Camila Lopes Veronez

Mycoplasma hyopneumoniae in vitro peptidase activities: Identification and cleavage of kallikrein-kinin system-like substrates

Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.

Homozygosity for a factor XII mutation in one female and one male patient with hereditary angio‐oedema

Hereditary angio‐oedema (HAE) with normal C1 inhibitor is associated with heterozygous mutations in the factor XII gene (FXII‐HAE). We report two Brazilian FXII‐HAE families segregating the mutation c.983 C>A (p.Thr328Lys). In each family, one patient with a homozygous mutation was found. The homozygous female patient in family 1 displayed a severe phenotype. However, this falls within the clinical phenotype spectrum reported for heterozygous female mutation carriers. The homozygous male patient in family 2 also showed a severe phenotype. This finding is intriguing, as to our knowledge, it is the first such report for a male FXII‐HAE mutation carrier. In the rare instances in which male mutation carriers are affected, a mild phenotype is typical. The present findings therefore suggest that homozygous FXII‐HAE mutation status leads to a severe phenotype in females and males, and to an increased risk of manifest symptoms in the latter.

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°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°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.

Targeted Next-Generation Sequencing in Brazilian Children With Nephrotic Syndrome Submitted to Renal Transplant

Background The aims of this study were to identify the genetic mutations profile in Brazilian children with nephrotic syndrome (NS) and to determine a genotype-phenotype correlation in this disease. Methods Next-generation sequencing and mutation analysis were performed on 24 genes related to NS in a cross-sectional study involving 95 children who underwent kidney transplantation due to NS, excluding congenital cases. Results A total of 149 variants were identified in 22 of 24 sequenced genes. The mutations were classified as pathogenic, likely pathogenic, likely benign and benign per the chance of causing the disease. NPHS2 was the most common mutated gene. We identified 8 (8.4%) patients with hereditary NS and 5 (5%) patients with probably genetically caused NS. COL4A3-5 variants were found as well, but it is not clear whether they should be considered isolated FSGS or simply a misdiagnosed type of the Alport spectrum. Considering the clinical results, hereditary NS patients presented a tendency to early disease onset when compared with the other groups (P = 0.06) and time to end stage renal disease (ESRD) was longer in this group (P = 0.03). No patients from hereditary NS group had NS recurrence after transplantation. Conclusions This is the first study in children with steroid-resistant NS who underwent kidney transplantation using next-generation sequencing. Considering our results, we believe this study has shed some light to the uncertainties of genotype-phenotype correlation in NS, where several genes cooperate to produce or even to modify the course of the disease.

New mutations in SERPING1 gene of Brazilian patients with hereditary angioedema.

Abstract Hereditary Angioedema is an autosomal dominant inherited disease leading to oedema attacks with variable severity and localization predominantly caused by C1-INH deficit. More than 400 mutations have been already identified, however no genetic analysis of a Brazilian cohort of HAE patients with C1-INH deficiency has been published. Our aim was to perform genetic analysis of C1-INH gene (SERPING1) in Brazilian HAE patients. We screened the whole SERPING1 coding region from 30 subjects out of 16 unrelated families with confirmed diagnosis of HAE due to C1-INH deficiency. Clinical diagnosis was based on symptoms and quantitative and/or functional analysis of C1-INH. We identified fifteen different mutations among which eight were not previously described according to databases. We found five small deletions (c.97_115del19; c.553delG; c.776_782del7; c.1075_1089del15 and c.1353_1354delGA), producing frameshifts leading to premature stop codons; seven missense mutations (c.498C>A; c.550G>C; c.752T>C; c.889G>A; c.1376C>A; c.1396C>T; c.1431C>A); one nonsense mutation (c.1480C>T), and two intronic alterations (c.51+1G>T; c.51+2T>C). Despite the small number of participants in this study, our results show mutations not previously identified in SERPING1 gene. This study represents the first Brazilian HAE cohort evaluated for mutations and it introduces the possibility to perform genetic analysis in case of need for differential diagnosis.

Genetic analysis of hereditary angioedema in a Brazilian family by targeted next generation sequencing.

Abstract 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.

Brazilian Guidelines for Hereditary Angioedema Management - 2017 Update Part 1: Definition, Classification and Diagnosis

Hereditary angioedema is an autosomal dominant disease characterized by recurrent angioedema attacks with the involvement of multiple organs. The disease is unknown to many health professionals and is therefore underdiagnosed. Patients who are not adequately diagnosed and treated have an estimated mortality rate ranging from 25% to 40% due to asphyxiation by laryngeal angioedema. Intestinal angioedema is another important and incapacitating presentation that may be the main or only manifestation during an attack. In this article, a group of experts from the “Associação Brasileira de Alergia e Imunologia (ASBAI)” and the “Grupo de Estudos Brasileiro em Angioedema Hereditário (GEBRAEH)” has updated the Brazilian guidelines for the diagnosis and treatment of hereditary angioedema.

Hereditary Angioedema with Normal C1 Inhibitor and F12 Mutations in 42 Brazilian Families.

BACKGROUND Hereditary angioedema (HAE) with normal C1 inhibitor (C1-INH) is a rare condition with clinical features similar to those of HAE with C1-INH deficiency. Mutations in the F12 gene have been identified in subsets of patients with HAE with normal C1-INH, mostly within families of European descent. OBJECTIVES Our aim was to describe clinical characteristics observed in Brazilians from 42 families with HAE and F12 gene mutations (FXII-HAE), and to compare these findings with those from other populations. METHODS We evaluated a group of 195 individuals, which included 102 patients clinically diagnosed with FXII-HAE and their 93 asymptomatic relatives. RESULTS Genetic analysis revealed that of the 195 subjects, 134 individuals (77.6% females) carried a pathogenic mutation in F12. The T328K substitution was found in 132 individuals, and the c.971_1018+24del72 deletion was found in 2 patients. The mean age at onset of symptoms in patients with FXII-HAE was 21.1 years. The most common symptoms were subcutaneous edema (85.8% of patients), abdominal pain attacks (69.7%), and upper airway edema (32.3%). Of male individuals carrying F12 mutations, 53.3% (16 of 30) were symptomatic. Compared with reports from Europe, fewer female patients (68.6%) reported an influence of estrogen on symptoms. CONCLUSIONS Our study included a large number of patients with FXII-HAE, and, as the first such study conducted in a South American population, it highlighted significant differences between this and other study populations. The high number of symptomatic males and patients with estrogen-independent FXII-HAE found here suggests that male sex and the absence of a hormonal influence should not discourage clinicians from searching for F12 mutations in cases of HAE with normal C1-INH.

A new mutation in the SERPING1 gene in a Brazilian family with hereditary angioedema

Background Hereditary angioedema (HAE) types I and II (due to quantitative and qualitative C1-INH deficiency, respectively) is a rare autosomal dominant condition in which more than 300 different mutations in the entire C1-INH gene (SERPING1) have been described. The objective of this study is to identify and characterize the mutation in the SERPING1 gene in a family of HAE outpatients from the allergy service of the University Hospital at Universidade Federal de Santa Catarina.