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Fabiano de Oliveira Poswar,1– 4 Johanna Henriques Nehm,1 Francyne Kubaski,5 Edina Poletto,6 Roberto Giugliani1– 4,7– 9 1Clinical Research Group in Medical Genetics, Clinical Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil; 2Medical Genetics Service, Hospital de Clinicas de Porto Alegre, Porto Alegre, RS, Brazil; 3Postgraduate Program in Genetics and Molecular Biology, UFRGS, Porto Alegre, RS, Brazil; 4DR Brasil Research Group, HCPA, Porto Alegre, RS, Brazil; 5Greenwood Genetic Center, Biochemical Genetics Laboratory, Greenwood, SC, USA; 6Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA; 7Department of Genetics, UFRGS, Porto Alegre, RS, Brazil; 8DASA Genômica, São Paulo, SP, Brazil; 9Casa dos Raros, Porto Alegre, RS, BrazilCorrespondence: Roberto Giugliani, Medical Genetics Service- HCPA / Dep Genet UFRGS, 2350 Ramiro Barcelos, Porto Alegre, RS, 90035-903, Brazil, Tel +55 51 3359 6338, Email rgiugliani@hcpa.edu.brAbstract: Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an ultra-rare lysosomal disease caused by a deficiency of the enzyme β-glucuronidase (GUS). The diagnosis is suspected based on a range of symptoms that are common to many other MPS types, and it is confirmed through biochemical and molecular studies. Besides supportive treatment, current and emerging treatments include enzyme replacement therapy, hematopoietic stem cell transplantation, and gene therapy. This review summarizes the clinical manifestations, diagnosis, and emerging treatments for MPS VII.Keywords: lysosomal disorders, mucopolysaccharidosis type VII, Sly syndrome, enzyme replacement therapy, hematopoietic stem cell transplantation, gene therapy

Shunji Tomatsu,1,2 Kazuki Sawamoto,1 Carlos J Alméciga-Díaz,3 Tsutomu Shimada,1 Michael B Bober,1 Yasutsugu Chinen,4 Hiromasa Yabe,5 Adriana M Montaño,6 Roberto Giugliani,7 Francyne Kubaski,1,8 Eriko Yasuda,1 Alexander Rodríguez-López,3 Angela J Espejo-Mojica,3 Oscar F Sánchez,9 Robert W Mason,1 Luis A Barrera,3 William G Mackenzie,1 Tadao Orii2 1Nemours/Alfred I duPont Hospital for Children, Wilmington, DE, USA; 2Department of Pediatrics, Gifu University, Gifu, Japan; 3Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia; 4Department of Pediatrics, Faculty of Medicine, University of the Ryukyus, Okinawa, 5Department of Cell Transplantation and Regenerative Medicine, Tokai University School of Medicine, Isehara, Japan; 6Department of Pediatrics, Saint Louis University, St Louis, MO, USA; 7Medical Genetics Service/HCPA and Department of Genetics/UFRGS, Porto Alegre, Brazil; 8Department of Biological Sciences, University of Delaware, Newark, DE, 9School of Chemical Engineering, Purdue University, West Lafayette, IN, USA Abstract: Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS IVA was treated with HSCT at 15 years of age and followed up for 10 years. Radiographs showed that the figures of major and minor trochanter appeared. Loud snoring and apnea disappeared. In all, 1 year after bone marrow transplantation, bone mineral density at L2–L4 was increased from 0.372 g/cm2 to 0.548 g/cm2 and was maintained at a level of 0.48±0.054 for the following 9 years. Pulmonary vital capacity increased approximately 20% from a baseline of 1.08 L to around 1.31 L over the first 2 years and was maintained thereafter. Activity of daily living was improved similar to the normal control group. After bilateral osteotomies, a patient can walk over 400 m using hip–knee–ankle–foot orthoses. This long-term observation of a patient shows that this treatment can produce clinical improvements although bone deformity remained unchanged. In conclusion, ERT is a therapeutic option for MPS IVA patients, and there are some indications that HSCT may be an alternative to treat this disease. However, as neither seems to be a curative therapy, at least for the skeletal dysplasia in MPS IVA patients, new approaches are investigated to enhance efficacy and reduce costs to benefit MPS IVA patients. Keywords: mucopolysaccharidosis IVA, ERT, HSCT, skeletal dysplasia, keratan sulfate

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidosis (MPS) disorders. However, false positives are observed due mainly to the presence of pseudodeficiencies. Our previous publications on glycosaminoglycan (GAG) biomarker levels in dried blood spots (DBS) for mucopolysaccharidoses demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we extend this approach to the analysis of a large number of false positives for MPS-I obtained from the Illinois, New York, and Tennessee NBS programs and from Greenwood Genetics Center. Results show that GAG levels measured by the Endogenous-Non-Reducing End method (Endogenous-NRE) are in the normal reference range for all samples. In a second study, we analyzed 166 samples that showed below-cutoff MPS-I enzymatic activity level after testing 384,144 newborns in the Ontario, Canada NBS program. Both genotype and Endogenous-NRE GAG levels were determined for all 166 samples. Newborns at high risk for MPS-I based on genotype also showed elevated GAG levels and were clinically confirmed to be symptomatic for MPS-I. All newborns with pseudodeficiency or carrier status by genotyping all showed normal levels of the appropriate GAG biomarker. Samples found to be inconclusive based on one or more variants of unknown significance (VUS) all showed normal GAG biomarker levels and were found to be clinically normal during follow-up. These studies show that the Endogenous-NRE GAG second-tier NBS method is preferred over second-tier DNA analysis for the NBS of MPS-I with minimal false positives., Competing Interests: Declaration of competing interest Z.M.H. is a member of GelbChem, LLC. M.H.G. is a co-founder and member of GelbChem, LLC. The other authors declare no conflicts of interest., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Geographic and sociodemographic aspects may influence the natural history and epidemiology of mucopolysaccharidoses (MPS). The main objective in this work was to evaluate the clinical, molecular, and geographic profile of MPS in a population from Ceará (Northeast Brazil). For this, we have performed a descriptive cross-sectional study based on clinical evaluation, interviews with patients and/or family members, and review of medical records of 76 MPS patients. MPS II was the most common type, with the most affected individuals presenting missense pathogenic variants. Patients with MPS I proved to be the most severe clinical phenotype, presenting the first symptoms (mean: 7.1 months; SD = 4.5) and being diagnosed earlier (2.2 years; SD = 2.1) in comparison with the other types. In addition, we have shown that 13 individuals with MPS VI were born of consanguineous marriages in small, nearby cities, in a place where geographical isolation, consanguinity, and clusters of genetic diseases were previously reported. Ten of these individuals (at least, seven different families) presented a rare pathogenic variant in the ARSB gene, c.1143-8T > G in homozygosity, previously reported only among Iberian and South American patients. The results presented here provide a comprehensive picture of MPS in an important state of the Brazilian Northeast, a region that concentrates many risk factors for rare genetic diseases, such as endogamy, inbreeding, and reproductive isolation. We discuss the possible evolutionary processes and biosocial dynamics that can help to explain this finding in terms of population medical genetics and public health., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

Gaucher disease (GD, OMIM 230800) is one of the most common lysosomal disorders, being caused by the deficient activity of the enzyme acid β-glucocerebrosidase (Gcase). Three clinical forms of Gaucher's disease (GD) are classified based on neurological involvement. Type 1 (GD1) is non-neuronopathic, while types 2 (GD2) and 3 (GD3) are neuronopathic forms. Gcase catalyzes the conversion of glucosylceramide (GlcCer) into ceramide and glucose. As GlcCer accumulates in lysosomal macrophages, it undergoes deacylation to become glycosylsphingosine (lyso-Gb1), which has shown to be a useful and reliable biomarker for the diagnosis and monitoring of treated and untreated patients with GD. Multiple myeloma (MM) is one of the leading causes of cancer-related death among patients with GD and monoclonal gammopathy of undetermined significance (MGUS) is a non-neoplastic condition that can be a telltale sign of a B clonal proliferation caused by the chronic activation of B cells. This study aimed to quantify Lyso-Gb1 levels in dried blood spots (DBS) and cerebrospinal fluid (CSF) as biomarkers for Gaucher disease (GD) and discuss the association of this biomarker with other clinical parameters. This is a mixed-methods study incorporating both cross-sectional and longitudinal elements within a cohort design with a convenience-sampling strategy. Data collection took place from January 2012 to March 2023. Lyso-Gb1 extraction from DBS involved the use of a methanol-acetonitrile-water mixture, followed by incubation and centrifugation. Analysis was performed using UPLC-MS/MS with MassLynx software version 4.2 and the control group for the DBS measurements included general newborns. CSF Lyso-Gb1 was extracted using ethyl acetate, analyzed by UPLC-MS/MS with a calibration curve, and expressed in pmol/L. Lysosomal activity in CSF was assessed by measuring chitotriosidase (Cht), and other lysosomal enzyme activities were assessed as previously described in the literature. Patients with metachromatic leukodystrophy (MLD) were used as controls. Thirty-two treated patients (twenty-nine GD1 and three GD3, all on ERT except for one GD type on SRT with eliglustat) and three untreated patients (one GD1, one GD2, and one GD3) were included. When analyzing only the treated GD1 group, a significant correlation was found between lyso-Gb1 and age (rho = -0.447, p = 0.001), ChT, and IgG levels (rho = 0.73, p < 0.001; and rho = 0.36, p = 0.03, respectively). Five GD1 patients (three females, mean age 40 years) also had their CSF collected and analyzed. The average measurement of lyso-Gb1 in CSF was 94 pmol/L (range: 57.1-157.9 pmol/L) versus <6.2 pmol/L in the control group (MLD). This is the first time, to the best of our knowledge, that lyso-Gb1 has been associated with IgG levels. While this finding reflects a risk for MGUS or MM and not only chronic plasma B-cell activation, it still requires further studies. Moreover, the analysis of CSF lyso-Gb1 levels in GD1 patients was demonstrated to be significantly higher than the control group. This raises the hypothesis that CSF lyso-Gb1 may serve as a valuable indicator for neurological involvement in GD, providing insights into the potential implications for neurological manifestations in GD, including GD1. The correlation between lyso-Gb1 and ChT levels in treated GD1 patients further underscores the interconnectedness of lysosomal markers and their relevance in monitoring.

Background and Aim: Mucopolysaccharidosis type III (MPS III) is a rare autosomal recessive lysosomal storage disease (LSD) caused by a deficiency of lysosomal enzymes required for the catabolism of glycosaminoglycans (GAGs), mainly in the central nervous system. Trehalose has been proposed as a potential therapeutic agent to attenuate neuropathology in MPS III. We conducted a single- arm, open-label study to evaluate the efficacy of trehalose treatment in patients with MPS IIIA and MPS IIIB., Methods: Five patients with MPS III were enrolled. Trehalose was administrated intravenously (15 g/week) for 12 weeks. Health-related quality of life and cognitive function, serum biomarkers, liver, spleen, and lung imaging were assessed to evaluate trehalose efficacy at baseline and trial end (week 12)., Results: TNO-AZL Preschool children Quality of Life (TAPQOL) scores increased in all patients, and the mean scores for quality of life were increased after the intervention. Serum GAG levels were reduced in all treated patients (however, the differences were not statistically significant). Alanine aminotransferase (ALT) levels were reduced in all patients post-treatment (p=0.0039). The mean levels of aspartate transaminase (AST) were also decreased after 12 weeks of treatment with Trehalose. Decreased serum pro-oxidant-antioxidant balance and increased GPX activity were observed at the end of the study. Decreases in mean splenic length were observed, whereas the liver volume did not change., Conclusion: Improvements in health-related quality of life and serum biomarkers (GAGs, liver aminotransferase levels, antioxidant status), as well as liver and spleen size, were found following 3 months of trehalose administration in patients with MPS IIIA and MPS IIIB., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Its classic motor symptoms may be preceded by non-motor symptoms (NMS). Population studies have identified GBA variants as risk factors for idiopathic PD. The increased risk of PD has also been suggested in other Lysosomal Storage Disorders (LSDs)., Objective: To assess the evolution of the prevalence of NMS compatible with PD in a cohort of South Brazilian adult patients with Gaucher Disease (GD) type 1, already evaluated 3 years ago (2018). Cerebrospinal Fluid (CSF) was collected to assess the levels of LSD enzymes (beta-hexosaminidases, beta-glucuronidase) and biomarker of macrophage activation (chitotriosidase, ChT), compared to controls (metachromatic leukodystrophy, MLD). Cognition was evaluated by the Montreal Cognitive Assessment (MoCA) questionnaire, daytime sleepiness by the Epworth Sleepiness Scale (ESS), depression by Beck´s Inventory, constipation by the Unified Multiple System Atrophy Rating Scale (UMSARS) scale, and REM sleep behavior disorder by the single-question screen. Hyposmia was assessed with Sniffin' Sticks (SST)., Results: Nineteen patients completed the follow-up (mean age of the sample was 44 years; range, 26-71). The patient with the highest number of NMS at the baseline (4 including the lowest SST score) was diagnosed with PD four years later. Apart from an improvement in the ESS score, no other statistical significance was found between the number of NMS between the first and second evaluation, nor between patients with one L444P variant (n = 5) and the rest of the cohort. CSF was collected in five patients (mean age of the sample was 40 years, range 30-53. A significant difference was found in the mean CSF activity levels of beta-hexosaminidases and beta-glucuronidase between GD1 and MLD patients. Mean ChT (CSF) was 62 nmol/h/mL in GD patients and 142 in MLD (n = 6) patients., Conclusions: The patient with the highest number of NMS in our 2018 cohort was the one that developed PD, corroborating with the importance of this longitudinal follow-up. CSF and plasma analysis might allow a better understanding of the neurodegenerative processes connecting PD and the lysosomal environment. Further analysis is needed to understand this relationship., (© 2023. Institut National de la Santé et de la Recherche Médicale (INSERM).)

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., Competing Interests: Declaration of Competing Interest The authors declare no confliefhe writing of the manuscript, or in the decision to publish the results., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidoses (MPSs). Our previous publications on glycosaminoglycan (GAG) biomarker levels in DBS for mucopolysaccharidosis type 1 (MPS-I) and MPS-II demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we evaluate two methods for measuring GAG biomarkers in seven MPS types and GM1 gangliosidosis. We obtained newborn DBS from patients with MPS-IIIA-D, -IVA, -VI, -VII, and GM1 gangliosidosis. These samples were analyzed via two GAG mass spectrometry methods: (1) The internal disaccharide biomarker method; (2) The endogenous non-reducing end (NRE) biomarker method. This study supports the use of second-tier GAG analysis of newborn DBS by the endogenous NRE biomarker method, as part of NBS to reduce the false positive rate., Competing Interests: Declaration of Competing Interest Z.M.H. is a member of GelbChem, LLC. M.H.G. is a co-founder and member of GelbChem, LLC. The other authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

Background: Acid sphingomyelinase deficiency (ASMD) is a lysosomal disorder caused by deficiency of acid sphingomyelinase (ASM) leading to the accumulation of sphingomyelin (SM) in a variety of cell types. Lysosphingomyelin (LysoSM) is the de-acetylated form of SM and it has been shown as a biomarker for ASMD in tissues, plasma, and dried blood spots (DBS) and lysosphingomyelin-509 (LysoSM509) is the carboxylated analogue of LysoSM. High levels of Lysosphingomyelin 509 (LysoSM509) have also been shown in ASMD patients. In this study, we report the utility of the quantification of LysoSM and LysoSM509 in DBS of patients from Latin America with ASMD by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)., Results: DBS samples from 14 ASMD patients were compared with 15 controls, and 44 general newborns. All patients had their diagnosis confirmed by the quantification of ASM and the measurement of the activity of chitotriosidase. All patients had significantly higher levels of lysoSM and lysoSM509 compared to controls and general newborns., Conclusions: The quantification of lysosphingolipids in DBS is a valuable tool for the diagnosis of ASMD patients and lysoSM can be useful in the differential diagnosis with NPC. This method is also valuable in the ASMD newborn screening process., (© 2022. The Author(s).)

Background and Objectives: Fragile X syndrome (FXS) is a neurodevelopmental disorder, identified as the most common cause of hereditary intellectual disability and monogenic cause of autism spectrum disorders (ASDs), caused by the loss of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein, a regulator of translation that plays an important role in neurodevelopment, and its loss causes cognitive and behavioral deficits. MicroRNAs (miRNAs) are small molecules that regulate gene expression in diverse biological processes. Previous studies found that the interaction of FMRP with miR-125b and miR-132 regulates the maturation and synaptic plasticity in animal models and miRNA dysregulation plays a role in the pathophysiology of FXS. The present study aimed to analyze the expression of miR-125b-5p and miR-132-3p in the serum of patients with FXS., Methods: The expressions of circulating miRNAs were studied in the serum of 10 patients with FXS and 20 controls using the real-time quantitative retrotranscribed method analyzed by relative quantification. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated to assess the diagnostic values of the miRNAs., Results: We found that both miR-125b and miR-132 were increased in the serum of patients with FXS compared with controls and likely involved with FMRP loss. The AUC (95% confidence interval) of miR-125b and miR-132 was 0.94 (0.86-1.0) and 0.89 (0.77-1.0), respectively. Databases allowed for the identification of possible target genes for miR-125b and miR-132, whose products play an important role in the homeostasis of the nervous system., Discussion: Our results indicate that serum miR-125b and miR-132 may serve as potential biomarkers for FXS. The increased expression of circulating miR-125b and miR-132 seems to be associated with the genotype of FXS. Predicted gene targets of the differentially regulated miRNAs are involved in cognitive performance and ASD phenotype., Classification of Evidence: This study provides Class III evidence that miR-125b and miR-132 distinguish men with FXS from normal controls., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Introduction: Thromboembolic events occur due to an imbalance in the hemostasis and some factors associated with this condition can be inherited. In order to evaluate the frequency of genotypes considered to be common hereditary risk factors for thrombophilia associated with venous thrombosis (g.1691G > A and g.20210G > A) and hyperhomocysteinemia (g.677C > T and g.1298A > C), samples from voluntary healthy blood donors at the Hospital de Clínicas de Porto Alegre were tested., Methods: We examined 325 blood samples from blood donors collected from October 2017 to July 2018. Blood was collected on filter paper and the DNA was extracted for single nucleotide polymorphisms (SNPs) analysis using the qualitative real time polymerase chain reaction., Results: The calculated frequencies of each genetic variant in heterozygosity were 4% for the FV gene (g.1691G > A), 4% for the F2 gene (g.20210G > A) and 42% and 39% for methylenetetrahydrofolate reductase (MTHFR), g.677C > T and g.1298A > C, respectively. Only the genetic variants of MTHFR were found in homozygosity, with frequencies of 14% and 6% (g.677C > T and g.1298A > C), respectively., Discussion: Altogether, these results describe the frequencies of genetic variants associated with venous thrombosis and hyperhomocysteinemia in the analyzed group and are important to enhance our current knowledge about the genetic profiles of Brazilian blood donors., (Copyright © 2021 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. All rights reserved.)

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs.

Aromatic l-amino acid decarboxylase (AADC, EC 4.1.1.28) deficiency is a rare genetic disorder characterized by developmental delay, oculogyric crises, autonomic dysfunction and other problems, caused by biallelic mutations in the DDC gene leading to deficient activity of aromatic l-amino acid decarboxylase, an enzyme involved in the formation of important neurotransmitters, such as dopamine and serotonin. A clinical development program of gene therapy for AADC deficiency is ongoing. An important step for the success of this therapy is the early and precise identification of the affected individuals, but it has been estimated that around 90% of the cases remain undiagnosed. The availability measurement of the AADC activity is mandatory for an accurate biochemical diagnosis. Based on these statements, our objectives were to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method suitable for the determination of the AADC activity, and to evaluate its capacity to confirm the deficiency of AADC in potential patients in Brazil. The AADC activities were measured in plasma samples of seven AADC deficient patients and 35 healthy controls, after enzymatic reaction and LC-MS/MS analysis of dopamine, the main reaction product. The results obtained showed clear discrimination between confirmed AADC deficient patients and healthy controls. The method presented here could be incorporated in the IEM laboratories for confirmation of the diagnosis of when a suspicion of AADC deficiency is present due to clinical signs and/or abnormal biomarkers, including when an increased level of 3- O -methyldopa (3-OMD) is found in dried blood spots (DBS) samples from high-risk patients or from newborn screening programs., (© 2022 The Authors.)

Background: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice., Methods: Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated., Results: Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage., Conclusion: Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders., (© 2022 John Wiley & Sons, Ltd.)

Mucopolysaccharidosis type IIIB is a rare autosomal recessive disorder characterized by deficiency of the enzyme N-acetyl-alpha-d-glucosaminidase (NAGLU), caused by biallelic pathogenic variants in the NAGLU gene, which leads to storage of heparan sulfate and a series of clinical consequences which hallmark is neurodegeneration. In this study clinical, epidemiological, and biochemical data were obtained from MPS IIIB patients diagnosed from 2004-2019 by the MPS Brazil Network ("Rede MPS Brasil"), which was created with the goal to provide an easily accessible and comprehensive investigation of all MPS types. One hundred and ten MPS IIIB patients were diagnosed during this period. Mean age at diagnosis was 10.9 years. Patients were from all over Brazil, with a few from abroad, with a possible cluster of MPS IIIB identified in Ecuador. All patients had increased urinary levels of glycosaminoglycans and low NAGLU activity in blood. Main clinical symptoms reported at diagnosis were coarse facies and neurocognitive regression. The most common variant was p.Leu496Pro (30% of alleles). MPS IIIB seems to be relatively frequent in Brazil, but patients are diagnosed later than in other countries, and reasons for that probably include the limited awareness about the disease by health professionals and the difficulties to access diagnostic tests, factors that the MPS Brazil Network is trying to mitigate., (© 2021 Wiley Periodicals LLC.)

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder caused by deficiency of arylsulfatase A (ARSA), leading to an accumulation of sulfatides. Sulfatides have been quantified in urine, dried blood spots (DBS), and tissues of patients with MLD. Newborn screening (NBS) for MLD has already been proposed based on a two-tier approach with the quantification of sulfatides in DBS followed by the quantification of ARSA by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Prenatal screening for MLD is also crucial, and sulfatide quantification in amniotic fluid (AF) can aid diagnosis. The prenatal study was initiated due to a family history of MLD at 19 weeks of gestation. ARSA was quantified in cultured amniocytes. C16:0 sulfatide was quantified by LC-MS/MS in the supernatant of AF. Molecular analysis of the ARSA gene was performed in cultured amniocytes. ARSA was deficient in fetal cells, and C16:0 sulfatides were significantly elevated in comparison to age-matched controls (3-fold higher). Genetic studies identified the c.465+1G>A variant in homozygosis in the ARSA gene. Our study shows that sulfatides can be quantified in the supernatant of AF of MLD fetuses, and it could potentially aid in a faster and more accurate diagnosis of MLD patients., Competing Interests: Francyne Kubaski, Maira Graeff Burin, Kristiane Michelin‐Tirelli, Franciele B. Trapp, Rejane Gus, Alice B. O. Netto, Ana Carolina Brusius‐Facchin, Sandra Leistner‐Segal, Maria Teresa Sanseverino, Carolina Moura Fischinger de Souza, Matheus V. M. B. Wilke, Thiago Oliveira, Jose A. A. Magalhães, Roberto Giugliani declare that they have no conflict of interest. Zackary M. Herbst is a consultant for GelbChem, LLC., (© 2022 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background and Aims: Niemann-Pick disease (NPD) types A (NPA) and B (NPB) are caused by deficiency of the acid sphingomyelinase enzyme, which is encoded by the SMPD1 gene, resulting in progressive pathogenic accumulation of lipids in tissues. Trehalose has been suggested as an autophagy inducer with therapeutic neuroprotective effects. We performed a single-arm, open-label pilot study to assess the potential efficacy of trehalose treatment in patients with NPA and NPB patients., Methods: Five patients with NPD type A and B were enrolled in an open-label, single-arm clinical trial. Trehalose was administrated intravenously (IV) (15 g/week) for three months. The efficacy of trehalose in the management of clinical symptoms was evaluated in patients by assessing the quality of life, serum biomarkers, and high-resolution computed tomography (HRCT) of the lungs at the baseline and end of the interventional trial (day 0 and week 12)., Results: The mean of TNO-AZL Preschool children Quality of Life (TAPQOL) scores increased in all patients after intervention at W12 compared to the baseline W0, although the difference was not statistically significant. The serum levels of lyso-SM-509 and lyso-SM were decreased in three and four patients out of five, respectively, compared with baseline. Elevated ALT and AST levels were decreased in all patients after 12 weeks of treatment; however, changes were not statistically significant. Pro-oxidant antioxidant balance (PAB) was also decreased and glutathione peroxidase (GPX) activity was increased in serum of patients at the end of the study. Imaging studies of spleen and lung HRCT showed improvement of symptoms in two patients., Conclusions: Positive trends in health-related quality of life (HRQoL), serum biomarkers, and organomegaly were observed after 3 months of treatment with trehalose in patients with NPA and NPB. Although not statistically significant, due to the small number of patients enrolled, these results are encouraging and should be further explored.

The COVID-19 pandemic led to the reorganization of health care in several countries, including Brazil. Inborn Errors of Metabolism (IEM) are a group of rare and difficult to diagnose genetic diseases caused by pathogenic variants in genes that code for enzymes, cofactors, or structural proteins affecting different metabolic pathways. The aim of this study was to evaluate how COVID-19 affected the diagnosis of patients with IEM during the first year of the pandemic in Brazil comparing two distinct periods: from March 1st, 2019 to February 29th, 2020 (TIME A) and from March 1st, 2020 to February 28th, 2021 (TIME B), by the analysis of the number of tests and diagnoses performed in a Reference Center in South of Brazil. In the comparison TIME A with TIME B, we observe a reduction in the total number of tests performed (46%) and in the number of diagnoses (34%). In both periods analyzed, mucopolysaccharidoses (all subtypes combined) was the most frequent LD suspected and/or confirmed. Our data indicates a large reduction in the number of tests requested for the investigation of IEM and consequently a large reduction in the number of diagnoses caused by the COVID-19 pandemic leading to a significant underdiagnosis of IEM.

Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oliveira Netto, Brusius-Facchin, Leistner-Segal, Kubaski, Josahkian and Giugliani.)

Mucopolysaccharidoses (MPS) are lysosomal storage diseases (LSDs) caused by the deficiency of enzymes essential for the metabolism of extracellular matrix components called glycosaminoglycans (GAGs). To understand the physiopathology and alterations due to the lysosomal accumulation resulting from enzymatic deficiencies and their secondary outcomes can improve the diagnosis and treatment of rare genetic diseases. This work presents a database for differentially expressed genes from different public MPS data. We developed our database, including 13 studies previously deposited in the GEO (https://www.ncbi.nlm.nih.gov/geo/). The website is hosted in the UFRGS data processing center (CPD) and is available at . The site was constructed in PHP, and the analyses were performed in R. The organisms represented by the datasets are Canis lupus familiaris, Homo sapiens, Mus musculus, and Rattus norvegicus. The user can search for the differentially expressed genes and ontologies by species, MPS type, or tissue type. For each comparison, a heatmap with the 50 top differentially expressed genes is available as well as dot plots for the 30 top ontologies divided by biological process, cellular component, KEGG pathways, and molecular function. This data is also fully available in tables. There are 54 possible comparisons involving about 5000 to 10,000 genes each. This website is the only specific database for MPS with filtering and presenting their results in a one-click approach to the best of our knowledge. The development of such analytical and automated strategies accessible to health professionals is essential for fostering MPS research. The MPSBase is a web user-friendly, comprehensive repository of differentially expressed genes and ontologies regarding the MPS data., Competing Interests: Declaration of Competing Interest The authors declare they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aromatic L-amino acid decarboxylase (AADCD) deficiency is an autosomal recessive neurometabolic disorder, caused by biallelic mutations in the DDC gene, that impairs the synthesis or metabolism of neurotransmitters leading to severe motor dysfunction. The main clinical signs are oculogyric crisis, hypotonia, hypokinesia, and dystonia. The biochemical diagnosis can be performed in cerebrospinal fluid by neurotransmitter analysis, which requires an invasive lumbar puncture, and the sample needs to be shipped frozen to a reference laboratory, usually across a country border. Measurement of AADC activity in plasma is also possible, but available in a few labs globally. 3- O -methyldopa (3-OMD) is a catabolic product of L-dopa and it is elevated in patients with AADC deficiency. The quantification of 3-OMD can be performed in dried blood spots (DBS), a sample that could be shipped at room temperature. 3-OMD levels of AADCD patients and controls were quantified in DBS by liquid chromatography tandem mass spectrometry. DBS samples from 7 Brazilian patients previously diagnosed with AADCD were used to validate the 3-OMD quantification as a screening procedure for this condition. All AADCD patients had at least a four-fold increase of 3-OMD. Thus, 3-OMD seems to be a reliable marker for AADCD, with potential use also in the newborn screening of this disease., (© 2021 The Authors. Published by Elsevier Inc.)

Newborn screening enables the diagnosis of treatable disorders at the early stages, and because of its countless benefits, conditions have been continuously added to screening panels, allowing early intervention, aiming for the prevention of irreversible manifestations and even premature death. Mucopolysaccharidoses (MPS) are lysosomal storage disorders than can benefit from an early diagnosis, and thus are being recommended for newborn screening. They are multisystemic progressive disorders, with treatment options already available for several MPS types. MPS I was the first MPS disorder enrolled in the newborn screening (NBS) panel in the USA and a few other countries, and other MPS types are expected to be added. Very few studies about NBS for MPS in Latin America have been published so far. In this review, we report the results of pilot studies performed in Mexico and Brazil using different methodologies: tandem mass spectrometry, molecular analysis, digital microfluidics, and fluorimetry. These experiences are important to report and discuss, as we expect to have several MPS types added to NBS panels shortly. This addition will enable timely diagnosis of MPS, avoiding the long diagnostic odyssey that is part of the current natural history of this group of diseases, and leading to a better outcome for the affected patients.

Precision medicine (PM) is an emerging approach for disease treatment and prevention that accounts for the individual variability in the genes, environment, and lifestyle of each person. Lysosomal diseases (LDs) are a group of genetic metabolic disorders that include approximately 70 monogenic conditions caused by a defect in lysosomal function. LDs may result from primary lysosomal enzyme deficiencies or impairments in membrane-associated proteins, lysosomal enzyme activators, or modifiers that affect lysosomal function. LDs are heterogeneous disorders, and the phenotype of the affected individual depends on the type of substrate and where it accumulates, which may be impacted by the type of genetic change and residual enzymatic activity. LDs are individually rare, with a combined incidence of approximately 1:4000 individuals. Specific therapies are already available for several LDs, and many more are in development. Early identification may enable disease course prediction and a specific intervention, which is very important for clinical outcome. Driven by advances in omics technology, PM aims to provide the most appropriate management for each patient based on the disease susceptibility or treatment response predictions for specific subgroups. In this review, we focused on the emerging diagnostic technologies that may help to optimize the management of each LD patient and the therapeutic options available, as well as in clinical developments that enable customized approaches to be selected for each subject, according to the principles of PM.

Mucopolysaccharidoses (MPS) are a subtype of lysosomal storage disorders (LSDs) characterized by the deficiency of the enzyme involved in the breakdown of glycosaminoglycans (GAGs). Mucopolysaccharidosis type I (MPS I, Hurler Syndrome) was endorsed by the U.S. Secretary of the Department of Health and Human Services for universal newborn screening (NBS) in February 2016. Its endorsement exemplifies the need to enhance the accuracy of diagnostic testing for disorders that are considered for NBS. The progression of MPS disorders typically incudes irreversible CNS involvement, severe bone dysplasia, and cardiac and respiratory issues. Patients with MPS have a significantly decreased quality of life if untreated and require timely diagnosis and management for optimal outcomes. NBS provides the opportunity to diagnose and initiate treatment plans for MPS patients as early as possible. Most newborns with MPS are asymptomatic at birth; therefore, it is crucial to have biomarkers that can be identified in the newborn. At present, there are tiered methods and different instrumentation available for this purpose. The screening of quick, cost-effective, sensitive, and specific biomarkers in patients with MPS at birth is important. Rapid newborn diagnosis enables treatments to maximize therapeutic efficacy and to introduce immune tolerance during the neonatal period. Currently, newborn screening for MPS I and II has been implemented and/or in pilot testing in several countries. In this review article, historical aspects of NBS for MPS and the prospect of newborn screening for MPS are described, including the potential tiers of screening.

Lysosomal storage disorders (LSDs) are a group of genetic disorders characterized by deficiency of specific lysosomal enzymes. In general, patients are clinically normal at birth, and progressively develop severe signs and symptoms. Diagnosis is usually made several years after onset of manifestations, preventing patients to have the benefits of the early treatment. Newborn screening programs are being considered for LSDs to allow early diagnosis and treatment. The present study evaluated the feasibility of a customized screening approach based on modified fluorometric assays with reduced amounts of reagents, substrates and samples for: mucopolysaccharidosis (MPS) type I (MPS I), MPS VI, Fabry, Gaucher, and Pompe diseases. We also evaluated the advantages of including blood chitotriosidase and urinary glycosaminoglycans in the protocol. By the measurement of the specific disease-associated enzymes (plus blood chitotriosidase and urinary glycosaminoglycans) we analyzed 834 de-identified DBS of unselected newborns. No positive case was detected, and the false-positive rates were low. Taking into consideration the limitations of this methodology, we believe that, after defining proper cutoffs, it could be a viable alternative to provide NBS for LSDs by laboratories that may not be able to afford the commercial methods available.

The mucopolysaccharidoses (MPSs) include 11 different conditions caused by specific enzyme deficiencies in the degradation pathway of glycosaminoglycans (GAGs). Although most MPS types present increased levels of GAGs in tissues, including blood and urine, diagnosis is challenging as specific enzyme assays are needed for the correct diagnosis. Enzyme assays are usually performed in blood, with some samples (as leukocytes) providing a final diagnosis, while others (such as dried blood spots) still being considered as screening methods. The identification of variants in the specific genes that encode each MPS-related enzyme is helpful for diagnosis confirmation (when needed), carrier detection, genetic counseling, prenatal diagnosis (preferably in combination with enzyme assays) and phenotype prediction. Although the usual diagnostic flow in high-risk patients starts with the measurement of urinary GAGs, it continues with specific enzyme assays and is completed with mutation identification; there is a growing trend to have genotype-based investigations performed at the beginning of the investigation. In such cases, confirmation of pathogenicity of the variants identified should be confirmed by measurement of enzyme activity and/or identification and/or quantification of GAG species. As there is a growing number of countries performing newborn screening for MPS diseases, the investigation of a low enzyme activity by the measurement of GAG species concentration and identification of gene mutations in the same DBS sample is recommended before the suspicion of MPS is taken to the family. With specific therapies already available for most MPS patients, and with clinical trials in progress for many conditions, the specific diagnosis of MPS as early as possible is becoming increasingly necessary. In this review, we describe traditional and the most up to date diagnostic methods for mucopolysaccharidoses.

Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of α-l-iduronidase, leading to the storage of dermatan and heparan sulfate. There is a broad phenotypical spectrum with the presence or absence of neurological impairment. The classical form is known as Hurler syndrome, the intermediate form as Hurler-Scheie, and the most attenuated form is known as Scheie syndrome. Phenotype seems to be largely influenced by genotype. Patients usually develop several somatic symptoms such as abdominal hernias, extensive dermal melanocytosis, thoracolumbar kyphosis odontoid dysplasia, arthropathy, coxa valga and genu valgum, coarse facial features, respiratory and cardiac impairment. The diagnosis is based on the quantification of α-l-iduronidase coupled with glycosaminoglycan analysis and gene sequencing. Guidelines for treatment recommend hematopoietic stem cell transplantation for young Hurler patients (usually at less than 30 months of age). Intravenous enzyme replacement is approved and is the standard of care for attenuated-Hurler-Scheie and Scheie-forms (without cognitive impairment) and for the late-diagnosed severe-Hurler-cases. Intrathecal enzyme replacement therapy is under evaluation, but it seems to be safe and effective. Other therapeutic approaches such as gene therapy, gene editing, stop codon read through, and therapy with small molecules are under development. Newborn screening is now allowing the early identification of MPS I patients, who can then be treated within their first days of life, potentially leading to a dramatic change in the disease's progression. Supportive care is very important to improve quality of life and might include several surgeries throughout the life course.

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach "hard-to-treat" tissues to ensure better systemic and neurological effects.

Mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders which can lead to degenerative and irreversible skeletal, cardiovascular, pulmonary, and neurological damage. Current treatments, including hematopoietic stem cell transplantation and enzyme replacement therapy, have been found most effective if administered before clinical symptoms are present, highlighting the urgent need for the development of newborn screening. This study analyzed 18,222 dried blood spot samples from newborns for both enzyme activity and glycosaminoglycan (GAG) concentration levels. GAG levels were measured using liquid chromatography tandem mass spectrometry. Results were compared to our previously established cutoff values for three subtypes of GAGs: dermatan sulfate (DS) and heparan sulfate (HS0S and HSNS). Samples that were high for two of the three GAGs were identified and screened a second time. Samples were also measured for iduronate-2-sulfatase and alfa-L-iduronidase activity. A total of 300 samples were above the established cutoff values for at least two of the three GAGs after the first screening. One sample was determined through clinical and genetic testing to be a true positive for MPS II. The false positive rate after the first GAG screening was 1.64%. A Cochran's formula test showed that the samples available for the second screening were representative samples (p = .0000601). False positive rate after second GAG screening, extrapolated from the representative sample was 0.4%. False positive rate after enzyme activity assay by fluorimetry for IDUA and IDS enzymes was 0.21% and 0.18%. A combination of GAG and enzyme assays provided no false positive and false negative samples. Two-tier screening involving a combination of enzyme activity and multiple GAGs should be considered the gold standard for the diagnosis of MPS patients., (© 2020 The Authors.)

Background: Mucopolysaccharidosis type II (Hunter syndrome, or MPS II) is an X-linked lysosomal disorder caused by the deficiency of iduronate-2-sulfatase, which leads to the accumulation of glycosaminoglycans (GAGs) in a variety of tissues, resulting in a multisystemic disease that can also impair the central nervous system (CNS)., Objective: This review focuses on providing the latest information and expert opinion about the therapies available and under development for MPS II., Methods: We have comprehensively revised the latest studies about hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT - intravenous, intrathecal, intracerebroventricular, and intravenous with fusion proteins), small molecules, gene therapy/genome editing, and supportive management., Results and Discussion: Intravenous ERT is a well-established specific therapy, which ameliorates the somatic features but not the CNS manifestations. Intrathecal or intracerebroventricular ERT and intravenous ERT with fusion proteins, presently under development, seem to be able to reduce the levels of GAGs in the CNS and have the potential of reducing the impact of the neurological burden of the disease. Gene therapy and/or genome editing have shown promising results in preclinical studies, bringing hope for a "one-time therapy" soon. Results with HSCT in MPS II are controversial, and small molecules could potentially address some disease manifestations. In addition to the specific therapeutic options, supportive care plays a major role in the management of these patients., Conclusion: At this time, the treatment of individuals with MPS II is mainly based on intravenous ERT, whereas HSCT can be a potential alternative in specific cases. In the coming years, several new therapy options that target the neurological phenotype of MPS II should be available., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Allogenic hematopoietic stem cell transplantation (HSCT) has proven to be a viable treatment option for a selected group of patients with mucopolysaccharidoses (MPS), including those with MPS types I, II, IVA, VI, and VII. Early diagnosis and timely referral to an expert in MPS are critical, followed by a complete examination and evaluation by a multidisciplinary team, including a transplantation physician. Treatment recommendations for MPS are based on multiple biological, sociological, and financial factors, including type of MPS, clinical severity, prognosis, present clinical signs and symptoms (disease stage), age at onset, rate of progression, family factors and expectations, financial burden, feasibility, availability, risks and benefits of available therapies such as HSCT, enzyme replacement therapy (ERT), surgical interventions, and other supportive care. International collaboration and data review are critical to evaluating the therapeutic efficacy and adverse effects of HSCT for MPS. Collaborative efforts to assess HSCT for MPS have been ongoing since the first attempt at HSCT in a patient with MPS reported in 1981. The accumulation of data since then has made it possible to identify early outcomes (ie, transplantation outcomes) and long-term disease-specific outcomes resulting from HSCT. The recent identification of predictive factors and the development of innovative regimens have significantly improved the outcomes of both engraftment failure and transplantation-related mortality. Assessment of long-term outcomes has considered a variety of factors, including type of MPS, type of graft, age at transplantation, and stage of disease progression, among others. Studies on long-term outcomes are considered a key factor in the use of HSCT in patients with MPS. These studies have shown the effects and limitations of HSCT on improving disease manifestations and quality of life. In this review, we summarize the efficacy, side effects, risks, and cost of HSCT for each type of MPS., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders that impair degradation of glycosaminoglycans (GAG). The specific GAGs that accumulate depend on the type of MPS, leading to unique characteristic clinical features. Development of guidelines for treatment of MPS has traditionally been multifaceted and largely based on palliative care. In the last three decades, hematopoietic stem cell transplantation and enzyme replacement therapy have been developed based on experimental and clinical studies. Guidelines have been established with the accumulation of the clinical data from natural history of the disease and therapeutic consequences, mainly sponsored by pharmaceutical companies. In recent years, committees in three countries, Australia (2015), Japan (2017), and Brazil (2018) have adopted guidelines for the treatment of MPS II, sponsored and authorized by each government. As novel treatments for MPS including substrate reduction therapy, pharmacological chaperone therapy, and gene therapy become clinically available, it is increasingly necessary to establish the optimal guideline for each type of MPS, considering multiple factors including therapeutic efficacy, adverse effects, age, disease stage, prognosis, feasibility and availability of access to treatment, and cost- performance. In this article, we discuss the historical guidelines for specific MPS types and the most recently adopted guidelines for MPS II and propose the development of future guidelines without conflict of interest and bias leading to mutual benefits to all parties including patients and families, professionals, tax payers, and governments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rare genetic disorders are currently in the spotlight due to the elevated number of different conditions and significant total number of affected patients. The study of these disorders is extremely helpful for the elucidation of physiological processes related with complex disorders. Isolated populations are instrumental for the study of genetic disorders, considering their homogeneity and high proportion of affected patients in a small geographic area. These favorable conditions lead to the creation of a new discipline, known as "population medical genetics", which integrates medical genetics, population genetics, epidemiological genetics and community genetics. In order to develop practical activities in this new discipline, the National Institute of Population Medical Genetics (INaGeMP) was created in 2008 in Brazil. INaGeMP has developed several tools and funded numerous research activities. In this review, we highlight three successful projects developed in the first 10 years of INaGeMP activities (2008-2018): a newborn screening pilot study for MPS VI in Northeast Brazil, the study of Machado-Joseph disease in Brazilian families with Azorian ancestry, and the high twinning rate in a small town in southern Brazil. The results of these projects in terms of scientific output and contributions to the affected communities highlight the success and importance of INaGeMP.

Lysosomal diseases (LDs), also known as lysosomal storage diseases (LSDs), are a heterogeneous group of conditions caused by defects in lysosomal function. LDs may result from deficiency of lysosomal hydrolases, membrane-associated transporters or other non-enzymatic proteins. Interest in the LD field is growing each year, as more conditions are, or will soon be treatable. In this article, we review the diagnosis of LDs, from clinical suspicion and screening tests to the identification of enzyme or protein deficiencies and molecular genetic diagnosis. We also cover the treatment approaches that are currently available or in development, including hematopoietic stem cell transplantation, enzyme replacement therapy, small molecules, and gene therapy.

Mucopolysaccharidoses (MPS) are estimated to affect1 in 25,000 live births although specific rates vary between the ethnic origin and country. MPS are a group of lysosomal storage disorders, which cause the buildup of GAG(s) due to insufficient or absent GAG-degrading enzymes. With seven types of MPS disorders and eleven subtypes, the MPS family presents unique challenges for early clinical diagnosis due to the molecular and clinical heterogeneity between groups and patients. Novel methods of early identification, particularly newborn screening through mass spectrometry, can change the flow of diagnosis, allowing enzyme and GAG quantification before the presentation of clinical symptoms improving outcomes. Genetic testing of patients and their families can also be conducted preemptively. This testing enables families to make informed decisions about family planning, leading to prenatal diagnosis. In this review, we discuss the clinical symptoms of each MPS type as they initially appear in patients, biochemical and molecular diagnostic methods, and the future of newborn screening for this group of disorders., (Copyright © 2018. Published by Elsevier Inc.)

Objectives: Current evidence supports participation of neurotrophic and inflammatory factors in the pathogenesis of major depressive disorder (MDD). Some studies reported an association between the Val66Met polymorphism (rs6265) of brain-derived neurotrophic factor (BDNF) gene with MDD and peripheral BDNF levels. However, no previous studies have examined the association of this polymorphism with inflammation. The present study assessed the association of the Val66Met polymorphism with serum levels of BDNF and inflammatory markers among depressed outpatients., Methods: All participants (n = 73) met DSM-IV criteria for a unipolar depressive episode. The serum levels of BDNF and inflammatory biomarkers (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) were compared between individuals presenting with at least one Met allele (Met-carriers) and those homozygous for the Val allele., Results: In our sample (84.9% female, mean age 52.4 ± 10.3 years), 24.7% (n = 18) were Met-carriers. After Bonferroni correction, the Met allele was significantly associated with higher BDNF and lower TNF-α. These associations persisted after adjusting for potential confounders., Conclusions: The pattern of low BDNF and high inflammation in MDD may be influenced by the Val66Met polymorphism. The association of a polymorphism in the BDNF gene with inflammatory markers in addition to BDNF levels suggests an interaction between these systems.

Background: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34 + cells as a predictor with data from other reports, this study analyzed the implementation of this routine., Methods: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed., Results: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34 + quantification. A moderate positive correlation was observed between peripheral blood CD34 + cell count and total CD34 + cell count/kg ( r  = 0.596; p -value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34 + cells/μL in the peripheral blood, a mean increase of 0.38 × 10 6  CD34 +  cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34 + cell count in this sample., Conclusions: As observed in other centers, quantification of peripheral blood CD34 + progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.

Lysosomal disorders have been an area of interest since intravenous enzyme replacement therapy was successfully introduced for the treatment of Gaucher's disease in the early 1990s. This treatment approach has also been developed for several other lysosomal disorders, including Fabry's disease, Pompe's disease, lysosomal acid lipase deficiency, and five types of mucopolysaccharidosis. Despite the benefits of enzyme replacement therapy, it has limitations-most importantly, its ineffectiveness in treating the neurological components of lysosomal disorders, as only a small proportion of recombinant enzymes can cross the blood-brain barrier. Development of strategies to improve drug delivery to the CNS is now the primary focus in lysosomal disorder research. This Review discusses the neurological manifestations and emerging therapies for the CNS component of these diseases. The therapies in development (which are now in phase 1 or phase 2 clinical trials) might be for specific lysosomal disorders (enzyme replacement therapy via intrathecal or intracerebroventricular routes or with fusion proteins, or gene therapy) or applicable to more than one lysosomal disorder (haemopoietic stem cell transplantation, pharmacological chaperones, substrate reduction therapy, or stop codon readthrough). The combination of early diagnosis with effective therapies should change the outlook for patients with lysosomal disorders with neurological involvement in the next 5-10 years., (Copyright © 2018 Elsevier Ltd. All rights reserved.)