Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV.
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.
Chipman
S D
SD
Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Shapiro
J R
JR
McKinstry
M B
MB
Stover
M L
ML
Branson
P
P
Rowe
D W
DW
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
D011347:Procollagen; D003094:Collagen; D004247:DNA; D000469:Alkaline Phosphatase