Estudo do perfil genético e molecular da cascata de formação da bradicinina e de vias de regulação da permeabilidade do endotélio vascular em famílias brasileiras com angioedema hereditário
Data
2024-07-22
Tipo
Tese de doutorado
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Introdução: O angioedema hereditário (AEH) é uma doença genética rara, com herança autossômica dominante, potencialmente fatal e frequentemente subdiagnosticada. É caracterizado por recorrentes crises de edema sem urtica nos tecidos subcutâneo e submucoso, devido principalmente à desregulação da cascata de formação de bradicinina, que promove a produção de óxido nítrico por células endoteliais, o que provoca aumento da permeabilidade vascular. É causado por variantes patogênicas no gene SERPING1, resultando em deficiência do inibidor de C1 (AEH C1-INH), e nos genes F12, ANGPT1, PLG, KNG1, MYOF e HS3ST6, relacionados ao AEH com C1-INH normal (AEH nC1-INH). No entanto, em muitos indivíduos com AEH nC1-INH, a causa genética é desconhecida (AEH-U). Objetivo: Avaliar o perfil genético e molecular dos componentes envolvidos na cascata de formação de bradicinina e em vias de regulação da permeabilidade do endotélio vascular em indivíduos com sintomas de AEH. Métodos: Um total de 310 indivíduos de 150 famílias não relacionadas foram selecionados. A análise molecular do SERPING1 foi realizada em 87 indivíduos de 29 famílias, e do exon 9 do F12 em 157 indivíduos de 122 famílias. A análise de genes possivelmente relacionados ao AEH por Sequenciamento Completo do Exoma (WES) foi realizada em 23 casos-índice com AEH nC1-INH. Para investigar o impacto de variantes no gene NOS3, encontradas em algumas famílias analisadas em nosso trabalho, na disfunção endotelial, foi realizada mutagênese sítio-dirigida, seguida por transfecção em células EA.hy926. Resultados: Em 53 indivíduos (31 mulheres e 22 homens) de 23 famílias com AEH C1-INH, 20 diferentes variantes patogênicas ou provavelmente patogênicas foram identificadas no SERPING1, incluindo principalmente variantes dos tipos missense e nonsense, além de duas variantes, p.Met441_Gln442delinsIle* e c.551-825_685+396del, descritas pela primeira vez. A variante patogênica p.Thr328Lys no F12 foi identificada em 61 indivíduos (44 mulheres e 17 homens) de 40 famílias com AEH nC1-INH. Não foram identificadas variantes patogênicas no exon 9 do F12 em 80 casos-índice (67 mulheres e 13 homens). A média de tempo entre o início dos sintomas e o diagnóstico genético foi de 23,9 e 11,5 anos para pacientes com AEH C1-INH e AEH-FXII, respectivamente. Além disso, o WES identificou 55 variantes raras em 35 genes. Destas, 34 variantes em 23 genes foram validadas por sequenciamento de Sanger. Algumas variantes, encontradas nos genes ADGRE2, CPN1, HS3ST6, KNG1, NOS3, PLG, e SYTL2 podem ser candidatas a estarem envolvidas na patogênese do AEH. Apesar de serem consideradas como variantes raras, as análises, por ferramentas computacionais e por ensaios funcionais, do efeito patogênico das alterações p.Ala422Thr, p.Ala423Thr e p.Arg885Met identificadas no NOS3 mostraram-se inconclusivas. Conclusões: Nossos resultados evidenciam a diversidade de variantes patogênicas no SERPING1 e que 33% das famílias de AEH nC1-INH são portadoras da variante patogênica que afeta a atividade funcional do FXII, enquanto a maioria não apresenta diagnóstico genético confirmado. Elucidar os mecanismos de modulação das crises de angioedema, a partir da investigação de novas variantes, pode ampliar o conhecimento da fisiopatologia do AEH, contribuindo para o diagnóstico, geração de estratégias terapêuticas específicas e aconselhamento genético nas famílias afetadas.
Introduction: Hereditary angioedema (HAE) is a rare genetic disease, with autosomal dominant inheritance, life-threatening, and frequently underdiagnosed. It is characterized by recurrent subcutaneous and submucosal tissue swelling without hives, mainly due to dysregulation of bradykinin-forming cascade, which promotes production of nitric oxide by endothelial cells, leading to increased vascular permeability. It is caused by pathogenic variants in SERPING1 gene, resulting in deficiency (HAE C1-INH) of the C1 inhibitor (C1-INH), and in F12, ANGPT1, PLG, KNG1, MYOF and HS3ST6 genes, related to HAE with normal C1-INH (AEH nC1-INH). However, in several individuals with HAE nC1-INH, the genetic cause is unknown (HAE-U). Objective: To evaluate the genetic and molecular profile of components involved in bradykinin-forming cascade and pathways regulating vascular endothelium permeability in individuals with symptoms of HAE. Methods: A total of 310 individuals from 150 unrelated families were selected. Molecular analysis of SERPING1 was performed in 87 individuals of 29 families, and of exon 9 of F12 in 157 individuals of 122 families. In 23 HAE nC1-INH probands, Whole Exome Sequencing (WES) was performed, targeting genes possibly related to HAE. To investigate the impact of variants in NOS3 gene, found in some families analyzed in our study, on endothelial dysfunction, site-directed mutagenesis was performed, followed by transfection in EA.hy926 cells. Results: In 53 individuals (31 females and 22 males) of 23 HAE C1-INH families, 20 different pathogenic or probably pathogenic variants were identified in SERPING1, including mainly missense and nonsense variants, as well as two variants, p.Met441_Gln442delinsIle* and c.551-825_685+396del, described for the first time. The pathogenic variant p.Thr328Lys in F12 was identified in 61 individuals (44 females and 17 males) of 40 HAE nC1-INH families. No pathogenic variants were identified in exon 9 of F12 in 80 probands (67 females and 13 males). The average time between symptom onset and genetic diagnosis was 23.9 and 11.5 years for patients with HAE C1-INH and HAE-FXII, respectively. Furthermore, WES identified 55 rare variants in 35 genes. Of these, 34 variants in 23 genes were validated by Sanger sequencing. Some of them, found in ADGRE2, CPN1, HS3ST6, KNG1, NOS3, PLG and SYTL2 genes may be candidate variants to be involved in the pathogenesis of HAE. Despite being considered rare variants, according to analyses, by computational tools and functional assays, of the pathogenic effect of p.Ala422Thr, p.Ala423Thr and p.Arg885Met variants identified in NOS3 showed to be inconclusive. Conclusions: Our results highlight the diversity of pathogenic variants in SERPING1 and that 33% of HAE nC1-INH families carry the pathogenic variant that affects FXII function, while the majority do not have a confirmed genetic diagnosis. Elucidating the mechanisms of modulation of angioedema attacks, with the investigation of new variants, may expand knowledge of the pathophysiology of HAE, contributing to diagnosis, generation of specific therapeutic strategies and genetic counseling in affected families.
Introduction: Hereditary angioedema (HAE) is a rare genetic disease, with autosomal dominant inheritance, life-threatening, and frequently underdiagnosed. It is characterized by recurrent subcutaneous and submucosal tissue swelling without hives, mainly due to dysregulation of bradykinin-forming cascade, which promotes production of nitric oxide by endothelial cells, leading to increased vascular permeability. It is caused by pathogenic variants in SERPING1 gene, resulting in deficiency (HAE C1-INH) of the C1 inhibitor (C1-INH), and in F12, ANGPT1, PLG, KNG1, MYOF and HS3ST6 genes, related to HAE with normal C1-INH (AEH nC1-INH). However, in several individuals with HAE nC1-INH, the genetic cause is unknown (HAE-U). Objective: To evaluate the genetic and molecular profile of components involved in bradykinin-forming cascade and pathways regulating vascular endothelium permeability in individuals with symptoms of HAE. Methods: A total of 310 individuals from 150 unrelated families were selected. Molecular analysis of SERPING1 was performed in 87 individuals of 29 families, and of exon 9 of F12 in 157 individuals of 122 families. In 23 HAE nC1-INH probands, Whole Exome Sequencing (WES) was performed, targeting genes possibly related to HAE. To investigate the impact of variants in NOS3 gene, found in some families analyzed in our study, on endothelial dysfunction, site-directed mutagenesis was performed, followed by transfection in EA.hy926 cells. Results: In 53 individuals (31 females and 22 males) of 23 HAE C1-INH families, 20 different pathogenic or probably pathogenic variants were identified in SERPING1, including mainly missense and nonsense variants, as well as two variants, p.Met441_Gln442delinsIle* and c.551-825_685+396del, described for the first time. The pathogenic variant p.Thr328Lys in F12 was identified in 61 individuals (44 females and 17 males) of 40 HAE nC1-INH families. No pathogenic variants were identified in exon 9 of F12 in 80 probands (67 females and 13 males). The average time between symptom onset and genetic diagnosis was 23.9 and 11.5 years for patients with HAE C1-INH and HAE-FXII, respectively. Furthermore, WES identified 55 rare variants in 35 genes. Of these, 34 variants in 23 genes were validated by Sanger sequencing. Some of them, found in ADGRE2, CPN1, HS3ST6, KNG1, NOS3, PLG and SYTL2 genes may be candidate variants to be involved in the pathogenesis of HAE. Despite being considered rare variants, according to analyses, by computational tools and functional assays, of the pathogenic effect of p.Ala422Thr, p.Ala423Thr and p.Arg885Met variants identified in NOS3 showed to be inconclusive. Conclusions: Our results highlight the diversity of pathogenic variants in SERPING1 and that 33% of HAE nC1-INH families carry the pathogenic variant that affects FXII function, while the majority do not have a confirmed genetic diagnosis. Elucidating the mechanisms of modulation of angioedema attacks, with the investigation of new variants, may expand knowledge of the pathophysiology of HAE, contributing to diagnosis, generation of specific therapeutic strategies and genetic counseling in affected families.