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

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

Mutantes Do Receptor B2 De Cininas: Análise Da Interação Droga-Receptor E Sua Apllcabilldade No Estudo Do Angioedema Heredit Ário

Introduction: bradykinin (8K) is an important pressure regulating peptide arterial, nociception, inflammation and is associated with the pathophysiology of angioedema (HAE). The main clinical aspects of HAE are mediated by 8K, which exercise by the activation of receptor 82 (82R), a member of the family of G protein coupled receptors (GPCR). The objective of the present study was to evaluate if 82R mutations found in HAE patients could alter the profile pharmacological effect of 8K signal transduction, leading to relevant for the pathophysiology of the disease. Materials and methods: we use G-and-3-arrestin biosensors to perform 8RET analyzes and evaluate functional profiles of mutant 82Rs. All the experiments were carried out in transiently transfected HEK 293T cells, either with the wild-type or with the human 82R mutants. Competitive binding assays using 8K radiolabeled (3H-8K) were performed to estimate the 8K affinity for mutant receptors. The 8RET assays, used to evaluate the activation of G protein and the recruitment of β-arrestin 1 and 2, were performed using a method adapted from that described by Quoyer et al. (2013). Results: the binding assays showed that 8K retains a similar affinity at mutant receptors R14C, W344C, G354E and V376M, when compared to the wild-type receptor. In analysis, 8K triggered the coupling of G protein and mobilization intracellular calcium with similar potency and efficacy at R14C and G354E compared to WT. On the other hand, these receptors were not able to to recruit! 3-arrestinas. In the W344C and V376M mutants, the potency and efficacy of 8K was reduced on activation of all of the signaling pathways tested. Conclusions: These findings provide strong evidence for a biased agonist profile endogenous 8K G protein by activating mutants R14C and G354E 82R identified in patients with HAE. On the other hand, mutants W344C and V376M showed a decrease in the pharmacological responses induced by 8K. We believe that the current data contribute to revealing the mechanisms molecules involved in HAE and thus opening up new perspectives and approaches to study and treat this disease.Introdução: a bradicinina (8K) é um importante peptídeo regulador da pressão arterial, nocicepção, inflamação e é associada com a fisiopatologia do angioedema hereditário (AEH). Os principais aspectos clínicos do AEH são mediados pela 8K, que exerce suas ações pela ativação do receptor 82 (82R), um membro da família de receptores acoplados à proteína G (GPCR). O objetivo do presente trabalho foi avaliar se mutações do 82R encontradas em pacientes com AEH poderiam alterar o perfil farmacológico de transdução de sinal da 8K, levando a consequências relevantes para a fisiopatologia da doença. Materiais e métodos: utilizamos construções de biossensores de proteína G e !3-arrestinas para realizar análises de 8RET e avaliar perfis funcionais dos 82R mutantes. Todos os experimentos foram realizados em células HEK 293T transientemente transfectadas, quer com o receptor selvagem ou com os mutantes do 82R humano. Os ensaios de ligação competitiva utilizando 8K radiomarcada (3H-8K) foram realizados para estimar a afinidade de 8K para receptores mutantes. Os ensaios de 8RET, utilizados para avaliar a ativação da proteína G e o recrutamento de !3-arrestina1 e 2, foram realizados usando um método adaptado do descrito por Quoyer et aI. (2013). Resultados: os ensaios de ligação mostraram que a 8K mantém uma afinidade semelhante nos receptores mutantes R14C, W344C, G354E e V376M, quando comparados ao receptor selvagem. Em análises funcionais, o 8K desencadeou o acoplamento da proteína G e mobilização de cálcio intracelular com potências e eficácias similares nos receptores R14C e G354E em comparação com o WT. Por outro lado, esse receptores não foram capazes de recrutar !3-arrestinas. Já nos mutantes W344C e V376M, a potência e a eficácia da 8K foi reduzida na ativação de todas as vias de sinalização testadas. Conclusões: Esses achados fornecem evidências fortes para um perfil de agonismo tendencioso de proteína G de 8K endógena ao ativar mutantes os mutantes R14C e G354E do 82R identificados em pacientes com AEH. Por outro lado, os mutantes W344C e V376M apresentaram uma diminuição das respostas farmacológicas induzidas pela 8K. Acreditamos que os dados atuais contribuem para revelar os mecanismos moleculares envolvidos no AEH e, portanto, abrindo novas perspectivas e abordagens para estudo e tratamento desta doença.Dados abertos - Sucupira - Teses e dissertações (2017

Evidence that kinin B-2 receptor expression is upregulated by endothelial overexpression of B-1 receptors

Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B-2 (B2R) and B-1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie(2)B(1))) exclusively in the endothelium. in another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie(2)B(1)) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie(2)B(1)) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R. (c) 2013 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Nephrol, BR-04023062 São Paulo, BrazilJohns Hopkins Univ, Sch Med, Baltimore, MD USAUniversidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Nephrol, BR-04023062 São Paulo, BrazilFAPESP: 2009/08336-2FAPESP: 2010/05255-9CNPq: 300247/2010-9Web of Scienc

Novel Complex ABCA4 Alleles in Brazilian Patients With Stargardt Disease: Genotype-Phenotype Correlation

PURPOSE. To analyze the presence of complex alleles of the ABCA4 gene in Brazilian patients with Stargardt disease and to assess the correlation with clinical features. METHODS. This was an observational cross-sectional study. Patients with a diagnosis of Stargardt disease who presented three pathogenic variants of the ABCA4 gene or who had variants previously described as complex alleles were included. The relatives of these probands were evaluated in the segregation analysis. The patients were evaluated based on age at symptom onset and visual acuity, and the clinical characteristics were classified according to the findings observed on autofluorescence examination. RESULTS. Among the 47 families analyzed, approximately 30% (14/47) presented complex alleles. The segregation analysis in 14 families with cases of Stargardt disease identified three novel complex alleles and one previously described complex allele. The known complex allele p.[Leu541Pro; Ala1038Val] was identified in two families. The novel complex alleles identified were p.[Leu541Pro; Arg1443His] in five families, p.[Ser1642Arg; Val1682 Val1686-dell in seven families, and p. [Pro1761Arg; Arg2106Cys] in one family. Furthermore, four new variants (p.Lys22Asn, p.Asp915Asn, p. Glu1447Val, and p. Pro1761Arg) were identified in the second allele of the ABCA4 gene. CONCLUSIONS. Segregation analysis is important in order to confirm the molecular diagnosis of patients with Stargardt disease, given the frequency of complex alleles in the ABCA4 gene. The various pathogenic variation combinations observed in this study were associated with different phenotypes.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fed Univ Sao Paulo UNIFESP, Dept Ophthalmol & Visual Sci, Sao Paulo, BrazilFed Univ Sao Paulo UNIFESP, Dept Biophys, Sao Paulo, BrazilOregon Hlth & Sci Univ, Casey Eye Inst, Mol Diagnost Lab, Portland, OR 97201 USAMol Vis Lab, Hillsboro, OR USAFed Univ Sao Paulo UNIFESP, Dept Ophthalmol & Visual Sci, Sao Paulo, BrazilFed Univ Sao Paulo UNIFESP, Dept Biophys, Sao Paulo, BrazilFAPESP: 2012/50454-5Web of Scienc