Hidden Aberrant Transcripts in TTC37 Cause Trichohepatoenteric Syndrome.

The patient had clinical suspicion of THES. Complex genetic analyzes using WES, WGS were performed without success in the diagnosis. Further molecular analyzes using RNA and protein was necessary to reach the final correct THES diagnosis.

The Burden of Hereditary Angioedema in a Large Family in Brazil.

INTRODUCTION: Hereditary angioedema (HAE) is a rare genetic disease characterized by submucosal and subcutaneous edema with high morbidity and possibility of mortality. This study presents the sociodemographic characteristics of a large Brazilian family with HAE. METHODS: Descriptive cross-sectional study with patients from two family branches coming from the same city and HAE diagnosis was carried out. Clinical, laboratory, and treatment data of patients have been collected. Genetic testing was performed on some individuals. Correlation tests and comparisons between variables were applied using IBM SPSS Statistics® 2.0 program. RESULTS: We provide a detailed characterization of two families affected by HAE due to C1-INH deficiency, residing in a small town in southern Brazil. These families harbor an identified mutation in the SERPING1 gene (c.1104del, p.Asp369ThrfsTer2). The mean age at HAE diagnosis was 16.7 (±14.0) years, with the mean onset of symptoms at 6.0 (±6.1) years of age. A correlation was observed between patients' current age and age at HAE diagnosis, with older patients being diagnosed later than younger individuals (p < 0.0001). On average, there were 16.8 emergency visits in the past year (±24.8), and 53.5% of patients reported at least one lifetime hospitalization. Notably, treatment modalities often diverged from consensus recommendations regarding optimal prophylaxis and management of HAE attacks. CONCLUSIONS: This study describes one of the largest known families with HAE in Brazil and highlights the significant impact of unfavorable social conditions on disease control.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

OBJECTIVE: We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. METHODS: We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). RESULTS: DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. CONCLUSION: Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.

Aerobic physical training reduces severe asthma phenotype involving kinins pathway.

INTRODUCTION: Aerobic physical training (APT) reduces eosinophilic airway inflammation, but its effects and mechanisms in severe asthma remain unknown. METHODS: An in vitro study employing key cells involved in the pathogenesis of severe asthma, such as freshly isolated human eosinophils, neutrophils, and bronchial epithelial cell lineage (BEAS-2B) and lung fibroblasts (MRC-5 cells), was conducted. Additionally, an in vivo study using male C57Bl/6 mice, including Control (Co; n = 10), Trained (Exe; n = 10), house dust mite (HDM; n = 10), and HDM + Trained (HDM + Exe; n = 10) groups, was carried out, with APT performed at moderate intensity, 5x/week, for 4 weeks. RESULTS: HDM and bradykinin, either alone or in combination, induced hyperactivation in human neutrophils, eosinophils, BEAS-2B, and MRC-5 cells. In contrast, IL-10, the primary anti-inflammatory molecule released during APT, inhibited these inflammatory effects, as evidenced by the suppression of numerous cytokines and reduced mRNA expression of the B1 receptor and ACE-2. The in vivo study demonstrated that APT decreased bronchoalveolar lavage levels of bradykinin, IL-1ß, IL-4, IL-5, IL-17, IL-33, TNF-α, and IL-13, while increasing levels of IL-10, klotho, and IL-1RA. APT reduced the accumulation of polymorphonuclear cells, lymphocytes, and macrophages in the peribronchial space, as well as collagen fiber accumulation, epithelial thickness, and mucus accumulation. Furthermore, APT lowered the expression of the B1 receptor and ACE-2 in lung tissue and reduced bradykinin levels in the lung tissue homogenate compared to the HDM group. It also improved airway resistance, tissue resistance, and tissue damping. On a systemic level, APT reduced total leukocytes, eosinophils, neutrophils, basophils, lymphocytes, and monocytes in the blood, as well as plasma levels of IL-1ß, IL-4, IL-5, IL-17, TNF-α, and IL-33, while elevating the levels of IL-10 and IL-1RA. CONCLUSION: These findings indicate that APT inhibits the severe asthma phenotype by targeting kinin signaling.

Kinins: Locally formed peptides during inflammation with potential use in tissue regeneration.

Kinins are a set of peptides present in tissues and involved in cardiovascular regulation, inflammation, and pain. Here, we briefly comment on recent key findings on the use of kinins in regenerative medicine.

Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors.

OBJECTIVE AND DESIGN: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. MATERIAL AND METHODS: To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated. RESULTS: Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals. CONCLUSIONS: We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.

ACE2, ACE, DPPIV, PREP and CAT L enzymatic activities in COVID-19: imbalance of ACE2/ACE ratio and potential RAAS dysregulation in severe cases.

OBJECTIVE AND DESIGN: Circulating enzymatic activity and RAAS regulation in severe cases of COVID-19 remains unclear, therefore we measured the serum activity of several proteases as potential targets to control the SARS-CoV-2 infection. MATERIAL OR SUBJECTS: 152 patients with COVID-19-like symptoms were grouped according to the severity of symptoms (COVID-19 negative, mild, moderate and severe). METHODS: Serum samples of COVID-19 patients and controls were subjected to biochemical analysis and enzymatic assays of ACE2, ACE, DPPIV, PREP and CAT L. One-way ANOVA and multivariate logistic regression analysis were used. Statistical significance was accepted at p < 0.05. RESULTS: We detected a positive correlation among comorbidities, higher C-reactive protein (CRP) and D-dimer levels with disease severity. Enzymatic assays revealed an increase in serum ACE2 and CAT L activities in severe COVID-19 patients, while ACE, DPPIV and PREP activities were significantly reduced. Notably, analysis of ACE2/ACE activity ratio suggests a possible imbalance of ANG II/ANG(1-7) ratio, in a positive association with the disease severity. CONCLUSION: Our findings reveal a correlation between proteases activity and the severity of COVID-19. These enzymes together contribute to the activation of pro-inflammatory pathways, trigger a systemic activation of inflammatory mediators, leading to a RAAS dysregulation and generating a significant damage in several organs, contributing to poor outcomes of severe cases.

Exploring the Potential of Olfactory Receptor Circulating RNA Measurement for Preeclampsia Prediction and Its Linkage to Mild Gestational Hypothyroidism.

Gestational hypothyroidism may lead to preeclampsia development. However, this pathophysiological is unknown. We expect to find a shared mechanism by comparing hypothyroidism and preeclampsia. From our transcriptome data, we recognized olfactory receptors as that fingerprint. The reduction of taste and smell in hypothyroid patients has been known for a long time. Therefore, we decided to look to the olfactory receptors and aimed to identify genes capable of predicting preeclampsia (PEC). Methods: An Ion Proton Sequencer (Thermo Fisher Scientific, Waltham, MA, USA) was used to construct the transcriptome databases. RStudio with packages Limma v.3.50.0, GEOquery v.2.62.2, and umap v.0.2.8.8 were used to analyze the differentially expressed genes in GSE149440 from the Gene Expression Omnibus (GEO). The 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for RT-qPCR amplification of OR6X1 and OR4E2. Results: Our transcriptomic datasets analysis revealed 25.08% and 26.75% downregulated olfactory receptor (ORs) in mild nontreated gestational hypothyroidism (GHT) and PEC, respectively. In the GSE149440 GEO dataset, we found OR5H1, OR5T3, OR51A7, OR51B6, OR10J5, OR6C6, and OR2AG2 as predictors of early-onset PEC. We also evaluate two chosen biomarkers' responses to levothyroxine. The RT-qPCR demonstrated a difference in OR6X1 and OR4E2 expression between GHT and healthy pregnancy (p < 0.05). Those genes presented a negative correlation with TSH (r: -0.51, p < 0.05; and r: -0.44, p < 0.05), a strong positive correlation with each other (r: 0.89; p < 0.01) and the levothyroxine-treated group had no difference from the healthy one. We conclude that ORs could be used as biomarkers at the beginning of gestation, and the downregulated ORs found in GHT may be improved with levothyroxine treatment.

Transcriptomic and histological analysis of exposed facial skin areas wrinkled or not and unexposed skin.

BACKGROUND: Skin aging involves genetic, environmental and hormonal factors. Facial wrinkles also depend on muscular activity. Gene expression investigation may be useful for new anti-aging products. METHODS AND RESULTS: To evaluate structure and gene expression differences among exposed and unexposed skin in menopausal women. Cross-sectional study, including 15 menopausal women, 55-65 years, phototype III; photo-exposed, periorbital wrinkles (A1), preauricular, not wrinkled (A2), and unexposed gluteal (A3) areas were described and compared by non-invasive measures, histology, immunohistochemistry and gene expression (RNASeq); participants mean age was 61yo, presenting moderate periorbital wrinkles and light facial photodamage. Higher roughness, wrinkles number and echogenicity were observed in A1 and A2 versus A3. Decreased epidermal thickness and dermal collagen IV were demonstrated in A1 versus A2 and A3. Exposed areas impacted different pathways compared to unexposed. Exposed wrinkled skin (A1) showed impact on cell movement with decreased inflammatory activation state. Pathways related to lipid and aminoacids metabolism were modulated in non-wrinkled exposed (A2) compared to unexposed (A3) skin. CONCLUSIONS: Expected histological findings and gene expression differences among areas were observed. Photoaging in menopausal women may modulate lipid and aminoacids metabolism and decrease inflammatory and keratinization pathways, cellular homeostasis, immune response, fibrogenesis and filament formation. These findings may help development of new therapies for skin health and aging control.

The Challenges in the Follow-Up and Treatment of Brazilian Children with Hereditary Angioedema.

INTRODUCTION: Hereditary angioedema (HAE) with C1 inhibitor (C1-INH) deficiency is a rare autosomal dominant disease. Although the first symptoms can appear in childhood, the diagnosis's delay has a strong impact on the patient's quality of life. We analyzed clinical and laboratory characteristics and the drug therapy of pediatric patients with HAE in Brazil. METHODS: Medical records from 18 reference centers of HAE patients under 18 years of age were evaluated after confirmed diagnosis was performed by quantitative and/or functional C1-INH. RESULTS: A total of 95 participants (51 M:44 F; mean age: 7 years old) out of 17 centers were included; 15 asymptomatic cases were identified through family history and genetic screening. Angioedema attacks affected the extremities (73.5%), gastrointestinal tract (57%), face (50%), lips (42.5%), eyelids (23.7%), genitals (23.7%), upper airways (10%), and tongue (6.3%). Family history was present in 84% of patients, and the mean delay in the diagnosis was 3.9 years. Long-term prophylaxis (51/80) was performed with tranexamic acid (39/80) and androgens (13/80); and short-term prophylaxis (9/80) was performed with tranexamic acid (6/80) and danazol (3/80). On-demand therapy (35/80) was prescribed: icatibant in 7/35, fresh frozen plasma in 16/35, C1-INH plasma-derived in 11/35, and tranexamic acid in 12/35 patients. CONCLUSIONS: This is the first study on HAE pediatric patients in Latin America. Clinical manifestations were similar to adults. Drugs such as androgens and tranexamic acid were indicated off-label, probably due to restricted access to specific drugs. Educational programs should address pediatricians to reduce late diagnosis and tailored child therapy.

APOL1 in an ethnically diverse pediatric population with nephrotic syndrome: implications in focal segmental glomerulosclerosis and other diagnoses.

BACKGROUND: APOL1 high-risk genotypes (HRG) are associated with increased risk of kidney disease in individuals of African ancestry. We analyzed the effects of APOL1 risk variants on an ethnically diverse Brazilian pediatric nephrotic syndrome (NS) cohort. METHODS: Multicenter study including 318 NS patients, categorized as progressors to advanced CKD [estimated glomerular filtration rate (eGFR)] < 30 mL/min/1.73 m2] and slow/non-progressors (eGFR > 30 mL/min/1.73 m2 through the study). We employed Cox regression with progression time as the outcome and APOL1 genotype as the independent variable. We tested this association in the entire cohort and three subgroups; (1) focal segmental glomerulosclerosis (FSGS), (2) steroid-resistant NS (SRNS), and (3) those who underwent kidney biopsy. RESULTS: Nineteen patients (6%) had an HRG. Of these, 47% were self-reported White. Patients with HRG manifested NS at older ages and presented higher frequencies of FSGS and SRNS. HRG patients progressed to advanced CKD more often than low-risk-genotype (LRG) children in the whole NS cohort (p = 0.001) and the three subgroups. In SRNS and biopsied patients, a single risk variant was associated with trends of higher CKD progression risk. CONCLUSIONS: Novel discoveries include a substantial prevalence of HRG among patients self-reported White, worse kidney outcomes in HRG versus LRG children in the FSGS subgroup, and a trend of higher CKD progression risk associated with a single risk variant in the SRNS cohort. These findings suggest APOL1-associated NS extends beyond patients self-reported non-White, the HRG effect is independent of FSGS, and a single risk variant may have a detrimental impact in children with NS.

Fabry disease: GLA deletion alters a canonical splice site in a family with neuropsychiatric manifestations.

Fabry disease (FD) is a rare X-linked glycosphingolipidosis caused by mutations in GLA, a gene responsible for encoding α-galactosidase A, an enzyme required for degradation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in all cells of the body. FD patients present a broad spectrum of clinical phenotype and many symptoms are shared with other diseases, making diagnosis challenging. Here we describe a novel GLA variant located in the 5' splice site of the intron 3, in four members of a family with neuropsychiatric symptoms. Analysis of the RNA showed the variant promotes alteration of the wild type donor site, affecting splicing and producing two aberrant transcripts. The functional characterization showed absence of enzymatic activity in cells expressing both transcripts, confirming their pathogenicity. The family presents mild signs of FD, as angiokeratoma, cornea verticillata, acroparesthesia, tinnitus, vertigo, as well as accumulation of plasma lyso-Gb3 and urinary Gb3. Interestingly, the man and two women present psychiatric symptoms, as depression or schizophrenia. Although psychiatric illnesses, especially depression, are frequently reported in patients with FD and studies have shown that the hippocampus is an affected brain structure in these patients, it is not clear whether the Gb3 accumulation in the brain is responsible for these symptoms or they are secondary. Therefore, new studies are needed to understand whether the accumulation of Gb3 could produce neuronal alterations leading to psychiatric symptoms.

Interactions between carboxypeptidase M and kinin B1 receptor in endothelial cells.

INTRODUCTION: Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. AIMS: We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. METHODS: Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. RESULTS: CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. CONCLUSIONS: Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.

Chemotherapy-induced fatigue is associated with changes in gene expression in the peripheral blood mononuclear cell fraction of patients with locoregional breast cancer.

PURPOSE: Chemotherapy-induced fatigue (CIF) is a frequent symptom that impairs patient functioning and quality of life. We aimed to evaluate whether systemic chemotherapy can induce a specific gene expression profile in peripheral blood mononuclear cells (PBMNC) of patients with locoregional breast cancer (LRBC) who develop CIF. METHODS: PBMNC were collected from 3 patients who developed CIF before and after their initial cycle of chemotherapy, and RNA-seq was performed in an Ion Torrent™ System. A total of 12.345 transcripts were sequenced, of which 26 were selected out of 71 that had significantly different expression before and after chemotherapy. The RNA-seq results were validated by RT-qPCR in a different group of 28 patients with LRBC who developed CIF after their first cycle of chemotherapy and in six patients who also received chemotherapy but did not develop CIF (controls). We assessed CIF according the BFI and Chalder Questionnaires. RESULTS: We observed a significant increase in expression of DUSP18 and RHOBTB1 and decreased expression of NCAN and RAET1G in patients who developed CIF after chemotherapy. Control patients only exhibited a significant decrease in NCAN expression. CONCLUSION: CIF induces specific changes in gene expression in the PBMNC of LRBC patients. Some of these changes, such as downregulation of NCAN expression, may reflect direct effects of chemotherapy since they are also observed in the controls. Furthermore, CIF may involve downregulation of skeletal muscle genes (RHOBT1, DUSP18) and immune systems (RAETG1), whereas NCAN downregulation may underlie the adverse cognitive effects of chemotherapy.

Variants in the ABCA4 gene in a Brazilian population with Stargardt disease.

Purpose: The aim of this study was to analyze and report pathogenic variants in the ABCA4 gene in Brazilian patients with a clinical diagnosis of Stargardt disease. Methods: This retrospective study evaluated variants in the ABCA4 gene in Brazilian patients with Stargardt disease. The patients' visual acuity and age of symptom onset were obtained from previous medical records. The patients were classified according to the autofluorescence patterns. Results: Fifty patients aged between 10 and 65 years from 44 families were included in the study. Among these cases, the mean age of symptom onset was 14 years (range, 5-40 years). ABCA4 gene sequencing was conclusive in 40 patients (80%), negative in two patients (4%), and inconclusive in eight patients (16%). Four families carried homozygous pathogenic variants. Segregation analysis results were available for 23 families. One novel variant was found: p.Ala2084Pro. The most frequent pathogenic variant in this group was p.Arg602Trp (12/100 alleles). Based on the phenotypic characteristics assessed with fundus autofluorescence imaging, 12 patients were classified as having type I phenotype, 16 as having type II, and 18 patients as having type III. The cases classified as type III phenotype included patients who were homozygous for the p.Asn96Asp and p.Arg2030* variants. One patient with a type I phenotype carried the homozygous intronic variant c.3862+1G>A. Conclusions: Next-generation sequencing was effective for the molecular diagnosis of genetic diseases and specifically allowed a conclusive diagnosis in 80% (40/50) of the patients. As the ABCA4 gene does not show a preferential region for pathogenic variants, the diagnosis of Stargardt disease depends on broader analysis of the gene. The most common pathogenic variants in the ABCA4 gene described in the literature were also found in these Brazilian patients. Although some genotype-phenotype correlations were found, more studies regarding the progression of Stargardt disease will help increase our understanding of the pathogenicity of these gene variants.

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

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.

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.