Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC-UVa), Valladolid, Spain.
Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), Madrid, Spain.
J Pathol. 2022 Sep;258(1):83-101. doi: 10.1002/path.5979. Epub 2022 Jul 15.
The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
共济失调毛细血管扩张症突变（ATM）蛋白是 DNA 损伤反应途径的主要协调蛋白。ATM 功能丧失变异与乳腺癌风险增加 2 倍相关。我们旨在识别和分类在大规模测序项目 BRIDGES 中检测到的剪接体 ATM 变体。在内含子-外显子边界共鉴定出 381 个变体，其中 128 个被预测为剪接变体。经过进一步筛选，我们最终选择了 56 个变体进行剪接分析。构建了跨越外显子 4-9、11-17、25-29 和 49-52 的四个功能迷你基因（mgATM）。选择的变体被遗传工程构建到四个构建体中，并在 MCF-7/HeLa 细胞中进行测定。48 个变体（85.7%）干扰剪接，其中 32 个变体没有全长（FL）转录的任何痕迹。共鉴定出 43 个转录本，最常见的事件是外显子/多外显子跳跃。27 个转录本预测会截断 ATM 蛋白。一种基于美国医学遗传学与基因组学学院/分子病理学协会（American College of Medical Genetics and Genomics/Association for Molecular Pathology）的暂定分类方案，该方案整合了 mgATM 数据，使我们能够将 29 个 ATM 变体归类为致病性/可能致病性，将 7 个变体归类为可能良性。有趣的是，可能致病性变体 c.1898+2T>G 由于使用了非典型 GG-5'-剪接位点（人类供体位点的 0.014%），产生了 13%的 minigene FL-转录本。在三个 ATM 变体中（我们的 mgATM 分析与临床数据一起揭示的渗漏性）的间接证据为剂量敏感表达模型提供了一些支持，其中产生≥30%FL-转录本的变体被预测为良性，而产生≤13%FL-转录本的变体可能是致病性的。
