Niemann–Pick type C disease: Novel NPC1 mutations and characterization of the concomitant acid sphingomyelinase deficiency

Abstract Niemann–Pick type C (NPC) disease is an inherited lipid storage disorder characterized by the lysosomal accumulation of free cholesterol in affected cells. Three novel mutations in the NPC1 gene (c.3615delA, c.2000C > T, and c.2240delT) were detected in two unrelated patients with the severe phenotype of NPC. The analyses showed that the c.2240delT mutation, which causes a premature stop at codon 748, resulted in nonsense-mediated decay of the mutant transcripts. Immunoblotting analyses for the NPC1 protein did not detect the mutant proteins in COS-1 cells transiently transfected with the two mutant NPC1 cDNA constructs (c.3615delA and c.2000C > T). In NPC cells, sphingomyelin accumulates with cholesterol, leading to an identical subcellular distribution of both lipids. Acid sphingomyelinase (ASM), which is responsible for the lysosomal hydrolysis of sphingomyelin, is partially reduced in NPC fibroblasts. Therefore, NPC fibroblasts were studied to determine if ASM activity was perturbed due to the accumulation of cholesterol. However, these studies demonstrated that the subcellular localization of ASM was preserved, suggesting that the high content of lysosomal cholesterol was not responsible for the decreased ASM activity.