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. 2006 Apr;78(4):691-701.
doi: 10.1086/501532. Epub 2006 Feb 15.

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation

Anne Puel  1 , Janine Reichenbach, Jacinta Bustamante, Cheng-Lung Ku, Jacqueline Feinberg, Rainer Döffinger, Marion Bonnet, Orchidée Filipe-Santos, Ludovic de Beaucoudrey, Anne Durandy, Gerd Horneff, Francesco Novelli, Volker Wahn, Asma Smahi, Alain Israel, Tim Niehues, Jean-Laurent Casanova
Affiliations

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation

Anne Puel et al. Am J Hum Genet. 2006 Apr.

Abstract

Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause X-dominant incontinentia pigmenti, which is lethal in males in utero, whereas hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with immunodeficiency, a complex developmental disorder and life-threatening primary immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates the most-upstream premature translation termination codon (at codon position 49) of any known NEMO mutation. Surprisingly, this mutation is associated with a pure immunodeficiency. We solve this paradox by showing that a Kozakian methionine codon located immediately downstream from the insertion allows the reinitiation of translation. The residual production of an NH(2)-truncated NEMO protein was sufficient for normal fetal development and for the subsequent normal development of skin appendages but was insufficient for the development of protective immune responses.

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Figures

Figure 1
Figure 1
Sequencing of the NEMO gene in the patient and his relatives. A, Automated reverse-sequencing profile showing the 110_111insC mutation in genomic DNA extracted from peripheral blood cells from the hemizygous patient, compared with a control (WT) with the corresponding amino-acid sequence, and the M38 reinitiation site (boxed). B, Pedigree of the family. Each generation is designated by a roman numeral (I–III) and each individual is designated by an arabic numeral (1–3); the hemizygous patient is shown in black. Heterozygous carriers are shown with a black dot in the center. C, X-inactivation pattern in blood cells from a healthy female control, from a female patient with IP carrying the common IP NEMO ex4_10del mutation, and from the patient’s mother.
Figure 2
Figure 2
Schematic representation of NEMO with all reported mutations. A, Schematic representation of the coding-region domain organization: coil-coiled (CC), leucine-zipper (LZ), and zinc-finger (ZF) domains, and published mutations of NEMO. B, Schematic representation of all known premature stop codons in NEMO due to nonsense and splice mutations, frameshift insertions, and deletions. For the mutations leading to a frameshift, the position of the premature stop codon is indicated, with the position of the first amino acid affected by the mutation in parentheses. The mutations associated with XD-IP are shown in black, with XR-OL-EDA-ID in blue, with XR-EDA-ID without OL in red, with incomplete XR-I-EDA-ID in pink, and with XR-ID without EDA in green.
Figure 3
Figure 3
NF-κB signaling in SV40-transformed fibroblasts. A, Time-course analysis of IκBα protein degradation and resynthesis detected by western blot after stimulation with IL-1β for 20, 40, 60, and 120 min; the antibody against p38 served as a control for protein loading. B, NF-κB DNA-binding activity, measured by EMSA after stimulation for 20 and 40 min with IL-1β. γ-Activating–factor DNA-binding activity after 20 min of stimulation with IFN-γ and competition with unlabeled NF-κB–specific probe (*) served as internal activation control and specificity control, respectively. C, IL-6 production after 18 h of exposure to IL-1β, TNF-α, and PMA/ionomycin, in fibroblasts from a healthy control, from the patient, from a patient with XR-OL-EDA-ID (X420W), and from a patient with XD-IP (ex4_10del). Results shown are representative of two to three independent experiments.
Figure 4
Figure 4
NF-κB signaling in fibroblasts. Composition of the NF-κB dimers bound to the κB probe, as measured by EMSA in SV40-transformed fibroblasts from a healthy control, from the patient, from a patient with OL-EDA-ID, and from a patient with IP, after exposure to IL-1β for 40 min, and as determined using antibodies directed against p50 and p65.
Figure 5
Figure 5
Whole blood cell activation. A, IL-10 production. B, IL-6 production. C, TNF-α production. D–E, IFN-γ and IL-12p40 production, respectively, in the culture supernatants of whole blood cells from a control and from the patient after 48 h of activation with TNF-α, IL-1β, LPS, heat-killed S. aureus, and PMA/ionomycin; after 24 h of stimulation with agonists of TLR1-9 and whole bacteria (E. coli and M. tb); and after 24 or 48 h with BCG, BCG plus IL-12, or BCG plus IFNγ. Cytokine production was normalized for 106 peripheral blood mononuclear cells (PBMC). F–G, B-cell proliferation and B-cell immunoglobulin class switching of PBMC from a control and the patient on IL-4, soluble recombinant CD154, and CD154 plus IL-4. When indicated, means and SDs have been calculated from three to five different controls, and the patient has been tested two to four times.
Figure 6
Figure 6
NEMO production in SV40-transformed fibroblasts. A, NEMO production assessed by western blot analysis, with the use of antibodies directed against the N- or C-terminal part of NEMO (amino acids 28–33 and 356–414, respectively), antibodies against p38 and STAT-2 being used as controls for protein loading. B, Intracellular staining with an antibody directed against amino acids 278–396 of NEMO (black) or an isotype-control antibody (gray) of fibroblasts from a control, from the patient, from a patient with XR-OL-EDA-ID, and from a patient with XD-IP. Results shown are representative of three independent experiments.
Figure 7
Figure 7
Expression of various NEMO constructs in vitro. A, WT sequence of the 5′ end of NEMO showing the first methionine site involved in the initiation of translation (M1, boxed), the position of the 110_111insC mutation, the second methionine site (M38) involved in translation reinitiation, and the IP nonsense mutation at Arg 62 (R62X). B, Western blot analysis with an anti-V5 antibody of 293T HEK cells transfected with insert-free V5-tagged vector (mock) or with expression vectors carrying various NEMO alleles: WT, 110_111insC (the patient’s mutation), M38A (the methionine 38 to alanine mutation, blocking reinitiation of translation at M38) with or without the 110_111insC mutation, X420W, and R62X. Cells were cotransfected with 3 μg of each vector and 1 μg of a myc-tagged expression vector encoding β-galactosidase, as a transfection-efficiency control. An antibody against p38 was used to control for protein loading.
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References

Web Resources

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