A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide in a patient with Ehlers-Danlos syndrome type VII.

An unusual splicing mutation has been characterized in the pro alpha 1(I) collagen gene of a sporadic case of Ehlers-Danlos Syndrome Type VII. Cloning of primer extended cDNA in conjunction with R-looping experiments established that nearly half of the pro alpha 1(I) collagen gene transcripts are abnormally spliced, for they lack exon 6 sequences. Analysis of cloned genomic fragments revealed that one of the proband's alleles displays the substitution of an A for a G in the last nucleotide of exon 6. The change converts the normal Met (ATG) codon to Ile (ATA) and, in addition, obliterates a NcoI restriction site. The latter event was exploited to demonstrate the de novo nature of the mutation since DNA from the unaffected parents was fully digested with the enzyme, after in vitro amplification by the polymerase chain reaction. Further confirmation of the missplicing was obtained by transient expression into animal cells of allelic minigene constructs. Finally, Western blot analysis of cyanogen bromide cleaved collagen and nucleotide sequencing of appropriately selected cDNA clones demonstrated the production of relatively low amounts of correctly spliced molecules harboring the Ile substitution, as well.