Osteogenesis imperfecta is a heritable disease of connective tissue that results from a wide variety of mutations of type I collagen. This grant outlines a series of protocols designed to define these abnormalities at the molecular level. It is based on the assumption that it is easier to define the mutations in collagen by studying the mRNA rather than its very complex gene. Fibroblasts derived from patients with 0I will be used as a source of RNA. Structural mutation of the alpha 1 or alpha 2 collegen mRNA will be localized using a series of end-labeled S1 probes that encompass the entire mRNA. The probes will be derived from single-stranded cDNAs cloned in M-13. The exact mutation will be defined by dideoxy sequencing of an extended cDNA initiated from a synthetic oligonucleotide primer complementary to a region 3' to the region suggested of having the abnormality as dictated by the S-1 protection experiments. Another oligo-nucleotide coding for the abnormal sequence will be constructed and used as a mutation specific hybridization probe to precisely localize the mutation to the gene. Mutations which alter the steady state level of collagen mRNA will be tested for an error of RNA processing using a novel application of the M-13 derived hybridization probe. This will involve an S-1 protection protocol to detect an accumulation of a specific unspliced mRNA, measurement of half-life of a normal and mutant cytoplasmic mRNA and solution hybridization of radiolabeled RNA from in vitro transcribed muclei. The methods that are developed in this grant will have general applicability to a wide variety of mutations involving type I collagen. The mutation-specific oligonucleotides provide the means to rapidly detect a subtle mutation in genomic DNA. It has the potential to become a very powerful adjuvant for the clinical geneticist who must counsel a family with a connective tissue disorder.
