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Optimising mRNA integrity and read-through of CFTR bearing PTC mutations
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Orientador(es)
Resumo(s)
Fibrose quística (FQ) é uma doença monogénica complexa que afeta várias populações no mundo, sendo
mais frequente entre os Caucasianos. Esta condição é causada por mutações no gene CFTR, um dos maiores
genes humanos que codifica uma proteína com vários domínios, a qual é responsável pelo fluxo de Cl através da membrana apical voltada para o lúmen das células epiteliais e quando mutada pode causar
disfunção do pâncreas, glândulas sudoríparas, trato gastrointestinal, trato reprodutivo e pulmões.
Atualmente existem cerca de 2000 variantes conhecidas que podem causar FQ e estas estão divididas em
sete classes de mutações de acordo com o defeito no CFTR e a correspondente terapia.
Apesar de não haver cura para a FQ, existem vários tratamentos disponíveis para diferentes genótipos
da mutação do CFTR. Infelizmente, este não é o caso das mutações nonsense, que estão incluídas na classe
I e representam 8.4% das variantes identificadas. Estas introduzem um codão de terminação prematuro (do
inglês, “premature termination codon”; PTC) no mRNA do CFTR e estão associadas a sintomas severos,
embora ainda não haja terapias especificas aprovadas. O PTC quando presente no mRNA causa o término
precoce da tradução, e isto levaria há produção de uma proteína CFTR truncada que provavelmente não
seria funcional ou poderia ainda ser tóxica para a célula se não fosse pelo nonsense mediated decay (NMD),
um mecanismo de vigilância pós-transcricional responsável pela redução de transcritos que possuem PTC.
Este mecanismo pode influenciar a eficácia de um dos potenciais tratamentos para as mutações PTC,
nomeadamente os agentes read-through que permitem a tradução completa da proteína CFTR mesmo com
a presença do PTC. O NMD reduz a abundância de mRNA que possuem PTCs, e, por conseguinte, diminuí
a eficácia destes agentes.
Para melhor entender as repercussões que a presença do PTC tem na degradação do mRNA do CFTR, o
presente estudo avaliou os efeitos de três mutações nonsense sob a estabilidade dos transcritos do mRNA
do CFTR fazendo uso de uma transcriptional shutdown assay e RT-qPCR. Os nossos resultados indicam
que a presença do PTC influência a degradação do mRNA do CFTR ao longo do tempo, influenciando os
níveis de abundância do transcrito não só de uma forma dependente da mutação, mas também de forma
distinta entre as extremidades 5’ e 3’. Este foi também o caso após o tratamento com o agente de supressão
G418 e o inibidor do NMD SMG1i. Dados como estes sobre a estabilidade dos transcritos de mRNA podem
ser importantes no sucesso do desenvolvimento de terapias de PTC read-through que sejam específicas em
relação à mutação e, portanto, com aplicabilidade para a medicina personalizada, considerando que
diferentes variantes PTC podem responder de forma distinta à mesma abordagem.
Cystic Fibrosis (CF) is a complex monogenic disease that affects several populations around the world, being most frequent among Caucasians. This condition is caused by mutations in the CFTR gene, one of the largest human genes which encodes a multidomain protein responsible for Cl flow across the apical lumen facing membrane of epithelial cells and which when mutated can cause dysfunction of the pancreas, sweat glands, gastrointestinal tract, reproductive tract and lungs. Currently, there are around 2000 known potentially disease causing CF variants and these are divided into seven classes of mutations according to the CFTR defect and fitting therapeutic approaches. Although there is no cure for CF, there are several approved treatments that cover a number of CFTR mutation genotypes. Unfortunately, that is not the case for nonsense mutations, that are included in class I and represent 8.4% of the identified variants. These introduce an in-frame premature termination codon (PTC) into the CFTR mRNA and are associated with severe CF symptoms, but still have no specific approved therapies. A PTC present in the mRNA leads to premature termination of translation, thereby producing a truncated CFTR protein that would most likely be a non-functional protein or even be toxic for the cell if not for nonsense mediated decay (NMD), the posttranscriptional surveillance mechanism responsible for the reduction of PTC transcripts in the cytoplasm. This mechanism may influence the efficacy of one of the potential treatments for PTC mutations, namely the use of PTC read-through agents that allow translation of full-length CFTR protein even with the presence of a PTC. NMD reduces the abundance of PTC bearing mRNAs, and may thereby lower the efficacy of such agents. To better understand the repercussions of the presence of a PTC on CFTR mRNA decay, the present study measured the effect of three different nonsense mutations on the stability of CFTR mRNA transcripts using a transcriptional shutdown assay and RT-qPCR. Our results indicate that the presence of a PTC influences the degradation of CFTR mRNA over time, influencing transcript abundance levels not only in a mutation dependent fashion, but also to a different extent at the 5’ and 3’ends. This was also shown to be the case following treatment with the read-through agent G418 and the NMD inhibitor SMG1i. Such data on the stability of mRNA transcripts may be important in designing successful approaches to PTC read through therapy in a mutation specific manner and therefore with applicability to personalized medicine, considering that distinct PTC variants might respond differently to the same approaches
Cystic Fibrosis (CF) is a complex monogenic disease that affects several populations around the world, being most frequent among Caucasians. This condition is caused by mutations in the CFTR gene, one of the largest human genes which encodes a multidomain protein responsible for Cl flow across the apical lumen facing membrane of epithelial cells and which when mutated can cause dysfunction of the pancreas, sweat glands, gastrointestinal tract, reproductive tract and lungs. Currently, there are around 2000 known potentially disease causing CF variants and these are divided into seven classes of mutations according to the CFTR defect and fitting therapeutic approaches. Although there is no cure for CF, there are several approved treatments that cover a number of CFTR mutation genotypes. Unfortunately, that is not the case for nonsense mutations, that are included in class I and represent 8.4% of the identified variants. These introduce an in-frame premature termination codon (PTC) into the CFTR mRNA and are associated with severe CF symptoms, but still have no specific approved therapies. A PTC present in the mRNA leads to premature termination of translation, thereby producing a truncated CFTR protein that would most likely be a non-functional protein or even be toxic for the cell if not for nonsense mediated decay (NMD), the posttranscriptional surveillance mechanism responsible for the reduction of PTC transcripts in the cytoplasm. This mechanism may influence the efficacy of one of the potential treatments for PTC mutations, namely the use of PTC read-through agents that allow translation of full-length CFTR protein even with the presence of a PTC. NMD reduces the abundance of PTC bearing mRNAs, and may thereby lower the efficacy of such agents. To better understand the repercussions of the presence of a PTC on CFTR mRNA decay, the present study measured the effect of three different nonsense mutations on the stability of CFTR mRNA transcripts using a transcriptional shutdown assay and RT-qPCR. Our results indicate that the presence of a PTC influences the degradation of CFTR mRNA over time, influencing transcript abundance levels not only in a mutation dependent fashion, but also to a different extent at the 5’ and 3’ends. This was also shown to be the case following treatment with the read-through agent G418 and the NMD inhibitor SMG1i. Such data on the stability of mRNA transcripts may be important in designing successful approaches to PTC read through therapy in a mutation specific manner and therefore with applicability to personalized medicine, considering that distinct PTC variants might respond differently to the same approaches
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2022
Palavras-chave
Fibrose quística mutações PTC Nonsense mediated decay read-through agents Teses de mestrado - 2022