Nova abordagem no estudo da Distrofia Muscular das Cinturas tipo 2A

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Molecular e Celular, realizada sob a orientação científica da Doutora Márcia Oliveira, Assistente Principal de Genética do Centro de Genética Médica Doutor Jacinto de Magalhães – INSA, IP e co-orientação da Professora Doutora Adelaide Almeida, Professora Auxiliar do Departamento de Biologia da Universidade de Aveiro.Muscular dystrophies comprise a group of heterogeneous genetic diseases characterized by progressive muscle weakness. Until now, eighteen genes have been associated to a specific subgroup of muscular dystrophies called limb-girdle muscular dystrophies (LGMD). Mutations in the CAPN3 gene are responsible for calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A), one of the most frequent form of LGMD. The gene CAPN3 codes for calpain 3 (or CAPN3), a muscle-specific calcium-dependent cysteine protease functionally regulated via its autolytic activity. The pathogenesis mechanism of LGMD2A still continues to be unresolved, however functional defects of CAPN3 seem to be the major cause of the pathology. With the present work, it is proposed a new approach for the LGMD2A diagnostic analysis, combining molecular genetics and protein analysis methodologies in order to contribute to a differential diagnostic essential for global characterization of patients, specifically among patients with non-specific muscular dystrophy symptoms. Muscle biopsy samples of 9 LGMD2A patients, already studied at the molecular genetics level, were subjected to protein analysis and functional tests by western blotting and densitometry analyses. Additionally, gene expression studies were performed using real-time quantitative PCR. A global analysis of the data obtained allowed us: (a) to complement the previous molecular genetics study in patients 2-7, including the identification of a CAPN3 functional defect in patient 3; (b) to confirm the clinical diagnostic in patient 8 (with only one mutation in heterozygous state) also caused by a protein functional defect; and (c) to exclude the CAPN3 implication in the other two patients which presented one mutated allele in heterozygous state, because of the detection of an overexpression of the CAPN3 gene in comparison to control samples.As distrofias musculares são um grupo heterogéneo de doenças genéticas caracterizadas por fraqueza muscular progressiva. Até agora, foram mapeados 18 genes associados a um subgrupo específico de distrofias musculares denominado distrofias musculares das cinturas (limb-girdle muscular dystrophies, LGMD). As mutações no gene CAPN3 são responsáveis pelo fenótipo de calpainopatia ou distrofia muscular das cinturas tipo 2A (LGMD2A), uma das formas mais frequentes de LGMD. O gene CAPN3 codifica a proteína calpaína 3 (ou CAPN3), uma protease de cisteína dependente de cálcio, específica do músculo esquelético, regulada funcionalmente através da sua actividade autolítica. O mecanismo de patogénese da LGMD2A ainda não se encontra esclarecido, mas pensa-se que em grande parte esteja associado a défices funcionais da CAPN3. Com o presente trabalho, propõe-se uma nova abordagem de estudo no diagnóstico da LGMD2A combinando técnicas moleculares e análise de proteínas, no sentido de contribuir para um diagnóstico diferencial essencial na caracterização global de doentes com calpainopatia, especificamente nos casos de doentes com sintomas de distrofia muscular não específicos. Amostras de biópsia muscular de 9 doentes com LGMD2A, previamente estudados molecularmente, foram submetidos à análise de proteínas e testes funcionais através de western blotting e densitometria. Adicionalmente, foram efectuados estudos de expressão génica utilizando PCR em tempo real quantitativo. Uma análise global dos resultados obtidos permitiu: (a) completar o diagnóstico molecular efectuado nos doentes 2-7, incluindo a identificação de um defeito funcional da CAPN3 no caso do doente 3; (b) confirmar o diagnóstico clínico no doente 8 (com apenas uma mutação em heterozigotia), também causado pelo défice funcional da proteína; e (c) excluir o envolvimento da CAPN3 nos restantes 2 doentes com apenas um alelo mutado em heterozigotia, devido à elevada expressão observada para o gene CAPN3 comparativamente com as amostras controlo.Muscular dystrophies comprise a group of heterogeneous genetic diseases characterized by progressive muscle weakness. Until now, eighteen genes have been associated to a specific subgroup of muscular dystrophies called limb-girdle muscular dystrophies (LGMD). Mutations in the CAPN3 gene are responsible for calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A), one of the most frequent form of LGMD. The gene CAPN3 codes for calpain 3 (or CAPN3), a muscle-specific calcium-dependent cysteine protease functionally regulated via its autolytic activity. The pathogenesis mechanism of LGMD2A still continues to be unresolved, however functional defects of CAPN3 seem to be the major cause of the pathology. With the present work, it is proposed a new approach for the LGMD2A diagnostic analysis, combining molecular genetics and protein analysis methodologies in order to contribute to a differential diagnostic essential for global characterization of patients, specifically among patients with non-specific muscular dystrophy symptoms. Muscle biopsy samples of 9 LGMD2A patients, already studied at the molecular genetics level, were subjected to protein analysis and functional tests by western blotting and densitometry analyses. Additionally, gene expression studies were performed using real-time quantitative PCR. A global analysis of the data obtained allowed us: (a) to complement the previous molecular genetics study in patients 2-7, including the identification of a CAPN3 functional defect in patient 3; (b) to confirm the clinical diagnostic in patient 8 (with only one mutation in heterozygous state) also caused by a protein functional defect; and (c) to exclude the CAPN3 implication in the other two patients which presented one mutated allele in heterozygous state, because of the detection of an overexpression of the CAPN3 gene in comparison to control samples