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

The Involvement of Proteoglycans in the Human Plasma Prekallikrein Interaction with the Cell Surface

Introduction: the aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen (H-kininogen), influence this interaction.Methods: We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type) and CHO-745 (deficient in proteoglycans). Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule.Results: At 37 degrees C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4 +/- 0.010 and 1,070.3 +/- 0.001 pixels/cell, respectively, for ECV304 and 1,319.1 +/- 0.003 and 631.3 +/- 0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein colocalized with LysoTracker in the absence and presence of exogenous H-kininogen at levels of 76.0% and 88.5%, respectively, for ECV304 and at levels of 40.7% and 57.0%, respectively, for CHO-K1. After assembly on the cell surface, a plasma kallikrein fragment of 53 kDa was predominant in the incubation buffer of all the cell lines studied, indicating specific proteolysis; plasma kallikrein fragments of 48-44 kDa and 34-32 kDa were also detected in the incubation buffer, indicating non-specific cleavage. Bradykinin free H-kininogen internalization was not detected in CHO-K1 or CHO-745 cells at 37 degrees C.Conclusion: the prekallikrein interaction with the cell surface is temperature-dependent and independent of exogenously applied H-kininogen, which results in prekallikrein endocytosis promoted by proteoglycans. Prekallikrein proteolysis/activation is influenced by H-kininogen/glycosaminoglycans assembly and controls plasma kallikrein activity.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilUniv Bandeirante São Paulo, Biomat & Biotechnol Res Grp, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilFAPESP: FAPESP 09/51319-1FAPESP: 09/13160-0FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6CNPq: CNPq 472403/2007-9Web of Scienc

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

Genética do angioedema hereditário: do diagnóstico molecular à caracterização clínica e genética da população brasileira

Hereditary angioedema (HAE) is a rare autosomal dominant disease in which C1 inhibitor or coagulation factor XII codifying genes are affected, leading to the deregulation of plasma kallikrein-kinin system and increased bradykinin (BK) production. BK causes vasodilation and consequent sporadic localized angioedema without wheals episodes. The duration, severity and frequency of the attacks are widely variable even in kindred. However, there are only few studies concerning the genetic profile of different proteins involved in BK release pathway. The molecular diagnosis was performed by Sanger sequencing of SERPING1 and F12 from 321 Brazilian patients and their relatives. Seventy nine patients presented mutations in SERPING1 (47 women and 32 men), from 21 families, in a total of 19 mutations, 12 of them described for the first time in the literature (c.51+2T>C, c.97_115del19, c.195delG, c.553delG, c.686-1G>A, c.752T>C, c.776_782del7, c.1075_1089del15, c.1104delA, c.1369G>C, c.1376C>A and c.1480C>T). Sixty two patients presented specific mutations in F12 (49 women and 13 men) from 23 families, whereby a partial genetic characterization of HAE Brazilian population was achieved. Pathogenic mutations were absent in 69 symptomatic individuals distributed in 45 families classified as HAE-unknown. In order to search for genetic modulators of HAE symptoms, 15 genes related to BK release pathway were analyzed by nextgeneration sequencing in 87 subjects from different countries. Although no significant genotypephenotype association was found, a list with more than 200 alterations was identified, expanding our knowledge about variation in important genes for HAE pathophysiology and broadening perspectives for future studies.O angioedema hereditário (AEH) é uma doença genética autossômica dominante na qual os genes codificadores do C1 inibidor ou fator XII podem ser afetados, gerando um desbalanço no sistema calicreína-cininas plasmático e produção excessiva de bradicinina (BK). A BK causa vasodilatação, levando os pacientes a episódios esporádicos e localizados de angioedema sem urticária, cuja duração, gravidade e frequência é bastante variável, mesmo entre familiares. Ainda assim, poucos estudos até o momento se propuseram a analisar o perfil genético de outras proteínas diretamente envolvidas na via de liberação da BK. Foi realizado o diagnóstico molecular do AEH, baseado no sequenciamento de SERPING1 e F12 pelo método de Sanger, de 321 pacientes e familiares brasileiros, sendo diagnosticados 79 pacientes portadores de mutação em SERPING1 (47 mulheres e 32 homens), distribuídos em 21 famílias, em um total de 19 alterações, das quais 12 foram descritas pela primeira vez na literatura (c.51+2T>C, c.97_115del19, c.195delG, c.553delG, c.686-1G>A, c.752T>C, c.776_782del7, c.1075_1089del15, c.1104delA, c.1369G>C, c.1376C>A e c.1480C>T). Mutações específicas no F12 foram encontradas em 62 pacientes (49 mulheres e 13 homens), distribuídos em 23 famílias, permitindo a caracterização genética de parte de nossa população com AEH. Não foram identificadas mutações patogênicas em 69 indivíduos sintomáticos (45 famílias) classificados como portadores de AEH idiopático. Análises por sequenciamento de nova geração nos permitiram avaliar 15 genes envolvidos na via de liberação da BK em busca de possíveis moduladores dos sintomas em 87 indivíduos de diferentes nacionalidades. Embora não tenha sido encontrada qualquer associação genótipo/fenótipo, uma lista de mais de duzentas alterações foi identificada, ampliando nosso conhecimento sobre modificações em genes importantes no AEH e passíveis de serem explorados experimentalmente em projetos futuros.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Cellular interaction and activation of human plasma prekallikrein

O sistema calicreina-cinina plasmatico humano e formado pela calicreina plasmatica (huPK), que hidrolisa o seu substrato natural, o cininogenio de alta massa molecular (HK), liberando a bradicinina (BK) e esta relacionado a biologia vascular interagindo com os sistemas da coagulacao, fibrinolise e complemento. A huPK participa da regulacao da pressao sanguinea pela liberacao de BK, do processo fibrinolitico, da via de contato da coagulacao e da via alternativa do sistema do complemento. A pro-calicreina plasmatica (PK) e o HK circulam complexados e recentes estudos sugerem que o zimogenio PK apresenta atividade enzimatica na presenca de seu cofator HK. O objetivo do presente trabalho foi investigar a interacao da PK com a superficie celular e sua atividade como zimogenio. Na presenca das linhagens celulares endoteliais ECV304 e RAEC, e epiteliais tumorais CHO-K1 e CHO-745, a PK interagiu com as celulas, na presenca ou ausencia do HK, a 37C e foi internalizada e direcionada vesiculas endociticas acidas, exceto pelas celulas CHO-745 que produzem niveis reduzidos de proteoglicanos comparado as CHO-K1. Na interacao com essas linhagens celulares, a PK sofreu proteolise em fragmentos de 52 kDa, 48 kDa e 36 kDa, identificados pelo anticorpo U-691.10, e hidrolisou o HK no mesmo perfil que a huPK. A atividade da PK foi avaliada em solucao pela interacao com HK, na presenca de inibidores de proteases ou do peptideo SDD31, atraves da imunodeteccao dos seus fragmentos, dos fragmentos do HK e pela hidrolise de substratos especificos para huPK, alem da dosagem de BK por radioimunoensaio. A PK apresentou atividade enzimatica, pois hidrolisou o HK e liberou BK, porem foi parcialmente convertida em huPK e apresentou velocidade mais lenta quando comparada a huPK. Os resultados indicam que a PK/huPK pode interagir com a superficie celular nao so atraves do HK, mas pela presenca de glicosaminoglicanos ou outras proteinas de membrana e ser internalizada; a PK pode sofrer proteolise na superficie celular, e em solucao na presenca do HK pode ocorrer uma mudanca conformacional na PK sem proteolise o que permite sua atividade. A internalizacao da PK, as alteracoes estruturais na presenca ou ausencia do HK, na superficie celular ou em solucao caracterizam um mecanismo de controle fisiologico da PK/huPK alem de seus inibidores naturaisBV UNIFESP: Teses e dissertaçõe

A rare mutation in the F12 gene in a patient with ACE inhibitor-induced angioedema

Sao Paulo Research Foundation-FAPESPUniv Fed Sao Paulo, Dept Biophys, Sao Paulo, SP, BrazilEscola Super Ciencias Santa Casa Misericor Vitoria, Sch Med, Vitoria, ES, BrazilUniv Fed Sao Paulo, Dept Biophys, Sao Paulo, SP, BrazilFAPESP: 2011/24142-3FAPESP: 2013/02661-4Web of Scienc

Hereditary Angioedema: Impact of COVID-19 pandemic stress upon disease related morbidity and well-being.

Background: Individuals with hereditary angioedema (HAE) experience stress-related sequelae, including enhanced disease morbidity and reduced quality of life. The pervasive societal strain that surround the coronavirus disease 2019 (COVID-19) pandemic may theoretically pose a disproportionate risk for patients with HAE. Objective: To dissect the interrelationship(s) among the COVID-19 pandemic, stress, and HAE disease-related morbidity and overall well-being. Methods: Subjects with HAE (either due to C1-inhibitor deficiency or with normal C1 inhibitor) as well as non-HAE household members (normal controls) completed online questionnaires that covered the impact of the COVID-19 pandemic on attack frequency, observed effectiveness of HAE medications, stress, and perceived quality of life and/or well-being. The subjects scored each of the questions to reflect their current status as well as their status before being aware of the pandemic. Results: Disease morbidity and psychologic stress outcomes were significantly worse in patients with HAE during the pandemic compared with before they were aware of the pandemic. A COVID-19 infection further increased attack frequency. Control subjects also experienced deterioration of well-being and optimism. A comorbid diagnosis of anxiety, depression, or posttraumatic stress disorder (PTSD) was generally associated with worse outcomes. Women consistently showed greater decrements in wellness during the pandemic compared with men. Women also reported higher levels of comorbid anxiety, depression, or PTSD than men and experienced a higher rate of job loss during the pandemic. Conclusion: The results implicated a deleterious impact of stress in the aftermath of COVID-19 awareness on HAE morbidity. The female subjects were universally more severely affected then were the male subjects. Overall well-being and/or quality of life, and optimism for the future deteriorated after awareness of the COVID-19 pandemic for the subjects with HAE and non-HAE household controls

Genetic Analysis Of Hereditary Angioedema In A Brazilian Family By Targeted Next Generation Sequencing

Hereditary angioedema (HAE) is accompanied by an overproduction of bradykinin (BK) as the primary mediator of swelling. Although many proteins may be involved in regulating the wide spectrum of HAE symptoms, most studies have only focused on C1-INH and FXII. For the first time, a next generation sequencing (NGS) method was applied to develop a robust, time- and cost-effective diagnostic and research tool to analyze selected genes related to HAE. The entire coding region and the exon-intron boundaries of 15 genes from 23 subjects of a Brazilian family, nine of whom were symptomatic, were analyzed by NGS. One new mutation found uniquely in the nine symptomatic patients, p.Ala457Pro in the SERPING1 gene, was estimated as likely to be pathogenic -(PolyPhen-2 software analysis) and is the main candidate to be responsible for HAE in these patients. Alterations identified in a few asymptomatic individuals but also found in almost all symptomatic patients, such as p.Ile197Met (HMWK), p. Glu298Asp (NOS3) and p.Gly354Glu (B2R), may also be involved in modulating patient-specific symptoms. This NGS gene panel has proven to be a valuable tool for a quick and accurate molecular diagnosis of HAE and efficient to indicate modulators of HAE symptoms.3974315322International Meeting on Kinin System and Peptide ReceptorsJUN 28-JUL 01, 2015Sao Paulo, BRAZI