Case Report: German Family with Unspecified Antibody Deficiency

The index patient (II-1, female, born in 1985) suffered from upper recurrent tonsillitis and severe sinusitis since childhood, requiring intravenous antibiotic therapy approximately twice per year. At the age of 10, she developed arthritis in the right ankle joint, however serum rheumatoid antibodies were not identified. Hence, this was classified as an episode of seronegative arthritis, which resolved without treatment at age 11. She had no further episodes of autoimmunity. A tonsillectomy was performed at 10.5 years of age, however, this did not lead to an improvement in the upper respiratory tract infection frequency or severity. She did not suffer from recurrent viral or opportunistic infections. At 15 years of age, her IgG level was found to be 5.9 g/l, while her IgA, IgM and IgE levels remained within the normal range. IgG1, IgG2 and IgG3 subclasses were reduced. Specific antibody levels to tetanus, diphtheria, and Haemophilus influenzae type B were above the protective threshold. Pneumococcal vaccine responses were not assessed. Due to recurrent upper respiratory infections, immunoglobulin replacement therapy was started at that time. At 28 years of age, the immune phenotype of the patient was largely normal, with normal proportions of T- and B- cells and very slightly reduced NK cells. CD4 + and CD8 + T cell subsets, including regulatory T cells (Treg) and circulating T follicular helper cells were unremarkable but effector memory CD8 + T cells were reduced. B cell subpopulations showed reduced unswitched and switched memory B cells (Table 1). Two years later, her IgM dropped below the normal reference range (0.25 g/L). Her CRP remained persistently elevated at regular follow-up intervals between 10 and 40 ng/l, without clinical symptoms of infection or inflammation. A CT Thorax performed at 38 years of age showed no signs of bronchiectasis and was otherwise unremarkable.

Table 1 Laboratory features of subject I-1 (father of index patient) and patient II-1 (index patient)

The patient is currently 40 years old and does not suffer from recurrent infections under immunoglobulin replacement therapy. Since commencing immunoglobulin replacement therapy, her IgG trough level remained within protective levels between 8 and 10 g/l, except during a short episode of a treatment administration lapse at 30 years of age (Table 1). At regular clinical follow-up intervals, the patient denied symptoms of endocrine dysfunction, such as fatigue, unexplained weight changes, dizziness or hypotension. Therefore, no endocrine laboratory screening was performed.

To test whether a genetic defect accounts for the disease phenotype, the index patient (II-1) underwent routine genetic testing by Next Generation Sequencing (NGS) as previously described [36, 37]. Whole exome sequencing (WES) revealed a heterozygous missense variant c.781C>T in NFKB2, predicting the substitution of one amino acid p.Arg261Trp (R261W) within the Rel-homology domain of p100/p52 (Fig. 1A). Most of the known pathogenic NFKB2 variants affect the C-terminal phospho-degron domain of p100, rendering the precursor non-processable and are, amongst other symptoms, associated with early-onset primary immunodeficiency, antibody deficiency and profoundly impaired B-cell differentiation and frequent T-cell mediated autoimmunity as well as endocrinological abnormalities [4, 7,8,9,10,11]. Compared to published NFKB2 cases, disease expression in our index patient was relatively milder suggesting a rather ‘non-typical’ p100/p52 defect (if any), consistent with the affected amino acid position far from the C-terminal end. Subsequent Sanger sequencing (Fig. 1B-C) showed that she inherited the NFKB2 variant from her father (I-1; born in 1959), who was clinically asymptomatic. The father’s laboratory assessment showed marginally reduced IgG levels, IgA and IgM levels within reference range and an impaired polysaccharide pneumococcal vaccine response. His immunological phenotyping showed a reduction in memory and early effector CD8⁺ T cells, along with an increase in late effector CD8⁺ T cells (Table 1). Memory B cells were decreased, with a more pronounced reduction in IgA⁺ B cells, as well as a decrease in plasmablasts. He was commenced on immunoglobulin replacement therapy as a prophylactic measure. The elder of the two daughters (III-1 born in 2011) of the index patient carries the same NFKB2 variant and has so far remained clinically unaffected. No detailed immunological phenotyping was performed. The younger daughter (III-2 born in 2015), her mother and her brother are wildtype for NFKB2 (Fig. 1C) as confirmed by Sanger sequencing.

Fig. 1

Segregation of the heterozygous NFKB2 missense variant c.781C>T in a German family with hypogammaglobulinemia is associated with reduced p52 levels and impaired NF-κB2 signaling. A Domain structure of p100/p52 and localization of the missense variant p.Arg261Trp (R261W) within the C-terminal part of the Rel-homology domain. Protein domains of the p100 precursor (long horizontal double arrow) comprise the N-terminal Rel-homology domain (RHD), the nuclear localization sequence (NLS), the central glycine-rich region (GRR), and the C-terminal Ankyrin-repeat domain (ANK) and the death domain (DD). Pathway stimulation leads to NIK-dependent phosphorylation of two of the three Serin residues (S866 and S870) near the C-terminus and subsequent ubiquitination at K855. The mature p52 form (short horizontal double arrow), originates from limited proteasomal processing of the C-terminal half of p100. The ‘cleavage site’ which generates the C-terminal end of p52 is reported differently in the literature and is indicated by triple arrow heads. Amino acid positions are indicated by numbers. B Sanger sequencing confirms the heterozygous c.781C>T variant, predicting a single amino acid change p.Arg261Trp (R261W) in the index patient. C The pedigree of the affected family. Family members tested by Sanger sequencing are indicated. Circles, female; squares, male; filled black symbol, affected individual, divided symbol, unaffected carrier; open symbol, healthy member. The arrow points toward the index. D Western blot of whole cell lysates derived from EBV-transformed B lymphocytes shows p100 levels in the lower range and strongly reduced p52 in two independently generated lines from the index patient compared to three healthy donors. Relative normalized densitometric values are shown on the right. E Reduced overall NF-κB2 expression (p100 plus p52) in EBV-B cell lines derived from the index patient (red) compared to four healthy donor controls (black). Mean fluorescence intensity (MFI) values are indicated. The relative MFI are shown on the right. Identical cell preparations were used in D and E. F Ratio of the mean fluorescence intensities (MFI) stimulated and unstimulated for CD86, CD69, ICOS-L, CD83 and CD62L gated on naïve IgDpositive-CD27negative B cells after stimulation with BAFF or anti-IgM in three healthy donor controls (HD, blue) and the patient (red). In patient cells, CD86 and CD69 are slightly less increased after BAFF stimulation. The gating strategy and representative histograms are shown in Supplementary Figure S3

The index patient has previously been reported (alias: patient-171) by us to carry a copy number variant (CNV) of uncertain significance in the putative promoter region of TRIM22 (tripartite motif containing 22), comprising a 9.3 kb duplication of the first four exons, in addition to the NFKB2 missense variant [36]. TRIM22 variants have been associated with inflammatory bowel disease [44], yet TRIM22 is not known to be involved in B cell development or antibody deficiencies.

In addition, the index patient is compound heterozygous in trans [c.439G>A];[c.2391C>A] for two missense variants in exons 4 and 26 of VAV1 (vav guanine nucleotide exchange factor 1) both causing single amino acid changes [p.Asp147Asn];[p.Asp797Glu] ([D147N];[D797E]. She inherited the D147N allele from her father (I-1) which was inherited by her younger daughter (III-2), while the older daughter (III-1) inherited the D797E allele. D147N was listed in gnomAD (allele count: 5) and ClinVar (one carrier) and had moderate variant effect predictions (SIFT: deleterious; PolyPhen: possibly/probably damaging; CADD: 28.8; REVEL: 0.29.). D797E was not reported in population databases (SIFT deleterious; PolyPhen possibly damaging; CADD: 22; REVEL 0.186). Since haploinsufficiency of VAV-1 has previously been associated with T-cell driven CVID and mediates intracellular calcium release and activation of transcription factors such as NFAT and NF-κB in T and B cells, respectively [45, 46] we tested both, VAV1 expression and calcium flux in patient-derived PBMCs (Supplementary Figure S1). While Western blotting indicated reduced VAV1 levels in the index patient and her father, calcium release in stimulated B and T cells was within the normal range. We therefore do not consider the identified VAV-1 missense variants as pathogenic although we cannot exclude a modifying effect.

Identification of a Detrimental NFKB2 Missense Variant as the Genetic Cause of Hypogammaglobulinemia

The heterozygous NFKB2 missense variant (c.781C>T; p.Arg261Trp; R261W) within the Rel-homology domain affects both, the p100 precursor protein and its processing product, the mature p52 transcription factor subunit (Fig. 1A). The sequence change (dbSNP ID: rs769218279) is listed three times in gnomAD (https://gnomad.broadinstitute.org; dataset v.4.1.0; Variant ID: 10-102398226-C-T), and twice in ClinVar (www.ncbi.nlm.nih.gov/clinvar; Variation ID 546654) with uncertain significance (accessed April 23, 2026). One of the entries represents the current case. ACMG criteria were: PM1, PM2, PP3, (https://wintervar.wglab.org/). Variant effect predictions were: SIFT deleterious; PolyPhen: probably damaging; CADD: 34; REVEL 0.924. R261W has been shown to affect the surface of the dimerization domain of p52 [47] and has been identified as a somatic sequence variant in endometrial, kidney and lung carcinoma tissue samples (https://cancer.sanger.ac.uk/cosmic; Genomic Mutation ID COSV50045881).

A sequence alignment indicated Arg261 of the non-canonical p100/p52 to correspond to Arg284 of the canonical p105/p50, a residue known to be affected by the detrimental missense change R284P [33]. We therefore hypothesized that the single amino acid change R261W in NFKB2 might cause a similar defect with protein loss. Western blot analysis of unstimulated EBV-immortalized lymphoblastoid cells derived from the index patient showed moderately diminished levels of p100 (72.6 ± 21.4% of HD levels), while p52 was decreased to approximately half (51.0 ± 14.9% of HD levels) compared to healthy donor controls (Fig. 1D). Similarly, in CD40L-stimulated EBV-B cells, we found reduced p52 levels, while p100 and phospho-p100 were less affected (Supplementary Figure S2). However, various of our attempts to determine p100 and phospho-p100 levels by Western blotting mostly remained inconclusive, likely due to the variable expression in our cell lines, while reduced p52 was repeatedly examined in patient-derived cells (data not shown). When we used flow-cytometric analysis in patient-derived EBV lines, we observed the expression of NF-κB2 (total levels of p100 plus p52) to be reduced to 37–70% of the levels in healthy donor cells (Fig. 1E). Since Western blot analyses suggested that p52 is likely to account for a larger proportion of the protein loss than p100, we suspected that R261W might destabilize p100 levels, but primarily affects p52 in a destructive way, probably leaving very few (or no) mutated proteins which causes a shortage of the mature transcription factor subunits.

To address the functional capacity of B cells carrying the NFKB2 R261W variant in vitro, PBMCs were stimulated with BAFF to activate the alternative NF-κB2 pathway or with anti-IgM to selectively stimulate signaling via the canonical NF-κB1 pathway (Fig. 1F; Supplementary Fig. 3). Flow cytometry indicated that BAFF-dependent upregulation of CD86 and to lower extend of CD69 was reduced on naïve B cells derived from the patient compared to the healthy donor controls. However, both markers were normally induced after anti-IgM stimulation. The upregulation of ICOSL and CD83 as well as the downregulation of CD62L was not remarkably different from healthy controls. These findings confirm functional alterations in alternative NF-κB2 signaling while BCR-mediated signaling was normal. However, tests for reduced p100/p52 expression in primary cells did not yield clear results (data not shown).

The R261W Missense Variant Mitigates the Sustainability of the NF-κB2 Transcription Factor Precursor p100 and Causes Subnuclear Mis-localization of the Mature p52 Upon Overexpression

Because we obtained ambiguous results when analyzing p100 and p52 protein content in EBV cells and primary patient-derived cells, we sought for a better suited model to investigate the effects of the R261W variant on protein stability and function. Therefore, to confirm that R261W causes a severe protein damage, we followed previously introduced and approved in vitro protocols, enabling the functional characterization of mutant NF-κB proteins [33, 34]. The assay employs overexpression of EGFP-fusion wildtype and mutant NF-κB proteins in a routine cell culture model while any observed deviation from the functional properties of the wildtype control indicates a pathogenic protein defect and supports the diagnosis of a NF-κB-related disease. We first generated cDNA expression constructs for N-terminally EGFP-tagged derivates of wildtype p100 (the cytoplasmic precursor) and p52 (the nuclear transcription factor subunit) and subsequently introduced the missense variant via site-directed mutagenesis to obtain EGFP-p100-R261W and EGFP-p52-R261W. We then assessed the subcellular localization of the overexpressed proteins in HEK293T cells by fluorescence microscopy. Both EGFP-p100wt and EGFP-p100-R261W were confined to the cytoplasmic compartment in all transfected cells (Fig. 2A), with the mutant protein gaining markedly weaker intensities (Supplementary Figure S4), which suggests reduced protein sustainability.

Fig. 2

The NF-κB2 missense variant R261W causes sub-nuclear mis-localization and aggregation of p52. Confocal fluorescence microscopy of HEK293T cells transiently transfected with expression vectors (300ng) encoding EGFP-tagged wildtype or R261W-mutant p100 or p52 as indicated (green). A non-processable truncated variant R853X and an empty EGFP-vector control were included as shown in Supplementary Figure S4. DIC-overlay images are shown in Supplementary Figure S5. Nuclei were stained with Hoechst33342 (blue). A scale bar is indicated in the upper left panel. Paradigmatic images are shown representing the most prominent aspects. A Both, p100-wt and p100-R261W localize exclusively to the cytoplasm. Please note: p100-R261W typically gains weaker intensities compared to the wildtype control (not shown), suggestive of lowered stability. Fluorescence intensities were adjusted for best visibility and do not reflect the relative expression levels (please see low-resolution images in Supplementary Figure S4). B Co-expression of wildtype or R261W-mutant p100 together with a constitutively active variant of the NF-κB-inducing kinase (GOF-NIK) to enforce p100-to-p52 processing. Upon GOF-NIK-mediated processing of its cytoplasmic EGFP-p100 precursor, the endogenously generated EGFP-p52-wt localizes with a homogeneous distribution within the nuclei. In contrast, the endogenously generated EGFP-p52-R261W mutant accumulates in dense aggregates in the centers of the nuclei. 3D-reconstructed Z-scans are shown in Supplementary Figure S6. C The artificially expressed nuclear wildtype EGFP-p52 (short-cutting the generation of nuclear p52 from its cytoplasmic p100 precursor), like its endogenously generated counterpart, shows a homogeneous subnuclear localization, while the directly expressed EGFP-p52-R261W mutant proteins again accumulate within dense structures inside the nuclei. Please note the unique subnuclear localization patterns of the endogenously generated and the ectopic EGFP-p52-R261W. Please also see the 3D-images in Supplementary Figure S6

To promote p100-to-p52 processing, we adapted a firmly-established and elegant experimental maneuver [26], and co-transfected trace amounts of a vector encoding a constitutively active variant of the NF-κB-inducing kinase [38], denominated here as GOF-NIK, together with the vectors encoding the wildtype or mutant p100 precursors (Fig. 2B and Supplementary Figures S4 and S5). Upon overexpression of the EGFP-fused wildtype p100, the co-expressed GOF-NIK caused a relocation of the fluorescence signal from the cytoplasm to the nucleus in a high proportion of the transfected cells, indicating efficient, GOF-NIK-dependent conversion of cytoplasmic p100 precursor proteins into mature nuclear p52 subunits (please see the sections below). While the nuclear fluorescence of the wildtype protein was uniformly distributed, the R261W-mutant p52 proteins, generated from their mutant precursors by the ectopic GOF-NIK activity, accumulated in dense aggregates in the centers of the nuclei (please see the following two paragraphs for details), thereby displacing the genomic DNA to the periphery of the nuclei (Fig. 2B and Supplementary Figures S4 and S5). The cytoplasmic localization and expression level of the C-terminally truncated, non-processable variant p.Arg853* (R853X), which was included as a representative control of the most common types of pathogenic NFKB2 variants, was indistinguishable from the wildtype control while the EGFP alone had no specific subcellular localization. Both proteins largely remained unaffected when GOF-NIK was co-expressed (Supplementary Figure S4) further confirming the high specificity of the assay.

Upon immediate expression of the EGFP-fused wildtype p52 (to by-pass the NIK-mediated processes which generate p52 from its precursor), the homogeneously distributed fluorescence signals were exclusively detected within the nuclei of all transfected cells (Fig. 2C; Supplementary Figures S5 and S6). In contrast, the missense mutant EGFP-p52-R261W accumulated in high intense spot-like sub-nuclear structures in all cells gaining high overexpression levels, again occupying the centers of the nuclei, and caused morphological aberrations of the nuclei.

The altered expression and localization of the R261W-mutant NF-κB2 proteins resembles the typical pattern previously observed with protein-decaying pathogenic NFKB1 variants [2, 33, 34, 48]. Formation of characteristic subnuclear aggregates upon overexpression in vitro has previously been established as the most-reliable indicator of protein-damaging missense variants and certain types of truncating variants in NFKB1 and is hitherto a main diagnostic criterion for pathogenicity. The dynamically accumulating fluorescence signals can result in huge subnuclear structures and is typically seen in cells with high over-expression levels of severely damaged p50 and, as we demonstrate here, of p52 as well. We therefore hypothesized that the R261W variant in NFKB2 similarly causes intensified protein loss of p100 but particularly of p52.

The R261W Missense Variant Prevents Durable p52 Expression

To further characterize the deleterious effects of the R261W missense change, we analyzed the overexpressed mutant proteins in transfected HEK293T cells by Western blotting (Fig. 3). The EGFP-fused wildtype p100 gained robust protein levels, consistent with our microscopic observations. In contrast, the p100-R261W variant only reached moderate levels (54.9 ± 3.1% of the wildtype protein), compatible with injurious effects, while expression of the truncated R853X variant (which was included as a non-processable control) was comparable to its wildtype counterpart (Fig. 3; left). Constitutively generated EGFP-p52 - produced from the transfected EGFP-fused precursor proteins by cell-intrinsic processes - was observed in moderate amounts upon overexpression of EGFP-tagged wildtype p100 (5.08 ± 1.55% compared to its precursor), but was only weakly present (1.26 ± 1.72% compared to its precursor and only 19.52 ± 9.64% of wildtype levels) or in some cases undetectable with the EGFP-p100-R261W variant (Fig. 3 left and Supplementary Figure S7). Minor amounts of EGFP-p52 were also generated from the overexpressed EGFP-R853X mutant.

Fig. 3

The R261W missense variant causes a deleterious defect with protein loss. HEK293T cells were transiently transfected with expression vector constructs (300ng) either encoding EGFP-fused p100 derivates alone (left) or together with untagged GOF-NIK (12.5ng) to promote p100-to-p52 conversion (middle), or encoding wildtype or mutant p52 (right). The truncated variant R853X and the empty EGFP-vector serve as controls. A Proteins were simultaneously detected in whole cell lysates by Western blotting using an antibody directed against an N-terminal epitope of p100 and p52 (both green). An anti-β-actin antibody was used as loading control (red). Representative results of multiple experimental repeats as indicated in Methods are shown. (left) In transfected cells, a small portion of wildtype (and to a much lesser extent of the truncated R853X) but not of the R261W-mutant EGFP-p100 is constitutively converted to EGFP-p52 by endogenous precursor processing. EGFP-p100-R261W typically gained weakened expression levels and could not be elevated using saturating DNA amounts (Supplementary Figure S7). (middle) Co-expression of GOF-NIK strongly promoted processing of the wildtype EGFP-p100 whereas the mutant precursor forms either are only weakly processed or release only unsustainable p52 (R261W) or largely remain unprocessed (R853X). (right) The limited expression level gained with the EGFP-p52-R261W expression construct indicates the deleterious character of the missense variant. Please note: the p52 proteins (still retaining their N-terminal EGFP-tag) generated by endogenous mechanisms are smaller in size than the EGFP-fused 447 amino acid p52 used for ectopic expression (which is likewise further processed into a smaller form). Comparable results were obtained with shorter p52 versions (415 and 405 amino acids; Supplementary Figures S4 and S8) verifying that the mis-localization and decay is due to the R261W missense change but not the extension at the C-terminal end. B Automated microscopic scan of the duplicate transfection samples in 48-well format prior to cell harvest to obtain whole cell lysates for the Western blot shown above or nuclear extracts for EMSAs as shown in Fig. 4 and relative fluorescence intensities (MFI) normalized to the EGFP-p100wt samples. EGFP-p100-R261W gained (0.73 ± 0.14)-fold and EGFP-p100-R261W plus GOF-NIK (0.55 ± 0.03)-fold MFI values compared to the wildtype counterpart in 4 and 7 independent experiments, respectively. Please note: The magnitude of any observed deviation may vary depending on the experimental conditions and should not be regarded as a ‘threshold’ for pathogenic effects

Strongly intensified p100-to-p52 processing (with p52 reaching 146.6 ± 19.3% of p100 levels) mediated by co-delivery of GOF-NIK, was only observed with the EGFP-tagged wildtype p100, whereas only marginal amounts of EGFP-p52-R261W (reaching only 14.1 ± 2.4% of its parental protein and only 6.8 ± 2.0% of the wildtype levels) were generated from its mutant p100 precursor (Fig. 3; middle and Supplementary Figure S7). A minor increase (if at all) was detectable with the truncated R853X variant, confirming its resistance against NIK-mediated activation. Compared to the EGFP-fused wildtype p52, immediate expression of the nuclear EGFP-p52-R261W variant gained substantially lower (21.2 ± 5.5% of wildtype) protein levels (Fig. 3; right), indicating a damaging effect, which is compatible with subnuclear disposition as observed in the microscopic analyses above.

To experimentally short-cut the p100-to-p52 conversion by immediate expression of p52, we used an artificial 447 amino acids protein [24, 25], which (most likely due to an extension at the C-terminal end) has a slightly higher molecular weight than the p52 form generated by endogenous processes (Fig. 3). However, except for reduced expression levels, we did not observe any relevant difference (Supplementary Figures S4 and S8) when we repeated our experiments with 415 and 405 amino acids versions of p52 (415aa wildtype: 82.4 ± 15.6%; 405aa wildtype: 82.4 ± 19.1; 415aa mutant: 46.1 ± 9.1%; 405aa mutant: 47.2 ± 12.6% compared to the 447aa versions each), which better match the size of endogenously generated protein [47, 49, 50]. The even longer 454 amino acids variant (Uniprot Q00653) has not been tested.

Our in vitro results, involving CMV-promoter-driven protein over-expression in HEK293T cells, suggest that the missense mutant p100-R261W precursors have reduced sustainability, which limits the generation of mutant and probably harmful p52 transcription factors. The mutant mature p52-R261W subunits, however, are unsustainable and appear to undergo augmented decay and/or subnuclear deposition. Alternatively, if R261W only affects the stability of p52, permanent processing of p100 to supply and to maintain baseline p52 levels, would limit the total amount of p100. Therefore, in either case, a disease-causing mechanism most likely originates from reduced amounts of p100 and particularly p52, rather than from the presence of dysfunctional proteins.

Cell-intrinsic Processing of p100-R261W Does not Produce Nuclear DNA-binding Activity Although DNA-binding Ability of Ectopically Expressed p52-R261W Remains Preserved

We then tested whether the missense variant R261W prevents the generation of p52-dependent DNA-binding activities (which involves dimerization and nuclear translocation) when p52 is generated via intracellular processing of overexpressed mutant p100 precursor proteins. This could, if binding partners from endogenous pools are available, allow the assembly of any hetero- or homo-dimeric NF-κB transcription factor species. In addition, we investigated whether the amino acid change itself interferes with the DNA binding ability of p52. When overexpressed, nuclear p52 is constitutively present in excess and, if dimerization is enabled, the assembly of homo-dimers is likely favored due to stoichiometry. Using electrophoretic mobility shift assays (EMSA) no DNA-binding activity was detectable in nuclear protein extracts when either wildtype or R261W-mutant EGFP-fused p100 alone was overexpressed in HEK293T cells (Fig. 4 left), indicating that constitutive precursor processing is too low or absent (as determined by Western blotting; please see Fig. 3) and/or that nuclear translocation of newly generated p52 is prevented by excess cytoplasmic IκB-activity (most likely mediated by their own parental p100 proteins). No DNA-binding was observed with the truncated R853X variant.

Fig. 4

The R261W missense variant abolishes the generation of p52-dependent DNA-binding activities via endogenous precursor processing, although DNA-binding itself is retained in ectopically expressed mutant p52. HEK293T cells were transiently transfected with 300ng of the indicated EGFP-fused derivates of p100 or p52 as indicated (and as shown in Fig. 3). NF-κB-specific DNA-binding activities in nuclear protein extracts were analyzed by EMSA. Representative results (please see Methods for details) are shown. (left) In cells transfected with wildtype or mutant EGFP-p100 expression constructs, DNA-binding activity is undetectable, most likely because the processed EGFP-p52 proteins (both wildtype and mutant) are retained within the cytoplasm by excess IκB-activities (presumably mediated by their own overexpressed precursors) or because constitutive processing is low or absent (p100-R261W and R853X). (middle) Co-expression of GOF-NIK (12.5ng DNA) leads to increased p100-to-p52 processing of the wildtype EGFP-p100 (as shown in Fig. 3) and consequently to pronounced nuclear DNA-binding activity, also including endogenous NF-κB proteins. No DNA-binding activity is generated from the EGFP-fused p100-R261W-mutant precursor, even with saturating DNA amounts (please see Supplementary Figure S7). Please note: the smaller sized EMSA bands in EGFP-p100-R261W and the empty vector lanes, most likely correspond to endogenous NF-κB proteins (and are restrained in the cytoplasm by R853X). (right) Upon artificial expression of mature nuclear EGFP-p52 (i.e. not generated via processing of cytoplasmic precursor proteins), strong DNA-binding activities are detectable with both, the wildtype and the R261W-mutant. Comparable results were obtained with three differently-sized (447, 415 and 405 amino acids) ectopically expressed p52 variants (Supplementary Figure S8). Under non-saturated conditions, the magnitude of DNA-binding depends on the overexpression levels and protein input

When we co-expressed EGFP-p100 together with GOF-NIK to enhance precursor processing and EGFP-p52 generation, strong DNA binding activity was detectable, (gaining 96.5 ± 6.4% of the levels of the ectopically expressed EGFP-p52; lane 9) only in the wildtype control, but not with the EGFP-fused R261W-mutant precursors or the non-processable truncated variant (Fig. 4 middle). However, GOF-NIK expression was sufficient to increase the nuclear DNA-binding activity of cell-intrinsic NF-κB proteins, discernable by the smaller size of the shifted band. Interestingly, the truncated EGFP-R853X variant, which gains expression levels comparable to its wildtype counterpart, was able to completely block nuclear translocation of endogenous NF-κB proteins, while the low expressed EGFP-p100-R261W mutant was not.

On the other hand, robust DNA-binding activity was obtained upon direct overexpression of both, the EGFP-fused nuclear wildtype p52 and the mutant p52-R261W variant (gaining 61.4 ± 15.6% of wildtype levels), suggesting that the single amino acid change per se does not abolish the ability of the mutant transcription factor to bind to DNA (Fig. 4, right).

Together, these observations suggest a scenario in which R261W-mutant precursor proteins can be expressed, but are unsustainable, and are either not further processed to produce mutant p52 transcription factor subunits, or the processing products are instantly removed or sequestered by a protein quality control mechanism to protect the NF-κB system from dysfunctional signaling components. Both the overall mild phenotypes within the affected family and the low frequency of p52-decaying variants observed in IEI patients are consistent with previous reports [7,8,9,10,11], suggesting the heterozygous loss of p52 – or even of p100 and p52 - being usually associated with haplo-sufficiency (non-pathogenic), rather than insufficiency (pathogenic).