Niemann-Pick C Variant Detection by Altered Sphingolipid Trafficking and Correlation with Mutations within a Specific Domain of NPC1

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, approximately 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.

The National Niemann-Pick Type C1 Disease Database: correlation of lipid profiles, mutations, and biochemical phenotypes

“…Since identifi cation of the NPC1 gene, several studies have reported specifi c disease-causing mutations among different populations and ethnic groups ( 27,29,31,33,40,52,53 ). In the present study, the 52 patients that provided gene mutations ranged in age between 1.5 and 48.5 years and were at different stages and severity of disease.…”

The Spectrum of Niemann-Pick Type C Disease in Greece

“…In the NPC1 patients, 12 different mutations and nine different genotypes were identified. Eight of the mutations had been previously described: p.E1089K (3265G>A) (Sun et al 2001), p.F284Lfs*26 (c.852delT) (Fancello et al 2009), p. A1132P(c.3394G>C) (Mavridou et al 2014), del promoter region and exons 1-10 (Rodriguez-Pascau et al 2012), p. R1186H(c.3557G>A) (Carstea et al 1997), p.P1007A (c.3019C>G) (Greer et al 1999), p.Q92R(c.275A>G) (Ribeiro et al 2001), and p.S940L(c.2819C>T) (Greer et al 1999). The mutations p.A1132P(c.3394G>C) and the large deletion of the promoter region and of exons 1-10 have only been described in Greek patients (included in this report as #2, #3, #7).…”

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

“…The function of NPC1 is not fully understood, but NPC cells exhibit a trafficking defect at the late endosomal/lysosomal stage, as observed for cholesterol by using Filipin staining (32). Accumulation of Sph and lactosylceramide was previously visualized using fluorescent lipid analogs (33,34). Here, we used TFS to visualize Sph localization and trafficking.…”