Caracterização funcional de variantes presentes no gene GLA de pacientes com suspeita de Doença de Fabry
Arquivos
Data
2023-01-27
Autores
Mendes, Agatha Ribeiro 
Orientadores
Pesquero, João Bosco
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A Doença de Fabry é uma patologia rara causada por variantes presentes no gene GLA, localizado no braço longo do cromossomo X. Variantes patogênicas nesse gene levam a uma atividade nula ou reduzida em diferentes níveis da enzima alfa-galactosidase A (α-Gal A), causando um acúmulo de globotriaosilceramida (Gb3) nos lisossomos. Essa doença apresenta duas classificações de fenótipo, clássico e o não clássico, sendo o primeiro a forma mais grave e precoce. Devido aos sintomas serem multissistêmicos, o diagnóstico dessa doença é complexo, podendo demorar dez anos ou mais para ser efetivado. O diagnóstico preconizado trata-se da avaliação da atividade enzimática e análise molecular do gene GLA para pessoas do sexo masculino. Já para as pessoas do sexo feminino, devido a inativação aleatória do cromossomo X, é realizada apenas a análise molecular. No entanto, devido a elevada quantidade de variantes restritas às famílias, o diagnóstico molecular ainda assim pode não ser suficiente para a comprovação da patogenicidade das variantes, sendo necessária a realização de testes funcionais. Objetivo: Esse projeto visou a caracterização funcional de variantes encontradas no gene GLA quanto a sua potencial patogenicidade na Doença de Fabry. Métodos: Foram analisadas oito variantes encontradas em pacientes analisados em nosso laboratório, através da construção de vetores contendo as variantes e avaliação da atividade da enzima alfa-galactosidase obtida de células transfectadas com esses vetores. Além delas, foi incluída na análise uma variante já conhecida como causadora da doença. Resultado: O estudo observou uma atividade residual deficiente da enzima em todas as variantes estudadas, quando comparado a um controle selvagem, sendo que duas delas, a p.F273S e a p.Q279L apresentaram atividade residual com valores próximos ao controle positivo, causador da doença. Conclusão: Com esses dados, foi possível concluir que todas as variantes presentes nesse estudo são responsáveis por uma atividade deficiente ou nula da enzima e podem contribuir para o desenvolvimento da Doença de Fabry.
Fabry disease is a rare pathology caused by variants present in the GLA gene, located on the long arm of the X chromosome. Pathogenic variants in this gene lead to null or reduced activity at different levels of the enzyme alpha-galactosidase A (α-Gal A), causing an accumulation of globotriaosylceramide (Gb3) in lysosomes. This disease has two phenotype classifications, classic and non-classical, the first being the most severe and early form. Because the symptoms are multisystemic, the diagnosis of this disease is complex and may take ten years or more to be effective. The recommended diagnosis is the evaluation of enzymatic activity and molecular analysis of the GLA gene for male people. As for female people, due to random inactivation of the X chromosome, only molecular analysis is performed. However, due to the high number of variants restricted to families, molecular diagnosis may still not be sufficient to prove the pathogenicity of the variants, requiring functional tests. Objective: This project aimed at the functional characterization of variants found in the GLA gene regarding their potential pathogenicity in Fabry disease. Methods: Eight variants found in patients analyzed in our laboratory were analyzed, through the construction of vectors containing the variants and evaluation of the alpha-galactosidase enzyme activity obtained from cells transfected with these vectors. In addition to them, a variant already known to cause disease was included in the analysis. Result: The study observed a deficient residual activity of the enzyme in all variants studied, when compared to a wild-type control, and two of them, p.F273S and p.Q279L, showed residual activity with values close to the positive control, disease-causing. Conclusion: With these data, it was possible to conclude that all the variants present in this study are responsible for a deficient or null activity of the enzyme and can contribute to the development of Fabry Disease.
Fabry disease is a rare pathology caused by variants present in the GLA gene, located on the long arm of the X chromosome. Pathogenic variants in this gene lead to null or reduced activity at different levels of the enzyme alpha-galactosidase A (α-Gal A), causing an accumulation of globotriaosylceramide (Gb3) in lysosomes. This disease has two phenotype classifications, classic and non-classical, the first being the most severe and early form. Because the symptoms are multisystemic, the diagnosis of this disease is complex and may take ten years or more to be effective. The recommended diagnosis is the evaluation of enzymatic activity and molecular analysis of the GLA gene for male people. As for female people, due to random inactivation of the X chromosome, only molecular analysis is performed. However, due to the high number of variants restricted to families, molecular diagnosis may still not be sufficient to prove the pathogenicity of the variants, requiring functional tests. Objective: This project aimed at the functional characterization of variants found in the GLA gene regarding their potential pathogenicity in Fabry disease. Methods: Eight variants found in patients analyzed in our laboratory were analyzed, through the construction of vectors containing the variants and evaluation of the alpha-galactosidase enzyme activity obtained from cells transfected with these vectors. In addition to them, a variant already known to cause disease was included in the analysis. Result: The study observed a deficient residual activity of the enzyme in all variants studied, when compared to a wild-type control, and two of them, p.F273S and p.Q279L, showed residual activity with values close to the positive control, disease-causing. Conclusion: With these data, it was possible to conclude that all the variants present in this study are responsible for a deficient or null activity of the enzyme and can contribute to the development of Fabry Disease.