Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland.
Institute of Medical Genetics, University of Zurich, 8952 Schlieren, Switzerland.
Int J Mol Sci. 2024 Sep 3;25(17):9569. doi: 10.3390/ijms25179569.
The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.
据报道，剪接变异体对遗传性疾病分子诊断的贡献小于 10%。由于多种因素，包括预测此类变异体影响的难度、对功能检测的需求以及无法检测到它们（取决于它们的位置和使用的测序技术），这一数字可能被低估了。本研究旨在评估纳米孔测序在描述和量化异常剪接事件中的应用。为此，我们选择了 19 个候选剪接变异体，这些变异体是在遗传性视网膜营养不良患者中发现的。我们使用了几种计算工具来预测变异的性质，并估计其引起的异常剪接事件的幅度。我们使用微基因测定或全血衍生的 cDNA 来对变异体进行功能特征分析。使用针对微基因特异性 cDNA 或血液 cDNA 中靶基因的 PCR 扩增，结合纳米孔测序，可鉴定出相应的转录物。在这 19 个变异体中，有 13 个导致异常剪接事件，包括隐蔽剪接位点激活、外显子跳跃、假外显子包含或这些情况的组合。纳米孔测序可识别全长转录物并对其进行精确定量，这与计算预测通常一致。该方法可可靠地检测到低丰度转录物，而这是传统策略（如 RT-PCR 后进行 Sanger 测序）无法检测到的。
