Substitution of arginine for glycine 664 in the collagen alpha 1(I) chain in lethal perinatal osteogenesis imperfecta. Demonstration of the peptide defect by in vitro expression of the mutant cDNA.

Structurally abnormal type I collagen was identified in tissues and cultured fibroblasts from a case of lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I) chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution (Bateman, J. F., Mascara, T., Chan, D., and Cole, W. G. (1987) J. Biol. Chem. 262, 4445-4451). Sequencing of cloned alpha 1(I) cDNAs prepared using mRNA from the patient's fibroblasts demonstrated that one clone had a single base substitution of A for G which resulted in the substitution of arginine for glycine 664 within the alpha 1(I)CB7 peptide. To determine whether this mutation was responsible for the peptide map abnormality, in vitro transcription of mRNA from the mutant cDNA was performed using an SP6 vector system. The mRNA was then translated into mutant protein in a rabbit reticulocyte lysate. Peptide analysis of the protein produced from the mutant cDNA demonstrated the same altered charge distribution of the alpha 1(I)CB7 peptide as observed with tissue- and cell-derived mutant collagen peptides. This finding confirmed that the arginine for glycine 664 sequence abnormality defined in the cDNA clone was the mutation causing the observed protein peptide map defect. This mutation is consistent with the functional abnormalities of collagen observed in this case such as reduced helical stability, reduced secretion, increased degradation, and excessive posttranslational modification of lysine.