Desenvolvimento da técnica de quantificação de exons (TQE) para a detecção de grandes deleções e inserções no gene SERPING1 para o diagnóstico de angiodema hereditário

• Ano de defesa: 2017
• Autor(a) principal: Amorim, Priscila Nicolicht de [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Exons; Deleção de fragmentos do gene; Angioedema hereditário; Análise de fragmentos; Angioedemas hereditários; Reação em cadeia da polimerase; Serpingl; Angioedemas, hereditary; Multiplex polymerase chain reaction; Polymerase chain reaction
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3882484; http://repositorio.unifesp.br/handle/11600/48484

Hereditary angioedema (HAE) is a genetic disease caused by mutations in SERPING1 gene encoding the inhibitor of C1 esterase (C1-INH), determining the quantitative and/or qualitative deficiency in C1-INH, largely caused by point mutations and approximately 15 -20%, related to the major gene rearrangements. The intronic gene regions have 17 sequences of repetitive elements represented by Alu, making it prone to deletions and insertions gene/duplications. To enable a semiquantitative molecular diagnosis for SERPING1 gene with the capacity to detect large gene rearrangements, was developed and standardized one technique to quantity the exons (EOT). Through capillary electrophoresis was performed separation and quantification of different sized fragments amplified by Multiplex PCR-technique associated with the use of primers labeled with a fluorescent molecule. It was selected sample of blood from a family with clinical history of HAE, low serum C4 and C1-INH, and who were admitled to molecular analysis of SERPING1 gene by sequencing Sanger in our Center. Samples of this family showed no pathogenic known change in SERPING1 gene by Sanger sequencing technique. So, they were analyzed by TOE confirmed by PCR and MLPA Long Range. Th.[ough TOE was deteded deletion in exon 4 in the patient 12 and confirmed by the MLPA. The deleted fragment was separated by Long Range PCR and subsequently by Sanger sequencing, were found the location and size of the deletion. Therefore, the quantification technique Exons (TOE) proved to be an efficient method for the detection of large deletions or insertions/duplication with power efficiency is simple and fast implementation, in addition to a benefit cost. These data show that SO technique successfully developed for the deletion analysis involving SERPING1 gene, serving as support technique in routine laboratory diagnosis of HAE and may be further standardized and applied to other genetic diseases, contributing to the research and detection major gene rearrangements.