BACKGROUND Minigenes and in silico prediction tools are commonly used to assess the impact on splicing of CFTR variants. Exon skipping is often neglected though it could impact the efficacy of targeted therapies. The aim of the study was to identify exon skipping associated with CFTR variants and to evaluate in silico predictions of seven freely available software. METHODS CFTR basal exon skipping was evaluated on endogenous mRNA extracted from non-CF nasal cells and on two CFTR minigene banks. In silico tools and minigene systems were used to evaluate the impact of CFTR exonic variants on exon skipping. RESULTS Data showed that out of 65 CFTR variants tested, 26 enhanced exon skipping and that in silico prediction efficacy was of 50%-66%. Some in silico tools presented predictions with a bias towards the occurrence of splicing events while others presented a bias towards the absence of splicing events (non-detection including true negatives and false negatives). Classification of exons depending on their basal exon skipping level increased prediction rates up to 80%. CONCLUSION This study indicates that taking basal exon skipping into account could orientate the choice of the in silico tools to improve prediction rates. It also highlights the need to validate effects using in vitro assays or mRNA studies in patients. Eventually, it shows that variant-guided therapy should also target exon skipping associated with variants.

中文翻译：

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外显子同一性影响外显子变体诱导的剪接和计算机预测功效

背景 Minigenes 和计算机预测工具通常用于评估对 CFTR 变体剪接的影响。外显子跳跃经常被忽视，尽管它可能会影响靶向治疗的功效。该研究的目的是确定与 CFTR 变体相关的外显子跳跃，并评估七种免费软件的计算机预测。方法 CFTR 基础外显子跳跃对从非 CF 鼻细胞和两个 CFTR 小基因库中提取的内源性 mRNA 进行评估。计算机工具和 minigene 系统用于评估 CFTR 外显子变体对外显子跳跃的影响。结果 数据显示，在测试的 65 个 CFTR 变体中，26 个增强了外显子跳跃，并且计算机预测效率为 50%-66%。一些计算机工具提供的预测偏向于剪接事件的发生，而另一些则偏向于不存在剪接事件（未检测，包括真阴性和假阴性）。根据其基础外显子跳跃水平对外显子进行分类将预测率提高到 80%。结论 本研究表明，考虑到基础外显子跳跃可以引导选择计算机工具以提高预测率。它还强调了使用体外试验或 mRNA 研究在患者身上验证效果的必要性。最终，它表明变异引导治疗也应该针对与变异相关的外显子跳跃。根据其基础外显子跳跃水平对外显子进行分类将预测率提高到 80%。结论 本研究表明，考虑到基础外显子跳跃可以引导选择计算机工具以提高预测率。它还强调了使用体外试验或 mRNA 研究在患者身上验证效果的必要性。最终，它表明变异引导治疗也应该针对与变异相关的外显子跳跃。根据其基础外显子跳跃水平对外显子进行分类将预测率提高到 80%。结论 本研究表明，考虑到基础外显子跳跃可以引导选择计算机工具以提高预测率。它还强调了使用体外试验或 mRNA 研究在患者身上验证效果的必要性。最终，它表明变异引导治疗也应该针对与变异相关的外显子跳跃。