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

Xiaofeng Sun, David L. Marks, Walter D. Park, Christine L. Wheatley, Vishwajeet Puri, John F. O’Brien, Daniel L. Kraft, Patrick A. Lundquist, Marc C. Patterson, Richard E. Pagano, Karen Snow

Research output: Contribution to journalArticle

105 Scopus citations

Abstract

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with 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. This is a surprising result, since, in general, ∼90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Original languageEnglish (US)
Pages (from-to)1361-1372
Number of pages12
JournalAmerican journal of human genetics
Volume68
Issue number6
DOIs
StatePublished - Jun 2001

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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    Sun, X., Marks, D. L., Park, W. D., Wheatley, C. L., Puri, V., O’Brien, J. F., Kraft, D. L., Lundquist, P. A., Patterson, M. C., Pagano, R. E., & Snow, K. (2001). Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1. American journal of human genetics, 68(6), 1361-1372. https://doi.org/10.1086/320599