A Pathogenic Missense Variant in NFKB1 Causes Common Variable Immunodeficiency Due to Detrimental Protein Damage

Abstract

In common variable immunodeficiency (CVID), heterozygous damaging NFKB1 variants represent the most frequent monogenic cause. NFKB1 encodes the precursor p105, which undergoes proteasomal processing to generate the mature NF-κB transcription factor subunit p50. The majority of NFKB1 sequence changes comprises missense variants of uncertain significance (VUS), each requiring functional evaluation to assess causality, particularly in families with multiple affected members presenting with different phenotypes. In four affected members of a German family, all diagnosed with CVID, we identified a previously uncharacterized heterozygous NFKB1 missense variant (c.1049A>G; p.Tyr350Cys). The clinical phenotypes varied markedly regarding onset, frequency and severity of infections. Consistent immunologic findings were hypogammaglobulinemia with normal specific antibody response to protein- and polysaccharide-based vaccinations, reduced switched memory B cells and decreased lymphocyte proliferation upon stimulation with the B cell mitogen SAC. To assess the pathogenicity of the NFKB1 missense variant, we employed immunophenotyping and functional analyses in a routine in vitro cell culture model. Following site-directed mutagenesis to introduce the variant into overexpression vectors encoding EGFP-fused p105 or p50, we analyzed transiently transfected HEK293T cells by confocal imaging and Western blotting. The cytoplasmic p105-Tyr350Cys precursor gained only weak expression levels indicating accelerated decay. The missense change disabled processing of the precursor to prevent the generation of mutant p50. Unlike the wildtype p50, the overexpressed mutant p50-Tyr350Cys was also not sustainable and showed a conspicuous subnuclear mislocalization with accumulation in dense aggregates instead of a homogenous distribution. Electrophoretic mobility shift assays, fluorescence-based reporter gene analyses and co-transfection experiments however demonstrated, that the DNA-binding activity of p50-Tyr350Cys and the interaction with RelA(p65), IκBα and wildtype p50 were preserved. Mutation carriers had reduced p105 and p50 levels, indicating insufficient protein amounts as the most likely primary defect. In conclusion, the missense variant c.1049A>G caused a detrimental defect, preventing the persistent expression of both, the p105-Tyr350Cys precursor and the mature p50-Tyr350Cys. The variable clinical phenotypes among affected family members sharing an identical pathogenic NFKB1 variant support a disease mechanism provoked by a p105/p50 (haplo)insufficient condition.

Establishing the Molecular Diagnoses in a Cohort of 291 Patients With Predominantly Antibody Deficiency by Targeted Next-Generation Sequencing: Experience From a Monocentric Study

“…The latter patient additionally carried the known p.Cys104Arg mutation in TNFRSF13B. Although functional in vitro testing, as previously described (23), indicated that none of these four variants cause a detrimental protein loss, we could not exclude a hypomorphic reduction of protein function (data not shown). Using reporter assays, a recent study demonstrated a loss-of-function for p.Arg57Cys, whereas a functional defect associated with p.Tyr90Ser, p.Arg214Gln, and p.Met216Val, remained obscure (63).…”

“…Levels of Adenosine deaminase 1 or 2 (ADA and ADA2) were evaluated by measuring the specific enzyme activity in Michael S. Hershfield's lab at Duke University School of Medicine (USA) and at the Advanced Diagnostic Unit, University of Freiburg (Germany), respectively. NFKB1 variants were analyzed as described previously (23).…”

B- and T-Cell Subset Abnormalities in Monogenic Common Variable Immunodeficiency

“…Patients with this defect manifest hypogammaglobulinemia due to decreased switched memory B cells, and diminished lymphocyte proliferation after stimulation with B cell mitogens. It seems that the patients usually have a normal response to T-dependent and T-independent antigens ( 86 ).…”

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

“…In addition, frameshift variants in the central part of p105, predict immediate expression of p50-like proteins, which by-pass the precursor stage ( 19 , 20 ), as well as the sporadically occurring C-terminal truncations ( 21 , 22 ), are also considered to have a pathogenic effect, although the disease-causing mechanisms are still unknown for both entities. The pathogenic significance of most (51/54 and 31/33) of the identified NFKB1 missense alterations, however, remained uncertain ( 7 , 10 ) unless experimental confirmation of their causality became available ( 18 , 23 ). Amino acid changes localizing to the N-terminal half (aa 1-433) affect both the p105 precursor and the p50 subunit.…”

“…In a recent report, we used a standard cell culture model, based on transient transfection of HEK293T cells with EGFP-fused p105 and p50 constructs, enabling functional assessments of NFKB1 missense variants ( 23 ). Subsequent analyses, including expression and subcellular localization of the overexpressed proteins as well as p50-dependent DNA-binding and RelA-mediated reporter gene activation, confirmed a detrimental protein loss caused by a single amino acid change (p.Tyr350Cys) in a CVID family with p105/p50 haploinsufficiency.…”