Positional cloning utilizing genomic DNA microarrays: The Niemann-Pick type C gene as a model system

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig, Here we describe the application of DNA microarray technology to a defined genomic region (:physical map) to identify: ii) exons without a priori sequence data and (ii) the disease ge ne based on differential gene expression in a recessive disorder. The feasi bility was tested using resources from the positional cloning of the Neiman n-Pick Type C: (NP-C) disease gene, NPC1. To identify NPC1 exons and optimi ze the technology, an array was generated from genomic fragments of the 110 -kb bacterial artificial chromosome, 108N2, which encodes NPC1, First, as a test case fur blindly identifying exons, fluorescently labeled NPC1 cDNA i dentified 108N2 fragments that contained NPC1 exons, many of which also con tained intronic sequences and could be used to determine part of the NPC1 g enomic structure, Second, to demonstrate that the NPC1 disease gene could b e identified based upon differential gene expression, subarrays of 108N2 fr agments were hybridized with fluorescently labeled cDNA probes generated fr om total RNA from hamster cell lines differentially expressing NPC1, A prob e derived from the NP-C cell line CT60 did not detect NPC1 exons or other g enomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell Line 911D5A13, which was derive d from CT60, and expressed NPC1 as a consequence of stable transduction wit h a YAC that contains N1DC1 and encompasses 108N2, Thus, the array technolo gy identified NPC1 as a candidate gene based on a physical contig and diffe rential NPC1 expression between NP-C and non-NP-C cells, This technique sho uld facilitate gene identification when a physical contig exists For a regi on of interest and mutations result in changes in the mRNA level of the dis ease gene or portions thereof. (C) 2000 Academic Press.