Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene
Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene
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DOI：
10.1136/jmg.2006.042317
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发表时间：
2006-12-01
影响因子：
4
通讯作者：
Matsuo, Masafumi
中科院分区：
文献类型：
--
作者：
Tran, Van Khanh;Takeshima, Yasuhiro;Matsuo, Masafumi
背景：已知外显子剪接增强子序列的突变会导致剪接错误。虽然外显子剪接增强子已被确定为一段富含嘌呤的序列，但很难精确地确定这些序列中的决定核苷酸。本文报道了肌营养不良蛋白基因38外显子4 bp的缺失在体内诱导了38外显子的完全跳变。此外，在编码突变外显子的杂交小基因的体内剪接分析中，缺失的第三个核苷酸被证明是外显子剪接增强子活性的决定因素。方法：对一名血清肌酸激酶水平升高的2岁男孩进行基因组DNA分析，发现在肌营养不良蛋白基因（c. 5434-5437del TTCA）第38外显子39末端上游11 bp处有一个4 bp的缺失，破坏了一个预测的sc35结合位点。结果：有趣的是，他的肌营养不良蛋白mRNA完全缺乏38外显子（382外显子转录本）。由于外显子382转录本编码截断的肌营养不良蛋白，该外显子跳变被确定为其表型的修饰因素。在体内剪接实验中，一个编码外显子38的4 bp缺失的杂交迷你基因被证明可以诱导完整的外显子38跳变，证实了缺失区域是剪接增强子序列。缺失序列的定点突变表明，完整的38号外显子跳变是由缺失的第三个核苷酸位置（C5436）突变引起的，而其他三个核苷酸位置的突变引起部分外显子跳变。结论：我们的研究结果强调了了解外显子剪接增强子序列的调控和外显子跳过治疗杜氏肌营养不良的潜力。
Background: Mutations in exonic splicing enhancer sequences are known to cause splicing errors. Although exonic splicing enhancers have been identified as a stretch of purine-rich sequences, it has been difficult to precisely pinpoint the determinant nucleotides in these sequences. This article reports that a 4-bp deletion in exon 38 of the dystrophin gene induced complete exon 38 skipping in vivo. Moreover, the third nucleotide of the deletion was shown to be determinant for the exonic splicing enhancer activity in in vivo splicing analysis of hybrid minigenes encoding mutant exons.Method: Genomic DNA analysis of a 2-year-old boy with a raised level of serum creatine kinase yielded a 4-bp deletion 11 bp upstream of the 39 end of exon 38 of the dystrophin gene (c. 5434-5437del TTCA), disrupting a predicted SC35-binding site.Result: Interestingly, his dystrophin mRNA was shown to completely lack exon 38 (exon 382 transcript). As the exon 382 transcript coded for a truncated dystrophin protein, this exon skipping was determined to be a modifying factor of his phenotype. In an in vivo splicing assay, a hybrid minigene encoding exon 38 with the 4-bp deletion was shown to induce complete exon 38 skipping, confirming the deleted region as a splicing enhancer sequence. Site-directed mutagenesis of the deleted sequence showed that the complete exon 38 skipping was caused by mutation of the third nucleotide position of the deletion (C5436), whereas mutations at the other three nucleotide positions induced partial exon skipping.Conclusion: Our results underline the potential of understanding the regulation of exonic splicing enhancer sequences and exon skipping therapy for treatment of Duchenne's muscular dystrophy.