Doença de Kawasaki e infecção por SARS-CoV-2 em crianças: avaliação clínica e de marcadores genéticos associados ao desenvolvimento da doença
Data
2024-05-09
Tipo
Dissertação de mestrado
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Objetivos: Este estudo tem como objetivo realizar análise genética de pacientes pediátricos diagnosticados com doença de Kawasaki (DK) após o início de pandemia por SARS-CoV-2, através de extração de DNA, sequenciamento e análise de regiões de interesse do exoma. É de grande relevância caracterizarmos o perfil genético dos pacientes no nosso meio e avaliarmos os grupos com potencial de maior gravidade de doença, podendo no futuro direcionar melhores opções de diagnóstico e de terapêutica. Casuística e método: Foram incluídos no estudo pacientes até 20 anos de idade com diagnóstico de DK completa ou incompleta de acordo com os critérios da American Heart Association, no período da pandemia da COVID-19. A coleta foi realizada entre março de 2020 e outubro de 2021. Dados clínicos e laboratoriais foram extraídos do prontuário. Foi realizada coleta de sangue dos pacientes, em seguida realizada extração de DNA com o Kit Qiagen segundo as instruções do fabricante; após extração, amostras foram armazenadas em temperatura de -80 °C no Laboratório de Pesquisas da Disciplina de Infectologia Pediátrica da Universidade Federal de São Paulo. O DNA genômico extraído sofreu uma fragmentação seguida de indexação, captura e enriquecimento com sondas desenhadas para o genoma humano com kit de preparo de biblioteca Nextera DNA Flex (Nextera DNA Library Preparation Kit). O sequenciamento de nova geração (NGS) das sequências-alvo foi realizado utilizando a plataforma NextSeq 550 (Illumina, San Diego, CA, EUA). Após revisão de literatura, genes de interesse relacionados à DK e desenvolvimento de MIS-C e DK associado à COVID-19 foram selecionados. As variantes encontradas foram analisadas nas principais bases de dados genéticos e classificadas em ‘patogênicas’, ‘provavelmente patogênicas’, ‘significado incerto’, ‘provavelmente benignas’, e 'benignas’ de acordo com critérios estabelecidos pelo Colégio Americano de Genética Médica e Genômica. Resultados: Foram selecionados 25 pacientes com diagnóstico de DK, dos quais 15 (60%) pacientes preencheram critérios para DK completo e 10 (40%), para incompleto. Além disso, 8 (32%) pacientes foram classificados como COVID+, pois apresentavam RT-PCR positivo para SARS‐CoV‐2 e/ou sorologia (IgM, IgG ou IgA) positiva. Foi realizada a extração e análise de exoma dos 25 pacientes e selecionadas zonas de interesse para análise de acordo com mutações identificadas em estudos anteriores. Foram encontradas duas mutações, no gene ABCA4 e no gene ABCC6, classificadas como provavelmente patogênicas, em dois pacientes, respectivamente. No total, 12 variantes de significado incerto (VUS) foram encontradas em 11 pacientes. Nos restantes 14 pacientes não foram encontradas mutações significativas. Conclusão: Dessa forma, no presente estudo, realizamos extração de DNA com realização de exoma e posterior análise de genes de interesse de 25 pacientes com diagnóstico de DK. Relatamos também suas características clínicas e laboratoriais. Foram encontradas duas mutações classificadas como provavelmente patogênicas em dois pacientes. No total, 12 VUS foram encontradas em 11 pacientes. Estudos posteriores se fazem necessários para que possamos evidenciar características genéticas próprias da população brasileira que possam influenciar no surgimento da DK.
Objective: This study aims to perform a genetic analysis of pediatric patients diagnosed with Kawasaki Disease (KD) after the start of the SARS-CoV-2 pandemic, through DNA extraction, sequencing and analysis of regions of interest in the exome. It is of great importance to characterize the genetic profile of patients in our environment and evaluate groups with the potential for greater severity of the disease, being able to direct better diagnostic and therapeutic options in the future. Methods: Patients up to 20 years of age diagnosed with complete or incomplete KD according to the American Heart Association criteria, during the COVID-19 pandemic, were included in the study. Collection was carried out between March 2020 and October 2021. Clinical and laboratory data were extracted from the medical records. Blood was collected from the patients, followed by DNA extraction using the Qiagen Kit according to the manufacturer's instructions; After extraction, samples were stored at -80 °C in the Research Laboratory of the Pediatric Infectious Diseases Discipline at the Federal University of São Paulo. The extracted genomic DNA underwent fragmentation followed by indexing, capture and enrichment with probes designed for the human genome with the Nextera DNA Flex library preparation kit (Nextera DNA Library Preparation Kit). Next-generation sequencing (NGS) of target sequences was performed using the NextSeq 550 platform (Illumina, San Diego, CA, USA). After reviewing the literature, genes of interest related to KD and the development of MIS-C and KD associated with COVID-19 were selected. The variants found were analyzed in the main genetic databases and classified as 'pathogenic', 'probably pathogenic', 'uncertain significance', 'probably benign', and 'benign' according to criteria established by the American College of Medical Genetics and Genomics. Results: 25 patients diagnosed with KD were selected, of which 15 (60%) patients met criteria for complete KD and 10 (40%) for incomplete KD. Furthermore, 8 (32%) patients were classified as COVID+, as they had a positive RT-PCR for SARS-CoV-2 and/or positive serology (IgM, IgG or IgA). Exome extraction and analysis of the 25 patients was performed and areas of interest were selected for analysis according to mutations identified in previous studies. Two mutations were found, in the ABCA4 gene and the ABCC6 gene, classified as probably pathogenic, in two patients, respectively. In total 12 variants of uncertain significance (VUS) were found in 11 patients. In the remaining 14 patients, no significant mutations were found. Conclusion: Therefore, in the present study, we performed DNA extraction with exome analysis and subsequent analysis of genes of interest from 25 patients diagnosed with KD. We also report their clinical and laboratory characteristics. Two mutations classified as probably pathogenic were found in two patients. In total, 12 VUS were found in 11 patients. Further studies are necessary so that we can highlight genetic characteristics specific to the Brazilian population that may influence the emergence of KD.
Objective: This study aims to perform a genetic analysis of pediatric patients diagnosed with Kawasaki Disease (KD) after the start of the SARS-CoV-2 pandemic, through DNA extraction, sequencing and analysis of regions of interest in the exome. It is of great importance to characterize the genetic profile of patients in our environment and evaluate groups with the potential for greater severity of the disease, being able to direct better diagnostic and therapeutic options in the future. Methods: Patients up to 20 years of age diagnosed with complete or incomplete KD according to the American Heart Association criteria, during the COVID-19 pandemic, were included in the study. Collection was carried out between March 2020 and October 2021. Clinical and laboratory data were extracted from the medical records. Blood was collected from the patients, followed by DNA extraction using the Qiagen Kit according to the manufacturer's instructions; After extraction, samples were stored at -80 °C in the Research Laboratory of the Pediatric Infectious Diseases Discipline at the Federal University of São Paulo. The extracted genomic DNA underwent fragmentation followed by indexing, capture and enrichment with probes designed for the human genome with the Nextera DNA Flex library preparation kit (Nextera DNA Library Preparation Kit). Next-generation sequencing (NGS) of target sequences was performed using the NextSeq 550 platform (Illumina, San Diego, CA, USA). After reviewing the literature, genes of interest related to KD and the development of MIS-C and KD associated with COVID-19 were selected. The variants found were analyzed in the main genetic databases and classified as 'pathogenic', 'probably pathogenic', 'uncertain significance', 'probably benign', and 'benign' according to criteria established by the American College of Medical Genetics and Genomics. Results: 25 patients diagnosed with KD were selected, of which 15 (60%) patients met criteria for complete KD and 10 (40%) for incomplete KD. Furthermore, 8 (32%) patients were classified as COVID+, as they had a positive RT-PCR for SARS-CoV-2 and/or positive serology (IgM, IgG or IgA). Exome extraction and analysis of the 25 patients was performed and areas of interest were selected for analysis according to mutations identified in previous studies. Two mutations were found, in the ABCA4 gene and the ABCC6 gene, classified as probably pathogenic, in two patients, respectively. In total 12 variants of uncertain significance (VUS) were found in 11 patients. In the remaining 14 patients, no significant mutations were found. Conclusion: Therefore, in the present study, we performed DNA extraction with exome analysis and subsequent analysis of genes of interest from 25 patients diagnosed with KD. We also report their clinical and laboratory characteristics. Two mutations classified as probably pathogenic were found in two patients. In total, 12 VUS were found in 11 patients. Further studies are necessary so that we can highlight genetic characteristics specific to the Brazilian population that may influence the emergence of KD.
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PINA, Ana Flávia da Silva. Doença de Kawasaki e infecção por SARS-CoV-2 em crianças: avaliação clínica e de marcadores genéticos associados ao desenvolvimento da doença. 2024. 88 f. Dissertação (Mestrado em Pediatria) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.