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In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene.
J Med Genet. 2009 Aug; 46(8):542-7.JM

Abstract

BACKGROUND

Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene.

OBJECTIVE

To identify elements determining alternative splicing pathways in intron +1G-->A mutations of the dystrophin gene.

RESULTS

We found that exon 25 is spliced out in the +1G-->A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G-->A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G-->A mutation of intron 25.

CONCLUSION

It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G-->A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

Authors+Show Affiliations

Department of Pediatrics, Kobe University Graduate School of Medicine, Chuo, Kobe 6500-017, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

19001018

Citation

Habara, Y, et al. "In Vitro Splicing Analysis Showed That Availability of a Cryptic Splice Site Is Not a Determinant for Alternative Splicing Patterns Caused By +1G-->A Mutations in Introns of the Dystrophin Gene." Journal of Medical Genetics, vol. 46, no. 8, 2009, pp. 542-7.
Habara Y, Takeshima Y, Awano H, et al. In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene. J Med Genet. 2009;46(8):542-7.
Habara, Y., Takeshima, Y., Awano, H., Okizuka, Y., Zhang, Z., Saiki, K., Yagi, M., & Matsuo, M. (2009). In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene. Journal of Medical Genetics, 46(8), 542-7. https://doi.org/10.1136/jmg.2008.061259
Habara Y, et al. In Vitro Splicing Analysis Showed That Availability of a Cryptic Splice Site Is Not a Determinant for Alternative Splicing Patterns Caused By +1G-->A Mutations in Introns of the Dystrophin Gene. J Med Genet. 2009;46(8):542-7. PubMed PMID: 19001018.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene. AU - Habara,Y, AU - Takeshima,Y, AU - Awano,H, AU - Okizuka,Y, AU - Zhang,Z, AU - Saiki,K, AU - Yagi,M, AU - Matsuo,M, Y1 - 2008/11/10/ PY - 2008/11/13/pubmed PY - 2009/11/3/medline PY - 2008/11/13/entrez SP - 542 EP - 7 JF - Journal of medical genetics JO - J Med Genet VL - 46 IS - 8 N2 - BACKGROUND: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. OBJECTIVE: To identify elements determining alternative splicing pathways in intron +1G-->A mutations of the dystrophin gene. RESULTS: We found that exon 25 is spliced out in the +1G-->A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G-->A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G-->A mutation of intron 25. CONCLUSION: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G-->A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy. SN - 1468-6244 UR - https://www.unboundmedicine.com/medline/citation/19001018/In_vitro_splicing_analysis_showed_that_availability_of_a_cryptic_splice_site_is_not_a_determinant_for_alternative_splicing_patterns_caused_by_+1G__>A_mutations_in_introns_of_the_dystrophin_gene_ L2 - https://jmg.bmj.com/lookup/pmidlookup?view=long&pmid=19001018 DB - PRIME DP - Unbound Medicine ER -