BACKGROUND Interpretation of genetic variants detected by sequencing of genomic DNA, which may cause splicing defects, regularly requires mRNA analysis. Usually, only bioinformatic testing is provided, because simple and non-invasive assay protocols are lacking. Furthermore, the detection of mis-splicing is often hampered by nonsense mediated mRNA decay (NMD). METHODS Starting from a case of Pompe disease with two potential splicing variants an assay for the analysis of splice defects in general was developed. We analyzed the transcripts from the gene of interest by standard methods after short-term culture of the patient's lymphocytes in the presence and absence of a NMD inhibitor. Variant and wild type transcript expression were quantified by allele specific PCR in the patient and both parents and the expression ratio with/without NMD inhibition was calculated for each transcript. RESULTS NMD detection in lymphocytes was optimized and evaluated by analyzing a naturally occurring NMD transcript. Several compounds inhibited NMD successfully, including potential therapeutic agents. Sample storage for up to 4 days at room temperature prior to lymphocyte isolation did not affect results. In a proof of concept we identified two candidate variants as severe splicing variants in a patient with Pompe disease, but the strategy can also be used to screen for any mis-spliced transcripts prone to NMD. CONCLUSIONS We developed a simple, non-invasive assay for the detection and characterization of potential splicing variants. This is essential, because early and near-term diagnosis and disease classification is required to facilitate therapy in many genetic diseases.

中文翻译：

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通过无意义的介导的衰变抑制来快速检测和表征异常mRNA剪接的非侵入性诊断测定。

背景技术通过对基因组DNA进行测序而检测到的遗传变异的解释可能会导致剪接缺陷，通常需要进行mRNA分析。通常，仅提供生物信息学测试，因为缺少简单且非侵入性的测定方案。此外，错误拼接的检测通常受到废话介导的mRNA衰变（NMD）的阻碍。方法从具有两个潜在剪接变体的庞贝病病例开始，开发了一种用于分析剪接缺陷的测定方法。在存在和不存在NMD抑制剂的情况下，对患者淋巴细胞进行短期培养后，我们通过标准方法分析了目标基因的转录本。通过等位基因特异性PCR对患者和父母双方的变体和野生型转录本表达进行定量，并计算每种转录本在有/无NMD抑制下的表达率。结果通过分析天然存在的NMD转录本，优化并评估了淋巴细胞中的NMD检测。几种化合物成功抑制NMD，包括潜在的治疗剂。在分离淋巴细胞之前，样品在室温下最多可保存4天，不会影响结果。在概念验证中，我们在庞贝病患者中确定了两个候选变体为严重的剪接变体，但该策略也可用于筛选任何容易发生NMD的错剪式转录本。结论我们开发了一种简单的非侵入性检测方法，用于检测和鉴定潜在的剪接变体。