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Background: N-glycosylation is a key post-translational modification critical for protein function and stability. Chitotriosidase-1 (CHIT1), belonging to glycoside hydrolase family 18, is clinically utilized as a biomarker of Gaucher disease. A G102S variant is common in some populations, but the implications of this missense mutation on CHIT1 function and in disease pathology are unknown. We have investigated the effects of the G102S mutation on the N-glycosylation, structure, and activity of CHIT1., Methods: Three recombinant CHIT1 proteins, wild-type (WT), G102S, and N100Q+G102S double mutants, were expressed, purified, and analyzed for glycosylation using SDS-PAGE, MALDI-MS, PNGase F treatment, and lectin blotting. NMR and LC-MS/MS were employed to characterize glycan structures. Enzymatic assays and molecular dynamics simulations were used to assess the effects of mutations on CHIT1 function and dynamics., Results: The G102S mutation introduced a new N-glycosylation site at N100, confirmed by SDS-PAGE and MALDI-MS, and the composition of the N-glycan structures was verified by lectin blotting, NMR, and MS. Both G102S and N100Q+G102S proteins exhibited reduced catalytic efficiency compared to WT. Molecular dynamics simulations suggested that G102S mutation induces significant structural changes and reduces stability, particularly without N-glycan, likely impairing substrate binding and enzymatic activity., Conclusion: Our findings indicate that the common G102S mutation affects the structure and function of CHIT1, partially by introducing a new N-glycosylation site. They provide a foundation for further research on the impact of N-glycosylation on its hydrolase activity and structural dynamics, with potential implications for understanding the role of CHIT1 in Gaucher disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

Homozygous mutations in the lysosomal glucocerebrosidase gene, GBA1, cause Gaucher's disease (GD), while heterozygous mutations in GBA1 are a strong risk factor for Parkinson's disease (PD), whose pathological hallmark is intraneuronal α-synuclein (asyn) aggregates. We previously reported that gba1 knockout (KO) medaka exhibited glucosylceramide accumulation and neuronopathic GD phenotypes, including short lifespan, the dopaminergic and noradrenergic neuronal cell loss, microglial activation, and swimming abnormality, with asyn accumulation in the brains. A recent study reported that deletion of GBA2, non-lysosomal glucocerebrosidase, in a non-neuronopathic GD mouse model rescued its phenotypes. In the present study, we generated gba2 KO medaka and examined the effect of Gba2 deletion on the phenotypes of gba1 KO medaka. The Gba2 deletion in gba1 KO medaka resulted in the exacerbation of glucosylceramide accumulation and no improvement in neuronopathic GD pathological changes, asyn accumulation, or swimming abnormalities. Meanwhile, though gba2 KO medaka did not show any apparent phenotypes, biochemical analysis revealed asyn accumulation in the brains. gba2 KO medaka showed a trend towards an increase in sphingolipids in the brains, which is one of the possible causes of asyn accumulation. In conclusion, this study demonstrated that the deletion of Gba2 does not rescue the pathological changes or behavioral abnormalities of gba1 KO medaka, and GBA2 represents a novel factor affecting asyn accumulation in the brains.

Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1 D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.

Background: Alglucerase enzyme replacement therapy was approved for Gaucher disease (GD) in the United States in 1991; imiglucerase in 1994. We report hematologic, visceral, bone pain, bone crisis, height, weight, and Body Mass Index (BMI) outcomes in patients treated for 20 (±3) years with subset analyses based on pre-treatment severity, genotype, and age at treatment initiation., Methods: GD type 1 (GD1) patients in the ICGG Gaucher Registry with complete sets of baseline, 10-year, and 20-year data are included (N = 475). Ten-year and 20-year data are compared to pre-treatment baseline, stratified by splenectomy status., Results: Non-splenectomized patients: Improvements observed at 10 years were maintained at 20 years for most outcomes. Mean changes from baseline at 10 and 20 years, respectively, were: spleen volume: 18.2 multiples of normal (MN) to 5.1 MN and 4.2 MN; liver volume: 1.8 MN to 1.0 MN and 1.0 MN; hemoglobin: 11.4 g/dL to 13.7 g/dL and 13.8 g/dL; platelet count: 91.6 × 10 9 /L to 168.0 × 10 9 /L and 169.1 × 10 9 /L; without bone crisis: 85.0% to 98.2% and 96.5%; without bone pain: 52.5% to 72.0% at 10 years, no significant change at 20 years (58.5%). Splenectomized patients: significant changes were observed in liver volume: 2.3 MN to 1.1 MN and 1.0 MN; hemoglobin: 11.7 g/dL to 13.3 g/dL and 13.4 g/dL; platelet count: 229.1 × 10 9 /L to 288.1 × 10 9 /L and 257.0 × 10 9 /L; without bone crisis: 52.2% to 91.3% and 100%; without bone pain: 16.3% to 30.6% (not significant) and 46.9%. Similar results were found in each of the subset analyses. Patients who start treatment during childhood have normal weight and height in young adulthood. Many treated adult patients are overweight or obese; however, this is consistent with BMI trends observed in the general population. After 1-2 years, the average biweekly imiglucerase dose is ~40 units/kg body weight., Conclusion: Imiglucerase is an effective, long-term treatment for GD1. In a long-term observational setting, improvements seen during early treatment years are sustained by continuing treatment for 20 years, except for bone pain in non-splenectomized patients. These results are consistent when analyzed by different patient subsets, including by disease severity., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Enzyme replacement therapy (ERT) can positively affect the visceral manifestations of lysosomal storage diseases (LSDs). However, the exclusion of the intravenous ERT agents from the central nervous system (CNS) prevents direct therapeutic effects., Methods: Using a neuronopathic Gaucher disease (nGD) mouse model, CNS-ERT was created using a systemic, non-invasive, and CNS-selective delivery system based on nanovesicles of saposin C (SapC) and dioleoylphosphatidylserine (DOPS) to deliver to CNS cells and tissues the corrective, functional acid β-glucosidase (GCase)., Findings: Compared to free GCase, human GCase formulated with SapC-DOPS nanovesicles (SapC-DOPS-GCase) was more stable in serum, taken up into cells, mostly by a mannose receptor-independent pathway, and resulted in higher activity in GCase-deficient cells. In contrast to free GCase, SapC-DOPS-GCase nanovesicles penetrated through the blood-brain barrier into the CNS. The CNS targeting was mediated by surface phosphatidylserine (PS) of blood vessel and brain cells. Increased GCase activity and reduced GCase substrate levels were found in the CNS of SapC-DOPS-GCase-treated nGD mice, which showed profound improvement in brain inflammation and neurological phenotypes., Interpretation: This first-in-class CNS-ERT approach provides considerable promise of therapeutic benefits for neurodegenerative diseases., Funding: This study was supported by the National Institutes of Health grants R21NS 095047 to XQ and YS, R01NS 086134 and UH2NS092981 in part to YS; Cincinnati Children's Hospital Medical Center Research Innovation/Pilot award to YS and XQ; Gardner Neuroscience Institute/Neurobiology Research Center Pilot award to XQ and YS, Hematology-Oncology Programmatic Support from University of Cincinnati and New Drug State Key Project grant 009ZX09102-205 to XQ., Competing Interests: Declaration of Competing Interest The authors claim a provisional patent application has been filed on March 10, 2020. Consistent with current Cincinnati Children's Hospital Medical Center policies, the development and commercialization of this technology has been licensed to Bexion Pharmaceuticals, LLC, in which X. Qi, holds (< 5%) equity interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m , where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg -1 , 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Fundal abnormalities, including preretinal and retinal changes, are a rare finding in patients with the autosomal recessive lysosomal storage disorder Gaucher disease, most often described in patients with the chronic neuronopathic form (type 3). We evaluated whether these ophthalmological findings correlated with other manifestations of type 3 Gaucher disease. Reviewing the records of 40 patients with type 3 Gaucher disease, we identified five with white vitreous opacities and reviewed their clinical course in depth. Each of the patients described decreased visual acuity and "floaters" obstructing their vision. The development and/or progression of these fluffy-appearing white opacities in each patient were tracked longitudinally in the context of their neurological and other clinical findings. It was noted that all five patients shared genotype p.L483P/p.L483P (L444P/L444P) and had significant neurological, oculomotor and bone involvement and two had undergone splenectomy. Enzyme replacement therapy with recombinant glucocerebrosidase did not prevent the development or progression of these ocular opacities. Since preretinal findings, in addition to other neuro-ophthalmological findings, can be a feature of Gaucher disease, it is recommended that patients be monitored by regular eye examinations., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Bone differentiation defects have been recently tied to Wnt signaling alterations occurring in vitro and in vivo Gaucher disease (GD) models. In this work, we provide evidence that the Wnt signaling multi-domain intracellular transducers Dishevelled 1 and 2 (DVL1 and DVL2) may be potential upstream targets of impaired beta glucosidase (GBA1) activity by showing their misexpression in different type 1 GD in vitro models. We also show that in Gba mutant fish a miR-221 upregulation is associated with reduced dvl2 expression levels and that in type I Gaucher patients single-nucleotide variants in the DVL2 3' untranslated region are related to variable canonical Wnt pathway activity. Thus, we strengthen the recently outlined relation between bone differentiation defects and Wnt/β-catenin dysregulation in type I GD and further propose novel mechanistic insights of the Wnt pathway impairment caused by glucocerebrosidase loss of function., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background Gaucher disease (GD) is a lysosomal storage disorder caused by autosomal recessive mutations in the glucocerebrosidase (GBA) gene, which encodes acid β-glucosidase. GD type 3c is a rare group characterised by cardiovascular involvement, and homozygous D448H is the most frequent mutation. Case presentation We describe two patients who had homozygous D448H mutations. The index patient had hepatosplenomegaly, liver insufficiency and cardiac involvement and her sister had severe cardiac involvement with cardiomyopathy and diffuse aortic calcification. The index case's liver was transplanted at the age of 6 months from a related donor and her sister who had severe cardiovascular disease died at the age of 12 years. Conclusions Our patients had clinical variability. We need to discuss whether liver involvement could be the initial signs in patients with GD type 3c.

Background: Chitotriosidase (ChT) is used as a biomarker for the follow-up of patients with Gaucher disease (GD), once his activity is extremely elevated and declines during ERT. However, some variants in the CHIT1 gene affect ChT activity., Methods: To assess association between ChT genotype, and clinical/biochemical features of GD were performed CHIT1 genotyping for: c.1049&#95;1072dup24, p.Gly102Ser, p.Gly354Arg, c.1155&#95;1156 + 2delGAGT, c.1156 + 5&#95;1156 + 8delGTAA, p.Ala442Val/Gly and the rearrangement delE/I-10., Results: Were evaluated 42 patients with GD from Southern Brazil. Pretreatment ChT activity was available for 32 patients. Allelic frequencies found for dup24, p.Gly102Ser and p.Ala442Gly were 0.14, 0.32 and 0.12, respectively. Only one patient presented reduced ChT activity (dup24 homozygous). Comparison between wild homozygous and heterozygous for dup24 showed that both differ in relation to the ChT activity before (15,230 vs 6936 nmol/h/mL, p < .001), but not after treatment (5212 vs 3045 nmol/h/mL, p = .227)., Conclusions: Pretreatment ChT activity was not correlated with clinical/biochemical features. There was a reduction of 63% in the ChT activity after 12 months on treatment (p < .001). There is no evidence that higher ChT levels are associated with a more severe symptomatology in untreated GD patients. The pretreatment ChT levels appear to be mainly dependent on the presence/absence of the dup24 allele., (Copyright © 2019. Published by Elsevier B.V.)

The lysosomal enzyme glucocerebrosidase-1 (GCase) catalyzes the cleavage of a major glycolipid glucosylceramide into glucose and ceramide. The absence of fully functional GCase leads to the accumulation of its lipid substrates in lysosomes, causing Gaucher disease, an autosomal recessive disorder that displays profound genotype-phenotype nonconcordance. More than 250 disease-causing mutations in GBA1 , the gene encoding GCase, have been discovered, although only one of these, N370S, causes 70% of disease. Here, we have used a knowledge-based docking protocol that considers experimental data of protein-protein binding to generate a complex between GCase and its known facilitator protein saposin C (SAPC). Multiscale molecular-dynamics simulations were used to study lipid self-assembly, membrane insertion, and the dynamics of the interactions between different components of the complex. Deep learning was applied to propose a model that explains the mechanism of GCase activation, which requires SAPC. Notably, we find that conformational changes in the loops at the entrance of the substrate-binding site are stabilized by direct interactions with SAPC and that the loss of such interactions induced by N370S and another common mutation, L444P, result in destabilization of the complex and reduced GCase activation. Our findings provide an atomistic-level explanation for GCase activation and the precise mechanism through which N370S and L444P cause Gaucher disease., Competing Interests: The authors declare no conflict of interest.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene. Enzyme replacement treatment is the most effective therapy available for type 1 GD patients, but it is very expensive and does not improve neurologic outcomes in type 2 and 3 GD patients. This study evaluated the effectiveness of an adeno-associated virus 9 (AAV9) vector expressing the Gba gene delivered systemically in GD mouse models. To detect the therapeutic effects of the AAV9-mediated Gba transfer on the systemic symptoms of GD, an inducible whole-body Gba knockout mouse was developed in which tamoxifen effectively induced whole-body Gba gene deletion, and the mice displayed systemic symptoms of GD. The AAV9-CMV-Gba vector, with the expression of Gba driven by the universal CMV promoter, restored GCase activity in multiple organs and prolonged the lifespan in tamoxifen-induced GD mice after intravenous injection. Mice with brain-specific Gba deletion were also included in this study as a model of neuropathic GD (nGD) and injected intraperitoneally on postnatal day 5 with the AAV9-SYN-Gba vector; this improved the GCase activity, ameliorated the neuropathological changes and extended the mean lifespan two-fold. This study demonstrates that AAV9-mediated gene transfer is a potentially effective treatment for GD.

Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba +/+ and gba -/- immortalized neurons and primary human wild-type and type 2 GD fibroblasts., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The major cellular clearance pathway for organelle and unwanted proteins is the autophagy-lysosome pathway (ALP). Lysosomes not only house proteolytic enzymes, but also traffic organelles, sense nutrients, and repair mitochondria. Mitophagy is initiated by damaged mitochondria, which is ultimately degraded by the ALP to compensate for ATP loss. While both systems are dynamic and respond to continuous cellular stressors, most studies are derived from animal models or cell based systems, which do not provide complete real time data about cellular processes involved in the progression of lysosomal storage diseases in patients. Gaucher and Fabry diseases are rare sphingolipid disorders due to the deficiency of the lysosomal enzymes; glucocerebrosidase and α-galactosidase A with resultant lysosomal dysfunction. Little is known about ALP pathology and mitochondrial function in patients with Gaucher and Fabry diseases, and the effects of enzyme replacement therapy (ERT). Studying blood mononuclear cells (PBMCs) from patients, we provide in vivo evidence, that regulation of ALP is defective. In PBMCs derived from Gaucher patients, we report a decreased number of autophagic vacuoles with increased cytoplasmic localization of LC3A/B, accompanied by lysosome accumulation. For both Gaucher and Fabry diseases, the level of the autophagy marker, Beclin1, was elevated and ubiquitin binding protein, SQSTM1/p62, was decreased. mTOR inhibition did not activate autophagy and led to ATP inhibition in PBMCs. Lysosomal abnormalities, independent of the type of the accumulated substrate suppress not only autophagy, but also mitochondrial function and mTOR signaling pathways. ERT partially restored ALP function, LC3-II accumulation and decreased LC3-I/LC3-II ratios. Levels of lysosomal (LAMP1), autophagy (LC3), and mitochondrial markers, (Tfam), normalized after ERT infusion. In conclusion, there is mTOR pathway dysfunction in sphingolipidoses, as observed in both PBMCs derived from patients with Gaucher and Fabry diseases, which leads to impaired autophagy and mitochondrial stress. ERT partially improves ALP function., Competing Interests: Ozlem Goker-Alpan is affiliated with Consulting Shire and Sanofi Genzyme pharmacological companies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Patients with Gaucher Disease (GD) exhibit three phenotypes, including type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic)., Aim: Identifying which GBA changes represent benign polymorphisms and which may result in disease-causing mutations is essential for diagnosis and genotype/phenotype correlations but is often challenging., Results: Here, we describe a patient with type 3 GD, presenting with drug-resistant epilepsy, who bears a set of GBA polymorphic variants including the novel c.363A > G (Gly82Gly) synonymous mutation. In silico predictions, mRNA and functional studies revealed that the new Gly82Gly mutation causes skipping of GBA exon 4, leading to a severe reduction of the wild type GBA mRNA. This is the first report of a synonymous change causing GD through loss of an exonic splicing enhancer sequence. The synonymous mutation is in trans with the Asn188Ser missense mutation, thus making the Asn188Ser responsible for the patient's phenotype and strengthening the association of Asn188Ser with the particular neurological phenotype of type 3 GD., Conclusion: We strengthen the association of Asn188Ser with the type 3 GD phenotype and progressive myoclonus epilepsy. Our data confirm that in silico predictions and mRNA analysis are mandatory in discriminating pathological mutations from the background of harmless polymorphisms, especially synonymous changes.

Background: Gaucher disease (GD) is one of the most common lysosomal storage diseases resulting from a deficiency of glucocerebrosidase. Three main types have been described, with type 2 being the most rare and severe form. Here we investigated the clinical symptoms and mutation spectrum in 20 unrelated type 2 GD patients., Method: The diagnosis of GD was based on the acid β-glucocerebrosidase (GBA) enzyme activity and direct sequencing of the GBA gene. GBA activity was measured in leukocytes and the GBA gene was amplified by a polymerase chain reaction (PCR). For patient 7, the GBA gene was analyzed by PCR as well as quantitative real-time PCR., Results: The age of onset was under 12 months for all patients. All patients experienced severe neurological involvement. A total of 19 different GBA gene mutations were identified, including 6 novel mutations: two were exonic point mutations, c.1127T > C (p.Phe376Ser), c.1418T > G (p.Val473Gly); one was splicing error, ISV7-1G > C; one was insertion, c.717&#95;718insACAG; and the other two were a gross deletion that involved exon 6 and a recombinant allele. The most prevalent mutation was Leu483Pro, which constituted 42.5% of all mutant alleles and was associated with a neurological form in Chinese GD patients as calculated by a Fisher's exact test., Conclusion: The clinical characteristics of Chinese type 2 GD were consistent with reports from other ethnic populations. We identified 6 novel mutations that contribute to the spectrum of GBA gene mutations. Our study confirmed that GD patients with the Leu483Pro allele were prone to experience neurological symptoms., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Objective: Plasma chitotriosidase is a documented biomarker for certain lysosomal storage disorders. However, its clinical utility for prenatal samples is not elucidated yet., Methods: We have established Reference intervals for amniotic fluid chitotriosidase using control amniotic fluids (n = 47) and compared the activity with amniotic fluids affected by lysosomal storage disorders (n = 25)., Results: The reference interval established was 0-6.76 nmol/h/ml. The amniotic fluids affected with LSDs exhibited elevation of chitotriosidase. The area under the curve (AUC) of receiver operating characteristic curve for affected vs. healthy was 0.987 indicating 98.6% accuracy of chitotriosidase in identifying pregnancies affected with LSDs. Among the different LSDs, Gaucher (202.00 ± 35.27 nmol/h/ml) and Niemann-pick A/B (60.33 ± 21.59 nmol/h/ml) showed very high levels of chitotriosidase., Conclusion: Amniotic fluid chitotriosidase has the potential to serve as a diagnostic marker for lysosomal storage disorders, more specifically for Gaucher and Niemann-Pick A/B., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Gaucher disease is a rare pan-ethnic disorder which occurs due to an increased accumulation of undegraded glycolipid glucocerebroside inside the cells' lysosomes. A beta-Glucosidase (GBA) gene defect results in glucocerebrosidase enzyme deficiency. Though the disease is mainly diagnosed in childhood, the adult manifestation is often missed or identified late due to the failure to recognize the heterogeneous clinical presentation. The present study includes seven unrelated Indian adult patients (age range: 20-40 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality., Methods: The biochemical investigation implicated measuring plasma chitotriosidase enzyme activity followed by confirmatory test of β-Glucosidase enzyme activity from the leukocytes. The molecular characterization involved patients' initial screening for the common Gaucher mutation (Leu444Pro). Later, all patients were subjected to whole GBA gene coding region study using bidirectional Sanger sequencing. The population screening for common Gaucher disease mutation (Leu444Pro) was executed in 1200 unrelated and healthy Indian subjects by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction technique. The allele frequency was calculated using Hardy-Weinberg formula., Results: The biochemical analysis revealed a significant reduction in the β-Glucosidase activity in all the patients. Also, an elevated level of plasma Chitotriosidase activity in five patients supported their diagnosis of Gaucher disease. Sanger sequencing established four patients with homozygous variation and three patients with compound heterozygous variation in GBA gene. This study uncovers two missense variants (Ala448Thr and Val17Gly) not previously reported in Gaucher disease patients. Also the known mutations like Leu444Pro, Arg329Cys, Asp315Asn, Ser125Arg, and Arg395Cys were identified in these patients. The homology modeling suggested the destabilization of the protein structure due to novel variants. The Leu444Pro mutation screening in the Indian population spotted two people as a carrier. This emerged the carrier frequency of 1:600 along with wild-type allele frequency 0.97113 and mutant allele frequency 0.02887., Conclusions: The study reports novel and known variants identified in the GBA gene in seven adult patients. The given study is the first report on the carrier frequency of the Leu444Pro mutant allele in an Indian population which will help understanding the burden and susceptibility of Gaucher disease to affect next generation in India.

Gaucher disease (GD) is an inherited disorder in which mutations in the GBA1 gene lead to deficient β-glucocerebrosidase activity and accumulation of its substrate glucosylceramide. Bone disease is present in around 84% of GD patients, ranging from bone loss including osteopenia and osteonecrosis to abnormal bone remodelling in the form of Erlenmeyer flask formation. The range of severity and variety of types of bone disease found in GD patients indicate the involvement of several mechanisms. Here we investigate the effects of exogenous sphingolipids on osteoclasts, osteoblasts, plasma cells and mesenchymal stem cells (MSC) and the interactions between these cell types. Osteoclasts were differentiated from the peripheral blood of Gaucher patients and control subjects. Osteoblasts were differentiated from mesenchymal stem cells isolated from bone marrow aspirates of Gaucher patients and control subjects. The human osteoblast cell line SaOS-2 was also investigated. Osteoclasts, osteoblasts and a human myeloma plasma cell line NCI-H929 were cultured with relevant exogenous sphingolipids to assess effects on cellular viability and function. Calcium deposition by osteoblasts differentiated from Gaucher patient MSC's was on average only 11.4% of that deposited by control subject osteoblasts. Culture with glucosylsphingosine reduced control subject MSC viability by 10.4%, SaOS-2 viability by 17.4% and plasma cell number by 40%. Culture with glucosylceramide decreased calcium deposition by control MSC-derived osteoblasts while increasing control subject osteoclast generation by 55.6%, Gaucher patient osteoclast generation by 37.6% and plasma cell numbers by up to 29.7%. Excessive osteoclast number and activity and reduced osteoblast activity may have the overall effect of an uncoupling between osteoclasts and osteoblasts in the GD bone microenvironment., (Copyright © 2018. Published by Elsevier Inc.)

Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a Nicotiana benthamiana root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A N -glycan analysis of purified GCase was performed using nano LC/MS. The Man₃XylFucGlcNAc₂ structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins.

A series of sp²-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d- gluco or l- ido ), the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 β-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N′ -octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase). The 1-deoxynojirimycin (DNJ)-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM). At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

Background: Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls., Methods: ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes., Results: We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen., Conclusions: The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The data on occurrence and pathogenetic mechanisms of glucosylceramide lipidosis (Gaucher disease), which is based on the hereditary deficiency of glucocerebrosidase activity -the enzyme involved in the processing of cellular metabolism products, is presented. Clinical and morphological manifestations of the disease, therapeutic tactics and prognosis are defined. A typical clinical case of Gaucher disease type 1 in two adult patients was analyzed. It is shown that timely diagnosis of Gaucher disease allows the prescription of adequate treatment by means of enzyme replacement therapy with cerezyme drug. That is why the correct and timely diagnosis of the disease can prolongate patient's life for many years. In this case, the patient should be under the care of doctors (pediatricians, general physicians, hematologists). Continued treatment of Gaucher disease with cerezyme stabilizes the pathological process completely, decreases significant changes in bones and parenchymal organs, improving patients' lives considerably. For that reason, the sooner the adequate therapy is started, the more effective the results will be.

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity., (© 2017 Wiley Periodicals, Inc.)

Eliglustat, an oral substrate reduction therapy, is a first-line treatment for adults with Gaucher disease type 1 (GD1) who are poor, intermediate, or extensive CYP2D6 metabolizers (>90% of patients). In the primary analysis of the Phase 3 ENGAGE trial (NCT00891202), eliglustat treatment for 9 months resulted in significant reductions in spleen and liver volumes and increases in hemoglobin concentration and platelet count compared with placebo. We report 18-month outcomes of patients who entered the trial extension period, in which all patients received eliglustat. Of 40 trial patients, 39 entered the extension period, and 38 completed 18 months. Absolute values and percent change over time were determined for spleen and liver volume, hemoglobin concentration, platelet count, bone mineral density, bone marrow burden, and Gaucher disease biomarkers. For patients randomized to eliglustat in the double-blind period, continuing treatment with eliglustat for 9 more months resulted in incremental improvement of all disease parameters. For patients randomized to placebo in the double-blind period, eliglustat treatment during the 9-month, open-label period resulted in significant decrease of spleen and liver volumes and significant increase of hemoglobin and platelets, with a similar rate of change to patients who had received eliglustat in the double-blind period. Eliglustat treatment was also associated with improvement in bone marrow burden score, bone mineral density, and established biomarkers of Gaucher disease, including reduction of the bioactive lipid, glucosylsphingosine. These findings underscore the efficacy of eliglustat in treatment-naïve patients. Eliglustat was well-tolerated, and there were no new safety concerns with longer-term exposure., (© 2017 The Authors American Journal of Hematology Published by Wiley Periodicals, Inc.)

Gaucher disease (GD) is caused by mutations on the gene encoding for the lysosomal enzyme glucocerebrosidase. Type I GD (GD1) patients present anemia, hepatosplenomegaly and bone alterations. In spite of treatment, bone alterations in GD patients persist, including poor bone mineral density (BMD). Mechanisms leading to bone damage are not completely understood, but previous reports suggest that osteoclasts are involved. Chitotriosidase (CHIT) is the most reliable biomarker used in the follow up of patients, although its correlation with bone status is unknown. The aim of this work was to study the pro-osteoclastogenic potential in patients and to evaluate its correlation with CHIT activity levels and clinical parameters. PBMCs from treated patients and healthy controls were cultured in the presence of M-CSF, and mature osteoclasts were counted. BMD, blood CHIT activity and serum levels of CTX, BAP, and cytokines were evaluated in patients. We found that blood CHIT activity and osteoclast differentiation were significantly increased in patients, but no correlation between them was observed. Interestingly, osteoclast numbers but not CHIT, presented a negative correlation with BMD expressed as Z-score. CTX, BAP and serum cytokines involved in bone remodeling were found altered in GD1 patients. These results show for the first time a correlation between osteoclast differentiation and BMD in GD1 patients, supporting the involvement of osteoclasts in the bone pathology of GD1. Our results also suggest that an altered immune response may play an important role in bone damage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gaucher disease (GD) is an inherited metabolic disorder that involves accumulation of glycolipid glucocerebroside in monocyte-macrophage cells, which can result in multiple organ damage. Enzyme replacement and substrate reduction therapies have improved the potential for early diagnosis and treatment. Determining the true incidence of this rare disease is critical for relevant policy establishment. Newborn screening allows for early diagnosis and an comparatively accurate incidence of GD. A fluorometric method to detect acid β-glucocerebrosidase (GBA) activity on a dried blood spot punch was developed. Validity and feasibility of the fluorometric method was demonstrated by examining 116 healthy controls, 19 confirmed GD patients and 19 obligate carriers. GBA activity was measured on dried blood spots of 80 855 newborns. Samples from positively screened newborns were reanalyzed by a leukocyte GBA activity test and GBA gene analysis. Plasma glucosylsphingosine level was determined as a biomarker of the pathophysiology of GD. GD patients were distinguished from healthy controls and obligate carriers using the fluorometric method. Mean GBA activity in newborn screening specimens was 145.69±44.76 μmol l -1  h -1 (n=80 844). Three children had low GBA activity, of which one child had low GBA activity on the second dried blood spot specimen. Leukocyte, genetic and biomarker analysis confirmed the diagnosis and indicated that this child was in the early stages of GD. In conclusion, the incidence of GD in Shanghai of China is approximately 1 in 80 855. Screening for GD by fluorometric analysis of GBA activity is an efficient and feasible technology in newborns.

Chitotriosidase, an enzyme secreted by activated macrophages, is widely used as a biomarker for therapeutic monitoring and patient follow-up in Gaucher disease (GD), a lysosomal disorder caused by an inherited deficiency of glucocerebrosidase. We analyzed the long-term evolution of chitotriosidase aiming to establish an accurate model that describes the influence of enzyme replacement therapy (ERT) and the impact of several covariates. A total of 55 patients with non-neuronopathic (type 1) GD were followed for almost 17 years (during a maximum of 7.57 and 8.96 years, before and after the onset of ERT, respectively). Plasma chitotriosidase activity, measured yearly before the onset of ERT and at 6-month intervals after the initiation of ERT, was analyzed as a function of several covariates (age at diagnosis and at ERT initiation, nature of the most frequent genotypes, spleen status and the occurrence of bone complications). The evolution of chitotriosidase was approximated by a sigmoidal function, which allows the calculation of predicted values, based on several parameters inferred from our data. Splenectomy and the occurrence of bone complications significantly delayed the decline in chitotriosidase activity and induced higher mean residual values after long-term (4-9 years) ERT. Likewise, patients who started ERT infusions under 15 years of age had significantly higher mean residual chitotriosidase activities. The influence of other covariates did not reach statistical significance. In conclusion, we propose a novel model describing the evolution of chitotriosidase, allowing more accurate treatment adjustments, according to the variations of this biomarker.

Purpose: The overall published experience with pediatric type 1 Gaucher disease (GD1) has been based on ascertainment through clinical presentation of the disease. We describe the longitudinal follow-up in a presymptomatic pediatric cohort., Methods: The cohort includes children diagnosed with GD1, either prenatally or postnatally by molecular genetic testing, and followed for clinical care at our center from 1998 to 2016. All patients' parents were GBA mutation carriers identified through carrier screening programs. Longitudinal clinical, laboratory, and imaging data were obtained through chart review., Results: Thirty-eight patients aged 1-18 years (mean at last visit 6.9 ± 4.1 years) were followed, including 32 p.N409S homozygotes and 6 p.N409S/p.R535H compound heterozygotes. At the last evaluation, a minority had hematological (5%), bone (15%), or linear growth (19%) issues. Only 12% had splenomegaly and 74% had moderate hepatomegaly. Chitotriosidase activity varied widely (6-5,640 nmol/hour/ml) and generally increased with age. Pediatric Gaucher severity scores (GSS) remained stable and within the mild-disease range for most (95%). Treatment for progressive disease during this period was recommended for four children., Conclusion: Most children with the p.N409S/p.N409S and p.N409S/p.R535H GD1 genotypes have minimal disease manifestations and progression during childhood and can be monitored using limited assessments. Those with other mutations may require additional monitoring. These data are valuable for newborn screening and counseling.Genet Med advance online publication 13 October 2016.

Background: Recently, the metabolite-likeness of the drug space has emerged and has opened a new possibility for exploring human metabolite-like candidates in drug discovery. However, the applicability of metabolite-likeness in drug discovery has been largely unexplored. Moreover, there are no reports on its applications for the repositioning of drugs to possible enzyme modulators, although enzyme-drug relations could be directly inferred from the similarity relationships between enzyme's metabolites and drugs., Methods: We constructed a drug-metabolite structural similarity matrix, which contains 1,861 FDA-approved drugs and 1,110 human intermediary metabolites scored with the Tanimoto similarity. To verify the metabolite-likeness measure for drug repositioning, we analyzed 17 known antimetabolite drugs that resemble the innate metabolites of their eleven target enzymes as the gold standard positives. Highly scored drugs were selected as possible modulators of enzymes for their corresponding metabolites. Then, we assessed the performance of metabolite-likeness with a receiver operating characteristic analysis and compared it with other drug-target prediction methods. We set the similarity threshold for drug repositioning candidates of new enzyme modulators based on maximization of the Youden's index. We also carried out literature surveys for supporting the drug repositioning results based on the metabolite-likeness., Results: In this paper, we applied metabolite-likeness to repurpose FDA-approved drugs to disease-associated enzyme modulators that resemble human innate metabolites. All antimetabolite drugs were mapped with their known 11 target enzymes with statistically significant similarity values to the corresponding metabolites. The comparison with other drug-target prediction methods showed the higher performance of metabolite-likeness for predicting enzyme modulators. After that, the drugs scored higher than similarity score of 0.654 were selected as possible modulators of enzymes for their corresponding metabolites. In addition, we showed that drug repositioning results of 10 enzymes were concordant with the literature evidence., Conclusions: This study introduced a method to predict the repositioning of known drugs to possible modulators of disease associated enzymes using human metabolite-likeness. We demonstrated that this approach works correctly with known antimetabolite drugs and showed that the proposed method has better performance compared to other drug target prediction methods in terms of enzyme modulators prediction. This study as a proof-of-concept showed how to apply metabolite-likeness to drug repositioning as well as potential in further expansion as we acquire more disease associated metabolite-target protein relations.

A library of dimers and heterodimers of both enantiomers of 2-O-alkylated iminoxylitol derivatives has been synthesised and evaluated on β-glucocerebrosidase (GCase), the enzyme responsible for Gaucher disease (GD). Although the objective was to target simultaneously the active site and a secondary binding site of the glucosidase, the (-)-2-iminoxylitol moiety seemed detrimental for imiglucerase inhibition and no significant enhancement was obtained in G202R, N370S and L444P fibroblasts. However, all compounds having at least one (+)-2-O-alkyl iminoxylitol are GCase inhibitors in the nano molar range and are significant GCase activity enhancers in G202R fibroblats, as confirmed by a decrease of glucosylceramide levels and by co-localization studies.

Aim: To provide more efficient and timely immunogenicity testing service to support routine patient care, the original complex testing algorithm for evaluation of anti-velaglucerase alfa antibodies has been simplified and individual methods (screen, confirm, titer, neutralizing antibody [NAb] and IgE) have been redeveloped/optimized and validated., Results: To compare the performance of different methods, 50 velaglucerase alfa-treated patient samples were analyzed using both old and new methods for the presence of antidrug antibodies (ADAs) and 31 ADA-positive samples were analyzed for neutralizing capacity. The ADA and NAb statuses are almost identical from both methods and both ADA and NAb titer results are highly correlated with a Spearman's correlation of 0.96 and 0.86, respectively., Conclusion: The original and new testing methods can be considered interchangeable for the measurement of total and neutralizing anti-velaglucerase alfa antibodies.

In the phase 3 Study of Eliglustat Tartrate (Genz-112638) in Patients With Gaucher Disease Who Have Reached Therapeutic Goals With Enzyme Replacement Therapy (ENCORE), at 1 year, eliglustat was noninferior to imiglucerase enzyme therapy in maintaining stable platelet counts, hemoglobin concentrations, and spleen and liver volumes. After this primary analysis period, patients entered a long-term extension phase in which all received eliglustat. Duration on eliglustat ranged from 2 to 5 years, depending on timing of enrollment (which spanned 2 years), treatment group to which patients were randomized, and whether they lived in the United States when commercial eliglustat became available. Here we report long-term safety and efficacy of eliglustat for 157 patients who received eliglustat in the ENCORE trial; data are available for 46 patients who received eliglustat for 4 years. Mean hemoglobin concentration, platelet count, and spleen and liver volumes remained stable for up to 4 years. Year to year, all 4 measures remained collectively stable (composite end point relative to baseline values) in ≥85% of patients as well as individually in ≥92%. Mean bone mineral density z scores (lumbar spine and femur) remained stable and were maintained in the healthy reference range throughout. Eliglustat was well tolerated over 4 years; 4 (2.5%) patients withdrew because of adverse events that were considered related to the study drug. No new or long-term safety concerns were identified. Clinical stability assessed by composite and individual measures was maintained in adults with Gaucher disease type 1 treated with eliglustat who remained in the ENCORE trial for up to 4 years. This trial was registered at www.clinicaltrials.gov as #NCT00943111., (© 2017 by The American Society of Hematology.)

Background: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by deficiency in acid beta-glucosidase. GD exhibits a wide clinical spectrum of disease severity with an unpredictable natural course. Plasma chitotriosidase activity and CC chemokine ligand 18 (CCL18) have been exchangeably used for monitoring GD activity and response to enzyme replacement therapy in conjunction with clinical assessment. Yet, a large-scale head-to-head comparison of these two biomarkers is currently lacking. We propose a collaborative systematic review with meta-analysis of individual participant data (IPD) to compare the accuracy of plasma chitotriosidase activity and CCL18 in assessing type I (i.e., non-neuropathic) GD severity., Methods: Eligible studies include cross-sectional, cohort, and randomized controlled studies recording both plasma chitotriosidase activity and CCL18 level at baseline and/or at follow-up in consecutive children or adult patients with type I GD. Pre-specified surrogate outcomes reflecting GD activity include liver and spleen volume, hemoglobin concentration, platelet count, and symptomatic bone events with imaging confirmation. Primary studies will be identified by searching Medline (1995 onwards), EMBASE (1995 onwards), and Cochrane Central Register of Controlled Trials (CENTRAL). Electronic search will be complemented by contacting research groups in order to identify unpublished relevant studies. Where possible, IPD will be extracted from published articles. Corresponding authors will be invited to collaborate by supplying IPD. The methodological quality of retrieved studies will be appraised for each study outcome, using a checklist adapted from the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The primary outcome will be a composite of liver volume >1.25 multiple of normal (MN), spleen volume >5 MN, hemoglobin concentration <11 g/dL, or platelet count <100 × 10 9 /L. Effect size estimates for biomarker comparative accuracy in predicting outcomes will be reported as differences in areas under receiver operating characteristic curves along with 95% confidence intervals. Effect size estimates will be reported as (weighted) mean differences along with 95% confidence intervals for each biomarker according to outcomes. IPD meta-analysis will be conducted with both one- and two-stage approaches., Discussion: Valid and precise accuracy estimates will be derived for CCL18 relative to plasma chitotriosidase activity in discriminating patients according to GD severity., Systematic Review Registration: PROSPERO 2015 CRD42015027243.

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Amphiphilic glycomimetics encompassing a rigid, undistortable nortropane skeleton based on 1,6-anhydro-l-idonojirimycin and a polyfluorinated antenna, when formulated as the corresponding inclusion complexes with β-cyclodextrin (βCD), have been shown to behave as pharmacological chaperones (PCs) that efficiently rescue lysosomal β-glucocerebrosidase mutants associated with the neuronopathic variants of Gaucher disease (GD), including the highly refractory L444P/L444P and L444P/P415R single nucleotide polymorphs, in patient fibroblasts. The body of work here presented includes the design criteria for the PC prototype, the synthesis of a series of candidates, the characterization of the PC:βCD complexes, the determination of the selectivity profiles toward a panel of commercial and human lysosomal glycosidases, the evaluation of the chaperoning activity in type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (adult neuronopathic) GD fibroblasts, the confirmation of the rescuing mechanism by immunolabeling, and the analysis of the PC:GCase binding mode by docking experiments.

Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease, whereas Gaucher disease (GD) is the most frequent lysosomal storage disorder caused by homozygous mutations in the glucocerebrosidase (GBA1) gene. Increased risk of developing PD has been observed in both GD patients and carriers. It has been estimated that GBA1 mutations confer a 20- to 30-fold increased risk for the development of PD, and that at least 7-10% of PD patients have a GBA1 mutation. To date, mutations in the GBA1 gene constitute numerically the most important risk factor for PD. The type of PD associated with GBA1 mutations (PD-GBA1) is almost identical to idiopathic PD, except for a slightly younger age of onset and a tendency to more cognitive impairment. Importantly, the pathology of PD-GBA1 is identical to idiopathic PD, with nigral dopamine cell loss, Lewy bodies, and neurites containing alpha-synuclein. The mechanism by which GBA1 mutations increase the risk for PD is still unknown. However, given that clinical manifestation and pathological findings in PD-GBA1 patients are almost identical to those in idiopathic PD individuals, it is likely that, as in idiopathic PD, alpha-synuclein accumulation, mitochondrial dysfunction, autophagic impairment, oxidative and endoplasmic reticulum stress may contribute to the development and progression of PD-GBA1. Here, we review the GBA1 gene, its role in GD, and its link with PD. The impact of glucocerebrosidase 1 (GBA1) mutations on functioning of endoplasmic reticulum (ER), lysosomes, and mitochondria. GBA1 mutations resulting in production of misfolded glucocerebrosidase (GCase) significantly affect the ER functioning. Misfolded GCase trapped in the ER leads to both an increase in the ubiquitin-proteasome system (UPS) and the ER stress. The presence of ER stress triggers the unfolded protein response (UPR) and/or endoplasmic reticulum-associated degradation (ERAD). The prolonged activation of UPR and ERAD subsequently leads to increased apoptosis. The presence of misfolded GCase in the lysosomes together with a reduction in wild-type GCase levels lead to a retardation of alpha-synuclein degradation via chaperone-mediated autophagy (CMA), which subsequently results in alpha-synuclein accumulation and aggregation. Impaired lysosomal functioning also causes a decrease in the clearance of autophagosomes, and so their accumulation. GBA1 mutations perturb normal mitochondria functioning by increasing generation of free radical species (ROS) and decreasing adenosine triphosphate (ATP) production, oxygen consumption, and membrane potential. GBA1 mutations also lead to accumulation of dysfunctional and fragmented mitochondria. This article is part of a special issue on Parkinson disease., (© 2016 International Society for Neurochemistry.)

Gaucher's disease is a common genetic disease caused by mutations in the β-glucocerebrosidase (GBA1) gene that have been also linked to increased risk of Parkinson's disease and Lewy body dementia. Stabilization of misfolded mutant β-glucocerebrosidase (GCase) represents an important therapeutic strategy in synucleinopathies. Here we report a novel class of GCase quinazoline inhibitors, obtained in a high throughput screening, with moderate potency against wild-type GCase. Rational design and a SAR study of this class of compounds led to a new series of quinazoline derivatives with single-digit nanomolar potency. These compounds were shown to selectively stabilize GCase when compared to other lysosomal enzymes and to increase N370S mutant GCase protein concentration and activity in cell assays. To the best of our knowledge, these molecules are the most potent noniminosugar GCase modulators to date that may prove useful for future mechanistic studies and therapeutic approaches in Gaucher's and Parkinson's diseases.

Background: Enzyme replacement therapy (ERT) is currently the standard treatment for patients with Gaucher disease type I (GD1), but the pharmacokinetics have hardly been studied. This study aimed to quantify in vivo enzyme activity in peripheral leukocytes from patients receiving long-term treatment with imiglucerase or velaglucerase for GD1, and set out to assess the process of enzymatic uptake by peripheral leukocytes., Methods: A prospective semi-experimental study was conducted. Four time points for blood withdrawal were planned per patient to quantify the intra-leukocyte enzymatic activity. In order to assess the uptake process, the rate of enzyme uptake by leukocytes (Rupt) and the rate of enzyme disappearance from the plasma (Rdis) were estimated., Results: Eight GD1 patients were included. Intra-leukocyte activity was 24.31 mU/mL [standard deviation (SD) 6.32 mU/mL; coefficient of variation (CV) 25.96 %] at baseline and 27.14 mU/mL (SD  6.96 mU/mL; CV 25.65 %) at 15 min post-perfusion. The relationships with the administered dose were linear. The Rupt value was 37.73 mU/mL/min [95 % confidence interval (CI) 25.63-49.84] and showed a linear correlation with the administered enzyme dose (p < 0.05), and the Rdis value was 189.43 mU/mL/min (95 % CI 80.31-298.55) and also showed a linear correlation with the dose (p < 0.05)., Conclusion: This was the first in vivo study to quantify the accumulated enzymatic activity in patients receiving ERT for GD1. It showed that intra-leukocyte activity at baseline and at 15 min post-perfusion could be used as a possible marker for therapeutic individualization in patients receiving ERT for GD1.

Small compound active site interactors receive considerable attention for their ability to positively influence the fold of glycosidases. Endoglycoceramidase II (EGCII) from Rhodococcus sp. is an endo-β-glucosidase releasing the complete glycan from ceramide in glycosphingolipids. Cleavage of the β-glycosidic linkage between glucose and ceramide is also catalyzed by glucocerebrosidase (GBA), the exo-β-glucosidase deficient in Gaucher disease. We demonstrate that established β-glucoside-configured cyclophellitol-type activity-based probes (ABPs) for GBA also are effective, mechanism-based, and irreversible inhibitors of EGCII. The stability of EGCII is markedly enhanced by formation of covalent complexes with cyclophellitol ABPs substituted with hydrophobic moieties, as evidenced by an increased melting temperature, resistance against tryptic digestion, changes in (15)N-(1)H transverse relaxation optimized spectroscopy spectra of the [(15)N]Leu-labeled enzyme, and relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic acid fluorescence. The stabilization of EGCII conformation correlates with the shape and hydrophobicity of the substituents of the ABPs. We conclude that the amphipathic active site binders with aliphatic moieties act as a "hydrophobic zipper" on the flexible EGCII protein structure.

Background: Gaucher disease (GD) is an autosomal recessive disorder produced by mutations in the glucocerebrosidase gene (GBA), causing storage of glucosylceramide in reticuloendothelial cells in multiple organs. Traditionally, the prediction of the phenotype based on the genotype has been reported to be limited., Subjects and Methods: We investigated the correlation between the enzymatic residual activity (ERA) and the phenotype at diagnosis of the disease in 45 GD Spanish patients (44 with type I and 1 with type III GD). The genotype involved two of the following previously expressed proteins: c.517A > C (T134P), 1%; c.721G > A (G202R), 17%; c.1090G > T (G325W), 13.9%; c.1208G > A (S364N), 4.1%; c.1226A > G (N370S), 17.8%; c.1246G > A (G377S), 17.6%; c.1289C > T (P391L), 8.5%; c.1448T > C (L444P), 3%; and c.1504C > T (R463C), 24.5%. Recombinant alleles, deletion of 55 bp in exon 9 and 84GG mutation were considered as mutations with no residual enzymatic activity., Results: The ERA showed a statistically significant correlation with chitotriosidase (P < 0.001), age (P < 0.001), spleen size (P < 0.001), 'Zimran's Severity Score Index' (P < 0.01) and the 'Gaucher Disease Severity Score Index-Type I' (P <  0.0001) at diagnosis of the disorder. Previous to any medical intervention, a comparison between the ERA and bone involvement, demonstrated a statistically significant relationship (P < 0.01) between the two variables., Conclusions: This study data allowed us to define a new criterion for prognostic assessment of the disease at diagnosis, called Protein Severity Index, which expresses the theoretical severity of the genotype presented by patients, according to the corresponding ERA., (© The Author 2016. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies., (© 2016. Published by The Company of Biologists Ltd.)

Multivalent iminosugars conjugated with a morpholine moiety and/or designed as prodrugs have been prepared and evaluated as new classes of pharmacological chaperones for the treatment of Gaucher disease. This study further confirms the interest of the prodrug concept and shows that the addition of a lysosome-targeting morpholine unit into iminosugar cluster structures has no significant impact on the chaperone activity on Gaucher cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sphingolipids represent a major class of lipids that are essential constituents of eukaryotic cells. They are predominantly located in plasma membrane microdomains, and play an important structural role in regulating membrane fluidity. They are also bioactive effectors involved in diverse key cellular functions such as apoptosis and proliferation. The implication of some sphingolipids in cancer is well established whereas that of some others is still a matter of intense investigation. Glucosylceramide is the backbone of more than 300 structurally different glycosphingolipids including gangliosides and sulfatides, and is essential for mammalian development. Therefore, glucosylceramidases (also named GBA1, GBA2 and GBA3 β-glucosidases), the enzymes that hydrolyse β-glucosylceramide, play important functions. GBA1 is a lysosomal hydrolase whose deficiency causes Gaucher disease, the most prevalent inherited lysosomal storage disorder. GBA2 is a ubiquitous non-lysosomal glucosylceramidase whose mutations have been associated with some forms of hereditary spastic paraplegia. GBA3 is a cytosolic β-glucosidase, mostly present in the kidney, liver, spleen, intestine and lymphocytes of mammals, the function of which is still unclear. Whereas glucosylceramide synthase is implicated in multidrug resistance, the role of glucosylceramide breakdown in cancer is not yet fully appreciated. Defective GBA1 enzyme activity in humans, i.e., Gaucher disease, is associated with an increased risk of multiple myeloma and other malignancies. Putative molecular links between Gaucher disease and cancer, which might implicate the malignant cell and/or its microenvironment, are reviewed. The functions of GBA2 and GBA3 in cancer progression are also discussed., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Plasma chitotriosidase originates from activated macrophages and is reported to be elevated in many Lysosomal Storage Disorders. Measurement of this enzyme activity has been an available tool for monitoring therapy of Gaucher disease. The degree of elevation of chitotriosidase is useful for differential diagnosis of Gaucher disease and Niemann Pick A/B. However the potential utility of this chitotriosidase assay depends on the frequency of deficient chitotriosidase activity in a particular population. We therefore aim to study the clinical utility of this assay Gaucher and Niemann Pick A/B diseases in the backdrop of chitotriosidase deficiency in our population. The study comprises 173 patients with clinical suspicion of either Gaucher disease (n=108) or Niemann Pick A/B (n=65) and 92 healthy controls. The plasma samples of controls, Gaucher disease, and Niemann Pick A/B showed chitotriosidase deficiency of 12%, 25% and 27% respectively. The degree of elevation of chitotriosidase in Gaucher disease and Niemann Pick A/B patients is 40-326 (11,325.7±6395.4nmol/h/ml) and 7-22 folds (1192.5±463.0nmol/h/ml) respectively. In view of these findings of distinguishable fold elevation of chitotriosidase in Gaucher disease or Niemann Pick A/B, it can be a potential surrogate differential diagnostic marker for these groups of diseases, except in the patients in whom this enzyme is deficient., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.