title: 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.
authors.affiliation: Thoracic Diseases Research Unit, Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: X
authors.initials: X
authors.lastname: Sun
authors.forename: D L
authors.initials: DL
authors.lastname: Marks
authors.forename: W D
authors.initials: WD
authors.lastname: Park
authors.forename: C L
authors.initials: CL
authors.lastname: Wheatley
authors.forename: V
authors.initials: V
authors.lastname: Puri
authors.forename: J F
authors.initials: JF
authors.lastname: O'Brien
authors.forename: D L
authors.initials: DL
authors.lastname: Kraft
authors.forename: P A
authors.initials: PA
authors.lastname: Lundquist
authors.forename: M C
authors.initials: MC
authors.lastname: Patterson
authors.forename: R E
authors.initials: RE
authors.lastname: Pagano
authors.forename: K
authors.initials: K
authors.lastname: Snow
mesh_terms: D000483:Alleles; D015703:Antigens, CD; D001692:Biological Transport; D001896:Boron Compounds; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008078:Cholesterol, LDL / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D007790:Lactosylceramides / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D010641:Phenotype; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D016037:Single-Blind Method; D013107:Sphingolipids / Q000378:metabolism*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015703:Antigens, CD; D001896:Boron Compounds; D002352:Carrier Proteins; D008078:Cholesterol, LDL; D047908:Intracellular Signaling Peptides and Proteins; D007790:Lactosylceramides; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D013107:Sphingolipids; C009744:CDw17 antigen; D003545:Cysteine