Hanenberg Helmut, Zhang Fan, Malev Nikita, Wiek Constanze, Klamer Brett G, Nassar Nicolas, Hesselbrock Tyler, Hanenberg Judith H, Aeilts Amber M, Hentschel Julia, Faust Ulrike, Gehrig Andrea, Engel Christoph, Hauke Jan, Niederacher Dieter, Toland Amanda E, Andreassen Paul R
Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Department of Otorhinolaryngology & Head/Neck Surgery, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
Clin Cancer Res. 2025 Jun 13;31(12):2426-2440. doi: 10.1158/1078-0432.CCR-24-3936.
ATM is a moderate-risk cancer susceptibility gene that harbors thousands of missense variants of uncertain significance (VUS) which limit the power of clinical genetic testing for cancer risk management and personalized medicine. Functional tests provide a valuable basis for testing the impact of variants but have been lacking for ATM.
We developed a systematic approach to functionally characterize missense ATM variants based on the correction of various DNA damage-related phenotypes via reexpression of ATM in either of two ATM-deficient human cell lines.
A pKAP1 phospho-flow assay for ATM VUS observed in patients with hereditary cancer was calibrated using 48 benign and pathogenic controls, achieving 100% specificity and 97% sensitivity. This system distinguished 82 of 88 (93%) missense ATM VUS of the FATKIN region as functionally neutral or deleterious. Importantly, for the clinical classification of VUS, functional results were incorporated into an American College of Medical Genetics points-based framework, also considering conservation and properties of amino acids/substitutions, along with genetic data; 79 of 88 (90%) were thereby reclassified as benign/likely benign or pathogenic/likely pathogenic. As additional validation of our approach, clinical characteristics from a database of 1,134 patients with breast cancer were distinct for carriers of neutral versus deleterious ATM variants. Also, utilizing our functional results, we identified hotspots for deleterious VUS and controls at amino acids 2702-2730 and 2891-2951 of ATM.
We have established functional assays as a reliable tool that will better interpret the clinical impact of ATM variants and guide improved cancer prevention measures for carriers.
ATM是一种中度风险的癌症易感基因，含有数千种意义未明的错义变异（VUS），这限制了癌症风险管理和个性化医疗中临床基因检测的作用。功能测试为检测变异的影响提供了有价值的基础，但ATM的功能测试一直缺乏。
我们开发了一种系统方法，通过在两种ATM缺陷的人类细胞系之一中重新表达ATM来校正各种DNA损伤相关表型，从而对ATM错义变异进行功能表征。
使用48个良性和致病性对照对遗传性癌症患者中观察到的ATM VUS进行了pKAP1磷酸化流式分析校准，特异性达到100%，灵敏度达到97%。该系统将FATKIN区域88个错义ATM VUS中的82个（93%）区分为功能中性或有害。重要的是，对于VUS的临床分类，功能结果被纳入美国医学遗传学学院基于积分的框架，同时考虑氨基酸/替代的保守性和特性以及遗传数据；88个中的79个（90%）因此被重新分类为良性/可能良性或致病性/可能致病性。作为我们方法的额外验证，来自1134例乳腺癌患者数据库的临床特征对于中性与有害ATM变异携带者是不同的。此外，利用我们的功能结果，我们在ATM的2702 - 2730和2891 - 2951氨基酸处鉴定了有害VUS和对照的热点。
我们已将功能测定确立为一种可靠工具，它将更好地解释ATM变异的临床影响，并指导为携带者改进癌症预防措施。
