A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency - PubMed (original) (raw)

Case Reports

. 2003 Oct;112(7):1108-15.

doi: 10.1172/JCI18714.

Asma Smahi, Janine Reichenbach, Rainer Döffinger, Caterina Cancrini, Marion Bonnet, Anne Puel, Christine Chable-Bessia, Shoji Yamaoka, Jacqueline Feinberg, Sophie Dupuis-Girod, Christine Bodemer, Susanna Livadiotti, Francesco Novelli, Paolo Rossi, Alain Fischer, Alain Israël, Arnold Munnich, Françoise Le Deist, Jean-Laurent Casanova

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Case Reports

A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency

Gilles Courtois et al. J Clin Invest. 2003 Oct.

Abstract

X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the gene encoding NEMO/IKKgamma, the regulatory subunit of the IkappaB kinase (IKK) complex. IKK normally phosphorylates the IkappaB-inhibitors of NF-kappaB at specific serine residues, thereby promoting their ubiquitination and degradation by the proteasome. This allows NF-kappaB complexes to translocate into the nucleus where they activate their target genes. Here, we describe an autosomal-dominant (AD) form of EDA-ID associated with a heterozygous missense mutation at serine 32 of IkappaBalpha. This mutation is gain-of-function, as it enhances the inhibitory capacity of IkappaBalpha by preventing its phosphorylation and degradation, and results in impaired NF-kappaB activation. The developmental, immunologic, and infectious phenotypes associated with hypomorphic NEMO and hypermorphic IKBA mutations largely overlap and include EDA, impaired cellular responses to ligands of TIR (TLR-ligands, IL-1beta, and IL-18), and TNFR (TNF-alpha, LTalpha1/beta2, and CD154) superfamily members and severe bacterial diseases. However, AD-EDA-ID but not XL-EDA-ID is associated with a severe and unique T cell immunodeficiency. Despite a marked blood lymphocytosis, there are no detectable memory T cells in vivo, and naive T cells do not respond to CD3-TCR activation in vitro. Our report highlights both the diversity of genotypes associated with EDA-ID and the diversity of immunologic phenotypes associated with mutations in different components of the NF-kappaB signaling pathway.

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Figures

Figure 1

Figure 1

Defective NF-κB activation in patient’s fibroblasts. (a) EMSA after exposure to 10 ng/ml TNF-α. The composition of the retarded species is indicated on the right and was determined as described in Smahi et al. (10). C, control fibroblasts; P, fibroblasts from patient under study; X-EDA-ID, patient with OL-EDA-ID fibroblasts. (b) Western blot analysis of cytoplasmic RelA, p105, and p50.(c) Analysis of IL-6 synthesis by primary fibroblasts from patients and a healthy control. Cells were treated overnight with 10 ng/ml of TNF-α or IL-1β, and IL-6 secretion was measured in the supernatant using ELISA. Results from one representative experiment are shown. (d) Analysis of IL-6 synthesis by SV-40-transformed fibroblasts from patients and a healthy control. Cells were treated for 24 hours with 50 ng/ml of LTα1/β2, and IL-6 secretion was measured in the supernatant using ELISA. Results from one representative experiment are shown.

Figure 2

Figure 2

Specific defect of IκBα degradation in patient’s fibroblasts treated with TNF-α (a) IKK kinase assay and Western blot analysis of IκBα degradation. Phosphorylated IκBα exhibits a retarded migration and is indicated on the right. GST-IκBα, a fusion between gluthatione-S-Transferase and the first 72 amino acids of IκBα, was used as a substrate for IKK. GST, gluthatione-S-transferase. (b) Western blot analysis of IKK subunits in fibroblasts from a healthy control, NEMO-mutated patient X-EDA-ID and IκBα-mutated patient P. (c) Western blot analysis of IκBα Ser32 phosphorylation after TNF-α stimulation. Circled P, phosphorylated. (d) Time course analysis of TNF-induced IκBα, IκBβ, and IκBε degradation, as detected by Western blot. Results from one representative experiment are shown.

Figure 3

Figure 3

Sequence of the IKBA gene in the patient and his relatives. (a) Schematic representation of IκBα. The various functional/structural domains of the protein are shown. NH2, N-terminal; rPEST, repeated peptidic sequence rich in proline, glutamic acide, serine, and threonine (PEST); Ile, isoleucine. (b) Phosphoacceptor sites of IκB molecules and location of the patient’s mutation S32I. The two conserved serine residues that are phosphorylated by IKK in IκBα, IκBβ, and IκBε are boxed. Mutated Ser32 of patient P is indicated by an arrow. (c) Automated sequencing profile of genomic DNA showing the heterozygous C/T polymorphism at position 89 and the heterozygous G/T (S32I) disease-causing mutation at position 94 in our patient. The two heterozygous positions from left (position 89) to right (position 94) appear as N nucleotides.

Figure 4

Figure 4

Dominant effect of the S32I IKBA mutation on NF-κB activation. HEK 293T cells were transfected with Igκ-luc, a reporter plasmid for NF-κB, and either an empty expression vector (control) or an expression vector encoding WT IκBα (WT), the S32I mutant or the S32A/S36A double mutant. Twenty-four hours later, the cells were stimulated with TNF-α. The relative expression of IκBα molecules is shown at the bottom, as detected by Western blotting.

Figure 5

Figure 5

PBMC and T cell analysis. (a) Peripheral blood cells from patient P, a healthy control, and patient X-EDA-ID with OL-EDA-ID were stimulated by PHA, PMA-ionomycin, IL-12, IL-12 + IL-1β, or PHA, and IFN-γ secretion was measured by ELISA. Results from one representative experiment are shown. (b) Peripheral blood cells were stimulated by PMA-ionomycin or LPS + IFN-γ, and TNF-α secretion was measured by ELISA. Results from one representative experiment are shown. (c) CD45RA and CD45RO expression on control (C) and patient (P) T cells at day 0 and day 10 of PHA + IL-2 stimulation in vitro, as detected by flow cytometry. (d) T cell proliferation (left) after 64 hours of stimulation with anti-CD3 alone, or anti-CD3 in combination with anti-CD28. Production of IFN-γ (right) in culture supernatants after 48 hours of stimulation with anti-CD3 alone or in combination with anti-CD28. Results from one representative experiment are shown. Data are normalized for 106 cells.

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References

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