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A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.
Mol Genet Metab. 2005 Jul; 85(3):213-9.MG

Abstract

The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518+5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32-98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518+5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518+5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence.

Authors+Show Affiliations

Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo, Kobe 650-0017, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Case Reports
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15979033

Citation

Thi Tran, Hoai Thu, et al. "A G-to-A Transition at the Fifth Position of Intron-32 of the Dystrophin Gene Inactivates a Splice-donor Site Both in Vivo and in Vitro." Molecular Genetics and Metabolism, vol. 85, no. 3, 2005, pp. 213-9.
Thi Tran HT, Takeshima Y, Surono A, et al. A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro. Mol Genet Metab. 2005;85(3):213-9.
Thi Tran, H. T., Takeshima, Y., Surono, A., Yagi, M., Wada, H., & Matsuo, M. (2005). A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro. Molecular Genetics and Metabolism, 85(3), 213-9.
Thi Tran HT, et al. A G-to-A Transition at the Fifth Position of Intron-32 of the Dystrophin Gene Inactivates a Splice-donor Site Both in Vivo and in Vitro. Mol Genet Metab. 2005;85(3):213-9. PubMed PMID: 15979033.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro. AU - Thi Tran,Hoai Thu, AU - Takeshima,Yasuhiro, AU - Surono,Agus, AU - Yagi,Mariko, AU - Wada,Hiroko, AU - Matsuo,Masafumi, PY - 2005/01/08/received PY - 2005/03/11/revised PY - 2005/03/12/accepted PY - 2005/6/28/pubmed PY - 2005/9/15/medline PY - 2005/6/28/entrez SP - 213 EP - 9 JF - Molecular genetics and metabolism JO - Mol Genet Metab VL - 85 IS - 3 N2 - The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518+5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32-98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518+5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518+5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence. SN - 1096-7192 UR - https://www.unboundmedicine.com/medline/citation/15979033/A_G_to_A_transition_at_the_fifth_position_of_intron_32_of_the_dystrophin_gene_inactivates_a_splice_donor_site_both_in_vivo_and_in_vitro_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7192(05)00102-2 DB - PRIME DP - Unbound Medicine ER -