An RT-PCR-SSCP screening strategy for detection of mutations in the gene encoding the alpha 1 chain of type I collagen: application to four patients with osteogenesis imperfecta.

A strategy has been developed to allow the screening for mutations in the cDNA of the type I collagen alpha 1 chain. The method involves polymerase chain reaction amplification of four overlapping segments covering the majority of the sequence coding for alpha 1(I) collagen. Four or five restriction endonuclease digestions are performed for each segment and digested samples subjected to single strand conformation polymorphism analysis. Mutations are finally defined by DNA sequence analysis. The application to the detection of mutations in four patients with lethal and non-lethal forms of osteogenesis imperfecta is described. In each of these patients, the heterozygous mutation identified resulted in substitution of different glycine residues with serines. In addition to these deleterious mutations, several novel silent variants were characterised. The strategy provides a rapid approach to screening for mutations and polymorphisms in alpha 1(I) collagen cDNA and is of general interest in scanning large regions of DNA for sequence changes.