While advances in molecular biology have led to better understanding and diagnosis of many genetic diseases, many human skeletal dysplasias remain a poorly understood group of disorders at both biochemical and molecular levels. This project will focus on the isolation of a murine X-linked locus, bare patches (Bpa), which produces a complex developmental phenotype and is likely homologous to human X-linked dominant chondrodysplasia punctata (CDPX2). Genetic mapping in an interspecific backcross and physical mapping using pulsed field gel (PFG) electrophoresis have identified loci, Gabra3 and CamLl, which flank Bpa and are 0.8 + 0.6 cM and <3 million base pairs apart. Overlapping yeast artificial chromosome (YAC) clones will be isolated that span the region to which Bpa maps using a murine YAC library prepared in the laboratory of Dr. Shirley Tilghman. Murine genomic clones will be isolated for existing probes in the region. Additional random murine probes will be generated from mouse X:human hybrids using the polymerase chain reaction (PCR) and DNA primers of dispersed murine repetitive sequences. Portions of genomic clones from the region will be sequenced to enable the design of PCR primers to screen the murine YAC library. The ends of YACs will be isolated and mapped on PFGs and the interspecific backcross to develop overlaps spanning the region between Gabra3 and CamLl. YACs that cosegregate with Bpa in the interspecific backcross will be screened for conservation among species (""""""""zoo blots"""""""") and patterns of expression on Northern blots. The normal and affected X chromosomes from Bpa mice and CDPX2 females separated in somatic cell hybrids will be examined for evidence of submicroscopic deletions which would further localize the gene(s). Final proof of the identity of the gene will rely on the demonstration of the mutation in the Bpa gene or cDNA. Once the murine locus is cloned, studies aimed at understanding the biology of the Bpa gene product will be initiated and attempts made to isolate the homologous human gene.
