Novel nonsense gain-of-function NFKB2 mutations associated with a combined immunodeficiency phenotype

Abstract

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.

Figure 1.

Genetics and pedigrees of families with NFKB2 mutations. (A) Pedigrees and Sanger sequencing of families with NFKB2 mutations. Mutation status of NFKB2 c.1252G>T for family A, NFKB2 c.1903C>T for family B, and NFKB2 c.2596A>C for family C are indicated for each individual. Pedigree with those affected shown in black filled symbols. (B) Schematic of NF-κB2 with the Rel homology domain (RHD), nuclear localization signal (N), glycin-rich region (GR), ankyrin repeat domain (ARD), and death domain (DD). The amino acid sequence and location for affected individuals are shown with arrow.

Figure 2.

NF-κB2 protein expression in PBMCs. (A-C) Immunoblot of wild-type and mutant NF-κB2 in PBMCs from healthy normal control and patients. Total PBMCs were treated with α-CD3 to stimulated NF-κB2 pathway for 48 hours. Cell lysates were prepared and analyzed for phosphorylation of p100 and for full-length (p100) and active form (p52), and mutant expression by western blotting. Immunoblotting of β-actin was used as a loading control. Red asterisks indicate mutant form of NF-κB2 in patient cells. Due to the similar size between mutant E418X and active form of NF-κB2 (p52), it was difficult to discriminated mutant (E418X) from active form of NF-κB2 in stimulated condition in patient A1.

Figure 3.

Protein expression and localization of mutant NF-κB2 in transfected cells. (A) HEK293T cells were transiently transfected with the indicated constructs in the presence and absence of NIK coexpression. Cell lysates were analyzed for phosphorylation of p100 and for p100, p52, and mutant expression by western blotting. E418X mutant size was closed to the active form p52. All other mutants are failed phosphorylation and processing to p52 in the presence of NIK expression. Molecular weight markers are indicated on the left size (kilodaltons). (B) HEK293T cells were transiently transfected indicated WT or mutant vector (pcDNA3-HA) with or without Flag-NIK. Nucleus was labeled with DAPI (blue). Original magnification ×175. Cells were stained with anti-Flag and anti-HA antibody, followed by Alexa 488-conjugated (for HA-NF-kB2, green) and Alexa 568-conjugated (for Flag-NIK, red) secondary antibody. Data shown are representative of 3 experiments.

Figure 4.

Increased interaction of RelB with mutant E418X, R635X in nuclear fraction. HEK293T cells were transiently transfected with the indicated constructs in the presence and absence of NIK coexpression. (A) Cytoplasmic and (B) nuclear fractions were prepared, and immunoprecipitated with an α-HA antibody. Cell lysates from IP samples were analyzed for the interaction of NF-κB2 (WT or mutants) with RelB. The faint band at 50 kDa in all lanes is immunoglobulin heavy chain. Immunoblotting of Lamin A/C and GAPDH was used as a marker for the nuclear and cytoplasmic fraction, respectively. Red asterisks indicate increased interaction of mutant E418X, R635X with RelB without NIK expression. Blue asterisks indicate increased translocation of mutant forms E418X, R635X, and active form of NF-κB2 (p52) to the nucleus. Data shown are representative of 3 experiments.

Figure 5.

Regulation of CXCL13 gene expression by NF-κB2 protein. (A-B) Indicated siRNA or pcDNA3-HA-NFKB2 (WT or mutants) were transfected into total PBMCs from healthy donor control. Next day, cells were washed and stimulated with Dynabeads-CD3/28 for 48 hours. RNA was prepared and CXCL13 gene expression was analyzed by real-time PCR. A fold change was calculated for NFKB2 siRNA or mutants with control vector (A) or WT (B) transfected cells, normalize to 1. CXCL13 was not detected in unstimulated samples. Decreased NF-κB2 (A) or overexpressed WT or mutant protein expression (B) was confirmed by western blot. Results shown are means + standard error of the mean of 3 independent experiments. (C) CD4 T cells were enriched using stem cell negative selection kit and cells were stimulated with Dynabeads-CD3/28 for 48 hours. Levels of CXCL13 mRNA in activated CD4 T cells from indicated patients were measured by real-time PCR using the probe for CXCL13 and normalized to 18S rRNA. CXCL13 expression was not detected in A1 due to low cell number. Data are means value of replicates from indicated patients. For relative gene expression, all data were normalized to the paired normal control.

Figure 6.

Increased canonical NF-κB signaling in patients with nonsense GOF NFKB2 mutation. (A) HEK293T cells growing in 24-well plates were cotransfected with pGL4.32-NF-κB and indicated NFKB2 expression vector. Next day, cells were treated with TNFα for 5 hours and luciferase activities were measured. Western blot analysis confirmed similar protein expression levels. The data were shown as fold change compared with TNFα-treated empty vector (pcDNA3-HA). Results shown are means + standard error of the mean of 4 experiments. *P = .0267, ***P = .0005 (Student t test). (B) Total PBMCs were stimulated with LPS (100 ng/mL) for 24 hours. Cytokine expression in supernatants was analyzed via multiplex bead assay (Luminex). Data shown are from 3 experiments with a heathy donor and indicated patients tested in pairs.