Discovery of CSF3R as a candidate germ line predisposition gene through the analysis of invariant variants on tumor-based NGS panels. (A) Pyramid chart showing the algorithm used to identify CSF3R as a candidate germ line hematologic malignancy predisposition gene. OncoPlus is a custom NGS panel of ∼1200 genes important in oncogenesis. Invariant variants are defined as variants present at consistent VAFs of about 50% in ≥2 OncoPlus tests conducted at different times from the same patient. (B) Histogram of the 31 genes found to have invariant variants with a VAF ≥0.4 in the 832 OncoPlus testing cohort displaying the number of such variants present within each of these genes. (C) Change in VAF across different testing time points for 33 genes found to be mutated among 632 patients who underwent OncoPlus testing. Time points were normalized to the date of each patient’s first test, set as time point 0 months. Variants in CSF3R remained at approximately the same VAF across multiple different testing time points. (D-F) Examples of variation in VAF across multiple testing time points for individual patients found to have variants in multiple different genes, including CSF3R. The CSF3R variants remained at a VAF of ∼0.5, whereas the VAFs of variants in most other genes fluctuated over time. p.Ala119Thr and p.Pro784Thr are not shown because the patients harboring these variants underwent a single OncoPlus test.