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Maroteaux-Lamy syndrome (MPS-VI) is a rare autosomal-recessive disorder with a wide spectrum of clinical manifestations, ranging from an attenuated to a rapidly progressive disease. It is caused by variants in ARSB, which encodes the lysosomal arylsulfatase B (ARSB) enzyme, part of the degradation process of glycosaminoglycans in lysosomes. Over 220 variants have been reported so far, with a majority of missense variants. We hereby report two siblings of Bedouin origin with a diagnosis of MPS-VI. Western blots in patient fibroblasts revealed total absence of ARSB protein production. Complete sequencing of the coding region of ARSB did not identify a candidate disease-associated variant. However, deep sequencing of the noncoding region of ARSB by whole genome sequencing (WGS) revealed a c.1142+581A to G variant. The variant is located within intron 5 and fully segregated with the disease in the family. Determination of the genetic cause for these patients enabled targeted treatment by enzyme replacement therapy, along with appropriate genetic counseling and prenatal diagnosis for the family. These results highlight the advantage of WGS as a powerful tool, for improving the diagnostic rate of rare disease-causing variants, and emphasize the importance of studying deep intronic sequence variation as a cause of monogenic disorders.

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase, also called arylsulfatase B (ARSB, EC 3.1.6.12). Dogs with MPS VI show progressive predominantly oculoskeletal signs homologous to those in human and feline patients. We report herein two pathogenic ARSB gene variants in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI and a genotyping survey in these breeds. All exons and adjacent regions of the ARSB gene were sequenced from three affected Miniature Pinschers and three affected Miniature Schnauzers. Allelic discrimination assays were used for genotyping. A missense variant (NM_001048133.1:c.910G>A) was found in exon 5 of MPS VI-affected Miniature Pinschers that is predicted to result in a deleterious amino acid substitution of a highly conserved glycine to arginine (NP_001041598.1:p.Gly304Arg). In MPS VI-affected Miniature Schnauzers, a 56 bp deletion (NM_001048133.1:c.-24_32del) was found at the junction of exon 1 and its upstream region, predicting no enzyme synthesis. All clinically affected Miniature Pinschers and Miniature Schnauzers were homozygous for the respective variants, and screened healthy dogs in each breed were either heterozygous or homozygous for the wt allele. Whereas the Miniature Pinscher variant seemed to occur commonly (0.133 allele frequency), the Miniature Schnauzer variant was presumed to be rare. In conclusion, two breed-specific pathogenic ARSB gene variants were identified in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI, allowing for genotyping and informed breeding to prevent the production of affected offspring.

Mucopolysaccharidosis VI is a rare disease characterized by the arylsulfatase B enzyme deficiency, which is responsible for different clinical manifestations. The treatment consists of enzyme replacement therapy with intravenous administration of galsulfase. Evaluate the effectiveness of the enzyme replacement therapy with galsulfase for the mucopolysaccharidosis VI treatment. Systematic review of observational studies. The databases of PubMed, Cochrane Library, Lilacs, and Journal of Inherited Metabolic Disease were reviewed. The selection of studies, data mining, and methodological quality assessment were independently conducted by two authors. Eighteen studies fulfilled the inclusion criteria. Two studies were cohorts, one was longitudinal study, one was cross-sectional, one was a case-control, eight were case series, and five were case reports. A total of 362 participants with mucopolysaccharidosis type VI were evaluated, and 14 different outcomes related to the treatment effect were identified. Seven outcomes showed positive results, characterized by the patient survival, quality of life, respiratory function, joint mobility, physical resistance, reduction of urinary glycosaminoglycans, and growth. The hearing function and the cognitive development were stable after the treatment. Other outcomes related to the cardiac function, visual acuity, sleep apnea, and the size of the liver and spleen presented inconclusive outcomes. Concerning safety, light adverse reactions of hypersensitivity were reported. This review provided a broader panoramic view of the outcomes related to mucopolysaccharidosis type VI. Regardless of the inherent limitations of observational studies, the outcomes indicate that the enzyme replacement therapy has a positive effect on most of the outcomes associated to the disease.

BACKGROUND Mucopolysaccharidosis (MPS) VI is a rare, autosomal recessive congenital metabolic disorder caused by deficient activity of the lysosomal metabolic enzyme, N-acetylgalactosamine 4-sulfatase. Enzyme replacement therapy (ERT) is the current treatment for MPS VI, although it involves limited compliance to the therapy and high cost. The aim of this study was to develop a new method of treatment by conducting an orthotopic liver transplantation (LTx) using an animal model of human MPS VI, and to evaluate and examine its effectiveness for treating MPS VI. METHODS LTx was carried out from normal unaffected to affected MPS VI rats (MPR), which were then killed after LTx, and tissues from the heart, spleen, and knee joint, as well as serum, collected for biological and morphologic evaluation. RESULTS Liver-transplanted (LTx) MPR had the same level of N-acetylgalactosamine 4-sulfatase activity in the liver and lungs as normal unaffected MPR, and the urinary secretion of mucopolysaccharides/glycosaminoglycan (GAG) in LTx MPR was significantly decreased. Furthermore, on histopathology, the spleens of LTx MPR showed elimination of vacuole cells. In the knee joints, growth plates became thinner, and on radiography the facial and cranial bones of LTx MPR were morphologically normal. CONCLUSIONS LTx from normal to affected MPR was effective for symptoms of MPS and accumulation of GAG, suggesting that LTx could be a promising alternative approach for MPS VI.

In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-β1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-β1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-β1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the glial scar.

Background In people, lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). Objective The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Methods Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children's Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 μL blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Results Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of β-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), α-mannosidase, α-galactosidase, α-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; α-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II, activities of lysosomal enzymes were markedly increased. Conclusions Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made.

The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) removes 4-sulfate groups from chondroitin-4-sulfate and dermatan sulfate and is required for the degradation of these sulfated glycosaminoglycans (sGAGs). Since these GAGs accumulate in patients with Cystic Fibrosis (CF), we investigated the activity of ARSB in leukocytes of patients with CF, to consider if reduced activity of ARSB might contribute to the pathophysiology of CF. Previous cell-based experiments had demonstrated that when the deficiency of the cystic fibrosis transmembrane regulator (CFTR) was corrected in bronchial epithelial cells, the ARSB activity increased significantly. De-identified, citrated blood samples were collected from 16 children with CF and 31 control subjects, seen in the Pediatric Clinic at Rush University Medical Center. Polymorphonuclear leukocytes (PMN) and mononuclear cell (MC) populations were separated by density gradient, and blinded determinations of ARSB activity were performed using the exogenous substrate 4-methylumbilliferyl sulfate. Interleukin-6 was measured in the plasma samples by ELISA. ARSB activity was significantly less in the PMN and MC from the CF patients than controls (P

Various sulfatase activities were assayed in cultured skin fibroblasts from patients with multiple sulfatase deficiency (MSD). MSD cell lines displayed deficiencies of arylsulfatase A and iduronate sulfatase, but activities of arylsulfatase B, N-acetylgalactosamine 6-sulfate sulfatase and N-acetylglucosamine 6-sulfate sulfatase were within normal ranges, but not consistently. Arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD cell lines had similar Km, pH optima, inhibitory or activator sensitivity to that of normal skin fibroblasts. Arylsulfatase B in MSD cell lines also had properties similar to that of normal skin fibroblasts, except an abnormal heat stability. From our results, we conclude that properties of arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD fibroblasts were intact. On the other hand, arylsulfatase B in MSD might be a functionally abnormal enzyme.

This paper presents data collected by a Brazilian center in a multinational multicenter observational study of patients with mucopolysaccharidosis type VI (MPS VI), aiming at determining the epidemiological, clinical, and biochemical profile of these patients. Twenty-eight south-American patients with MPS VI were evaluated through medical interview, physical exam, echocardiogram, electrocardiogram, ophthalmologic evaluation, quantification of glycosaminoglycans (GAGs) in urine, and measurement of the activity of N-acetylgalactosamine-4-sulfatase (ARSB) in leukocytes. 92.9% of patients were Brazilian. Mean age at diagnosis and at evaluation was 48.4 months and 97.1 months, respectively. 88% of patients had onset of symptomatology before the age of 36 months. Consanguinity was reported by 27% of the families. Mean weight and height at birth were 3.481 kg and 51.3 cm, respectively. The most frequently reported clinical manifestations were short stature, corneal clouding, coarse facial features, joint contractures, and claw hands. All patients presented with echocardiogram changes as well as corneal clouding. Mean ARSB activity in leukocytes was 5.4 nmoles/h/mg protein (reference values: 72-174), and urinary excretion of GAGs was on average 7.9 times higher than normal. The number of clinical manifestations did not show a significant correlation with the levels of urinary GAGs nor with the ARSB activity. Also, no significant correlation was found between the levels of urinary GAGs and the ARSB activity. It was concluded that MPS VI has high morbidity and that, when compared with data published in the literature, patients in our study were diagnosed later and presented with a higher frequency of cardiological findings.

Initial experiments in human colonic epithelial cell lines and in the arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB)-deficient mouse showed that reduction in ARSB and the resultant increase in chondroitin-4-sulfate (C4S) were associated with reduction in chondroitin-4-sulfotransferase expression and ~50% reduction in sulfotransferase activity with overall increase in total sulfated glycosaminoglycans and C4S. Our experiments have also shown that decline in ARSB leads to increase in Wnt9A expression in human colonic epithelial cells and mouse intestine. Since modeling of the extracellular matrix by Wnt and BMP4 involves their interactions with sulfated glycosaminoglycans, including chondroitin sulfate, we evaluated the interactions among ARSB, Wnt9A, BMP4, and chondroitin-4-sulfotransferase (CHST11). Following silencing of Wnt9A by siRNA, the CHST11 expression increased to ~3 times baseline and the sulfotransferase activity increased to ~1.6 times the baseline. In contrast, when BMP4 was neutral...

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?

Secreted and intracellular arylsulfatase B (ASB) activities were measured in normal and osteoarthritic (OA) human chondrocyte cultures in the absence and presence of monensin, ammonium chloride, and chloroquine. Of the three agents added, only monensin produced a significant stimulation of secreted enzyme activity. Osteoarthritic cells consistently exhibited a three-fold higher level of secreted specific ASB activity than did normal cells, with or without monensin. When compared with normal cells, OA cells also consistently exhibited a twofold heightened intracellular specific enzyme activity both in the absence or presence of monensin. With increasing dosage of monensin, secreted and intracellular ASB activity increased for both OA and normal cells. Total enzyme activity of secreted and intracellular ASB was found to be cell density dependent. No inhibition of secreted or intracellular ASB activity was observed for sparsely plated cultures. In contrast to sparse cultures, an inhibition of secreted ASB, with or without monensin, was observed in densely plated cultures. Intracellular total activity was not inhibited by high-density cultures. Secreted ASB activity was found to be time-dependent after passage. Enzyme activity was maximal at 6 h in both OA and normal cells and decreased by the end of 24 h both in serum-free medium and in serum-free medium with monensin. When compared with normal cells, OA cells expressed higher levels of ASB activity under all test conditions. This heightened activity therefore appears to be a property inherent in the OA chondrocyte.

Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions.

Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue-Sepharose affinity chromatography, DEAE-cellulose chromatography, hydrophobic chromatography on phenyl-Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis. The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction. The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.