Precocious puberty in Sanfilippo IIIA disease: diagnosis and follow-up of two new cases.

To date the association between mucopolysaccharidosis (MPS) IIIA and precocious puberty has been found in only three polish patients. We observed two children affected by MPS IIIA with central precocious puberty (CPP) both treated with GnRH agonists. The occurrence of CPP in both patients with MPS IIIA suggests that it is necessary to look for an association between the two conditions. The follow-up of our two patients leads us to believe also that GnRH agonist treatment can have a beneficial effect on final height and probably on the improvement of behavioural problems.

Effect of 'attenuated' mutations in mucopolysaccharidosis IVA on molecular phenotypes of N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2-36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55 degrees C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for 'attenuated' mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that 'attenuated' mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.

Limited transgene immune response and long-term expression of human alpha-L-iduronidase in young adult mice with mucopolysaccharidosis type I by liver-directed gene therapy.

Mucopolysaccharidosis type I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in the accumulation of glycosaminoglycans (GAGs) in many of the cells of affected patients. Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We have previously shown in a murine model the therapeutic potential of lentiviral IDUA vector-mediated gene therapy, in which human IDUA cDNA was driven by the cytomegalovirus promoter. However, the major limitation of this approach was the induction of an immune response against the therapeutic protein, which limited the efficacy and long-term duration of treatment. In this study, we evaluate the potential of liver-directed gene therapy, that is, programming of murine hepatocytes to secrete the enzyme with mannose 6-phosphate (M6P), which can be taken up by distant cells. Eight- to 10-week-old mice were injected via the tail vein with a lentiviral vector expressing human IDUA cDNA driven by the albumin gene promoter selectively expressed in hepatocytes. One month after treatment, IDUA activity was present in the liver and spleen of treated mice; an expression level of 1% normal IDUA activity was sufficient to reduce the GAG level in liver, spleen, kidney, heart, and lung. Interestingly, 6 months after a single injection of this vector, IDUA activity was detectable in several murine tissues; the level of enzyme activity was low but sufficient to maintain the decrease in GAG levels in liver, spleen, kidney, heart, and lung. Also, the level of enzyme-specific antibodies reached at 6 months postinjection was nearly null, and real-time polymerase chain reaction analysis showed high levels of vector DNA content in liver and spleen. Thus, these results show that the use of LV with the albumin gene promoter selectively expressed in hepatocytes limited the immune response to the transgene and allowed stable and prolonged expression of the IDUA enzyme and a partial correction of the pathology.

Analysis of Sanfilippo A gene mutations in a large pedigree.

Mucopolysaccharidosis type IIIA, also known as Sanfilippo A disease, results from mutations in the sulfamidase gene. To date, a total of 62 mutations have been described underlying this lysosomal disorder. Expression studies on missense mutations have shown that each alteration was disease-causing and helped to clarify the genotype-phenotype correlation. In this report we describe a large pedigree where the mutations have been identified in two second cousins: one with severe disease (E369K/R433Q) and the other with a mild form of the illness (E369K/P128L). This study places R433Q as a severe mutation underlying Sanfilippo A disease.

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.

The effect of four mutations on the expression of iduronate-2-sulfatase in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13). Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named DeltaS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype-phenotype correlation, prognosis and possible therapeutic intervention.

Extraneurologic symptoms as presenting signs of Sanfilippo disease.

Sanfilippo disease, or mucopolysaccharidosis type III, results from the deficiency of lysosomal hydrolases, which impairs heparan sulfate metabolism. Clinically, the disease is characterized by a mild somatic phenotype combined with early severe neurodegenerative illness with prominent behavioral disturbance. We report clinical and molecular findings of a child with Sanfilippo disease type B (alpha-N>-acetylglucosaminidase deficiency) who presented at age 18 months with marked systemic involvement and normal initial psychomotor development. These findings suggest that atypical mucopolysaccharidosis type III patients may present with early somatic changes preceding the onset of overt neurologic symptoms and ensuring an early diagnosis and possible therapeutic intervention.

Heparan N-sulfatase: cysteine 70 plays a role in the enzyme catalysis and processing.

Sulfatases are members of a highly conserved family of enzymes that catalyze the hydrolysis of sulfate ester bonds from a variety of substrates. The functional correlation reflects a high degree of amino acid sequence similarity along the entire length, in particular in the active site where the C(X)PSR consensus sequence is present. Cysteine undergoes an important co- or post-translation modification essential for the accomplishment of catalytic activity: conversion in formylglycine. In this work, the cysteine of heparan N-sulfatase (NS) was replaced either by a serine (C70S) or by a methionine (C70M) using site-directed mutagenesis. C70S and C70M mutant cDNAs were expressed and analyzed in COS cells; both mutations caused a loss of NS activity; however, while C70S showed a normal precursor form undergoing processing to a reduced mature form within the lysosomes, C70M was poorly synthesized and formed a complex with the molecular chaperone immunoglobulin binding protein.

Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites.

Heparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability.

Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK-paxillin interaction in fibronectin-adherent melanoma cells.

Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.

Expression of five iduronate-2-sulfatase site-directed mutations.

Five point mutations (R88H, R88P, T118I, 959delT, R468Q) previously identified in the iduronate-2-sulfatase (IDS) gene of Italian Hunter patients were expressed in COS cells to evaluate their functional consequence on enzyme activity, processing and intracellular localization. The 88 arginine residue belongs to the CXPSR pentapeptide conserved in all human sulfatases, where cysteine modification to formylglycine is required for enzyme activity. Substitution of arginine with histidine residue resulted in 13.7% residual enzyme activity, with an apparent K(m) value (133 microM) lower than that found for the normal enzyme (327 microM), indicating a higher affinity for the substrate; substitution of arginine with proline resulted in total absence of residual activity, in agreement with the phenotypes observed in patients carrying R88H and R88P mutations. For the four missense mutations, pulse-chase labelling experiments showed an apparently normal maturation; however, subcellular fractionation demonstrated poor transport to lysosomes. Therefore, residues 88, 118 and 468 appear to be not essential for processing but important for IDS conformation.

Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

Molecular defects in the alpha-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G-->A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G-->A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.

Bone marrow transplantation in a Hunter patient with P266H mutation.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a lysosomal disease caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS, EC 3.1.6.13). Affected patients show a wide spectrum of clinical phenotypes, from severe to mild. Mutational analysis on this disease resulted in the identification of more than 200 alterations. Bone marrow transplantation (BMT) is considered, at present, an appropriate therapy for MPS II subjects without severe neuropsychological impairment, however molecular analysis in BMT treated patients has been poorly studied. We describe here a patient subjected to BMT in 1995 whose IDS gene alteration, mutation P266H, was identified thereafter. The 4-year follow-up included clinical, biochemical and molecular parameters. DNA analysis showed, after BMT, coexisting host mutant and donor normal alleles, ensuring the effectiveness of the therapy and providing a fast and accurate tool to monitor the colonization of donor cells after treatment.

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Identification of molecular defects in Italian Sanfilippo A patients including 13 novel mutations.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by the deficiency of the enzyme heparin sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. Patients develop central nervous system degeneration resulting in progressive dementia, developmental delay, hyperactivity, and aggressive behaviour; subjects may present a wide spectrum of clinical severity. Here, we report the results from molecular analysis of 24 Italian MPS IIIA patients diagnosed over the last 15 years in our laboratory. Altogether, we were able to characterize 38 out of the 48 (79%) pathogenic alleles. We identified 16 molecular defects, 13 novel. The majority of alterations were missense mutations: on exon two (Y40N; A44T; S66W; R74C), on exon four (G122R; P128L; L146P; R150Q), on exon five (D179N; R182C), on exon six (P227R) and on exon eight (E369K; R377C). Single base pair deletions: on exon two (A52nt-1) and on exon eight (T360nt-1) and one base pair insertion on exon eight (V361nt+1) were also identified. Restriction enzyme or ARMS analyses were used to confirm each alteration. S66W represents the most common alteration in our patients population accounting for 33% of the total alleles. Interestingly, all six patients from Sardinia present this mutation, and five of them are homozygous for this change, suggesting that these subjects may have been derived from a common founder.