Abstract Massive sequencing platforms allow the identification of complex clinical phenotypes involving more than one autosomal recessive disorder. In this study, we report on an adult patient, born to a related couple (third degree cousins), referred for genetic evaluation due to ectopia lentis, deafness and previous diagnosis of juvenile idiopathic arthritis. He was biochemically diagnosed as having Classic Homocystinuria (HCU); Sanger sequencing of the CBS gene showed the genotype NM_000071.2(CBS):c.[833T>C];[833T>C], compatible with the diagnosis of pyridoxine-responsive HCU. As he also had symptoms not usually associated with HCU, exome sequencing was performed. In addition to the variants found in the Sanger sequencing, the following variants were identified: NM_001256317.1(TMPRSS3):c.[413C>A];[413C>A]; and the NM_005807.6(PRG4):c.[3756dup]:[3756dup], confirming the diagnosis of autosomal recessive nonsyndromic deafness and Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome (CACP), respectively. Genomic analysis allowed the refinement of the diagnosis of a complex case and improvement of the patient’s treatment. disorder study patient third cousins, cousins , cousins) lentis arthritis HCU (HCU) NM_000071.2CBSc.833T>C833T>C, NM0000712CBSc833TC833TC NMCBScTCTC NM_000071.2 c. 833T>C NM 000071 2 c 833T C T NM_000071.2(CBS):c.[833T>C];[833T>C] pyridoxineresponsive pyridoxine responsive performed identified NM_001256317.1TMPRSS3c.413C>A413C>A NM0012563171TMPRSS3c413CA413CA NMTMPRSScCACA NM_001256317.1 TMPRSS3 413C>A 001256317 1 413C A TMPRSS NM_001256317.1(TMPRSS3):c.[413C>A];[413C>A] NM_005807.6PRG4c.3756dup3756dup, NM0058076PRG4c3756dup3756dup NMPRGcdupdup NM_005807.6 PRG4 3756dup 005807 6 PRG dup NM_005807.6(PRG4):c.[3756dup]:[3756dup] CamptodactylyArthropathyCoxa Camptodactyly Arthropathy Coxa VaraPericarditis Vara Pericarditis CACP, CACP (CACP) respectively patients s treatment (HCU 2CBSc C833T CBSc NM_000071.2CBSc.833T>C833T>C TC NM0000712 NM_000071. 833TC 00007 NM_000071.2(CBS):c.[833T>C];[833T>C 1TMPRSS3c A413C CA NM0012563171 NM_001256317. 413CA 00125631 NM_001256317.1(TMPRSS3):c.[413C>A];[413C>A 6PRG4c 3756dup3756dup NM_005807.6PRG4c.3756dup3756dup NM0058076 NM_005807. 00580 NM_005807.6(PRG4):c.[3756dup]:[3756dup (CACP CT NM000071 NM_000071 0000 TMPRSSc AC NM001256317 NM_001256317 0012563 PRGc dupdup NM005807 NM_005807 0058 NM00007 NM_00007 000 NM00125631 NM_00125631 001256 NM00580 NM_00580 005 NM0000 NM_0000 00 NM0012563 NM_0012563 00125 NM0058 NM_0058 NM000 NM_000 0 NM001256 NM_001256 0012 NM005 NM_005 NM00 NM_00 NM00125 NM_00125 001 NM0 NM_0 NM0012 NM_0012 NM_ NM001 NM_001

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

Resumo Antecedentes A fenilcetonúria (PKU) é um erro inato do metabolismo causado pela atividade deficiente da fenilalanina hidroxilase. No Brasil, o Programa Nacional de Triagem Neonatal permite o tratamento precoce de pacientes com PKU, o que os impede de desenvolver danos neurológicos e deficiências intelectuais graves. No entanto, já foi descrito que de 20 a 30% dos pacientes tratados precocemente com PKU apresentam déficits cognitivos focais, incluindo déficits na memória de trabalho, velocidade de processamento e sintomas psiquiátricos como ansiedade, depressão e Transtorno de Déficit de Atenção e Hiperatividade (TDAH). Neste sentido, testes neuropsiquiátricos e cognitivos são componentes importantes no cuidado destes pacientes. Atualmente, não existe um compêndio científico ou recomendações de ferramentas validadas em português para avaliar a saúde mental em pacientes brasileiros com PKU. Objetivo Recomendar ferramentas validadas localmente para avaliar a qualidade de vida e aspectos neuropsicológicos de pacientes com PKU. Métodos Seis especialistas brasileiros discutiram as ferramentas mais apropriadas com base em suas experiências clínicas, a viabilidade de realizar as avaliações na rotina clínica, e o acesso às ferramentas na saúde pública. Antes da reunião, foi realizada uma revisão independente da literatura para identificar as ferramentas validadas no Brasil, utilizando as bases de dados MEDLINE e Scielo. Resultados Os especialistas recomendaram nove ferramentas para avaliar a qualidade de vida (Peds-QL, SF-36 ou WHOQOL-bref), função executiva (BRIEF ou Bayley-III), QI (SONR 2½-7[a] ou WASI) e TDAH (MTA-SNAP-IV e ASRS). Conclusões Estes instrumentos podem ser facilmente incorporados na prática clínica e melhorar a qualidade dos cuidados multidisciplinares dos pacientes com PKU. (PKU hidroxilase Brasil graves entanto 2 30 focais trabalho ansiedade TDAH. . (TDAH) sentido Atualmente clínicas pública reunião Scielo PedsQL, PedsQL Peds QL, QL (Peds-QL SF36 SF 36 SF-3 WHOQOLbref, WHOQOLbref WHOQOL bref , WHOQOL-bref) BRIEF BayleyIII, BayleyIII Bayley III Bayley-III) SONR 2½7a 2½ 7 2½-7[a WASI MTASNAPIV MTA SNAP IV ASRS. ASRS ASRS) 3 (TDAH SF3 SF- WHOQOL-bref Bayley-III

Abstract Background Phenylketonuria (PKU) is an inborn error of metabolism caused by deficient activity of phenylalanine hydroxylase. In Brazil, the National Neonatal Screening Program enables early treatment of patients with PKU, which prevents them from developing severe neurological damage and mental disabilities. However, between 20 and 30% of early-treated patients with PKU present focal cognitive deficits, including deficits in working memory, processing speed, and psychiatric symptoms such as anxiety, depression, and attention deficit hyperactivity disorder (ADHD). Therefore, age-specific neuropsychiatric and cognitive tests are important components of PKU patient care. To date, there are no officially approved guidelines or recommendations of tools in Portuguese validated for use in Brazil that could be applied to assess these parameters in patients with PKU. Objective To recommend tools validated for use in Brazil that can be used in daily clinical practice to assess quality of life and neuropsychological outcomes in patients with PKU. Methods Six Brazilian experts discussed about eligible tools based on their clinical experience, the feasibility of their use in clinical routines, and their availability in public health services. Before the meeting, an independent review of the literature was conducted to identify the currently validated tools in Brazil, using the MEDLINE and SciELO databases. Results The experts recommended nine tools to assess quality of life (Peds-QL, SF-36 or WHOQOL-bref), executive function (BRIEF or Bayley-III), IQ (SONR 2½-7[a] or WASI) and ADHD (MTA-SNAP-IV and ASRS). Conclusions These instruments may be easily incorporated into clinical practice and improve the quality of multidisciplinary care of patients with PKU. (PKU hydroxylase disabilities However 2 30 earlytreated treated memory speed anxiety depression ADHD. . (ADHD) Therefore agespecific age specific date experience routines services meeting databases PedsQL, PedsQL Peds QL, QL (Peds-QL SF36 SF 36 SF-3 WHOQOLbref, WHOQOLbref WHOQOL bref , WHOQOL-bref) BRIEF BayleyIII, BayleyIII Bayley III Bayley-III) SONR 2½7a a 2½ 7 2½-7[a WASI MTASNAPIV MTA SNAP IV ASRS. ASRS ASRS) 3 (ADHD SF3 SF- WHOQOL-bref Bayley-III

Abstract Mucolipidosis II and III (MLII and MLIII) are autosomal recessive diseases caused by pathogenic variants in GNPTAB and GNPTG genes that lead to defects in GlcNAc-1-phosphotransferase. This enzyme adds mannose 6-phosphate residues to lysosomal hydrolases, which allows enzymes to enter lysosomes. Defective GlcNAc-1-phosphotransferase causes substrate accumulation and inflammation. These diseases have no treatment, and we hypothesized that the use of substrate reduction therapy and immunomodulation may be beneficial at the cell level and as a future therapeutic approach. Fibroblasts from two patients with MLIII alpha/beta and 2 patients with MLIII gamma as well as from one healthy control were treated with 10 µM miglustat, 20 µM genistein, and 20 µM thalidomide independently. ELISA assay and confocal immunofluorescence microscopy were used to evaluate the presence of heparan sulfate (HS) and the impact on substrate accumulation. ELISA assay showed HS reduction in all patients with the different treatments used (p=0.05). HS reduction was also observed by immunofluorescence microscopy. Our study produced encouraging results, since the reduction in substrate accumulation, even partial, may offer benefits to the phenotype of patients with inborn errors of metabolism. MLII GlcNAc1phosphotransferase. GlcNAc1phosphotransferase GlcNAcphosphotransferase GlcNAc 1 phosphotransferase. phosphotransferase 6phosphate phosphate 6 hydrolases lysosomes inflammation treatment approach alphabeta alpha beta miglustat genistein independently (HS p=0.05. p005 p p=0.05 . 0 05 (p=0.05) results partial metabolism p00 p=0.0 (p=0.05 p0 p=0. (p=0.0 p=0 (p=0. p= (p=0 (p= (p

RESUMO Objetivo analisar os resultados de um instrumento que se propõe a auxiliar na identificação das dificuldades alimentares em crianças com Fenilcetonúria (PKU), em comparação a crianças sem a doença. Método estudo transversal, controlado, com amostra de conveniência composta por pacientes com PKU e por indivíduos hígidos, equiparados por idade e sexo. O convite para participação no estudo foi feito por meio de divulgação da pesquisa nas redes sociais. As respostas foram fornecidas pelos responsáveis, sendo que 46 controles e 28 pacientes participaram. Além desses, 13 responsáveis por pacientes em acompanhamento em um Ambulatório de Tratamento de Erros Inatos do Metabolismo foram convidados por ligação telefônica, sendo que 12 aceitaram o convite. Todos os participantes responderam a Escala Brasileira de Alimentação Infantil (EBAI) de forma eletrônica. Resultados foram incluídos no estudo 86 participantes, sendo 40 pacientes (mediana de idade, 2 anos; intervalo interquartil (IQR) = 2 - 4) e 46 controles (mediana de idade, 3,5 anos; IQR = 2 - 5,25). Dez (25%) pacientes e 13 (28,3%) controles apresentaram resultados compatíveis com dificuldades alimentares, demonstrando uma frequência semelhante entre os grupos. O estudo observou que os pacientes com PKU apresentaram menos autonomia alimentar (p = 0,005), foram menos amamentados (p = 0,002) e usaram mais mamadeira que os controles (p = 0,028). Conclusão a frequência de dificuldades alimentares referidas pelos cuidadores foi semelhante entre os grupos, porém as crianças com PKU demonstraram menos autonomia para se alimentar, foram menos amamentadas e usaram mais mamadeira quando comparadas com as crianças sem a doença. PKU, , (PKU) doença transversal controlado hígidos sexo sociais 4 participaram desses 1 telefônica EBAI (EBAI eletrônica 8 mediana anos (IQR 35 3 5 3, 5,25. 525 5,25 . 25 5,25) 25% (25% 28,3% 283 (28,3% grupos p 0,005, 0005 0,005 0 005 0,005) 0,002 0002 002 0,028. 0028 0,028 028 0,028) (PKU 52 5,2 (25 28,3 (28,3 000 0,00 00 0,02 02 5, (2 28, (28, 0,0 ( (28 0,

ABSTRACT Purpose to analyze the results of an instrument that aims to assist in the identification of feeding difficulties in children with Phenylketonuria (PKU), compared to children without the disease. Methods cross-sectional, controlled study with a convenience sample composed of patients with PKU and healthy individuals, matched for age and sex. The invitation to participate in the study was made through the dissemination of the research on social networks. The answers were provided by the guardians, 46 controls and 28 patients agreed to participate. In addition to these, 13 guardians of patients being followed up at an Outpatient Clinic for the Treatment of Inborn Errors of Metabolism were invited by phone call, and 12 accepted the invitation. All participants answered the Brazilian Infant Feeding Scale (in Portuguese Escala Brasileira de Alimentação Infantil (EBAI)) electronically. Results the study included 86 participants, 40 patients (median of age = 2 years; interquartile range (IQR) = 2 - 4) and 46 controls (median of age = 3.5 years; IQR = 2 - 5.25). Ten (25%) patients and 13 (28.3%) controls had suspicion of feeding difficulties, demonstrating a similar frequency of feeding difficulties between groups. The study found that PKU patients had less feed autonomy (p = 0.005), were less breastfed (p = 0.002) and used more baby’s bottle than controls (p = 0.028). Conclusion the frequency of feeding difficulties reported by caregivers was similar between the comparison groups, but children with PKU had less feed autonomy, were less breastfed and used more baby’s bottles when compared to children without the disease. PKU, , (PKU) disease crosssectional, crosssectional cross sectional, sectional cross-sectional individuals sex networks 4 these 1 call EBAI (EBAI) electronically 8 median years (IQR 35 3 5 3. 5.25. 525 5.25 . 25 5.25) 25% (25% 28.3% 283 (28.3% groups p 0.005, 0005 0.005 0 005 0.005) 0.002 0002 002 babys baby s 0.028. 0028 0.028 028 0.028) (PKU (EBAI 52 5.2 (25 28.3 (28.3 000 0.00 00 0.02 02 5. (2 28. (28. 0.0 ( (28 0.

Abstract Hyperhomocysteinemia and vitamin B12 deficiency have been reported in patients with phenylketonuria. In this study, total homocysteine (tHcy) and methylmalonic acid (MMA) levels were analyzed in samples from 25 phenylketonuria (PKU) patients. Comparisons were made between pre- and post-treatment values (n= 3); on treatment values, between periods with high and normal/low phenylalanine (Phe) levels (n= 20); and in women before, during and after pregnancy (n= 3). THcy levels decreased after treating PKU with metabolic formula (p=0.014). Except for a pregnant woman before pregnancy, none of the patients had tHcy values above the normal range. In fact, tHcy was < 5 μmol/L in 34% of the samples. We observed a decrease in Phe, tHcy, and tyrosine levels during pregnancy. MMA levels did not differ significantly, with values remaining in the normal range. These data indicate that there was no B12 deficiency in patients who adhere to the diet. In conclusion, in PKU patients treated with metabolic formula, tHcy is frequently not elevated, remaining even in the lower normal range in some patients. Thus, clinical follow-up and adherence to dietary treatment are crucial to prevent B12 deficiency. B B1 study (tHcy (MMA 2 (PKU pre posttreatment post n= n (n 3 3) normallow low Phe (Phe 20 20) 3. . p=0.014. p0014 p p=0.014 0 014 (p=0.014) fact μmolL μmol L 34 significantly diet conclusion elevated Thus followup follow up p001 p=0.01 01 (p=0.014 p00 p=0.0 (p=0.01 p0 p=0. (p=0.0 p=0 (p=0. p= (p=0 (p= (p

Abstract Reduced biotinidase activity is associated with a spectrum of deficiency ranging from total deficiency to heterozygous levels, a finding that is not always explained by the pathogenic variants observed in the BTD gene. The investigation of miRNAs, regulatory elements and variants in the 3’UTR region may present relevance in understanding the genotype-phenotype association. The aims of the study were to characterize the regulatory elements of the 3’UTR of the BTD gene and identify variants and miRNAs which may explain the discrepancies observed between genotype and biochemical phenotype. We evaluated 92 individuals with reduced biotinidase activity (level of heterozygotes = 33, borderline = 35, partial DB = 20 or total DB= 4) with previously determined BTD genotype. The 3’UTR of the BTD gene was Sanger sequenced. In silico analysis was performed to identify miRNAs and regulatory elements. No variants were found in the 3’UTR. We found 97 possible miRNAs associated with the BTD gene, 49 predicted miRNAs involved in the alanine, biotin, citrate and pyruvate metabolic pathways and 5 genes involved in biotin metabolism. Six AU-rich elements were found. Our data suggest variants in the 3'UTR of BTD do not explain the genotype-phenotype discrepancies found in Brazilian individuals with reduced biotinidase.

Abstract Gaucher disease (GD) is one of the most common lysosomal disorders, occurring in approximately 1 in 40,000 live births worldwide. Since 2014 enzyme replacement therapy (ERT) with taliglucerase alfa has been the treatment of choice for adult patients with GD in Brazil. The aim of this study was to evaluate the long-term efficacy and safety of taliglucerase alfa in a cohort of Brazilian patients treated at a referral center for inborn errors of metabolism. All patients who received at least one infusion of the enzyme at the study center were considered eligible to participate. Patients were followed for adverse reactions and events throughout the study period. Platelets, hemoglobin, chitotriosidase activity, bone marrow burden (BMB) score, bone mineral density, and the severity score index (SSI) were analyzed. For patients who were switched to taliglucerase alfa from imiglucerase, the same variables were compared before and after the switch. At 9-year follow-up, all parameters of interest had remained stable or improved. The overall rate of adverse events was lower than in other studies that evaluated long-term ERT with taliglucerase, and no serious adverse events were considered related to treatment. Based on our findings, ERT with taliglucerase alfa is an effective and safe approach for treatment of patients with GD.

Abstract The mainstay of management of phenylketonuria (PKU) is restriction of dietary phenylalanine (Phe) intake. The present study sought to assess the perception and understanding of health care providers and lay users (patients/family members/caregivers) regarding the national reference database for checking the Phe content of foods, provided by the Brazilian Health Regulatory Agency (Anvisa), whose data are presented in the Table of Phenylalanine Content of Foods (TCFA-Anvisa) and recently in the Phenylalanine Content of Foods Dashboard (PCCFA-Anvisa); and to identify factors which interfere with the usability of these resources. Two online questionnaires, one for providers (n=33) and another for lay users (n=194), were used to collect sociodemographic information, knowledge about dietary management of PKU, sources of information about the Phe content of foods, and perception and understanding of the Anvisa tools. TCFA-Anvisa and PCCFA-Anvisa were not used as main sources of information by either group. Among the participants who had used these tools (15 providers;35 lay users), most considered the PCCFA-Anvisa to be superior or partially superior to the TCFA-Anvisa. The main limitations reported were related to layout and limited variety of foods. We suggest that the limitations identified in this study be considered for future improvement of these resources.

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 GlcNAc-1-phosphotransferase is a hexameric complex formed by subunits α, β, and γ, where the first two are encoded by the GNPTAB gene and the third by the GNPTG gene. Pathogenic variants identified in the GNPTAB gene cause the diseases Mucolipidosis II and III alpha/beta, which are severe and characterized by an overflow of lysosomal hydrolases into the extracellular environment, and their absence in lysosomal compartments causes an accumulation of non-degraded macromolecules. Methodology: a retrospective study that included 32 unrelated Brazilian patients with a clinical and genetic diagnosis of Mucolipidosis II/III alpha/beta. The regional frequency of the altered alleles was determined. Results: The patients were from all regions of Brazil. The most prevalent variants were c.3503_3504del, associated with the severe form of the disease, and c.1208T>C, associated with the milder form. Variant c.3503_3504del is the most frequently found in the Midwest, Northeast, and Southeast regions of Brazil. In the South, 42.8% of the alleles present the c.1196C>T variant. Conclusions: From the perspective of all patients diagnosed with Mucolipidosis II/III in Brazil, it is possible to conclude that different regions present allelic frequencies of specific pathogenic variants, which can be explained by the occurrence of a founding effect or high inbreeding rates.

Abstract Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inborn error of fructose metabolism caused by pathogenic variants in the FBP1 gene. As gluconeogenesis is affected, catabolic episodes can induce ketotic hypoglycemia in patients. FBP1 analysis is the most commonly used approach for the diagnosis of this disorder. Herein, a Brazilian patient is reported. The proband, a girl born to a consanguineous couple, presented with severe hypoglycemia crisis in the neonatal period. At the age 17 months, presented a new crisis accompanied by metabolic acidosis associated with a feverish episode. Genetic analysis was performed by next-generation sequencing (NGS), identifying the NM_000507.3:c.611_614del variant in homozygosis in the FBP1 gene. In silico analysis and 3D modeling were performed, suggesting that this variant is associated with a loss of sites for substrate and Mg2+ binding and for posttranslational modifications of FBPase. The c.611_614del variant is located in a repetitive region of the FBP1 gene that appears to be a hotspot for mutational events. This frameshift creates a premature termination codon in the last coding exon which escapes the nonsense-mediated decay mechanism, according to in silico analysis. This variant results in an intrinsically disordered protein with loss of substrate recognition and post-translational modification sites.