SUBSTANTIAL NARROWING OF THE NIEMANN-PICK-C CANDIDATE INTERVAL BY YEAST ARTIFICIAL CHROMOSOME COMPLEMENTATION

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulatio n of unesterified cholesterol and delayed induction of cholesterol-med iated homeostatic responses. This cellular phenotype is identifiable c ytologically by filipin staining and biochemically by measurement of l ow-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellul ar phenotype, was used as the recipient for a complementation assay af ter somatic cell fusions with normal and RTP-C murine cells suggested that this Chinese hamster ovary cell fine carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interv al for NP-C, three overlapping yeast artificial chromosomes (YACs) spa nning the 1 centimorgan human NP-C interval were introduced stably int o CT60 cells and analyzed for correction of the cellular phenotype. On ly YAC 911D5 complemented the NP-C phenotype, as evidenced by cytologi cal 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 91 1D5 was integrated at a single site per CT60 genome. These data substa ntially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is t he first demonstration of YAC complementation as a valuable adjunct st rategy for positional cloning of a human gene.