Kevin T. Cavanagh

Human mucopolysaccharidosis IIID: clinical, biochemical, morphological and immunohistochemical characteristics

Mucopolysaccharidosis IIID (MPS IIID) is one of the rarest of the MPS-III syndromes. To date, the clinical manifestations of 10 patients have been reported, the deficient N-acetylglucosamine 6-sulfatase (G6S) enzyme has been purified, and the G6S gene has been cloned, sequenced and localized. However, morphological manifestations of this condition have not been reported and the pathogenesis of the severe neurological deficits remains an enigma. In this paper we describe and correlate the clinical, biochemical and pathological observations for 2 cases of MPS IIID. We used monoclonal antibodies against heparan sulfate (HS) and GMZ-ganglioside, thin layer chromatography, mass spectrometry, and morphological techniques to demonstrate the nature and the distribution of the uncatabolized substrates. The majority of the cells in various tissues showed morphological changes expected with lysosomal storage of HS. The central nervous system (CNS) was most severely affected because of the secondary storage of GM2 and GM3 gangliosides in addition to the primary accumulation of HS. The extent as well as the distribution of the diverse storage materials varied within and among different neurons as observed in MPS-III A, B, and C syndromes. This study supports the hypothesis that the neurological dysfunction and neurodegencration common to the Sanfilippo syndromes is, in part, due to the secondary metabolic perturbations induced by HS accumulation.

Caprine Mucopolysaccharidosis-IIID: Clinical, Biochemical, Morphological and Immunohistochemical Characteristics

Several animal models have been developed for the mucopolysaccharidoses (MPSs), a group of lysosomal storage disorders caused by lysosomal hydrolase deficiencies that disrupt the catabolism of glycosaminoglycans (GAG). Among the MPS, the MPS-III (Sanfilippo) syndromes lacked an animal counterpart until recently. In this investigation of caprine MPS-IIID, the clinical, biochemical, morphological, and immunohistochemical studies revealed severe and mild phenotypes like those observed in human MPS III syndromes. Both forms of caprine MPS HID result from a nonsense mutation and consequent deficiency of lysosomal N-acetylglucosamine 6-sulfatase (G6S) activity and are associated with tissue storage and urinary excretion of heparan sulfate (HS). Using special stains, immunohistochemistry, and electron microscopy, secondary lysosomes filled with GAG were identified in most tissues from affected goats. Primary neuronal accumulation of HS and the secondary storage of gangliosides were observed in the central nervous system (CNS) of these animals. In addition, morphological changes in the CNS such as neuritic expansions and other neuronal alterations that may have functional significance were also seen. The spectrum of lesions was greater in the severe form of caprine MPS HID and included mild cartilaginous, bony, and corneal lesions. The more pronounced neurological deficits in the severe form were partly related to a greater extent of CNS dysmyelination. These findings demonstrate that caprine MPS HID is a suitable animal model for the investigation of therapeutic strategies for MPS III syndromes.

Molecular defect of caprine N-acetylglucosamine-6-sulphatase deficiency. A single base substitution creates a stop codon in the 5'-region of the coding sequence.

Sanfilippo D syndrome (mucopolysaccharidosis type IIID, MPS IIID; McKusick 252940) is caused by the deficiency of N-acetylglucosamine-6-sulphatase ( (G6S; EC 3.1.6.14). A Nubian goat with this lysosomal storage disease has been identified (Thompson et al 1992). Towards further development of this animal model of MPS IIID, determination of the DNA defect was initiated. Southern analysis of restriction enzyme-digested goat genomic DNA indicated that there was no apparent deletion or rearrangement of G6S in the affected animal using both human and goat probes. Initial RT-PCR studies of RNA from affected animals indicated that G6S mRNA was present. For mutation analysis, a series of normal and affected caprine G6S RT-PCR amplicons, covering the entire coding region, was sequenced and compared. Either oligo(dT) or antisense primers were used for cDNA synthesis. Amplicons, 300400 bp long, were produced with primers based on caprine G6S clone sequences or human sequence (Robertson et al 1992). A nonsense mutation was found in the cDNA of the affected animal, changing a C to T in codon 102 of the 559-amino-acid G6S coding sequence. This mutation, in addition to truncating the protein, also introduces an AluI restriction site (underlined below) (recognition sequence AGCT) that will provide a convenient basis for carrier detection. Validation of a PCR-based test is now underway.

Caprine mucopolysaccharidosis IIID: A preliminary trial of enzyme replacement therapy

Mucopolysaccharidosis type IIID (MPS IIID) is a lysosomal storage disorder resulting from lack of activity of the lysosomal hydrolase N-acetylglucosamine 6-sulfatase (6S) (EC 3.1.6.14). The syndrome is associated with systemic and central nervous system (CNS) heparan sulfate glycosaminoglycan (HS-GAG) accumulation, secondary storage of lipids, and severe, progressive dementia. In this investigation, caprine MPS IIID, established as a large animal model for the human disease, was used to evaluate the efficacy of enzyme replacement therapy (ERT). Recombinant caprine 6S (rc6S) (1 mg/kg/dose) was administered intravenously to one MPS IIID goat kid at 2, 3, and 4 wks of age. Five days after the last dose, the uronic acid (UA) content and the composition of uncatabolized HS-GAG fractions in the brain of the ERT-treated MPS IIID kid were similar to those from a control, untreated MPS IIID animal. However, hepatic uronic acid levels in the treated MPS IIID kid were approximately 90% lower than those in the untreated MPS IIID control; whereas the composition of the residual hepatic HS-GAG was identical to that in the untreated animal. Marked reduction of lysosomal storage vacuoles in hepatic cells of the treated MPS IIID kid was observed, but ERT had no effect on CNS lesions. No residual 6S activity was detected in brain or liver. This preliminary investigation indicates that other treatment regimens will be necessary to ameliorate MPS III-related CNS lesions.

CLONING AND SEQUENCE ANALYSIS OF CAPRINE N-ACETYLGLUCOSAMINE 6-SULFATASE CDNA

Mucopolysaccharidosis IIID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the caprine N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence.

Biochemical and histochemical analysis of lysosomal enzyme activities in caprine β-mannosidosis

Goats affected with β-mannosidosis, and autosomal recessive disease of glycoprotein catabolism, have deficient tissue and plasma levels of the lysosomal enzyme β-mannosidase. Pathological characteristics include cytoplasmic vacuolation in the nervous system and viscera, and myelin deficits that demonstrate regional variation. This study was designed to determine the correlation between β-mannosidase activity in normal animals and the severity of lesions in affected goats, and to assess the regional changes in lysosomal enzyme activity in specific regions and cell types in affected animals. Although enzyme activity in normal organs (kidney, thyroid, brain) is correlated in general with the accumulation of uncatabolized substrate and with the extent of vacuolation, this correlation does not extend to assessment of specific region of the central nervous system (CNS). In affected goats, the activities of α-mannosidase, α-fucosidase, and β-hexosaminidase are elevated to a greater extent in all CNS regions than in organs. The results suggest cell-specific, organ-specific, and enzyme-specific regulation of changes in lysosomal enzyme activity in the presence of metabolic perturbations, such as deficiency of β-mannosidase activity.

Caprine mucopolysaccharidosis IIID: fetal and neonatal brain and liver glycosaminoglycan and morphological perturbations.

Mucopolysaccharidosis IIID (MPS IIID) is a lysosomal storage disease associated with deficient activity of the enzyme N-acetylglucosamine 6-sulfatase (EC 3.1.6.14), a lysosomal hydrolase in the heparan sulfate glycosaminoglycan (HS-GAG) degradation pathway. In caprine MPS IIID, enzyme replacement therapy reversed early postnatal systemic but not primary or secondary central nervous system (CNS) substrate accumulations. The caprine MPS IIID large animal model system was used in this investigation to define the developmental profile of morphological and biochemical perturbations to estimate a time frame for therapeutic intervention. Light and electron microscopy were used to compare the CNS, liver, and kidney of normal +/+, MPS IIID carrier +/-, and MPS IIID-affected -/- goat kids (kids), at 60, 113–114, 128–129, and 135 d gestation (dg) of a 150-d gestational period, at birth, and at 59–64 d of postnatal (d-pn) age. In the CNS of -/- kids, morphological correlations of HS-GAG and glycolipid accumulations were evident in early differentiating neurons at 60 dg. CNS and systemic developmental, regional, and cellular differences in -/-kids at all time points included more prominent and earlier accumulation of lucent, putative HS-GAG substrates in lysosomes of meningeal and perivascular macrophages and hepatic sinusoidal cells than in CNS, hepatic, or renal parenchymal cells. The amounts and compositions of HS-GAG substrates in the brain and liver of +/+, +/-, and -/- kids were determined at 60, 65, 113–114, and 128–135 dg, at birth, and 53–78 d-pn. In the CNS of -/- kids, HS-GAG concentrations were variable and exceeded those of age-matched control tissue samples in the third but not the second trimester. In contrast, hepatic HS-GAG levels in -/- kids exceeded control values at all time points evaluated and paralleled the progressive morphological alterations. CNS and hepatic HS-GAG compositions in -/- kids were similar to each other and were more complex at all pre- and postnatal ages than those from control kids. Based on the time frame of development of CNS lesions and biochemical perturbations, prenatal therapeutic intervention in caprine MPS IIID is likely to be necessary to prevent or ameliorate substantive CNS and systemic lesions.

Determination of Genotypic Frequency of Caprine Mucopolysaccharidosis IIID

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases caused by enzyme deficiencies in the degradative pathways of glycosaminoglycans (GAGs). Because of a specific enzyme deficiency, GAGs or partially degraded GAGs accumulate in the lysosomes and may be excreted in the urine. The 4 types of MPS III (Sanfilippo syndromes A–D), characterized by the inability to degrade heparan sulfate (HS), are clinically similar, but significant phenotypic variation is observed within and among MPS III subtypes.5 Mucopolysaccharidosis IIID (McKusick 252940) is caused by a deficiency in N-acetylglucosamine 6-sulfatase activity (G6S; EC 3.1.6.14). Clinical, biochemical, morphological, and immunohistochemical characterization of human and caprine MPS IIID has been reported.3,8 Heparan sulfate and N-acetylglucosamine 6-sulfate accumulate in the tissues and urine of individuals affected by MPS IIID.8 A secondary storage of gangliosides also occurs in the central nervous system. Caprine G6S cDNA has been cloned and sequenced,2 and the cDNA defect in caprine MPS IIID has been determined.1 A mutation test utilizing the polymerase chain reaction (PCR) has been established for carrier detection and prenatal screening for caprine G6S deficiency.4 Using the mutation test, 552 purebred Nubian goats (52 males and 500 females) from 20 herds in central lower Michigan (herd sizes of 1–210 goats and ages ranging from newborn to 10 years) were surveyed to determine the frequencies of the homozygous and heterozygous genotypes for MPS IIID. Efficacies of 2 DNA sampling techniques, collection of buccal epithelial cells (BECs) on cytology brushesa (a noninvasive procedure used for human PCR diagnosis6) and collection of white blood cells (WBC), were also compared. BECs were collected by swirling a cytology brush on the buccal mucosa of the lower lip. The brush was rinsed in 200 ml 50 mM NaOH. Cells were lysed by boiling for 10 minutes and then neutralized with 10 ml 2 M Tris-HCl, pH 8.0. Blood was collected in ethylene diaminetetraacetic acid (EDTA) collection tubes.b WBCs were isolated by combining 100 ml whole blood and 0.5 ml red cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.4), incubating the solution on ice for 10 minutes, centrifuging at 12,000 3 g for 30 seconds, and removing the supernatant. Cells were lysed as above. The previously described G6S PCR-based mutation test4 was used with the following minor modifications. One-microliter aliquots of the lysed cells were used as the DNA

Possible β-mannosidosis chimera. Altered expression of metabolic perturbations

SummaryAn aberrant β-mannosidosis phenotype in a 5-month-old triplet goat kid was characterized by a late postnatal onset of mild neurological symptoms. Necropsy examination revealed relatively normal myelination; however, the distribution of cytoplasmic vacuolation in the kidney and brain was similar to that observed in neonatal β-mannosidosis. Variable engraftment of donor stem cells, resulting from transplacental transfusion of stem cells from a normal sibling during the immunotolerant period, may have modified the expected severe β-mannosidosis phenotype. This investigation was designed to determine the effects of a possible chimeric state on organ-specific metabolic perturbations. Residual β-mannosidase enzyme activity was found in plasma, kidney, liver and spleen but not in brain. Other lysosomal enzyme activities were comparable to normal values. Immunoreactive β-mannosidase was estimated to be less than 10% of normal levels. Kidney, brain grey matter and brain white matter contained 33%, 12% and 4%, respectively, of the oligosaccharides expected in the organs of β-mannosidosis animals. There were no detectable oligosaccharides or cytoplasmic vacuolation in the liver or spleen. Studies of this possible chimera provided preliminary evidence for the efficacy of prenatal treatment of early-onset neurodegenerative disorders.

Bovine Plasma β-Mannosidase Activity and Its Potential use for β-Mannosidosis Carrier Detection

Plasma β-mannosidase activities were determined for Salers cattle from 8 herds as an evaluation of this method for detection of β-mannosidosis heterozygotes. Several biological factors, such as age, gender, herd, and risk of being a β-mannosidosis carrier, were considered in this study. The mean enzyme activity for obligate heterozygotes (n = 8) was 55 U/ml (range = 43–65 U/ml), which was 59% of the mean enzyme activity for cattle that were low risk for being a carrier. These data indicate that bovine β-mannosidosis is characterized by a gene dosage effect. The analytical and biological variation of plasma β-mannosidase activity that was observed necessitates limiting the test to adult fullblood/purebred Salers cattle within a herd. Plasma β-mannosidase analysis provides important information for intraherd selection of Salers cattle that are heterozygous for β-mannosidosis.