破坏的前mRNA剪接机制是遗传性肿瘤中常见的有害机制。我们的目标是通过剪接报告基因来功能分析乳腺癌易感基因CHEK2的候选剪接异常变体。我们使用MaxEntScan分析了来自Breast Cancer After Diagnostic Gene Sequencing (BRIDGES)项目 (https://cordis.europa.eu/project/id/634935) 的128个CHEK2剪接位点变异，并将其筛选为52个预测会影响剪接的变体。我们构建并验证了涵盖其15个外显子的三种CHEK2小基因。然后，我们将这52个选定的变体基因工程插入到小基因中，并在MCF-7（人乳腺腺癌）细胞中进行了检测。在52个变体中，有46个（88.5%）影响了剪接。其中一些导致了复杂的剪接模式，最多产生11种不同的转录本。34个变体引起了剪接异常，没有任何全长转录本的存在或只有微量。我们注释了总共89种不同的转录本，它们来源于不同的事件：单外显子或多外显子跳跃，可选择的位点使用，互斥外显子包含，内含子保留或上述事件的组合。其中59个转录本预测引入了提前终止密码子，7个保留了原始的开放阅读框，5个去除了翻译起始密码子，6个影响了5' UTR（非翻译区）且2个包含了错误变异。对变体c.684-2 A > G的分析揭示了非典型的TG受体位点的激活和对于其识别至关重要的第6外显子序列的影响。将小基因结果纳入基于ACMG / AMP (美国医学遗传学会/分子病理学协会) 的分类方案中，我们成功对32个CHEK2变异进行分类（27个致病/或可能致病，5个可能良性）。然而，20个变异（38%）仍然具有不确定的意义，部分原因是由于该基因的复杂剪接模式。 © The Author(s) 2023. Published by Oxford University Press on behalf of Association for Diagnostics & Laboratory Medicine.

Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes.A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells.Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5'UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition.Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.© The Author(s) 2023. Published by Oxford University Press on behalf of Association for Diagnostics & Laboratory Medicine.