Acta PaediatricaVolume 91, Issue s439 p. 148-148 Molecular analysis of a mild form of mucopolysaccharidosis VII: novel mutations and expression studies S Storch, S Storch University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorB Wittenstein, B Wittenstein University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorJ Grubb, J Grubb Hospital, Hamburg, Germany Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorK Ullrich, K Ullrich University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorWS Sly, WS Sly Hospital, Hamburg, Germany Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorT Braulke, T Braulke University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this author S Storch, S Storch University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorB Wittenstein, B Wittenstein University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorJ Grubb, J Grubb Hospital, Hamburg, Germany Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorK Ullrich, K Ullrich University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorWS Sly, WS Sly Hospital, Hamburg, Germany Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this authorT Braulke, T Braulke University of Hamburg, Children', Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO, USASearch for more papers by this author First published: 02 January 2007 https://doi.org/10.1111/j.1651-2227.2002.tb03179.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume91, Issues439November 2002Pages 148-148 RelatedInformation

BACKGROUND Carbonic anhydrase (CA) plays a fundamental role in regulation of systemic acid-base homeostasis by facilitating urinary acidification. Four CA isozymes (CA II, IV, XII, XIV) have been identified in kidney. Until now, luminal CA IV, a GPI-anchored isozyme, was thought to mediate most bicarbonate absorption. Although CA XIV mRNA has been demonstrated in mouse and human kidney, the localization of this newly discovered CA has not been established. METHODS RT-PCR and Western blot analyses were used to demonstrate CA XIV mRNA and protein in extracts of cortex and medulla of mouse kidney. Polyclonal antibodies against mouse CA XIV were utilized for immunofluorescence to examine the pattern of expression of CA XIV in the nephron of both rat and mouse kidney. RESULTS Immunofluorescence staining showed abundant expression of CA XIV in apical plasma membranes of the S1 and S2 segments of proximal tubules, and weaker staining in the basolateral membranes. Also, strong staining was seen in the initial portion of the thin descending limb of Henle. These results show that luminal CA XIV is strongly expressed in regions of the rodent nephron that have been thought to be important in urinary acidification. Staining for CA XIV and CA IV in the same sections showed some areas of co-expression, but also some areas where each was expressed without the other. CONCLUSIONS Luminal CA XIV may account for a substantial fraction of the bicarbonate reabsorption previously attributed to CA IV. If so, CA XIV and CA IV may be functionally redundant.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy.

We demonstrated previously that short term administration of recombinant beta-glucuronidase to newborn mice with mucopolysaccharidosis type VII reduced lysosomal storage in many tissues. Lysosomal storage accumulated gradually after cessation of enzyme replacement therapy. Mice alive at 1 yr of age had decreased bone deformities and less lysosomal storage in cortical neurons. Here we compare the effects of long term enzyme replacement initiated either at birth or at 6 wk of age, and of enzyme administration initiated at birth followed by syngeneic bone marrow transplantation (BMT) at 5 wk of age. Several mice from each treatment group lived to at least 1 yr of age. Liver and spleen samples had beta-glucuronidase levels ranging from 2.4 to 19.8% of normal and showed a parallel decrease in lysosomal storage. The combination of enzyme replacement therapy followed by BMT reduced lysosomal distension in meninges, corneal fibroblasts, and bone when compared with treatment with enzyme alone. Mice treated at birth had less lysosomal storage in some neurons of the brain and the skeletal dysplasia was less severe when compared to mice whose treatment was delayed until 6 wk of age. We conclude that both enzyme replacement alone and early enzyme replacement followed by BMT have long term positive effects on murine mucopolysaccharidosis type VII. In addition, treatment started at birth is far more effective than treatment initiated in young adults.

beta-Glucuronidase injected i.v. into newborn mucopolysaccharidosis VII mice was cleared from the circulation in less than 1 h and taken up by tissues in a distribution corresponding to the location of the mannose 6-phosphate receptor. One h after a 3.5-mg/kg beta-glucuronidase injection, beta-glucuronidase levels were equal to or greater than normal in every organ examined with the exception of the brain, where 31% normal activity was present. Enzyme was detectable histochemically in the major sites of pathology for mucopolysaccharidosis VII including bone, brain, heart, and fixed tissue macrophages. The half-life of recombinant beta-glucuronidase activity in various organs of injected mucopolysaccharidosis VII mice was 1.5 to 4.5 d. These studies show that recombinant beta-glucuronidase administered to newborn mice reaches the sites of clinically important storage in murine mucopolysaccharidosis VII.

Carbonic anhydrase (CA) I and II are soluble isozymes that represent the major nonhemoglobin proteins in the erythrocyte. We recently identified a deficiency of CA II as the enzymatic basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Virtual absence of the CA II peak on high-performance liquid chromatography, of CA II esterase activity, and of immunoprecipitable CA II were demonstrated on extracts of red cell lysates from all patients studied. Reduced levels of CA II were found in obligate heterozygotes. Here, we present evidence that CA II in red cell lysates can be quantitated by measuring CO2 hydratase activity in the presence of inhibitors that selectively inhibit the activity of CA I to a much greater extent than that of CA II. This was done with iodide (anion binding) and bromopyruvic acid (alkylation), and the respective assays evaluated as diagnostic tools for CA II deficiency in human red cells. These techniques greatly simplify the quantitation of CA II in hemolysates and should make genetic diagnosis and counseling for the newly described inborn error of metabolism due to CA II deficiency generally available. They also allow quantitation of CA I in red cell lysates.

This chapter examines how to obtain a skin biopsy and propagates cultured fibroblasts for biochemical studies from the source. Cell lines from patients with many human genetic disorders are available on request from the Human Genetic Mutant Cell Repository, Institute for Medical Research, Camden, New Jersey. This is a federally financed collection of human mutant cell lines developed under contract for the National Institute of General Medical Sciences. Its purpose is to provide material from patients with human genetic diseases to investigators who wish to study these diseases, but who may have difficulty gaining access to cells from affected patients. Mutant strains are established from skin biopsies solicited from around the world, grown up, tested for contamination, and stored frozen in liquid. Mutant strains are established from skin biopsies solicited from around the world, grown up, tested for contamination, and stored frozen in liquid.