Physical mapping of human 12p11.2-q13.1 and identification of lipoma breakpoints at 12q13

Abstract

One of the major objectives of the Human Genome Project is the development of physical maps for all human chromosomes with an average marker resolution between 100 and 150 kb. To contribute to this effort we have generated a physical map for human 12p11.2-q13.1, a region that encompasses 13.9 cM on the genetic map and that we estimate to cover 20 Mb of DNA and approximately 15% of human chromosome 12. Yeast artificial chromosomes (YACs) were assembled into contiguous sets of overapping clones by sequence-tagged site (STS)-content mapping. Utilization of a number of publicly available mapping resources and fluorescence in situ hybridization of individual YAC clones from the physical map integrated our map with three genetic maps, two radiation hybrid maps and the cytogenetic map of human chromosome 12. The physical map contains a total of 183 YACs and 198 markers, of which 171 were unambiguously resolved. The average marker resolution for the DNA covered by contigs is 87 kb, at an average clone depth per marker of 8.6. The resolution for the entire 20 Mb region is approximately 116 kb.;One of the major advantage of physical maps in positional cloning efforts is that they provide an immediate access to chromosomal regions, facilitating the identification and isolation of disease genes. We have taken advantage of the YAC contig map developed for human 12p11.2-q13.1 to determine whether the broad breakpoints assignment to 12q13-q15 of clinically and histologically identical lipomas was due to cytogenetic discrepancies or whether 12q rearrangements in these lesions have indeed molecularly distinct breakpoints. Fluorescence in situ hybridization (FISH) with individual YACs from the physical map enabled us to localize breakpoints to 12q13 in 4/14 (28%) lipomas and identify the corresponding YAC clones involved in these rearrangements. As a step towards the molecular definition of these breakpoints we constructed a high-resolution bacterial clone map in the breakpoint region of one of the four lipomas, T93608. FISH analysis of individual PAC and cosmid clones from this map identified one PAC to span the breakpoint. This PAC was subjected to gene isolation efforts in form of Alu-splice PCR and sample sequencing.