Caracterização funcional de variantes genéticas ligadas ao Angioedema Hereditário
Data
2024-12-17
Autores
Marins, Maryana Mara 
Orientadores
Pesquero, João Bosco
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Angioedema Hereditário (AEH) é uma doença genética rara não monogênica sendo os tipos I e II os mais prevalentes. Os tipos I e II tem como causa variantes genéticas patogênicas no gene SERPING1, o que resulta na deficiência quantitativa e ou funcional do inibidor C1 (C1-INH). A característica clínica clássica de ambos os tipos são crises de edema em diversos locais do corpo, com gravidade, duração e frequência variáveis, podendo ser letal. Objetivo: a caracterização de uma grande deleção, bem como o estudo funcional das variantes do tipo SNV p.G184R, p.A297T, p.A457P e p.A459D no gene SERPING1. Métodos: A caracterização da grande deleção foi realizada utilizando as técnicas de sequenciamento de Sanger, MLPA, TQE, dosagem de atividade sérica da C1 esterase, western blotting e estudo de segregação familiar. Para o estudo funcional das SNVs, plasmídeos contendo as variantes pontuais foram gerados por mutação sítio dirigida, clonados e transfectados na linhagem celular HepG2. Resultados: A caracterização da grande deleção mostrou que a mesma compreende aproximadamente 6.000pb, resultando na deleção completa dos éxons 5 e 6, que altera o quadro de leitura do SERPING1 e gera um códon de parada prematuro após 24 aminoácidos, denominada p.Asp229Glyfs*24. Essa variante é considerada patogênica e relacionada ao AEH do tipo I. Os mutantes das SNVs foram validados por sequenciamento de Sanger e após cultivo das células HepG2, a expressão dos C1-INHs mutantes foi avaliada por qPCR, western blotting e espectrofotometria (dosagem enzimática indireta do C1-INH). O estudo funcional das SNVs mostrou que as variantes p.G184R, p.A457P e p.A459D podem ser consideradas patogênicas. Com os testes realizados até o momento, ainda não se pode afirmar que a variante p.A297T, isoladamente, seja patogênica. Conclusão: Esse estudo destacou a importância da análise de variantes no gene SERPING1. Nossos achados identificaram variantes causadoras da doença, o que reforça que a incorporação de testes genéticos pode contribuir para avanços no aconselhamento genético, diagnóstico e tratamento do Angioedema Hereditário.
Hereditary Angioedema (HAE) is a rare, non-monogenic genetic disease, with types I and II being the most prevalent. These types are caused by pathogenic variants in the SERPING1 gene, leading to quantitative or functional deficiencies of the C1 inhibitor (C1-INH). The hallmark clinical feature of both types is recurrent edema episodes in various body regions, with variable severity, duration, and frequency, potentially life-threatening. Objective: To characterize a large deletion and functionally study the variants (SNVs) p.G184R, p.A297T, p.A457P, and p.A459D in the SERPING1 gene. Methods: The large deletion was characterized using the techniques of Sanger sequencing, MLPA, TQE, serum C1 esterase activity measurement, western blotting, and familial segregation analysis. Functional studies of SNVs were performed using site-directed mutagenesis to generate plasmids carrying the variants, which were cloned and transfected into HepG2 cells. Results: The large deletion was identified as encompassing approximately 6,000 bp, resulting in the deletion of exons 5 and 6, causing a frameshift and a premature stop codon after 24 amino acids, designated p.Asp229Glyfs*24. This variant is considered pathogenic and associated with type I HAE. SNV mutants were validated by Sanger sequencing, and expression of C1-INH mutants was evaluated by qPCR, western blotting, and spectrophotometry (indirect enzymatic assay of C1-INH). Functional studies showed that variants p.G184R, p.A457P, and p.A459D are pathogenic. Based on the tests conducted so far, it cannot yet be confirmed that the p.A297T variant is pathogenic. Conclusion: This study highlights the importance of analyzing variants in the SERPING1 gene. Our findings identified disease-causing variants, emphasizing the role of genetic testing in advancing genetic counseling, diagnosis, and treatment of Hereditary Angioedema.
Hereditary Angioedema (HAE) is a rare, non-monogenic genetic disease, with types I and II being the most prevalent. These types are caused by pathogenic variants in the SERPING1 gene, leading to quantitative or functional deficiencies of the C1 inhibitor (C1-INH). The hallmark clinical feature of both types is recurrent edema episodes in various body regions, with variable severity, duration, and frequency, potentially life-threatening. Objective: To characterize a large deletion and functionally study the variants (SNVs) p.G184R, p.A297T, p.A457P, and p.A459D in the SERPING1 gene. Methods: The large deletion was characterized using the techniques of Sanger sequencing, MLPA, TQE, serum C1 esterase activity measurement, western blotting, and familial segregation analysis. Functional studies of SNVs were performed using site-directed mutagenesis to generate plasmids carrying the variants, which were cloned and transfected into HepG2 cells. Results: The large deletion was identified as encompassing approximately 6,000 bp, resulting in the deletion of exons 5 and 6, causing a frameshift and a premature stop codon after 24 amino acids, designated p.Asp229Glyfs*24. This variant is considered pathogenic and associated with type I HAE. SNV mutants were validated by Sanger sequencing, and expression of C1-INH mutants was evaluated by qPCR, western blotting, and spectrophotometry (indirect enzymatic assay of C1-INH). Functional studies showed that variants p.G184R, p.A457P, and p.A459D are pathogenic. Based on the tests conducted so far, it cannot yet be confirmed that the p.A297T variant is pathogenic. Conclusion: This study highlights the importance of analyzing variants in the SERPING1 gene. Our findings identified disease-causing variants, emphasizing the role of genetic testing in advancing genetic counseling, diagnosis, and treatment of Hereditary Angioedema.
Descrição
Citação
MARINS, Maryana Mara. Caracterização funcional de variantes genéticas ligadas ao Angioedema Hereditário. 2024. 87 f. Tese (Doutorado em Ciências Biológica: Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.
ODS
Não se aplica