J Clin Immunol (2014) 34:916–921 DOI 10.1007/s10875-014-0097-1
ASTUTE CLINICIAN REPORT
Immunodeficiency Associated with a Nonsense Mutation of IKBKB Christian Nielsen & Marianne A. Jakobsen & Martin Jakob Larsen & Amanda C. Müller & Soren Hansen & Søren T. Lillevang & Niels Fisker & Torben Barington
Received: 2 June 2014 / Accepted: 1 September 2014 / Published online: 14 September 2014 # Springer Science+Business Media New York 2014
Abstract We report an infant of consanguineous parents of Turkish decent with a novel immunodeficiency associated with homozygosity for a nonsense mutation of the gene encoding Inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ). At five months, she presented with respiratory insufficiency and Pneumocystis jirovecii pneumonia which was successfully treated. At nine months, iatrogenic systemic infection with Mycobacterium bovis was found and eventually led to her death at age 14 months. Laboratory findings were reminiscent of hyper-IgM syndrome, but genetic testing gave no explanation before whole exome sequencing revealed a novel mutation abrogating signaling through the canonical NF-κB pathway.
Electronic supplementary material The online version of this article (doi: 10.1007/s10875-014-0097-1 ) contains supplementary material, which is available to authorized users. C. Nielsen : M. A. Jakobsen : A. C. Müller : S. T. Lillevang : T. Barington (*) Department of Clinical Immunology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark e-mail:
[email protected] M. J. Larsen Department of Clinical Genetics, Odense University Hospital, Odense, Denmark M. J. Larsen Human Genetics, Clinical Institute, University of Southern Denmark, Odense, Denmark S. Hansen Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark N. Fisker Hans Christian Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark
Keywords IKBKB . IKKβ . SCID . immunodeficiency . NF-κB . BCG vaccine
Introduction The Nuclear factor kappa-B (NF-κB) pathway plays a pivotal role in inflammatory and immune responses as well as stress responses, cell adhesion, and protection against apoptosis. Activation is elicited by signaling through receptors for antigen, many cytokines and microbial molecules [1]. Members of the NF-κB inhibitor (IκB) family restrain NF-κB-induced transcriptional activity by preventing nuclear translocation of NF-κB subunits. Canonical NF-κB pathway activation requires degradation of IκB proteins initiated by their phosphorylation by the IκB kinase (IKK) complex consisting of two catalytically active kinases, IKKα and IKKβ, and the regulatory subunit IKKγ (NEMO). Two hypermorphic gain-of-function mutations [2, 3] and haploinsufficiency of IκBα [4] as well as several hypomorphic mutations in NEMO [5] have been described as causes of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), a condition with several abnormalities of the skin (lack of sweat glands, reduced number of hair follicles, dry scaly skin) and dentition (conical teeth) combined with an immunodeficiency with increased susceptibility for pyogenic bacteria, systemic viral infections, and in some cases even fungal and atypical mycobacterial infections. The humoral immunity is often affected by reduced antibody responses to polysaccharides and low levels of IgA and IgG sometimes reminiscent of hyper IgM syndrome. Here, we describe an infant with a severe combined immunodeficiency but without signs of ectodermal dysplasia in whom IKKβ deficiency was identified using whole-exome sequencing.
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Case Report An apparently healthy girl born to consanguineous Turkish parents had uneventful BCG vaccination at age three months, but developed failure to thrive and progressive respiratory failure at five months. Pneumocystis jirovecii pneumonia was diagnosed and successfully treated with trimethoprimsulfamethoxazole. The patient had a small subcutaneous granuloma in the left deltoid region and two palpable axillary lymph nodes which subsequently diminished and were deemed to be consistent with previous vaccination and contained BCG infection. The patient’s chest X-ray showed no visible thymus and initial immunologic workup was consistent with Hyper IgM syndrome (IgM 0.59 g/l, IgA and IgG undetectable, normal lymphocyte subsets except for a complete absence of isotype-switched memory B cells and low numbers of CD45R0 positive memory T cells (Table 1). Routine blood tests indicated inflammation with raised LDH but normal liver enzymes and kidney function. Skin, skin appendages and sweat production appeared normal. Tooth eruption occurred at age 10 months, later ortopan tomography confirmed normal dentition where only the lower cuspids were identified as conical. Following recovery she was isolated at home with antimicrobial prophylaxis (immunoglobulin, fluconazole, acyclovir, and co-trimoxazole) pending genetic evaluation. At age nine months progressive signs of systemic/ multiple site infection developed with intermittent fever, a nodular rash with extended subcutaneous infiltrations, severe subcutaneous inflammation in the left thigh and left sided knee arthritis, splenomegaly with progressive focal hypodense areas, low level ascites and progressive wall thickening in the small and large intestines. Acid-fast bacteria, later identified as Mycobacterium bovis, were isolated from multiple sites. Initial improvement on antituberculous treatment (amikacin, Table 1 Lymphocyte subsets at 6 months of age
# Age matched 5–95 percentiles from ref. [6]
moxifloxacin, rifampicin, isoniazid, and ethambutol with supplementary IFN-γ therapy) occurred after a few weeks but was unfortunately transient. Systemic symptoms with fevers and progressive abdominal distension followed in a few weeks. Despite three and a half months of the above therapy with i.v. ethambutol administration, splenomegaly progressed to almost total necrosis, why splenectomy was performed at age 11 months. Viable mycobacteria were isolated from the spleen and another 6 weeks later also from the bone marrow. Due to the uncontrolled systemic mycobacterial infection despite relevant therapy, IFN-γ therapy was discontinued and experimental treatment with IL-2 was initiated. The rationale was that IL-2 supplementation corrected the impaired lymphocyte proliferation in vitro (see below) and previous case reports wherein IL-2 treatment seemed to protect against infection in selected primary immunodeficiency cases [7–9], although IL-2 treatment of severe ongoing systemic mycobacterial infection has not been reported. The child weighed 9 kg with an estimated body surface area 0.43 sqm and treatment was initiated with 10,000 IU subcutaneously once daily for five days and escalated to a final dosage of 270,000 IU three weeks later. During treatment the only suspected side effect showed to be intercurrent catheter-related Enterobacter bacteremia. The last dose was given for four days without obvious side effects. Several hours following the last administration, the patient unexpectedly had cardiac arrest. Resuscitation was unsuccessful and the patient died at 14 months.
Results Hyper IgM syndrome was excluded by sequencing of the AID, CD40, CD40L, UNG and NEMO genes using biAbsolute number (109/L)
Reference values (109/L)
33 % (leuco.)
5.7
1.9–12.4
81 % (CD3+) 16 % (CD3+) 89 % (CD3+CD4+) 8 % (CD3+CD4+) 4 % (CD3+)
3.6 0.7 3.2 0.3 0.2
0.9–4.1 0.1–0.7 – – –
Cell populations
% (of cell type)
Lymphocytes T cells CD3+CD4+ CD3+CD8+ CD3+CD4+CD45RA+ CD3+CD4+CD45R0+ CD3+HLA-DR+ B cells CD3-CD19+ CD19+CD27–IgD+ CD19+CD27+IgD+ CD19+CD27+IgDNK cells CD3-CD16+56+
Reference values# (%)
16 % (lymph.) 98 % (CD19+)
85.5–93.4
0.9 0.9
0.3–1.0 –
1 % (CD19+) 0 % (CD19+)
2.8–7.4 1.6–7.0
0.01 0.00
– –
0.11
0.1–1.1
2 % (lymph.)
918
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directional Sanger sequencing. Also TRAF2 and JAK3 were sequenced. No mutations were found. To search for potential causal mutations, a trio-based whole-exome sequencing approach was undertaken. DNA from the patient and her parents were subjected to exome capture using TruSeq Exome Enrichment Kit (Illumina), followed by sequencing on an Illumina HiSeq 1500 to a mean coverage of 84–90×. Raw reads were aligned using Novoalign v. 3.01 (Novocraft) and GATK Best Practice pipeline v. 2.7 was used for variant calling [10]. As autosomal recessive inherited disease was suspected due to parental consanguinity, only variants found to be homozygous in the child and heterozygous in both parents were considered. Filtering common variants (minor allele frequency>1 %) and synonymous variants resulted in one nonsense mutation and 26 missense mutations (Supplementary table 1). No frame-shift or in-frame deletions or insertions fulfilled the criteria. Functional prediction algorithms (Polyphen2, SIFT and MutationTaster) were applied to evaluate the significance of the identified missense variants. Five of the missense variants were predicted damaging by one tool; there was, however, no agreement between the three tools. Given the known critical role of IKKβ in the canonical NF-κB pathway the most promising candidate was a nonsense mutation (c.814C>T) in the IKBKB gene encoding IKKβ (R272X, numbering according to NM_001556.2) situated on chromosome 8p11 within a large 50 Mb region of homozygosity (Supplementary table 2). This mutation, confirmed by Sanger sequencing of the complete coding region (Fig. 1a), leads to a complete lack of the IKKβ protein. Protein levels of
A
IKBKB genomic DNA Sequence
B
IKKα and NEMO were normal as compared with control PBMCs (Fig. 1b). Activation of T cells through the T-cell receptor (TcR) is one of many processes dependent on the canonical NF-kB pathway which relies on functional complexes of IKKß and NEMO phosphorylating IkB subunit alpha leading to its ubiquitinylation and degradation allowing nuclear translocation of phosphorylated (serine 529) NF-kB p65 (relA). We therefore evaluated the effects of the IKBKB mutation on T cells collected before IL-2 therapy. Stimulation by anti-CD3/CD28 completely failed to phosphorylate p65 and phosphorylation was only partial after activation by PMA and ionomycin (Fig. 2). Correspondingly, T cells (gated as CD4+ or CD8+ lymphocytes) failed to proliferate in response to anti-CD3/CD28 during 6 days of culture (Fig. 3). Proliferation was, however, normal after addition of IL-2 and partial proliferative responses were seen to the T-cell mitogen PHA showing that other signaling pathways could bypass the canonical NF-kB pathway and induce proliferation.
Discussion We show that IKKβ deficiency abrogates the canonical NFkB signaling pathway and thereby cell proliferation in response to TcR engagement simulated by CD3- and CD28 co-receptor stimulation (Figs. 2 and 3). This confirms the results presented in a recent report of 4 IKKβ-deficient children of Northern Cree ancestry, caused by a homozygous duplication (c.1292dupG) in exon 13 of IKBKB [11]. Murine
Western blot of PBMCs
268 269 270 271 272 273 274 275
} } } } } } } }
Val Leu Ala Glu Arg Leu Glu Lys
IKKβ
Control:
IKKα
NEMO ERK2
Mother: R272X
Paent:
Fig 1 a: IKBKB sequence analysis in the patient compared with that in a healthy control and the mother; the patient was homozygous for a c.814C>T mutation leading to a premature stop-codon at amino acid 272. b: Western blot analysis of PBMCs from the patient, showing a complete lack of the IKKβ protein (using an anti-IKKβ antibody from Abcam directed against the N-terminal part of the molecule). Protein
levels of IKKα and NEMO (Abs from Abcam) were normal as compared with control PBMCs. The extracellular signal-regulated kinase 2 (ERK2) protein level served as a loading control. Equal amounts of total protein derived from the lysed PBMCs were loaded to each lane of the same gel, and the blot was divided into four for development with respective antibodies
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Unstim
+ PMA/ionomycin
+ anti-CD3/CD28 1%
48 %
90%
99 %
52 %
10 %
1%
1%
41%
99 %
99 %
59 %
p-NF-kB B p65 (S529)
Control:
Patient:
Fig. 2 NF-κB p65 phosphorylation at serine 529 in the activation domain in lymphocytes detected by intracellular staining after 10 min of stimulation as indicated above the contour plots
studies have shown that IKKß (rather than IKKα) binding to NEMO is essential for canonical NF-kB signaling [12]. Mice totally deficient of IKKß die in mid gestation from uncontrolled liver apoptosis. T lineage-specific deletion of IKKß leads to normal generation of naïve T cells, but failure of differentiation into memory T cells [13]. Similarly, in mice B cell lineage-specific deletion of IKKß demonstrated reduced numbers of B cells failing to differentiate into mature B cells [14]. Our case had normal liver function and normal numbers of naïve T- and B cells. A considerable reduction in the number of memory CD45R0 positive CD4 T cells were, however, noted and the B cells showed complete absence of isotype-switched memory B cells at six months of age. In line with Pannicke and colleagues [11] this indicates that in humans, IKKß is not critical for fetal development and also dispensable for the generation of T- and B cells, but critical for activation of T cells and differentiation of B cells. This readily explains our patient’s propensity for severe fungal (Pneumocystis jiroveci) and mycobacterial infections, a feature shared with already known immunodeficiencies of the NF-kB signaling pathway, namely hypermorphic mutations in IκBα and hypomorphic mutations in NEMO inhibiting phosphorylation of IκBα [2, 3, 5]. However, whereas NEMO and IκBα mutations usually are accompanied by ectodermal dysplasia (EDA-ID), our patient showed no signs of this. We therefore suggest that ectodermal dysplasia in NEMO deficiency is related to impaired signaling by IKKα-NEMO
complexes independent of IKKβ whereas the signaling defects of the TcR pathway yielding the immunodeficiency are related to IKKβ-NEMO or IKKα-IKKβ-NEMO complexes. This is supported by the finding that in vitro blockade of IKKβ signaling in purified human primary CD4+ T cells, either by exposure to the cell-permeable IKKβ inhibitor PS1145 or by genetic ablation of IKKβ, abrogates proliferation and activation of NF-kB transcription factors upon anti-CD3/ CD28 stimulation [15]. Moreover, IKKα has been shown to be dispensable for NF-kB activation [16] but indispensable for normal epidermal development because nonsense mutations have been found to lead to severe malformations of bone and epidermis and fetal loss [17]. In contrast to our findings, Pannicke and colleagues reported that IKKβ deficiency leads to decreased protein levels of NEMO and lack of IKKα protein in PBMCs [11]. This is unexpected and difficult to explain in light of our data showing that total absence of IKKβ does not preclude normal expression of IKKα and NEMO. In agreement with that, fetal cells in IKBKB knockout mice reveal normal levels of NEMO and IKKα [12]. Nor does siRNA-induced knock down of IKKβ in human cell lines affect IKKα expression in vitro [18]. It is possible that the discrepancy regarding the influence of IKKβ deficiency on NEMO and IKKα levels reflects differences in the experimental settings, e.g. the activation status of the cells studied. Both the mutation reported here, and the mutation reported by Pannicke and colleagues [11] may in principle lead to
920
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+ anti-CD3/CD28 ti CD3/CD28
+ +anti -CD3/CD28 CD3/CD28
+ PHA
Day 6
Day 6
+ rhIL-2
Day 4
# T cells
Control:
+ rhIL-2
Patient:
CFSE Fig. 3 Proliferation measured as carboxyfluorescein succinimidyl ester (CFSE) dilution after 4–6 days of stimulation of T cells defined as CD4+ or CD8+ lymphocytes. Red lines: unstimulated; blue lines: stimulated as
indicated above histograms; blue dotted lines: anti-CD3/CD28 stimulated+recombinant human IL-2 added at the beginning of culture. Green lines: unlabeled cells stimulated as indicated above histograms
formation of truncated proteins (271 and 492 amino acids, respectively) and it is conceivable that the c.1292dupG mutation may lead to formation of small amounts of a truncated protein interfering with the stability of IKKα and NEMO. It must be mentioned, however, that neither we nor Pannicke and colleagues found evidence for the presence of truncated versions of IKKβ using relevant antibodies. Moreover, the predicted 492 amino acids protein lacks most of the α-helical scaffold dimerization domain including the leucine-zipper and the helix-loop-helix domain as well as the NEMO-binding domain involved in binding to IKKα and NEMO. How such an interaction could occur therefore remains obscure. The fact that the c.814C>T mutation reported here did not interfere with IKKα or NEMO expression allows us to conclude that the case demonstrates the consequences of IKKβ deficiency alone and therefore elucidates the role of this factor in the NF-κB pathway in humans. Normal activation of naïve T cells depends on balanced engagement of NF-kB, NFAT, and AP-1 transcription factors. Lupino and colleagues [15] have shown that IKKβ independently of NF-kB is crucial for mediation of AP-1 signaling after TcR stimulation and that lack of IKKβ may lead to T-cell anergy. It is therefore likely that our patient had a more
pronounced T-cell deficiency than that usually seen in patients with NEMO mutations. On the other hand, IKKβ independent mechanisms that lead to NF-kB p65 phosphorylation and Tcell proliferation were intact in our patient as indicated by the PHA-induced proliferation of T cells bypassing TcR signaling (Fig. 3) and the PMA/ionomycin induced phosphorylation of the NF-kB p65 subunit (Fig. 2), respectively. Similarly, IL-2 was able to rescue proliferation induced by anti-CD3/CD28. Unfortunately, IL2 treatment of our patient did not demonstrate any clear clinical effect. The death of this infant stresses that in any BCG vaccinated infant subsequently diagnosed with cellular immunodeficiency evidence of active systemic BCG infection should be sought regardless of clinical presentation. Given that functional reconstitution experiments were not conducted, it cannot be definitively excluded that other mutations than the nonsense mutation in IKBKB contributed to the immunodeficiency. However, among the 26 missense mutations that our patient was homozygous for, none was known to cause disease and no consistent results with regard to potential pathogenicity were provided by three different disease prediction software tools. Two genes deserve special attention though: CHD7 and LPB. CHD7 is associated with CHAR
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GE syndrome (reviewed in ref. [19]) with malformations including eye, nose and heart and sometimes reduced T-cell numbers with immunodeficiency. However, CHARGE syndrome is usually dominantly inherited and our patient presented none of the malformations characteristic for that syndrome and had normal numbers of T cells. Similarly, the missense mutation in LPB which encodes the lipopolysaccharide binding protein was unlikely to contribute to our patient’s phenotype because gram negative infections were not a part of the clinical picture. We therefore find it highly likely that the IKKß deficiency was the principal cause of the immune phenotype of our patient. Our case suggests that deficiency of IKKß should be classified as a severe combined immunodeficiency and that it should be considered in children with clinical signs of severe immunodeficiency but normal lymphocyte counts.
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