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

Background

Human hypomorphic nuclear factor-κB 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-α or Toll-like receptor (TLR)–5 signals were evaluated for nuclear factor-κB 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-α, 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-α–induced NF-κB activation, L153R also increased TNF-α–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.

Key words: NEMO, immunodeficiency, genetic database, Jurkat reconstitution, NF-κB activation, A20

Abbreviations used: 7-AAD, 7-Amino actinomycin D, DC, Dendritic cell, EDA, Ectodermal dysplasia and anhidrosis, EMSA, Electrophoretic mobility shift assay, FACS, Fluorescence-activated cell sorting, GFP, Green fluorescent protein, IKK, IκB kinase, NEMO, Nuclear factor-κB essential modulator, NF-κB, Nuclear factor-κB, pNEMO, Parental nuclear factor-κB essential modulator, rNEMO, Wild-type reconstituted NEMO(-), TCR, T-cell receptor