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Advisor(s)
Abstract(s)
Introdução: A doença das cĂ©lulas falciformes constitui uma desordem sanguĂnea monogĂ©nica e hereditĂĄria, caracterizada pela manifestação de hemoglobina S, a variante mutante da hemoglobina mais prevalente a nĂvel mundial. A anemia falciforme resulta numa homozigotia de hemoglobina S, contribuindo com inĂșmeras complicaçÔes clĂnicas e podendo afetar a integridade do microbioma intestinal, que desempenha um papel fundamental na fisiopatologia da doença. A hidroxiureia tem vindo a ser apontada como um potencial modulador de caracterĂsticas da anemia falciforme podendo induzir determinados efeitos benĂ©ficos no microbioma intestinal. Objetivos: AnĂĄlise dos efeitos da hidroxiureia na composição do microbioma intestinal em doentes com anemia das cĂ©lulas falciformes. Mais ainda, pretende-se avaliar qual a regiĂŁo mais informativa (V1-V2 ou V3-V4) do gene 16S para o estudo da composição do microbioma intestinal.
Materiais e mĂ©todos: O procedimento baseou-se na anĂĄlise de amostras de material fecal recolhidas antes e depois da administração de hidroxiureia em 16 crianças com anemia das cĂ©lulas falciformes. Posteriormente estas amostras foram sequenciadas no equipamento Ilumina NextSeq 550 pela tĂ©cnica de Next Generation Sequencing, aplicando primers para amplificação das regiĂ”es pretendidas (com o total 64 amostras, 32 para cada regiĂŁo). Os resultados foram analisados com recurso ao software STAMP e SPSS. Estas amostras foram tambĂ©m analisadas quanto Ă presença da bactĂ©ria Clostridium difficile, identificada no decorrer da sequenciação. Resultados: Os resultados permitiram observar um aumento de frequĂȘncia de bactĂ©rias benĂ©ficas no microbioma intestinal apĂłs o tratamento, entre elas Blautia luti e Ruminococcus callidus. Verificou-se que os Ăndices de alfa diversidade diminuĂram ligeiramente apĂłs seis meses de administração de hidroxiureia e que a regiĂŁo V3-V4 mostrou ser a mais informativa. ConclusĂŁo: Os resultados demonstram que a terapĂȘutica com hidroxiureia altera o microbioma intestinal, promovendo a frequĂȘncia de espĂ©cies benĂ©ficas. Esta tĂ©cnica revela-se importante para o estudo do microbioma uma vez que Ă© um mĂ©todo mais rĂĄpido permitindo compreender com maior precisĂŁo a sua composição microbiana.
Introduction: Sickle cell disease is a monogenic and hereditary blood disorder characterized by the existence of haemoglobin S, the most prevalent mutant haemoglobin variant worldwide. Sickle cell anaemia results in a homozygotia of haemoglobin S, contributing with numerous clinical complications and affecting the integrity of the intestinal microbiome, which plays a fundamental role in the pathophysiology of the disease Hydroxyurea has been indicated as a potential modulator of sickle cell anaemia characteristics and may induce beneficial effects on the intestinal microbiome. Objectives: Analyse the effects of hydroxyurea therapy on the composition of the intestinal microbiome in patients with sickle cell anaemia. Moreover, it is intended to evaluate which is the most informative region (V1-V2 or V3-V4) of the 16S gene for the study of the gut microbiome composition. Materials and methods: The procedure was based on the analysis of samples of faecal material collected before and after hydroxyurea administration in 16 children with sickle cell anaemia. Subsequently, these samples were sequenced in the equipment Ilumina NextSeq 550 by the technique Next Generation Sequencing, by applying primers for amplification of the desired regions (with a total of 64 samples, 32 for each region), and then the results were processed using the STAMP and SPSS software. These samples were also analysed for the presence of the bacterium Clostridium difficile, identified during sequencing process. Results: The results allowed to observe an increase in the frequency of beneficial bacteria in the intestinal microbiome after treatment, among them Blautia luti and Ruminococcus callidus. Alpha diversity indexes decreased slightly after six months of hydroxyurea administration and that the V3-V4 region proved to be the most informative. Conclusion: The results show that hydroxyurea therapy changes the intestinal microbiome, promoting the frequency of beneficial species. This technique is important for the study of the microbiome as it is a faster method allowing a more precise understanding of the microbial composition.
Introduction: Sickle cell disease is a monogenic and hereditary blood disorder characterized by the existence of haemoglobin S, the most prevalent mutant haemoglobin variant worldwide. Sickle cell anaemia results in a homozygotia of haemoglobin S, contributing with numerous clinical complications and affecting the integrity of the intestinal microbiome, which plays a fundamental role in the pathophysiology of the disease Hydroxyurea has been indicated as a potential modulator of sickle cell anaemia characteristics and may induce beneficial effects on the intestinal microbiome. Objectives: Analyse the effects of hydroxyurea therapy on the composition of the intestinal microbiome in patients with sickle cell anaemia. Moreover, it is intended to evaluate which is the most informative region (V1-V2 or V3-V4) of the 16S gene for the study of the gut microbiome composition. Materials and methods: The procedure was based on the analysis of samples of faecal material collected before and after hydroxyurea administration in 16 children with sickle cell anaemia. Subsequently, these samples were sequenced in the equipment Ilumina NextSeq 550 by the technique Next Generation Sequencing, by applying primers for amplification of the desired regions (with a total of 64 samples, 32 for each region), and then the results were processed using the STAMP and SPSS software. These samples were also analysed for the presence of the bacterium Clostridium difficile, identified during sequencing process. Results: The results allowed to observe an increase in the frequency of beneficial bacteria in the intestinal microbiome after treatment, among them Blautia luti and Ruminococcus callidus. Alpha diversity indexes decreased slightly after six months of hydroxyurea administration and that the V3-V4 region proved to be the most informative. Conclusion: The results show that hydroxyurea therapy changes the intestinal microbiome, promoting the frequency of beneficial species. This technique is important for the study of the microbiome as it is a faster method allowing a more precise understanding of the microbial composition.
Description
Trabalho final de Mestrado para obtenção do grau de Mestre em Engenharia Biomédica
Keywords
Hemoglobina S Anemia das células falciformes Hidroxiureia Microbioma intestinal Gene 16S rRNA Haemoglobin S Sickle cell anaemia Hydroxyurea Gut microbiome 16S rRNA gene
Citation
FERNANDES, Maria Carolina de Olivença Dias - A influĂȘncia da hidroxiureia no microbioma intestinal em doentes com anemia das cĂ©lulas falciformes. Lisboa: Instituto Superior de Engenharia de Lisboa, 2022. Dissertação de Mestrado