Abstract Hepatic glycogen storage diseases (GSD) are characterized by recurrent episodes of hypoglycemia, and anemia has been recognized as a frequent complication of these disorders.This was a convenience cross-sectional study to evaluate hepcidin and IL-6 concentrations in patients with hepatic GSD and their association with anemia and other parameters of iron metabolism.Levels of hepcidin, IL-6, and markers of iron metabolism were measured in 32 patients receiving uncooked cornstarch therapy for GSD (GSD Ia= 18; Ib= 7; III= 3; IXa= 3; IXb= 1; median age 9.5 years). IL-6 concentrations were compared to those of 8 individuals heterozygous for GSD. Nine patients were anemic and five patients had hepatic adenomas. IL-6 levels were higher in patients than in heterozygotes. Eight patients had hyperferritinemia, and one had elevated transferrin saturation as well. Hepcidin correlated positively with ferritin levels. IL-6 correlated with hemoglobin, iron, transferrin, and transferrin saturation. There was no correlation between hepcidin and IL-6 levels. Patients with GSD Ib had the highest IL-6 levels.Anemia is a common finding in hepatic GSD, especially in GSD Ib, the type of GSD associated with the highest IL-6 levels. These findings suggest that inflammation is strongly associated with development of anemia in GSD Ib. hypoglycemia disordersThis disorders This crosssectional cross sectional IL6 IL 6 IL- metabolismLevels Levels IL6, 6, 3 Ia 18 7 III IXa IXb 1 95 9 5 9. years. years . years) adenomas heterozygotes hyperferritinemia well hemoglobin levelsAnemia Anemia

Abstract Objectives: To evaluate the bacterial microbiome found in tracheostomy cannulas of a group of children diagnosed with glossoptosis secondary to Robin Sequence (RS), and its clinical implications. Methods: Pediatric patients were enrolled in the study at the time of the cannula change in the hospital. During this procedure, the removed cannula was collected and stored for amplicon sequencing of 16s rRNA. DNA extraction was performed using DNeasy PowerBiofilm Kit (QIAGEN® - Cat nº 24000-50) while sequencing was performed with the S5 (Ion S5™ System, Thermo Fisher Scientific), following Brazilian Microbiome Project (BMP) protocol. Results: All 12 patients included in the study were using tracheostomy uncuffed cannulas of the same brand, had tracheostomy performed for over 1-year and had used the removed cannula for approximately 3-months. Most abundant genera found were Aggregatibacter, Pseudomonas, Haemophilus, Neisseria, Staphylococcus, Fusobacterium, Moraxella, Streptococcus, Alloiococcus, and Capnocytophaga. Individual microbiome of each individual was highly variable, not correlating to any particular clinical characteristic. Conclusion: The microbiome of tracheostomy cannulas is highly variable, even among patients with similar clinical characteristics, making it challenging to determine a standard for normality. © 2022 Associa¸c˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/). Objectives RS, RS , (RS) implications Methods hospital procedure s rRNA QIAGEN® QIAGEN (QIAGEN 2400050 24000 50 24000-50 S Ion System Scientific, Scientific Scientific) BMP (BMP protocol Results 1 brand 1year year 3months. 3months months 3 months. 3-months Aggregatibacter Pseudomonas Haemophilus Neisseria Staphylococcus Fusobacterium Moraxella Streptococcus Alloiococcus Capnocytophaga variable characteristic Conclusion characteristics normality 202 Associacao Associa c ao C´ervicoFacial. CervicoFacial C´ervico Facial. C ervico Facial C´ervico-Facial Ltda https//creativecommons.org/licenses/by/4.0/. httpscreativecommonsorglicensesby40 httpscreativecommonsorglicensesby https //creativecommons.org/licenses/by/4.0/ . creativecommons org licenses 4 0 (https://creativecommons.org/licenses/by/4.0/) (RS 240005 2400 5 24000-5 20 ervicoFacial C´ervicoFacial Cervico https//creativecommons.org/licenses/by/4.0/ httpscreativecommonsorglicensesby4 creativecommonsorglicensesby40 creativecommonsorglicensesby //creativecommons.org/licenses/by/4.0 (https://creativecommons.org/licenses/by/4.0/ 240 24000- 2 https//creativecommons.org/licenses/by/4.0 creativecommonsorglicensesby4 //creativecommons.org/licenses/by/4. (https://creativecommons.org/licenses/by/4.0 24 https//creativecommons.org/licenses/by/4. //creativecommons.org/licenses/by/4 (https://creativecommons.org/licenses/by/4. https//creativecommons.org/licenses/by/4 //creativecommons.org/licenses/by/ (https://creativecommons.org/licenses/by/4 https//creativecommons.org/licenses/by/ //creativecommons.org/licenses/by (https://creativecommons.org/licenses/by/ https//creativecommons.org/licenses/by (https://creativecommons.org/licenses/by

Abstract Background: Epidermolysis bullosa is characterized by cutaneous fragility and blistering. Historically, diagnosis is achieved by immunofluorescence mapping or transmission electron microscopy, both involving biopsy procedures. Genetic analysis, especially through next-generation sequencing, is an important tool for the diagnosis of this disease. In Brazil, access to diagnostic methods is limited, and consequently, most patients do not have an accurate diagnosis. Diagnosis allows the indication of prognosis and genetic counselling of the patient. Objectives: To evaluate the cost-effectiveness of a gene panel compared to immunofluorescence mapping and transmission electron microscopy by analyzing its benefits, limitations, and economic aspects. Methods: The gene panel included the 11 main genes associated with epidermolysis bullosa. The techniques were compared, assessing the average cost, advantages, and limitations, through a price survey and literature review. Results: Both immunofluorescence mapping and transmission electron microscopy require skin biopsy, are dependent on the investigator's expertise, and are subject to frequent inconclusive results. The gene panel is effective for the conclusive diagnosis of epidermolysis bullosa, presents high efficiency and accuracy, is economically feasible, and excludes the need for biopsy. The gene panel allows for prognosis, prenatal genetic diagnosis, and genetic counseling. Study limitations: It was not possible to find laboratories that perform transmission electron microscopy for epidermolysis bullosa diagnosis in Brazil. Conclusion: This study supports the gene panel as the first-choice method for epidermolysis bullosa diagnosis.

ABSTRACT Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by a disturbance in the metabolism of glucocerebroside in the macrophages. Most of its manifestations – hepatosplenomegaly, anemia, thrombocytopenia, and bone pain – are amenable to a macrophage-target therapy such as enzyme replacement. However, there is increasing evidence that abnormalities of the liver persist despite the specific GD treatment. In this work, we adapted histomorphometry techniques to the study of hepatocytes in GD using liver tissue of treated patients, developing the first morphometrical method for canalicular quantification in immunohistochemistry-stained liver biopsies, and exploring histomorphometric characteristics of GD. This is the first histomorphometric technique developed for canalicular analysis on histological liver biopsy samples.

Abstract The effects of non-nutritive sweeteners (NNS) on the gut microbiota are an area of increasing research interest due to their potential influence on weight gain, insulin resistance, and inflammation. Studies have shown that mice and rats fed saccharin develop weight gain and metabolic alterations, possibly related to changes in gut microbiota. Here, we hypothesized that chronic exposure to a commercial NNS would change the gut microbiota composition in Wistar rats when compared to sucrose exposure. To test this hypothesis, Wistar rats were fed either NNS- or sucrose-supplemented yogurt for 17 weeks alongside standard chow (ad libitum). The gut microbiome was assessed by 16S rDNA deep sequencing. Assembly and quantification were conducted using the Brazilian Microbiome Project pipeline for Ion Torrent data with modifications. Statistical analyses were performed in the R software environment. We found that chronic feeding of a commercial NNS-sweetened yogurt to Wistar rats, within the recommended dose range, did not significantly modify gut microbiota composition in comparison to sucrose-sweetened yogurt. Our findings do not support the hypothesis that moderate exposure to NNS is associated with changes in gut microbiota pattern compared to sucrose, at least in this experimental model.

RESUMO As paraparesias espásticas hereditárias (PEH) são um grupo de doenças genéticas caracterizado por espasticidade dos membros inferiores com ou sem características neurológicas adicionais. A disfunção da deglutição é pouco estudada nas PEH e sua presença pode levar a complicações respiratórias e nutricionais significativas. Objetivo: O objetivo deste estudo foi avaliar a frequência e a caracterização clínica da disfagia em diferentes tipos de PEH. Métodos: Foi realizado um estudo transversal em dois centros. Os pacientes com PEH confirmados geneticamente foram avaliados pelo Northwestern Dysphagia Patient Check Sheet e pela Escala Funcional de Ingestão Oral. Além disso, a autopercepção da disfagia foi avaliada pelo Eat Assessment Tool-10 e pelo Swallowing Disturbance Questionnaire. Resultados: Trinta e seis pacientes com paraplegia espástica tipo 4 (SPG4), cinco com SPG11, quatro com SPG5, quatro com xantomatose cerebrotendinosa (CTX), três com SPG7 e dois com SPG3A foram avaliados. Disfagia orofaríngea leve a moderada estava presente em 3/5 (60%) dos pacientes com SPG11 e 2/4 (50%) dos pacientes com CTX. Um único SPG4 (2%) e um único SPG7 (33%) apresentaram disfagia orofaríngea leve. Todos os outros pacientes avaliados apresentaram deglutição normal ou funcional. Conclusão: Disfagia orofaríngea clinicamente significativa estava presente apenas nas formas complicadas de PEH. A SPG11 e CTX apresentaram maiores riscos de disfagia, sugerindo que a avaliação da deglutição deve fazer parte do manejo dos pacientes com essas condições.

ABSTRACT Hereditary spastic paraplegias (HSP) are a group of genetic diseases characterized by lower limb spasticity with or without additional neurological features. Swallowing dysfunction is poorly studied in HSP and its presence can lead to significant respiratory and nutritional complications. Objectives: The aim of this study was to evaluate the frequency and clinical characteristics of dysphagia in different types of HSP. Methods: A two-center cross-sectional prevalence study was performed. Genetically confirmed HSP patients were evaluated using the Northwestern Dysphagia Patient Check Sheet and the Functional Oral Intake Scale. In addition, self-perception of dysphagia was assessed by the Eat Assessment Tool-10 and the Swallowing Disturbance Questionnaire. Results: Thirty-six patients with spastic paraplegia type 4 (SPG4), five with SPG11, four with SPG5, four with cerebrotendinous xanthomatosis (CTX), three with SPG7, and two with SPG3A were evaluated. Mild to moderate oropharyngeal dysphagia was present in 3/5 (60%) of SPG11 and 2/4 (50%) of CTX patients. A single SPG4 (2%) and a single SPG7 (33%) patient had mild oropharyngeal dysphagia. All other evaluated patients presented with normal or functional swallowing. Conclusions: Clinically significant oropharyngeal dysphagia was only present in complicated forms of HSP Patients with SPG11 and CTX had the highest risks for dysphagia, suggesting that surveillance of swallowing function should be part of the management of patients with these disorders.

Abstract Pathogenic variants in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for cystic fibrosis (CF), the commonest monogenic autosomal recessive disease, and CFTR-related disorders in infants and youth. Diagnosis of such diseases relies on clinical, functional, and molecular studies. To date, over 2,000 variants have been described on CFTR (~40% missense). Since few of them have confirmed pathogenicity, in silico analysis could help molecular diagnosis and genetic counseling. Here, the pathogenicity of 779 CFTR missense variants was predicted by consensus predictor PredictSNP and compared to annotations on CFTR2 and ClinVar. Sensitivity and specificity analysis was divided into modeling and validation phases using just variants annotated on CFTR2 and/or ClinVar that were not in the validation datasets of the analyzed predictors. After validation phase, MAPP and PhDSNP achieved maximum specificity but low sensitivity. Otherwise, SNAP had maximum sensitivity but null specificity. PredictSNP, PolyPhen-1, PolyPhen-2, SIFT, nsSNPAnalyzer had either low sensitivity or specificity, or both. Results showed that most predictors were not reliable when analyzing CFTR missense variants, ratifying the importance of clinical information when asserting the pathogenicity of CFTR missense variants. Our results should contribute to clarify decision making when classifying the pathogenicity of CFTR missense variants.

Abstract Mucolipidosis II and III (ML II and III) alpha/beta and ML III gamma are lysosomal diseases caused by GlcNAc-1-phosphotransferase deficiency. Previous data indicate that MLII patients have functionally impaired immune system that contributes to predisposition to infections.We evaluated the immunological phenotype of three Brazilian patients with ML III gamma. Our data suggest that the residual activity of GlcNAc-1-phosphotransferase in patients with ML III gamma is enough to allow the targeting of the lysosomal enzymes required for B-cell functions maintenance.

Abstract Lysosomal storage disorders (LSDs) constitute a heterogeneous group of approximately 50 genetic disorders. LSDs diagnosis is challenging due to variability in phenotype penetrance, similar clinical manifestations, and a high allelic heterogeneity. A powerful tool for the diagnosis of the disease could reduce the “diagnostic odyssey” for affected families, leading to an appropriate genetic counseling and a better outcome for current therapies, since enzyme replacement therapies have been approved in Brazil for Gaucher, Fabry, and Pompe diseases, and are under development for Niemann-Pick Type B. However, application of next-generation sequencing (NGS) technology in the clinical diagnostic setting requires a previous validation phase. Here, we assessed the application of this technology as a fast, accurate, and cost-effective method to determine genetic diagnosis in selected LSDs. We have designed two panels for testing simultaneously 11 genes known to harbor casual mutations of LSDs. A cohort of 58 patients was used to validate those two panels, and the clinical utility of these gene panels was tested in four novel cases. We report the assessment of a NGS approach as a new tool in the diagnosis of LSDs in our service.

Abstract Mucopolysaccharidosis (MPS) are a group of rare genetic disorders caused by deficiency in the activity of specific lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs). A defect in the activity of these enzymes will result in the abnormal accumulation of GAGs inside the lysosomes of most cells, inducing progressive cellular damage and multiple organ failure. DNA samples from 70 patients with biochemical diagnosis of different MPSs genotypes confirmed by Sanger sequencing were used to evaluate a Next Generation Sequencing (NGS) protocol. Eleven genes related to MPSs were divided into three different panels according to the clinical phenotype. This strategy led to the identification of several pathogenic mutations distributed across all exons of MPSs-related genes. We were able to identify 96% of all gene variants previously identified by Sanger sequencing, showing high sensitivity in detecting different types of mutations. Furthermore, new variants were not identified, representing 100% specificity of the NGS protocol. The use of this NGS approach for genotype identification in MPSs is an attractive option for diagnosis of patients. In addition, the MPS diagnosis workflow could be divided in a two-tier approach: NGS as a first-tier followed by biochemical confirmation as a second-tier.

Abstract Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

Abstract Untreated vitamin B12 deficiency manifests clinically with hematological abnormalities and combined degeneration of the spinal cord and polyneuropathy and biochemically with elevated homocysteine (Hcy) and methylmalonic acid (MMA). Vitamin B12 metabolism involves various cellular compartments including the lysosome, and a disruption in the lysosomal and endocytic pathways induces functional deficiency of this micronutrient. Gaucher disease (GD) is characterized by dysfunctional lysosomal metabolism brought about by mutations in the enzyme beta-glucocerebrosidase (Online Mendelian Inheritance in Man (OMIM): 606463; Enzyme Commission (EC) 3.2.1.45, gene: GBA1). In this study, we collected and examined available literature on the associations between GD, the second most prevalent lysosomal storage disorder in humans, and hampered vitamin B12 metabolism. Results from independent cohorts of patients show elevated circulating holotranscobalamin without changes in vitamin B12 levels in serum. Gaucher disease patients under enzyme replacement therapy present normal levels of Hcy and MMA. Although within the normal range, a significant increase in Hcy and MMA with normal serum vitamin B12 was documented in treated GD patients with polyneuropathy versus treated GD patients without polyneuropathy. Thus, a functional deficiency of vitamin B12 caused by disrupted lysosomal metabolism in GD is a plausible mechanism, contributing to the neurological form of the disorder but this awaits confirmation. Observational studies suggest that an assessment of vitamin B12 status prior to the initiation of enzyme replacement therapy may shed light on the role of vitamin B12 in the pathogenesis and progression of GD.

Abstract The clinical utility of serum ferritin as a biomarker of disease severity and prognosis in Gaucher disease (GD) is still debated. Here, we aimed to evaluate ferritin and its relation to clinicolaboratory parameters of GD patients seen at the Reference Center for Gaucher Disease of Rio Grande do Sul, Brazil, so as to gather evidence on the utility of ferritin as a biomarker of this condition. A retrospective chart review was performed collecting pre-and posttreatment data from GD patients. Eighteen patients with ferritin levels available before and after treatment were included in the study. Nine of these participants were males, and seventeen had type I GD. All patients were given either enzyme replacement (n = 16) or substrate reduction therapy (n = 2), and ferritin was found to decrease from 756 [318-1441] ng/mL at baseline to 521 [227-626] ng/mL (p=0.025) after 28.8 month soft treatment. Serum ferritin levels did not correlate with measures of disease severity, but showed an association with age at onset of treatment (ρ= 0.880; n = 18; p < 0.001). In conclusion, although serum ferritin did not correlate with disease severity, after a median 28.8 months of treatment, clinical outcomes had clearly improved, and ferritin levels had decreased.

OBJETIVO: Avaliar a contribuição da análise molecular do gene cystic fibrosis transmembrane conductance regulator (CFTR, regulador da condutância transmembrana na fibrose cística) na investigação diagnóstica da fibrose cística em pacientes com suspeita de fibrose cística (FC) leve ou atípica. MÉTODOS: Estudo transversal em adolescentes e adultos (idade ≥ 14 anos). Os voluntários foram submetidos à avaliação clínica, laboratorial e radiológica; espirometria, microbiologia do escarro, ecografia hepática, teste do suor e análise molecular do gene CFTR. Compararam-se as características dos pacientes divididos em três grupos, segundo o número de mutações identificadas (duas ou mais, uma e nenhuma). RESULTADOS: Foram avaliados 37 pacientes com achados fenotípicos de FC, com ou sem confirmação pelo teste do suor. Houve predomínio do sexo feminino (75,7%), e a média de idade dos participantes foi de 32,5 ± 13,6 anos. A análise molecular contribuiu para o diagnóstico de FC em 3 casos (8,1%), todos esses com pelo menos duas mutações. Houve a identificação de uma e nenhuma mutação, respectivamente, em 7 (18,9%) e 26 pacientes (70,3%). Nenhuma característica clínica estudada se associou com o diagnóstico genético. A mutação p.F508del foi a mais comum, encontrada em 5 pacientes. A associação de p.V232D e p.F508del foi encontrada em 2 pacientes. Outras mutações encontradas foram p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H e p.T351S. CONCLUSÕES: A análise molecular da região codificadora do gene CFTR apresentou uma contribuição limitada para a investigação diagnóstica desses pacientes com suspeita de FC leve ou atípica. Além disso, não houve associações entre as características clínicas e o diagnóstico genético.

OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.