Search Results (8)

MPS I (Mucopolysaccharidosis type I) is a rare lysosomal storage disease originating from the deficiency of the enzyme alpha-L-iduronidase, encoded by the IDUA gene, which impairs the degradation of glycosaminoglycans (GAGs) and diminishes biological functioning across several organs. Background: Out of the eleven MPS disorders, MPS I includes three syndromes, of which the first, named Hurler syndrome, affects the most. Methods: Several in silico tools were used, such as ConSurf (73 variants), Mutation Assessor (69 variants), PredictSNP, MAPP, PhDSNP, Polyphen-1, Polyphen-2, SIFT, SNAP, PANTHER, MetaSNP (24 variants); Missense 3D-DB (11 variants) and AlignGVGD (eight variants) for physicochemical properties; and I-Mutant, Mupro, CUPSAT, and INPS for stability predictions (four variants). Results: A molecular docking study was performed for the two variants: L238Q and P385R scored −7.22 and −7.05 kcal/mol, respectively, and the native scored −7.14 kcal/mol with IDR as the ligand. Molecular dynamics anticipated how these molecules fluctuate over a period of 100 nanoseconds. Conclusions: Alpha-L-iduronidase enzyme has a critical role in the lysosomal degradation of glycosaminoglycans. According to the comparative analysis of the three structures by MDS, P385R had the least stability in all aspects of the plots. Our study demonstrates that the mutation significantly alters protein stability and binding efficiency with the ligands. Full article

GBA variants increase the risk of Parkinson’s disease (PD) by 10 times. The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase). The p.N370S substitution causes a violation of the enzyme conformation, which affects its stability in the cell. We studied the biochemical characteristics of dopaminergic (DA) neurons generated from induced pluripotent stem cells (iPSCs) from a PD patient with the GBA p.N370S mutation (GBA-PD), an asymptomatic GBA p.N370S carrier (GBA-carrier), and two healthy donors (control). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we measured the activity of six lysosomal enzymes (GCase, galactocerebrosidase (GALC), alpha-glucosidase (GAA), alpha-galactosidase (GLA), sphingomyelinase (ASM), and alpha-iduronidase (IDUA)) in iPSC-derived DA neurons from the GBA-PD and GBA-carrier. DA neurons from the GBA mutation carrier demonstrated decreased GCase activity compared to the control. The decrease was not associated with any changes in GBA expression levels in DA neurons. GCase activity was more markedly decreased in the DA neurons of GBA-PD patient compared to the GBA-carrier. The amount of GCase protein was decreased only in GBA-PD neurons. Additionally, alterations in the activity of the other lysosomal enzymes (GLA and IDUA) were found in GBA-PD neurons compared to GBA-carrier and control neurons. Further study of the molecular differences between the GBA-PD and the GBA-carrier is essential to investigate whether genetic factors or external conditions are the causes of the penetrance of the p.N370S GBA variant. Full article

Mucopolysaccharidoses (MPS) are rare genetic disorders belonging to the lysosomal storage diseases. They are caused by mutations in genes encoding lysosomal enzymes responsible for degrading glycosaminoglycans (GAGs). As a result, GAGs accumulate in lysosomes, leading to impairment of cells, organs and, consequently, the entire body. Many of the therapies proposed thus far require the participation of chaperone proteins, regardless of whether they are therapies in common use (enzyme replacement therapy) or remain in the experimental phase (gene therapy, STOP-codon-readthrough therapy). Chaperones, which include heat shock proteins, are responsible for the correct folding of other proteins to the most energetically favorable conformation. Without their appropriate levels and activities, the correct folding of the lysosomal enzyme, whether supplied from outside or synthesized in the cell, would be impossible. However, the baseline level of nonspecific chaperone proteins in MPS has never been studied. Therefore, the purpose of this work was to determine the basal levels of nonspecific chaperone proteins of the Hsp family in MPS cells and to study the effect of normalizing GAG concentrations on these levels. Results of experiments with fibroblasts taken from patients with MPS types I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX, as well as from the brains of MPS I mice (Idua^−/−), indicated significantly reduced levels of the two chaperones, Hsp70 and Hsp40. Interestingly, the reduction in GAG levels in the aforementioned cells did not lead to normalization of the levels of these chaperones but caused only a slight increase in the levels of Hsp40. An additional transcriptomic analysis of MPS cells indicated that the expression of other genes involved in protein folding processes and the cell response to endoplasmic reticulum stress, resulting from the appearance of abnormally folded proteins, was also modulated. To summarize, reduced levels of chaperones may be an additional cause of the low activity or inactivity of lysosomal enzymes in MPS. Moreover, this may point to causes of treatment failure where the correct structure of the enzyme supplied or synthesized in the cell is crucial to lower GAG levels. Full article

Mucopolysaccharidosis type I (MPS I) is an inherited autosomal recessive disease resulting from mutation of the α-l-Iduronidase (IDUA) gene. New unknown mutated nucleotides of idua have increasingly been discovered in newborn screening, and remain to be elucidated. In this study, we found that the z-Idua enzymatic activity of zebrafish idua-knockdown embryos was reduced, resulting in the accumulation of undegradable metabolite of heparin sulfate, as well as increased mortality and defective phenotypes similar to some symptoms of human MPS I. After microinjecting mutated z-idua-L346R, -T364M, -E398-deleted, and -E540-frameshifted mRNAs, corresponding to mutated human IDUA associated with MPS I, into zebrafish embryos, no increase in z-Idua enzymatic activity, except of z-idua-E540-frameshift-injected embryos, was noted compared with endogenous z-Idua of untreated embryos. Defective phenotypes were observed in the z-idua-L346R-injected embryos, suggesting that failed enzymatic activity of mutated z-Idua-L346R might have a dominant negative effect on endogenous z-Idua function. However, defective phenotypes were not observed in the z-idua-E540-frameshifted-mRNA-injected embryos, which provided partial enzymatic activity. Based on these results, we suggest that the z-Idua enzyme activity assay combined with phenotypic observation of mutated-idua-injected zebrafish embryos could serve as an alternative platform for a preliminary assessment of mutated idua not yet characterized for their role in MPS I. Full article

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder caused by deleterious mutations in the α-L-iduronidase (IDUA) gene. Until now, MPS I in Vietnamese has been poorly addressed. Five MPS I patients were studied with direct DNA sequencing using Illumina technology confirming pathogenic variants in the IDUA gene. Clinical characteristics, additional laboratory results, and family history were collected. All patients have presented with the classical characteristic of MPS I, and α-L-iduronidase activity was low with the accumulation of glycosaminoglycans. Three variants in the IDUA gene (c.1190-10C>A (Intronic), c.1046A>G (p.Asp349Gly), c.1862G>C (p.Arg621Pro) were identified. The c.1190-10C>A variant represents six of the ten disease alleles, indicating a founder effect for MPS I in the Vietnamese population. Using biochemical and genetic analyses, the precise incidence of MPS I in this population should accelerate early diagnosis, newborn screening, prognosis, and optimal treatment. Full article

Lysosomal storage diseases (LSDs) are a heterogeneous group of approximately 70 monogenic metabolic disorders whose diagnosis represents an arduous challenge for clinicians due to their variability in phenotype penetrance, clinical manifestations, and high allelic heterogeneity. In recent years, the approval of disease-specific therapies and the rapid emergence of novel rapid diagnostic methods has opened, for a set of selected LSDs, the possibility for inclusion in extensive national newborn screening (NBS) programs. Herein, we evaluated the clinical utility and diagnostic validity of a targeted next-generation sequencing (tNGS) panel (called NBS_LSDs), designed ad hoc to scan the coding regions of six genes (GBA, GAA, SMPD1, IDUA1, GLA, GALC) relevant for a group of LSDs candidate for inclusion in national NBS programs (MPSI, Pompe, Fabry, Krabbe, Niemann Pick A-B and Gaucher diseases). A standard group of 15 samples with previously known genetic mutations was used to test and validate the entire flowchart. Analytical accuracy, sensitivity, and specificity, as well as turnaround time and costs, were assessed. Results showed that the Ion AmpliSeq and Ion Chef System-based high-throughput NBS_LSDs tNGS panel is a fast, accurate, and cost-effective process. The introduction of this technology into routine NBS procedures as a second-tier test along with primary biochemical assays will allow facilitating the identification and management of selected LSDs and reducing diagnostic delay. Full article

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR < 0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients. Full article

The lysosomal storage disorder, mucopolysaccharidosis I (MPSI), results from mutations in IDUA, the gene that encodes the glycosaminoglycan-degrading enzyme α-L-iduronidase. Newborn screening efforts for MPSI have greatly increased the number of novel IDUA variants identified, but with insufficient experimental evidence regarding their pathogenicity, many of these variants remain classified as variants of uncertain significance (VUS). Defining pathogenicity for novel IDUA variants is critical for decisions regarding medical management and early intervention. Here, we describe a biochemical platform for the characterization of IDUA variants that relies on viral delivery of IDUA DNA into IDUA-deficient HAP1 cells and isolation of single cell expression clones. The relative specific activity of wild-type and variant α-iduronidase was determined using a combination of Western blot analysis and α-iduronidase activity assays. The specific activity of each variant enzyme was consistent across different single cell clones despite variable IDUA expression and could be accurately determined down to 0.05–0.01% of WT α-iduronidase activity. With this strategy we compared the specific activities of known pseudodeficiency variants (p.His82Gln, p.Ala79Thr, p.Val322Glu, p.Asp223Asn) or pathogenic variants (p.Ser633Leu, p.His240Arg) with variants of uncertain significance (p.Ser586Phe, p.Ile272Leu). The p.Ser633Leu and p.His240Arg variants both show very low activities consistent with their association with Scheie syndrome. In our experiments, however, p.His240Arg exhibited a specific activity five times higher than p.Ser633Leu in contrast to other reports showing equivalent activity. Cell clones expressing the p.Ser586Phe and p.Ile272Leu variants had specific activities in the range of other pseudodeficiency variants tested. Our findings show that pseudodeficiency and pathogenic variants can be distinguished from each other with regard to specific activity, and confirms that all the pseudodeficiency variants variably reduce α-iduronidase activity. We envision this platform will be a valuable resource for the rigorous assessment of the novel IDUA variants emerging from the expansion of newborn screening efforts. Full article