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The molecular defect in a patient with a moderately severe form of osteogenesis imperfecta was characterized by nuclease S1 mapping. Single-stranded 5' and 3' end-labeled DNA probes coding for 80% of the carboxyl-propeptide of the pro alpha 2(I) collagen gene were hybridized to mRNA isolated from cultured fibroblasts of the patient and his parents. Nuclease S1 digestion revealed a homozygous mutation in the patient and a heterozygous pattern in the consanguineous parents. As a result of the defect in the gene, none of the pro alpha 2(I) chains synthesized by the patient's fibroblasts were incorporated into a type I procollagen heterotrimer consisting of two pro alpha 1(I) chains and one pro alpha 2(I) chain. Cultured skin fibroblasts from the patient have previously been shown to secrete only pro alpha 1(I) trimers. As shown here, fibroblasts from both parents, who do not have osteogenesis imperfecta, secrete both pro alpha 1(I) trimers and normal type I procollagen. A further observation was that synthesis of pro alpha 2(I) chains was decreased in fibroblasts from the patient and his parents. The decrease in the synthesis of pro alpha 2(I) chains is not caused by decreased transcription of the pro alpha 2(I) collagen alleles, since the pro alpha 1(I)/pro alpha 2(I) mRNA ratios were normal in the patient and his parents.

Osteogenesis imperfecta (OI), a brittle-bone disorder, constitutes a major group of the inherited diseases of connective tissue. We have been studying an autosomal recessive form of OI in which the severely affected patient has inherited two abnormal pro-alpha 2(I) collagen alleles from consanguinous parents. Previously, nuclease S1 mapping was employed to localize a defect in the mRNA coding for the pro-alpha 2(I) collagen carboxyl-propeptide. The mutation prevents incorporation of pro-alpha 2(I) chains into the normal type I procollagen heterotrimer resulting in secretion of only pro-alpha 1(I) homotrimers. Here we report complete characterization of the corresponding region of the altered gene. Polyacrylamide gel electrophoresis and Southern blot hybridization showed a small homozygous deletion in the pro-alpha 2(I) collagen gene of the patient and a heterozygous pattern in both parents. Genomic cloning of the patient's DNA revealed a four nucleotide frameshift deletion in exon 1 near the end of translation which apparently instigates use of a new termination codon four nucleotides 3' to the original site. The mutation identified in this OI patient directly demonstrates the critical role of the carboxyl-propeptides in chain selection and assembly during the biosynthesis of procollagen.