Publicação
Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis
| Resumo: | Cystic Fibrosis (CF) is the most common autosomal disease in Caucasians, with an estimated incidence of 1:6000 births in Portugal. The most relevant clinical aspect of its classic manifestation is chronic lung disease, which is the main cause of morbidity and mortality. Other symptoms include, pancreatic dysfunction, male infertility and high concentrations of chloride (Cl-) in sweat. However, even though the classic form of the disease is well defined, its pathophysiology is not completely understood with this pleiotropic disease having highly variable manifestations of clinical phenotypes. Novel therapies aim to correct the basic defect, specifically focusing on the rescue of Cystic fibrosis transmembrane conductance regulator (CFTR) function in CF airways. Most of these CFTR modulator strategies target the F508del, the most common mutation. Nevertheless, widespread evidence has demonstrated that a significant number of CFcausing mutations affect splicing efficiency and the stability of mRNA molecules. Here, we propose to elucidate the regulatory mechanisms underlying these CF-associated mutations. To this end, our aims are: 1) to identify CFTR gene mutations in individuals with non-CF chronic lung diseases, namely chronic obstructive pulmonary disease (COPD), asthma and disseminated bronchiectasis (DB); 2) to identify CFTR gene mutations in individuals with a suspicion of CF, followed by the analysis of CFTR expression in their native tissues to characterize the impact of CFTR splicing or premature termination codon (PTC) mutations in the structure and levels of mRNA; 3) to identify key factors in the nonsense/mediated decay (NMD) pathway by automated microscopy screens using a cell model expressing a novel CFTR NMD-PTC/read-through mini-gene reporter; and 4) to screen for novel compounds suppressing PTC mutations by automated microscopy screens using the previous cell-based model, as potential corrective therapies for CF. The expected results will provide knowledge on RNA-processing dysfunction and on the efficacy of novel RNA modulator compounds towards a "personalised medicine" approach. Regarding the first objective, our data show that 7 (out of 136) patients with non-CF respiratory diseases presented CFTR mutations in one allele, in contrast with the control group, in which no mutations were detected. We analysed the association of CFTR gene mutations with each of the three respiratory diseases considered. For asthma our data did not show an increase in mutation frequency when compared to the control group. For DB, we found an increase in the frequency of CFTR gene mutations, albeit with no statistical significance, which is in agreement with previous reports. For COPD however, we found a statistically significant increase in CFTR gene mutation incidence, relative to the control group. Our data reinforce the importance of characterizing CFTR gene mutations on non-CF respiratory diseases in Portuguese patients, to gain a better understanding of the epidemiology and etiology of these diseases. The results also lead to the identification of groups of patients who may benefit from the new therapeutic compounds currently under development to correct the basic CFTR protein defect in CF. Concerning the second objective, we have developed a novel RNA-based approach to detect unknown CFTR mutations [Felício et al (2017) Clin Genet 91: 476-481]. We are currently using this protocol for patients with a suspicion of CF and none or just one CFTR mutation identified. With this method we identified one mutation (711+3A>T) which had been previously reported but had not been characterized. We can conclude that this is a rapid, robust and inexpensive method to detect rare mutations, and therefore a method that can be easily used after a first screen. Regarding the third objective, we used this CFTR-NMD reporter to identify novel NMD factors by screening a previously validated shRNA library – a subset of The RNAi Consortium (TRC) – which is enriched in shRNAs targeting genes with a known or predicted involvement in transcript processing (425 genes), using HT microscopy. We selected the 24 top hits for the confirmation: 11 genes with NMD score ≥ 2 and more than 2 shRNAs with the same phenotype; 2 genes from the screen that showed read-through activity; and 11 other genes resulting from a high-throughput screen (HTS) aimed at the identification of CFTR splicing regulators (unpublished data). We chose these genes related to splicing because this is a process known to be required for NMD to occur and thus the knock-down of such genes can lead to NMD inhibition. The confirmation screen was performed using a library of siRNAs targeting the previously selected genes, however the results were inconclusive due to the low NMD score obtained. We have identified 4 genes with higher values but NMD score ≤ 1, three (eIF4A3, SREK1 and RPS19) are related to splicing, elF4A3 is directly and RPS19 is indirectly related to NMD, SREK1 is related only to splicing, with the fourth gene, ADIRF, having unknown functional properties. Some of the hits identified within this screen may be potential drug targets by their effects in inhibiting NMD, when knocked-down. With the results obtained in the confirmation screen we decided to follow the 24 genes identified in the primary screen for validation studies using two different techniques: WB and qRT-PCR (study in progress). Lastly, the fourth objective was to restore functional protein production to PTC mutations using read-through compounds, with the ultimate aim of CF patient treatment. The CFTRNMD construct used has the G542X nonsense mutation, mCherry at the N terminus and eGFP fused at the CFTR C-terminus. Through the screen of the library, we identified new small-molecule compounds that induced PTC read-through. To confirm the read-through efficiency future experiments are needed using additional techniques, such as, WB and transcript analysis by semi-quantitative PCR and qRT-PCR. Finally, to validate the top hit compounds, it is necessary to test them in patient’s materials, including nasal primary cells for functional activity and intestinal organoids to determine a dose response and to test these compounds in combination with potentiators and correctors. The studies presented in this dissertation had the overall aims of advancing the current knowledge on RNA-processing dysfunction and of identifying novel RNA modulator compounds towards a "personalised medicine" approach. The results obtained have indeed provided new insights into: 1) the relationship of other respiratory diseases with the presence of CFTR mutations; 2) new approaches to detect CFTR mutations in DNA and RNA; and 3) our understanding of key factors in NMD and read-through activity in relation to CFTR nonsense mutations. |
|---|---|
| Autores principais: | Felício, Verónica Manuela Rôxo |
| Assunto: | Teses de doutoramento - 2018 |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| _version_ | 1868467354714767360 |
|---|---|
| author | Felício, Verónica Manuela Rôxo |
| author_facet | Felício, Verónica Manuela Rôxo |
| author_role | author |
| contributor_name_str_mv | Amaral, Margarida, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Felício, Verónica Manuela Rôxo\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Amaral, Margarida, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| datacite.creators.creator.creatorName.fl_str_mv | Felício, Verónica Manuela Rôxo |
| datacite.date.Accepted.fl_str_mv | 2018-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2019-05-17T10:21:54Z |
| datacite.date.embargoed.fl_str_mv | 2019-05-17T10:21:54Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Teses de doutoramento - 2018 |
| datacite.titles.title.fl_str_mv | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| dc.contributor.none.fl_str_mv | Amaral, Margarida, 1958- Repositório Científico de Acesso Aberto da ULisboa |
| dc.creator.none.fl_str_mv | Felício, Verónica Manuela Rôxo |
| dc.date.Accepted.fl_str_mv | 2018-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2019-05-17T10:21:54Z |
| dc.date.embargoed.fl_str_mv | 2019-05-17T10:21:54Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10451/38258 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Teses de doutoramento - 2018 |
| dc.title.fl_str_mv | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_db06 |
| description | Cystic Fibrosis (CF) is the most common autosomal disease in Caucasians, with an estimated incidence of 1:6000 births in Portugal. The most relevant clinical aspect of its classic manifestation is chronic lung disease, which is the main cause of morbidity and mortality. Other symptoms include, pancreatic dysfunction, male infertility and high concentrations of chloride (Cl-) in sweat. However, even though the classic form of the disease is well defined, its pathophysiology is not completely understood with this pleiotropic disease having highly variable manifestations of clinical phenotypes. Novel therapies aim to correct the basic defect, specifically focusing on the rescue of Cystic fibrosis transmembrane conductance regulator (CFTR) function in CF airways. Most of these CFTR modulator strategies target the F508del, the most common mutation. Nevertheless, widespread evidence has demonstrated that a significant number of CFcausing mutations affect splicing efficiency and the stability of mRNA molecules. Here, we propose to elucidate the regulatory mechanisms underlying these CF-associated mutations. To this end, our aims are: 1) to identify CFTR gene mutations in individuals with non-CF chronic lung diseases, namely chronic obstructive pulmonary disease (COPD), asthma and disseminated bronchiectasis (DB); 2) to identify CFTR gene mutations in individuals with a suspicion of CF, followed by the analysis of CFTR expression in their native tissues to characterize the impact of CFTR splicing or premature termination codon (PTC) mutations in the structure and levels of mRNA; 3) to identify key factors in the nonsense/mediated decay (NMD) pathway by automated microscopy screens using a cell model expressing a novel CFTR NMD-PTC/read-through mini-gene reporter; and 4) to screen for novel compounds suppressing PTC mutations by automated microscopy screens using the previous cell-based model, as potential corrective therapies for CF. The expected results will provide knowledge on RNA-processing dysfunction and on the efficacy of novel RNA modulator compounds towards a "personalised medicine" approach. Regarding the first objective, our data show that 7 (out of 136) patients with non-CF respiratory diseases presented CFTR mutations in one allele, in contrast with the control group, in which no mutations were detected. We analysed the association of CFTR gene mutations with each of the three respiratory diseases considered. For asthma our data did not show an increase in mutation frequency when compared to the control group. For DB, we found an increase in the frequency of CFTR gene mutations, albeit with no statistical significance, which is in agreement with previous reports. For COPD however, we found a statistically significant increase in CFTR gene mutation incidence, relative to the control group. Our data reinforce the importance of characterizing CFTR gene mutations on non-CF respiratory diseases in Portuguese patients, to gain a better understanding of the epidemiology and etiology of these diseases. The results also lead to the identification of groups of patients who may benefit from the new therapeutic compounds currently under development to correct the basic CFTR protein defect in CF. Concerning the second objective, we have developed a novel RNA-based approach to detect unknown CFTR mutations [Felício et al (2017) Clin Genet 91: 476-481]. We are currently using this protocol for patients with a suspicion of CF and none or just one CFTR mutation identified. With this method we identified one mutation (711+3A>T) which had been previously reported but had not been characterized. We can conclude that this is a rapid, robust and inexpensive method to detect rare mutations, and therefore a method that can be easily used after a first screen. Regarding the third objective, we used this CFTR-NMD reporter to identify novel NMD factors by screening a previously validated shRNA library – a subset of The RNAi Consortium (TRC) – which is enriched in shRNAs targeting genes with a known or predicted involvement in transcript processing (425 genes), using HT microscopy. We selected the 24 top hits for the confirmation: 11 genes with NMD score ≥ 2 and more than 2 shRNAs with the same phenotype; 2 genes from the screen that showed read-through activity; and 11 other genes resulting from a high-throughput screen (HTS) aimed at the identification of CFTR splicing regulators (unpublished data). We chose these genes related to splicing because this is a process known to be required for NMD to occur and thus the knock-down of such genes can lead to NMD inhibition. The confirmation screen was performed using a library of siRNAs targeting the previously selected genes, however the results were inconclusive due to the low NMD score obtained. We have identified 4 genes with higher values but NMD score ≤ 1, three (eIF4A3, SREK1 and RPS19) are related to splicing, elF4A3 is directly and RPS19 is indirectly related to NMD, SREK1 is related only to splicing, with the fourth gene, ADIRF, having unknown functional properties. Some of the hits identified within this screen may be potential drug targets by their effects in inhibiting NMD, when knocked-down. With the results obtained in the confirmation screen we decided to follow the 24 genes identified in the primary screen for validation studies using two different techniques: WB and qRT-PCR (study in progress). Lastly, the fourth objective was to restore functional protein production to PTC mutations using read-through compounds, with the ultimate aim of CF patient treatment. The CFTRNMD construct used has the G542X nonsense mutation, mCherry at the N terminus and eGFP fused at the CFTR C-terminus. Through the screen of the library, we identified new small-molecule compounds that induced PTC read-through. To confirm the read-through efficiency future experiments are needed using additional techniques, such as, WB and transcript analysis by semi-quantitative PCR and qRT-PCR. Finally, to validate the top hit compounds, it is necessary to test them in patient’s materials, including nasal primary cells for functional activity and intestinal organoids to determine a dose response and to test these compounds in combination with potentiators and correctors. The studies presented in this dissertation had the overall aims of advancing the current knowledge on RNA-processing dysfunction and of identifying novel RNA modulator compounds towards a "personalised medicine" approach. The results obtained have indeed provided new insights into: 1) the relationship of other respiratory diseases with the presence of CFTR mutations; 2) new approaches to detect CFTR mutations in DNA and RNA; and 3) our understanding of key factors in NMD and read-through activity in relation to CFTR nonsense mutations. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| fulltext.url.fl_str_mv | https://repositorio.ulisboa.pt/bitstreams/fdb37dc9-6d2f-45d7-ae74-7f62215437d3/download |
| funding.funder.alternateName_str_mv | FCT |
| funding.funder.identifier_str_mv | http://doi.org/10.13039/501100001871 |
| funding.funder.name_str_mv | Fundação para a Ciência e a Tecnologia |
| id | ul_0c95ea9bb91c05dd7ea14642b30e4e10 |
| identifier.url.fl_str_mv | http://hdl.handle.net/10451/38258 |
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| institution | Universidade de Lisboa |
| instname_str | Universidade de Lisboa |
| language | eng |
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| network_name_str | Repositório da Universidade de Lisboa |
| oai_identifier_str | oai:repositorio.ulisboa.pt:10451/38258 |
| organization_str_mv | urn:organizationAcronym:ul |
| person_str_mv | Felício, Verónica Manuela Rôxo |
| publishDate | 2018 |
| reponame_str | Repositório da Universidade de Lisboa |
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| service_str_mv | urn:repositoryAcronym:ul |
| spelling | engpt_PTCystic Fibrosis (CF) is the most common autosomal disease in Caucasians, with an estimated incidence of 1:6000 births in Portugal. The most relevant clinical aspect of its classic manifestation is chronic lung disease, which is the main cause of morbidity and mortality. Other symptoms include, pancreatic dysfunction, male infertility and high concentrations of chloride (Cl-) in sweat. However, even though the classic form of the disease is well defined, its pathophysiology is not completely understood with this pleiotropic disease having highly variable manifestations of clinical phenotypes. Novel therapies aim to correct the basic defect, specifically focusing on the rescue of Cystic fibrosis transmembrane conductance regulator (CFTR) function in CF airways. Most of these CFTR modulator strategies target the F508del, the most common mutation. Nevertheless, widespread evidence has demonstrated that a significant number of CFcausing mutations affect splicing efficiency and the stability of mRNA molecules. Here, we propose to elucidate the regulatory mechanisms underlying these CF-associated mutations. To this end, our aims are: 1) to identify CFTR gene mutations in individuals with non-CF chronic lung diseases, namely chronic obstructive pulmonary disease (COPD), asthma and disseminated bronchiectasis (DB); 2) to identify CFTR gene mutations in individuals with a suspicion of CF, followed by the analysis of CFTR expression in their native tissues to characterize the impact of CFTR splicing or premature termination codon (PTC) mutations in the structure and levels of mRNA; 3) to identify key factors in the nonsense/mediated decay (NMD) pathway by automated microscopy screens using a cell model expressing a novel CFTR NMD-PTC/read-through mini-gene reporter; and 4) to screen for novel compounds suppressing PTC mutations by automated microscopy screens using the previous cell-based model, as potential corrective therapies for CF. The expected results will provide knowledge on RNA-processing dysfunction and on the efficacy of novel RNA modulator compounds towards a "personalised medicine" approach. Regarding the first objective, our data show that 7 (out of 136) patients with non-CF respiratory diseases presented CFTR mutations in one allele, in contrast with the control group, in which no mutations were detected. We analysed the association of CFTR gene mutations with each of the three respiratory diseases considered. For asthma our data did not show an increase in mutation frequency when compared to the control group. For DB, we found an increase in the frequency of CFTR gene mutations, albeit with no statistical significance, which is in agreement with previous reports. For COPD however, we found a statistically significant increase in CFTR gene mutation incidence, relative to the control group. Our data reinforce the importance of characterizing CFTR gene mutations on non-CF respiratory diseases in Portuguese patients, to gain a better understanding of the epidemiology and etiology of these diseases. The results also lead to the identification of groups of patients who may benefit from the new therapeutic compounds currently under development to correct the basic CFTR protein defect in CF. Concerning the second objective, we have developed a novel RNA-based approach to detect unknown CFTR mutations [Felício et al (2017) Clin Genet 91: 476-481]. We are currently using this protocol for patients with a suspicion of CF and none or just one CFTR mutation identified. With this method we identified one mutation (711+3A>T) which had been previously reported but had not been characterized. We can conclude that this is a rapid, robust and inexpensive method to detect rare mutations, and therefore a method that can be easily used after a first screen. Regarding the third objective, we used this CFTR-NMD reporter to identify novel NMD factors by screening a previously validated shRNA library – a subset of The RNAi Consortium (TRC) – which is enriched in shRNAs targeting genes with a known or predicted involvement in transcript processing (425 genes), using HT microscopy. We selected the 24 top hits for the confirmation: 11 genes with NMD score ≥ 2 and more than 2 shRNAs with the same phenotype; 2 genes from the screen that showed read-through activity; and 11 other genes resulting from a high-throughput screen (HTS) aimed at the identification of CFTR splicing regulators (unpublished data). We chose these genes related to splicing because this is a process known to be required for NMD to occur and thus the knock-down of such genes can lead to NMD inhibition. The confirmation screen was performed using a library of siRNAs targeting the previously selected genes, however the results were inconclusive due to the low NMD score obtained. We have identified 4 genes with higher values but NMD score ≤ 1, three (eIF4A3, SREK1 and RPS19) are related to splicing, elF4A3 is directly and RPS19 is indirectly related to NMD, SREK1 is related only to splicing, with the fourth gene, ADIRF, having unknown functional properties. Some of the hits identified within this screen may be potential drug targets by their effects in inhibiting NMD, when knocked-down. With the results obtained in the confirmation screen we decided to follow the 24 genes identified in the primary screen for validation studies using two different techniques: WB and qRT-PCR (study in progress). Lastly, the fourth objective was to restore functional protein production to PTC mutations using read-through compounds, with the ultimate aim of CF patient treatment. The CFTRNMD construct used has the G542X nonsense mutation, mCherry at the N terminus and eGFP fused at the CFTR C-terminus. Through the screen of the library, we identified new small-molecule compounds that induced PTC read-through. To confirm the read-through efficiency future experiments are needed using additional techniques, such as, WB and transcript analysis by semi-quantitative PCR and qRT-PCR. Finally, to validate the top hit compounds, it is necessary to test them in patient’s materials, including nasal primary cells for functional activity and intestinal organoids to determine a dose response and to test these compounds in combination with potentiators and correctors. The studies presented in this dissertation had the overall aims of advancing the current knowledge on RNA-processing dysfunction and of identifying novel RNA modulator compounds towards a "personalised medicine" approach. The results obtained have indeed provided new insights into: 1) the relationship of other respiratory diseases with the presence of CFTR mutations; 2) new approaches to detect CFTR mutations in DNA and RNA; and 3) our understanding of key factors in NMD and read-through activity in relation to CFTR nonsense mutations.application/pdfpt_PTCharacterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosisFelício, Verónica Manuela RôxoAmaral, Margarida, 1958-HostingInstitutionOrganizationalRepositório Científico de Acesso Aberto da ULisboae-mailmailto:repositorio@reitoria.ulisboa.ptrepositorio@reitoria.ulisboa.ptURNurn:tid:1015046832019-05-17T10:21:54Z201820182018-01-01T00:00:00ZHandlehttp://hdl.handle.net/10451/38258http://purl.org/coar/access_right/c_abf2open accessTeses de doutoramento - 20183686338 bytesFundação para a Ciência e a TecnologiaCHARACTERIZATION OF RNA DYSFUNCTIONAL MECHANISMS ASSOCIATED WITH THE GENETIC DISEASE CYSTIC FIBROSISCrossref Funder IDhttp://doi.org/10.13039/501100001871literaturehttp://purl.org/coar/resource_type/c_db06doctoral thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorio.ulisboa.pt/bitstreams/fdb37dc9-6d2f-45d7-ae74-7f62215437d3/download |
| spellingShingle | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis Felício, Verónica Manuela Rôxo Teses de doutoramento - 2018 |
| status | SINGLETON |
| subject.fl_str_mv | Teses de doutoramento - 2018 |
| title | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| title_full | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| title_fullStr | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| title_full_unstemmed | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| title_short | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| title_sort | Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis |
| topic | Teses de doutoramento - 2018 |
| topic_facet | Teses de doutoramento - 2018 |
| url | http://hdl.handle.net/10451/38258 |
| visible | 1 |