title: Order of intron removal influences multiple splice outcomes, including a two-exon skip, in a COL5A1 acceptor-site mutation that results in abnormal pro-alpha1(V) N-propeptides and Ehlers-Danlos syndrome type I.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Takahara
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schwarze
authors.forename: Yasutada
authors.initials: Y
authors.lastname: Imamura
authors.forename: Guy G
authors.initials: GG
authors.lastname: Hoffman
authors.forename: Helga
authors.initials: H
authors.lastname: Toriello
authors.forename: Lynne T
authors.initials: LT
authors.lastname: Smith
authors.forename: Peter H
authors.initials: PH
authors.lastname: Byers
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Greenspan
mesh_terms: D000483:Alleles; D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D002675:Child, Preschool; D024062:Collagen Type V / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D004535:Ehlers-Danlos Syndrome / Q000145:classification / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005091:Exons / Q000235:genetics*; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology; D005787:Gene Frequency; D006801:Humans; D007438:Introns / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011110:Polymorphism, Genetic; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics
chemical_list: D024062:Collagen Type V; D011498:Protein Precursors; D022821:RNA Splice Sites