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

Thoracic Diseases Research Unit, Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
The American Journal of Human Genetics (Impact Factor: 10.99). 07/2001; 68(6):1361-72. DOI: 10.1086/320599
Source: PubMed

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.

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Available from: Marc Patterson, Jul 17, 2014
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    • "Mutations in the NPC1 gene occur in 95% of cases [3] [4]. Niemann-Pick disease is fatal [5], hereditary, and autosomal recessive [2] [6] [7]. The disease occurs in 1/120,000 live births [4]. "
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    ABSTRACT: Objective. This case report describes the changes in caries risk and activity and dental treatment of a 9-year-old patient who presented with signs and symptoms of Niemann-Pick disease type C (NPC). Treatment. The preventive dental treatment included instructions to caregivers for oral hygiene and diet. A calcium hydroxide pulpotomy and restorative dental treatments were performed in a dental office with desensitization techniques and behavioral management. The patient was attended every 3 months for the control of dental plaque biofilm, for topical fluoride application, and for observing the pulpotomized tooth. Results. The bacterial plaque biofilm was being adequately controlled by the caregiver. After 2 years, the clinical and radiographic examination of the pulpotomized tooth showed the absence of internal root resorption and bone rarefaction, and clinical examination showed tooth sensitivity, dental pain, and gingival swelling. Conclusion. The pulpotomy prevented clinical and radiographic success. Dentists must be aware of and be able to identify systemic and local aspects associated with caries risk of children with NPC disease. Furthermore, dentists must employ stringent preventive measures and provide instructions to caregivers to reduce caries risk.
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    • "Moreover, in a more recent study, the IGL of a Npc1 −/− cat was found to be thinner compared to that of wild-type controls, opening the possibility that GNs were generated in a lower number during postnatal cerebellar development or died because of an increased level of apoptosis (Vite et al., 2008). An additional line of evidence enlightening the possible contribution of a GN defect to NPC neuropathology derives from a recent study showing that the synaptic transmission from parallel fibers to PCs is increased and that long-term depression (LTD) is deficient in 3- week-old Npc1 −/− mice (Sun et al., 2001). "
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    ABSTRACT: In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1−/− mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%–25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8–PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1−/− mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1−/− mice. A single injection of hydroxypropyl-β-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.
    Neurobiology of Disease 06/2014; 70(100). DOI:10.1016/j.nbd.2014.06.012 · 5.20 Impact Factor
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    • "(Runz et al. 2008), most of them are present in single families. Only three relative frequent mutations have been found within distinct patient groups: the p.G992W, almost exclusively seen in Acadian patients from Nova Scotia (Greer et al. 1998), the p.P1007A found in about 15% of mutated alleles in different European populations (Ribeiro et al. 2001; Fancello et al. 2009) and the p.I1061T that accounts for 15–20% of mutated alleles in Western Europe and USA (Millat et al. 1999; Sun et al. 2001; Park et al. 2003; Millat et al. 2005). However, a study performed in 44 Italian NPC patients showed that the p.I1061T mutation is much less common in Italy, representing only a 4.7% of the NPC1 alleles (Fancello et al. 2009). "
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    ABSTRACT: Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F, p.V1165M, and p.I1061T, the increment of NPC1 mutant protein resulted in an improvement of the intracellular trafficking of cholesterol and GM1. These findings showed that it is possible to correct the NPC cellular phenotype by increasing the amount of endogenous NPC1 mutated protein, suggesting that at least some NPC1 mutations might be potentially rescued by small molecules-based chaperone therapy.
    01/2012; 2:59-69. DOI:10.1007/8904_2011_49
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