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: D Weil, M Bernard, N Combates, M K Wirtz, D W Hollister, B Steinmann, F Ramirez
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 263 Issue 18 Pg. 8561-4 (Jun 25 1988) ISSN: 0021-9258 [Print] United States
• PMID: 2454224 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• RNA Precursors
• RNA, Messenger
• Poly A
• RNA
• Collagen

Topics

• Alleles
• Amino Acid Sequence
• Base Sequence
• Cells, Cultured
• Collagen (genetics)
• Ehlers-Danlos Syndrome (genetics, metabolism)
• Exons
• Fibroblasts (metabolism)
• Genes
• Genetic Variation
• Humans
• Molecular Sequence Data
• Mutation
• Poly A (genetics, isolation & purification)
• RNA (genetics, isolation & purification)
• RNA Precursors (genetics)
• RNA Splicing
• RNA, Messenger
• Skin (metabolism)