Background/Objectives: The non-canonical GC-5′ splice sites (5′ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5′ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5′ss of the breast/ovarian cancer susceptibility genes,ATM(exon 50),BRIP1(exon 1), andPALB2(exon 12), and their dysregulation induced by DNA variants.Methods: Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5′ss.Results: A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals,ATMc.7335_7344del,PALB2c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14ATMand 9PALB2variants (Hexplorer score < −40) located at these intervals that were assayed in MCF-7 cells. NineATMand threePALB2variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. TheATMspliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55–59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms.Conclusions:ATMexon 50 andPALB2exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals.
背景/目的：非经典GC-5′剪接位点是经典GT/AG剪接规则中最常见的例外情况（约占1%）。它们构成弱5′剪接位点，并受剪接因子调控，因此对导致相应外显子错误识别的遗传变异尤为敏感。本研究旨在探讨乳腺癌/卵巢癌易感基因ATM（外显子50）、BRIP1（外显子1）和PALB2（外显子12）的GC-5′剪接位点及其受DNA变异诱导的调控异常。方法：通过构建微型基因mgATM_49-52、mgBRIP1_1-2和mgPALB2_5-12进行剪接实验，研究特定GC-5′剪接位点的调控机制。结果：通过重叠外显子微缺失构建的剪接调控元件功能图谱揭示了三个关键区域：ATMc.7335_7344del、PALB2c.3229_3258del和c.3293_3322del，这些区域可能是致剪接异常调控元件变异的靶点。随后选取位于这些区域的14个ATM变异和9个PALB2变异（Hexplorer评分<-40），在MCF-7细胞中进行功能验证。其中9个ATM变异和3个PALB2变异显著影响剪接过程，分别损害了外显子50和外显子12的识别。因此，这些变异可能破坏了参与成熟mRNA中外显子保留的活性剪接调控元件。DeepCLIP预测提示多种剪接因子参与外显子识别，包括SRSF1、SRSF2和SRSF7等已知参与其他GC位点识别的因子。ATM致剪接异常变异c.7336G>T（p.(Glu2446Ter)）和c.7340T>A（p.(Leu2447Ter)）产生大量（55-59%）包含提前终止密码子的全长转录本，表明其可能通过剪接破坏和蛋白质截断双重机制导致ATM功能失活。结论：ATM外显子50和PALB2外显子12需要特定序列才能被剪接机制有效识别。在微型基因中绘制富含剪接调控元件的功能区域，是识别致剪接异常变异的有效方法，其性能优于任何预测软件。本研究在关键功能区间内成功鉴定出12个致剪接异常的调控元件变异。
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