TRACING THE PATHWAY BETWEEN MUTATION AND PHENOTYPE IN OSTEOGENESIS IMPERFECTA - ISOLATION OF MINERALIZATION-SPECIFIC GENES

The brittleness of bone in people with lethal (type II) osteogenesis i mperfecta, a heritable disorder caused by mutations in the type I coll agen genes, arises from the deposition of abnormal collagen in the bon e matrix, The inability of the abnormal collagen to participate in min eralization may be caused by its failure to interact with other bone p roteins, Here, we have designed a strategy to isolate the genes import ant for mineralization of collagen during bone formation, Cells isolat ed from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks, Chick skin fibroblasts seeded and cultured under the same conditions did no t mineralize. Using RT-PCR, we prepared short cDNAs (similar to 300 bp ) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells, Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvaria l cells but not to fibroblasts. Screening of 100,000 plaques of a chic k bone ZAP Express cDNA library with this pool of mineralizing-specifi c cDNAs identified ten clones which comprised full-length cDNAs for th e bone proteins osteopontin (eight of the ten positives), bone sialopr otein II (one of the ten positives), and cystatin (one of the ten posi tives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen, The pool of short cDNAs is lik ely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones. (C) 199 6 Wiley-Liss, Inc.