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[1-3H]Galactitol-6-sulfate, N- [1-3H]acetylgalactosaminitol-6-sulfate, N-[1-3H]acetylglucosaminitol-6-sulfate, N-acetylglucosamine-6-sulfate, and 6-sulfated tetrasaccharides from chondroitin-6-sulfate have been used for the measurement of 6-sulfatase activity of extracts of normal skin fibroblasts and of fibroblasts cultured from patients with genetic mucopolysaccharidoses. With these substrates, extracts of fibroblasts derived from Morquio patients lack or have greatly reduced activities for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and 6-sulfated tetrasaccharides but have normal activity for N-acetylglucosamine-6-sulfate and its alditol; those derived from a patient with a newly discovered mucopolysaccharidosis have greatly reduced activity for N-acetylglucosamine-6-sulfate and its alditol but normal activity for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and the 6-sulfated tetrasaccharides. These findings demonstrate the existence of two different hexosamine-6-sulfate sulfatases, specific for the glucose or galactose configuration of their substrates. Their respective deficiencies, causing inability to degrade keratan sulfate and heparan sulfate in one case and keratan sulfate and chondroitin-6-sulfate in the other, are responsible for different clinical phenotypes.

We have prepared a new substrate (o-beta-D-sulfo-galactosyl-(1-4)-beta-D-6-sulfo-2-acetamido-2-deoxyglucosyl- (1-4)-D-[1-3H]galactitol), from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, we found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Our results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides.

A 14-year-old girl with a unique type of progressive spondyloepiphyseal dysplasia, corneal clouding, and no evidence of neurological abnormality, was found to have a remarkable deficiency of acid beta-galactosidase activity in cultured skin fibroblasts and in leucocyte preparations. In fibroblasts, ganglioside GM1 beta-galactosidase activity averaged 7% of the normal mean while asialofetuin beta-galactosidase and 4-methylumbe lifery-beta-galactosidase averaged 1.4% and 3.5%, respectively. Activities for all three substrates in leucocytes from both her parents were close to 50% of the normal mean indicating that the patient is homozygous for a mutation (or mutations) affecting GM1 beta-galactosidase.

The activity of N-acetyl galactosamine-6-sulfate sulfatase was studied for the first time in the liver and brain of a patient with a clinically typical case of Morquio syndrome with keratosulfaturia. As has been demonstrated in the fibroblasts of patients with this syndrome, this enzymatic activity was markedly decreased in both organs. Neuropathological examination revealed moderately swollen neurons containing PAS-positive, coarse globular inclusions in the cerebral cortex, Ammon's horn, basal ganglia, and thalamic nuclei. Ultrastructurally, the inclusions consisted of stacked, straight or loose, wavy membranes of various lengths, often associated with pale or moderately electron dense homogeneous "lipid droplets." These ultrastructural features of the inclusions were closely similar to the granulomembranous bodies of Hurler syndrome and the inclusions described in type B of the Sanfilippo syndrome. Unlike those mucopolysaccharidoses, however, no abnormalities were found in the gangliosides in the brain of the patient with Morquio syndrome.

The tritiated disulphated trisaccharide 6-sulpho-N-acetylgalactosamine-glucuronic acid-6-sulpho-N-acetyl-[1-3H]galactosaminitol was prepared from chondroitin 6-sulphate for use as a substrate for N-acetylgalactosamine 6-sulphate sulphatase. The reaction product was separated on ECTEOLA cellulose rather than Dowex 1 X 2. The mean activities of normal fibroblasts, leukocytes and amniotic cells were 8.43, 2.59 and 3.14 nmol h-1 mg protein-1, respectively. Fibroblasts from five patients with classical Morquio's disease (mucopolysaccharidosis IVA; MPSIVA) and one patient with neonatal multiple sulphatase deficiency displayed activities of less than 5% of control mean. Activity in amniotic cells from a pregnancy where the fetus was affected with MPS IVA was 6% of control mean. Activity was also found to be present in normal specimens of chorionic villi (mean value 1.21 nmol h-1 mg protein-1), demonstrating the feasibility of first trimester prenatal diagnosis of MPS IVA by assay of activity in this tissue.