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.
|Original language||English (US)|
|Number of pages||13|
|Journal||Journal of Bone and Mineral Research|
|State||Published - Jul 1992|
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Orthopedics and Sports Medicine