An alpha 2(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 bioch emically and molecularly to characterize the defect in a patient affected b y lethal Osteogenesis Imperfecta. The SDS-Urea-PAGE of procollagen and coll agen revealed a broad alpha 1(I) band, a normal alpha 2(I) and another alph a 2(I) band migrating equidistant between al and a2. When synthesized in th e presence of alpha alpha'-dipyridyl, an inhibitor of prolyl and lysyl hydr oxylation, procollagen and collagen of media and cell layers contained both normal and slower alpha 2(I), but only normal alpha 1(I). The persistence of the two forms of alpha 2(I) chains suggested a mutation in a COL1A2 gene . CNBr cleavage of collagen yielded overmodified alpha 1(I) CB3 and CB7 pep tides and delayed migration of the alpha 2(I) CB3-5 peptide. A delayed CB3- 5 was also found after alpha,alpha'-dipyridyl treatment. These data localiz ed the mutation between aa 353 and 551 in alpha 2(I) (CB3-5). Sequencing th e subcloned alleles in this region revealed a G-->A transition at nt 1671 i n one allele, changing Gly 421 to Asp in an alpha 2(I) chain. The mutation was demonstrated to occur on the paternally derived allele, using a common G-->A polymorphism at alpha 2(I) nt 1585 and by the presence of a rare vari ant, Arg618-->Gln (Phillips et al., 1990), in the paternal genomic DNA and the proband's mutant allele. Procollagen processing was normal. The T-m of the slow alpha 2(I) collagen was 2 degrees C lower than the control, indica ting decreased triple helix stability. Mutant collagen was incorporated in the extracellular matrix deposited by cultured fibroblasts. The dramatic delay in alpha 2(I) electrophoretic mobility must be induced b y the Gly-->Asp substitution, since the Arg-->Gln variant causes only mild electrophoretic delay. Substantial delay in gel mobility even in the absenc e of overmodification suggested the presence of a kink in the mutated alpha 2(I) chains. Rotary shadowing electron microscopy of secreted fibroblast p rocollagen confirmed the presence of a kink in the region of the helix cont aining the glycine substitution. The kinking of the collagen helix occurs i n the absence of dimer formation. Kinking may interfere with normal helix f olding, as well as with the interactions of collagen fibrils with the colla genous and non-collagenous extracellular matrix proteins.