Expression of mutant α(I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV

Abstract

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of α₂(D-collagen. The phenotype of the bone cell cultures was defined by a 3–4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened α₂(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened α₁(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast α-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of α₂(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T→G point mutation in the second position of the 5′ splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant α₂(I)-collagen allele compared to dermal fibroblasts.