title: Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant.
abstract: Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.
authors: Department of Microbiology and Immunology, Morse Institute of Molecular Genetics, State University of New York, Health Science Center at Brooklyn 11203.
authors: D
authors: D
authors: Weil
authors: M
authors: M
authors: Bernard
authors: N
authors: N
authors: Combates
authors: M K
authors: MK
authors: Wirtz
authors: D W
authors: DW
authors: Hollister
authors: B
authors: B
authors: Steinmann
authors: F
authors: F
authors: Ramirez
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D004535:Ehlers-Danlos Syndrome / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D005796:Genes; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger; D012867:Skin / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D003094:Collagen