Dieter Niederacher

Quantification for pCHK2 and pP53 immunoblots shown in Fig. 1D.

Insights into structural effects of selected deleterious variants in the N- and C-terminal lobes within the Kinase domain of human ATM.

Schematic diagram of key steps and a timeline for functional testing of ATM variants from the final cloning step to generate the lentiviral-ATMco expression construct to phospho-flow functional assays and quality assurance measures.

Optimization of the timepoint after exposure to IR for collection of cells for pKAP1 flow cytometric assays.

Immunoblots demonstrating expression of WT ATM, and benign and stable pathogenic missense controls, in ATM-deficient cells, and their activities against multiple substrates.

Purpose: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.Experimental Design: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.Results: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.Conclusions: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.

Immunoblots displaying expression levels for all VUS functionally tested in this study in comparison to WT ATM.

Immortalization with SV40 lg T-antigen does not affect pKAP1 phospho-flow assays in A-T and non A-T cell lines.

Immunoblots for nptII demonstrate instability of each of the 9 pathogenic truncating ATM controls.

Parameters of variants and assignment of ACMG points for controls and VUS that were functionally tested in our study.