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Abstract(s)
A febre Q é uma zoonose causada por Coxiella burnetii, na qual cerca de 60% dos doentes
apresentam quadros clínicos de infecção aguda, com recuperação após intervenção
clínica. No entanto, nos casos de febre Q crónica, a infecção culmina em complicações
graves, frequentemente endocardites. Mesmo com medidas preventivas, a incidência da
febre Q tem vindo a aumentar, principalmente na Península Ibérica. A fisiopatologia da
infecção por C. burnetii assemelha-se à do Mycobacterium tuberculosis. Em ambas as
infecções, a via interleucina12—interferão gama (IL12-IFN?) desempenha um papel
importante na sobrevivência e replicação intracelular bacteriana. As doenças causadas
por micobactérias apresentam homogeneidade fisiológica inerente à deficiência de
interferão gama (IFN?), o que é fundamentado pela identificação de determinantes
genéticos dessas infecções na via IL12-IFN?. No entanto, esta via permanece
praticamente inexplorada em termos de associação genética com a febre Q crónica,
enaltecendo a necessidade destes estudos. Assim, nesta investigação procurou-se
identificar variantes associadas a febre Q crónica através de uma abordagem por estudos
populacionais de associação genótipo fenótipo com base em genes candidatos
(Candidate Gene Association Studies - CGAS).
Esta abordagem insere-se numa arquitetura de estudo caso-controlo. Selecionaram-se
genes candidatos e optou-se pela utilização de uma estratégia de sequenciação de nova
geração (Next Generation Sequencing – NGS) para a genotipagem das regiões 5’UTR e
os exões dos genes candidatos. Após consentimento informado, foram recrutados 34
indivíduos saudáveis e 60 doentes com febre Q (43 formas agudas e 17 formas crónicas).
Os genes do receptor ß1 de interleucina-12 (IL12RB1), receptor 1 de interferão gama
(IFN?R1) e receptor 2 de interferão gama (IFN?R2), com fragmentos de 2kb a 17kb,
foram amplificados por polimerase de reação em cadeia (PCR) de longo alcance com
ácido desoxirribonucleico (ADN) polimerase Takara LA Taq® para construção de
bibliotecas Nextera e sequenciação com tecnologia da Illumina®. Uma vez que a NGS
trabalha com milhares de milhões de dados em simultâneo requer a aplicação de várias
métricas de controlo de qualidade de modo a garantir resultados fidedignos. Para além
destas métricas foram ainda implementadas métricas específicas para CGAS que têm por
objectivo diminuir a obtenção de erros por falsos positivos na análise estatística.
Do presente trabalho de investigação resultou uma lista de polimorfismos de nucleótido
único (Single Nucleotide Polymorphism - SNP) dos genes IL12RB1, IFN?R1 e IFN?R2
em indivíduos com febre Q crónica, aguda e numa amostra de conveniência de indivíduos saudáveis. Estes dados têm a necessária qualidade para constituir uma mais valia,
podendo ser aplicados a diferentes estudos.
Futuramente, esta lista de SNPs será usada em estudos de associação genótipo-fenótipo
para compreender se existem variantes na via IL12-IFN? associadas à apresentação
clínica doença. Assim, estes resultados poderão vir a ser um importante avanço clínico
não só para antecipar formas crónicas da doença como para o desenvolvimento de
estratégias de prevenção e para prestação de melhores cuidados de saúde a estes doentes.
Q fever is a zoonosis caused by Coxiella burnetii, in which about 60% of the affected patients develop acute Q fever, recovering after clinical intervention. However, in chronic Q fever cases, the disease is accompanied with serious complications, mainly manifested as endocarditis. Even with preventive measures, the incidence of Q fever has been increasing, namely in the Iberian Peninsula. The pathophysiology of C. burnetii infection resembles that of Mycobacterium tuberculosis, both characterized by granuloma formation. In this context, the interleukin 12 - interferon ? (IL12-IFN?) pathway plays a major role in intracellular bacterial survival and replication. Diseases caused by mycobacteria display physiological homogeneity inherent to interferon ? (IFN-?) deficiency, since genetic determinants of these infections have been identified in the IL12-IFN? pathway. Despite of the above mentioned, the association between genetic variants this pathway and chronic Q fever remains practically unexplored. Thus, in this investigation we seek to establish a list of variants within the IL12-IFN? pathway with pathogenic potential associated with the development of chronic Q fever. therefore, we sought to identify variants associated with chronic Q fever resorting to candidate-gene, population association analysis in a Candidate Gene Association Studies (CGAS) approach. This approach is part of a case-control study architecture. Candidate genes were selected and a Next Generation Sequencing (NGS) strategy was chosen for the genotyping of the 5’UTR region and exons of candidate genes. After informed consent, 34 healthy individuals and 60 Q fever patients (43 acute and 17 chronic forms) were recruited. The genes interleukin-12 receptor ß1 (IL12RB1), interferon ? receptor 1 (IFN?R1) and interferon ? receptor 2 (IFN?R2), were amplified in 2kb to 17kb fragments, by long-range polymerase chain reaction (PCR) with Takara LA Taq® deoxyribonucleic acid (DNA) polymerase for Nextera library construction and Illumina® sequencing. Since NGS works simultaneously with billions of data points, it requires the application of several quality control metrics in order to guarantee reliable results. In addition to these metrics, specific metrics for CGAS were also implemented to reduce possible false positives in the statistical analysis of the data. The present research work provided a list of single nucleotide polymorphisms (SNPs) of the IL12RB1, IFN?R1 and IFN?R2 genes, observed in patients with chronic Q fever, acute Q fever and in a convenience sample of healthy individuals. These data have the necessary quality to constitute an asset and can be applied to different studies. In the future, this list of SNPs will be used in genotype-phenotype association studies to understand whether there are variants in the IL12-IFN? pathway associated with the clinical presentation of the disease. Thus, these results could prove to be an important clinical advance not only to anticipate chronic forms of the disease but also to develop prevention strategies and to provide better health care to these patients.
Q fever is a zoonosis caused by Coxiella burnetii, in which about 60% of the affected patients develop acute Q fever, recovering after clinical intervention. However, in chronic Q fever cases, the disease is accompanied with serious complications, mainly manifested as endocarditis. Even with preventive measures, the incidence of Q fever has been increasing, namely in the Iberian Peninsula. The pathophysiology of C. burnetii infection resembles that of Mycobacterium tuberculosis, both characterized by granuloma formation. In this context, the interleukin 12 - interferon ? (IL12-IFN?) pathway plays a major role in intracellular bacterial survival and replication. Diseases caused by mycobacteria display physiological homogeneity inherent to interferon ? (IFN-?) deficiency, since genetic determinants of these infections have been identified in the IL12-IFN? pathway. Despite of the above mentioned, the association between genetic variants this pathway and chronic Q fever remains practically unexplored. Thus, in this investigation we seek to establish a list of variants within the IL12-IFN? pathway with pathogenic potential associated with the development of chronic Q fever. therefore, we sought to identify variants associated with chronic Q fever resorting to candidate-gene, population association analysis in a Candidate Gene Association Studies (CGAS) approach. This approach is part of a case-control study architecture. Candidate genes were selected and a Next Generation Sequencing (NGS) strategy was chosen for the genotyping of the 5’UTR region and exons of candidate genes. After informed consent, 34 healthy individuals and 60 Q fever patients (43 acute and 17 chronic forms) were recruited. The genes interleukin-12 receptor ß1 (IL12RB1), interferon ? receptor 1 (IFN?R1) and interferon ? receptor 2 (IFN?R2), were amplified in 2kb to 17kb fragments, by long-range polymerase chain reaction (PCR) with Takara LA Taq® deoxyribonucleic acid (DNA) polymerase for Nextera library construction and Illumina® sequencing. Since NGS works simultaneously with billions of data points, it requires the application of several quality control metrics in order to guarantee reliable results. In addition to these metrics, specific metrics for CGAS were also implemented to reduce possible false positives in the statistical analysis of the data. The present research work provided a list of single nucleotide polymorphisms (SNPs) of the IL12RB1, IFN?R1 and IFN?R2 genes, observed in patients with chronic Q fever, acute Q fever and in a convenience sample of healthy individuals. These data have the necessary quality to constitute an asset and can be applied to different studies. In the future, this list of SNPs will be used in genotype-phenotype association studies to understand whether there are variants in the IL12-IFN? pathway associated with the clinical presentation of the disease. Thus, these results could prove to be an important clinical advance not only to anticipate chronic forms of the disease but also to develop prevention strategies and to provide better health care to these patients.
Description
Keywords
Controlo de Qualidade de Dados
Genotípicos Coxiella Burnetii Estudos Populacionais de Associação Genótipo-Fenótipo Com Base Em Genescandidatos Febre Q Genética Humana da Suscetibilidade à Infecção Imunidade Inata Sequenciação de Nova Geração Variação Genética do Hospedeiro Humano Via Biológica Il12-
Ifn?