Ganglioside loading of cultured fibroblasts: a provocative method for the diagnosis of the G_M2 gangliosidoses

Confirmation of deficient β-hexosaminidase activity in suspected cases of G_M2 gangliosidosis may be difficult with available assay systems if the residual activity is high (as in many juvenile cases and genetic compounds). Hexosaminidase activity is normal in the AB-variant (activator protein deficiency), although G_M2-ganglioside (G_M2) catabolism is severely impaired. We therefore examined ganglioside degradation in intact fibroblasts in culture. Intracellular ganglioside levels were determined in cultured human skin fibroblasts grown in standard tissue culture medium or medium supplemented with mixed bovine brain gangliosides. Cellular uptake and catabolism of the added gangliosides were manifested by modest increases in the intracellular concentrations of gangliosides G_T + G_D and G_M1 in all cells tested, and marked accumulation of G_M2 in fibroblasts from patients with G_M2 gangliosidoses. Intracellular G_M2 increased five-to fifteen-fold in all of the G_M2 gangliosidosis cell lines tested, including those from patients with infantile Tay-Sachs disease (TSD), Sandhoff disease, late infantile and juvenile variants of TSD with high residual enzyme activity, adult onset G_M2 gangliosidosis, and the AB-variant. Significant GM2 accumulation did not occur in fibroblasts from patients with G_M2 gangliosidosis grown in standard medium, or in normal fibroblasts grown in ganglioside enriched medium. Our method of ganglioside feeding employs commercially available materials and no special equipment. It should be useful for the confirmation of impaired G_M2 catabolism in a variety of settings.