title: Expression of mutant alpha (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 alpha 2(I)-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 alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-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 alpha 2(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 alpha 2(I)-collagen allele compared to dermal fibroblasts.
authors.affiliation: Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: S D
authors.initials: SD
authors.lastname: Chipman
authors.forename: J R
authors.initials: JR
authors.lastname: Shapiro
authors.forename: M B
authors.initials: MB
authors.lastname: McKinstry
authors.forename: M L
authors.initials: ML
authors.lastname: Stover
authors.forename: P
authors.initials: P
authors.lastname: Branson
authors.forename: D W
authors.initials: DW
authors.lastname: Rowe
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism*; D010013:Osteogenesis Imperfecta / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000096:biosynthesis*; D014158:Transcription, Genetic
chemical_list: D011347:Procollagen; D003094:Collagen; D004247:DNA; D000469:Alkaline Phosphatase