Endophenotypes of Duchenne muscular dystrophy and characterisation of human and experimental myofibrose
Subphénotypes de la maladie de Duchenne et caractérisation de la myofibrose dystrophique humaine et expérimentale
Résumé
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 <2yrs" with "mental retardation" reliably assigned patients to group A (sensitivity 0.93, specificity 0.98). Combination of "lower limb MMT score>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>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<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<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 ?m 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
La dystrophie musculaire de Duchenne (DMD) est la maladie neuromusculaire la plus fr?quente de l'enfant. Son ?volution progressive inexorable conduit habituellement au d?c?s dans la troisi?me d?cade. La DMD constitue cependant une affection h?t?rog?ne pour la s?v?rit? de l'atteinte musculaire, cognitive et cardiaque, et cette h?t?rog?n?it? n'est pas totalement expliqu?e par la localisation des mutations dans le g?ne de la dystrophine. Ma th?se comporte trois volets: (1) une analyse clinique multivari?e d'une cohorte de DMD suivie ? long terme qui nous a permis de d?finir 4 ph?notypes distincts de DMD; (2) une ?tude de corr?lation clinico-pathologique qui a identifi? la fibrose endomysiale pr?coce comme seul facteur histologique pr?dictif de s?v?rit? motrice; (3) la mise au point d'un mod?le murin original de myofibrose dystrophique chez la souris mdx d?ficiente en dystrophine. 1- ?tude multiparam?trique clinique. La saisie par la m?me ?quipe des donn?es fonctionnelles musculaires, cardiaques, respiratoires et cognitives de 75 patients atteints de DMD (tous g?notyp?s et pr?sentant une absence compl?te de dystrophine musculaire), suivis pendant >10 ans, a permis d'?tablir un mod?le multiparam?trique satisfaisant ? deux dimensions principales, cognitive et motrice, et de d?finir 4 clusters ph?notypiques : (i) DMD cognitive et motrice cong?nitale (20%), (ii) DMD classique (28%), (iii) DMD motrice pure mod?r?e (22%), (iv) DMD motrice pure s?v?re (30%). La corr?lation g?notypeph?notype ?tait restreinte ? la seule atteinte cognitive. Des indicateurs pronostics pr?coce ont ?t? identifi?s et valid?s sur une 2?me s?rie de 34 patients. 2- ?tude histopathologique. Les variations de s?v?rit? de l'atteinte musculaire n'?tant pas expliqu?es par la g?n?tique mol?culaire, nous avons cherch? ? corr?ler les param?tres moteurs et la biopsie musculaire pr?lev?e dans le quadriceps ? un stade pr?coce (3-7 ans) chez 25 patients (analyse st?r?ologique des images num?ris?es pour les param?tres ?l?mentaires: n?crose/r?g?n?ration, fibres hypercontract?es, adipocytes, fibrose endomysiale et p?rimysiale). Seule la fibrose endomysiale ?tait associ?e ? un pronostic moteur d?favorable (p<0.002) attest? par l'?ge de perte de marche, la force du quadriceps et le testing musculaire global ? 10 ans. Cette fibrose endomysiale dissociait les capillaires des myofibres (?cartement x 2.5), et s'accompagnait d'une augmentation s?lective des macrophages CD206+ activ?s dans la voie alterne (M2) et d'une diminution relative des cellules satellites musculaires (p<0.0001). Ces donn?es sugg?rent un r?le cl? de la fibrose endomysiale (et des macrophages M2 profibrosant) et dans la s?v?rit? clinique de la DMD. 3- ?tude exp?rimentale. Ces ?l?ments rendent n?cessaire la mise au point d'un mod?le exp?rimental de myofibrose dystrophique, la souris mdx pr?sentant peu de fibrose et un d?ficit moteur mod?r? et tardif. Nous avons mis au point une nouvelle m?thode de l?sion musculaire focale profibrosante du tibialis ant?rieur chez la souris mdx (piq?res multiples quotidiennes pendant 15 jours). Une fibrose endomysiale attest?e par un fort immunomarquage du collag?ne I (? 8, 30, 60 et 90 jours) a ?t? quantifi?e et corr?l?e ? la perte de la force musculaire dans la patte l?s?e (compar?e au muscle contralat?ral). Ces r?sultats l?gitiment et pr?parent les futures strat?gies th?rapeutiques "anti-fibrosantes" dans la DMD
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