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BACKGROUND. Genetic defects of innate immunity impairing intestinal bacterial sensing are linked to the development of Inflammatory Bowel Disease (IBD). Although much evidence supports a role of the intestinal virome in gut homeostasis, most studies focus on intestinal viral composition rather than on host intestinal viral sensitivity. AIM. To demonstrate the association between the development of Very Early Onset IBD (VEOIBD) and variants in the IFIH1 gene which encodes MDA5, a key cytosolic sensor for viral nucleic acids. METHODS. Whole exome sequencing (WES) was performed in two independent cohorts of children with VEOIBD enrolled in Italy (n=18) and USA (n=24). Luciferase reporter assays were employed to assess MDA5 activity. An enrichment analysis was performed on IFIH1 comparing 42 VEOIBD probands with 1527 unrelated individuals without gastrointestinal or immunological issues. RESULTS. We identified rare, likely loss-of-function (LoF), IFIH1 variants in eight patients with VEOIBD from a combined cohort of 42 children. One subject, carrying a homozygous truncating variant resulting in complete LoF, experienced neonatal-onset, pan-gastrointestinal, IBD-like enteropathy plus multiple infectious episodes. The remaining seven subjects, affected by VEOIBD without immunodeficiency, were carriers of one LoF variant in IFIH1. Among these, two patients also carried a second hypomorphic variant, with partial function apparent when MDA5 was weakly stimulated. Furthermore, IFIH1 variants were significantly enriched in children with VEOIBD as compared to controls (p=0.007). CONCLUSIONS. Complete and partial MDA5 deficiency is associated with VEOIBD with variable penetrance and expressivity, suggesting a role for impaired intestinal viral sensing in IBD pathogenesis.

Purpose: This study aims to demonstrate the possibility of detecting segmental uniparental isodisomy (iUPD) using a next-generation sequencing gene panel by reporting a Leber congenital amaurosis (LCA) case caused by a homozygous pathogenic variant in RPE65 (c.1022 T > C:p.Leu341Ser) inherited exclusively from the proband's mother.Methods: Samples from the trio (proband, mother, and father) were sequenced with a next-generation sequencing (NGS) retinopathy gene panel (224 genes) and the VCF file containing all variants was used in order to determine single nucleotide variant (SNV) counts from each sample across all chromosomes.Results: Trio analysis showed that of 81 Chr1 inherited variants 41 were exclusively maternal, including 21 homozygous. The other 40 variants were common to both parents. On remaining autosomal chromosomes (Chr2-22) 645 inherited variants were found, 147 of them were exclusively maternal and 132 exclusively paternal. Based on these NGS data, it was possible to note that the proband's chromosomes 1 are more similar to his mother's chromosome 1 than his father's, suggesting the pathogenic homozygous variant found in this patient was inherited exclusively from the mother due to uniparental maternal isodisomy.Conclusions: This study presents a secondary analysis pipeline to identify responsible variants for a phenotype and the correct inheritance pattern, which is a critical step to the proper and accurate genetic counseling of all family members. In addition, this approach could be used to determine iUPD in different Mendelian disorders if the sequencing panel identifies variants spread throughout the genome.

The deficiency or absence of the lysosomal hydrolase α-Galactosidase A results in Fabry disease (FD), a rare and underdiagnosed X-linked disorder. The symptoms caused by FD have a direct relation with the variant present in the gene coding α-Galactosidase A (GLA) and enzyme residual activity, and it can vary drastically between men and women of the same family. Here, we present four novel variants found in patients with suspicion of FD. The patients were screened for FD by enzymatic activity and/or DNA sequencing, which showed four novel GLA missense variants. To confirm the potential pathogenicity of these variants, we employed site-directed mutagenesis. GLA wild-type and mutant plasmids were transfected into mammalian cells; RNA and proteins were extracted for expression and enzymatic activity analysis. The patients presented the variants p.Ile133Asn, p.Lys140Thr, p.Lys168Gln and p.Pro323Thr in the GLA. In vitro analysis showed pathogenic potential of three variants and one tolerated variant. The variants p.Ile133Asn and p.Lys168Gln showed no residual activity and, therefore, leading to classical phenotype, and the variant p.Lys140Thr, which presented 22% of residual activity, was considered a mild variant leading to non-classical phenotype. The variant p.Pro323Thr presented 66.7% of residual activity and alone, it is not enough to cause FD.

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.

Purpose: In Mycobacterium tuberculosis endemic regions, BCG vaccine is administered early after birth to confer protection against severe form of tuberculosis disease. Previous reports suggest that BCG adverse events, even localized ones (BCGitis), can be the first manifestation of immunodeficiency. We investigated children with a history of BCGitis who needed drug treatment looking for possibly pathogenic variants in inborn errors of immunity genes (IEI-genes). Methods: Forty-four probands were evaluated. The exome sequences obtained by Next-Generation Sequencing were filtered for variants in the 344 IEI-genes described by the International Union of Immunological Societies (IUIS) and classified according to the recommendations of the American College of Medical Genetics. The identified candidate variants were validated by Sanger sequencing. Results: Out of the 44 probands, 36 were sporadic cases and 8 were familial cases. Thirty-one in 44 (70.5%) presented immunoallergic or other infectious clinical conditions besides BCGitis; 19 in 44 (43.2%) presented variants classified as pathogenic or likely pathogenic in 17 different IEI-genes, of which 35.3% were genes related to defects in intrinsic and innate immunity, including Mendelian Susceptibility to Mycobacterial Disease (MSMD) genes (IRF8, IFNGR1, JAK1, STAT1, TLR3 and TBK1). Remaining genes were distributed in another five IUIS classifications groups (CARD14, CFH, CHD7, FOXN1, NFAT5, NLRP3, NOD2, PMS2, STAT3, TNFRSF13B and TNFSF12). Conclusion: The high prevalence of pathogenic or likely pathogenic variants found in IEI-genes may be associated with BCGitis, which should be considered a sign of an inborn error of immunity.

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. To evaluate structure and gene expression differences among exposed and unexposed skin in menopausal women. Cross-sectional study, including 15 menopausal women, 55-65yo, 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. 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.

Background With the advent of whole exome (ES) and genome sequencing (GS) as tools for disease gene discovery, rare variant filtering, prioritization and data sharing have become essential components of the search for disease genes and variants potentially contributing to disease phenotypes. The computational storage, data manipulation, and bioinformatic interpretation of thousands to millions of variants identified in ES and GS, respectively, is a challenging task. To aid in that endeavor, we constructed PhenoDB, GeneMatcher and VariantMatcher. Results PhenoDB is an accessible, freely available, web-based platform that allows users to store, share, analyze and interpret their patients’ phenotypes and variants from ES/GS data. GeneMatcher is accessible to all stakeholders as a web-based tool developed to connect individuals (researchers, clinicians, health care providers and patients) around the globe with interest in the same gene(s), variant(s) or phenotype(s). Finally, VariantMatcher was developed to enable public sharing of variant-level data and phenotypic information from individuals sequenced as part of multiple disease gene discovery projects. Here we provide updates on PhenoDB and GeneMatcher applications and implementation and introduce VariantMatcher. Conclusion Each of these tools has facilitated worldwide data sharing and data analysis and improved our ability to connect genes to phenotypic traits. Further development of these platforms will expand variant analysis, interpretation, novel disease-gene discovery and facilitate functional annotation of the human genome for clinical genomics implementation and the precision medicine initiative.

Environment is crucial for manifestation and development of schizophrenia, bringing to light epigenetic mechanisms involved, such as post-transcriptional control of gene expression by miRNA, in this disorder. MiRNAs participate in physiological processes such as neurogenesis, behavioral manifestations as well as in adverse conditions likepsychiatric disorders. Experimental models are essential to investigate the development of schizophrenia from early life, especially its molecular aspects. Spontaneously hypertensive rats (SHR) are considered a good model forschizophrenia, since they show behavior that can be related to SCZ symptoms in humans and respond to treatment with anti-psychotic drugs. We investigated miRNA profile in prefrontal cortex, hippocampus and sperm of SHR through next generation sequencing (NGS). SHR showed differential expression (DE) of miRNAs related to neurological processes and schizophrenia in sperm, prefrontal cortex and hippocampus. Gene ontology and enrichment analyses showed that the majority of DE miRNA target genes are involved in pathways related to behavior, neurodevelopmentand synaptic processes. Among these genes, 12 are dysregulated in schizophrenia patients, what reinforces SHR as a good model for epigenetic and molecular studies of this disorder. Finally, altered miRNA expression in sperm suggests that SHR model may be useful for future studies on epigenetic inheritance of schizophrenia.

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.

Background Fabry disease is a rare X-linked inherited disorder caused by deficiency of α-Galactosidase A. Hundreds of mutations and non-coding haplotypes in the GLA gene have been described; however, many are variants of unknown significance, prompting doubts about the diagnosis and treatment. The α-Galactosidase A enzymatic activity in dried blood spot (DBS) samples are widely used for screening purposes; however, even when values below the normal are found, new tests are required to confirm the diagnosis. Here we describe an analysis of GLA variants and their correlation with DBS α-Galactosidase A enzymatic activity in a large Brazilian population with Fabry disease symptoms. Results We analyzed GLA variants by DNA sequencing of 803 male patients with suspected Fabry disease or belonging to high-risk populations; in 179 individuals, 58 different exonic variants were detected. From these, 50 are variants described as pathogenic and eight described as variants of unknown significance. The other individuals presented complex non-coding haplotypes or had no variants. Interestingly, the enzymatic activity in DBS was different among pathogenic variants and the other genotypes, including variants of unknown significance; the first presented mean of 12% of residual activity, while the others presented levels above 70% of the activity found in healthy controls. Conclusion The activity of α-Galactosidase A in DBS was markedly reduced in males with known pathogenic variants when compared with subjects presenting variants of unknown significance, non-coding haplotypes, or without variants, indicating a possible non-pathogenic potential of these latter genotypes. These findings bring a better understanding about the biochemical results of α-Galactosidase A in DBS samples, as well as the possible non-pathogenic potential of non-coding haplotypes and variants of unknown significance in GLA gene. These results certainly will help clinicians to decide about the treatment of patients carrying variants in the gene causing this rare but life-threatening disease.

A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T&gt, C (p.Phe83Leu) and c.560G&gt, A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

Inherited retinal dystrophies are characterized by progressive retina degeneration and mutations in at least 250 genes have been associated as disease-causing. CRB1 is one of many genes analyzed in molecular diagnosis for inherited retinal dystrophy. Crumbs homolog-1 protein encoded by CRB1 is important for cell-to-cell contact, polarization of epithelial cells and the morphogenesis of photoreceptors. Pathogenic variants in CRB1 lead to a huge variety of phenotypes ranging from milder forms of inherited retinal dystrophy, such as retinitis pigmentosa to more severe phenotypes such as Leber congenital amaurosis. In this study, seven novel likely-pathogenic variants were identified: four missense variants (p.Leu479Pro, p.Ala921Pro, p.Cys948Arg and p.Asp1031Asn), two frameshift deletions (c.2536_2542del7 and c.3460_3461delTG) and one frameshift indel variant (c.276_294delinsTGAACACTGTAC). Furthermore, two patients with cone-rod dystrophy due to mutations in CRB1 were reported, supporting previous data, in which mutations in CRB1 can also cause cone-rod dystrophy. Finally, our data suggested there was a direct relation between phenotype severity and the mutation effect on protein functionality in 15 Brazilian CRB1 patients.

Vertebral malformations (VMs) are caused by alterations in somitogenesis and may occur in association with other congenital anomalies. The genetic etiology of most VMs remains unknown and their identification may facilitate the development of novel therapeutic and prevention strategies. Exome sequencing was performed on both the discovery cohort of nine unrelated probands from the USA with VMs and the replication cohort from China (Deciphering Disorders Involving Scoliosis & COmorbidities study). The discovery cohort was analyzed using the PhenoDB analysis tool. Heterozygous and homozygous, rare and functional variants were selected and evaluated for their ClinVar, HGMD, OMIM, GWAS, mouse model phenotypes, and other annotations to identify the best candidates. Genes with candidate variants in three or more probands were selected. The replication cohort was analyzed by another in-house developed pipeline. We identified rare heterozygous variants in KIAA1217 in four out of nine probands in the discovery cohort and in five out of 35 probands in the replication cohort. Collectively, we identified 11 KIAA1217 rare variants in 10 probands, three of which have not been described in gnomAD and one of which is a nonsense variant. We propose that genetic variations of KIAA1217 may contribute to the etiology of VMs.

Kallikrein-Kinin System; Genetic variation; Hereditary angioedema Sistema calicreina-cinina; Variació genètica; Angioedema hereditari Sistema calicreina-cinina; Variación genética; Angioedema hereditario 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.

G protein-coupled receptors (GPCRs) are the largest family of membrane protein playing an important role in cellular signal transduction. GPCRs interact with different molecules acting as ligands capable to trigger responses on signaling pathway. Those molecules present specific binding profiles in which, usually, are determined by methods based on radioactive labeled ligands. Here we present an alternative method based on time-resolved fluorescent labeled ligand, specific customized for angiotensin II receptors (AGTR1 and AGTR2) and kinin receptors (BDKRB1 and BDKRB2) wherein, their natural ligands were labeled with the lanthanide europium to generate the Eu

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B2 receptor, a family A G-protein coupled receptor. Using a strategy which allows systematical monitoring of BK R1 and R9 residues and B2 receptor acidic residues Glu5.35(226) and Asp6.58(298), our study aims at clarifying the BK interaction profile with the B2 receptor [receptor residue numbers are normalized according to Ballesteros and Weinstein, Methods Neurosci. 25 (1995), pp. 366–428) followed by receptor sequence numbering in brackets]. N- and C-terminal analogs of BK (-A1, -G1, -K1, -E1 and BK-A9) were tested against wild type B2, Glu5.35(226)Ala and Asp6.58(298)Ala B2 mutant receptors for their affinity and capability to elicit responses by mechanical recordings of isolated mice stomach fundus, measuring intracellular calcium mobilization, and competitive fluorimetric binding assays. BK showed 2- and 15-fold decreased potency for Glu5.35(226) and Asp6.58(298) B2 mutant receptors, respectively. In B2-Glu5.35(226)Ala BK analogs showed milder reduction in evaluated parameters. On the other hand, in the B2-Asp6.58(298)Ala mutant, no N-terminal analog was able to elicit any response. However, the BK-A9 analog presented higher affinity parameters than BK in the latter mutant. These findings provide enough support for defining a novel interaction role of BK-R9 and Asp6.58(298) receptor residues.

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.

Among the Brazilian population, the frequency rates of inherited retinal dystrophies and their causative genes are underreported. To increase the knowledge about these dystrophies in our population, we retrospectively studied the medical records of 1,246 Brazilian patients with hereditary retinopathies during 20 years of specialized outpatient clinic care. Of these patients, 559 had undergone at least one genetic test. In this cohort, the most prevalent dystrophies were non-syndromic retinitis pigmentosa (35%), Stargardt disease (21%), Leber congenital amaurosis (9%), and syndromic inherited retinal dystrophies (12%). Most patients had never undergone genetic testing (55%), and among the individuals with molecular test results, 28.4% had negative or inconclusive results compared to 71.6% with a conclusive molecular diagnosis. ABCA4 was the most frequent disease-causing gene, accounting for 20% of the positive cases. Pathogenic variants also occurred frequently in the CEP290, USH2A, CRB1, RPGR, and CHM genes. The relative frequency rates of different inherited retinal dystrophies in Brazil are similar to those found globally. Although mutations in more than 250 genes lead to hereditary retinopathies, only 66 genes were responsible for 70% of the cases, which indicated that smaller and cheaper gene panels can be just as effective and provide more affordable solutions for implementation by the Brazilian public health system.

Autonomic dysreflexia (AD), a syndrome caused by loss of supraspinal control over sympathetic activity and amplified vascular reflex upon sensory stimuli below injury level, is a major health problem in high-level spinal cord injury (SCI). After supraspinal sympathetic control of the vasculature below the lesion is lost, the renin-angiotensin system (RAS) is thought to be involved in AD by regulating blood pressure and vascular reactivity. In this study, we aimed to assess the role of different RAS receptors during AD following SCI. Therefore, we induced AD by colorectal distention (CRD) in wild-type mice and mice deficient in the RAS components angiotensin (Ang) II type 1a receptor (AT1a) (Agtr1a-/-) and Ang-(1-7) receptor Mas (Mas-/-) four weeks after complete transection of spinal cord at thoracic level 4 (T4). Systemic blood pressure measurements and wire myography technique were performed to assess hemodynamics and the reactivity of peripheral arteries, respectively. CRD increased mean arterial blood pressure (MAP) and decreased heart rate (HR) in all three animal groups. However, we found less increases in MAP in Mas-/- mice compared to control mice after CRD, whereas AT1a deficiency did not affect the hemodynamic response. We found that the reactivity of wild-type and Mas-/- mesenteric arteries, which are innervated from ganglia distal but close to thoracic level T4, was diminished in response to Ang II in AD after T4-SCI, but this difference was not observed in the absence of AT1a receptors. CRD did not influence the reactivity of femoral arteries which are innervated from ganglia more distal to thoracic level T4, in response to Ang II in AD. In conclusion, we identified a specific role of the Ang-(1-7) receptor Mas in regulating the systemic blood pressure increase in AD in T4-SCI mice. Furthermore, AT1a signaling is not involved in this hemodynamic response, but underlies increased vascular reactivity in mesenteric arteries in response to Ang II, where it may contribute to adaptive changes in regional blood flow.

Although the pathogenicity of the prominin-1 (PROM1) gene has already been described as associated with autosomal dominant Stargardt disease, little is known about sequence variations in this gene.The aim of this study was to evaluate PROM1 gene sequence variations in patients with macular dystrophy.This retrospective study evaluated variations in the PROM1 gene detected by next-generation sequencing test in patients with macular dystrophy and Stargardt disease.Of 25 medical records of patients with Stargardt disease, three records of patients with PROM1 gene sequence variations were selected for the study. The p.Asp776Val and p.Asp829Asn variants were detected in cases 1 and 2, respectively, and predicted to be pathogenic; they were probably responsible for macular dystrophy in these patients. Case 3 showed a p.Ala643Gly variant in the PROM1 gene and a single variation in the ABCA4 gene, but molecular testing results were inconclusive.In cases of Stargardt disease, where molecular testing results are inconclusive for pathogenic variations in the ABCA4 gene, variations in the PROM1 gene may occur and be considered responsible for the disease in the molecular analysis. This study described three cases in which variations in PROM1 gene may play a role in the pathogenesis of macular dystrophy or be associated with both autosomal recessive and autosomal dominant inheritance.

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) 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. Fed Univ Sao Paulo UNIFESP, Dept Ophthalmol & Visual Sci, Sao Paulo, Brazil Fed Univ Sao Paulo UNIFESP, Dept Biophys, Sao Paulo, Brazil Oregon Hlth & Sci Univ, Casey Eye Inst, Mol Diagnost Lab, Portland, OR 97201 USA Mol Vis Lab, Hillsboro, OR USA Fed Univ Sao Paulo UNIFESP, Dept Ophthalmol & Visual Sci, Sao Paulo, Brazil Fed Univ Sao Paulo UNIFESP, Dept Biophys, Sao Paulo, Brazil FAPESP: 2012/50454-5 Web of Science

Angiotensin (Ang) I–converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N -acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

Medulloblastoma (MB) is a malignant childhood brain tumor from cerebellum. Four principal MB molecular subgroups were established: Sonic Hedgehog (SHH), Wingless (WNT), group 3 and group 4. The present study reports the results of the first Brazilian single-institution investigation of pediatric MB subgroups-related genes. We developed an AmpliSeq(TM) custom panel for DNA sequencing, comprising CTNNB1, PTCH1, DDX3X, TERT and TP53 genes. We selected 28 MB treatment-naïve tumor samples. We observed two variants in exon 3 of CTNNB1, and two non-described variants in DDX3X gene. PTCH1 gene presented the highest number of variants observed (74% of samples). TP53 variants were present in 15% of samples, occurring concomitantly with PTCH1 mutations, and was related to worst overall survival (p=0.0005). Previous studies about pediatric MB genome were carried out from cohorts of North American and European populations, resulting in the identification of several genes variants. However, these variants might not be representative for other populations, mainly Brazilian population, due to the history of the current Brazilian population formation. This particularity might contribute to a very specific combination of rare and common variation, making this population interesting and enhancing the importance of the present screening study. MB occurring in pediatric patients requires deregulation of a minimal number of driver genes, and, variants that leads to deregulation of these genes may not be the same between different populations. Finally, we reinforce that new molecular diagnostic approaches involving PTCH1 and TP53 genes screening, concomitantly, can collaborate for the better clinical management of infancy and childhood MB.

e13623Background: Lynch syndrome (LS) is the most common inherited colorectal cancer syndrome (CRC), comprising 2-4% of CRC, that may cause others cancers. Individuals with LS have at least one MMR...

Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC 1 -AngII and TOAC 0 -BK) and internal (TOAC 3 -AngII and TOAC 3 -BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT 1 and B 2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC 3 -AngII or TOAC 3 -BK), the TOAC 1 -AngII and TOAC 0 -BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT 1 or B 2 receptors, respectively. In addition, TOAC 1 -AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC 0 -BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC 3 -AngII or TOAC 3 -BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide–receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT 1 and B 2 receptors.

Angiotensin II (AngII) and kinins (bradykinin (BK) and des-Arg9-bradykinin (DBK)), are potent agents involved in the maintenance of blood pressure and several biological activities, and their better understanding is important to produce new drugs aimed to control arterial blood pressure. Previous studies on ligand-receptor binding have been based on radioactive methods, which led us to study a new method based on the fluorimetric method. A lanthanide attached to the N-terminal segment of the peptide (AngII, BK and DBK), which produces a time-resolved-fluorescent ligand, was used in a binding test with CHO cells expressing the AT1, AT2, B1 or B2 receptors in comparison with the same cell line tested with the radioactive ligand. Our findings indicated that the non-radioactive method provided a comparable result for the angiotensin receptors. On the other hand, the kinin receptors showed a slight reduction in the binding affinity, probably due to the linkage at the N-terminal segment and/or to the lower biological stability associated to the high temperature (37°C) used for the fluorimetric method, while the radioactive one was at 4°C. We can conclude that a time-resolved fluorescence assay would provide a sensitive method as an alternative tool for receptor studies.

Mutant forms of kinin B(1) receptor (B(1)R) and analogs of the full agonist des-Arg(9)-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys(100)-Cys(650)), the N-terminal (N(t)) and the EC3 loop C-terminal (C(t)) segments of angiotensin II (AngII) receptor 1 (AT(1)R) have been identified to form an extracellular site for binding the agonist N(t) segment (Asp(1) and Arg(2) residues). Asp(712) residue at the receptor EC3 loop binds the peptide Arg(2) residue. By homology, a similar site might be considered for DABK binding to B(1)R since this receptor contains the same structural elements for composing the site in AT(1)R, namely the disulfide bond and the EC3 loop Asp(712) residue. DABK, Ala(n)-DABK analogs (n=Ala(1)-, Ala(2)-, Ala(3)-, Ala(4)-, Ala(5)-, Ala(6)-, Ala(7)-, Ala(8)-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B(1)R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT(1)R system may be applied to DABK binding to B(1)R. The most crucial similarity in the two cases is that the N(t) segments of peptides equally bind to the homologous Asp(712) residue of both AT(1)R and B(1)R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B(1)R extracellular site as EC3 loop Asp(712) and Cys(100) caused the same modifications in biological assays observed in AT(1)R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B(1)R receptors.

Bradykinin (BK) and des-Arg 9 -bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B 2 (B 2 R) and B 1 (B 1 R) receptors, respectively. It was already shown that the deletion of kinin B 1 R or of B 2 R induces upregulation of the remaining receptor subtype [10] , [12] , [16] , [28] , [36] . However studies on overexpression of B 1 R or B 2 R 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 B 1 R (TGR(Tie 2 B 1 )) exclusively in the endothelium. In another study, mice overexpressing B 1 R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B 2 R was investigated in the thoracic aorta isolated from TGR(Tie 2 B 1 ) rats overexpressing the B 1 R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B 2 R 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 B 2 R associated with the overexpression of the B 1 R.

Previous research showed that disruption of the Cys18-Cys274bond in the angiotensin II (AngII) AT1receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar1Leu8]-AngII, specific AT1receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT1receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP3. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT1receptor leads to a conformational structure similar to that of the active mode of the AT1receptor, favoring its internalization in the absence of the agonist.

Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT₁ receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu⁸]-AngII, specific AT₁ receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT₁ receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP₃. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT₁ receptor leads to a conformational structure similar to that of the active mode of the AT₁ receptor, favoring its internalization in the absence of the agonist.

Bradykinin (BK) is a vasorelaxant, algesic and inflammatory agent. Angiotensin II (AngII) is known to control vascular tone and promote growth, inflammation and artherogenesis. There is evidence for cross talking between BK and AngII receptors. Therefore, the effect of lack of kinin receptors was assessed in mice with genetic disruption of B1 or B2 and both receptors. Responsiveness of abdominal aortic rings to BK and AngII as well as the receptor gene expression of both peptides were analysed. Although no specific phenotype was displayed in the normotensive and healthy mice lacking the kinin receptors, a decreased expression level of the remaining kinin receptor mRNA was observed. AT1 receptor mRNA level was also reduced, indicating that kinin receptors regulate AngII receptors. Downregulation of the receptors was well correlated with reduction in the reactivity of both agonists to induce contraction of aortic rings, but other signal regulations must be sought in these transgenic mice. We conclude that cross talk between kinin and AngII receptors occurs in mouse abdominal aorta and that both peptides may regulate the initiation and progression of important pathophysiological processes, such as hypertension and inflammation.