This patient presented with a unique NEMO
mutation and phenotype, identified primarily because of the presenting infection with Pneumocystis
. For most male patients presenting with Pneumocystis
, a diagnosis of severe combined immunodeficiency disease or X-linked hyper-IgM syndrome is considered. As a rule, patients with severe combined immunodeficiency disease have severe T-cell lymphopenia, hypogammaglobulinemia, and impaired proliferative responses to mitogens and specific antigens, unlike this patient. Patients with X-linked hyper-IgM syndrome almost uniformly have hypogammaglobulinemia, and not a single patient in the US registry series of 79 patients presented with an IgG level greater than 300 mg/dL.19
Although reported as a pathogen in patients with NEMO,6,20–22 Pneumocystis
has been less common in the earlier reports. There are too few NEMO patients who have had Pneumocystis
infection to determine whether susceptibility is associated with specific NEMO genotypes. Therefore, until more patient natural histories are collected, Pneumocystis
infection should be considered in patients who have NEMO deficiency. Conversely, NEMO deficiency should be considered in patients with otherwise unexplained Pneumocystis
Unlike many reportedNEMOcases, this patient had essentially normal B-cell numbers and serum immunoglobulin levels, in keeping with a normal capacity for class switching as well as the capacity to make specific antibody. This distinguishes him from the usual patient described with NEMO deficiency or any of the forms of hyper-IgM syndrome. The antigen-induced maturation, survival, and activation of B cells through the B-cell receptor is known to use NF-κB in the signaling cascade; however, this patient’s NEMO
mutation did not seem to affect the numbers and distribution of B cells or immunoglobulin isotypes. The functional interaction between CD40 and CD40L, which is essential for class switching and B-cell proliferation, requires NF-κB activity. B-cell function, however, has been reported in patients with a NEMO mutation,5
which is likely to be a feature of either the specific NEMO hypomorphism or contributions from alternative costimulatory mechanisms. Regarding the former, there is a clear association with a complete lack of class switch recombination in patients with extreme c-terminal NEMO hypomorphisms.3
In terms of the latter, it is speculated that B-lymphocyte stimulators/B cell–activating factor-induced and a B-cell proliferation–inducing ligand-induced costimulation, which can bypass canonical NF-κB signaling, may be relevant.23
This may also explain why certain NEMO patients who have a more typical hyper-IgM phenotype can have elevated levels of IgA.4
In our patient, class switching may have occurred through these pathways, but more likely indicates that the NEMO D113N hypomorphism does not impair CD40 and B-cell receptor–induced signals.
Most patients with hypomorphic mutations of NEMO experience pyogenic bacterial or mycobacterial infections,3
but the patient described here presented with Pneumocystis
and chronic cytomegalovirus viremia. These infections are typically found in patients with a profound T-cell defect. This patient did have normal T-cell numbers and distribution of subsets but lacked antigen-specific lymphocyte proliferation. Although this may have been a result of his relatively young age, it is most likely an indication of a defect of antigen-specific T-cell function. This was determined directly by evaluating IκB degradation after TCR stimulation (). Patient T cells had decreased TCR-induced activation of NF-κB and thus demonstrate the effect of this NEMO mutation. In experimental systems, it has been demonstrated that after ligation, TCR signals are transmitted to NF-κB through CARMA proteins Bcl-10 and Malt1.16
It is likely that the ability of this upstream complex to interact with NEMO in the IKK complex is affected by the D113N hypomorphism. The resulting disturbance of antigen-specific T-cell function is potentially informative about how TCR signaling links to NEMO and is likely related to the patient’s presenting infection. Interestingly, the NEMO region between residues 50 and 100 has been defined as critical in interacting with CARMA1-Bcl10 complexes.24
The CARMA1-Bcl10-ubiquitin-conjugating enzyme 13 complex affects NEMO ubiquitination at the NEMO zinc finger domain. NEMO ubiquitination is necessary for the full activation of the IKK complex in response to relevant signals.25,26
This patient’s mutation is in the first coiled-coil domain. The first coiled-coil domain is near the site where NEMO binds to IKKβ, and given the proximity of this critical region to the patient’s mutation, it is possible that the significant charge27
alteration resulting from D113N may disrupt NEMO binding to the CARMA-containing, TCR-induced complex, and/or IKK kinase activity in response to upstream stimuli.
A defect in antigen-specific TCR function could also explain the patient’s unusual susceptibility to cytomegalovirus. Susceptibility to cytomegalovirus has been previously described in NEMO patients10,19
and in at least 1 case was associated with some normal cytomegalovirus-specific adaptive immune responses. 10
Thus, a number of innate responses against cytomegalovirus could be defective in NEMO deficiency and contribute to susceptibility to infection. Two hypomorphic mutations that have been previously defined as defective in patients with NEMO impair TLR responses and NK cell cytotoxicity. The latter has been reported in a patient with a NEMO hypomorphism and severe cytomegalovirus infection.10
Both NK cells and TLR responses are believed to be important in defense against cytomegalovirus.17,28
NK cells are valuable in defense against cytomegalovirus, in part because they are capable of recognizing cells with downregulated MHCclass I—a specific evasion mechanism used by the virus. As a result, many human NK cell deficiency states are associated with susceptibility to cytomegalovirus disease.15
NK cells were present in the patient, but cytotoxicity was decreased. This pattern has been found in patients with a variety of NEMO hypomorphisms, one of which was a missense mutation of the N-terminal portion of NEMO and thus more similar to our patient.4,10
Thus, it is possible that the patient’s persistent cytomegalovirus infection, rather than being secondary to compromised TLR or adaptive immune function, may be related to reduced NK cytotoxicity. At a minimum, the combination of decreased NK cell cytotoxicity and impaired antigen-specific T-cell responses may have created an immunologic deficiency resulting in cytomegalovirus susceptibility.
Recently, Fusco et al29
described the NEMO D113N as a common polymorphism seen in 1 of 120 normal control alleles. However, this finding could not be confirmed by Aradhya et al,30
who did not identify any single nucleotide polymorphisms in the NEMO coding sequence in their analysis of 700 normal chromosomes. In an author’s laboratory, NEMO sequencing is offered for diagnostic purposes, and they have also not observed a polymorphism in the NEMO coding region in a panel of normal controls (200 chromosomes; A. Jain, unpublished data). Taken together, these findings highlight the importance of an undisrupted NEMO gene sequence. They also compared the NEMO sequence in several species. The nucleotide in question (337G) is 100% conserved among Bos taurus, Canis familiaris, Gallus gallus, Macaca mulatta, Monodelphis domestica, Mus musculus, Ornithorhynchus anatinus
, and Pan troglodytes
. Moreover, codon 113 is highly conserved among species in that it encodes either an aspartate (D) or a glutamate (E); both are negatively charged acidic amino acids. The D113N mutation is an acidic to an uncharged amino acid substitution. Such a mutation is not biochemically neutral and would be expected to have a functional consequence. In keeping with this hypothesis, we demonstrated abnormalities in functional assays such as NK cell cytotoxicity, T-cell proliferation, and impaired NF-κB activation after ligation of the TCR. Nevertheless, we were unable to assess directly the effect of the NEMO D113N mutation with complementation experiments due to poorly characterized in vitro
experimental models. Despite the lack of accessible human complementation systems to validate the effect of NEMO D113N mutation in the conserved domain of NEMO, the lack of sequence variation at this location in a multispecies comparison as well as a large panel of normal human controls implicates a major contribution for NEMO in this patient’s phenotype.
Because the TLR system can recognize herpes viruses through TLR917
and has been described as impaired in all NEMO patients reported to date, a TLR function defect could have also contributed to the patient’s infectious susceptibility. Repeated evaluations of the patient’s PBMCs, however, demonstrated intact TLR function, and therefore this case is contrary to all previous reported cases of NEMO. TLR signaling, with the exception of TLR3 and TLR4, occurs through the MyD88-dependent pathway and NF-κB. The normal TLR function in this patient most likely signifies the dispensability of the region including NEMO residue 113 in TLR signaling.
Because the infectious, immunologic, and ectodermal characteristics of NEMO vary significantly, it is likely that particular NEMO hypomorphisms will provide instructive insights into how NEMO participates in particular signaling pathways and contribute to host defense. Another instructive finding in this patient is his lack of ectodermal dysplasia. Ectodermal dysplasia results from impaired NF-κB activation during development in response to ligation of the ectodysplasin A receptor, a TNF superfamily receptor, by its ligand ectodysplasin A. Patients with ectodermal dysplasia have impaired development of ectodermderived tissues with hypohidrosis (caused by lack of eccrine sweat glands), conical teeth, delayed or incomplete dentition, and sparse hair. Although a lack of ectodermal dysplasia is now reported in an increasing number of patients with NEMO hypomorphisms, this patient’s NEMO D113N alterations appear to have a common theme of not impairing TNF superfamily receptor function. Mechanistic biochemical experiments to evaluate the utility of this region of NEMO signaling pathways exclusive to non-TNF superfamily and non-TLR receptors that activate NF-κB will likely be informative and represent an important lead provided by this patient.
In summary, this patient presents with a novel and informative NEMO mutation. He demonstrated essentially normal activation of cells after TLR ligation, further distinguishing his defect from other NEMO defects that have been evaluated. He did manifest defects in antigen-specific T-cell function, and TCR-induced NF-κB activation, as well as decreased NK cell cytotoxicity, which most likely explains an immune defect underlying his susceptibility to Pneumocystis and cytomegalovirus. These results suggest the dispensability of amino acid 113 for some immunoreceptors, but not for signaling downstream of NK cell activation receptors and the TCR, or the defense against opportunistic infection.
Patients with NEMO mutations demonstrate phenotypic and immunologic heterogeneity. A novel mutation in the coiled-coil region of the NEMO protein is described that retains normal TLR function but abnormal TCR activation and infection susceptibility.