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Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases
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Advisor(s)
Abstract(s)
[PT] Algumas das mutações que ocorrem em células somáticas ou na linha germinal podem
resultar na formação de codões de terminação prematura da tradução (CTPs ou
codões nonsense). Um terço das mutações associadas a doenças genéticas resulta na
produção de CTPs. Em geral, a introdução dum CTP num transcrito induz a terminação
prematura da tradução e a activação do mecanismo de decaimento do mRNA mediado
por mutações nonsense (nonsense-mediated mRNA decay; NMD). O NMD é um
mecanismo de controlo de qualidade que impede a produção de proteínas truncadas
na extremidade C-terminal tóxicas para a célula.
No decorrer dos últimos anos, tem sido desenvolvida uma nova terapia, designada
como terapia de supressão, que utiliza compostos de baixo peso molecular para
induzir a maquinaria de tradução a alterar um CTP para um codão sense. O objectivo
da terapia de supressão é estimular a capacidade de competição de tRNAs aminoacil
com o complexo de terminação para a ligação ao CTP no local A do ribossoma, de
forma a suprimir especificamente a terminação em CTPs e não em codões de
terminação naturais. Embora a talassémia seja uma das doenças genéticas mais
comuns e as mutações nonsense no gene da beta-globina (β-globina) estejam muito
frequentemente associadas à beta-talassémia (β-talassémia), a utilização da terapia de
supressão nestes casos ainda não foi investigada de forma detalhada. Resultados
preliminares obtidos pelo nosso laboratório indicam que o aminoglicósido G418 tem a
capacidade de suprimir uma mutação nonsense no codão 39 do mRNA da β-globina,
em células eritróides em cultura. De forma a confirmar o princípio de que a terapia de
supressão pode restaurar os níveis de β-globina, testámos se o composto G418 tem a
capacidade de induzir níveis significativos de supressão em células HeLa transfectadas
com plasmídeos que contêm o gene da β-globina humana normal (βWT) ou o
equivalente com mutação nonsense no codão 39 (β39).
Segundo os resultados obtidos, há um ligeiro aumento nos níveis de transcrito β39 o
que pode ser indicador de um possível efeito da droga. Como não nos foi possível
verificar o efeito da droga nos níveis de proteína é necessário trabalho futuro para
avaliar o seu efeito nos níveis de expressão proteica.
[ENG] Premature termination codons (PTCs or nonsense codons) can arise from various types of mutations in germ or somatic cells. PTCs promote premature translational termination and the induction of nonsense-mediated mRNA decay (NMD). NMD is a surveillance system that prevents the synthesis of C-terminally truncated proteins toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. In recent years, a novel therapeutic approach called suppression therapy has been developed that utilizes low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon. Beta-thalassaemia (β-thalassaemia) is one of the most common genetic diseases worldwide, and nonsense mutations occurring in the beta-globin (β-globin) are among the most frequent mutations associated with β-thalassaemia. Some studies have shown that aminoglycosides, low molecular weight compounds, and non-aminoglycosides can suppress PTCs in cystic fibrosis and Duchenne’s muscular dystrophy, but it remains unclear whether β- thalassaemia would also be responsive to a similar drug treatment. Preliminary results obtained in our lab have shown that the aminoglycoside G418 can suppress a nonsense mutation at codon 39 of the human β-globin mRNA, although at low levels in cultured erythroid cells. To investigate if suppression therapy can restore enough β- globin protein to correct the disease manifestations of β-thalassaemia, we tested whether G418 is able to induce efficient levels of suppression in a dose-dependent and time-course manner in HeLa cells transfected with plasmids containing the human β- globin wild type gene (βWT) or the counterpart carrying a nonsense mutation at codon 39 (β39). Our results show a slight increase in the levels of mRNA of the β39 transcript which may indicate a possible effect by the drug. Seen as we were unable to test the effect of the drug on a protein level, in the future, it would be of great interest to evaluate its effect on protein expression levels.
[ENG] Premature termination codons (PTCs or nonsense codons) can arise from various types of mutations in germ or somatic cells. PTCs promote premature translational termination and the induction of nonsense-mediated mRNA decay (NMD). NMD is a surveillance system that prevents the synthesis of C-terminally truncated proteins toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. In recent years, a novel therapeutic approach called suppression therapy has been developed that utilizes low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon. Beta-thalassaemia (β-thalassaemia) is one of the most common genetic diseases worldwide, and nonsense mutations occurring in the beta-globin (β-globin) are among the most frequent mutations associated with β-thalassaemia. Some studies have shown that aminoglycosides, low molecular weight compounds, and non-aminoglycosides can suppress PTCs in cystic fibrosis and Duchenne’s muscular dystrophy, but it remains unclear whether β- thalassaemia would also be responsive to a similar drug treatment. Preliminary results obtained in our lab have shown that the aminoglycoside G418 can suppress a nonsense mutation at codon 39 of the human β-globin mRNA, although at low levels in cultured erythroid cells. To investigate if suppression therapy can restore enough β- globin protein to correct the disease manifestations of β-thalassaemia, we tested whether G418 is able to induce efficient levels of suppression in a dose-dependent and time-course manner in HeLa cells transfected with plasmids containing the human β- globin wild type gene (βWT) or the counterpart carrying a nonsense mutation at codon 39 (β39). Our results show a slight increase in the levels of mRNA of the β39 transcript which may indicate a possible effect by the drug. Seen as we were unable to test the effect of the drug on a protein level, in the future, it would be of great interest to evaluate its effect on protein expression levels.
Description
Dissertação de mestrado em Biologia (Biologia Humana e Ambiente, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2014.
Luísa Romão, Ph.D. (INSA)
Luísa Romão, Ph.D. (INSA)
Keywords
Genómica Funcional e Estrutural Biologia Molecular Doenças Genéticas Terapia de Supressão Nonsense-mediated mRNA Decay (NMD) mRNA Translation Genetic Diseases Suppression Therapy G418 Decaimento do mRNA Mediado por Mutações Nonsense (NMD) Tradução do mRNA