Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and mu ltiple neurological symptoms, such as vertical supranuclear ophthalmoplegia , progressive ataxia, and dementia. More than 90% of cases of NPC are due t o a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane p rotein that plays a role in cholesterol transport or homeostasis. Biochemic al diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholest erol esterification and a cytological technique-filipin staining-to demonst rate the intracellular accumulation of cholesterol. A small percentage of p atients, referred to as "NPC variants," present with clinical symptoms of N PC but show near-normal results of these biochemical tests, making laborato ry confirmation of NPC disease problematic. Here, we demonstrate that NPC-v ariant fibroblast samples can be detected as sphingolipid storage disease c ells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. Thi s lipid accumulated in endosomes/lysosomes in variant cells preincubated wi th LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. Thi s is a surprising result, since, in general, similar to 90% of patients wit h NPC possess defects in NPC1. Our findings should be useful for the detect ion of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.