Rozprawy doktorskie na temat „LGMDR8”

Ageing is a natural process, which is characterised by progressive decline in physiological functions and increased susceptibility to disease and death. Brain is particularly susceptible to structural and functional changes, which is more evident in disorders associated with ageing such as Alzheimer disease (AD). Copper is necessary for the protection against oxidative stress, energy production and neurotransmitter processing in the brain. However, higher copper levels can increase oxidative stress, resulting in neuronal damage. In order to avoid copper induced cytotoxicity, cells have to regulate copper levels through distribution into three intracellular pathways. By identifying changes in the copper pathways in the healthy and AD brain and by estimating the effects of copper chelation or supplementation in model cell line a better understanding of copper function in the brain will be obtained. In order to accomplish that copper, activity and protein levels of cytochrome c oxidase (COX) and superoxide dismutase (SOD) were measured in the healthy, AD brain and in HEK293 cell treated with copper chelators or supplemented with copper. Copper concentration was significantly decrease by more than 40% in healthy ageing brain and in the AD brain. Copper loss did not seem to affect the activity or protein level of the COX and SOD, since their levels were significantly increased in the ageing and AD brain. On the other hand, cells treated with copper chelators for three days faced a more than 75% decrease in intracellular copper concentration, which led to a more than 85% inhibition of the COX and SOD activity. Copper levels should be regulated properly in order to meet body’s metabolic demands and avoid cytotoxicity. Brain seems to have a mechanism where its energy demands have to be fulfilled even under low copper concentrations. Whereas, the prolonged and severe copper loss can dramatically affect the energy production and antioxidant defence systems which could be fatal to the cells.

Les dystrophies musculaires des ceintures (lgmd ou limb girdle muscular dystrophy) sont un groupe de maladies genetiques cliniquement heterogenes et caracterisees par une atrophie et une faiblesse progressive des muscles des ceintures. La premiere localisation d'une forme recessive avait ete determinee en 15q15. 1-q21. 1 grace a l'etude d'un groupe de familles reunionnaises. Cette these relate l'identification du gene correspondant, realisee par clonage positionnel. Apres construction de cartes physiques, restriction de l'intervalle candidat par analyse genetique et recherche de genes presents dans la region, l'identification de mutations chez des patients lgmd2a a permis de demontrer l'implication du gene codant pour une protease intracellulaire, la calpaine 3 specifique du muscle squelettique comme etant le site genetique responsable de cette forme de dystrophie des ceintures. L'analyse systematique de ce gene a conduit a l'identification de 50 mutations differentes dans des familles de toutes origines, demontrant la grande variete des defauts moleculaires. Afin de pouvoir entreprendre des etudes chez l'animal, nous avons ensuite caracterise la sequence de l'adnc de souris et d'une partie de l'organisation genomique du gene correspondant. Ces informations ont servi a la construction d'un modele murin de la maladie par recombinaison homologue. Ces travaux ont demontre, par l'implication de canp3 dans lgmd2a, l'existence d'un nouveau mecanisme conduisant a un processus dystrophique par opposition a des defauts dans des proteines structurales de la membrane musculaire presents dans les autres myopathies

Master of Science
Biochemistry and Molecular Biophysics Interdepartmental Program
Erika Rae Geisbrecht
The E3 ubiquitin ligase TRIM32 is a member of tripartite motif (TRIM) family of proteins involved in various processes including differentiation, cell growth, muscle regeneration and cancer. TRIM32 is conserved between vertebrates (humans, mouse) and invertebrates (Drosophila). The N-terminus of this protein is characterized by a RING domain, B-box domain, and Coiled-Coil region, while the C-terminus contains six NHL repeats. In humans, mutations that cluster in the NHL domains of TRIM32 result in the muscle disorders Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM). Mutations in the B-box region cause Bardet-Biedl Syndrome (BBS), a clinically separate disorder that affects multiple parts of the body. A comprehensive genetic analysis in vertebrate models is complicated by the ubiquitous expression of TRIM32 and neurogenic defects in TRIM32-/- mutant mice that are independent of the muscle pathology associated with LGMD2H. The model organism Drosophila melanogaster possesses a TRIM32 [dTRIM32/Thin (Tn)/Abba] homolog highly expressed in muscle tissue. We previously showed that dTRIM32 is localized to Z-disk of the sarcomere and is required for myofibril stability. Muscles form correctly in Drosophila tn mutants, but exhibit a degenerative muscle phenotype once contraction ensues. Mutant or RNAi knockdown larvae are also defective in locomotion, which mimics clinical features associated with loss of TRIM32 in LGMD2H patients. It is predicted that mutations in the NHL domain either affect protein structure or are involved in protein-protein interactions. However, the molecular mechanism by which these mutations affect the interaction properties of dTRIM32 is not understood. Biochemical pulldown assays using the bait fusion protein GST-dTRIM32-NHL identified numerous dTRIM32 binding proteins in larval muscle tissue. Many key glycolytic enzymes were present in the dTRIM32 pulldowns and not in control experiments. Glycolytic genes are expressed in the developing Drosophila musculature and are required for myoblast fusion. Strikingly, many glycolytic proteins are also found at the Z-disk, consistent with dTRIM32 localization. Our biochemical and genetic studies provide evidence that there is direct interaction between dTRIM32 and glycolytic proteins (Aldolase and PGLYM). dTRIM32 also regulates glycolytic enzyme levels and protein localization at their sites of action. These data together suggest a role for dTRIM32 in coordinating glycolytic enzyme function, possibly for localized ATP production or to maintain muscle mass via glycolytic intermediates.

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity, myogenic potential, M2 macrophage polarization and decreases NF-κB inflammation in dysferlin-deficient models. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, and myoblast migration in dysferlin-deficient myotubes. Furthermore, rHsGal-1 showed an increased membrane repair capacity of dysferlin-deficient myotubes. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggests mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-l significantly reduces canonical NF-κB inflammation through TAK 1, P65, P50. Lastly we find 2.7 mg/kg in vivo rHsGal-1 treatment in BLA/J mice supports an M2 cyto-regenerative macrophage populations. Together our novel results reveal Gal-1 remediates disease pathologies in LGMD2B through changes in integral myogenic protein expression, mechanical membrane stabilization, immune modulation, and reducing canonical NF-κB inflammation.

La dystrophie musculaire des ceintures est une myopathie hereditaire transmise sur un mode autosomal et presentant une heterogeneite clinique. Il existe deux formes: dominante et recessive. Un gene responsable de la forme recessive, lgmd2, a ete localise sur le bras long du chromosome 15 en 1991. Le travail presente ici concerne la cartographie de la region du chromosome 15 contenant lgmd2. Compte tenu de l'absence de donnees cytogenetiques, biochimiques ou autres pour cette myopathie, l'approche gene candidat n'a pu etre appliquee et l'identification du gene a ete envisagee selon la demarche de clonage positionnel qui fait appel a des techniques de cartographies genetique et physique. La cartographie genetique a permis de demontrer l'heterogeneite non allelique de ce phenotype clinique. Par ailleurs, l'intervalle contenant lgmd2 a ete precise et evalue a 7 cm. Parallelement, en vue du clonage du gene, la construction de la carte physique de la region a ete entreprise a partir des marqueurs l'encadrant. Cette region etant pauvre en stss, trois sortes de stss ont ete developpes pour cribler la banque de yacs du ceph: (i) ceux derives de genes deja sequences, (ii) des marqueurs microsatellites localises apres genotypage sur les familles de malades a proximite de la region lgmd2, (iii) des sequences des extremites de yacs ou des segments de la pcr inter-alu. Pres de 150 yacs ont ete identifies et une partie d'entre eux caracterises pour leur taille et leur chimerisme. Differentes approches ont ete pratiquees jusqu'a l'etablissement d'un continuum ininterrompu de yacs, de 15q15. 1 a 15q21. 1, soit 10-12 mb. La construction de la carte physique de la region a permis le developpement cible de nouveaux marqueurs. Cette strategie sera poursuivie jusqu'a restriction suffisante de l'intervalle par des approches genetiques avant de debuter une recherche systematique des transcrits

Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L’analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n’apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L’anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L’anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d’un phénotype normal. La compensation de DYSF par ANO5 n’est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l’ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d’un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L’efficacité du SMaRT dans un contexte endogène s’est en revanche révélée faible, et n’a pas permis la restauration d’une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l’observation de l’expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l’utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies
Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin’s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies

Les dystrophies musculaires des ceintures representent un groupe heterogene de myopathies hereditaires transmises selon un mode autosomique dominant (lgmd1) ou recessif (lgmd2). L'analyse de la forme recessive lgmd2a a conduit a la demonstration d'une heterogeneite genetique au sein de la communaute amish de l'etat d'indiana aux etats-unis, suggerant l'existence d'un nouveau locus, appele lgmd2e, implique dans l'etiologie des formes recessives de dystrophie des ceintures. Une demarche de clonage positionnel a ete adoptee en vue de l'identification du gene, conduisant a la localisation primaire du locus morbide dans la region pericentromerique du chromosome 4. L'analyse genetique a permis la definition d'un intervalle contenant le locus lgmd2e, estime a 3 cm, sur lequel a ete entreprise la construction d'une carte physique sous forme d'un continuum de clones de yacs. Parallelement a cette etude, le gene de la -sarcoglycane, une glycoproteine de 43 kda du complexe associe a la dystrophine, a ete clone et caracterise. Ce gene, localise en 4q12, representait un excellent candidat pour cette myopathie. Des travaux communs ont permis d'identifier une mutation segregeant a l'etat homozygote dans le gene de la -sarcoglycane chez tous les patients amish etudies et de reveler une reduction concomitante de la proteine au niveau du sarcolemme, confirmant son implication dans la dystrophie des ceintures de forme lgmd2e. La determination de la sequence complete du gene de la -sarcoglycane a ensuite ete effectuee par sequencage aleatoire d'un cosmide. L'assemblage des sequences, effectue par le programme xbap du package staden, a conduit a l'etablissement d'un continuum unique de 36159 pb. L'organisation genomique du gene de la -sarcoglycane a ete deduite de la comparaison de cette sequence consensus avec les sequences d'adnc publiees. Six exons ont ete mis en evidence, representant une sequence codante de 954 paires de bases.

Les dystrophies musculaires des ceintures sont un groupe heterogene de myopathies autosomiques presentant une definition nosologique floue, un diagnostic incertain, une heterogeneite clinique et genetique. Afin d'identifier un locus responsable d'une forme recessive, lgmd2a, une strategie de clonage positionnel a ete appliquee. Consecutivement a l'assignation du locus lgmd2a dans la region 15q15. 1 - q21. 1, la construction d'un continuum ininterrompu de clones de yacs chevauchants couvrant cet intervalle de 10 a 12 mb a ete entreprise, en parallele avec l'etablissement d'une cartographie genetique fine de cette region de 7 cm. Les clones de yacs de la carte physique ont ete utilises pour generer de facon ciblee de nouveaux marqueurs polymorphes de type microsatellite. L'analyse genetique de lgmd2a a aboutit a deux resultats inattendus: (1) la demonstration d'une heterogeneite genetique au sein d'une communaute amish de l'etat d'indiana, impliquant l'existence d'un autre locus responsable d'une forme recessive de dystrophie musculaire des ceintures ; (2) l'observation d'une heterogeneite allelique dans des familles originaires de l'ile de la reunion, en contradiction avec l'hypothese d'un effet fondateur unique sur laquelle l'etude genetique etait basee. L'identification de nouveaux evenements de recombinaison dans les familles de malades a permis de definir une nouvelle region de 1 cm contenant le locus de la maladie, correspondant approximativement a 3 - 4 mb. Par la suite, des etudes de desequilibre de liaison entre les marqueurs polymorphes de la region et le locus de la maladie ont permis de definir une region de 1. 6 mb consideree comme localisation preferentielle du gene lgmd2a

Limb Girdle Muscular Dystrophy R3 (LGMDR3), previously known as LGMD2D, is a rare autosomal recessive primary myopathy, clinically characterized by progressive involvement of the pelvic and shoulder girdles, and genetically by mutations in the αsarcoglycan gene (SGCA) coding for α-sarcoglycan (SG). The clinical course of LGMDR3 presents a great variability, ranging from severe form with onset in the first decade of life and rapid progression, to milder form with later onset. Currently, physical therapy and prevention of secondary cardiac, pulmonary or orthopedic complications are the only possible care interventions and no disease-specific therapies are yet available. As other muscular dystrophies, LGMDR3 muscle histology is characterized by myofibernecrosis and regeneration, reactive fibrosis, and inflammatory infiltrates. However, to date, no data exploring the role of immune response on disease progression have been published. The primary objective of this PhD thesis was therefore to deepen the contribution of inflammatory processes to the severity of α-sarcoglycanopathy. AIM1. -to correlate the inflammatory infiltrates extension with the age at onset, the clinical severity, and the muscle involvement at MRI, in a cohort of patients followed at Istituto Giannina Gaslini, Genova In a cohort of 8 LGMDR3 patients, we characterized the clinical course, the MRI muscle pattern, and the histological parameters, focusing on inflammatory features. AIM2.-to immunophenotype the composition of muscular immune infiltrates in different sarcoglycanopathies and verify whether differences would be detectable between distinct subtypes of sarcoglycan-related LGMDs In a multicenter study involving Italian tertiary centers for neuromuscular disorders, we analysed muscle expression of inflammatory markers in two forms of sacoglycanopathies LGMDR3 and LGMDR5, the latter due to deficiency of γ-sarcoglycan. AIM3.-to study the in vivo effect of anti-purinergic molecules on disease progression in the Sgca null mouse In two pre-clinical studies, we explored the role of the P2X7 receptor, a ionotrophic receptor involved in the inflammasome pathway which mediates the release of pro-inflammatory cytokines, in a mouse model lacking the α-sarcoglycan gene (Sgca-null).

Les dysferlinopathies sont des dystrophies musculaires qui se manifestent par la dystrophie musculaire des ceintures de type 2B (LGMD2B) ou la myopathie de Miyoshi (MM). Elles sont causées par des mutations dans le gène dysferline. La dysferline est une protéine membranaire exprimée dans le muscle squelettique, responsable de la réparation des microlésions du sarcolemme. L’absence d’une telle réparation de la membrane entraîne une atrophie musculaire progressive. Ce travail de thèse explore le potentiel thérapeutique d'une stratégie de modulation d'épissage pour le traitement de la LGMD2B causée par la mutation faux-sens c4022T>C dans l'exon 38 du gène dysferline. Des oligonucléotides et des petits ARN U7 délivrés par un vecteur viral de type adéno-associé ont été utilisés comme outils antisens pour induire un saut d'exon in vitro et in vivo. Ce projet de thèse étudie également la capacité de la dysferline tronquée à se localiser de façon appropriée à la membrane et ainsi la réparer
Dysferlinopathy is a muscular dystrophy that manifests as two major phenotypes: limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy (MM). It is caused by mutations in the dysferlin gene. Dysferlin is a membrane protein expressed in skeletal muscle. It is responsible for the repair of sarcolemma microlesions produced by muscle contractions. A compromised membrane repair leads to slowly progressing muscle wasting. This thesis explores the therapeutic potential of an antisense mediated splice switching strategy in LGMD2B caused by the missense mutation c4022T>C in the exon 38 of the dysferlin gene. Antisense oligonucleotides and U7 snRNAs delivered by an adeno-associated viral vector were used as antisense tools to trigger exon skipping in vitro and in vivo. The thesis investigates also if the truncated dysferlin maintainsa proper membrane localization and its membrane repair ability

Sarcoglycanopathy is the collective name of four rare autosomal recessive diseases belonging to the limb-girdle muscular dystrophies type 2 (LGMD2C-F), due to mutations in SGCG, SGCA, SGCB, SGCD genes coding for γ-, α-, β- and δ- sarcoglycan (SG) respectively. SGs form a tetrameric sub-complex on the sarcolemma, closely linked to the dystrophin associated protein complex (DAPC) and play an essential role in assuring membrane integrity during muscle contraction (Bushby, 2009). Mutations in each SG gene cause the loss/reduction of the mutated protein as well as of the wild type (WT) partners with an alteration of the DAPC structural properties and an increased fragility of the sarcolemma. Most of the known disease-causing mutations are missense mutations that results in a folding defective SG, recognized and prematurely degraded by the endoplasmic reticulum associated degradation (ERAD) through the proteasome (Gastaldello et al., 2008). The knowledge of the pathological mechanism opens new avenues to two small molecules-based pharmacological interventions. By inhibiting the mutant degradation or assisting its folding the whole SG complex is recovered on the membrane, suggesting that many mutants are still functional although structurally defective (Bianchini et al., 2014; Carotti et al., 2018). Now for the subsequent preclinical test, these in vitro data need to be validated in vivo. To accomplish this task, in the absence of suitable rodent models, zebrafish models carrying missense mutations in SGs are needed to mimic the human condition. Among SGs, β- and δ-SG are the most conserved, therefore they were chosen to mimic sarcoglycanopathy in zebrafish. The nearly identical muscle structure with the human one, the ease to set up functional and drug screening test, make this vertebrate an ideal choice for our task. The proof of concept of the suitability of the model came from the knock-down of δ-SG by using the morpholino technique. Morphants showed altered morphology and compromised swimming ability. On these promising data, we decided to generate novel zebrafish lines in which β- or δ-SG genes are modified by using the CRISPR/Cas9 technique. β- and δ-SG Knock-out (KO) animals have been generated and characterized. The absence of the protein led to the progressive alteration of the muscle structure and impairment of the swimming ability. Presently, KO animals are used as the background for the injection of the WT or mutated sequence of the corresponding SG, of human or zebrafish origin. The injection of the WT sequence should allow the rescue of the phenotype, whereas that of the mutated forms will permit to evaluate the ability of the zebrafish ERAD to recognize and degrade a folding defective SG. This data will be of utmost importance to verify the suitability of zebrafish in mimicking those forms of sarcoglycanopathy due to missense mutations. β-SGT145R/T145R and δ-SGE264K/E264K Knock-in (KI) are presently under production. Since the efficacy of the homologoy direct repair is very low, much effort has been devoted to set up a selective procedure to screen the positive fish. Actually, the screening of the somatic recombinants for the introduction of the T145R mutation in z-sgcb gene was positive. We are presently waiting for the sexual maturation of the F0 population to perform the outcross with WT in order to identify the founder animals, in which the recombination occurred in the germline. If successfully modelling the sarcoglycanopathy conditions, these KI lines will represent a fundamental tool for testing the proposed pharmacological approach and will be a valuable boost for both basic and translational research.
Le sarcoglicanopatie sono quattro rare patologie autosomiche recessive appartenenti alla famiglia delle distrofie muscolari di tipo 2 (LGMD2C-F). Esse sono causate da mutazioni nei geni SGCG, SGCA, SGCB, SGCD codificanti rispettivamente per γ-, α-, β- e δ- sarcoglicano (SG). I SG formano un complesso tetramerico nel sarcolemma, connesso a quello delle glicoproteine associate alla distrofina (DAPC), essenziale per garantire l'integrità della membrana durante la contrazione muscolare (Bushby, 2009). Mutazioni dei SG causano la perdita/riduzione della proteina mutata e dei partner wild type (WT), alterando le proprietà strutturali del DAPC e aumentando la fragilità del sarcolemma. La maggior parte delle mutazioni sono di tipo missenso e, causando un problema nel folding, portano alla prematura degradazione della proteina mutata effettuata dalla via ERAD (degradazione associata al reticolo endoplasmatico) attraverso il proteasoma (Gastaldello et al., 2008; Bartoli et al., 2008). La conoscenza del meccanismo patologico ha aperto nuove strade a due possibili interventi farmacologici: inibendo la degradazione o assistendo il processo di folding dei mutanti, il complesso dei SG è recuperato riducendo così la fragilità della membrana (Bianchini et al., 2014; Carotti et al., 2018). Questi dati prodotti in vitro suggeriscono che molti mutanti sono ancora funzionali, sebbene strutturalmente difettosi. Tuttavia, per la messa a punto di un approccio terapeutico, questi dati necessitano di una validazione in vivo, attraverso modelli animali per mimare la malattia umana e recanti quindi mutazioni missenso nei SG. In assenza di modelli murini, un’alternativa promettente per lo studio di molte malattie, fra cui i disturbi muscolari, è rappresentata dallo zebrafish (D.rerio). Tra gli ortologhi dei SG umani, β- e δ-SG sono i più conservati e quindi sono stati scelti per produrre linee knock-out (KO) e knock-in (KI). La bontà dell’uso di questo vertebrato nel mimare la sarcoglicanopatia nasce da uno studio preliminare di Knock-Down con morfolino contro il δ-SG. I morfanti ottenuti hanno mostrato: alterazioni morfologiche, danni alla struttura muscolare e compromissione delle capacità motorie. Grazie a queste premesse, il sistema CRISPR/Cas9 è stato usato per generare modelli KO di β- e δ-SG. In entrambi i casi, l'assenza della proteina provoca la progressiva alterazione della struttura muscolare e delle capacità motorie. Attualmente stiamo usando i KO come background per l'iniezione della sequenza WT o mutata dei SG, sia di origine umana sia di zebrafish. La prima dovrebbe consentire il recupero del fenotipo, mentre quella delle forme mutate permetterà di valutare la capacità dello zebrafish di riconoscerle e degradarle. Questi esperimenti sono della massima importanza per verificare l'idoneità dello zebrafish nel mimare le forme di sarcoglicanopatia causate da mutazioni missenso. La generazione di due modelli KI in zebrafish, β-SGT145R/T145R e δ-SGE264K/E264K, prevede l’attivazione della via di riparazione HDR (riparazione omologa diretta). Poiché l'efficienza della ricombinazione omologa è molto bassa, è necessario l’uso di un metodo altamente selettivo per identificare i pesci positivi alla voluta mutazione. Lo screening delle mutazioni somatiche per l'introduzione della mutazione T145R nel gene z-sgcb è stato positivo. Attualmente stiamo aspettando la maturazione sessuale della popolazione F0 per identificare quei pesci in cui ricombinazione sia avvenuta nella linea germinale. Se le linee KI, una volta caratterizzate, mimeranno la patologia umana, rappresenteranno uno strumento fondamentale per testare l'approccio farmacologico proposto in vitro, e costituiranno un valido stimolo per la ricerca di base e traslazionale.

Aim: The overall aim of this thesis, using a salutogenic framework, was to develop knowledge about experiences and perceptions of living with recessive limb-girdle muscular dystrophy and its influences on health, from the affected young adults’ and their parents’ perspectives. Methods: A qualitative explorative and descriptive study design was used. Semi-structured interviews were held with 14 young adults diagnosed with recessive limb-girdle muscular dystrophy, aged 20–0 years, and 19 parents. Data analyses were conducted using content analysis (I, II, III) and phenomenography (IV). In order to mirror the interview data, the participants also answered the 13-item sense of coherence questionnaire. Findings: Recessive limb-girdle muscular dystrophy has a major impact on the affected young adults’ and their parents’ lives as the disease progresses. Health described in terms of well-being was thus perceived to be influenced, not only by physical, emotional and social consequences due to the disease and worry about disease progression but also by external factors, such as accessibility to support provided by society and other people’s attitudes. There was, however, a determination among the participants to try to make the best of the situation. The importance of being able to mobilize internal resources, having social support, meaningful daily activities, adapted environment, the young adult being seen as a person and having support from concerned professionals, including personal assistance when needed, was thereby described. Self-rated sense of coherence scores varied. Those who scored above or the same as median among the young adults (≥56) and the parents (≥68) expressed greater extent satisfaction regarding social relations, daily activities and external support than those who scored less than median. Conclusion: This thesis highlights the importance of early identification of personal perceptions and needs to enable timely health-promoting interventions. Through dialogue, not only support needed for the person to comprehend, manage and find meaning in everyday life can be identified, but also internal and external resources available to enhance health and well-being, taking into account the person’s social context as well as medical aspects.

The myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. In particular, an expansion of CTG triplet in the 3'UTR of the DMPK gene (myotonic dystrophy protein kinase) is responsible for myotonic dystrophy type 1 (DM1; MIM # 160900). DM1 is an autosomal dominant multisystemic desease, involving various systems of the human body such as the endocrine system, heart, skeletal muscle, eye and brain. It might present a congenital form, as well as the involvement of the central nervous system. MicroRNAs (miRNA) are short non coding RNA molecules with single stranded sequence, that can regulate gene expression at the post transcriptional level. They are involved in many biological processes such as embryonic development, differentiation and proliferation. There are some miRNAs expressed in the muscular tissue (myomiRs), in particular miR-1 and miR-133a/b, expressed in skeletal and cardiac muscle, and miR-206, expressed in skeletal muscle. The main objective is to study the expression profile of these miRNAs in skeletal muscle and serum of patients with myotonic dystrophy type 1. The results of the study in 12 biopsies shows a decrease of miR-1 and miR-133a in patients with DM1 compared with controls, while miR-133b doesn’t show significant changes, and miR-206 is significantly increased in the muscle of DM1 patients. These data were compared with the histopathological score of muscle biopsies of these patients. There was no correlation between alterations of miRNA expression and histopatological impairment of muscle. We analyzed the levels of miRNAs in the serum of patients DM1 before and after a period of 8 weeks of rehabilitation, to verify the role of these molecules and their involvement in muscle regeneration. At time zero (admission in hospital) there was a decrease in the expression of miR-1, miR-133b and miR-206 compared to controls, instead miR-133a showed increased level. After rehabilitation treatment (time one) there was a significant decrease of all four miRNAs analyzed
La Distrofia Miotonica (DM) è la più comune forma di distrofia muscolare nell’adulto. In particolare un’espansione della tripletta CTG nella regione 3’UTR del gene DMPK (myotonic dystrophy protein kinasi) è causativa della distrofia miotonica di tipo 1 (DM1; MIM# 160900), una malattia a trasmissione autosomica dominante, multisistemica che coinvolge diversi apparati del corpo umano come il sistema endocrino, il cuore, i muscoli scheletrici, oculari, il cervello. La DM1 presenta forme congenite e il coinvolgimento del sistema nervoso centrale. I microRNA sono corte molecole di RNA a singolo filamento non codificanti, in grado di regolare l’espressione genica a livello post trascrizionale. Sono coinvolti in molti processi biologici, come lo sviluppo embrionale, il differenziamento e la proliferazione cellulare. Esistono dei microRNA espressi a livello muscolare (myomiRs), in particolare il miR-1 e il miR133a/b, espressi nel muscolo scheletrico e cardiaco, e il miR-206, espresso nel muscolo scheletrico. L’obiettivo principale è di studiare il profilo di espressione di questi microRNA nel muscolo scheletrico e nel siero di pazienti affetti da distrofia miotonica di tipo 1. I risultati dello studio nel muscolo hanno mostrato una diminuzione di miR-1 e miR-133a nei pazienti DM1 rispetto ai controlli, miR-133b non mostra significative variazioni, mentre miR-206 aumenta significativamente nel muscolo di pazienti DM1. Questi dati sono stati confrontati con la valutazione semiquantitativa del grado di compromissione istopatologico delle biopsie muscolari, senza però trovare una correlazione tra alterazione dell’espressione dei microRNA e compromissione del muscolo. Si sono indagati i livelli dei microRNA nel siero di pazienti DM1 prima e dopo un periodo di riabilitazione, allo scopo di comprendere il ruolo di queste molecole a livello muscolare e in particolare il loro coinvolgimento in processi di ipertrofia e rigenerazione muscolare. Le analisi svolte sul siero al tempo zero (ingresso all’ospedale) evidenziano una diminuzione nell’espressione di miR-1, miR-133b e miR-206 rispetto ai controlli, al contrario miR-133a mostra un livello di espressione più elevato. Dopo trattamento riabilitativo (tempo 1) c’è un abbassamento significativo di tutti e quattro i microRNA analizzati

碩士
高雄醫學大學
醫學研究所碩士班
95
Dysferlin（DYSF） gene encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B（LGMD 2B）, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF gene. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 11 unrelated LGMD 2B patients, including 7 suspected LGMD 2B Taiwanese patients, and 4 Japanese patients with 6 confirmed mutations in DYSF gene. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using denaturing high performance liquid chromatography（DHPLC）of PCR products of each of 55 exons of the gene was followed by sequencing whenever a variation was detected. In 7 Taiwanese patients, 17 sequence variations were identified in DYSF, 3 of which predict single amino-acid substitution and are novel, while the other 14 changes are known SNPs (Single nucleotide polymorphisms). The three novel sequence variations, G486、A3472G、C334T, was not detected in 150 normal individuals and was identified as potential pathogenic mutation (SNP<1%). These three mutations were widely spread in the coding sequence of the gene.

Limb girdle muscular dystrophies (LGMDs) are a clinically and genetically heterogeneous group of myopathies characterized by weakness and wasting of the proximal musculature. There are currently seventeen loci associated with different LGMDs, seven with an autosomal dominant mode of inheritance (LGMD1A–1G) and 10 with an autosomal recessive mode of inheritance (LGMD2A– 2J). The cumulative worldwide prevalence of LGMD is thought to be ~1/15,000. In the Hutterite population of North America there is an over-representation of autosomal recessive LGMD with a prevalence estimated to be >1/400. The objective of this work was to delineate the genetic basis of LGMD in this large genetically isolated population. A genome-wide scan was performed on Hutterite LGMD patients and their families in order to locate the mutant gene. This allowed us to identify a novel locus at chromosome region 9q31-33 that was named LGMD2H. Extensive haplotyping and mutation screening led to the discovery of c.1459G>A in TRIM32 as the causative mutation of LGMD2H. We then found that this same mutation was the cause of another previously described myopathy in the Hutterites, sarcotubular myopathy (STM)[reference awaiting publishers decision]. Analysis of the TRIM32 gene product revealed that it is a potential E3-ubiquitin ligase, is expressed in many human tissues including muscle and brain, and has a punctate cytoplasmic distribution. During the analysis of the LGMD2H region, it became apparent that there were Hutterite LGMD patients not linked to the LGMD2H locus. In order to identify the causative gene(s) in the remaining families, we performed a genome-wide scan. A locus at chromosome 19q13 was found to correspond to disease inheritance, the site of a previously described LGMD locus, LGMD2I. No causative gene had yet been identified at this locus so haplotyping and mutation screening was performed. We were able to identify c.826C>A in FKRP as the causative mutation in our remaining cohort of LGMD patients. The same mutation has since been found in many other populations, and is apparently a relatively common cause of LGMD. We obtained DNA from 19 non-Hutterite LGMD2I patients of diverse origins with c.826C>A and determined that it is an old founder mutation. There is no further evidence of any other loci causing autosomal recessive myopathy in the Hutterites. With the identification of c.1459G>A in TRIM32 and c.826C>A in FKRP we appear to have delineated the genetic cause of all myopathies of increased prevalence in the Hutterite population. To date, we have been able to provide accurate, non-invasive, diagnosis to over 70 patients and have provided carrier testing to approximately 120 at-risk family members. This kind DNA-based approach is not feasible in the general population due the enormous amount of locus, allelic, and clinical heterogeneity among myopathy patients.
May 2005

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: As disferlinopatias são doenças musculares de transmissão autossómica recessiva provocadas por mutações no gene da disferlina (DYSF). Os fenótipos mais comuns são a Miopatia de Myoshi (MM) e a Distrofia Muscular das Cinturas 2B (DMC 2B).Objectivos: Descrever as características clínicas, laboratoriais, moleculares e a evolução clínica de quinze doentes com disferlinopatia.Metodologia: Registaram-se dados demográficos, clínicos, laboratoriais e moleculares de quinze doentes com diagnóstico de disferlinopatia. Foi avaliada a marcha, existência de sintomatologia cardiorrespiratória e resultados de electrocardiograma, provas de função respiratória e radiografia torácica.Resultados: Quinze doentes (oito do género masculino), com idade média actual de 47+/-16 anos. A idade média dos primeiros sintomas foi 24+/-14 anos e o tempo médio até ao diagnóstico molecular de 12+/-12 anos. Na observação inicial, o fenótipo DMC 2B observou-se em oito doentes, o de MM em três, hipercreatinémia (hiperCK) isolada em dois, proximodistal num doente e Miopatia Distal do Compartimento Anterior (MDCA) noutro doente. Atualmente, oito doentes apresentam generalização da fraqueza muscular (um DMC 2B e sete proximodistais) e sete doentes mantêm o fenótipo inicial (quatro DMC 2B, um hiperCK, um MDCA, um proximodistal). No momento do diagnóstico, todos os doentes tinham marcha autónoma, sendo que nove perderam capacidade de marcha e três fazem atualmente marcha com apoio (tempo médio até perda de marcha autónoma de 17+/-9 anos). Sete doentes referiram dispneia e/ou ortopneia. 18% das radiografias torácicas apresentaram aumento do índice cardiotorácico (ICT) e foram encontradas anomalias em 50% das provas de função respiratória (PFR) e 54% dos electrocardiogramas (ECG). Valores séricos de creatina quinase (CK) estavam elevados em todos os doentes. Observaram-se treze mutações diferentes no gene DYSF, dez em homozigotia e cinco em heterozigotiaConclusões: O reconhecimento da heterogeneidade fenotípica e molecular e da evolução clínica das disferlinopatias evidenciada nesta série poderá contribuir para o diagnóstico mais precoce e dirigido desta doença.
Introduction: Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF). Dysferlin deficiency leads to several phenotypes, with Myoshi Myopathy (MM) and Limb Girdle Muscular Dystrohy 2B (LGMD 2B) being the most common ones. Aim: Describe the clinical, laboratorial and molecular findings of fifteen patients with muscle disease caused by pathogenic mutations in the DYSF gene, as well as the progression of the disease. Methods: A total of fifteen patients with molecular confirmed dysferlinopathy were clinical and laboratory assessed. The initial and actual pattern of muscle weakness, the gait, the rate of progression and distribution of the muscle weakness, the presence of cardiorespiratory symptoms and the results of electrocardiogram, thoracic radiography and respiratory function were evaluated. Results: Fifteen patients (eight males) with a mean age at the present evaluation of 47±16 years old. The mean age of onset of the disease was 24±14 years and the mean time until the molecular confirmation was 12±12 years. At the initial evaluation, five different phenotypes were identified: eight patients with LGMD2B, three with MM, two with HiperCK, one with proximodistal phenotype (Mixed type) and one with distal anterior compartment myopathy (DACM). At the last clinical evaluation, eight of them presented generalization of the weakness (one LGMD2B and seven proximodistal phenotype) and seven maintained their initial phenotype (four LGMD2B, one hiperCK, one DACM and one proximodistal). At the first clinical examination, all patients were able to walk without support and presently nine became wheelchair-bound and three needed walking assistance (mean time to loss of autonomous walk of 17 ±9 years). Seven patients referred dyspnea and/or orthopnea. 18% of thoracic x-rays presented an enlargement of the cardiothoracic index and abnormalities were found in 50% of the respiratory function tests and in 54% of the electrocardiograms. All patients had elevated serum creatine kinase (CK) levels. Thirteen sequence variations were identified in DYSF gene. Ten patients carried a single homozygous mutation; five patients had two compound heterozygous mutations. Conclusions: Dysferlinopathies are clinical and genetic heterogeneous muscle diseases with progression in the majority of the patients. The recognition of this features evidenced in this series, may contribute to the earlier and directed diagnosis of this disease.

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.