Sanfilippo Syndrome, Type D: A Spectrophotometric Assay with Prenatal Diagnostic Potential

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Abstract
Sanfilippo syndrome, type D (MPS IIID), is characterized by moderate physical abnormalities, progressive mental deterioration, and deficient activity of N-acetylglucosamine 6-sulfate sulfatase, a lysosomal hydrolase involved in the degradation of heparin, keratan sulfate, and heparan sulfate. To date, demonstration of the enzyme deficiency typically relies on a radiolabeled trisaccharide substrate derived from heparan sulfate. In our study, we have developed a spectrophotometric assay for the determination of N-acetylglucosamine 6-sulfate sulfatase activity using the monosaccharide, N-acetylglucosamine 6-sulfate, as substrate. The reaction mixture was incubated for 6 h at 37°C and, after Dowex chromatography, released N-acetylglucosamine was measured by a modification of the method of Reissig. Assay conditions were optimized for cultured skin fibroblasts and primary cultures of amniotic fluid cells. The pH optimum for each was 5.5. The assay was linear for 24 h and up to 0.1 absorbance units. Activities of the three known MPS IIID skin fibroblast cell lines were more than 4 SD below the skin fibroblast control mean and more than 5 SD below the control mean for amniotic fluid cells. An enzyme deficiency in cultured amniotic fluid cells of the same magnitude as the skin fibroblasts of the known patients would be detectable and, therefore, prenatal diagnosis by this method is feasible.