Garanto Alejandro, Duijkers Lonneke, Collin Rob W J
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands.
Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
Int J Mol Sci. 2015 Mar 9;16(3):5285-98. doi: 10.3390/ijms16035285.
A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo.
CEP290基因第26内含子的一个突变（c.2991 + 1655A>G）是莱伯先天性黑蒙（LCA）最常见的遗传病因，LCA是一种严重的遗传性视网膜变性。该突变产生了一个隐蔽的剪接供体位点，导致在所有CEP290转录本的约50%中插入一个异常外显子（外显子X）。具有这种突变的人源化小鼠模型并未重现LCA患者中观察到的异常CEP290剪接，这表明不同物种对隐蔽剪接位点的识别存在差异。为了进一步评估这一现象，我们构建了两个CEP290小基因构建体，一个带有内含子突变，一个不带，然后将它们转染到不同物种的细胞系中。逆转录聚合酶链反应（RT-PCR）分析显示，外显子X在人类和非人类灵长类动物细胞系中能被剪接机制很好地识别。有趣的是，在狗和啮齿动物等物种来源的细胞系中，这种识别能力下降，而在果蝇细胞系中则完全不存在。此外，在不同细胞系中还不同程度地观察到了与CEP290第26内含子序列相对应的其他隐蔽剪接事件。总之，这些结果凸显了不同物种间剪接位点识别的复杂性，并表明在构建动物模型以模拟体内剪接位点突变时需谨慎行事。
