Hypomorphic nuclear factor-κB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity

Hypomorphic NEMO mutations. Each asterisk represents an individual patient, and mutation types are color-coded. Structural predictions indicate an extended α-helix structure with 2 coiled coils, a leucine zipper, and zinc finger motifs. The minimal oligomerization domain, serine phosphorylation (p-S), ubiquitination (U), sumoylation (S), ubiquitin binding (NUB), and IKK binding/NEMO dimerization regions are shown. αH, Alpha helix; CC, coiled coil; LZ, leucine zipper; ZF, zinc finger.

NEMO phenotype maps. The following phenotypes are shown: ectodermal dysplasia (A), lymphedema/osteopetrosis (B), inflammatory disease (C), pyogenic infection (D), mycobacterial infection (E), TNF-α response (F), hyper-IgM phenotype/CD40 (G), IL-1/TLR response (H), TCR response (I), and mortality (J). Each oval represents the reported presence (shaded) or absence (dashed) of the indicated phenotype, and is intended to reflect the protein region affected.

Phenotype frequency of shared mutations. Each column represents a mutation that occurred in more than 1 individual. Frequency is depicted by quartile and is color-coded: high (red), intermediate (yellow), and low (green) phenotype presence. IBD, Inflammatory bowel disease; SGA, small for gestational age; Spec. Ab, specific antibody.

Expression levels of reconstituted NEMO are equivalent by anti-NEMO Western blot, intracellular FACS, and GFP FACS. A, Cells from reconstituted lines were lysed and probed with anti-NEMO mAb specific for the NEMO leucine zipper. Actin blotting demonstrates equal loading. FACS to determine GFP expression was performed on NEMO reconstituted cells lines (B), which was evaluated by intracellular staining (n = 2) (C). The gray shaded area demonstrates fluorescence of isotype-control stained cells.

Decreased NF-κB reporter expression after stimulation with TNF-α and flagellin in reconstituted NEMO(-) cells and impaired IκB degradation in the L153R but not C417R cell line. A, Cells were stained with rat-Thy-1phycoerytherin and analyzed by FACS. Decreased levels of expression indicate decreased NF-κB activation in response to TNF-α and flagellin in L153R and C417R. Replicates of experiments indicate significant differences compared to rNEMO; means, SDs, and P values are shown in the box above the histogram. ΔMFI denotes the difference in mean fluorescence intensity between stimulated and unstimulated cells. B, Western blot of IκB levels from the various cell lines after TNF-α activation. Densitometry measurements of IκBα/actin normalized to time = 0 for each cell line are indicated below individual bands.

Apoptosis in TNF-α stimulated cells and A20 expression. A, Cell lines were activated with TNF-α, and apoptosis was measured by annexin-V and 7-AAD. B, Replicates (N = 3) and statistical evaluation of repeated apoptosis assays. C, A20 transcripts were quantified by using real-time PCR, and fold induction of A20 expression is reduced in L153R reconstituted NEMO(-) cells. The result is representative of 2 independently conducted experiments.

Electrophoregram of NEMO G1000T after sequencing of the gel-purified NEMO gene–specific long range PCR product, demonstrating hemizygous presence in the IKBKG gene-specific sequence of the male karyotype Jurkat cell line, resulting in stop codon and predicted L334X protein.

Western blot of the parental Jurkat cell line (pNEMO) and NEMO-deficient line (NEMO[-]). Membranes were probed with rabbit polyclonal (left) and mouse monoclonal (right) antibodies, indicating the presence of only a specific band at the expected molecular weight. P, polyclonal; M, monoclonal.

Model of innate immune signaling through NEMO. Signaling occurs through different groups of functionally related proteins downstream of TNFR and TLR5, which activate the IKK complex and lead to IκB degradation (1). IκB processing leads to nuclear NF-κB translocation (2) and gene transcription (3). Simultaneously, programmed cell death pathways are activated (4) and suppressed (5) by NF-κB–dependent (right orange line) and classical NF-κB–independent (left orange line) gene transcription, such as A20. Thus, a NEMO-dependent but NF-κB–independent pathway uncovered by NEMO-C417R may exist (6). IRAK, IL-1 receptor–associated kinase; TRAF, TNF receptor–associated family of proteins; TAB, TAK-binding protein; TAK, TGF-β activated kinase; MyD, myeloid differentiation; FADD, Fas-associated death domain-containing protein; TRADD, TNF receptor–associated death domain-containing protein; RIP, receptor-interacting protein; cyt. C, cytochrome C.