs) are a popular, immersive genre of computer game. For some disabled users, eye-gaze offers the only input modality with the potential for sufficiently high bandwidth to support the range of time-critical interaction tasks required to play. Although, there has been much research into gaze interaction techniques for computer interaction over the past twenty years, much of this has focused on 2D desktop application control. There has been some work that investigates the use of gaze interaction as an additional input device for gaming but very little on using gaze on its own. Further, configuration of these techniques usually requires expert knowledge often beyond the capabilities of a parent, carer or support worker. The work presented in this thesis addresses these issues by the investigation of novel gaze-only interaction techniques. These are to enable at least a beginner level of game play to take place together with a means of adapting the techniques to suit an individual. To achieve this, a collection of novel gaze based interaction techniques have been evaluated through empirical studies. These have been encompassed within an extensible software architecture that has been made available for free download. Further, a metric of reliability is developed that when used as a measure within a specially designed diagnostic test, allows the interaction technique to be adapted to suit an individual. Methods of selecting interaction techniques based upon game task are also explored and a novel methodology based on expert task analysis is developed to aid selection.

Les dystrophies musculaires des ceintures (ou limb-girdle muscular dystrophy, LGMD) sont un groupe hétérogène de dystrophies musculaires chez l’adulte et sont définies par une atrophie et une faiblesse progressive qui surviennent dans les muscles proximaux. Chez une cohorte canadienne-française, nous avons précédemment décrit une nouvelle forme récessive, désignée LGMD2L et marquée par une atrophie asymétrique du quadriceps, que nous avions cartographiée au chromosome 11p12-p13 grâce à des analyses de liaison. L’objectif de ce projet de thèse était de raffiner l’intervalle candidat, puis d’identifier et de caractériser le gène muté responsable de la LGMD2L. Grâce à une cartographie par homozygotie de polymorphismes de nucléotide simple (SNPs) réalisée sur une grande famille consanguine, nous avons redéfini l’intervalle candidat à une région du chromosome 11p14.3-p15.1. Par séquençage de l’ADN génomique et complémentaire au gène Anoctamine 5 (ANO5) inclus dans cet intervalle, nous avons identifié trois mutations, chez autant de familles: une substitution créant un site d’épissage aberrant, une insertion d’un nucléotide et une mutation faux-sens. Les deux premières mutations étaient associées à une hausse de la dégradation de l’ARN messager médiée par une troncation prématurée. Nous avons également identifié des mutations ANO5 chez une seconde dystrophie musculaire de type distal cartographiant au même locus que la LGMD2L, nommée MMD3, et dont la manifestation initiale était une faiblesse des mollets, mais qui pouvait progresser vers une atrophie des quadriceps. Une réparation membranaire défective avait été observée chez les fibroblastes de deux patients MMD3, suggérant un rôle pour ANO5 dans ce mécanisme. La localisation et la fonction d’ANO5 dans le muscle sont inconnues, mais cette protéine fait partie d’une famille conservée de protéines à huit domaines transmembranaires, les Anoctamines, dont certains membres sont des transporteurs chloriques activés par le calcium. Les résultats de nos études d’immunofluorescence suggèrent qu’ANO5 se localise peu au sarcolemme, mais plutôt à une structure intracellulaire qui suit la ligne Z des myofibrilles. De façon étonnante, cette localisation était préservée chez un patient LGMD2L porteur homozygote de la mutation d’épissage, en dépit du fait que cette dernière était considérée comme une mutation nulle. Néanmoins, nous avons identifié un épissage alternatif de l’exon 15 qui se produisait sur une proportion des transcrits porteurs de la mutation d’épissage, ce qui rétablirait le cadre de lecture, soulignant la complexité de la régulation de l’épissage d’ANO5 et laissant croire que la LGMD2L pourrait être causée par une perte de fonction partielle, et non complète, d’ANO5. Des études subséquentes par des groupes européens ont montré que les anoctaminopathies 5 sont une cause fréquente de dystrophies musculaires des ceintures chez l’adulte. Notre découverte de mutations au gène Anoctamine 5 a mis en évidence une nouvelle classe de protéines importantes pour la biologie du muscle et a ouvert la voie à de nouvelles pistes pour étudier les mécanismes par lesquels un défaut de réparation membranaire progresse en une dystrophie musculaire. / Limb-girdle muscular dystrophies (LGMD) encompass a broad spectrum of muscular dystrophies in which the initial weakness arises in proximal muscles. We previously described in French-Canadian (FC) families a new form of LGMD characterized by asymmetrical quadriceps femoris atrophy, named LGMD2L, which we mapped to chromosome 11p12-p13 using linkage analyses. The objectives of this thesis project were to refine the candidate interval, identify and characterize the LGMD2L gene. Using single nucleotide polymorphisms (SNPs) homozygosity mapping in a large consanguineous family, we narrowed down the LGMD2L candidate interval to a region on chromosome 11p14.3-p15.1, and identified three mutations in the Anoctamin 5 (ANO5) gene located in the interval. These mutations consisted of a missense, a one-bp duplication and a splice site mutation. We demonstrated that the latter two triggered the nonsense-mediated RNA decay pathway. In addition, we identified ANO5 mutations in cases affected by a non-dysferlin Miyoshi muscular dystrophy mapped also to chromosome 11, termed MMD3. In two MMD3 families of European descent, patients presented with calf weakness as the initial symptoms, sometimes evolving to quadriceps atrophy. Fibroblasts from one MMD3 family were shown to be defective for membrane repair. ANO5 localization and function in muscle are unknown, but it is a member of the conserved Anoctamin family of proteins with eight transmembrane domains, of which some function as calcium-activated chloride channel. Our immunofluorescence studies on longitudinal muscle sections suggest that ANO5 is not importantly localized to the sarcolemma, but rather to a structure following the Z-line. To our surprise, this localization was preserved for a LGMD2L patient homozygous for the splice site mutation, previously considered as a null mutation. By studying the splicing isoforms in this patient, we observed that skipping of exon 15 occurs on a proportion of transcripts, in addition to the aberrant splicing caused by the mutation. This alternative splicing event would recover the reading frame, thus underlining the complexity of ANO5 splicing and suggesting that LGMD2L could be the consequence of a partial, rather than complete, loss-of-function. Subsequent studies by other groups have shown that anoctaminopathies 5 are a common cause of adult-onset LGMD. Our discovery of ANO5 mutations has shed light on a new class of proteins important for the muscle biology and opened new research avenues to study how defective membrane repair progresses into muscular dystrophies.

Our research is focused on using orthotic devices in patients with Duchenne muscular dystrophy (DMD), particularly in the ambulant phase of the disease. DMD is the most common hereditary muscle disorder in childhood. The typical symptoms are progressive muscle weakness and contractures that lead to loss of ability of independent walking, typically among the age of 9 to 13 years. The theoretical part is focused on pattern of standing and walking in these patients, the posibilities of useing the orthotic devices in various stages of the disease, and on the problems of contractures and deformities. The other teoretical part of the work is devoted to certain physiotherapy interventions and to associated physical activities. The practical part of the research is based on assesment of effect of physiotherapy and use of the nigt orthesis AFO (Ankle- Foot-Orthesis) in group of 10 DMD boys in the average age 9,1 ± 2,7 years. All boys were examined before and after 6 month of therapy. In examinations the following tests were used: NSAA (North Star Ambulatory Assessment), BI (Barthel index) measurements of muscle strength by hand held myometr and measurement of PROM (passive range of motion). The practical part also includes analysis of questionnaire datas from 19 patients with DMD collected in year 3/2013, questions...

Duchenne muscular dystrophy is a progressive muscle disease that is still incurable. The disease affects all systems in the body and is therefore a need for a comprehensive approach. It manifests itself in childhood muscle weakness. Over time, there is a postural and respiratory changes affecting posture and the respiratory system. Depending on the changes of these functions leads to certain complications. Among these complications rank poorly stereotype walk, spinal deformities such as scoliosis, bad stereotype affect breathing, leading to vital lung function and many more. These complications are present in a particular time sequence and can be assigned to a specific patient age. This thesis is focused on the monitoring of postural and respiratory function in these patients. The work is the evaluation of these changes when they occur and in which the patient's age. Patients are rehabilitated life and physiotherapy is an integral part of treatment. It focuses possibilities of surgical treatment of scoliosis. The work also includes a questionnaire aimed at rehabilitation and physical therapy patients. Powered by TCPDF (www.tcpdf.org)

Durant ma thèse, mes recherches se sont principalement portées sur l'identification des propriétés modulaires de la dysferline, une protéine impliquée dans des dystrophies musculaires, afin d'identifier les approches thérapeutiques les plus prometteuses. Mon travail s’est donc orienté selon deux axes de recherche, une approche par « mini-protéines » et une approche par «saut d’exon », toutes deux basées sur des preuves de principe obtenues chez des patients. Nous avons tout d'abord testé une approche de saut d'exon pour l'exon 32 de DYSF. Nous avons pu établir la fonctionnalité de cette protéine tronquée en permettant son expression dans des cellules de patients déficients en dysferline. Ceci a suggéré que le domaine C2D (encodé par les exons 31 à 34) n'est pas essentiel pour la dysferline puisque l'absence d'une partie de celui-ci n'empêche pas sa fonctionnalité. Nous avons dans le même temps analysé les caractéristiques d'autres domaines de la dysferline, en créant des miniprotéines contenant différentes combinaisons de domaines. Nous avons montré que la partie C-terminale de la dysferline (composée des deux derniers domaines C2 et du passage transmembranaire) était essentielle et suffisante pour la fonctionnalité de la dysferline dans le muscle. L'ensemble de ces résultats démontre que certains domaines de la dysferline sont dispensables, ouvrant ainsi la voie à l'étude d'approches de thérapie génique basé sur les minigènes ou le saut d'exon pour les dysferlinopathies. / During my thesis, my researches have mainly concerned the modular properties of dysferlin, a protein involved in muscular dystrophies, in order to identify the most promising therapeutic approaches.My work has been oriented within two research axes, a mini-proteins approach and an exon-skipping approach, both based on proofs of concept obtained in patients. We first tested an exon-skipping approach for the exon 32 of DYSF, based on the identification of a protein lacking the encoded part of this exon, in an asymptomatic person. We have established the functionality of this truncated protein by allowing its expression in dysferlin-deficient patients' cells. This suggests that the C2D domain (encoded by exons 31 to 34) is not essential for dysferlin since the absence of a part of it don't block its functionality.In the same time we analyzed the characteristics of the others domains of dysferlin, by creating miniproteins containing different combinations of domains. By studying the abilities of this constructs, we have showed that the C-ter part of dysferlin (composed of the last C2 domains and the transmembrane domain) was essential and sufficient for the functionality of dysferlin in muscle. All these results demonstrate that several domains of dysferlin are dispensable, paving the way for studying gene therapy approaches, based on minigenes or exon-skipping for dysferlinopathies.

A distrofia muscular de Duchenne (DMD) é uma doença hereditária, degenerativa e progressiva dos músculos esqueléticos. É causada pela ausência da proteína distrofina e caracterizada pela perda progressiva da força muscular e deterioração da capacidade funcional. Alterações na regulação da homeostase do cálcio, proteólise e alterações metabólicas, especialmente mitocondriais, são parte da patogênese da doença. A coenzima Q (CoQ10), potente antioxidante que participa da atividade da cadeia respiratória, tem sido utilizada em ensaios clínicos, entretanto, não há estudos que evidencie seu comprometimento na DMD. O objetivo deste estudo foi avaliar a CoQ10 e a da atividade da cadeia respiratória em fragmentos de biópsia muscular de pacientes com DMD e sua correlação com parâmetros clínicos e a capacidade funcional. O estudo constitui de uma etapa retrospectiva, onde foram analisadas 22 biópsias musculares de pacientes com DMD, e outra prospectiva, onde foram avaliados dez pacientes com DMD. Dez pacientes controles foram utilizados nas duas etapas do estudo. A concentração da CoQ10 foi realizada através da técnica de cromatografia líquida de alta performance de fase reversa. As atividades das enzimas da cadeia respiratória foram medidas através de técnicas espectrofotométricas. A capacidade funcional foi mensurada através das escalas Medida da Função Motora (MFM) e Escala de Avaliação para deambulantes North Star (NSAA), e dos testes cronometrados: tempo para percorrer 10 metros (T10), tempo para realizar a manobra de Gowers (TGowers) e teste da caminhada dos 6 minutos (TC6min). Fase retrospectiva: a média de idade dos pacientes com DMD foi de 6,9 anos, (DP ±2,4) e controles de 8 anos, (DP ±2,69). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 8,6 µg/g de tecido (DP ±3,9) e nos fragmentos dos controles foi de 31,6 µg/g de tecido (DP ±6,9). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 27,3% (DP ±14,2%) e nos controles foi de 89,2% (DP ±3,3%). Evidenciou-se alta correlação entre aquantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,767 e p= 0,016). As atividades dos complexos enzimáticos da cadeia respiratória dos pacientes com DMD não demonstraram deficiência. Já o resultado do ensaio conjunto dos complexos II+III, encontra-se significativamente reduzido nos pacientes com DMD. Etapa prospectiva: a média de idade dos pacientes com DMD foi de 6,5 anos, (DP ±2,4). A dosagem média de CoQ10 nos fragmentos de pacientes com DMD foi de 12,6 µg/g de tecido (DP ±5,1). A média da área ocupada por fibras musculares nos pacientes com DMD foi de 40,3% (DP ±20,4%). Houve alta correlação entre a quantidade de CoQ10 e a área relativa ocupada por fibras musculares (r= 0,690 e p= 0,058). A correlação da dosagem da CoQ10 com os instrumentos de avaliação da capacidade funcional foi alta com o TGowers e moderada com MFM total e dimensões 1 e 2, NSAA, T10 e TC6min. Em relação à área relativa de fibras musculares, houve moderada correlação com a dimensão 1 da MFM e com o TGowers. Não houve correlação da CoQ10 e da área relativa ocupada por fibras musculares com os parâmetros clínicos: idade no momento da biópsia, idade do início dos sintomas e tempo de evolução da doença. No presente estudo, concluímos que existe uma deficiência secundária de CoQ10 em pacientes com DMD, a qual contribui para entender a fisiopatologia da doença e com grande relevância para as propostas terapêuticas. / Duchenne muscular dystrophy (DMD) is a hereditary, degenerative and progressive skeletal muscles disease. It is caused by the absence of the protein dystrophin and characterized by progressive loss of muscle strength and deterioration of functional capacity. Alterations in the regulation of calcium homeostasis, proteolysis and metabolic abnormalities, especially mitochondrial dysfunction, are part of the pathogenesis of the disease. Coenzyme Q (CoQ10), a potent antioxidant that participates in respiratory chain activity, has been used in clinical trials, however, there are no studies showing its involvement in DMD. The purpose of this study was to investigate CoQ10 content and respiratory chain activity in muscle biopsy of patients with DMD and its correlation with clinical parameters and functional capacity. The study consisted of a retrospective phase, in which 22 muscle biopsies from patients with DMD were analyzed, and a prospective phase, where ten patients with DMD were evaluated. The same control group of ten patients were used in the two phases of the study. The concentration of CoQ10 was measured using the reverse phase high performance liquid chromatography technique. Activities of the respiratory chain enzymes were measured by spectrophotometry. The functional capacity was evaluated using the Motor Function Measurement (MFM) and North Star Ambulatory Assessment (NSAA) and the following timed tests: to run 10 meters (T10), to perform the Gowers maneuver (TGowers) and the 6-minute walk test (6MWT). Retrospective phase: the mean age of patients with DMD was 6.9 years (SD ± 2.4) and of controls was 8 years (SD ± 2.69). The mean CoQ10 content in fragments from patients with DMD was 8.6 ?g / g tissue (DP ± 3.9) and in fragments from controls was 31.6 ?g / g tissue (DP ± 6.9). The mean area occupied by muscle fibers in patients with DMD was 27.3% (SD ± 14.2%) and in controls was 89.2% (SD ± 3.3%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.767 and p= 0.016). The activities of respiratory chain enzymes from patients with DMD were not deficient. On the other hand, the results of the combined analysis of complexes II + III were significantly reduced in patients with DMD. Prospective phase: the mean age of patients with DMD was 6.5 years (SD ± 2.4). The mean CoQ10 content in fragments from patients with DMD was 12.6 ?g / g tissue (SD ± 5.1). The mean area occupied by muscle fibers in patients with DMD was 40.3% (SD ± 20.4%). There was a high correlation between the amount of CoQ10 and the relative area occupied by muscle fibers (r= 0.690 and p= 0.058). The correlation between the amount of CoQ10 and the functional capacity assessment instruments was high forTGowers and moderate for MFM total and dimensions 1 and 2, NSAA, T10 andd TC6min. Regarding the relative area of muscle fibers, there was a moderate correlation with MFM dimension 1 (standing position and transfers) and TGowers. There was no correlation between CoQ10 and relative area occupied by muscle fibers with clinical parameters: age at time of biopsy, age of onset of symptoms and time of disease progression. In the present study, we conclude that there is a secondary deficiency of CoQ10 in patients with DMD, which contributes for the understanding of its physiopathology and is relevant for therapy.

La dystrophie musculaire de Duchenne (DMD) est la maladie neuromusculaire la plus fréquente de l'enfant. Son évolution progressive inexorable conduit habituellement au décès dans la troisième décade. La DMD constitue cependant une affection hétérogène pour la sévérité de l'atteinte musculaire, cognitive et cardiaque, et cette hétérogénéité n'est pas totalement expliquée par la localisation des mutations dans le gène de la dystrophine. Ma thèse comporte trois volets: (1) une analyse clinique multivariée d'une cohorte de DMD suivie à long terme qui nous a permis de définir 4 phénotypes distincts de DMD; (2) une étude de corrélation clinico-pathologique qui a identifié la fibrose endomysiale précoce comme seul facteur histologique prédictif de sévérité motrice; (3) la mise au point d'un modèle murin original de myofibrose dystrophique chez la souris mdx déficiente en dystrophine. 1- Étude multiparamétrique clinique. La saisie par la même équipe des données fonctionnelles musculaires, cardiaques, respiratoires et cognitives de 75 patients atteints de DMD (tous génotypés et présentant une absence complète de dystrophine musculaire), suivis pendant &gt;10 ans, a permis d'établir un modèle multiparamétrique satisfaisant à deux dimensions principales, cognitive et motrice, et de définir 4 clusters phénotypiques : (i) DMD cognitive et motrice congénitale (20%), (ii) DMD classique (28%), (iii) DMD motrice pure modérée (22%), (iv) DMD motrice pure sévère (30%). La corrélation génotypephénotype était restreinte à la seule atteinte cognitive. Des indicateurs pronostics précoce ont été identifiés et validés sur une 2ème série de 34 patients. 2- Étude histopathologique. Les variations de sévérité de l'atteinte musculaire n'étant pas expliquées par la génétique moléculaire, nous avons cherché à corréler les paramètres moteurs et la biopsie musculaire prélevée dans le quadriceps à un stade précoce (3-7 ans) chez 25 patients (analyse stéréologique des images numérisées pour les paramètres élémentaires: nécrose/régénération, fibres hypercontractées, adipocytes, fibrose endomysiale et périmysiale). Seule la fibrose endomysiale était associée à un pronostic moteur défavorable (p&lt;0.002) attesté par l'âge de perte de marche, la force du quadriceps et le testing musculaire global à 10 ans. Cette fibrose endomysiale dissociait les capillaires des myofibres (écartement x 2.5), et s'accompagnait d'une augmentation sélective des macrophages CD206+ activés dans la voie alterne (M2) et d'une diminution relative des cellules satellites musculaires (p&lt;0.0001). Ces données suggèrent un rôle clé de la fibrose endomysiale (et des macrophages M2 profibrosant) et dans la sévérité clinique de la DMD. 3- Étude expérimentale. Ces éléments rendent nécessaire la mise au point d'un modèle expérimental de myofibrose dystrophique, la souris mdx présentant peu de fibrose et un déficit moteur modéré et tardif. Nous avons mis au point une nouvelle méthode de lésion musculaire focale profibrosante du tibialis antérieur chez la souris mdx (piqûres multiples quotidiennes pendant 15 jours). Une fibrose endomysiale attestée par un fort immunomarquage du collagène I (à 8, 30, 60 et 90 jours) a été quantifiée et corrélée à la perte de la force musculaire dans la patte lésée (comparée au muscle contralatéral). Ces résultats légitiment et préparent les futures stratégies thérapeutiques "anti-fibrosantes" dans la DMD</abstract> / The clinical heterogeneity of Duchenne muscular dystrophy (DMD) may prove a major obstacle to the interpretation of therapeutic trials but has not been subjected to systematic analysis. In a first part, we present a statistical analysis on two series of steroid-free patients with complete lack of dystrophin determined by Western blot. Series 1 consisting of 75 patients longitudinally evaluated for motor, respiratory, cardiac and cognitive functions by the same team (median follow-up: 10.5yrs) was subjected to exploratory data analysis whose main conclusion were confirmed by exploration of data obtained from 34 routinely evaluated patients (series 2). Main outcome measures were age at loss of ambulation and of onset of contractures, manual muscle testing (MMT), cardiac and respiratory functional tests, general intelligence assessment (IQ), educational level. Multivariate exploratory analysis of series 1 classified 70/75 patients into 4 clusters with distinctive intellectual and motor outcomes: A (congenital DMD, 20%): markedly poor intellectual and motor outcome; B (classical DMD, 28%): intermediate intellectual and poor motor outcome; C (moderate pure motor DMD, 22%): normal intelligence and delayed motor impairment; and D (severe pure motor DMD, 30%): normal intelligence and poor motor outcome. Group A patients had the most severe respiratory and cardiac involvement. Frequency of mutations upstream to exon 30 increased from group A to D, but genotype/phenotype correlations were restricted to cognition. Diagnostic accuracy tests showed that combination of "clinical onset &lt;2yrs" with "mental retardation" reliably assigned patients to group A (sensitivity 0.93, specificity 0.98). Combination of "lower limb MMT score&gt;6 at 8yrs" with "normal or borderline mental status" reliably assigned patients to group C (sensitivity: 1, specificity: 0.94). These early criteria were also predictive of "congenital DMD" and "moderate pure motor DMD" in series 2. In a second part, we included 25 steroid-free DMD patients in a multiparametric analysis plotting initial histological alterations in quadriceps muscle with 13 relevant clinical data collected by the same team over a long-term follow-up (mean&gt;10yrs). Elementary histological parameters (fiber size, hypercontracted fibers, necrotic/basophilic fibers, edema, endomysial and perimysial fibrosis, and fatty degeneration) were assessed by morphometry. Endomysial fibrosis was the sole myopathological parameter significantly correlated with poor motor outcome, assessed by quadriceps muscle strength, manual muscle testing of upper and lower limbs at 10yrs, and age at ambulation loss (all p&lt;0.002). Motor outcome and fibrosis were not correlated with genotype. Myofibers exhibited oxidative stress-induced protein alterations and became separated from capillaries by fibrosis, which was associated with both increase of CD206+ alternatively activated macrophages and relative decrease of CD56+ satellite cells (both p&lt;0.0001). This study provides firm basis for antifibrotic therapeutic strategies in DMD, and supports the view that alternatively activated macrophages, known to inhibit myogenesis while promoting collagen-producing cell formation, play a key role in myofibrosis. In the third experimental part, experiments were conducted on mdx mice at 6-8 weeks of age. Fifteen micropunctures were made daily in the right tibialis anterior (TA) muscle with micropins used for entomology (150 µm diameter) during 2 weeks. Our data suggest that repeated microinjuries of muscle deficient in dystrophin protein triggers a endomysial fibrotic process that stabilizes with time well associated with muscle strenght decrease. Endomysial fibrosis seems to be one of the target of therapy in DMD. This satisfactory model will be usefull to study the mechanisms of fibrosis process in dystrophin deficient muscle and to evaluate antifibrotic treatment