Selective screening for lysosomal storage disorders in a large cohort of minorities of African descent shows high prevalence rates and novel variants.

Population studies point to regional and ethnicity-specific differences in genetic predisposition for some lysosomal storage disorders (LSDs). The aim of the study was to determine the prevalence of the three treatable forms of lysosomal storage disorders (Gaucher disease [GD], Pompe disease [PD], and Fabry disease [FD]) in a cohort of mostly urban-dwelling individuals of African ancestry, a previously unknown genetic landscape for LSDs. Large-scale selective multistep biochemical and genetic screening was performed in patients seeking healthcare for various health concerns. Fluorimetric enzyme assays for GD, PD, and FD were performed on dried blood spots. Targeted gene sequencing was performed on samples that showed significantly lower enzyme activities (<10% of control mean) after two tiers of enzymatic screening. A total of 5287 unique samples representing a cross section of patients who visited Howard University Hospital and College of Medicine from 2015 to 2017 were included in the study. Study samples were obtained from a population where ~90% reported as African-American, ~5% Hispanic, and <5% Caucasian or other. Regarding GD, three subjects had either homozygous or heterozygous mutations in the GBA gene. As to PD, eight subjects were either homozygous or compound heterozygous for GAA mutations, including three novel mutations: (a) c.472 A > G; p.T158A, (b) c.503G > T; p.R168L, (c) c.1985del. Regarding FD, two subjects had pathogenic GLA mutations, and four had single nucleotide polymorphisms in the 5'UTR, previously implicated in modulating gene expression. The findings highlight a higher incidence of abnormal enzyme levels and pathogenic mutations in the target population reflecting ancestry-based specific genotype and phenotype variations.

Impaired autophagic and mitochondrial functions are partially restored by ERT in Gaucher and Fabry diseases.

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.

Gaucheromas: When macrophages promote tumor formation and dissemination.

Deficiency of the lysosomal enzyme, ß-glucocerebrosidase, and accumulation of its substrate in cells of the reticuloendothelial system affects multiple organ systems in patients with Gaucher disease (GD). Lipid laden macrophages turn into Gaucher cells (GC) which are the pathological characteristic of GD. GC focally accumulate in the liver, spleen and at extraosseous sites to form benign lesions called Gaucheromas. Gaucheromas pose diagnostic and therapeutic challenges. We studied the pathophysiology of extraosseous Gaucheroma formation in a cohort of patients with GD. Among 63 patients followed at a single center, 3 patients with genotypes L444P/L444P and N370S/N370S, were diagnosed with extraosseous Gaucheromas. Flow cytometry revealed a higher expression of CD16+/CCR4+ non-classical monocytes in blood of GD patients who have developed Gaucheromas. A biopsy showed infiltration of GC, which reactivity against CD163, CD68 and VEGF. The cell proliferative marker Ki67 and CCL2, a factor anti-tumor activity, were negative. Our study indicates that extraosseous Gaucheromas are comprised of cellular elements with characteristics of tumor-associated macrophages, the major players in cancer related inflammation. The occurrence of non-classical CD16+/CCR4+ monocytes reflect the underlying cause for the accumulation of the macrophages capable of migrating to distant sites outside the reticuloendotheial system, and giving rise to tumor-like Gaucheromas.

Individualized screening for chaperone activity in Gaucher disease using multiple patient derived primary cell lines.

The knowledge of individual response to a therapy, which can be assesed by in vitro screening, is essential for the development of therapeutics. Chaperone therapy is based on the ability of small molecules to fold the mutant protein to recover its function. As a novel approach for the treatment of Gaucher disease (GD), ambroxol was recently identified as a chaperone for GD, caused by the pathogenic variants in GBA gene, resulting in lysosomal enzyme glucocerebrosidase (GCase) deficiency. Since ambroxol activity is mutation-dependent, the assessment of the chaperone action requires adaptation of a cell model with genetic format identical to the patient. We compared the chaperone activity of ambroxol using different primary cells derived from GD patients with different GBA genotypes. Ambroxol enhanced GCase activity in cells with wild type GBA and in those, compound heterozygous for N370S, but was ineffective in cell lines with complex GBA alleles. In cells from patients with neuropathic GD and L444P/L444P genotype, the response to ambroxol was varied. We conclude that chaperone activity depends on diverse factors in addition to a particular GBA genotype. We showed that PBMCs and macrophages are the most relevant cell-based methods to screen the efficacy of ambroxol therapy. For pediatric patients, a non-invasive source of primary cells, urine derived kidney epithelial cells, have a vast potential for drug screening in GD. These findings demonstrate the importance of personalized screening to evaluate efficacy of chaperone therapy, especially in patients with neuronopathic GD.