La pathogénicité de nombreuses maladies génétiques humaines peut être liée à la reconnaissance de la protéine mutante mal repliée par le système de contrôle de la qualité des cellules (CQ), conduisant à leur dégradation. Néanmoins, un certain nombre de ces protéines mutées ont conservé une activité biologique, suggérant que le sauvetage de la dégradation peut réduire la pathologie et ouvrir la porte à des stratégies thérapeutiques pour traiter ces maladies.Les dystrophies musculaires des ceintures (LGMD) sont des maladies musculaires caractérisés par une atrophie progressive de la ceinture pelvienne et scapulaire. Des études menées par nous et par d'autres groupes ont montré que le mécanisme pathologique de certaines LGMD (sarcoglycanopathies et dystroglycanopathies) est associé à une dégradation prématurée des protéines mal repliées par le CQ. Les sarcoglycanopathies (LGMD2C-F) sont causées par des mutations dans l'un des 4 sarcoglycanes (SG). Ces protéines transmembranaires font partie du complexe dystrophine-glycoprotéine (DGC), qui lie le cytosquelette à la matrice extracellulaire (ECM) et est crucial pour la résistance mécanique des fibres musculaires. Les dystroglycanopathies sont un groupe de maladies associées à l'hypoglycosylation de l'alpha-dystroglycane (a-DG). Au DGC, a-DG est en contact direct avec l'ECM par une glycosylation complexe générée par l'action de plusieurs enzymes. Notre laboratoire se concentre sur l'une de ces enzymes, la protéine liée à la fukutine (FKRP), dont les mutations mènent à LGMD2I.Dans le but d'identifier des molécules candidates capables de sauver les protéines mutantes sarcoglycanes et FKRP, un criblage à haute-débit de composés pharmacologiques validés a été envisagé. Pour ce type de criblage, des modèles cellulaires in vitro appropriés ont été générés. Grâce à une approche candidate et à une criblage, nous avons identifié plusieurs molécules capables de sauver et de localiser correctement les mutants alpha-SG à la membrane cellulaire. Dans le but de tester l'efficacité et la sécurité des molécules in vivo, nous avons généré un nouveau modèle de souris portant une mutation T151R b-SG, car le modèle précédent portant une mutation correspondant à la mutation humaine R77C ne présentait pas de pathologie. Le nouveau modèle ne présentait pas non plus de phénotype dystrophique, la protéine bêta-SG mutée étant correctement présente à la membrane de la fibre musculaire, ce qui indique que le système de CQ est différent entre les deux espèces.En ce qui concerne FKRP, nous avons caractérisé certaines protéines mutantes in vitro et identifié deux différentes classes de mutants: des mutants retenus dans l'ER mais néanmoins capables de trafiquer vers le Golgi où ils ont montré une fonctionnalité. D'autres mutations FKRP correctement adressées au Golgi ont cependant perdu leur fonctionnalité. Les patients affectés par ces mutations ne peuvent pas bénéficier de stratégies pharmacologiques ciblant le CQ et pourraient être des candidats pour des approches de thérapie génique. En utilisant les mutants FKRP qui pourraient être sauvés de la dégradation et être fonctionnels, nous avons généré de nouveaux modèles cellulaires pour les criblages à haut-débit qui sont actuellement en cours de validation.Globalement, ce projet a permis la génération de modèles in vitro pertinents pour le test de médicaments pour le sauvetage de protéines mutantes faux-sens menant à deux maladies musculaires pour lesquelles aucun traitement curatif actuel n'est disponible. / The pathogenicity of many human genetic diseases can be related to the recognition of the mutant misfolded protein by the cell quality control (QC) system, leading to their degradation. Interestingly, a number of these mutated proteins have nevertheless retained biological activity, suggesting that rescue from degradation can reduce the pathology and open the door for therapeutic strategies to treat these diseases.Limb-girdle muscular dystrophies (LGMDs) are muscular disorders characterized by progressive wasting of the pelvic and shoulder muscles. Previously studies by us and other groups showed that the pathological mechanism of some LGMDs (sarcoglycanopathies and dystroglycanopathies) is associated with premature degradation of misfolded proteins through QC. Sarcoglycanopathies (LGMD2C-F) are caused by mutations in any of the 4 sarcoglycans (SGs). These transmembrane proteins are part of the dystrophin-glycoprotein complex (DGC), which connects the cytoskeleton to the extracellular matrix (ECM) and is crucial for the mechanical resistance of the muscle fibers. Dystroglycanopathies are a group of diseases associated with hypoglycosylation of alpha-dystroglycan (a-DG). At the DGC, a-DG is in direct contact with the ECM through a complex glycosylation generated by the action of several enzymes. Our lab focuses on one of these enzymes, Fukutin-related protein (FKRP), whose mutations lead to LGMD2I.With the aim of identifying candidate molecules able to rescue the sarcoglycan and FKRP mutant proteins, a high-content screen of approved and validated pharmacological compounds was envisaged. For this type of screen, appropriate in vitro cellular models were generated. Through a candidate approach and high-content screen, we identified several molecules able to rescue and correctly localize alpha-SG mutants to the cell membrane. For the purpose of testing the efficacy and safety of the molecules in vivo, we generated a new mouse model carrying a T151R b-SG mutation, as the previous model carrying a mutation corresponding to the human R77C mutation failed to reproduce the pathology. The new model also did not present a dystrophic phenotype, with the mutated beta-SG protein correctly present at the muscle fiber membrane, indicating that the QC system is different between the two species.As for FKRP, we characterized a number of mutant proteins in vitro showing two different classes of mutants: some mutants were retained in the ER but could nonetheless overcome this retention and be allowed to traffic to the Golgi where they showed functionality. Other FKRP mutations are correctly addressed to the Golgi but are functionally impaired. Patients affected by these mutations may not benefit from QC-targeting pharmacological strategies and could be candidates for gene therapy approaches. Utilizing the FKRP mutants that could be rescued of degradation and be functional, we have generated new cellular models for high-content screens which are currently being validated.Altogether, this project allowed the generation of relevant in vitro models for identification of drugs allowing the rescue of missense mutant proteins leading to two muscular diseases for which no current curative treatment is available.

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

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.

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.