Systematic functional validation of IKAROS variants from patients and laboratory-generated mutations

The transcription factor IKAROS plays an important role in lymphocyte development, differentiation, and as a tumor suppressor. To date, >70 IKAROS germ line heterozygous variants have been reported in patients with primary immunodeficiency (PID)/inborn errors of immunity (IEI) and leukemia, and this number continues to grow. Germ line 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 germ line 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.

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Publication Details

Journal
Blood Advances
Published
2025-12-11
DOI
https://doi.org/10.1182/bloodadvances.2025017243
Primary Topic
Acute Lymphoblastic Leukemia research
Type
article
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0.00

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article

Systematic functional validation of IKAROS variants from patients and laboratory-generated mutations

Joshua D. Milner, Winnie Ip, Michael D. Claiborne, Rodman G. Miller et al.
Blood Advances
Acute Lymphoblastic Leukemia research
article

Systematic functional validation of IKAROS variants from patients and laboratory-generated mutations

Joshua D. Milner, Winnie Ip, Michael D. Claiborne, Rodman G. Miller, Lennart Hammarström, Hye Sun Kuehn, Jonathan J. Lyons, Patrick O’Young, David P. Hoytema van Konijnenburg, Juan Carlos Aldave Becerra, James Verbsky, Fabian Hauck, John M. Routes, P. Mendez, Sathish Kumar Loganathan, Constance Bindernagel, Dinakantha Kumararatne, Carlos Rodríguez‐Gallego, Ariharan Anantharachagan, Xavier Solanich, Agustin A. Gil Silva, Brian D. Modena, Ashley V. Geerlinks, Tamar Rubin, Evelien Zonneveld‐Huijssoon, João Farela Neves, Kaan Boztuǧ, Manish J. Butte, Sergio D. Rosenzweig, Craig D. Platt, Hassan Abolhassani, S. Cindy, Kelli W. Williams, Andrew White, Luís M. Allende, Reem Elfeky, Julie E. Niemela, William J. Griffiths, Abraham Rutgers, Harry Lesmana, Ana Esteve‐Solé, Ana García-Soidán, Hey Chong, Diana Cabanillas, Stuart G. Tangye, Aubrey D.N.J. de Grey, Natchanun Klangkalya, Thomas A. Fleisher, Jennifer Stoddard, Ali Doroudchi, Serena Shaffren, Mario Framil Seoane, Shubham Goel
article en

Abstract

The transcription factor IKAROS plays an important role in lymphocyte development, differentiation, and as a tumor suppressor. To date, >70 IKAROS germ line heterozygous variants have been reported in patients with primary immunodeficiency (PID)/inborn errors of immunity (IEI) and leukemia, and this number continues to grow. Germ line 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 germ line 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.

Blood AdvancesVol. 10(5)
Universidad de Las Palmas de Gran Canaria (ES), Manitoba Health (CA), Western University (CA), Boston Children's Hospital (US), Lancashire Teaching Hospitals NHS Foundation Trust (GB), Morgan Stanley Children's Hospital (US), National Institutes of Health (US), La Jolla Institute for Immunology (US), Scripps Health (US), Cleveland Clinic (US), University Medical Center Groningen (NL), Garvan Institute of Medical Research (AU), Johns Hopkins University (US), Medical University of South Carolina (US), University of California, Los Angeles (US), Christian Medical College, Vellore (IN), Medical College of Wisconsin (US), University of South Florida (US), University Hospital Bonn (DE), Columbia University Irving Medical Center (US), Royal Prince Alfred Hospital (AU), UCLA Health (US), Great Ormond Street Hospital (GB), London Health Sciences Centre (CA), The Royal Free Hospital (GB), Bellvitge University Hospital (ES), Cambridge University Hospitals NHS Foundation Trust (GB), Westmead Hospital (AU), Karolinska Institutet (SE), UNSW Sydney (AU), University of California San Diego (US), Cleveland Clinic Lerner College of Medicine (US), Australasia Paediatric Endocrine Group (AU), Universidad Fernando Pessoa Canarias (ES), Hospital Nacional Cayetano Heredia (PE), Hospital Universitario de Gran Canaria Doctor Negrín (ES), Johns Hopkins All Children's Hospital (US), Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (ES), Atlanta Allergy and Asthma Clinic (US), VA San Diego Healthcare System (US), Hospital Universitario 12 De Octubre (ES), La Jolla Alcohol Research (US), École Nationale des Greffes (FR), Christian Medical College (IN), Scripps Clinic Medical Group (US), Children's Medical Center (IR), Nova Medical (United States) (US), Hospital Clínic de Barcelona (ES), Hospital de Dona Estefânia (PT), Cancer Research Institute of the Slovak Academy of Sciences (SK), St Anna Children's Hospital (AT), Children's Hospital of Pittsburgh (US), Hospital de Niños Superiora Sor María Ludovica (AR), Ramathibodi Hospital (TH), Children's Hospital of Western Ontario (CA), Boston Children's Museum (US), Fundación Canaria de Investigación Sanitaria (ES), National Institutes of Health Clinical Center (US), Addenbrooke's Hospital (GB), St Martins Hospital (GB), St. Anna Children's Cancer Research Institute, University College London (GB), University of Manitoba (CA), Tehran University of Medical Sciences (IR), Ludwig-Maximilians-Universität München (DE), Universidade Nova de Lisboa (PT)
U.S. Department of Health and Human Services, Ministerio de Ciencia e Innovación, National Institutes of Health, Instituto de Salud Carlos III, Agencia Estatal de Investigación, NIH Clinical Center
Openalex Percentile: Top 49%
Acute Lymphoblastic Leukemia research
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