A Brazilian Rare-Disease Center's Experience with Glucosylsphingosine (lyso-Gb1) in Patients with Gaucher Disease: Exploring a Novel Correlation with IgG Levels in Plasma and a Biomarker Measurement in CSF.

Gaucher disease (GD, OMIM 230800) is one of the most common lysosomal disorders, being caused by the deficient activity of the enzyme acid ß-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.

Follow-up of pre-motor symptoms of Parkinson's disease in adult patients with Gaucher disease type 1 and analysis of their lysosomal enzyme profiles in the CSF.

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.

Pathogenic variants in GNPTAB and GNPTG encoding distinct subunits of GlcNAc-1-phosphotransferase differentially impact bone resorption in patients with mucolipidosis type II and III.

PURPOSE: Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. METHODS: We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. RESULTS: The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. CONCLUSION: The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.

SARS-CoV-2 pandemic in the Brazilian community of rare diseases: A patient reported survey.

The COVID-19 pandemic has led to a reorganization of health systems to prioritize the fight against the virus. The adoption of social distancing interfered with the flow of existing policies, and may thus negatively affect the most vulnerable groups, such as the rare disease community. Aimming at characterizing the perception of the impact of COVID-19 on the health care of the Brazilian rare disease community, an online questionnaire addressed to patients with rare diseases and their caregivers was disseminated in the Brazilian territory between June 1st to July 5th, 2020. The questions dealt with the sanitary measures adopted; access to medical services; and mental suffering during the pandemic. The survey was answered by 1,466 participants (<18 yo = 53.3%) representing 192 rare diseases. Regarding physical distancing, 1,372 (93.6%) participants did not leave their residence, or did so only when essential; 1,321 (90.1%) always wore masks when leaving home. 1,042 (71.1%) and 995 (67.9%) participants, respectively, referred medical genetics appointments and rehabilitation therapies were postponed/canceled. Telemedicine was experienced by 1,026 (70%), and 68.3% agreed this is a good strategy for health care. Patients with Inborn Errors of Metabolism (IEM, n = 624, 42.5%) appear to have more access to information and ability to overcome difficulties, and feel less threatened, lonely and depressed than the non-IEM group (p < .05). There was an increment of the rare disease patients' vulnerability in the pandemic scenario. The cooperation of patients/caregivers along with adaptation of the health system is crucial and may be so even post-pandemic.

A decade of molecular diagnosis of Mucolipidosis II and III in Brazil: a pooled analysis of 32 patients

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.

Prevalence of the most common pathogenic variants in three genes for inborn errors of metabolism associated with sudden unexpected death in infancy: a population-based study in south Brazil.

Citrullinemia type 1 (CTLNI), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), and mut0 methylmalonic acidemia (mut0 MMA) are inborn errors of metabolism (IEMs) associated with sudden unexpected death in infancy (SUDI). Its most common pathogenic variants are: c.1168G>A (CTLNI, ASS1 gene), c.1528G>C (LCHADD, HADHA gene), c.655A>T and c.1106G>A (mut0 MMA, MUT gene). Considering the absence of estimates regarding the incidence of these diseases in Brazil, this study sought to investigate the prevalence of its main pathogenic variants in a healthy population in the southern region of the country. A total of 1,000 healthy subjects from Rio Grande do Sul were included. Genotyping was performed by real-time PCR. Individuals found to be heterozygous for c.1528G>C underwent further acylcarnitine profile analysis by tandem mass spectrophotometry. Allele and genotype frequencies were calculated considering Hardy-Weinberg equilibrium. The c.1528G>C variant was detected in heterozygosity in two subjects (carrier frequency = 1:500; allele frequency = 0.001; minimum prevalence of LCHADD = 1: 1,000,000), whose acylcarnitine profiles were normal. Variants c.1168G>A, c.655A>T, and c.1106G>A were not identified. These results denote the rarity of these IEMs in Southern Brazil, highlighting the need to expand the investigation of IEMs in relation to infant morbidity and mortality within the country.