Biochemical analysis of type I collagen synthesized by cultured skin fibroblasts was performed to characterized the defect in a patient affected by lethal Osteogenesis Imperfecta (OI), a dominant heritable brittle bone disorder caused by mutations in one of the two genes coding for the α1 and α2 chains of type I collagen. The SDS-urea-PAGE of procollagen and collagen revealed a broad α1(I) chain, a normal α2(I) and anα2(I) migrating half way between α1 and α2. Procollagen and collagen of media and cell layers, synthesized in the presence ofαα′-dipyridyl, revealed both normal and slower α2(I) suggesting an insertion in COL1A2 gene. CNBr peptide digestion of collagen yielded overmodified α1(I) CB3 and CB7 peptides and a delayed migration of the α2(I) CB3-5 peptide. A delayed CB3-5 persisted also afterαα′-dipyridyl treatment, supporting an alteration ofα2(I) size. The mutation was localized, by this data, between aa 353-551 in α2(I) (CB 3-5). The sequence of this region revealed a G->A transition at nt 1671 in one allele, changing Gly421->Asp in oneα2(I) chain. Procollagen processing was normal. The Tm of the slowα2(I) collagen was 2¡C lower than the control, indicating a decrease in triple helix stability. The mutant protein was incorporated in the extracellular matrix synthesized by long term cultured fibroblasts.

In agreement with the regional model associating OI genotype and phenotype for the α2(I) chain, this mutation localizes at the boundary between the first non-lethal and lethal regions and further defines the 5′-end of the lethal region at the middle of exon 26. This is the first case, inα2(I) chain, of a single amino acid substitution resulting in a structural alteration of the collagen independent of excess posttranslational modification, suggesting the presence of a kink in the mutated α2 chains. This may compromise the helix folding and the interations of collagen fibrils with other extracellular matrix proteins.