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

Tran, Hoai Thu Thi; Takeshima, Yasuhiro; Surono, Agus; Yagi, Minoru; Wada, Hiroyoshi; Matsuo, Masafumi

doi:10.1016/j.ymgme.2005.03.006

Cited by 50 publications

(25 citation statements)

References 21 publications

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“…Putative small mutations were identified in 15 of 16 patients (table 3); most were either nonsense or frameshift mutations causing truncation of dystrophin. Intronic variant in patient 6 (c.4518+5G>A) was demonstrated previously to cause aberrant splicing in vivo and in vitro 25. Novel nonsense (c.1006G>T, p.Glu336X) and missense (c.1005G>A, p.Met335Ile) variants were found in patient 15.…”

Section: Resultsmentioning

confidence: 64%

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

Lim

Lee

Shin

et al. 2011

Journal of Medical Genetics

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Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/ duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology. Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication. Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction. Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.

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Section: Resultsmentioning

confidence: 64%

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

Lim

Lee

Shin

et al. 2011

Journal of Medical Genetics

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“…More than 30 point mutations have been reported in the consensus sequences at donor splice sites of the dystrophin gene,6 of which mutations changing G to A at the first nucleotide of the relevant intron (+1G→A) are the most common. Even within this limited subset, the resulting splicing pathways can differ from mutation to mutation, leasing to either exon skipping or cryptic splice-site activation.…”

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confidence: 99%

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

Habara

Takeshima

Awano

et al. 2008

Journal of Medical Genetics

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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.

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“…Data 1; for all online suppl. material, see www.karger.com/doi/10.1159/000484209) [4, 5]. Each test sequence was obtained to amplify the control sample and genomic DNA from the proband by PCR using primers that corresponded to introns 36 and 38, and included Nhe I and Bam HI restriction enzyme recognition sites, respectively (In36F-Nhe: 5-GCA GC TAGCTTCTCCTAGTAAAAAGCAAGAACTG-3 and In38R-Bam: 5-CGTGGATCCTGCCATATCTATAGAGAGCTGAG T G-3).…”

Section: Methodsmentioning

confidence: 99%

“…One microgram of total RNA was subjected to reverse transcription using RNA to cDNA EcoDry premix (doubled primed; Takara) and PCR was performed using a forward primer corresponding to a segment of upstream exon A and a reverse primer complementary to a segment of downstream exon B, as previously described (Fig. 1) [4, 5]. PCR products were analyzed using electrophoresis on a 2% agarose gel and with direct sequencing.…”

Section: Methodsmentioning

confidence: 99%

Detection of a Splice Site Variant in a Patient with Glomerulopathy and Fibronectin Deposits

Tsuji

Nozu

Sofue

et al. 2017

Nephron

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Background/Aims: Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in FN1 have been detected. Methods: We found a pathogenic intronic variant in intron 36 (c.5888–2A>G) located at the heparin-binding site. To determine whether this mutation influences splicing processes, we conducted RT-PCR analysis and an in vitro splicing assay using minigene construction. Results: RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of FN1 mRNA in leukocytes. We conducted in vitro functional splicing analysis using minigenes and found that c.5888–2A>G caused a 12 bp deletion at exon 37 by the activation of a novel splicing acceptor site within exon 37. We were able to detect the same abnormal transcript in mRNA extracted from the patient’s urinary sediment and confirmed the pathogenicity of c.5888–2A>G by both RT-PCR using the patient sample and an in vitro splicing assay. Conclusion: Intronic variants can cause GFND. Minigene analysis is useful for determining the pathogenicity of the intronic variants and could be used for all inherited kidney diseases.

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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

Cited by 50 publications

References 21 publications

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

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

Detection of a Splice Site Variant in a Patient with Glomerulopathy and Fibronectin Deposits

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