A de novo G+1 → a mutation at the α2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI Type IV proband

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Abstract
We have investigated the procollagen, collagen, α2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two α2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast α2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of α,α′‐dipyridyl. The α2(I) cyanogen bromide peptide CB 4‐2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating α2(I) chain was reduced 6°C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to α2(I) nt 236–1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16‐19. The region which hybridized to the riboprobe was amplified using RNA‐PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16‐17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15‐17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+1 → A mutation at the exon 16 donor site in one allele. The mutation eliminates a Styl site. Digestion of PCR fragments amplified from the proband and parental WBC DNA revealed that only the proband had the undigested mutant fragment.