Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant

D. Weil, M. Bernard, N. Combates, Mary Wirtz, D. W. Hollister, B. Steinmann, F. Ramirez

Research output: Contribution to journalArticle

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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-α2(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-α2(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-α2(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.

Original languageEnglish (US)
Pages (from-to)8561-8564
Number of pages4
JournalJournal of Biological Chemistry
Volume263
Issue number18
StatePublished - 1988
Externally publishedYes

Fingerprint

Ehlers-Danlos Syndrome
RNA Precursors
Exons
Collagen
Mutation
Base Pairing
RNA Splice Sites
Complementary DNA
Genes
Fibroblasts
Introns
Clone Cells
Procollagen
Messenger RNA
Cloning
Gene Deletion
Polymorphism
Oligomers
Organism Cloning
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Weil, D., Bernard, M., Combates, N., Wirtz, M., Hollister, D. W., Steinmann, B., & Ramirez, F. (1988). Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant. Journal of Biological Chemistry, 263(18), 8561-8564.

Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant. / Weil, D.; Bernard, M.; Combates, N.; Wirtz, Mary; Hollister, D. W.; Steinmann, B.; Ramirez, F.

In: Journal of Biological Chemistry, Vol. 263, No. 18, 1988, p. 8561-8564.

Research output: Contribution to journalArticle

Weil, D, Bernard, M, Combates, N, Wirtz, M, Hollister, DW, Steinmann, B & Ramirez, F 1988, 'Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant', Journal of Biological Chemistry, vol. 263, no. 18, pp. 8561-8564.
Weil, D. ; Bernard, M. ; Combates, N. ; Wirtz, Mary ; Hollister, D. W. ; Steinmann, B. ; Ramirez, F. / Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant. In: Journal of Biological Chemistry. 1988 ; Vol. 263, No. 18. pp. 8561-8564.
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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-α2(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-α2(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-α2(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.",
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AU - Ramirez, F.

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