The overall objective of the proposed research is to identify the gene defect responsible for pycnodysostosis (Pycno) and to delineate the normal role of that gene in bone formation and metabolism. Pycno, a rare, autosomal recessive skeletal dysplasia, is characterized by short stature, osteosclerosis, acro-osteolysis, bone fragility, clavicular dysplasia and skull deformities. Ultrastructural studies of bone suggest a functional abnormality in organic matrix resorption by osteoclasts. However, the etiology of this debilitating disease remains unknown. Therefore, a positional cloning strategy was undertaken by the applicants. In those studies, the Pycno gene was localized to a 4 cM region at 1q21 between D1S442 and D1S305; it has been further narrowed to a 1 cM region with supplemental data. To facilitate the isolation of the Pycno gene, the present application proposes to: 1) further refine the critical region by genotyping these families with additional simple tandem repeats (STRs), by developing novel STRs from YACs cosmids, and by finding and analyzing new Pycno families; 2) find genes which localize to the Pycno critical region by dbEST searches, exon trapping and direct cDNA selection, and then evaluate them for mutations by molecular analyses (Southern, Northern, SSCP and exon sequencing); 3) complete the YAC contig by end-cloning using the vectorette method, by mapping new markers (STRs, ESTs and STSs) including the YAC end-clones, and by assaying for pericentromeric alpha-repeats, and identify cosmid clones mapping to the Pycno region which are needed for STR development and gene discovery; 4) probe peripheral monocytes by assaying cytokine secretion, phagocytosis and kill function, and relevant protease activities; and 5) assay collagen protein production and secretion from skin fibroblasts. The latter two Specific Aims seek to identify the putative functional Pycno defect, which will inform the search for the Pycno gene. The identification of the gene defect causing Pycno should/may provide insights into its pathophysiology, as well as the normal mechanism(s) of bone resorption and metabolism.
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