Abstract
A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.
Highlights
A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNAA. nalysis of type I collagen synthesized by the proband’s fibroblasts showed excessive post-translational modification of cwl(1) chains along the entire length of the helix
Cell Culture and Biochemical Studies-Skin fibroblasts were obtained from the patient, his parents, and normal controls with informed consent and were grown under standard conditions
Osteogenesis imperfecta (01)‘is a genetic disease charac- incubated with ascorbic acid, P-aminopropionitrile, [3H]proline, and terized by fragility of bones
Summary
Structure of the Collagen Molecules Produced by the Proband's Fibroblasts-The proband's cells produced a population of normal type I collagen chains and a population of al(1)and a2(I) chains that migrated more slowly on SDS-. RNase A Mapping of a Mutation in the Pro-al(I, GeneFive overlapping subclones were constructed covering the helical and carboxyl-telopeptide regions of thepro-al(1) cDNA clones Hf404 and Hf677. These were used to generate antisense RNA probes for a study of mapping mutations in cases of perinatal lethal osteogenesis imperfecta. One subclone BX887 (Fig. 2a) when used as a probe in RNase A mapping experiments with RNA from the proband described here gave, inaddition to the 887-base-long fully protected fragment, two cleavage products of approximately 545 and 335 bases (Fig. 2b, lanes A ) , indicating a mismatch between the probe and the targeRt NA.
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