Tatsuo Nishioka

Heparan Sulfate Levels in Mucopolysaccharidoses and Mucolipidoses

SummaryGlycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Development and Testing of New Screening Method for Keratan Sulfate in Mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA), a progressive lysosomal storage disease, causes skeletal dysplasia through excessive storage of keratan sulfate (KS). We developed an ELISA-sandwich assay that used a MAb specific to KS. Forty-five blood and 59 urine specimens from MPS IVA patients (ages 1–65 y) were analyzed to determine whether KS concentration is a suitable marker for early diagnosis and longitudinal assessment of disease severity. Blood specimens were obtained from patients categorized as phenotypically severe (n = 36) and milder (n = 9). Urine specimens were also analyzed from patients categorized as severe (n = 56) and milder (n = 12), respectively. Blood KS levels (101–1525 ng/mL) in MPS IVA patients were two to eight times higher than those in age-matched controls (15–323 ng/mL). It was found that blood KS level varied with age and clinical severity. Blood KS levels in both MPS IVA and controls peaked between 5 and 10 y of age (mean, 776 versus 234 ng/mL, respectively). Blood levels in severe MPS IVA were 1.5 times higher than in the milder form. In contrast to blood, urine KS levels in both MPS IVA and controls peaked between 1 and 5 y (15.3 versus 0.26 mg/g creatinine), and thereafter declined with age. Urine KS level also varied with age and clinical severity, and the severe MPS IVA phenotype was associated with 6.7 times greater urine KS excretion than the milder one. These findings indicate that the new assay for blood or urine KS may be suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors

Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR−/− MPS VII mice. The availability of both MR+/+ and MR−/− mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human β-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.

Acidic amino acid tag enhances response to enzyme replacement in mucopolysaccharidosis type VII mice.

We have tested an acidic oligopeptide-based targeting system for delivery of enzymes to tissues, especially bone and brain, in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy is based upon tagging a short peptide consisting of acidic amino acids (AAA) to N terminus of human beta-glucuronidase (GUS). The pharmacokinetics, biodistribution, and the pathological effect on MPS VII mouse after 12 weekly infusions were determined for recombinant human untagged and tagged GUS. The tagged GUS was taken up by MPS VII fibroblasts in a mannose 6-phosphate receptor-dependent manner. Intravenously injected AAA-tagged enzyme had five times more prolonged blood clearance compared with the untagged enzyme. The tagged enzyme was delivered effectively to bone, bone marrow, and brain in MPS VII mice and was effective in reversing the storage pathology. The storage in osteoblasts was cleared similarly with both enzyme types. However, cartilage showed a little response to any of the enzymes. The tagged enzyme reduced storage in cortical neurons, hippocampus, and glia cells. A highly sensitive method of tandem mass spectrometry on serum indicated that the concentration of serum dermatan sulfate and heparan sulfate in mice treated with the tagged enzyme decreased more than the untagged enzyme. These preclinical studies suggest that this AAA-based targeting system may enhance enzyme-replacement therapy.

Identification of a common mutation in mucopolysaccharidosis IVA: correlation among genotype, phenotype, and keratan sulfate

AbstractMucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS was performed by genomic PCR and direct sequence analyses in 20 MPS IVA patients from Latin America. In this study, 12 different gene mutations including nine unreported ones were identified in 16 severe and four attenuated patients and accounted for 90.0% of the unrelated mutant alleles. The gene alterations were missense mutations except one insertion. Six recurrent mutations, p.A75G, p.G116S, p.G139S, p.N164T, p.R380S, and p.R386C, accounted for 5.0, 10.0, 5.0, 7.5, 5.0, and 32.5% of the unrelated mutant alleles, respectively. The p.R386C mutation was identified in all Latin American populations studied. Eleven mutations correlated with a severe form, while one mutation, p.R380S, was associated with an attenuated form. MPS IVA patients had an elevation of urine and plasma keratan sulfate (KS) concentrations compared with those of the age-matched control. KS concentrations in severe patients were higher than those in attenuated patients. These data provide evidence for extensive allelic heterogeneity and presence of a common mutation in Latin American patients. Accumulation of mutations with clinical description and KS concentration will lead us to predict clinical severity of the patient more precisely.

Differences in methylation patterns in the methylation boundary region of IDS gene in hunter syndrome patients: implications for CpG hot spot mutations

Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.