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Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone fragility. Most cases of severe OI result from mutations in the coding region of the COL1A1 or COL1A2 genes yielding an abnormal collagen alpha chain. In contrast, many patients with mild OI show evidence of a null allele due to a premature stop mutation in the mutant RNA transcript. We have previously described a null allele arising from a splice donor mutation where the transcript containing the included intron was sequestered in the nucleus. Here we demonstrate that transcripts from null alleles arising from premature stop mutations are also present in the nucleus and absent in the cytoplasm. Using reverse transcriptase-PCR and single-strand conformational polymorphism of COL1A1 mRNA from patients with mild OI, we describe three patients with distinct null producing mutations identified from the mutant transcript within the nuclear compartment. A fourth patient with a Gly--->Arg expressed point mutation exhibits the mutant transcript in both compartments. Defining the distribution of allelic variants of COL1A1 mRNA in the nuclear and cytoplasmic compartments gives further insight into cell biology of OI and provides a strategy for investigating potential causes of a null allele.

Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COL1A1 gene. We studied fibroblasts established from a child with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1 (I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population was confined to the nuclear compartment of the cell, and contained the entire sequence of intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A transition was also identified in the genomic DNA. The retained intron contained an in-frame stop codon and introduced an out-of-frame insertion within the collagen mRNA producing stop codons downstream of the insertion. These changes probably account for the failure of the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript, this mutation did not result in production of a defective collagen pro alpha 1 (I) chain. Instead, the mild nature of the disease in this case reflects failure to process the defective mRNA and thus the absence of a protein product from the mutant allele.

Mutations affecting the pro alpha 1(I) or pro alpha 2(I) collagen genes have been identified in each of the major clinical types of osteogenesis imperfecta. This study reports the presence of a heritable connective tissue disorder in a family with an osteopenic syndrome which has features of mild osteogenesis imperfecta but was considered idiopathic osteoporosis in the proband. At age 38, while still premenopausal, she was found to have osteopenia, short stature, hypermobile joints, mild hyperelastic skin, mild scoliosis, and blue sclerae. There was no history of vertebral or appendicular fracture. Hip and vertebral bone mineral density measurements were consistent with marked fracture risk. Delayed reduction SDS-PAGE of pepsin-digested collagens from dermal fibroblast cultures demonstrated an anomalous band migrating between alpha 1(I) and alpha 1(III). This band merged with the normal alpha-chains upon prereduction, indicating an unexpected cysteine residue. Cyanogen bromide peptide mapping suggested that the mutation was in the smaller NH2-terminal peptides. cDNA was reverse transcribed from mRNA and amplified by the polymerase chain reaction. A basepair mismatch between proband and control alpha 1(I) cDNA hybrids was detected by chemical cleavage with hydroxylamine:piperidine. The cysteine substitution was thus localized to alpha 1(I) exon 9 within the cyanogen bromide 4 peptide. Nucleotide sequence analysis localized a G----T point mutation in the first position of helical codon 43, replacing the expected glycine (GGT) residue with a cysteine (TGT). The prevalence of similar NH2-terminal mutations in subjects with this phenotype which clinically overlaps idiopathic osteoporosis remains to be determined.