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Am J Hum Genet. Apr 2006; 78(4): 691–701.
Published online Feb 15, 2006. doi:  10.1086/501532
PMCID: PMC1424680

The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation

Abstract

Amorphic mutations in the NF-κB 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 NH2-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.

The human IKBKG locus is located on chromosome Xq28 and encodes NEMO. Amorphic NEMO mutations are associated with a complete lack of NF-κB activation via the classical pathway. They are responsible for incontinentia pigmenti (IP), an X-linked dominant disorder that is lethal in hemizygous males in utero and is characterized by abnormalities in ectoderm-derived tissues, including the skin, eyes, CNS, and teeth, in heterozygous females. About 85% of patients with IP who have NEMO mutations carry a complex rearrangement of the NEMO gene that results in a frameshift deletion of exons 4–10 and encodes a putative truncated protein consisting of the first 133 N-terminal amino acids. A number of other IP-causing mutations have been identified in exons 2–10, including mutations associated with premature stop codons (Smahi et al. 2000; Aradhya et al. 2001b; Fusco et al. 2004). Blood leukocytes and fibroblasts expressing the mutated X-chromosome are selectively eliminated around the time of birth, leading to skewed X-inactivation in female carriers (Parrish et al. 1996).

Other NEMO mutations are hypomorphic, since they impair but do not abolish NF-κB activation via the classical pathway. They are associated with two closely related but distinct X-recessive (XR) forms of anhidrotic ectodermal dysplasia (EDA) with immunodeficiency (ID). A mutation of the stop codon (X420W) results in the more severe syndrome of osteopetrosis (O), lymphoedema (L), and EDA-ID (XR-OL-EDA-ID) (Smahi et al. 2000; Döffinger et al. 2001; Mansour et al. 2001; Dupuis-Girod et al. 2002). Most of the other mutations, including a few mutations creating premature stop codons, cause XR-EDA-ID without OL (Abinun et al. 1996; Zonana et al. 2000; Aradhya et al. 2001a; Döffinger et al. 2001; Jain et al. 2001; Kosaki et al. 2001; Mansour et al. 2001; Dupuis-Girod et al. 2002; Orange et al. 2002; Carrol et al. 2003; Jain et al. 2004; Nishikomori et al. 2004; Orange et al. 2004a, 2004b; Puel et al. 2004; Ku et al. 2005a, 2005b). The clinical features of EDA typically include the poor development of skin appendages, resulting in hypo- or anhidrosis with dry skin and heat intolerance; hypo- or atrichosis with sparse hair, eyebrows, and eyelashes; and hypo- or anodontia with conical incisors (Priolo and Lagana 2001; Ku et al. 2005a).

Patients with XR-EDA-ID suffer less from developmental defects than from severe infections, most of which are caused by pyogenic bacteria and mycobacteria (Abinun et al. 1996; Smahi et al. 2000; Zonana et al. 2000; Aradhya et al. 2001a; Döffinger et al. 2001; Jain et al. 2001; Dupuis-Girod et al. 2002; Orange et al. 2002, 2004a; Carrol et al. 2003; Niehues et al. 2004; Nishikomori et al. 2004; Ku et al. 2005a). In a few cases, infections with fungi (Zonana et al. 2000; Döffinger et al. 2001; Dupuis-Girod et al. 2002; Orange et al. 2004a) and viruses (Döffinger et al. 2001; Orange et al. 2002, 2004a) have been documented. Almost half the known patients with XR-EDA-ID died of infection. The most consistent immunological abnormality found in these patients is a poor serum-antibody response to polysaccharide antigens (reviewed by Puel et al. [2004]; Ku et al. [2005b]). Two unrelated patients bearing mutations in NEMO, with pure IDs and no apparent developmental defect—ID without EDA—were recently described (Niehues et al. 2004; Orange et al. 2004b). The first patient carries a splice mutation 1056(−1)G→A (Orange et al. 2004b), which results in the skipping of exon 9, with a premature stop codon inserted at position 373. We report here the characterization of the 110_111insC mutation in exon 2 in the second patient (Niehues et al. 2004).

The patient’s clinical features have been reported in detail elsewhere (Niehues et al. 2004). This patient, a boy, was the only child of healthy nonrelated Polish parents living in Germany. From the age of 15 mo, he suffered from multiple episodes of disseminated Mycobacterium avium disease (affecting mostly lymph nodes and bones). At the age of 7 years, he was diagnosed with bronchiectasis caused by Haemophilus influenzae and Streptococcus pneumoniae. At the age of 11 years, the patient presented extra-intestinal disease caused by Salmonella enteritidis. He presented severe autoimmune hemolytic anemia at the age of 12 years and died from herpes simplex virus 1 (HSV-1) meningo-encephalitis at the age of 12 years and 10 mo.

The immunological work-up of this patient, from the age of 2 years, showed low IgA and IgG levels, with markedly high serum IgM levels. No serum antibodies against diphtheria toxoid and tetanus toxoid were detected, and titers of antibodies against H. influenzae were low despite complete routine vaccination. The patient also presented low serum titers of antibodies against S. pneumoniae and had no allo-hemagglutinin antibodies. Known molecular causes of hyper IgM syndrome, including defects in CD40, CD154, and activation-induced cytidine deaminase, were excluded. Because of the patient’s low serum IgG levels, prophylactic intravenous immunoglobulin replacement therapy was initiated at 2 years of age and continued until his death. The numbers of T, B, and NK lymphocytes and the proportions of the CD4+ and CD8+ T lymphocyte subsets were within normal ranges at the ages of 2 and 4 years. However, the patient lacked memory B cells, since all his B cells had the naive IgD+CD27 phenotype. The proliferative responses of T lymphocytes to mitogens (PHA and PWM) were normal.

Genomic DNA was isolated from whole blood cells, granulocytes, SV40-transformed fibroblasts, and Epstein Barr virus (EBV)–transformed B cells by a standard phenol/chloroform extraction method. Total RNA was isolated from SV40-transformed fibroblasts by use of Trizol (GibcoBRL Life Technologies, Invitrogen SARL). Both cDNA and individual IKBKG exons were amplified using specific primers (primers and conditions for PCR amplification and sequencing are available on request), were sequenced using the Big Dye Terminator cycle sequencing kit (Applied Biosystems), and were analyzed on an ABI Prism 3700 apparatus (Applied Biosystems).

The X-chromosome inactivation status was evaluated by the analysis of DNA methylation at the human androgen receptor gene (HUMARA), as described elsewhere (Allen et al. 1992). In brief, genomic DNA (200 ng) extracted from whole blood was digested with 10 IU of HpaII (Invitrogen SARL) at 37°C overnight, in 50 μl of final reaction mixture. After digestion, the reaction was stopped by incubation of the mixture at 95°C for 6 min. A mock sample as control was prepared simultaneously without the HpaII. Twenty-five microliters of each digested product was then used in a 50-μl PCR containing 200 μM of dinucleotide triphosphate (Invitrogen SARL), 1 IU of Taq polymerase (AmpliTaq; Applied Biosystems), 1.5 mM of MgCl2, 50 mM of KCl, 10 mM of Tris-HCl (pH 8.3), and 20 μM of each primer. The primers used were AR-F 5′-GCTGTGAAGGTTGCGTTCCTCAT-3′ and AR-R 5′-TCCAGAATCTGTTCCAGAGCGTGC-3′; the AR-F was fluorescent. After an initial denaturation step at 95°C, samples were amplified for 30 cycles (1 min at 94°C, 1 min at 62°C, and 1 min at 72°C), with a final extension step for 10 min. Two microliters of PCR products (mock and HpaII) were then added to a mixture containing 10 μl of deionized formamide and 0.1 μl molecular weight standard GENESCAN 400HD (ROX). The samples were denatured for 6 min at 95°C, were cooled on ice, and were resolved using the 3100 ABI Prism Genetic Analyzer (Applied Biosystems). The percentage of inactivation was calculated as reported elsewhere (Chitnis et al. 2000).

Total or nuclear cell extracts were prepared from SV40-transformed fibroblasts or 293T HEK cells left unstimulated or stimulated with TNF-α (20 ng/ml; R&D Systems Europe) or IL-1β (20 ng/ml; R&D Systems Europe). Electrophoresis, western blot, and electrophoretic mobility shift assay (EMSA) were performed as described elsewhere (Döffinger et al. 2001; Picard et al. 2003). The rabbit anti-human IKKα (H-744, sc-7218), mouse anti-human IKKβ (H-4, sc-8014), mouse anti-human STAT-2 (A-7, sc-1668), rabbit anti-mouse p38 (C-20, sc-535), mouse anti-human IKKγ (B-3, sc-8032), goat anti-human IKKγ (M-18, sc-8256), and rabbit anti-human IκBα (C-21, sc-37) antibodies were purchased from SantaCruz Biotechnology (Tebu-Bio S.A.). The mouse anti-human IKKγ/NEMO (611306) was purchased from BD Transduction Laboratories (Becton Dickinson France S.A.S.), the mouse anti-human IKKγ/NEMO (557383) was purchased from BD Pharmingen (Becton Dickinson France S.A.S.), the rabbit anti-human IKKγ/NEMO (3328) and anti-IκBepsilon (1775) antibodies were kindly provided by G. Courtois and A. Israel, and the mouse anti-V5 monoclonal antibody (5960-25) was purchased from Invitrogen (Invitrogen SARL). The EMSA was performed by incubating 10 μg of nuclear extract with a 32P-labeled double-stranded NF-κB–specific oligonucleotide κB probe (5′-GATCATGGGGAATCCCCA-3′ and 5′-GATCTGGGGATTCCCCAT-3′). Supershift experiments were performed with goat anti-human p50 (C-19, sc-1190) and rabbit anti-human p65 (C-20, sc-372) antibodies from Santa Cruz Biotechnology (Tebu-Bio S.A.).

Cytokine production was assessed in whole blood diluted 1:2 in RPMI 1640 (GibcoBRL Life Technologies) and activated with TNF-α (20 ng/ml; R&D Systems Europe), IL-12p70 (20 ng/ml; R&D Systems Europe), IL-1β (20 ng/ml; R&D Systems Europe), IL-18 (50 ng/ml; R&D Systems Europe), and IL-12 plus IL-18 or phorbol 12-myristate 13-acetate (PMA) plus ionomycin (10−7 M and 10−5 M, respectively) (Sigma-Aldrich Chimie SARL) for 48 h. For toll-like receptor (TLR)–agonist activation, whole blood diluted 1:5 in RPMI 1640 (GibcoBRL) was incubated with polymyxin B (10 μg/ml; Sigma-Aldrich) (except for stimulation by gram-negative bacteria and lipopolysaccharide [LPS]) at 37°C for 30 min. Cells were then activated with TLR ligands: PAM3CSK4 (100 ng/ml; EMC Microcollections), which mimics gram-negative bacteria, recognized by TLR1 and TLR2; PAM2CSK4 (100 ng/ml; EMC Microcollections), which mimics gram-positive Mycoplasma lipoprotein; zymosan A from yeast (125 μg/ml; Molecular Probes, Invitrogen SARL) and peptidoglycan from Staphylococcus aureus (30 μg/ml; Sigma-Aldrich), recognized by TLR2 and TLR6; lipoteichoic acid from S. aureus (50 μg/ml; Sigma-Aldrich), recognized by TLR2; poly(I:C) double-stranded RNA (50 μg/ml; Sigma-Aldrich), which mimics RNA viruses, recognized by TLR3; LPS from Salmonella minnesota R595 (10 μg/ml; Sigma-Aldrich), recognized by TLR4; flagellin from the flagellated gram-negative bacterium Salmonella typhimurium (10 ng/ml; provided by K. D. Smith, Institute for Systems Biology), recognized by TLR5; imidazoquinoline 3M-13, recognized by TLR7 (provided by 3M Pharmaceuticals and 3M Innovative Properties); and CpG oligonucleotide DNA (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) (20 μg/ml; Research Genetics, Invitrogen SARL), recognized by TLR9. Several TLR agonists were kindly provided by Adrian Ozinsky. Whole bacteria were also used: heat-killed S. aureus (5×106 particles/ml; ATCC, LGC Promochem), Escherichia coli (5×106 particles/ml; ATCC), and H37Rv strain of Mycobacterium tuberculosis (M. tb) (1 μg/ml; provided by J. Belisle, TB Research Materials and Vaccine Testing Contract, NIH, NIAID NO1 AI-75320). For Bacille Calmette-Guérin (BCG) activation, whole blood was diluted 1:2 in RPMI 1640 (GibcoBRL Life Technologies) and was incubated for 12 or 48 h with live BCG (Mycobacterium bovis BCG, Pasteur substrain) at a multiplicity of infection of 20:1, BCG plus IFN-γ (5,000 IU/mL), and BCG plus recombinant IL-12p70 (20 ng/ml; R&D Systems Europe). Supernatants were collected after 12 or 48 h, and the secretion of IL-12p40, IFNγ, IL-6, IL-10, and TNF-α was measured using human IL-12p40 (R&D Systems Europe), IFNγ, IL-6, IL-10, and TNF-α (Sanquin, Mast Diagnostic) ELISA kits, respectively, in accordance with the manufacturer’s instructions. For SV40-transformed fibroblast activation, cells were activated by incubation for 18 h with TNF-α or IL-1β (10 ng/ml; R&D Systems Europe), and IL-6 production was measured by ELISA (Sanquin, Mast Diagnostic). In vitro B-cell proliferation was measured in peripheral blood lymphocytes stimulated by incubation for 5 d with sCD154 (1 μg/ml; Amgen France), by the addition of 3H-thymidine for the final 18 h (1 μCi). IgE production was measured by ELISA in the supernatants of peripheral blood lymphocytes stimulated for 12 d with sCD154 plus IL-4 (100 U/ml; R&D Systems Europe).

The full-length human IKBKG cDNA, inserted between the HindIII and NotI sites of the expression vector pcDNA3, was a gift from G. Courtois. We generated various IKBKG mutations, using the QuikChange Site-Directed Mutagenesis kit (Stratagene) in accordance with the manufacturer’s procedure. The patient’s 110_111insC mutation was generated using the primers P-CCTCTGGGGAAGCCAGCCCATGCTGCACCTGCCTTC and P-GAAGGCAGGTGCAGCATGGGCTGGCTTCCCCAGAGG, the IP mutation R62X using the primers P-GAGAATCAAGAGCTCTGAGATGCCATCCGGCAG and P-CTGCCGGATGGCATCTCAGAGCTCTTGATTCTC, the XL-OL-EDA-ID mutation X420W using the primers P-CATGGAGTGCATTGAGTGGGGCCGGCCAGTGCAAGG and P-CCTTGCACTGGCCGGCCCCACTCAATGCACTCCATG, the mutation M38A (the hypothetical translation reinitiation methionine) using the primers P-CCTCTGGGGAAGCCAGCCGCGCTGCACCTGCCTTCAGAAC and P-GTTCTGAAGGCAGGTGCAGCGCGGCTGGCTTCCCCAGAGG, the double mutation 110_111insC/M38A using the primers P-CCTCTGGGGAAGCCAGCCCGCGCTGCACCTGCCTTCAGAAC and P-GTTCTGAAGGCAGGTGCAGCGCGGGCTGGCTTCCCCAGAGG, and the mutation M94A (the second hypothetical translation reinitiation methionine) using the primers P-GAGGAGAAGGAGTTCCTCGCGTGCAAGTTCCAGGAGGCC and P-GGCCTCCTGGAACTTGCACGCGAGGAACTCCTTCTCCTC. The mutated fragments were then subcloned into the pcDNA3.1/V5-His-directional TOPO expression vector (Invitrogen SARL), in accordance with the manufacturer’s instructions.

The various pcDNA3.1/V5-His-TOPO expression vectors encoding wild-type (WT), 110_111insC, 110_111insC/M38A, M38A, M94A, R62X, and X420W NEMO or the insert-free vector were used to transfect SV40-transformed IP fibroblasts or 293T HEK cells, in the presence of Lipofectamine 2000 (Invitrogen SARL) or calcium phosphate (Invitrogen SARL), in accordance with the manufacturer’s recommendations.

For intracellular NEMO staining, we fixed 2×105 fibroblasts or EBV-transformed B cells, permeabilized them using IntraStain kit (DakoCytomation S.A.S. Trappes Cedex), and incubated them with a mouse anti-human IKKγ/NEMO IgG1 (611306) from BD Transduction Laboratories (Becton Dickinson France S.A.S.) or an isotype control mouse IgG1,κ, (555746) from BD Pharmingen (Becton Dickinson France S.A.S.). Cells were washed and were labeled with an Alexa Fluor 488 goat anti-mouse IgG (A-11029) from Molecular Probes (Invitrogen SARL), as described elsewhere (Nishikomori et al. 2004). Cells were analyzed by flow cytometry on a FACScan apparatus (Becton Dickinson).

The sequencing of genomic DNA and cDNA obtained from whole blood cells, granulocytes, SV40-transformed fibroblasts, and EBV-transformed B cells revealed a frameshift insertion in codon 37 of NEMO exon 2 (110_111insC) generating a premature stop codon 12 residues downstream at codon position 49 (M38fsX48) (figs. 1A, 2A2A, and and2B).2B). The mother, maternal aunt, and maternal grandmother of this patient were heterozygous for the mutation (fig. 1B). This previously unreported mutation was not found in 305 Caucasian controls (190 females and 115 males; 495 chromosomes), suggesting that it was pathogenic. It creates the furthest upstream of the known premature stop codons in NEMO—most others being associated with XD-IP, and the remaining few, mostly located in the 3′ region of the coding region, being associated with XR-EDA-ID (fig. 2A and and2B).2B). Paradoxically, the 110_111insC mutation was associated with viable fetal development and a lack of detectable developmental phenotype in the affected hemizygous boy. Moreover, the mother presented no clinical signs of XD-IP and displayed a random pattern of X-chromosome inactivation (75% and 25%) in her blood cells (fig. 1C).

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 ...
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 ...

We investigated whether the 110_111insC mutation was amorphic (as in loss-of-function), as predicted from genetic findings, or hypomorphic, as predicted from clinical findings, by assessing NF-κB activation in SV40-transformed fibroblasts from a healthy control, from the patient studied, from a patient with XR-OL-EDA-ID (bearing the NEMO X420W mutation), and from a fetus with XD-IP (bearing the ex4-10del NEMO mutation). Upon stimulation with TNF-α and IL-1β, IκBα degradation and resynthesis were normal in the patient’s cells, whereas they were impaired in XR-OL-EDA-ID cells and were completely abolished in XD-IP cells (fig. 3A and data not shown). However, the κB DNA-binding activity of the patient’s cells, measured by EMSA, was approximately one-third that of control fibroblasts—a decrease similar to that observed with XR-OL-EDA-ID fibroblasts. No detectable DNA-binding activity was observed with XD-IP fibroblasts (figs. 3B and and4).4). Finally, IL-6 production was impaired in the patient’s fibroblasts and, to a greater extent, in XR-OL-EDA-ID fibroblasts, in response to both IL-1β and TNF-α, whereas it was completely abolished in XD-IP fibroblasts (fig. 3C). Thus, the NEMO 110_111insC mutation, despite creating a stop codon at position 49, was hypomorphic, since it impaired but did not abolish NF-κB activation and was even associated with a somewhat milder cellular phenotype than the X420W NEMO mutation.

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 ...
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 ...

We evaluated the immunological impact of the NEMO mutation by stimulating the patient’s blood cells (fig. 5) with TNF-α, IL-1β, or agonists of TLRs. The response of the patient’s blood cells to TNF-α was reproducibly impaired, in terms of IL-10 production. In contrast, the response to IL-1β, LPS (an agonist of TLR-4), or other TLR agonists—such as heat-killed S. aureus Cowan, E. coli, and M. tb—was normal, in terms of IL-6 or TNF-α production. Because the main infectious disease of this patient was caused by M. avium, a weakly virulent environmental mycobacterial species, we also investigated the IL-12/23-IFN-γ circuit (Casanova and Abel 2002). We observed no response to live M. bovis BCG, BCG plus IL-12, or BCG plus IFN-γ, in terms of IFN-γ, IL-12p40, or IL-12p70 production in the patient’s blood cells. Finally, we tested CD40-mediated NF-κB activation by measuring B-cell proliferation and Ig isotype switching upon stimulation with CD40L (CD154) in vitro. The patient’s cells displayed impaired proliferation and IgE production in response to CD154, alone or in combination with IL-4. These results indicate that the patient’s NEMO mutation was hypomorphic in blood cells and provide an explanation for the patient’s immunological phenotype.

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 ...

We then assessed NEMO production in SV40-transformed fibroblasts from the patient. No protein was detected in the patient’s fibroblasts with the use of a serum directed against N-terminal amino acids 28–33 of NEMO (serum 3328), whereas two proteins of ~48 and 45 kDa were detected in control cells (fig. 6A, left panel), and a protein of ~51 kDa appeared with prolonged exposure in the OL-EDA-ID fibroblasts (data not shown). In contrast, with the use of antibodies directed against amino acids 278–396 (611306) (data not shown) and 356–414 (M-18) (fig. 6A, right panel), small amounts of a protein of ~45 kDa were detected in the control and patient’s fibroblasts. Neither the 48-kDa nor the 45-kDa protein was detected with either antibody in XD-IP fibroblasts, suggesting that the NEMO gene encoded both proteins. Residual NEMO production was detected by flow cytometry, with use of antibody 611306 in fibroblasts (fig. 6B) and EBV-transformed B cells (not shown) from the patient and from the patient with XR-OL-EDA-ID. Again, no NEMO was detected in XD-IP fibroblasts. Thus, the 110_111insC mutant allele encodes an N-terminally truncated NEMO protein that is produced in small amounts. The 110_111insC mutation probably allows reinitiation of the translation process downstream from the insertion.

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 ...

An almost canonically Kozakian methionine AUG start codon (Kozak 2000, 2002b) is located at nucleotide positions 112–114, corresponding to methionine 38 (fig. 7A). The likelihood of this AUG codon acting as a translation initiation site was estimated at 0.27 and increased to 0.51 in the sequence context found in the patient, versus 0.63 for the first AUG codon (Prediction of Translation Initiation ATG Web site). Such a reinitiation of translation would result in the synthesis of a protein ~45 kDa in size. We, therefore, transfected 293T HEK cells with V5-tagged expression vectors carrying WT and various mutant NEMO alleles: M38A (methionine 38 replaced by alanine, preventing reinitiation at codon 38) with or without the 110_111insC, R62X (the most-upstream nonsense mutation responsible for XD-IP), and X420W. With an anti-V5 antibody, a protein of ~48 kDa was detected in WT- and M38A-transfected cells. Smaller amounts of a protein of ~45 kDa were detected in WT- and 110_111insC-transfected cells, but not in M38A- or 110_111insC/M38A–transfected cells. A protein of ~51 kDa was detected in X420W-transfected cells, and no specific proteins were detected in mock- and NEMO R62X-transfected cells. These results confirmed that the WT NEMO allele encodes two proteins: the predominant 48-kDa protein and an N-terminally truncated protein of ~45 kDa produced in smaller amounts and translated from methionine 38. This second protein is the only isoform detected in the patient’s cells.

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 ...

This description of the most-upstream premature stop codon (at amino-acid position 49, M38fsX48) of any known NEMO mutation, in a child with ID without EDA, shows that the location of the premature stop codon is not correlated with the severity of the clinical phenotype. Nonetheless, the ID appeared to be particularly severe, since the patient developed fatal HSV-1 meningo-encephalitis (Niehues et al. 2004), identifying NEMO deficiency as the second genetic etiology of HSV-1 encephalitis (Dupuis et al. 2003) and suggesting that the NF-κB–mediated induction of IFN-α/β is essential for HSV-1 control. The 110_111insC mutation, therefore, had a highly deleterious effect on immunity to infection but a much weaker than expected effect on development, with normal fetal development and no signs of EDA in childhood. Studies on the patient’s cells showed that the 110_111insC NEMO mutation was hypomorphic because of the residual production of an N-terminally truncated protein resulting from the reinitiation of translation at an internal AUG codon located immediately downstream from the mutation. This process also occurs in control cells. The small amounts of the resulting NEMO protein were sufficient to trigger normal responses to some NF-κB dependent signaling pathways, preventing both fetal death and developmental disorders in childhood (Smahi et al. 2002).

This rare mechanism has been reported in other inherited disorders of leukocytes—such as Omenn’s syndrome (RAG1) (Santagata et al. 2000), Fanconi anemia (FAC) (Yamashita et al. 1996), and Nijmegen breakage syndrome (NBS1) (Maser et al. 2001)—and in other fields of human genetics (Yamashita et al. 1996; Ozisik et al. 2003; Pittis et al. 2004), upstream premature stop codons being typically involved (Santagata et al. 2000). Nevertheless, this compensatory mechanism is only partial, because (1) reinitiation at a secondary AUG is less efficient than use of the first AUG (Kozak 2002a), (2) truncated proteins are potentially unstable, and (3) truncated proteins are likely to be less functional. There is currently no documented example of full biological or clinical complementation of a premature stop codon by reinitiation of translation. However, some compensation does occur, the cellular and clinical phenotypes being milder than predicted from the genotype alone (Yamashita et al. 1996; Santagata et al. 2000; Maser et al. 2001). This report therefore adds a further level of complexity to studies of pathogenic mutations in human NEMO and provides further evidence that the reinitiation of translation is a potentially important compensatory mechanism in human genetics, with profound cellular and clinical consequences.

Acknowledgments

This work was supported in part by grants from the Institut Universitaire de France and the Schlumberger and BNP-Paribas Foundations. Anne Puel was supported by a European grant (QLK2-CT-2002-00846), and Janine Reichenbach was supported by the Lise Meitner program. We thank the patient and his family for their willingness to participate in this study, and we thank the numerous clinicians and scientists who collaborated with us. We thank Tony Leclerc, for expert technical assistance; Arnold Munnich, Laurent Abel, and all members of the Laboratory of Human Genetics of Infectious Diseases, for helpful discussions; Adrian Ozinsky (University of Washington), Richard Miller (3M Pharmaceuticals), and Franck Barrat (Dynavax), for helpful discussions and for the TLR agonists; and Sandra Weller, for B-cell subpopulation studies. Jean-Laurent Casanova is an International Scholar of the Howard Hughes Medical Institute.

Web Resources

The URL for data presented herein is as follows:

Prediction of Translation Initiation ATG, http://www.hri.co.jp/atgpr/

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