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Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50

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 immunodeficie...

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Autores principales: Fliegauf, Manfred, Kinnunen, Matias, Posadas-Cantera, Sara, Camacho-Ordonez, Nadezhda, Abolhassani, Hassan, Alsina, Laia, Atschekzei, Faranaz, Bogaert, Delfien J., Burns, Siobhan O., Church, Joseph A., Dückers, Gregor, Freeman, Alexandra F., Hammarström, Lennart, Hanitsch, Leif Gunnar, Kerre, Tessa, Kobbe, Robin, Sharapova, Svetlana O., Siepermann, Kathrin, Speckmann, Carsten, Steiner, Sophie, Verma, Nisha, Walter, Jolan E., Westermann-Clark, Emma, Goldacker, Sigune, Warnatz, Klaus, Varjosalo, Markku, Grimbacher, Bodo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9465457/
https://www.ncbi.nlm.nih.gov/pubmed/36105815
http://dx.doi.org/10.3389/fimmu.2022.965326
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author Fliegauf, Manfred
Kinnunen, Matias
Posadas-Cantera, Sara
Camacho-Ordonez, Nadezhda
Abolhassani, Hassan
Alsina, Laia
Atschekzei, Faranaz
Bogaert, Delfien J.
Burns, Siobhan O.
Church, Joseph A.
Dückers, Gregor
Freeman, Alexandra F.
Hammarström, Lennart
Hanitsch, Leif Gunnar
Kerre, Tessa
Kobbe, Robin
Sharapova, Svetlana O.
Siepermann, Kathrin
Speckmann, Carsten
Steiner, Sophie
Verma, Nisha
Walter, Jolan E.
Westermann-Clark, Emma
Goldacker, Sigune
Warnatz, Klaus
Varjosalo, Markku
Grimbacher, Bodo
author_facet Fliegauf, Manfred
Kinnunen, Matias
Posadas-Cantera, Sara
Camacho-Ordonez, Nadezhda
Abolhassani, Hassan
Alsina, Laia
Atschekzei, Faranaz
Bogaert, Delfien J.
Burns, Siobhan O.
Church, Joseph A.
Dückers, Gregor
Freeman, Alexandra F.
Hammarström, Lennart
Hanitsch, Leif Gunnar
Kerre, Tessa
Kobbe, Robin
Sharapova, Svetlana O.
Siepermann, Kathrin
Speckmann, Carsten
Steiner, Sophie
Verma, Nisha
Walter, Jolan E.
Westermann-Clark, Emma
Goldacker, Sigune
Warnatz, Klaus
Varjosalo, Markku
Grimbacher, Bodo
author_sort Fliegauf, Manfred
collection PubMed
description 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-κB 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.
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spelling pubmed-94654572022-09-13 Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50 Fliegauf, Manfred Kinnunen, Matias Posadas-Cantera, Sara Camacho-Ordonez, Nadezhda Abolhassani, Hassan Alsina, Laia Atschekzei, Faranaz Bogaert, Delfien J. Burns, Siobhan O. Church, Joseph A. Dückers, Gregor Freeman, Alexandra F. Hammarström, Lennart Hanitsch, Leif Gunnar Kerre, Tessa Kobbe, Robin Sharapova, Svetlana O. Siepermann, Kathrin Speckmann, Carsten Steiner, Sophie Verma, Nisha Walter, Jolan E. Westermann-Clark, Emma Goldacker, Sigune Warnatz, Klaus Varjosalo, Markku Grimbacher, Bodo Front Immunol Immunology 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-κB 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. Frontiers Media S.A. 2022-08-29 /pmc/articles/PMC9465457/ /pubmed/36105815 http://dx.doi.org/10.3389/fimmu.2022.965326 Text en Copyright © 2022 Fliegauf, Kinnunen, Posadas-Cantera, Camacho-Ordonez, Abolhassani, Alsina, Atschekzei, Bogaert, Burns, Church, Dückers, Freeman, Hammarström, Hanitsch, Kerre, Kobbe, Sharapova, Siepermann, Speckmann, Steiner, Verma, Walter, Westermann-Clark, Goldacker, Warnatz, Varjosalo and Grimbacher https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Fliegauf, Manfred
Kinnunen, Matias
Posadas-Cantera, Sara
Camacho-Ordonez, Nadezhda
Abolhassani, Hassan
Alsina, Laia
Atschekzei, Faranaz
Bogaert, Delfien J.
Burns, Siobhan O.
Church, Joseph A.
Dückers, Gregor
Freeman, Alexandra F.
Hammarström, Lennart
Hanitsch, Leif Gunnar
Kerre, Tessa
Kobbe, Robin
Sharapova, Svetlana O.
Siepermann, Kathrin
Speckmann, Carsten
Steiner, Sophie
Verma, Nisha
Walter, Jolan E.
Westermann-Clark, Emma
Goldacker, Sigune
Warnatz, Klaus
Varjosalo, Markku
Grimbacher, Bodo
Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title_full Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title_fullStr Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title_full_unstemmed Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title_short Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50
title_sort detrimental nfkb1 missense variants affecting the rel-homology domain of p105/p50
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9465457/
https://www.ncbi.nlm.nih.gov/pubmed/36105815
http://dx.doi.org/10.3389/fimmu.2022.965326
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