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

Abstract

Background: Human hypomorphic nuclear factor-kappaB essential modulator (NEMO) mutations cause diverse clinical and immunologic phenotypes, but understanding of their scope and mechanistic links to immune function and genotype is incomplete.

Objective: We created and analyzed a database of hypomorphic NEMO mutations to determine the spectrum of phenotypes and their associated genotypes and sought to establish a standardized NEMO reconstitution system to obtain mechanistic insights.

Methods: Phenotypes of 72 individuals with NEMO mutations were compiled. NEMO L153R and C417R were investigated further in a reconstitution system. TNF-alpha or Toll-like receptor (TLR)-5 signals were evaluated for nuclear factor-kappaB activation, programmed cell death, and A20 gene expression.

Results: Thirty-two different mutations were identified; 53% affect the zinc finger domain. Seventy-seven percent were associated with ectodermal dysplasia, 86% with serious pyogenic infection, 39% with mycobacterial infection, 19% with serious viral infection, and 23% with inflammatory diseases. Thirty-six percent of individuals died at a mean age of 6.4 years. CD40, IL-1, TNF-alpha, TLR, and T-cell receptor signals were impaired in 15 of 16 (94%), 6 of 7 (86%), 9 of 11 (82%), 9 of 14 (64%), and 7 of 18 (39%), respectively. Hypomorphism-reconstituted NEMO-deficient cells demonstrated partial restoration of NEMO functions. Although both L153R and C417R impaired TLR and TNF-alpha-induced NF-kappaB activation, L153R also increased TNF-alpha-induced programmed cell death with decreased A20 expression.

Conclusion: Distinct NEMO hypomorphs define specific disease and genetic characteristics. A reconstitution system can identify attributes of hypomorphisms independent of an individual's genetic background. Apoptosis susceptibility in L153R reconstituted cells defines a specific phenotype of this mutation that likely contributes to the excessive inflammation with which it is clinically associated.

Figure 1. Hypomorphic NEMO mutations

Each asterisk represents an individual patient, and mutation types are color-coded. Structural predictions indicate an extended alpha 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.

Figure 2. 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), 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.

Figure 3. Phenotype frequency of shared mutations

Each column represents a mutation which occurred in more than one individual. Frequency is depicted by quartile and is color coded: High (red), intermediate (yellow), and low (green) phenotype presence.

Figure 4. 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 monoclonal antibody specific for the C-terminus. Actin blotting demonstrates equal loading. B, FACS to determine GFP expression was performed on NEMO reconstituted cells lines, C, which was evaluated by intracellular staining (n=2).

Figure 5. 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-1PE 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, SD and p values are shown. B, Western blot of IκB levels from the various cell lines following TNF-α activation. Densitometry measurements of IκBα/actin normalized to time=0 for each cell line are indicated below individual bands.

Figure 6. Apoptosis in TNF-α stimulated cells and A20 expression

A, Cell lines were activated with TNF-a and apoptosis was measured by Annexin-V and 7-AAD. B, Replicates and statistical evaluation of repeated apoptosis assays. C, A20 transcripts were quantified using real-time PCR and fold induction of A20 expression is reduced in L153R reconstituted NEMO(−) cells, the result is representative of two independently conducted experiments.