Estudo de mutações somáticas no gene APC em doentes com cancro do cólon e recto em idade jovem

Tese de mestrado, Biologia (Biologia Humana e Ambiente), 2007, Universidade de Lisboa, Faculdade de CiênciasO cancro colorectal (CCR) é uma das doenças malignas mais comuns no mundo ocidental, sendo mais frequente após os 50 anos, embora também possa surgir em pessoas com idade mais jovem. Existem três padrões específicos do CCR: hereditário, familiar e esporádico. O Hereditary non-polyposis colon cancer (HNPCC) e a Polipose Adenomatosa Familiar (PAF) são provocados por mutações germinais, nos genes de reparação mismatch (MMR) e no gene supressor tumoral Adenomatous Polyposis Coli (APC), respectivamente. O cancro do cólon esporádico, sem qualquer predisposição familiar ou hereditária, pode ser causado, em 80% dos casos, por mutações somáticas no APC. A perda alélica do APC ou a hipermetilação do seu promotor podem também estar na origem desta doença. Alterações noutros genes, como o BRAF, K-ras e ß-catenina, podem também provocar um aumento da sinalização Wnt, ocorrendo em cerca 10%, 30-40% e em menos de 5% dos CCR. O objectivo deste trabalho foi encontrar uma possível causa genética para o desenvolvimento precoce de CCR esporádico. Para tal o trabalho experimental foi efectuado em dois grupos de indivíduos, com idade ≤ 45 anos e ≥ 50 anos, nos quais foi feita a pesquisa de mutações somáticas no gene APC, análise de perda alélica análise do estado de metilação do promotor do APC, e a pesquisa de mutações nos hot-spots mutacionais dos genes K-ras, BRAF e ß-catenina. Os resultados observados indicam que a incidência de mutações no APC é inferior nos indivíduos mais jovens, sendo que a maioria das mutações que ocorrem nos indivíduos mais jovens originam proteínas truncadas que retêm mais de 2 domínios de regulação da ß-catenina. Nos indivíduos jovens foram também observados níveis de instabilidade de microssatélites mais elevados, e maior incidência de mutações no K-ras e na ß-catenina, embora em níveis mais baixos do que os observados noutros estudos, sendo a frequência de indivíduos com alterações no APC e noutros genes mais elevada, comparativamente aos indivíduos de idade mais avançada. Isto sugere que alterações no APC ou nos restantes genes analisados neste estudo, por si só, não estão na origem de CCR esporádico em idade jovem, mas levanta a hipótese de que a origem precoce desta doença poderá estar na ocorrência de mutações simultâneas em vários genes, que resulte numa maior agressividade na progressão maligna, ou eventualmente em alterações noutro gene não analisado neste estudoColorectal cancer (CRC) is one of the most common malignancies in the western world, mostly affecting patients over 50 years old, but it can also affect younger people. There are three specific patterns of CRC: hereditary, familial and sporadic. Hereditary non-polyposis colon cancer (HNPCC) and Familial Adenomatous Poliposis (FAP) are caused by germinal mutations, in mismatch repair genes (MMR) and in the tumour suppressor gene Adenomatous Polyposis Coli (APC), respectively. Sporadic CRC, with no hereditary or familial predisposition, can be caused, in 80% of the cases, by somatic mutations in the APC gene. Allelic loss of APC gene or hipermethylation of its promoter region can also cause this disease. BRAF, K-ras and ß-catenin mutations can also increase Wnt signalling and may occur in 10%, 30-40% and less than 5% of CRC, respectively. The aim of this study was to evaluate the profile of somatic APC gene alterations in patients with early-onset CRC. Tumour and mucosal samples from patients aged lower 45 and from patients aged over 50 were studied for APC truncating mutation, allelic loss, APC promoter hipermethylation, and BRAF, K-ras and ß-catenin mutations. The results show that younger patients have lower incidence of APC mutations, and these occur mainly in a region that origins truncated proteins with more than 2 ß-catenin regulating domains. Younger patients also have higher microssatellite instability, and higher frequency of K-ras and ß-catenin mutations, though at lower levels than in other studies. This group also has a higher frequency of mutations in APC together with other genes, comparing with older patients. These results suggest that APC mutations per se are not the main cause for early on-set sporadic CRC, neither mutations in the other genes analysed here. However, it raises the hypothesis that early on-set sporadic CRC may result from mutations in more than one gene, so leading to a more aggressive malignant progression, or can result from mutations in other gene that was not analysed in this stud

CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. ERC_2015_StG _ 680156 _ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Scientific Stimulus Grant ( CEECIND/03482/2018 ). J.A. is a PhD fellow from FCT ( SFRH/BD/145110/2019 ). R.B.C. was funded by FCT ( ON2201403-CTO-BPD ) and EMBO (Short-Term Fellowship). We thank Joaquin Letelier, Ana Novoa, and Moises Mallo for important advice establishing the current protocol. We also thank Joana Marques for constant assistance in establishing the protocol

Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2015-StG-680156-ZPR and ERC-2016-AdG-740041-EvoLand to J.L.G.-S.). J.B. is supported by an FCT CEEC grant (CEECIND/03482/2018). J.L.G.-S. is supported by the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P), the Marató TV3 Fundacion (Grant 201611) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). R.B.C. was funded by FCT (ON2201403-CTO-BPD), IBMC (BIM/04293-UID991520-BPD) and EMBO (Short-Term Fellowship). J.Tx. (SFRH/BD/126467/2016), M.D. (SFRH/BD/135957/2018), A.E. (SFRH/BD/147762/2019), and F.J.F. (PD/BD/105745/2014) are PhD fellows from FCT. M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation programme (grant 668981). This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020”

Relatório estágio profissional

Relatório final do estágio profissionalizante do 6.º an

Cancer syndromes and therapy by stop-codon readthrough

Several hereditary cancer syndromes are associated with nonsense mutations that create premature termination codons (PTC). Therapeutic strategies involving readthrough induction partially restore expression of proteins with normal function from nonsense-mutated genes, and small molecules such as aminoglycosides and PTC124 have exhibited promising results for treating patients with cystic fibrosis and Duchenne muscular dystrophy. Transgenic expression of suppressor-tRNAs and depleting translation termination factors are, among others, potential strategies for treating PTC-associated diseases. In this review, the potential of using readthrough strategies as a therapy for cancer syndromes is discussed, and we consider the effect of nonsense-mediated decay and other factors on readthrough efficiency.This work was supported by the Calouste Gulbenkian Foundation through the project ‘Mutated suppressor tRNAs as a therapeutic tool for cancer associated syndromes: HDGC as a model’ and the Portuguese Foundation for Science and Technology (FCT) [PhD grant: SFRH/BD/46462/2008-RBC; salary support to C.O. from POPH – QREN/Type 4.2, European Social Fund and Portuguese Ministry of Science and Technology (MCTES)]. IPATIMUP and INEB are Associate Laboratories of the Portuguese Ministry of Science, Technology and Higher Education and are partially supported by FCT. The authors acknowledge Hugo Pinheiro, Denisa Mateus, and Cecília Durães for critical reading of the manuscript.publishe

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

© 2020 The Authors.The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.Amorim et al. find that Nog2 is expressed in the zebrafish notochord by the action of a tissue-specific enhancer, and it diffuses to the pancreatic domain and controls its size. The identification of Nog enhancers in other vertebrate lineages suggests a conserved mechanism for pancreas development in vertebrates.This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant ERC2015StG680156ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Investigator position (grant IF/00654/2013). J.T. and J.P.A. are PhD fellows from FCT (grant SFRH/BD/126467/2016 to J.T. and grant SFRH/BD/145110/2019 to J.P.A.). M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation program (grant 668981). The authors acknowledge the support of i3S Scientific Platform Advanced Light Microscopy, a member of the national infrastructure PPBI (Portuguese Platform of BioImaging) (supported by POCI010145FEDER022122)

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/E-cadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin- deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs ß cell differentiation, compatible with the antagonistic role of Bmp in ß cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs ß cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.We thank José Luís Gómez-Skarmeta and Paulo Pereira for helpful suggestions and critical reading of the manuscript, Vitor Silva for support in the phylogenetic tree design, and Marta Duque for help in designing the graphical abstract. This study was supported by the European Research Council ( ERC ) under the European Union’s Horizon 2020 research and innovation program (grant ERC2015StG680156ZPR ). J.B. acknowledges Fundação para a Ciência e a Tecnologia ( FCT ) for an FCT Investigator position (grant IF/00654/2013 ). J.T. and J.P.A. are PhD fellows from FCT (grant SFRH/BD/126467/2016 to J.T. and grant SFRH/BD/145110/2019 to J.P.A.). M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation program (grant 668981 ). The authors acknowledge the support of i3S Scientific Platform Advanced Light Microscopy, a member of the national infrastructure PPBI ( Portuguese Platform of BioImaging ) (supported by POCI010145FEDER022122 ). We acknowledge the I3S hpc facility, used for processing the 4C-seq data, and André Torres for the useful support. We also thank Francesco Argenton for providing us with a vector for generating an insulin-targeting probe. Finally, we thank Yolanda Roncero and Isabel Guedes for helping to screen and maintain the nog2 ED301 zebrafish line and Tania Medeiros for preliminary work on the function of the notochord in pancreas development