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

Abstract

ABSTRACT. Sanfilippo syndrome, type D (MPS IIID), is characterized by moderate physical abnormalities, progressive mental deterioration, and deficient activity of Nacetylglucosamine 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. (Pediatr Res 26:462-466, 1989)

Direct Profiling of Multiple Enzyme Activities in Human Cell Lysates by Affinity Chromatography/Electrospray Ionization Mass Spectrometry: Application to Clinical Enzymology

“…The application of our ACESIMS system to the biochemical testing of enzyme deficiencies in the Sanfilippo syndrome has several advantages over status quo testing methods. Current analytical methods used in clinical laboratories to assay the Sanfilippo enzymes rely on a variety of strategies, including radiometry, 29 colorimetry, 30 and fluorometry. 31 Several assays involve cumbersome separation techniques such as thin paper electrophoresis of radiometric mixtures.…”

Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model

“…Enzyme assay testing of cultured skin fibroblasts and leukocytes for enzyme deficiency are also used to provide definitive diagnosis of MPS III B (5). Prenatal diagnosis can be performed as early as 9– 16 weeks gestation using chorionic villus biopsies and amniotic fluid cells to ascertain whether the fetus has Sanfilippo type A (6), B (7, 8), C (9), or D (10).…”

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID)

“…The conventional GlcNAc-6S sulphatase assay uses radiolabelled oligosaccharides, derived from heparan sulphate (Kresse et al 1980;Freeman and Hopwood 1992); this is still the most widely used assay. Nawakowski et al (1989) have described a GlcNAc-6S sulphatase assay with the monosaccharide GlcNAc-6S. This method requires separation of substrate and free GlcNAc and is based on the colorimetric determination of GlcNAc.…”