Rapid Identification of New Biomarkers for the Classification of GM1 Type 2 Gangliosidosis Using an Unbiased 1H NMR-Linked Metabolomics Strategy.

Biomarkers currently available for the diagnosis, prognosis, and therapeutic monitoring of GM1 gangliosidosis type 2 (GM1T2) disease are mainly limited to those discovered in targeted proteomic-based studies. In order to identify and establish new, predominantly low-molecular-mass biomarkers for this disorder, we employed an untargeted, multi-analyte approach involving high-resolution 1H NMR analysis coupled to a range of multivariate analysis and computational intelligence technique (CIT) strategies to explore biomolecular distinctions between blood plasma samples collected from GM1T2 and healthy control (HC) participants (n = 10 and 28, respectively). The relationship of these differences to metabolic mechanisms underlying the pathogenesis of GM1T2 disorder was also investigated. 1H NMR-linked metabolomics analyses revealed significant GM1T2-mediated dysregulations in ≥13 blood plasma metabolites (corrected p < 0.04), and these included significant upregulations in 7 amino acids, and downregulations in lipoprotein-associated triacylglycerols and alanine. Indeed, results acquired demonstrated a profound distinctiveness between the GM1T2 and HC profiles. Additionally, employment of a genome-scale network model of human metabolism provided evidence that perturbations to propanoate, ethanol, amino-sugar, aspartate, seleno-amino acid, glutathione and alanine metabolism, fatty acid biosynthesis, and most especially branched-chain amino acid degradation (p = 10-12-10-5) were the most important topologically-highlighted dysregulated pathways contributing towards GM1T2 disease pathology. Quantitative metabolite set enrichment analysis revealed that pathological locations associated with these dysfunctions were in the order fibroblasts > Golgi apparatus > mitochondria > spleen ≈ skeletal muscle ≈ muscle in general. In conclusion, results acquired demonstrated marked metabolic imbalances and alterations to energy demand, which are consistent with GM1T2 disease pathogenesis mechanisms.

Pre-diagnosing and managing patients with GM1 gangliosidosis and related disorders by the evaluation of GM1 ganglioside content.

GM1 ganglioside, a monosialic glycosphingolipid and a crucial component of plasma membranes, accumulates in lysosomal storage disorders, primarily in GM1 gangliosidosis. The development of biomarkers for simplifying diagnosis, monitoring disease progression and evaluating drug therapies is an important objective in research into neurodegenerative lysosomal disorders. With this in mind, we established fluorescent imaging and flow-cytometric methods to track changes in GM1 ganglioside levels in patients with GM1 gangliosidosis and in control cells. We also evaluated GM1 ganglioside content in patients' cells treated with the commercially available Miglustat, a substrate inhibitor potentially suitable for the treatment of late-onset GM1 gangliosidosis. The flow-cytometric method proved to be sensitive, unbiased, and rapid in determining variations in GM1 ganglioside content in human lymphocytes derived from small amounts of fresh blood. We detected a strong correlation between GM1 ganglioside content and the clinical severity of GM1 gangliosidosis. We confirm the ability of Miglustat to act as a substrate reduction agent in the patients' treated cells. As well as being suitable for diagnosing and managing patients with GM1 gangliosidosis this method could be useful in the diagnosis and management of other lysosomal diseases, such as galactosialidosis, Type C Niemann-Pick, and any other disease with pathologic variations of GM1 ganglioside.

Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases.

To date, several genetic variants that lead to a deficiency of chitotriosidase activity have been described. The duplication of 24 bp (dup24bp) in exon 10 of the CHIT1 gene, which causes a complete loss of enzymatic activity of the gene product, is the most common among the European population. The aim of the study was to evaluate the possibility of using chitotriosidase activity as an additional biomarker in diagnosis of lysosomal storage diseases (LSDs) in Ukraine, to determine this parameter in blood plasma of the patients with various lysosomal diseases and to assess the effect of the presence of dup24bp in the CHIT1 gene on this parameter. It has been shown that chitotriosidase activity in blood plasma is a convenient additional biochemical marker in the diagnosis of some LSDs, namely Gaucher disease, Niemann-Pick disease A, B, C and GM1-gangliosidosis. Reference ranges of the normal chitotriosidase activity were determined in blood plasma of Ukrainian population and found to be 8.0-53.1 nmol 4-methylumbelliferone/h·ml of plasma. The total allele frequency of the dup24bp in the CHIT1 gene in Ukrainian population was determined, which amounted to 0.26 (323/1244) that is higher than in European population. It was indicated that moleculargenetic screening of dup24bp in the CHIT1 gene is a necessary stage in a protocol for the laboratory diagnosis of Gaucher disease, Niemann-Pick disease A, B, C as well as GM1-gangliosidosis to avoid incorrect diagnosis.

Role of beta-galactosidase and elastin binding protein in lysosomal and nonlysosomal complexes of patients with GM1-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of beta-galactosidase (GLB1). The GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), involved in elastic fiber deposition. GLB1 forms a complex with protective protein cathepsin A (PPCA), alpha neuraminidase (NEU1), and galactosamine 6-sulphate sulfatase (GALNS) inside lysosomes, while EBP binds to PPCA and NEU1 on the cell surface. We investigated the function of the GLB1 and EBP mutated proteins by analyzing the clinical, genetic, and cellular data of 11 G(M1)-gangliosidosis patients. Their molecular analysis, followed by expression studies, lead to the identification of four new and 10 known GLB1 mutations. Some common amino acid substitutions [c.1445G>A (p.Arg482H), c.622C>T (p.Arg208His), c.175C>T (p.Arg59Cys) and c.176G>A (p.Arg59His)] were present in the GLB1 enzyme of several patients, all of Mediterranean origin, suggesting a common origin. Western blotting analyses against GLB1, EBP, and PPCA proteins showed that the identified mutations affect GLB1 enzyme activity and/or stability. The c.1445G>A (p.Arg482His), c.175C>T (p.Arg59Cys), c.733+2T>C, c.1736G>A (p.Gly579Asp), and c.1051C>T (p.Arg351X) GLB1 mutations, affect the stabilization of PPCA probably because they hamper the interaction between GLB1/EBP and PPCA within the multiprotein complex. The amount of EBP was normal, but the detection of impaired elastogenesis in such patients suggests an alteration in its function. We conclude that the presence of genetic lesions in both GLB1 and EBP coding region does not directly predict impaired elastogenesis and that elastic fiber assembly has to be evaluated specifically in each case. Nevertheless, the degree of EBP involvement may be linked to specific clinical findings.

GM1 gangliosidosis in adults: clinical and molecular analysis of 16 Japanese patients.

Clinical findings were compared with the results of molecular analysis in 16 Japanese patients from 10 unrelated families with the adult/chronic form of GM1 gangliosidosis. Age of onset ranged from 3 to 30 years. Major clinical manifestations were gait and speech disturbances caused by persistent muscle hypertonia. Dystonic postures and movements, facial grimacing, and parkinsonian manifestations were commonly seen. Cerebellar signs, myoclonus, severe intellectual impairment, dysmorphism, or visceromegaly were not observed. A common single-base substitution, 51Ile(ATC)----Thr(ACC), reported in a previous study of ours, was confirmed in 14 patients by the Bsu36I restriction site analysis; one was a compound heterozygote with another mutation (457Arg[CGA]----Gln[CAA]) and the others were homozygotes of this mutation. Clinically, the compound-heterozygous patient showed more severe neurological manifestations and a more rapid clinical course than those of homozygotes. The homozygotes showed considerable variations in the age of onset and subsequent clinical course. The 51Ile----Thr mutant allele expressed a significant amount of beta-galactosidase activity, whereas the 457Arg----Gln mutant allele expressed extremely low activity in human GM1 gangliosidosis fibroblasts. We conclude that these gene mutations causing different residual enzyme activities are related to the severity of clinical manifestations, but some other genetic or environmental factors contribute to clinical heterogeneity. The Bsu36I restriction site analysis was performed in 7 families and provided clear results for the diagnosis of heterozygotes as well as homozygotes of this specific clinical form of GM1 gangliosidosis. The technique is applicable to prenatal diagnosis and genetic counseling.

Clinical and molecular heterogeneity in hereditary beta-galactosidase deficiency.

Results of a molecular analysis of GM1-gangliosidosis and galactosialidosis in our laboratory are briefly reviewed. A common single base substitution was found in adult/chronic form of GM1-gangliosidosis among heterogeneous beta-galactosidase gene mutations, and restriction site analysis was successfully performed for diagnosis of homozygotes and heterozygotes. All adult galactosialidosis patients had a common mutation at a splice junction which caused skipping of an exon of the protective protein/carboxypeptidase gene. An artificial restriction site was introduced in this case and applied to diagnosis of this disease. The heterogeneous gene mutations were compared and correlated with phenotypic manifestations in these two diseases.