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
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
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DOI：
10.1016/j.ymgme.2005.03.006
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发表时间：
2005-07-01
影响因子：
3.8
通讯作者：
Matsuo, M
中科院分区：
文献类型：
--
作者：
Tran, HTT;Takeshima, Y;Matsuo, M
前体mRNA的剪接模式在剪接供体位点的共有序列内具有单核苷酸变化的基因中是不可预测的。在肌营养不良蛋白基因中，在内含子-32的第五位置处（4518 + 5G > A）从G到A的转变已被报道为在杜氏肌营养不良症（DMD）中鉴定的共有序列内的多态性或突变。在这里，我们报告在体内和体外的证据表明，这种突变引起的剪接供体位点的失活。在一个日本DMD病例中，在患者的淋巴细胞中鉴定出两种新的肌营养不良蛋白mRNA，一种具有外显子32的3'端的98 bp缺失（dys 32 - 98），另一种具有保留在外显子32和33之间的28 bp内含子（dys 32 + 28）。基因组测序结果显示，在内含子32的第5位（4518 + 5G > A）有一个由G变为A的单核苷酸改变。为了证明在体外失活的剪接供体位点的这种核苷酸的变化，微型肌营养不良蛋白基因，包括三个外显子窝藏正常或突变的内含子-32序列在HeLa细胞中表达，并通过逆转录PCR扩增的剪接产物进行了分析。从正常构建体获得由三个外显子组成的正常转录物。从突变体中，我们获得了一个在外显子32的3'端含有98 bp缺失的产物，表明天然剪接供体位点完全失活。因此，体内和体外实验都证明4518 + 5G > A引起剪接错误，导致转录终止;它的行为不像沉默多态性。我们的研究结果表明，在体外剪接系统是一个强大的工具，用于确定潜在的致病突变的剪接共识序列的机制。(c)2005年爱思唯尔公司All rights reserved.
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 polymorphisrn. 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. (c) 2005 Elsevier Inc. All rights reserved.