Development and validation of a new genotype-phenotype correlation for Niemann-Pick disease type C1.

Hundreds of NPC1 variants cause highly heterogeneous phenotypes. This study aims to explore the genotype-phenotype correlation of NPC1, especially for missense variants. In a well-characterized cohort, phenotypes are graded into three clinical forms: mild, intermediate, and severe. Missense residue structural location was stratified into three categories: surface, partially, and fully buried. The association of phenotypes with the topography of the amino acid substitution in the protein structure was investigated in our cohort and validated in two reported cohorts. One hundred six unrelated NPC1 patients were enrolled. A significant correlation of genotype-phenotype was found in 81 classified individuals with two or one (the second was null variant) missense variant (p < 0.001): of 25 patients with at least one missense variant of surface (group A), 19 (76%) mild, six (24%) intermediate, and none severe; of 31 cases with at least one missense variant of partially buried without surface variants (group B), 11 (35%) mild, 16 (52%) intermediate, and four (13%) severe; of the remaining 25 patients with two or one buried missense variants (group C), eight (32%) mild, nine (36%) intermediate, and eight (32%) severe. Additionally, 7-ketocholesterol, the biomarker, was lower in group A than in group B (p = 0.024) and group C (p = 0.029). A model was proposed that accurately predicted phenotypes of 72 of 90 (80%), 73 of85 (86%), and 64 of 69 (93%) patients in our cohort, Italian, and UK cohort, respectively. This study proposed a novel genotype-phenotype correlation in NPC1, linking the underlying molecular pathophysiology with clinical phenotype and aiding genetic counseling and evaluation in clinical practice.

Antigen-encapsulating host extracellular vesicles derived from Salmonella-infected cells stimulate pathogen-specific Th1-type responses in vivo.

Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections.

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

Investigation of GALNS variants and genotype-phenotype correlations in a large cohort of patients with mucopolysaccharidosis type IVA.

Mucopolysaccharidosis type IVA (MPS IVA) is a rare autosomal recessive disorder resulting from the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) caused by pathogenic variants in the GALNS gene. A systematic analysis for genotype-phenotype correlation is essential due to hundreds of variants generating different levels of residual GALNS activity and causing a wide degree of clinical manifestation effects. Here, we retrospectively analyzed clinical and genetic data of 108 unrelated patients with MPS IVA to investigate the variants spectrum of GALNS and assess their clinical effects. In this cohort, 82 patients were classified as severe, 14 as intermediate, and 12 as mild. One hundred and one GALNS variants were identified, of which 47 were novel. Most patients with at least one GALNS null variant were classified as severe phenotype (92%, 33/36). Missense variants mapped to different residues of GALNS protein resulted in different phenotypes in patients with MPS IVA. Ninety-two percent of patients with two missense variants mapped to buried residues were classified as severe (92%, 24/26), while at least one missense variant mapped to surface residues was identified in patients with biallelic missense variants presenting intermediate MPS IVA (78%, 7/9) and presenting mild MPS IVA (86%, 6/7). Our study contributes to a better understanding of the molecular spectrum of GALNS variants and their clinical implications. Based on the data herein reported, we generated a systematic flowchart correlating the GALNS variants to assist in phenotype prediction and classification of patients with MPS IVA.

Clinical, biochemical, and genotype-phenotype correlations of 118 patients with Niemann-Pick disease Types A/B.

Niemann-Pick disease Types A and B (NPA/B) are autosomal recessive disorders caused by variants in the sphingomyelin phosphodiesterase-1 (SMPD1) gene. This study aimed to describe and characterize a cohort of 118 patients diagnosed with NPA/B based on clinical, biochemical, and molecular findings, and to identify sound correlations between laboratory findings and clinical presentations. Decreased peripheral leukocyte acid sphingomyelinase activity levels and increased plasma 7-ketocholesterol levels were significantly correlated with disease onset and severity of the clinical course. We identified 92 different sequence SMPD1 variants, including 41 novel variants, in 118 NPA/B patients (19 NPA, 24 intermediate type, 75 NPB). The most prevalent mutation was p.Arg602His, which accounted for 9.3% of the alleles. Patients homozygous for p.Arg602His or p.Asn522Ser showed a late-onset form of the NPB phenotype. The homozygous SMPD1 variant p.Tyr500His correlated with the early-onset NPB clinical form. Additionally, homozygous variants p.His284SerfsX18, p.Phe465Ser, and p.Ser486Arg were associated with the neuronopathic NPA clinical form. The homozygous variant p.Arg3AlafsX74 was associated with the intermediate clinical form. Our study contributes to the understanding of the natural history of NPA/B and assists in the development of efficacious treatments for patients afflicted with this devastating lysosomal storage disorder.

Ambroxol improves skeletal and hematological manifestations on a child with Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disease caused by the deficiency of glucocerebrosidase characterized by a broad spectrum of clinical manifestations including hepatosplenomegaly, bone infiltration, and cytopenia, and even central nervous system involvement. Bone manifestations are typical of the GD-I and partially responded to mainstay therapy. Ambroxol (ABX), an approved cough-suppressant, was identified as an enzyme-enhancement agent of the residual activity of glucocerebrosidase mutants derived from different misfolding-mutations in the GBA gene. Here, we describe the early beneficial effects of ABX on skeletal and hematological manifestations of a child suffering with progressive GD-I.

Microphthalmia and linear skin defects syndrome: Precise diagnosis guides prognosis.

Microphthalmia and linear skin defects syndrome (MLS) is a rare X-linked dominant disorder characterized by microphthalmia and linear atrophic plaques of the face and neck. The diagnosis of MLS can be challenging secondary to both its rarity and to clinical overlap with Goltz syndrome. Whereas the skin lesions of MLS are more likely to improve in appearance with age, the lesions of Goltz are typically persistent.

Recurrent Muscle Weakness with Rhabdomyolysis, Metabolic Crises, and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations.

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3-9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3-9. Additionally, a homozygous exons 4-6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3-9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.

Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease.

Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of ß-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders.

Newborn Screening for 6 Lysosomal Storage Disorders in China.

Importance: Newborn screening (NBS) for lysosomal storage disorders (LSDs) is becoming an increasing concern in public health. However, the birth prevalence of these disorders is rarely reported in the Chinese population, and subclinical forms of diseases among patients identified by NBS have not been evaluated. Objective: To evaluate the birth prevalence of the 6 LSDs in the Shanghai population and determine subclinical forms based on clinical, biochemical, and genetic characteristics. Design, Setting, and Participants: This cohort study included 50 108 newborns recruited from 41 hospitals in Shanghai between January and December 2021 who were screened for 6 LSDs using tandem mass spectrometry (MS/MS). Participants with screen-positive results underwent molecular and biochemical tests and clinical assessments. Data were analyzed from January 2021 through October 2022. Exposures: All participants were screened for Gaucher, acid sphingomyelinase deficiency (ASMD), Krabbe, mucopolysaccharidosis type I, Fabry, and Pompe diseases using dried blood spots. Main Outcomes and Measures: Primary outcomes were the birth prevalence and subclinical forms of the 6 LSDs in the Shanghai population. Disease biomarker measurements, genetic testing, and clinical analysis were used to assess clinical forms of LSDs screened. Results: Among 50 108 newborns (26 036 male [52.0%]; mean [SD] gestational age, 38.8 [1.6] weeks), the mean (SD) birth weight was 3257 (487) g. The MS/MS-based NBS identified 353 newborns who were positive. Of these, 27 newborns (7.7%) were diagnosed with 1 of 6 LSDs screened, including 2 newborns with Gaucher, 5 newborns with ASMD, 9 newborns with Krabbe, 8 newborns with Fabry, and 3 newborns with Pompe disease. The combined birth prevalence of LSDs in Shanghai was 1 diagnosis in 1856 live births, with Krabbe disease the most common (1 diagnosis/5568 live births), followed by Fabry disease (1 diagnosis/6264 live births), and ASMD (1 diagnosis/10 022 live births). Biochemical, molecular, and clinical analysis showed that early-onset clinical forms accounted for 3 newborns with positive results (11.1%), while later-onset forms represented nearly 90% of diagnoses (24 newborns [88.9%]). Conclusions and Relevance: In this study, the combined birth prevalence of the 6 LSDs in Shanghai was remarkably high. MS/MS-based newborn screening, combined with biochemical and molecular genetic analysis, successfully identified and characterized newborns who were screen-positive, which may assist with parental counseling and management decisions.

A high-throughput screening assay using Krabbe disease patient cells.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by ß-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here, we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD because they are more prone to cross the blood-brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen has been shown to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from an SV40-transformed GLD patient fibroblast was measurable in high-density microplates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration-response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live GLD patient cells to identify therapeutic agents that can potentially be used for the treatment of this progressive neurodegenerative disease.

Novel proton MR spectroscopy findings in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease.

Integrating whole-genome sequencing and transcriptomic findings in the diagnosis and management of Coffin-Siris syndrome.

INTRODUCTION: Although the whole-exome sequencing (WES) approach has been widely used in clinic, many rare diseases with syndromic and nonsyndromic neurological manifestations remain undiagnosed. Coffin-Siris syndrome (CSS) is a rare autosomal dominant genetic disease characterized by neurodevelopmental delay. A suspected diagnosis can be made based on the typical CSS clinical features; however, molecular genetic testing is necessary for a confirmed diagnosis. OBJECTIVES: Three CSS-like patients with negative results in the WES and chromosomal microarray analysis (CMA) were recruited in this study. METHODS: We used whole-genome sequencing (WGS) technology to sequence the peripheral blood of the three families. To further explore the possible pathogenesis of CSS, we performed RNA-sequencing (RNA-seq). RESULTS: WGS identified the three CSS patients were carrying de novo copy number variants of the ARID1B gene, which have not been reported before. RNA-seq identified 184 differentially expressed genes (DEGs), with 116 up-regulated and 68 down-regulated. Functional annotation of DEGs showed that two biological processes (immune response, chemokine activity) and two signaling pathways (cytokine-cytokine receptor interaction, chemokine activity) were highlighted. We speculated that ARID1B deficiency might trigger abnormal immune responses, which may be involved in the pathophysiologic mechanisms of CSS. CONCLUSION: Our research provided further support for WGS application in CSS diagnosis and made an investigational approach for the underlying mechanisms of CSS.

USP8 inhibition regulates autophagy flux and controls Salmonella infection.

Introduction: Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica remains unknown. Methods: To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during Salmonella infection. Results: Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon Salmonella infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during Salmonella infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. Discussion: The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during Salmonella infection.

Use of Ambroxol as Therapy for Gaucher Disease.

Importance: Ambroxol was identified as an enhancer of stability and residual activity of several misfolded glucocerebrosidase variants in 2009. Objectives: To assess hematologic and visceral outcomes, biomarker changes, and safety of ambroxol therapy for patients with Gaucher disease (GD) without disease-specific treatment. Design, Setting, and Participants: Patients with GD who could not afford enzyme replacement therapy were enrolled and received oral ambroxol from May 6, 2015, to November 9, 2022, at Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China. Thirty-two patients with GD (29 with GD type 1, 2 with GD type 3, and 1 with GD intermediate types 2-3) were enrolled. Of those, 28 patients were followed up for longer than 6 months; 4 were excluded due to loss of follow-up. Data analyses were performed from May 2015 to November 2022. Intervention: An escalating dose of oral ambroxol (mean [SD] dose, 12.7 [3.9] mg/kg/d). Main Outcomes and Measures: Patients with GD receiving ambroxol were followed up in a genetic metabolism center. Biomarkers of chitotriosidase activity and glucosylsphingosine level, liver and spleen volumes, and hematologic parameters were measured at baseline and various time points throughout the ambroxol treatment. Results: A total of 28 patients (mean [SD] age, 16.9 [15.3] years; 15 male patients [53.6%]) received ambroxol for a mean (SD) duration of 2.6 (1.7) years. Two patients with severe symptoms at baseline experienced deterioration of hematologic parameters and biomarkers and were deemed nonresponders; clinical response was observed in the other 26 patients. After 2.6 years of ambroxol treatment, the mean (SD) hemoglobin concentration improved from 10.4 (1.7) to 11.9 (1.7) g/dL (mean [SD], 1.6 [1.7] g/dL; 95% CI, 0.8-2.3 g/dL; P < .001), and the mean (SD) platelet count improved from 69 (25) to 78 (30) × 103/µL (mean [SD], 9 [22] × 103/µL; 95% CI, -2 to 19 × 103/µL; P = .09). The mean (SD) spleen volume decreased from 17.47 (7.18) to 12.31 (4.71) multiples of normal (MN) (mean [SD], -5.16 [5.44] MN; 95% CI, -10.19 to -0.13; P = .04), and the mean (SD) liver volume decreased from 1.90 (0.44) to 1.50 (0.53) MN (mean [SD], -0.39 [0.42] MN; 95% CI, -0.75 to -0.04; P = .03). Biomarker median percentage changes from baseline were -43.1% for chitotriosidase activity (from 14 598 [range, 3849-29 628] to 8312 [range, 1831-16 842] nmol/mL/h; z = -3.413; P = .001) and -34.1% for glucosylsphingosine level (from 251.3 [range, 73.6-944.2] to 165.7 [range, 21.3-764.8] ng/mL; z = -2.756; P = .006). Patients were divided into subgroups according to age when initiating treatment; those who received treatment at a younger age (mean [SD] age, 6.3 [2.7] years) experienced more rapid improvements: hemoglobin concentration increased by 16.5% (from 10.3 [1.5] to 12.0 [1.5] g/dL; mean [SD] change, 1.6 [1.6] g/dL; 95% CI, 0.7-2.5 g/dL; P = .002), and platelet count increased by 12.0% (from 75 [24] to 84 [33] × 103/µL; mean [SD] change, 9 [26] × 103/µL; 95% CI, -5 to 24 × 103/µL; P = .17); whereas chitotriosidase activity decreased by 64.0% (from 15 710 [range, 4092-28 422] to 5658 [range, 1146-16 843] nmol/mL/h; z = -2.803; P = .005), and glucosylsphingosine level decreased by 47.3% (from 248.5 [range, 122.8-674.9] to 131.0 [range, 41.1-448.5] ng/mL; z = -2.385; P = .02). Three of the 28 patients experienced mild and transient adverse events. Conclusions and Relevance: In this case series of ambroxol repurposing among patients with GD, long-term treatment with ambroxol was safe and associated with patient improvement. Improvements in hematologic parameters, visceral volumes, and plasma biomarkers were larger among patients with relatively mild symptoms of GD and patients who received initial treatment at younger ages.

Fluorescent Labeling of Small Extracellular Vesicles (EVs) Isolated from Conditioned Media.

Extracellular vesicles (EVs), such as exosomes, are produced by all known eukaryotic cells, and constitute essential means of intercellular communication. Recent studies have unraveled the important roles of EVs in migrating to specific sites and cells. Functional studies of EVs using in vivo and in vitro systems require tracking these organelles using fluorescent dyes or, alternatively, transfected and fluorescent-tagged proteins, located either intravesicularly or anchored to the EV bilayer membrane. Due to design simplicity, the fluorescent dye might be a preferred method if the cells are difficult to modify by transfection or when the genetic alteration of the mother cells is not desired. This protocol describes techniques to label cultured cell-derived EVs, using lipophilic DiR [DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)] fluorophore. This technique can be used to study the cellular uptake and intracellular localization of EVs, and their biodistribution in vivo , which are crucial evaluations of any isolated EVs.

Potential Disease-Modifying Effects of Lithium Carbonate in Niemann-Pick Disease, Type C1.

Background: Niemann-Pick disease type C1 (NP-C1) is a rare, autosomal-recessive neurodegenerative disorder with no United States Food and Drug Administration (FDA)-approved drug. Lithium has been shown to have considerable neuroprotective effects for neurological disorders such as bipolar disorder, Alzheimer's disease and stroke and has been tested in many clinical trials. However, the pharmacological effect of lithium on NP-C1 neurodegenerative processes has not been investigated. The aim of this study was to provide an initial evaluation of the safety and feasibility of lithium carbonate in patients with NP-C1. Methods: A total of 13 patients diagnosed with NP-C1 who met the inclusion criteria received lithium orally at doses of 300, 600, 900, or 1,200 mg daily. The dose was reduced based on tolerance or safety observations. Plasma 7-ketocholesterol (7-KC), an emerging biomarker of NP-C1, was the primary endpoint. Secondary endpoints included NPC Neurological Severity Scores (NNSS) and safety. Results: Of the 13 patients with NP-C1 (12-33 years) enrolled, three withdrew (discontinuation of follow-up outpatient visits). The last observed post-treatment values of 7-KC concentrations (128 ng/ml, SEM 20) were significantly lower than pretreatment baselines values (185 ng/ml, SEM 29; p = 0.001). The mean NNSS was improved after lithium treatment at 12 months (p = 0.005). Improvement in swallowing capacity was observed in treated patients (p = 0.014). No serious adverse events were recorded in the patients receiving lithium. Conclusion: Lithium is a potential therapeutic option for NP-C1 patients. Larger randomized and double-blind clinical trials are needed to further support this finding. Clinical Trial Registration: ClinicalTrials.gov, NCT03201627.

CNS-Targeting Therapies for Lysosomal Storage Diseases: Current Advances and Challenges.

During the past decades, several therapeutic approaches have been developed and made rapidly available for many patients afflicted with lysosomal storage disorders (LSDs), inborn organelle disorders with broad clinical manifestations secondary to the progressive accumulation of undegraded macromolecules within lysosomes. These conditions are individually rare, but, collectively, their incidence ranges from 1 in 2,315 to 7,700 live-births. Most LSDs are manifested by neurological symptoms or signs, including developmental delay, seizures, acroparesthesia, motor weakness, and extrapyramidal signs. The chronic and later-onset clinical forms are at one end of the continuum spectrum and are characterized by a subtle and slow progression of neurological symptoms. Due to its inherent physiological properties, unfortunately, the blood-brain barrier (BBB) constitutes a significant obstacle for current and upcoming therapies to achieve the central nervous system (CNS) and treat neurological problems so prevalent in these conditions. To circumvent this limitation, several strategies have been developed to make the therapeutic agent achieve the CNS. This narrative will provide an overview of current therapeutic strategies under development to permeate the BBB, and address and unmet need for treatment of the progressive neurological manifestations, which are so prevalent in these inherited lysosomal disorders.