Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1.
Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1.
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DOI：
10.1007/s10157-020-01876-x
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发表时间：
2020-07
影响因子：
2.3
通讯作者：
Nozu K
中科院分区：
文献类型：
--
作者：
Inoue T;Nagano C;Matsuo M;Yamamura T;Sakakibara N;Horinouchi T;Shibagaki Y;Ichikawa D;Aoto Y;Ishiko S;Ishimori S;Rossanti R;Iijima K;Nozu K
近年来，剪接异常作为遗传性疾病的原因的阐明取得了进展。然而，在登特病病例中，没有关于CLCN5基因的疑似剪接变异的全面报道。我们通过诱变复制基因突变，将突变基因插入微型基因载体，并对这些变异的致病性和发病机制进行了研究。我们对CLCN5中的6个疑似剪接变体（C . 105g > a, C .105+5G>C, C .106−17T>G, C .393+4A>G, C .517−8A>G, C .517−3C> a）进行了功能剪接实验。我们从人类基因突变数据库中提取了这些变异的信息。我们复制了带有疑似剪接变体的相关外显子插入的迷你基因载体。然后将这些迷你基因载体转染到培养细胞中，提取mRNA并进行分析。此外，我们进行了硅分析，以确认我们的微量基因测定结果。我们成功地确定了这六种变异中的五种是通过产生剪接异常而致病的。其中一株仅显示正常转录产物，因此怀疑不具有致病性（c.106−17T>G）。我们发现5个CLCN5变异破坏了原始剪接位点，导致剪接异常。由于mRNA的易碎性或其在外周血白细胞中的低表达水平，有时很难从患者样本中获得mRNA。我们的体外系统可以作为体内试验的替代方法，以确定可疑剪接变异体的致病性。本文的在线版本（10.1007/s10157-020-01876-x）包含补充材料，可供授权用户使用。
In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants. The online version of this article (10.1007/s10157-020-01876-x) contains supplementary material, which is available to authorized users.
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影响因子：
6.8
作者：
Cai L;Fisher AL;Huang H;Xie Z
通讯作者：
Xie Z
影响因子：
5.3
作者：
Nozu, Kandai;Iijima, Kazumoto;Matsuo, Masafumi
通讯作者：
Matsuo, Masafumi
影响因子：
3.9
作者：
Mansour-Hendili, Lamisse;Blanchard, Anne;Vargas-Poussou, Rosa
通讯作者：
Vargas-Poussou, Rosa
影响因子：
64.8
作者：
Piwon, N;Günther, W;Jentsch, TJ
通讯作者：
Jentsch, TJ
DOI：
10.1007/978-1-4939-8651-4_1
发表时间：
2018-01-01
期刊：
EXON SKIPPING AND INCLUSION THERAPIES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Lim, Kenji Rowel Q.;Yokota, Toshifumi
通讯作者：
Yokota, Toshifumi