The same IkappaBalpha mutation in two related individuals leads to completely different clinical syndromes

Abstract

Both innate and adaptive immune responses are dependent on activation of nuclear factor kappaB (NF-kappaB), induced upon binding of pathogen-associated molecular patterns to Toll-like receptors (TLRs). In murine models, defects in NF-kappaB pathway are often lethal and viable knockout mice have severe immune defects. Similarly, defects in the human NF-kappaB pathway described to date lead to severe clinical disease. Here, we describe a patient with a hyper immunoglobulin M-like immunodeficiency syndrome and ectodermal dysplasia. Monocytes did not produce interleukin 12p40 upon stimulation with various TLR stimuli and nuclear translocation of NF-kappaB was impaired. T cell receptor-mediated proliferation was also impaired. A heterozygous mutation was found at serine 32 in IkappaBalpha. Interestingly, his father has the same mutation but displays complex mosaicism. He does not display features of ectodermal dysplasia and did not suffer from serious infections with the exception of a relapsing Salmonella typhimurium infection. His monocyte function was impaired, whereas T cell function was relatively normal. Consistent with this, his T cells almost exclusively displayed the wild-type allele, whereas both alleles were present in his monocytes. We propose that the T and B cell compartment of the mosaic father arose as a result of selection of wild-type cells and that this underlies the widely different clinical phenotype.

Figure 1.

Production of IL-12p40 in whole blood of a healthy control, the mother, the father, and the child. (A) Whole blood was stimulated with LPS in the presence or absence of IFN-γ. (B) Whole blood was stimulated with 25 μg/ml M. avium sonicate, 100 μg/ml zymosan A, S. aureus (5 × 10⁶ heat-killed bacteria/ml), and 200 ng/ml PAM3CSK4. Supernatants were collected after 18 h and IL-12p40 was measured by ELISA.

Figure 2.

Production of other cytokines in whole blood. Whole blood was stimulated with LPS (A, B, and C) in the presence or absence of IFN-γ (A). Supernatants were collected after 18 h and TNF-α (A), IL-6 (B), and IL-1β (C) was measured by ELISA.

Figure 3.

IFN-γ responsiveness of monocytes. PBMCs of a healthy control, the mother, the child, and the father were incubated for 18 h with increasing amounts of IFN-γ, and CD64 expression on CD14⁺ monocytes was measured by FACS analysis. Data are expressed as the ratio of median fluorescence of cells cultured in the presence of IFN-γ over cells cultured in the absence of IFN-γ. The median fluorescence of unstimulated monocytes of the healthy control, the mother, the son, and the father was 66, 66, 145, and 90, respectively.

Figure 4.

NF-κB translocation in monocyte-derived macrophages. Monocyte-derived macrophages were incubated with 1,000 ng/ml LPS (A, B, and C) or 200 U/ml TNF-α (D) for 45 min, and p65 translocation to the nucleus was analyzed (reference 14). Cells were stained with a p65-specific antibody, after which a Rhodamine RedX–conjugated goat anti–rabbit antibody was added. Nuclei were stained with DAPI and coverslips were analyzed by fluorescence microscopy (B). Translocation of p65 to the nucleus was scored by enumerating the number of cells that had no nuclear staining (−), cells that displayed intermediate nuclear staining (±), or cells that displayed bright nuclear staining (+).

Figure 5.

(A) Expression of wild-type versus mutated allele in whole blood, T cells, and monocytes. mRNA was isolated, cDNA was generated, and exon 1 of IκB was amplified by PCR. The PCR product was digested with MspA1, which only digests the wild-type allele. (B) Presence of wild-type versus mutated allele in monocytes, CD45RA, and CD45 RO cells of father and child. Genomic DNA was isolated from selected cell populations and exon 1 of IκB was amplified by PCR. The PCR product was digested with MspA1, which only digests the wild-type allele. Note that the size of the bands is different when DNA or RNA is used as a template for PCR.