A 5' SPLICE-SITE MUTATION AFFECTING THE PRE-MESSENGER-RNA SPLICING OF2 UPSTREAM EXONS IN THE COLLAGEN COL1A1 GENE - EXON-8 SKIPPING AND ALTERED DEFINITION OF EXON-7 GENERATES TRUNCATED PRO-ALPHA-1(I) CHAINS WITH A NONCOLLAGENOUS INSERTION DESTABILIZING THE TRIPLE-HELIX

A heterozygous de novo G to A point mutation in intron 8 at the +5 pos ition of the splice donor site of the gene for the pro alpha 1(I) chai n of type I procollagen, COLIA1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next u pstream intron, intron 7, leading to re-definition of the 3' limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resul ted in the maintenance of the correct codon reading frame, the resulta nt pro alpha 1(I) chain contained a short non-collagenous 32-amino-aci d sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequen ce motif. At the protein level, the mutant alpha 1(I) chain was reveal ed by digestion with pepsin, which cleaved the mutant procollagen with in the protease-sensitive noncollagenous insertion, producing a trunca ted alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-t erm culture with ascorbic acid, which stimulates the formation of a ma ture crosslinked collagen matrix, and in tissues, there was no evidenc e of the mutant chain, suggesting that during matrix formation the mut ant chain was unable to be stably incorporated into the matrix and was degraded proteolytically.