Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly 
inherited, genetically heterogeneous connective-tissue disorder. Mutations in 
the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified 
in several individuals. Most mutations affect either the triple-helical domain 
of the protein or the expression of one COL5A1 allele. We identified a novel 
splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of 
COL5A1, in one patient with EDS type I. The outcome of this mutation was 
complex: In the major product, both exons 5 and 6 were skipped; other products 
included a small amount in which only exon 5 was skipped and an even smaller 
amount in which cryptic acceptor sites within exon 5 were used. All products 
were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted 
and were incorporated into extracellular matrix, and the mutation resulted in 
dramatic alterations in collagen fibril structure. The two-exon skip occurred in 
transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, 
leaving a large (270 nt) composite exon that can be skipped in its entirety. The 
transcripts in which only exon 5 was skipped were derived from those in which 
intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in 
exon 5 occurred in transcripts in which intron 4 was removed subsequent to 
introns 5 and 6. These findings suggest that the order of intron removal plays 
an important role in the outcome of splice-site mutations and provide a model 
that explains why multiple products derive from a mutation at a single splice 
site.