https://orcid.org/0000-0002-2154-3966
Key Points
Deleterious germline mutations in IKAROS can cause immunodeficiency and leukemia; functional validation is critical to assign causality
Systematic multi-functional evaluation of IKAROS variants helped to confirm, exclude or anticipate IKAROS-associated diseases
The transcription factor IKAROS plays an important role in lymphocyte development, differentiation, and as a tumor suppressor. To date, more than 70 IKAROS germline heterozygous variants have been reported in patients with primary immunodeficiency (PID)/inborn errors of immunity (IEI) and leukemia, and this number continues to grow. Germline IKAROS loss- and gain-of-function mutations have been linked to immunodeficiency, immune dysregulation, and hematologic malignancies, with a broad spectrum of clinical manifestations. Routine next-generation-sequencing approaches in patients with PID/IEI have facilitated the identification of IKAROS variants, including several cases with variants of uncertain significance (VUS). To determine the VUS' functional behavior, we systematically generated constructs recapitulating those changes and tested IKAROS functions in vitro. We also conducted an in-depth examination of the C-terminal dimerization domain using alanine-scanning mutagenesis to identify amino acids critical for dimerization and other functions. This work provides a comprehensive description of the biologic impact of 81 previously unreported and/or untested IKAROS variants, including 33 patient-detected germline VUS and 48 laboratory-generated mutations in the dimerization domain. Among them, 15 of the patient-detected variants, primarily mapping to IKAROS DNA-binding or dimerization domains, and at least 21 of the laboratory-generated mutations, impaired IKAROS function and could explain or result in human disease. VUS located in between IKAROS DNA binding and dimerization domains were less likely to be functionally deleterious. Of note, both positive and negative functional data herein generated can be relevant for patients carrying these IKAROS variants, helping to establish a diagnosis and guide treatment decisions.
