De novo NFKBIA variants within the N-terminal hotspot: consistent immunophenotype and divergent clinical...

Germline monoallelic gain-of-function (GOF) variants in NFKBIA, encoding IκBα, cause a rare immunodeficiency syndrome classically described as autosomal-dominant anhidrotic ectodermal dysplasia with immunodeficiency. However, the pathogenic spectrum of variants within the N-terminal hotspot and the extent to which distinct alleles converge on shared immunologic phenotypes are not fully defined.

We studied four unrelated patients with de novo heterozygous NFKBIA variants (p.G33D, p.M37R, p.M37K, p.D31H), including two novel alleles (p.G33D and p.D31H). Clinical and immunological phenotyping, T-cell and B-cell subset analysis, and CFSE-based lymphocyte proliferation assays were performed. Functional consequences were assessed by TNF-α-induced IκBα degradation in patient PBMCs and by NF-κB dual-luciferase reporter assays in HEK293T cells expressing wild-type or mutant IκBα. A comprehensive literature review of all previously reported NFKBIA GOF cases was performed.

Clinical severity ranged from recurrent sinopulmonary infections onset in adolescence to severe infantile multisystem disease with bacterial, fungal, and opportunistic infections. All patients exhibited ectodermal abnormalities, and one had autoantibodies. Despite marked clinical heterogeneity, all four patients showed a qualitatively convergent lymphocyte phenotype characterized by expanded naïve T-cell and B-cell compartments and reduced memory and effector subsets. PHA-induced CD4⁺ and CD8⁺ T-cell proliferation was preserved in P1 and P3, whereas anti-CD3/CD28-induced T-cell proliferation, assessed only in P3, was impaired, while B-cell proliferation was preserved in the tested patients. Patient PBMCs exhibited markedly delayed or minimal TNF-α-induced IκBα degradation, and all four mutant proteins more strongly suppressed TNF-α-induced NF-κB reporter activity compared to wild-type IκBα. Baseline expression of the IκBα-EGFP fusion proteins was comparable across wild-type and all four mutant constructs.

These findings broaden the clinical and genotypic spectrum of N-terminal IκBα GOF disease, identify a consistent immune phenotype characterized by expanded naïve and contracted memory lymphocyte compartments, and support defective regulated IκBα degradation and impaired lymphocyte maturation as shared features of N-terminal IκBα GOF disease.