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Roberto Giugliani - One of the best experts on this subject based on the ideXlab platform.
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the natural history of juvenile or subacute gm2 gangliosidosis 21 new cases and literature review of 134 previously reported
Pediatrics, 2006Co-Authors: Gustavo Maegawa, Don J Mahuran, Michael B. Tropak, Tracy L Stockley, Susan Blaser, Brenda Banwell, Roberto Giugliani, Joe T. R. ClarkeAbstract:Juvenile gm2 gangliosidosis (jGM2), also called subacute GM2 gangliosidosis (Online Mendelian Inheritance in Man [OMIM] No. 230700), is a rare and heterogeneous autosomal recessive disorder characterized by progressive neurologic deterioration that mainly affects motor and spinocerebellar function. It is a lysosomal storage disease caused by deficiency of β-Hexosaminidase A, combined deficiency of β-Hexosaminidases A and B, or deficiency of the noncatalytic GM2 activator. The enzyme β-Hexosaminidase is comprised of 2 major isoenzymes, β-Hexosaminidase A and β-Hexosaminidase B. β-Hexosaminidase A is made up of 2 nonidentical subunits, α and β, that are encoded by the genes HEXA (15q23-q24) and HEXB (5q13), respectively, which are associated into the heterodimeric isoenzyme. It catalyzes the removal of the β-N-acetylgalactosamine residue from the nonreducing terminal of the oligosaccharide of GM2 ganglioside. β-Hexosaminidase B comprises 2 identical β subunits (β2). It does not catalyze the degradation of GM2 ganglioside. Mutations of the HEXA gene encoding the α subunit cause deficiency of the β-Hexosaminidase A and result in the well-known form of GM2 gangliosidosis called Tay-Sachs disease (OMIM No. 272800). Mutations of the HEXB gene, encoding the β-subunit, cause deficiency of both enzymes (β-Hexosaminidase A and β-Hexosaminidase B), leading to Sandhoff disease (OMIM No. 268800), which is, in clinical aspects, virtually indistinguishable from Tay-Sachs disease. Deficiency of the GM2 activator protein, which mediates the interaction between the water-soluble β-Hexosaminidase A and its membrane-embedded substrate, GM2 ganglioside, causes the AB variant of GM2 gangliosidosis (OMIM No. 272750).1,2 In the general population, the Tay-Sachs variant (TSV) of GM2 gangliosidosis is rare, with a prevalence of 1 in 201 000 live births and incidence of 1 in 222 000 live births.3 The Sandhoff variant (SV) prevalence and incidence rates have been reported as 1 in 384 000 and 1 in 422 000 live births, respectively.3 The TSV carrier frequency is much higher in the Ashkenazi Jewish (1 in 30) and eastern Quebec French Canadian (1 in 14) populations compared with that in the general population (1 in 300).4 Community-based TSV carrier screening programs in these at-risk populations have had a dramatic effect on birth prevalence, which is now lower than that in the general population.5,6 The classical infantile form of GM2 gangliosidosis is characterized by the onset of symptoms before the age of 6 months and progresses rapidly to death by 3 to 5 years of age.7 Juvenile or subacute and the adult, also called late-onset or chronic, subtypes of this condition present later in childhood or in adulthood and progress more slowly.1,8–22 The juvenile and adult forms of GM2 gangliosidosis differ from each other primarily by the impact of the disease on intelligence, which is minimal through much of the course of the adult or chronic variant. Several case reports and a small number of case series have been reported.11,13,20,21 However, few studies have provided accurate descriptions of the natural clinical course of jGM2 in larger groups of patients.21,23 We report here a series of 21 cases of juvenile or subacute GM2 gangliosidosis followed at 2 medical centers. We also review a collection of 134 previously reported cases of jGM2.* The focus of our analysis is on initial symptomatology, age of onset, and severity of symptoms during the course of the disease. We also report on the spectrum of HEXA and HEXB mutations identified in patients with the TSV and SV, respectively, and make some generalizations concerning genotype-phenotype correlations.
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twelve different enzyme assays on dried blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases
Clinica Chimica Acta, 2006Co-Authors: Gabriel Civallero, Kristiane Michelin, Marli Viapiana, Janice C Coelho, Jurema Fatima De Mari, Roberto GiuglianiAbstract:Abstract Background Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. Methods Fluorometric methods were used to evaluate the activities of arylsulfatase B, α- N -acetylglucosaminidase, chitotriosidase, α and β-galactosidases, β-glucosidase, β-glucuronidase, total Hexosaminidases, Hexosaminidase A, α-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 °C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. Results Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. Conclusions Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.
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Twelve different enzyme assays on dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases.
Clinica chimica acta; international journal of clinical chemistry, 2006Co-Authors: Gabriel Civallero, Kristiane Michelin, Jurema De Mari, Marli Viapiana, Janice C Coelho, Roberto GiuglianiAbstract:Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. Fluorometric methods were used to evaluate the activities of arylsulfatase B, alpha-N-acetylglucosaminidase, chitotriosidase, alpha and beta-galactosidases, beta-glucosidase, beta-glucuronidase, total Hexosaminidases, Hexosaminidase A, alpha-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 degrees C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.
Aldo Orlacchio - One of the best experts on this subject based on the ideXlab platform.
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Widespread distribution of β-Hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol
Gene Therapy, 2003Co-Authors: C Bourgoin, Aldo Orlacchio, L. Poenaru, R A Gravel, C Emiliani, E J Kremer, A Gelot, B Tancini, C Drugan, C CaillaudAbstract:Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the β-subunit of Hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G_M2 ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the β-subunit of β-Hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.
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Widespread distribution of beta-Hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol
Gene Therapy, 2003Co-Authors: C Bourgoin, Aldo Orlacchio, L. Poenaru, R A Gravel, C Emiliani, E J Kremer, A Gelot, B Tancini, C Drugan, C CaillaudAbstract:Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the beta-subunit of Hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G(M2) ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the beta-subunit of beta-Hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.
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β-Hexosaminidase, α-d-mannosidase, and β-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I
Clinica Chimica Acta, 2000Co-Authors: Tommaso Beccari, Aldo Orlacchio, F Mancuso, Egidia Costanzi, C. Tassi, Rita Barone, Agata Fiumara, Maria Cristina AisaAbstract:Abstract The activity of β-Hexosaminidase, determined with 4-methylumbelliferyl-β- N -acetylglucopyranoside substrate, and of β- d -mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of β-Hexosaminidase, determined using 4-methylumbelliferyl-β- N -acetylglucopyranoside-6-sulphate as substrate, and the activity of α- d -mannosidase. Using DEAE-cellulose chromatography, a greater amount of Hexosaminidase B than Hexosaminidase A was detected in CDG serum. In CDG serum, Hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of α- d -mannosidase and β- d -mannosidase was identified in control and CDG sera. α- d -Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas β- d -mannosidase isoenzyme was eluted in the same position.
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Calcium ionophore A-23187 inhibits the secretion of β-Hexosaminidase from the GG2EE mouse macrophage cell line
Biochemistry international, 1992Co-Authors: Tommaso Beccari, Aldo Orlacchio, Alessandro Datti, Senia Farinelli, Elisabetta BlasiAbstract:Secretion of the lysosomal enzyme beta-N-acetylHexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of Hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two Hexosaminidase isoenzymes corresponding to Hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to Hexosaminidase S.
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β-Hexosaminidase expression in chick embryo fibroblasts in vitro
The International journal of biochemistry, 1991Co-Authors: T. Beccari, Furio Pezzetti, Maria Bodo, Ennio Becchetti, G. Bellachioma, Aldo OrlacchioAbstract:Abstract 1. 1. Two forms of β-Hexosaminidase, similar to Hexosaminidase A and Hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro . 2. 2. β-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro . 3. 3. Concanavalin-A treatment determines the increase of the neutral form, Hexosaminidase C, during development. 4. 4. Concanavalin-A reduces the specific activity of β-Hexosaminidase during development.
Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
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Hexosaminidase assays
Glycoconjugate Journal, 2009Co-Authors: Michaela Wendeler, Konrad SandhoffAbstract:β-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal β-glycosidically bound N -acetylglucosamine and N -acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited β-Hexosaminidase deficiency. More recently, aberrant Hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered Hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient Hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.
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the x ray crystal structure of human β Hexosaminidase b provides new insights into sandhoff disease
Journal of Molecular Biology, 2003Co-Authors: Timm Maier, Konrad Sandhoff, Norbert Strater, Christina G Schuette, Ralf Klingenstein, Wolfram SaengerAbstract:Abstract Human lysosomal β-Hexosaminidases are dimeric enzymes composed of α and β-chains, encoded by the genes HEXA and HEXB. They occur in three isoforms, the homodimeric Hexosaminidases B (ββ) and S (αα), and the heterodimeric Hexosaminidase A (αβ), where dimerization is required for catalytic activity. Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay–Sachs disease and Sandhoff disease, respectively. Here, we present the crystal structure of a complex of human β-Hexosaminidase B with a transition state analogue inhibitor at 2.3 A resolution (pdb 1o7a). On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by β-Hexosaminidase B is proposed. In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation. The structure reported here is a valid template also for the dimeric structures of β-Hexosaminidase A and S.
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evidence for the involvement of glu 355 in the catalytic action of human beta Hexosaminidase b
Journal of Biological Chemistry, 1997Co-Authors: Mark Pennybacker, Konrad Sandhoff, Christina G Schuette, Bernd Liessem, Stefan T Hepbildikler, Jennifer A Kopetka, Matthew R Ellis, Rachel Myerowitz, Richard L. ProiaAbstract:Abstract In a previous study the photoactivable affinity probe, 3-azi-1-[([6-3H]2-acetamido-2-deoxy-1-β-D-galactopyranosyl)thio]-butane, was used to identify the active site of β-Hexosaminidase B, a β-subunit dimer (Liessem, B., Glombitza, G. J., Knoll, F., Lehmann, J., Kellermann, J., Lottspeich, F., and Sandhoff, K. (1995) J. Biol. Chem. 270, 23693-23699). The probe predominately labeled Glu-355, a highly conserved residue among Hexosaminidases. To determine if Glu-355 has a role in catalysis, β-subunit mutants were prepared with the Glu-355 codon altered to either Ala, Gln, Asp, or Trp. After expression of mutant proteins using recombinant baculovirus, the enzyme activity associated with the β-subunits was found to be reduced to background levels. Although catalytic activity was lost, the mutations did not otherwise affect the folding or assembly of the subunits. The mutant β-subunits could be isolated using substrate affinity chromatography, indicating they contained intact substrate binding sites. As shown by cross-linking with disuccinimidyl suberate, the mutant β-subunits were properly assembled. They could also participate in the formation of functional β-Hexosaminidase A activity as indicated by activator-dependent GM2 ganglioside degradation activity produced by co-expression of the mutant β-subunits with the α-subunit. Finally, the mutant subunits showed normal lysosomal processing in COS-1 cells, demonstrating that a transport-competent protein conformation had been attained. Collectively the results provide strong support for the intimate involvement of Glu-355 in β-Hexosaminidase B-mediated catalysis.
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mice lacking both subunits of lysosomal β Hexosaminidase display gangliosidosis and mucopolysaccharidosis
Nature Genetics, 1996Co-Authors: Kazunori Sango, Cynthia J. Tifft, Michael P. Mcdonald, Jacqueline N. Crawley, Alexander Hoffmann, Michelle L. Mack, Elisa Skop, Christopher M. Starr, Konrad SandhoffAbstract:The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (α-subunit) and HEXB (β-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, (β-Hexosaminidase A (αβ), as well as for the homodimers β-Hexosaminidase B (ββ) and S (αα). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs1 (Hexa−/−) and Sandhoff2 (Hexb-/-) disease model mice. Lacking both the α and β-subunits these ‘double knockout’ mice displayed a total deficiency of all forms of lysosomal β-Hexosaminidase including the small amount of β-Hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients3,4,5. This result demonstrates that glycosaminoglycans are crucial substrates for β-Hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the β-Hexosaminidase enzyme system.
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identification of domains in human beta Hexosaminidase that determine substrate specificity
Journal of Biological Chemistry, 1996Co-Authors: Mark Pennybacker, Konrad Sandhoff, Cynthia J. Tifft, Bernd Liessem, Heidi Moczall, Richard L. ProiaAbstract:Abstract The lysosomal β-Hexosaminidases are dimers composed of α and β subunits. β-Hexosaminidase A (αβ) is a heterodimer, whereas Hexosaminidase B (ββ) and S (αα) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the β-subunit primarily degrades neutral substrates, whereas the α-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only β-Hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human β-Hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both α- and β-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous α-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the β-subunit. Chimeric constructs containing only one of these regions linked to the β-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing α amino acids 1-191 fused with β amino acids 225 to 556, when co-expressed with the wild-type α-subunit, showed activity comparable with that of recombinant β-Hexosaminidase A formed by the co-expression of the α- and β-subunits. This result indicates that the β-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of β-Hexosaminidase A.
Livia Poenaru - One of the best experts on this subject based on the ideXlab platform.
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adenoviral gene therapy of the tay sachs disease in Hexosaminidase a deficient knock out mice
Human Molecular Genetics, 1999Co-Authors: Jacquesemmanuel Guidotti, Livia Poenaru, Alexandre Mignon, G Haase, Catherine Caillaud, N Mcdonell, A KahnAbstract:The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-Hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, Hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-Hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.
Gabriel Civallero - One of the best experts on this subject based on the ideXlab platform.
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twelve different enzyme assays on dried blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases
Clinica Chimica Acta, 2006Co-Authors: Gabriel Civallero, Kristiane Michelin, Marli Viapiana, Janice C Coelho, Jurema Fatima De Mari, Roberto GiuglianiAbstract:Abstract Background Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. Methods Fluorometric methods were used to evaluate the activities of arylsulfatase B, α- N -acetylglucosaminidase, chitotriosidase, α and β-galactosidases, β-glucosidase, β-glucuronidase, total Hexosaminidases, Hexosaminidase A, α-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 °C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. Results Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. Conclusions Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.
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Twelve different enzyme assays on dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases.
Clinica chimica acta; international journal of clinical chemistry, 2006Co-Authors: Gabriel Civallero, Kristiane Michelin, Jurema De Mari, Marli Viapiana, Janice C Coelho, Roberto GiuglianiAbstract:Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. Fluorometric methods were used to evaluate the activities of arylsulfatase B, alpha-N-acetylglucosaminidase, chitotriosidase, alpha and beta-galactosidases, beta-glucosidase, beta-glucuronidase, total Hexosaminidases, Hexosaminidase A, alpha-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 degrees C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.
