Genetic Variation of Kallikrein-Kinin System and Related Genes in Patients With Hereditary Angioedema

Kallikrein-Kinin System; Genetic variation; Hereditary angioedemaSistema calicreina-cinina; Variació genètica; Angioedema hereditariSistema calicreina-cinina; Variación genética; Angioedema hereditarioHereditary angioedema (HAE) is an autosomal dominant disease caused by C1-INH deficiency due to mutations in SERPING1 (C1-INH-HAE) in most of the cases, or by specific mutations in factor XII gene, F12 (F12-HAE). Identification of polymorphisms in the genes encoding proteins from key pathways driving HAE can help to understand how genetic diversity contributes to its phenotypic variability. Here, 15 genes related to the Kallikrein-Kinin System (KKS) were analyzed by next generation sequencing in 59 patients with C1-INH-HAE or F12-HAE from Brazil, Denmark and Spain, and 19 healthy relatives in a total of 31 families. We identified 211 variants, from which 23 occurred only in Danish subjects and 79 were found only in Brazilian individuals, resulting in 109/211 variations in common between European and Brazilian population in the HAE families analyzed. BDKRB2 and CPM presented a large number of variants in untranslated regions, 46/49 and 19/24, respectively; whereas ACE (n = 26), SERPING1 (n = 26), CPM (n = 24), and NOS3 (n = 16) genes presented the higher number of variants directly affecting amino acid sequence. Despite the large amount of variants identified, the lack of association between genotype and phenotype indicates that the modulation of HAE symptom requires a more complex regulation, probably involving pathways beyond the KKS, epigenetics and environmental factors. Considering the new HAE types recently described, molecules involved in the regulation of vasculature and in plasminogen activation become promising targets for future genetic studies

Hereditary Angioedema-Associated Acute Pancreatitis in C1-Inhibitor Deficient and Normal C1-Inhibitor Patients: Case Reports and Literature Review

Abdominal pain due to intestinal swellings is one of the most common manifestations in hereditary angioedema (HAE). Bowel swellings can cause severe abdominal pain, nausea, vomiting, and diarrhea, which may lead to misdiagnosis of gastrointestinal disorders. In rare cases, HAE abdominal attacks can be accompanied by acute pancreatitis. Here, we report 3 patients with HAE and acute pancreatitis and present a literature review of similar cases. Patients with confirmed diagnosis of HAE secondary to C1-inhibitor (C1-INH) deficiency (n = 2) and HAE with normal C1-INH and F12 mutation (F12-HAE) (n = 1) were included. Pancreatitis was diagnosed based on clinical symptoms and high lipase and amylase levels. Three HAE patients were diagnosed with acute pancreatitis based on increased amylase levels during severe abdominal swelling episodes. Two were previously diagnosed with HAE type I and one with F12-HAE. Pancreatitis was efficiently treated in two patients using Icatibant, with pain relief within hours. When conservatively treated, pancreatitis pain took longer time to resolve. Eighteen pancreatitis cases in HAE with C1-INH deficiency were previously reported and none in F12-HAE. Most patients (12/18) underwent invasive procedures and/or diagnostic methods. Although rare, severe abdominal HAE attacks could cause pancreatitis; HAE-specific treatments may be efficient for HAE-associated pancreatitis. HAE should be considered as a differential diagnosis of acute idiopathic pancreatitis. To our knowledge, this is the first report of HAE-associated pancreatitis in a F12-HAE patient treated with Icatibant

Genetic Variation of Kallikrein-Kinin System and Related Genes in Patients With Hereditary Angioedema

Hereditary angioedema (HAE) is an autosomal dominant disease caused by C1-INH deficiency due to mutations in SERPING1 (C1-INH-HAE) in most of the cases, or by specific mutations in factor XII gene, F12 (F12-HAE). Identification of polymorphisms in the genes encoding proteins from key pathways driving HAE can help to understand how genetic diversity contributes to its phenotypic variability. Here, 15 genes related to the Kallikrein-Kinin System (KKS) were analyzed by next generation sequencing in 59 patients with C1-INH-HAE or F12-HAE from Brazil, Denmark and Spain, and 19 healthy relatives in a total of 31 families. We identified 211 variants, from which 23 occurred only in Danish subjects and 79 were found only in Brazilian individuals, resulting in 109/211 variations in common between European and Brazilian population in the HAE families analyzed. BDKRB2 and CPM presented a large number of variants in untranslated regions, 46/49 and 19/24, respectively; whereas ACE (n = 26), SERPING1 (n = 26), CPM (n = 24), and NOS3 (n = 16) genes presented the higher number of variants directly affecting amino acid sequence. Despite the large amount of variants identified, the lack of association between genotype and phenotype indicates that the modulation of HAE symptom requires a more complex regulation, probably involving pathways beyond the KKS, epigenetics and environmental factors. Considering the new HAE types recently described, molecules involved in the regulation of vasculature and in plasminogen activation become promising targets for future genetic studies

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

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.O Angioedema Hereditário (AEH) é uma doença genética decorrente de mutações no gene SERPING1 que codifica o inibidor de C1 esterase (C1-INH), determinando deficiência quantitativa e/ou qualitativa no C1-INH, causado em maior parte por mutações pontuais e, aproximadamente 15-20%, referentes a grandes rearranjos gênicos. As regiões intrônicas do gene apresentam 17 sequências de elementos repetitivos representados por A/u, tornando o gene propenso a deleções e inserções/duplicações. Para viabilizar um diagnóstico molecular semiquantitativo para o gene SERPING1 com capacidade de detecção de grandes rearranjos gênicos foi desenvolvida e padronizada uma Técnica de Ouantificação de Exons (TOE). Através da eletroforese capilar, foi realizada a separação e quantificação dos fragmentos de diferentes tamanhos amplificados pela técnica de PCR-Multiplex associada à utilização de prímers marcados com molécula fluorescente. Foi selecionada amostra de sangue de uma família com histórico clínico de AEH, níveis séricos baixos de C4 e C1-INH, e que deram entrada para análise molecular do gene SERPING1 por sequenciamento de Sanger em nosso Centro. As amostras dessa família não apresentaram nenhuma alteração patogênica conhecida no gene .> SERPING1 pela técnica de sequenciamento de Sanger. Sendo assim, foram analisados por TOE, confirmados por MLPA e PCR de Longo Alcance. Através do TOE foi detectado deleção no exon 4 no paciente nO12 e confirmado pelo MLPA. O fragmento deletado foi separado por PCR Longo Alcance e, posteriormente, pelo sequenciamento de Sanger, foram localizados o local e o tamanho da deleção. Portanto, a Técnica de Ouantificação de Exons (TOE) mostrou ser um método eficiente na detecção de grandes deleções ou inserções/duplicações com poder de eficiência sendo simples e rápida na execução, além de ter um custo benefício. Estes dados revelam que a técnica de TOE foi desenvolvida com sucesso para a análise de deleções envolvendo o gene SERPING1, servindo como técnica de suporte na rotina laboratorial no diagnóstico de AEH, podendo ser ainda padronizada e aplicada para outras doenças genéticas, contribuindo na pesquisa e detecção de grandes rearranjos gênicos. XIVDados abertos - Sucupira - Teses e dissertações (2013 a 2016

Hereditary angioedema: development of a method molecular diagnostics based on the SERPING1 gene and an in vitro cellular model of the blood-brain barrier

Introdução: O angioedema hereditário é uma doença genética decorrente de variantes patogênicas no gene SERPING1, que codifica o inibidor de C1 esterase, cuja prevalência mundial estimada é de 1:50.000 a 1:100.000. A deficiência do inibidor de C1 esterase causa exacerbação das cascatas do complemento, fibrinolítica, da coagulação e das calicreínas-cininas e aumento da produção de mediadores inflamatórios, principalmente bradicinina, pela ação da enzima calicreína plasmática. O aumento de bradicinina ocasiona vasodilatação e aumento da permeabilidade vascular levando ao extravasamento de fluído dos vasos para os tecidos, causando edema. O angioedema hereditário é caracterizado clinicamente por episódios recorrentes de edema local ou generalizado, além de oferecer risco de morte por asfixia. Apesar da generalização da ocorrência de edema, o sistema nervoso central parece ser um local protegido, pois os episódios de edema cerebral são raros ou inexistentes no angioedema hereditário. Com base nesse fenômeno, nossa hipótese é que as células endoteliais da microvasculatura cerebral expressem um inibidor da calicreína plasmática, impedindo a geração de bradicinina no local. Objetivos: Os objetivos desta tese foram i) desenvolver um método para detectar grandes deleções e inserções no gene SERPING1 para o diagnóstico de angioedema hereditário e, ii) estabelecer um protocolo simplificado com alto rendimento celular para o isolamento e purificação de células endoteliais da microvasculatura cerebral de camundongo neonato. Métodos: i) A Técnica de Quantificação de Éxons (TQE) foi desenvolvida utilizando o DNA total extraído de sangue de pacientes de duas famílias brasileiras com histórico clínico de angioedema hereditário, baixos níveis plasmáticos de C4 e C1-INH e nenhuma alteração patogênica em SERPING1 analisada por sequenciamento de Sanger; ii) Descrevemos um protocolo simplificado usando camundongos recém-nascidos C57BL/6 no dia pós-natal (P1) para isolar, purificar e cultivar monocamadas de células endoteliais da microvasculatura cerebral em dois substratos diferentes (lamínulas e insertos). Resultados: i) O uso da TQE permitiu detectar duas grandes deleções diferentes no éxon 4, uma de 1356 pb e outra de 1804 pb, que resultaram da recombinação de dois elementos Alu presentes nos íntrons 3 e 4 do gene SERPING1, ii) Testamos a função de células endoteliais da microvasculatura cerebral usando um modelo de privação de oxigênio-glicose. A caracterização e validação in vitro da cultura primária das células endoteliais incluiu microscopia de luz, imunomarcação e análise do perfil de expressão gênica. A medida da resistência elétrica transendotelial (TEER) foi usada como um ensaio funcional dos complexos de junção aderente. Conclusão: Com o desenvolvimento e utilização da técnica de quantificação de éxons foi possível estabelecer o diagnóstico molecular de duas famílias afetadas com o angioedema hereditário. Além disso, estabelecemos um modelo in vitro da barreira hematoencefálica que poderá ser utilizado para testar a hipótese da existência de um possível inibidor da calicreína plasmática expresso localmente nas células endoteliais da microvasculatura cerebral. A caracterização desse inibidor poderá ser um passo importante para o conhecimento e desenvolvimento de novas terapias para o tratamento do angioedema hereditário, para doenças neurológicas e neurodegenerativas.Introduction: Hereditary angioedema is a genetic disease resulting from pathogenic variants in the SERPING1 gene, which encodes the C1 esterase inhibitor whose estimated worldwide prevalence is 1:50,000 to 1:100,000. C1 esterase inhibitor deficiency causes exacerbation of complement, fibrinolytic, coagulation and kallikrene-kinin cascades and increased production of inflammatory mediators, mainly bradykinin, through the action of plasma kallikrein. The increase in bradykinin causes vasodilation and increased vascular permeability leading to leakage of fluid from the vessels into the tissues, causing edema. Hereditary angioedema is clinically characterized by recurrent episodes of local or generalized edema, in addition to being life-threatening by asphyxia. Despite the generalized occurrence of edema, the central nervous system appears to be a protected site, as episodes of cerebral edema are rare or non-existent in hereditary angioedema. Based on this phenomenon, our hypothesis is that the endothelial cells of the brain microvasculature express an inhibitor of plasma kallikrein, preventing the generation of bradykinin at the site. Objectives: The objectives of this thesis were i) to develop a method and detect large deletions and insertions in the SERPING1 gene for the diagnosis of hereditary angioedema and, ii) to establish a simplified protocol with high cell throughput for the isolation and purification of endothelial cells from the brain microvasculature of newborn mouse. Methods: i) The Exon Quantification Technique (EQT) was developed using the total DNA extracted from the blood of patients from two Brazilian families with a clinical history of hereditary angioedema, low plasma levels of C4 and C1-INH and no pathogenic alteration in SERPING1 analyzed by Sanger sequencing; ii) We describe a simplified protocol using postnatal day (P1) C57BL/6 newborn mice to isolate, purify and culture brain microvasculature endothelial cell monolayers on two different substrates (coverslips and inserts). Results: i) By using EQT two different large deletions could be found in exon 4, one of 1356 bp and the other of 1804 bp, which resulted from the recombination of two Alu elements present in introns 3 and 4 of the SERPING1 gene, ii) We tested the function of endothelial cells of the cerebral microvasculature using a model of oxygen-glucose deprivation. The characterization and in vitro validation of the primary culture of endothelial cells included light microscopy, immunostaining and analysis of the gene expression profile. The measurement of transendothelial electrical resistance (TEER) was used as a functional assay of adherent junction complexes. Conclusion: With the development and use of the exon quantification technique, it was possible to establish the molecular diagnosis of two families affected with hereditary angioedema. Furthermore, we established an in vitro model of the blood-brain barrier that can be used to test the hypothesis of the existence of a possible plasma kallikrein inhibitor expressed locally in the endothelial cells of the brain microvasculature. The characterization of this inhibitor could be an important step towards the knowledge and development of new therapies for the treatment of both hereditary angioedema and neurological and neurodegenerative diseases.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)141907/2018-