AbstractA single base mismatch was detected by a chemical cleavage method in heteroduplexes formed between patient mRNA and a control collagen α2(I) cDNA probe in a case of osteogenesis imperfecta type II. The region of the mRNA mismatch was amplified using the polymerase chain reaction, cloned and sequenced. A heterozygous point mutation of G to C at base pair 1,774 of the collagen α2(I) mRNA resulted in the substitution of glycine with arginine at amino acid position 457 of the helix. Type I collagen of α1(I)‐ and α2(I)‐chains from the patient migrated slowly on electrophoresis due to increased levels of posttranslational modification of lysine. The parents' fibroblast collagen did not contain the mRNA mismatch and the collagens showed normal electrophoretic behaviour. Two‐dimensional electrophoresis of the CNBr peptides from the patient's collagen confirmed the excessive posttranslational modification of the α1(I)‐ and α2(I)‐chains in the CNBr peptides N‐terminal to the mutation due to disruption of the obligatory Gly‐X‐Y triplet repeat of the helix. The mutation led to reduced procollagen secretion and helix destabilization as evidenced by a decreased thermal stability. These data lend further support to the accumulating evidence that type I collagen α2(I) glycine substitution mutations result in the same spectrum of clinical severity as those in the α1(I)‐chain. The disruptive effect of the glycine mutations seems to be largely dependent on the nature of the substituted amino acid, the position in the α‐chain of the mutation, and the nature of the local surrounding amino acid sequence, rather than whether the mutation resides in the α1(I)‐ or α2(