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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.

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Mucopolysaccharidosis type IIIB (MPS IIIB) is caused by deficiency of alpha-N-acetylglucosaminidase, leading to storage of heparan sulphate. The disease is characterized by intellectual disability and hyperactivity, among other neurological and somatic features. Here we studied retrospective data from a total of 19 MPS IIIB patients from Brazil, aiming to evaluate disease progression. Mean age at diagnosis was 7.2 years. Speech delay was one of the first symptoms to be identified, around 2-3 years of age. Behavioral alterations include hyperactivity and aggressiveness, starting around age four. By the end of the first decade, patients lost acquired abilities such as speech and ability to walk. Furthermore, as disease progresses, respiratory, cardiovascular and joint abnormalities were found in more than 50% of the patients, along with organomegaly. Most common cause of death was respiratory problems. The disease progression was characterized in multiple systems, and hopefully these data will help the design of appropriate clinical trials and clinical management guidelines.

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Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by pathogenic variants in GBA1 which result in the deficient activity of glucocerebrosidase (GCase). There are few data on the genetic characterization of Brazilian GD patients. This study aimed at characterizing the genotype of 72 unrelated Brazilian GD patients (type I = 63, type II = 4, type III = 5; male = 31). Forty patients were from South Brazil (SB), and 32 were from other regions of Brazil (Others). The exons and exon/intron junctions of GBA1 were analyzed by Sanger sequencing in 8 patients, or by massive parallel sequencing followed by Sanger of exons 9 and 10 in 64 patients. In total, 31 pathogenic variants were identified. The most frequent allele found was N370S (p.(Asn409Ser)) (41.0%), and the most frequent genotype was N370S/RecNciI p.[Asn409Ser];[Leu483Pro;Ala495Pro;Val499=](23.6%). Three variants (N370S - in exon 9, and RecNciI and L444P (p.(Leu483Pro), in exon 10) correspond to 76.3% of total alleles in SB and 59.4% in Others. Two novel variants were described: c.326del(p.(Gln109Argfs*9)) and c.690G>A (p.(?)). Although sequencing all the exons of GBA1 is the gold-standard method for the genetic analysis of GD patients, a step analysis can be proposed for Brazilian patients, starting with analysis of exons 9 and 10. The N370S allele is the most frequently associated with GD in Brazil.

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BACKGROUND: Lysosomal diseases (LDs) are progressive life-threatening disorders that are usually asymptomatic at birth. Specific treatments are available for several LDs, and early intervention improves patient's outcomes. Thus, these diseases benefit from newborn screening (NBS). We have performed a pilot study for six LDs in Brazil by tandem mass spectrometry. METHODS: Dried blood spot (DBS) samples of unselected newborns were analyzed by the Neo-LSD™ kit (Perkin-Elmer) by MS/MS. Samples with low enzyme activity were submitted to the evaluation of specific biomarkers by ultra-performance liquid chromatography tandem-mass spectrometry as the second-tier, and were analyzed by a next-generation sequencing (NGS) multi-gene panel as the third-tier. All tests were performed in the same DBS sample. RESULTS: In 20,066 newborns analyzed, 15 samples showed activity of one enzyme below the cutoff. Two newborns had biochemical and molecular results compatible with Fabry disease, and five newborns had biochemical results and pathogenic variants or variants of unknown significance (VUS) in GAA. CONCLUSIONS: This study indicates that the use of enzyme assay as the first-tier test gives an acceptably low number of positive results that requires second/third tier testing. The possibility to run all tests in a DBS sample makes this protocol applicable to large-scale NBS programs.

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INTRODUCTION: Hepatic Glycogen Storage Diseases (GSD) are rare genetic disorders in which the gluconeogenesis pathway is impaired. Cytokines control virtually every aspect of physiology and may help to elucidate some unsolved questions about phenotypes presented by GSD patients. METHODS: This was an exploratory study in which 27 GSD patients on treatment (Ia = 16, Ib = 06, III = 02, IXα = 03) and 24 healthy age- and sex-matched subjects had plasma samples tested for a panel of 20 cytokines (G-CSF,GM-CSF, IL-1α,IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17A, GRO, IP-10/CXCL10, MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, MDC/CCL22, IFN-γ, TNF-α, TNF-ß, VEGF) through a multiplex kit and analyzed in comparison to controls and among patients, regarding to clinical features as anemia, hepatic adenocarcinoma and triglyceride levels. RESULTS: Patients (GSD-Ia/III/IX) presented reduced levels of IL-4 (p = 0.040), MIP-1α/CCL3 (p = 0.003), MDC/CCL22 (p < 0.001), TNF-ß (p = 0.045) and VEGF (p = 0.043) compared to controls. When different types of GSD were compared, G-CSF was higher in GSD-Ib than -Ia (p < 0.001) and than -III/IX (p = 0.033) patients; IL-10 was higher in GSD-Ib than in GSD-Ia patients (p = 0.019); and GSD-III/IX patients had increased levels of IP-10/CXCL10 than GSD-Ib patients (p = 0.019). When GSD-I patients were gathered into the same group and compared with GSD-III/IX patients, IP10/CXCL10 and MCP-1 were higher in the latter group (p = 0.005 and p = 0.013, respectively). GSD-I patients with anemia presented higher levels of IL-4 and MIP-1α in comparison with patients who had not. Triglyceride level was correlated with neutrophil count and MDC levels on GSD-Ia patients without HCA. CONCLUSION: Altogether, altered levels of cytokines in GSD-I patients reflect an imbalance in immunoregulation process. This study also indicates that neutrophils and some cytokines are affected by triglyceride levels, and future studies on the theme should consider this variable.

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Genetic testing is being considered the first-step in the investigation of hereditary myopathies. However, the performance of the different testing approaches is little known. The aims of the present study were to evaluate the diagnostic yield of a next-generation sequencing panel comprising 39 genes as the first-tier test for genetic myopathies diagnosis and to characterize clinical and molecular findings of families from southern Brazil. Fifty-one consecutive index cases with clinical suspicion of genetic myopathies were recruited from October 2014 to March 2018 in a cross-sectional study. The overall diagnostic yield of the next-generation sequencing panel was 52.9%, increasing to 60.8% when including cases with candidate variants. Multi-gene panel solved the diagnosis of 12/25 (48%) probands with limb-girdle muscular dystrophies, of 7/14 (50%) with congenital muscular diseases, and of 7/10 (70%) with muscular dystrophy with prominent joint contractures. The most frequent diagnosis for limb-girdle muscular dystrophies were LGMD2A/LGMD-R1-calpain3-related and LGMD2B/LGMD-R2-dysferlin-related; for congenital muscular diseases, RYR1-related-disorders; and for muscular dystrophy with prominent joint contractures, Emery-Dreifuss-muscular-dystrophy-type-1 and COL6A1-related-disorders. In summary, the customized next-generation sequencing panel when applied in the initial investigation of genetic myopathies results in high diagnostic yield, likely reducing patient's diagnostic odyssey and providing important information for genetic counseling and participation in disease-specific clinical trials.

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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.

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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.

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INTRODUCTION: Gaucher disease (GD) is one of the most prevalent lysosomal disorders, with an estimated incidence of 1 in 40,000 live births worldwide. Skeletal involvement is one of the main features of GD, causing morbidity and impacting long-term quality of life in patients with type 1 GD. OBJECTIVES: To characterize bone marrow infiltration in patients with type 1 GD followed at the Gaucher Disease Referral Center of Porto Alegre, Brazil, and to assess whether the Bone Marrow Burden score (BMB) correlates with clinical or laboratory parameters. We also evaluated whether the BMB score is a suitable parameter for long-term follow-up of patients with type 1 GD. METHODS: All included patients underwent magnetic resonance imaging for BMB score calculation at baseline, 1 year, and every other year thereafter or as clinically indicated from 2012 to 2018. RESULTS: The BMB score tended to decrease during the first 5 years of treatment, at a rate of -1.08 points per year; after the 5-year mark, BMB tended to remain stable. CONCLUSIONS: The BMB score is useful for response monitoring in the first five years of treatment. We recommend that, after 5 years of treatment, MRI for BMB evaluation should only be performed in non-adherent patients or in those who develop symptoms of acute skeletal disease.

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Gaucher disease (GD), one of the most common lysosomal disorders, is caused by deficiency of ß-glucocerebrosidase. Based on the presence and severity of neurological complications, GD is classified into types I, II (the most severe form), and III. Abnormalities in systemic markers of vitamin B12 (B12 ) metabolism have been reported in GD type I patients, suggesting a higher prevalence of B12 deficiency in these patients. A 2-month-old male with GD type II was admitted to the hospital presenting jaundice, hepatosplenomegaly, and ichthyosis. At admission, cholestasis and ascites, abnormal liver function enzymes, prolonged prothrombin time, and high levels of B12 were confirmed. Analysis of biomarkers of B12 status revealed elevated B12 and holo-transcobalamin (holo-TC) levels. The B12 profile found in our patient is the opposite to what is described for GD type I patients. Holo-TC may increase in inflammatory states or due to liver diseases. In GD, the accumulation of glucocerebroside may be a trigger that initiates a systemic inflammatory reaction, characterized by macrophage activation. We suggest higher levels of holo-TC could be associated with a more severe (neuronopathic) GD, and be a biomarker of GD type II.

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