An alpha2(I) glycine to aspartate substitution is responsible for the presence of a kink in type I collagen in a lethal case of osteogenesis imperfecta.

Type I collagen synthesized by cultured skin fibroblasts was analyzed biochemically and molecularly to characterize the defect in a patient affected by lethal Osteogenesis Imperfecta. The SDS-Urea-PAGE of procollagen and collagen revealed a broad alpha1(I) band, a normal alpha2(I) and another alpha2(I) band migrating equidistant between alpha1 and alpha2. When synthesized in the presence of alphaalpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylation, procollagen and collagen of media and cell layers contained both normal and slower alpha2(I), but only normal alpha1(I). The persistence of the two forms of alpha2(I) chains suggested a mutation in a COL1A2 gene. CNBr cleavage of collagen yielded overmodified alpha1(I) CB3 and CB7 peptides and delayed migration of the alpha2(I) CB3-5 peptide. A delayed CB3-5 was also found after alpha,alpha'-dipyridyl treatment. These data localized the mutation between aa 353 and 551 in alpha2(I) (CB3-5). Sequencing the subcloned alleles in this region revealed a G-->A transition at nt 1671 in one allele, changing Gly 421 to Asp in an alpha2(I) chain. The mutation was demonstrated to occur on the paternally derived allele, using a common C-->A polymorphism at alpha2(I) nt 1585 and by the presence of a rare variant, Arg618-->Gln (Phillips et al., 1990), in the paternal genomic DNA and the proband's mutant allele. Procollagen processing was normal. The Tm of the slow alpha2(I) collagen was 2 degrees C lower than the control, indicating decreased triple helix stability. Mutant collagen was incorporated in the extracellular matrix deposited by cultured fibroblasts. The dramatic delay in alpha2(I) electrophoretic mobility must be induced by the Gly-->Asp substitution, since the Arg-->Gln variant causes only mild electrophoretic delay. Substantial delay in gel mobility even in the absence of overmodification suggested the presence of a kink in the mutated alpha2(I) chains. Rotary shadowing electron microscopy of secreted fibroblast procollagen confirmed the presence of a kink in the region of the helix containing the glycine substitution. The kinking of the collagen helix occurs in the absence of dimer formation. Kinking may interfere with normal helix folding, as well as with the interactions of collagen fibrils with the collagenous and non-collagenous extracellular matrix proteins.