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Case Reports
. 2016 Oct;101(10):e392-e396.
doi: 10.3324/haematol.2016.145136. Epub 2016 Jun 30.

Specific antibody deficiency and autoinflammatory disease extend the clinical and immunological spectrum of heterozygous NFKB1 loss-of-function mutations in humans

Cyrill Schipp  1 , Schafiq Nabhani  1 , Kirsten Bienemann  1 , Natalia Simanovsky  2 , Shlomit Kfir-Erenfeld  3 , Nathalie Assayag-Asherie  3 , Prasad T Oommen  1 , Shoshana Revel-Vilk  4 , Andrea Hönscheid  1 , Michael Gombert  1 , Sebastian Ginzel  5 , Daniel Schäfer  1 , Hans-Jürgen Laws  1 , Eitan Yefenof  3 , Bernhard Fleckenstein  6 , Arndt Borkhardt  1 , Polina Stepensky  4 , Ute Fischer  7
Affiliations
Case Reports

Specific antibody deficiency and autoinflammatory disease extend the clinical and immunological spectrum of heterozygous NFKB1 loss-of-function mutations in humans

Cyrill Schipp et al. Haematologica. 2016 Oct.
No abstract available

Keywords: Fas; NFKB; antibody deficiency; autoimmune lymphoproliferative syndrome; autoinflammatory disease; immunodeficiency.

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Figures

Figure 1.
Figure 1.
Two novel heterozygous NFKB1 mutations detected in two families decrease the protein levels of p105 and p50. (A) Upper left: Whole exome sequencing identified a heterozygous NFKB1 mutation (A/−) in patient 1. The patient is the only carrier of the mutation in the family pedigree (indicated by “+”) and the only diseased family member (indicated by a filled circle). Lower left: Capillary sequencing using genomic DNA confirmed an NFKB1 frameshift mutation (c.A137del, p.I47YfsX2) in patient 1. Representative chromatograms of patient 1 and a healthy control (HC) are shown. Upper right: Patient 2 descended from consanguineous parents and harbors an inherited heterozygous NFKB1 mutation (C/T). The patient is the only diseased family member. The father and two siblings carry the same mutation but are not affected. Sanger sequencing of NFKB1 confirmed the heterozygous missense mutation (c.C469T, p.R157X) in patient 2. Representative chromatograms are shown. (B) Schematic drawing of the proteins p105 and p50 and their domains which are both encoded by the NFKB1 gene. The mutations in the Rel homology domain (RHD) identified in the two patients (red arrows) lead to early truncation of both proteins. Previously reported heterozygous germline mutations associated with CVID are indicated on top (black arrow and brackets). ANK, ankyrin repeats; DD, death domain; P, PEST domain enriched for proline (P), glutamic acid (E), serine (S), and threonine (T) residues. (C, D) Expression of p105 and p50 proteins is decreased in the affected patients. (C) Primary T cells of patient 1 and healthy controls were activated by phytohemagglutinin in the presence of interleukin-2. Protein and RNA extracts were prepared. Western blot analysis was carried out employing a specific p105/p50 antibody using β-actin as a loading control (left panel). NFKB1 mRNA expression was measured by real-time polymerase chain reaction (right panel). The fold-change in cells of the patient compared to a representative healthy control is shown, GAPDH and β-actin expression were used as internal standards. Mean values of representative experiments performed in triplicates and corresponding SDs are shown. Sanger sequencing using reverse transcribed mRNA of the patient demonstrates the presence of mutated NFKB1 transcripts (lower panel). (D) Epstein-Barr virus-transformed B cells of patient 2 were used for protein and RNA extraction. Analysis of NFKB1 protein and RNA expression was carried out as described in (C). Capillary sequencing of cDNA from patient 2 failed to detect the NFKB1 mutation indicating that the mutation leads to mRNA instability (lower panel). (E) Upper panel: axial high resolution chest computer tomography image of patient 2 at the level of lung bases demonstrating multiple areas of bronchiectasis (white arrows) and consolidation with an atelectatic component surrounding bronchiectases in the right middle lobe (black dashed arrow). Mosaic pattern of perfusion of the lung parenchyma is noted, with multiple areas of low attenuation in the right low lobe (arrowheads). Lower panel: axial computer tomography image of patient 2 at the level of the upper abdomen demonstrating the enlarged spleen.
Figure 2.
Figure 2.
NF-κB-mediated signaling is affected in the patients. (A) Upregulation of NFKB1 mRNA expression in response to NF-κB activating stimuli is deficient in NFKB1-mutated cells. Epstein-Barr virus (EBV)-immortalized B cells of patient 2 and of a representative healthy control (HC) were treated for 16 h with LPS (5 μg/mL), tumor necrosis factor α (TNFα) (50 ng/mL) or IL-1β (10 ng/mL). Fold change of NFKB1 mRNA expression in untreated compared to treated samples was determined by real-time polymerase chain reaction (PCR). The relative expression was normalized to the respective untreated controls (=1). GAPDH and β-actin expression were used as internal standards. Mean values of representative experiments performed in triplicate and corresponding standard deviations are shown. (B) Expression of the NF-κB target gene CFLAR (synonymous for cFLIP) is not induced by LPS in the NFKB1-mutated patients’ cells. EBV-immortalized B cells of patient 2 and healthy control cells were treated for 16 h with LPS (5 μg/mL). Fold change of mRNA expression of CFLAR was determined by real-time PCR as described in Figure 1C. (C) Differential expression of NF-κB target genes in NFKB1-mutated primary T cells of patient 1 and two healthy wild-type controls. Baseline expression of a panel of NF-κB target genes was analyzed by real-time PCR using predesigned arrays (NF-κB signaling targets RT Profiler PCR arrays, Qiagen, Hilden, Germany). Mean values of two independent assays are shown. High gene expression is indicated in light color, low gene expression in dark red. The analysis was supervised and results are shown scaled. (D) LPS does not protect NFKB1 mutant primary T cells from apoptosis. Primary T cells of patients 1 and 2 and healthy controls were activated by phytohemagglutinin and IL2. Cells were treated with 100 ng/mL recombinant Fas ligand to induce apoptosis in the presence or absence of 100 ng/mL LPS or 10 ng/mL TNFα. Apoptosis was determined by flow cytometric measurement of phosphatidylserine exposure indicated by binding of annexin V-FITC. Dead cells were detected with propidium iodide. Significance was tested using two-way ANoVA (*P<0,05; ***P<0,001).
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