Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Abstract

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.Niemann-Pick disease type C (NP-C) is an autosomal recessive neurodegenerative disorder with heterogeneous manifestations. The classic clinical profile includes variable hepatosplenomegaly, vertical supra-nuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia (1). The metabolic lesion is characterized by lysosomal sequestration of low-density lipoprotein (LDL)-derived cholesterol leading to delayed cholesterol-mediated homeostatic responses (2, 3). Lysosomal cholesterol accumulation can be depicted cytochemically by the cholesterol-specific fluorescent dye, filipin. Normally, endocytosed LDL-derived cholesterol is mobilized from lysosomes to the endoplasmic reticulum for esterification. As a result, there is little free cholesterol accumulation in lysosomes detectable by filipin staining in normal cells. In contrast, in NP-C cells the lysosomal accumulation of the endocytosed LDL-derived free cholesterol results in a specific perinuclear filipin-staining pattern. Biochemically, the NP-C phenotype can most conveniently be monitored by LDL-induced cholesterol ester synthesis. Cholesterol ester synthesis is markedly stimulated by LDL in normal cells, but not in NP-C cells.

Role of Niemann-Pick Type C1 Protein in Intracellular Trafficking of Low Density Lipoprotein-derived Cholesterol

“…Pentchev and colleagues (1,2) undertook the positional cloning strategy and discovered the human NPC1 gene, thus providing a major breakthrough toward understanding the NPC disease at the molecular and cellular level (6). The final cloning work involved the identification of a 300-kb human genomic DNA containing the candidate NPC1 gene (7). This unique DNA was identified by its ability to complement the defect of a previously isolated Chinese hamster ovary (CHO) cholesterol-trafficking cell mutant, CT60 (8).…”

Niemann–Pick Type C Mutations Cause Lipid Traffic Jam

“…For these studies, NPC1 was transiently expressed in CT60 cells, a Chinese hamster ovary cell mutant in the same complementation class as NPC1 fibroblasts, which store LDL-cholesterol in perinuclear lysosomes (47,48). A plasmid expressing enhanced green fluorescent protein was co-transfected to identify transfected cells (34,40).…”

Localization of Niemann–Pick C1 protein in astrocytes: Implications for neuronal degeneration in Niemann– Pick type C disease

“…Chinese hamster ovary (CHO) cells from the mutant cell line CT60, which display an NP-C phenotype, were generously provided by T. Y. Chang (Dartmouth University, Hanover, NH). CT60 cells transfected with yeast artificial chromosome 911D5 (designated ''D5B5'' cells), which contains the NPC1 gene which has been shown to correct the NP-C phenotype, have been described (11). All cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin͞streptomycin.…”