Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.
Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.
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DOI：
10.1002/humu.22624
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发表时间：
2014-10
期刊：
影响因子：
3.9
通讯作者：
Cutting, Garry R.
中科院分区：
文献类型：
--
作者：
Sharma, Neeraj;Sosnay, Patrick R.;Ramalho, Anabela S.;Douville, Christopher;Franca, Arianna;Gottschalk, Laura B.;Park, Jeenah;Lee, Melissa;Vecchio-Pagan, Briana;Raraigh, Karen S.;Amara, Margarida D.;Karchin, Rachel;Cutting, Garry R.
在诊断实验室中，评估剪接位点附近变异的功能后果是一项主要挑战。为了解决这个问题，我们创建了表达迷你基因(EMG)来确定与囊性纤维化(CF)有关的剪接位点变异(n=10)产生的RNA和蛋白质产物。实验结果与8个硅胶工具中的剪接预测进行了比较。含有全长囊性纤维化跨膜电导调节因子(CFTR)编码序列和侧翼内含子序列的EMGS在人胚胎肾脏(HEK293)和CFBE410-细胞中产生了野生型转录本和完全加工的蛋白质。用片段分析和焦磷酸测序对变异诱导的异常mRNA异构体进行定量是一致的。C.1585−1G>；A和c.2657+5G>；A的剪接模式与报道的携带这些变异的个体的原代细胞中的剪接模式相似。生物信息学预测与实验结果一致，9/10变异体(MES)、8/10变异体(NNSplice)和7/10变异体(SSAT和Sroogle)。估计错误剪接后果的程序预测了11/16(HSF和ASSEDA)和10/16(FSplice和SplicePort)，并在实验中观察到了mRNA亚型。EMGS为剪接位点变异的临床解释和In Silicon o工具的改进提供了一种可靠的实验方法。
Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools.
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影响因子：
16.2
作者：
Iossifov I;Ronemus M;Levy D;Wang Z;Hakker I;Rosenbaum J;Yamrom B;Lee YH;Narzisi G;Leotta A;Kendall J;Grabowska E;Ma B;Marks S;Rodgers L;Stepansky A;Troge J;Andrews P;Bekritsky M;Pradhan K;Ghiban E;Kramer M;Parla J;Demeter R;Fulton LL;Fulton RS;Magrini VJ;Ye K;Darnell JC;Darnell RB;Mardis ER;Wilson RK;Schatz MC;McCombie WR;Wigler M
通讯作者：
Wigler M
影响因子：
14.9
作者：
Desmet FO;Hamroun D;Lalande M;Collod-Béroud G;Claustres M;Béroud C
通讯作者：
Béroud C
影响因子：
3.9
作者：
Aissat, Abdel;de Becdelievre, Alix;Hinzpeter, Alexandre
通讯作者：
Hinzpeter, Alexandre
影响因子：
17.1
作者：
Jacob, Howard J.;Abrams, Kelly;Worthey, Elizabeth A.
通讯作者：
Worthey, Elizabeth A.
影响因子：
3.9
作者：
Houdayer, Claude;Caux-Moncoutier, Virginie;Stoppa-Lyonnet, Dominique
通讯作者：
Stoppa-Lyonnet, Dominique