Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
Por:
Fliegauf M, Kinnunen M, Posadas-Cantera S, Camacho-Ordonez N, Abolhassani H, Alsina L, Atschekzei F, Bogaert DJ, Burns SO, Church JA, Dückers G, Freeman AF, Hammarström L, Hanitsch LG, Kerre T, Kobbe R, Sharapova SO, Siepermann K, Speckmann C, Steiner S, Verma N, Walter JE, Westermann-Clark E, Goldacker S, Warnatz K, Varjosalo M and Grimbacher B
Publicada:
29 ago 2022
Ahead of Print:
29 ago 2022
Resumen:
Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-kappa B subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.
Filiaciones:
Fliegauf M:
Institute for Immunodeficiency (IFI), Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
CIBSS - Centre for Integrative Biological Signalling Studies, Freiburg, Germany
Kinnunen M:
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Posadas-Cantera S:
Institute for Immunodeficiency (IFI), Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Camacho-Ordonez N:
Institute for Immunodeficiency (IFI), Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Faculty of Biology, University of Freiburg, Freiburg, Germany
Abolhassani H:
Department of Biosciences and Nutrition, NEO, Karolinska Institutet, Huddinge, Sweden
Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
Alsina L:
Clinical Immunology and Primary Immunodeficiencies Unit, Pediatric Allergy and Clinical Immunology Department, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, Barcelona, Spain
Department of Surgery and Surgical Specializations, Facultat de Medicina i Ciències de la Salut, Barcelona, Spain
Atschekzei F:
RESIST - Cluster of Excellence 2155 to Hanover Medical School , Satellite Center Freiburg, Freiburg, Germany
Department for Clinical Immunology and Rheumatology, Hannover Medical School, Hanover, Germany
Bogaert DJ:
Department of Pediatrics, Division of Pediatric Hemato-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
Primary Immunodeficiency Research Lab, Center for Primary Immunodeficiency Ghent, Jeffrey Modell Diagnosis and Research Center, Ghent University Hospital, Ghent, Belgium
Burns SO:
Department of Immunology, Royal Free London NHS Foundation Trust, London, United Kingdom
Institute of Immunity and Transplantation, University College London, London, United Kingdom
Church JA:
Department of Pediatrics, Keck School of Medicine, University of Southern California and Children's Hospital Los Angeles, Los Angeles, CA, United States
Dückers G:
HELIOS Children's Hospital, Krefeld, Germany
Freeman AF:
Laboratory of Clinical Immunology and Microbiology, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States
Hammarström L:
Department of Biosciences and Nutrition, NEO, Karolinska Institutet, Huddinge, Sweden
Hanitsch LG:
Department of Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Kerre T:
Department of Hematology, Ghent University Hospital, Ghent, Belgium
Kobbe R:
Institute for Infection Research and Vaccine Development (IIRVD), University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Sharapova SO:
Research Department, Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus
Siepermann K:
HELIOS Children's Hospital, Krefeld, Germany
Speckmann C:
Institute for Immunodeficiency (IFI), Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Center for Pediatrics and Adolescent Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Steiner S:
Department of Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Verma N:
Department of Immunology, Royal Free London NHS Foundation Trust, London, United Kingdom
Walter JE:
Division of Allergy and Immunology, Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
Division of Allergy/Immunology, Department of Pediatrics Johns Hopkins All Children's Hospital, St. Petersburg, FL, United States
Division of Allergy and Immunology, Massachusetts General Hospital for Children, Boston, MA, United States
Westermann-Clark E:
Division of Allergy and Immunology, Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
Division of Allergy and Immunology, Department of Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States
Goldacker S:
Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Department of Rheumatology and Clinical Immunology, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Warnatz K:
Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Department of Rheumatology and Clinical Immunology, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Varjosalo M:
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
Proteomics Unit, University of Helsinki, Helsinki, Finland
Grimbacher B:
Institute for Immunodeficiency (IFI), Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
CIBSS - Centre for Integrative Biological Signalling Studies, Freiburg, Germany
RESIST - Cluster of Excellence 2155 to Hanover Medical School , Satellite Center Freiburg, Freiburg, Germany
DZIF - German Center for Infection Research, Satellite Center Freiburg, Freiburg, Germany
Green Published, Green Submitted, gold
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