De novo variants identified by trio whole exome sequencing of bladder exstrophy epispadias complex.

Bladder exstrophy epispadias complex (BEEC) encompasses a spectrum of conditions ranging from mild epispadias to the most severe form: omphalocele-bladder exstrophy-imperforate anus-spinal defects (OEIS). BEEC involves abnormalities related to anatomical structures that are proposed to have a similar underlying etiology and pathogenesis. In general, BEEC, is considered to arise from a sequence of events in embryonic development and is believed to be a multi-etiological disease with contributions from genetic and environmental factors. Several genes have been implicated and mouse models have been generated, including a knockout model of p63, which is involved in the synthesis of stratified epithelium. Mice lacking p63 have undifferentiated ventral urothelium. MNX1 has also been implicated. In addition, cigarette smoking, diazepam and clomid have been implied as environmental factors due to their relative association. By in large, the etiology and pathogenesis of human BEEC is unknown. We performed de novo analysis of whole exome sequencing (WES) of germline samples from 31 unrelated trios where the probands have a diagnosis of BEEC syndrome. We also evaluated the DECIPHER database to identify copy number variants (CNVs) in genes in individuals with the search terms "bladder exstrophy" in an attempt to identify additional candidate genes within these regions. Several de novo variants were identified; however, a candidate gene is still unclear. This data further supports the multi-etiological nature of BEEC.

An observational human study investigating the effect of anabolic androgenic steroid use on the transcriptome of skeletal muscle and whole blood using RNA-Seq.

BACKGROUND: The effects of Anabolic Androgenic Steroids (AAS) are largely illustrated through Androgen Receptor induced gene transcription, yet RNA-Seq has yet to be conducted on human whole blood and skeletal muscle. Investigating the transcriptional signature of AAS in blood may aid AAS detection and in muscle further understanding of AAS induced hypertrophy. METHODS: Males aged 20-42 were recruited and sampled once: sedentary controls (C), resistance trained lifters (RT) and resistance trained current AAS users (RT-AS) who ceased exposure ≤ 2 or ≥ 10 weeks prior to sampling. RT-AS were sampled twice as Returning Participants (RP) if AAS usage ceased for ≥ 18 weeks. RNA was extracted from whole blood and trapezius muscle samples. RNA libraries were sequenced twice, for validation purposes, on the DNBSEQ-G400RS with either standard or CoolMPS PE100 reagents following MGI protocols. Genes were considered differentially expressed with FDR < 0.05 and a 1.2- fold change. RESULTS: Cross-comparison of both standard reagent whole blood (N = 55: C = 7, RT = 20, RT-AS ≤ 2 = 14, RT-AS ≥ 10 = 10, RP = 4; N = 46: C = 6, RT = 17, RT-AS ≤ 2 = 12, RT-AS ≥ 10 = 8, RP = 3) sequencing datasets, showed that no genes or gene sets/pathways were differentially expressed between time points for RP or between group comparisons of RT-AS ≤ 2 vs. C, RT, or RT-AS ≥ 10. Cross-comparison of both muscle (N = 51, C = 5, RT = 17, RT-AS ≤ 2 = 15, RT-AS ≥ 10 = 11, RP = 3) sequencing (one standard & one CoolMPS reagent) datasets, showed one gene, CHRDL1, which has atrophying potential, was upregulated in RP visit two. In both muscle sequencing datasets, nine differentially expressed genes, overlapped with RT-AS ≤ 2 vs. RT and RT-AS ≤ 2 vs. C, but were not differentially expressed with RT vs. C, possibly suggesting they are from acute doping alone. No genes seemed to be differentially expressed in muscle after the long-term cessation of AAS, whereas a previous study found long term proteomic changes. CONCLUSION: A whole blood transcriptional signature of AAS doping was not identified. However, RNA-Seq of muscle has identified numerous differentially expressed genes with known impacts on hypertrophic processes that may further our understanding on AAS induced hypertrophy. Differences in training regimens in participant groupings may have influenced results. Future studies should focus on longitudinal sampling pre, during and post-AAS exposure to better control for confounding variables.

Disruption of the HIF-1 pathway in individuals with Ollier disease and Maffucci syndrome.

Ollier disease (OD) and Maffucci Syndrome (MS) are rare disorders characterized by multiple enchondromas, commonly causing bone deformities, limb length discrepancies, and pathological fractures. MS is distinguished from OD by the development of vascular anomalies. Both disorders are cancer predisposition syndromes with malignancies developing in ~50% of the individuals with OD or MS. Somatic gain-of-function variants in IDH1 and IDH2 have been described in the enchondromas, vascular anomalies and chondrosarcomas of approximately 80% of the individuals with OD and MS. To date, however, no investigation of germline causative variants for these diseases has been comprehensively performed. To search for germline causative variants, we performed whole exome sequencing or whole genome sequencing of blood or saliva DNA in 94 unrelated probands (68 trios). We found that 7 had rare germline missense variants in HIF1A, 6 had rare germline missense variants in VHL, and 3 had IDH1 variants including 2 with mosaic IDH1-p.Arg132His variant. A burden analysis using 94 probands assigned as cases and 2,054 unrelated individuals presenting no OD- or MS-related features as controls, found that variants in HIF1A, VHL, and IDH1 were all significantly enriched in cases compared to controls. To further investigate the role of HIF-1 pathway in the pathogenesis of OD and MS, we performed RNA sequencing of fibroblasts from 4 probands with OD or MS at normoxia and at hypoxia. When cultured in hypoxic conditions, both proband and control cells showed altered expression of a subset of HIF-1 regulated genes. However, the set of differentially expressed genes in proband fibroblasts included a significantly reduced number of HIF-1 regulated genes compared to controls. Our findings suggest that germline or early post-zygotic variants identified in HIF1A, VHL, and IDH1 in probands with OD and MS underlie the development of the phenotypic abnormalities in a subset of individuals with OD and MS, but extensive functional studies are needed to further confirm it.

Functional characterization of novel variants found in patients with suspected Fabry disease.

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.

Variant-level matching for diagnosis and discovery: Challenges and opportunities.

Here we describe MyGene2, Geno2MP, VariantMatcher, and Franklin; databases that provide variant-level information and phenotypic features to researchers, clinicians, healthcare providers and patients. Following the footsteps of the Matchmaker Exchange project that connects exome, genome, and phenotype databases at the gene level, these databases have as one goal to facilitate connection to one another using Data Connect, a standard for discovery and search of biomedical data from the Global Alliance for Genomics and Health (GA4GH).

The impact of GeneMatcher on international data sharing and collaboration.

GeneMatcher (genematcher.org) is a tool designed to connect individuals with an interest in the same gene. Now used around the world to create collaborations and generate the evidence needed to support novel disease gene identification, GeneMatcher is a founding member of the Matchmaker Exchange (MME; matchmakerexchange.org) and strongest possible advocate for global data sharing including those in resource-limited environments. As of October 1, 2021, there are 12,531 submitters from 94 countries who have submitted 58,134 submissions with 13,498 unique genes in the database. Among these genes, 8970 (64%) have matched at least once and the total number of matches is 378,806, growing by about 10,000 per month. GeneMatcher submitters increase by 80-120 each month and submissions grow by >800 per month, while unique genes and gene matches continue to grow steadily at rate of about 80 per month. The number of genes without a match peaked at 4371 in February of 2019 and despite the increase in the number of new submissions, the number of unique genes without a match continues to slowly decline, currently standing at 4,016. All submissions in GeneMatcher are available for matching across the MME.

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.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

PhenoDB, GeneMatcher and VariantMatcher, tools for analysis and sharing of sequence data.

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.

Germline ERBB2/HER2 Coding Variants Are Associated with Increased Risk of Myeloproliferative Neoplasms.

Familial cases of myeloproliferative neoplasms (MPN) are relatively common, yet few inherited risk factors have been identified. Exome sequencing of a kindred with a familial cancer syndrome characterized by both MPN and melanoma produced a germline variant in the ERBB2/HER2 gene that co-segregates with disease. To further investigate whether germline ERBB2 variants contribute to MPN predisposition, the frequency of ERBB2 variants was analyzed in 1604 cases that underwent evaluation for hematologic malignancy, including 236 cases of MPN. MPN cases had a higher frequency of rare germline ERBB2 coding variants compared to non-MPN hematologic malignancies (8.9% vs. 4.1%, OR 2.4, 95% CI: 1.4 to 4.0, p = 0.0028) as well as cases without a blood cancer diagnosis that served as an internal control (8.9% vs. 2.7%, OR 3.5, 95% CI: 1.4 to 8.3, p = 0.0053). This finding was validated via comparison to an independent control cohort of 1587 cases without selection for hematologic malignancy (8.9% in MPN cases vs. 5.2% in controls, p = 0.040). The most frequent variant identified, ERBB2 c.1960A > G; p.I654V, was present in MPN cases at more than twice its expected frequency. These data indicate that rare germline coding variants in ERBB2 are associated with an increased risk for development of MPN. The ERBB2 gene is a novel susceptibility locus which likely contributes to cancer risk in combination with additional risk alleles.

IFIH1 loss-of-function variants contribute to very early-onset inflammatory bowel disease.

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. 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. 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. 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). 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.

Analysis of an NGS retinopathy panel detects chromosome 1 uniparental isodisomy in a patient with RPE65-related leber congenital amaurosis.

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.

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.

KIAA1217: A novel candidate gene associated with isolated and syndromic vertebral malformations.

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.

Correlation between GLA variants and alpha-Galactosidase A profile in dried blood spot: an observational study in Brazilian patients.

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.

Pathogenicity Reclasssification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy.

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>C (p.Phe83Leu) and c.560G>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.

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.

Non-radioactive binding assay for bradykinin and angiotensin receptors.

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 Eu3+-N1-DTT-ligands (AngII, BK, and DBK). Competitive binding profile is determined with a fixed concentration of labeled ligand competing with variable concentrations (10-5 to 10-12) of unlabeled ligand. This method is capable to determine binding profiles with comparable results with traditional one and present a reliable alternative to radioactive based methods usage.

Distinct roles of angiotensin receptors in autonomic dysreflexia following high-level spinal cord injury in mice.

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.

Relative frequency of inherited retinal dystrophies in Brazil.

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.