Structural and functional characterization of a splicing mutation in the pro-alpha 2(I) collagen gene of an Ehlers-Danlos type VII patient.

A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.