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

TY - JOUR

T1 - Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50

AU - Fliegauf, Manfred

AU - Kinnunen, Matias

AU - Posadas-Cantera, Sara

AU - Camacho-Ordonez, Nadezhda

AU - Abolhassani, Hassan

AU - Alsina, Laia

AU - Atschekzei, Faranaz

AU - Bogaert, Delfien J

AU - Burns, Siobhan O

AU - Church, Joseph A

AU - Dückers, Gregor

AU - Freeman, Alexandra F

AU - Hammarström, Lennart

AU - Hanitsch, Leif Gunnar

AU - Kerre, Tessa

AU - Kobbe, Robin

AU - Sharapova, Svetlana O

AU - Siepermann, Kathrin

AU - Speckmann, Carsten

AU - Steiner, Sophie

AU - Verma, Nisha

AU - Walter, Jolan E

AU - Westermann-Clark, Emma

AU - Goldacker, Sigune

AU - Warnatz, Klaus

AU - Varjosalo, Markku

AU - Grimbacher, Bodo

N1 - 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.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - DNA

KW - HEK293 Cells

KW - Humans

KW - Mutation, Missense

KW - NF-kappa B/metabolism

KW - NF-kappa B p50 Subunit/genetics

KW - Promoter Regions, Genetic

KW - Proto-Oncogene Proteins c-rel/metabolism

U2 - 10.3389/fimmu.2022.965326

DO - 10.3389/fimmu.2022.965326

M3 - SCORING: Journal article

C2 - 36105815

VL - 13

JO - FRONT IMMUNOL

JF - FRONT IMMUNOL

SN - 1664-3224

M1 - 965326

ER -