Exploring the Functional Impact of Individual DDX41 Variants With a Fast and Robust Cell-Based Method

Germline DDX41 variants are found in up to 4% of patients with acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDSs), with a higher prevalence among males and a late onset. In this study, we analyzed 647 Danish patients with suspected myeloid neoplasms to assess the frequency of germline DDX41 variants and the functional impact of variants of uncertain significance (VUSs) using a cell-based assay, CRISPR-Select. We identified 16 DDX41 variants in 30 patients, 14 of whom were confirmed or predicted as germline variants. Eight germline variants were classified as likely pathogenic/pathogenic (LP/P), and three as VUSs. We generated a monoallelic DDX41 K562 cell line and used it for CRISPR-Select assessment of variant effects on proliferation and survival. Pathogenic variants impaired proliferation, while benign variants did not. Missense variants had a more subtle effect than truncating ones, suggesting hypomorphic phenotypes. One DDX41 VUS had a significant negative effect on proliferation, suggesting it to be pathogenic, while the other two had no effect, suggesting them to be benign. These findings confirm a 2.4% frequency of LP/P DDX41 germline variants in Danish patients and demonstrate the value of CRISPR-Select in distinguishing pathogenic from benign variants.