Hybridization studies of fibroblasts from Hurler, Scheie, and Hurler/Scheie compound patients: support for the hypothesis of allelic mutants.

Heterokaryons were made by fusion of alpha-L-iduronidase-deficient fibroblasts from patients with the Hurler, Scheie, or Hurler/Scheie compound syndrome. The fused cell populations remained alpha-L-iduronidase deficient and accumulated 35S-labeled glycosaminoglycans (35S-GAG) to the same extent as the parental cells strains. Also, when 35S-GAG accumulation was studied by autoradiography at the level of single bi- and multinuclear hybrid cells, no evidence was found for genetic complementation. The results support the hypothesis that the mutations in the Hurler and Scheie syndromes are allelic, and they are compatible with the view that patients with intermediate phenotypes represent genetic compounds.

Pericellular glycosaminoglycans in cultured human cells. A possible source of error in prenatal diagnosis of mucopolysaccharidoses.

Normal fibroblasts and amniotic fluid cells, and cells from patients with Mucopolysaccharidosis type I and II, were cultured in the presence of 35SO4. After harvesting by trypsinization, the radioactivity was recovered from the intracellular and pericellular pool of glycosaminoglycans. The amount of incorporated radioactivity in these respective pools was 1 : 2.3 in normal fibroblasts and 1 : 7.2 in control amniotic fluid cells. Incorporation in the pericellular pool was not elevated in cells from patients with Mucopolysaccharidosis type I and II, in contrast to incorporation in the intracellular pool. Studies on different methods of harvesting showed that reliable prenatal analysis can be performed only if the pericellular pool is removed by trypsinization. Amniotic fluid cells from a pregnancy carrying a fetus affected with Hurler's disease revealed the expected increased level of 35SO4 incorporation if the cells were trypsinized, but the intracellular accumulation of glycosaminoglycans was obscured by the pericellular pool if the cells were harvested by scraping.

Autoradiographic detection of mucopolysaccharide accumulation in single fibroblasts.

A method is described for localizing acid mucopolysaccharides autoradiographically in cultured cells. Normal fibroblasts and fibroblasts, from patients suffering from Mucopolysaccharidosis II disease (MPS II), were cultured for six days in the presence of 35SO4 and one day in unlabelled medium. The cultured cells were transferred to plastic film dish and, after settling, they were rapidly quenched, freeze-dried, fixed in osmium tetroxide vapour and embedded in Epon. Grain counting after autoradiography in 2 mum sections revealed a significant difference (P greater than 0.001) in 35SO4 incorporation in the perinuclear cytoplasm of MPS II cells and control cells grown under the same conditions. Autoradiography was also performed after mixing MPS II cells and control fibroblasts in a ratio 1:1-8 prior to freezing and the same ratio was found between labelled and unlabelled fibroblasts. These results demonstrate the feasibility of the present autoradiographic technique for the detection of the acid mucopolysaccharide storage at the single cell level.