Polimorfismos bioquímicos humanos detectáveis em leucócitos : Genética populacional, formal e de ligação factorial

Dissertação de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Port

Screening of HBB*S in populations from Alentejo and implementation of a SNaPshot based system for HBB*S haplotyping

It is well demonstrated that the frequency of sickle cell allele, HBB*S, reaches the highest values in regions of endemic malaria, due to resistance to the malaria infection conferred by HBB*S. Its presence in Portugal has been explained as the consequence of past population migrations, which in part also explain the uneven distribution of HBB*S across the country, where it tends to increase in frequency from the north towards the south-central regions. In order to better understand how HBB*S was introduced in Portugal, the screening of HBB was performed in 266 individuals from Alentejo (Coruche, Serpa (Pias) and Alcácer do Sal). Then, to further infer the origin of the detected HBB*S chromosomes, their haplotypic background, encompassing the entire cluster of β-globin genes, was examined through a SNaPshot® Multiplex system that was purposely implemented as an alternative to the conventional RFLP-based method, and that allowed to interrogate the most informative positions defining the HBB*S haplotypes. 19 individuals harboured the c.20A>T mutation that underlies HBB*S, whose frequency was 2.2, 2.9 and 7.7% in Coruche, Alcácer do Sal and Serpa, respectively. The last value is the highest up to now reported in the country, where foci of high prevalence of HBB*S carriers have been previously identified in south regions. Among the chromosomes bearing HBB*S, 29% harboured the Benin haplotype, 13.1% the Senegal and 10.5% the Bantu. Remarkably, whereas in Alcáçer do Sal and Coruche only Senegal and Bantu haplotypes were found, in Serpa all haplotypes were Benin. Taking into account the global distribution of HBB*S haplotypes, the findings obtained reinforce a scenario before proposed positing that the introduction of HBB*S in south Portugal was mediated by gene influx events with distinct sources: one from the region encompassing the Mediterranean basin (captured by the Benin haplotype), and other from sub-Saharan Africa, likely afforded by the transatlantic slave trade (captured by the Senegal and Bantu haplotypes).info:eu-repo/semantics/publishedVersio

Assessing the potential of RNA-based therapeutics for a group of Lysosomal Storage Diseases with neurological involvement

During the first two decades of the 21st century, remarkable progresses have been achieved in the field of RNA-based therapeutics. From antisense RNA to RNA modification, the therapeutic potential of RNA-based technologies has nothing but increased. In our lab, we have been addressing the potential of different RNA-based drugs to either correct or ameliorate the sub-cellular phenotype of a number of severe, life-threatening diseases: the so-called Lysosomal Storage Disorders (LSDs). Among them, we are focusing our efforts on those which present with a predominant neurological phenotype, since there are virtually no approved treatments for any of them. Briefly, two major research lines are being pursued: the first relies on the design of mutation-specific approaches to correct abnormal splicing processes in LSD-related genes, whenever they underlie pathology. The second depends upon selective downregulation of genes involved in the biosynthethic cascades that give origin to the substrates that accumulate in each pathology. Here we present an overview on our results with both approaches on Sanfilippo syndrome, a sub-group of severe neurodegenerative LSDs. For the mutation-specific, splicing correction approach, we are using U1snRNA vectors to restore the splicing defect caused by the HGSNAT mutation c.234+1G>A, that leads to Sanfilippo C disease. We started by demonstrating in vitro that a modified U1snRNA vector designed to improve the definition of HGSNAT exon 2 could partially restore its normal splicing process. Now, we are evaluating its therapeutic potential in vivo, in mice expressing the human splicing defect. For the substrate reduction approach, we are using siRNAs. By acting over a specific biosynthethic cascade, siRNAs promote an overall decrease of the accumulating substrate. So far, we have already tested this approach in patients’ fibroblasts and observed a high inhibition of the target mRNAs and a decrease in storage. Overall, there are substantial differences between these two approaches but they also face common challenges and show equally promising results.FCT (SFRH/BPD/101965/2014; SFRH/BD/124372/2016)N/

Avaliação de alterações na parede celular de frutos de pêra ‘Rocha’ como mecanismo de defesa contra podridões de pós-colheita, em controlo biológico

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2015As doenças pós-colheita de frutos, causadas por fungos, reduzem drasticamente a produção comercializável, representando significativos prejuízos económicos. A utilização de métodos de controlo biológico, com recurso a microrganismos antagonistas, tem vindo a ser estudada como uma estratégia alternativa ao uso de fungicidas. Em pêra ‘Rocha’, a espécie Aureobasidium pullulans mostrou-se eficaz como agente de biocontrolo contra a podridão azul causada pelo fungo Penicillium expansum embora os mecanismos subjacentes à sua acção na indução de respostas de defesa no hospedeiro permaneçam por elucidar. Com o presente trabalho pretendeu-se investigar, a nível bioquímico e molecular, a hipótese da parede celular das células do fruto desempenhar um papel no mecanismo de defesa ao restringir a progressão da infecção, após biotratamentos envolvendo o A. pullulans. Através de 3 abordagens complementares - quantificação de i) capacidade antioxidante e de compostos fenólicos, ii) actividade de enzimas modificadoras da parede celular por espectrofotometria e viscosimetria, e iii) expressão de genes envolvidos em alterações da firmeza da polpa por PCR quantitativo em tempo real, os resultados obtidos sugerem a existência de uma resposta do hospedeiro, dependente da sua exposição ao agente antagonista. Foi observado um decréscimo diferencial de algumas moléculas com capacidade antioxidante, o que sugere um aumento da lenhificação dependente da condição ensaiada, sendo mais significativo em amostras inoculadas com o patogénio. Os resultados de actividades enzimáticas totais e de expressão génica sugerem uma redução de despolimerização da parede celular e/ou aumento de respostas de defesa através da libertação de moléculas sinalizadoras, promovidos pelo A. pullulans. Estes dados suportam-se ainda por observações morfológicas nos vários tratamentos, onde se constatou que, na presença dos fungos em sinergia nos tratamentos de biocontrolo, a área necrótica era restrita ao local da inoculação, sugerindo um reforço da parede celular, bloqueando assim a proliferação do patogénio nos tecidos do hospedeiro.Post-harvest diseases of fruit caused by fungi, drastically reduce marketable production, representing significant economic losses. The application of biological control methods, with the use of antagonist microorganisms has been studied as an alternative strategy to the use of fungicides. In pear 'Rocha', the species Aureobasidium pullulans was effective as a biocontrol agent against blue rot caused by the fungus Penicillium expansum although the mechanisms underlying its action in inducing defense responses in the host remain to be elucidated. The present study was intended to investigate the biochemical and molecular level, the hypothesis of cell walls of fruit cells play a role in defense mechanism to restrict the progression of infection after biotreatment involving A. pullulans. Through 3 complementary approaches – quantification of i) antioxidant capacity and phenolics ii) modifying enzyme activity of the cell wall by spectrophotometry and viscometry, and iii) expression of genes involved in changes of firmness by quantitative real-time PCR, the results obtained suggest the existence of a host response, dependent on its exposure to antagonist agent. A differential decrease of some molecules with antioxidant activity, suggesting an increased lignification dependent on the condition tested, being most significant for samples inoculated with the pathogen. The results of total enzyme activities and gene expression suggest a reduction in depolymerisation of the cell wall and / or increased defense responses through the release of signaling molecules promoted by A. pullulans. These data support itself by morphological features in the various treatments, where it was found that in the presence of the biocontrol fungi in synergy treatments, the necrotic area was restricted to the inoculation site, suggesting a strengthening of the cell wall, thus blocking proliferation the pathogen in the host tissues

Newborn screening of hemoglobinopathies in Bengo, Angola

Sickle cell anemia (SCA) is caused by the presence of the sickle cell allele-HBB*S, in homozygosity. In sub-Saharan Africa, HBB*S typically reaches high frequencies, especially in regions where malaria is endemic. Epidemiologic evidence indicates that the burden of SCA, which currently represents a dramatic public health concern in sub-Saharan Africa, will predictably increase in the future. In this sense, the determination of prevalence, as well as markers of disease severity, are necessary so that the Ministry of Health can develop correct programs to manage the disease. The aim of the study: The objective of this work was to screen by direct sequencing of the HBB gene in a sample of newborns from Bengo, Angola. Moreover, we aim to identify common haplotypes of SCA by Multiplex SNaPshot system.info:eu-repo/semantics/publishedVersio

Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studies

Mucolipidosis type II alpha/beta (ML II) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II. In a previous in vitro study in ML II patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 with AO at 25 mg/kg and groups 3 and 6 with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 or 7 days post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24/48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.FCTN/

Newborn screening of hemoglobinopathies in Bengo, Angola

Sickle cell anemia (SCA) is caused by the presence of the sickle cell allele-HBB*S, in homozygosity. In sub-Saharan Africa, HBB*S typically reaches high frequencies, especially in regions where malaria is endemic. Epidemiologic evidence indicates that the burden of SCA, which currently represents a dramatic public health concern in sub-Saharan Africa, will predictably increase in the future. In this sense, the determination of prevalence, as well as markers of disease severity, are necessary so that the Ministry of Health can develop correct programs to manage the disease. The aim of the study: The objective of this work was to screen by direct sequencing of the HBB gene in a sample of newborns from Bengo, Angola. Moreover, we aim to identify common haplotypes of SCA by Multiplex SNaPshot system.info:eu-repo/semantics/publishedVersio

Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.M.F. Coutinho is grantee from the FCT (SFRH/BPD/101965/2014).info:eu-repo/semantics/publishedVersio

Unverricht-Lundborg disease: development of splicing therapeutic approaches for a patient with an homozygous mutation in the cystatin B gene

Unverricht-Lundborg disease (ULD or EPM1) is the most common form of progressive myoclonic epilepsy (PME) worldwide. It is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion, missense, nonsense, frameshift and mutations that may lead to alternative splicing have been described as causal of EPM1. Recently, our group described an ULD patient who is homozygous for a new synonymous mutation (c.66G>A; p.Q22Q) located at the last nucleotide of exon 1. The transcriptional profile analysis allowed the identification of two CSTB splice variants, one of normal size with the G>A change and other with partial inclusion of intron 1 due to activation of a cryptic splice-site inside the intronic sequence. To correct the splice defect, here we developed antisense oligonucleotide and U1snRNA mediated therapeutic strategies. U1 is required for splice donor site (SDS) recognition of pre-mRNAs and initiates the splicing process. The mutation c.66G>A interferes with the recognition of the SDS by U1. In a first approach, to reduce missplicing from CSTB gene, we generated four U1 construct isoforms with increasing complementarity to the SDS. Transfection of patient-derived fibroblasts with different concentrations of the adapted U1 vectors did not allowed the correction of the aberrant transcript. In a second strategy, we have designed a specific lock nucleic-acid (LNA) oligonucleotide to block the activated cryptic splice-site in intron 1. Normal splicing pattern of a single transcript with the synonymous change G>A was successfully rescued after LNA transfection in patient cells. The therapeutic effect showed to be dose-dependent. These results suggest that antisense therapy might be a potential alternative or adjunct treatment strategy for patients holding splicing changes in CSTB gene. As far as we know this is the first report of a patient tailored therapy in cells of an ULD patient