X-linked inhibitor of apoptosis (XIAP) deficiency, caused by BIRC4 mutations, is described to cause X-linked lymphoproliferative disease (XLP) phenotypes. However, compared with XLP caused by SLAM-Associated Protein deficiency (SH2D1A mutation), XIAP deficiency was originally observed to be associated with a high incidence of hemophagocytic lymphohistiocytosis (HLH) and a lack of lymphoma, suggesting that classification of XIAP deficiency as a cause of XLP may not be entirely accurate. To further characterize XIAP deficiency, we reviewed our experience with 10 patients from 8 unrelated families with BIRC4 mutations. Nine of 10 patients developed HLH by 8 years of age. Most patients presented in infancy, and recurrent HLH was common. There were no cases of lymphoma. Lymphocyte defects thought to contribute to HLH development in SLAM-Associated Protein deficiency were not observed in XIAP deficiency. We conclude that XIAP deficiency is a unique primary immunodeficiency that is more appropriately classified as X-linked familial hemophagocytic lymphohistiocytosis.

Deficiency of X-linked inhibitor of apoptosis (XIAP), caused by BIRC4 gene mutations, was discovered to be associated with X-linked lymphoproliferative disease (XLP) phenotypes among 12 patients from 3 families by Rigaud et al in 2006.1  Before this, XLP was known only to be associated with mutations in SH2D1A, which encodes SLAM-Associated Protein (SAP).2-4  Patients with SAP deficiency commonly develop Epstein-Barr virus–associated hemophagocytic lymphohistiocytosis (HLH) (60%), hypogammaglobulinemia (30%), and lymphoproliferative disorders, including malignant lymphoma (30%), and other less common complications.5  In contrast to SAP deficiency, more than 90% of patients with XIAP deficiency developed HLH.1  Notably, HLH was recurrent in the majority of patients, which is not frequently observed in patients with SAP deficiency, in whom a single, frequently fatal episode is more commonly described. A Japanese patient has also recently been described to have recurrent HLH.6  There have been no cases of lymphoma associated with XIAP deficiency reported to date. We therefore hypothesized that the classification of XIAP deficiency as a cause of XLP may not be entirely accurate. To further characterize this disease, we studied clinical and laboratory findings among 10 patients with XIAP deficiency. We found that XIAP deficiency commonly presents with phenotypes consistent with familial HLH (FHLH), a collection of immune deficiencies, which feature HLH as the predominant disease manifestation.7 

Patients

All patients were treated at Cincinnati Children's Hospital, and Institutional Review Board approval was obtained for this retrospective study. Patient presentations are included in supplemental data (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Mutational analysis of the BIRC4 gene

BIRC4 mutational analysis was performed as previously described.8 

Analysis of XIAP expression

Western blot and flow cytometric analysis of XIAP was done as previously described.8 

NK-cell functional studies

Natural killer (NK)–cell cytotoxicity was measured by chromium 51 release assay as described.9 

NK-cell degranulation was quantified by measurement of surface CD107a up-regulation. Patient or control peripheral blood mononuclear cells (PBMCs) were incubated in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, 2mM glutamine, and penicillin/streptomycin (Invitrogen) with or without K562 cells in the presence of fluorochrome-conjugated anti-CD107a or isotype control antibody (BD Biosciences). Monensin (BD Biosciences, GolgiStop) was added 1 hour into the incubation. PBMCs were then stained with fluorochrome-conjugated surface antibodies against CD3, CD56, and CD8 (BD Biosciences). Cells were washed, fixed, and analyzed on a BD Biosciences FACSCalibur flow cytometer. NK cells were identified as CD3CD56+ lymphocytes. CD107a up-regulation was measured both as the percentage of NK cells positive for CD107a as well as the relative mean channel fluorescence with respect to isotype control staining.

Evaluation of T-lymphocyte Fas-induced and restimulation-induced cell death

Patient or control PBMCs were suspended at 0.5 to 1 × 106 cells/mL in RPMI 1640 medium supplemented as in the above paragraph. T cells were activated with 5 μg/mL concanavalin A (Sigma-Aldrich). After 4 days, activated T cells were washed and then cultured in medium supplemented with 100 U/mL recombinant human interleukin-2 for 11 to 23 days. Expanded T cells (1 × 105 cells/well) were then plated in duplicate in 96-well plates and treated with either APO-1-3 (Alexis Biochemicals) and Protein A (Sigma-Aldrich; 5-500 ng/mL) to evaluate Fas-mediated lymphocyte apoptosis, or the anti-CD3 monoclonal antibody OKT3 (Ortho Biotech Products; 5-500 ng/mL) to evaluate restimulation-induced cell death. Twenty-two hours after treatment, cells were stained with propidium iodide and analyzed on a FACSCanto flow cytometer (BD Biosciences). Cell death was quantified as follows: % cell loss = (1 − (% viable cells, treated/% viable cells, untreated)) × 100.

As summarized in Table 1, BIRC4 mutations were observed in 10 patients from 8 unrelated families, including whole exon deletions, nonsense, small insertion, and missense mutations, which resulted in truncated, decreased, or absent XIAP expression. Symptomatic patients developed HLH as defined by the “classic” features of fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, hyperferritinemia, elevated levels of soluble interleukin-2 receptor, decreased or absent NK-cell function, and hemophagocytosis observed on pathologic inspection of bone marrow or other tissues.10  Fifty percent of patients presented in infancy, with no known viral infection, and 60% developed recurrent HLH or recurrent HLH-like disease. HLH associated with Epstein-Barr virus occurred in 30% of patients, and HLH associated with cytomegalovirus occurred in 20%. Hemophagocytosis was observed in 40% of patients. One patient experienced symptomatic central nervous system (CNS) involvement, and CNS disease was documented in 2 of 7 evaluated patients (29%). The true incidence of CNS HLH may be higher given that not all patients were evaluated for CNS involvement. Hypogammaglobulinemia was observed in only 2 of 9 patients evaluated, probably resulting from immunosuppressive therapy at the time of evaluation given that normal IgG levels were observed in patients free of any treatment. Only 50% of patients had a family history suggestive of an X-linked disorder. Most patients received treatment with steroids, with or without calcineurin inhibitor, with or without etoposide. One patient required salvage therapy with alemtuzumab. One patient each received etanercept or rituximab. Interestingly, 3 patients were treated with only supportive care because of the lack of timely diagnosis of HLH, and survived. Seven patients have undergone allogeneic hematopoietic cell transplantation, with 3 deaths related to complications.

Table 1

Summary of patient data

Patient 1Patient 2*Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8*Patient 9Patient 10
BIRC4 mutation Deletion Exon 6 Deletion Exons 1–5 1445C>G (P482R) 1481T>A (I494N) 563G>A (G188E) 868–869insT (Y290fsX294) 997C>T (Q333X) Deletion Exons 1–5 310C>T (Q104X) 563G>A (G188E) 
XIAP expression Truncated, decreased Not detectable Decreased Not tested Decreased Not detectable Not detectable Not detectable Not detectable Decreased 
Age at initial HLH presentation Infancy Infancy Infancy Infancy Infancy 4 years 6 years 7 years 8 years Asymptomatic 
Recurrent HLH NA, prompt HCT NA, prompt HCT NA, prompt HCT Recurrent cytopenias, prolonged viral illnesses Recurrent fevers, prolonged viral illnesses  
Fever − 
Splenomegaly − 
Hepatitis Unknown − 
Bicytopenia − 
Hypertriglyceridemia Unknown Unknown − 
Hypofibrinogenemia Unknown Unknown − 
Hemophagocytosis − − − Unknown − − 
Hyperferritinemia Unknown Unknown − 
Elevated soluble IL2R Unknown Unknown − 
CNS symptoms − − + (seizure) − − − − − − − 
Pathologic CSF − − − NT NT NT − − 
EBV-associated HLH − − − − − − − 
CMV-associated HLH − − − − − − − − 
Hypogammaglobulinemia +§ § § § NT − − − − 
Treatment Liver transplantation; tacrolimus prednisone Prednisone; cyclosporine HLH 2004, including IT treatment HLH 2004 alemtuzumab HLH 2004 etanercept Supportive care Supportive care Supportive care Dexamethasone; rituximab − 
Allogeneic HCT − − − 
Current age 12 y 4 y Deceased 3 y Deceased 24 y 16 y 28 y Deceased 4 y 
Family history Yes Yes No No (No) No No Yes Yes Yes 
Patient 1Patient 2*Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8*Patient 9Patient 10
BIRC4 mutation Deletion Exon 6 Deletion Exons 1–5 1445C>G (P482R) 1481T>A (I494N) 563G>A (G188E) 868–869insT (Y290fsX294) 997C>T (Q333X) Deletion Exons 1–5 310C>T (Q104X) 563G>A (G188E) 
XIAP expression Truncated, decreased Not detectable Decreased Not tested Decreased Not detectable Not detectable Not detectable Not detectable Decreased 
Age at initial HLH presentation Infancy Infancy Infancy Infancy Infancy 4 years 6 years 7 years 8 years Asymptomatic 
Recurrent HLH NA, prompt HCT NA, prompt HCT NA, prompt HCT Recurrent cytopenias, prolonged viral illnesses Recurrent fevers, prolonged viral illnesses  
Fever − 
Splenomegaly − 
Hepatitis Unknown − 
Bicytopenia − 
Hypertriglyceridemia Unknown Unknown − 
Hypofibrinogenemia Unknown Unknown − 
Hemophagocytosis − − − Unknown − − 
Hyperferritinemia Unknown Unknown − 
Elevated soluble IL2R Unknown Unknown − 
CNS symptoms − − + (seizure) − − − − − − − 
Pathologic CSF − − − NT NT NT − − 
EBV-associated HLH − − − − − − − 
CMV-associated HLH − − − − − − − − 
Hypogammaglobulinemia +§ § § § NT − − − − 
Treatment Liver transplantation; tacrolimus prednisone Prednisone; cyclosporine HLH 2004, including IT treatment HLH 2004 alemtuzumab HLH 2004 etanercept Supportive care Supportive care Supportive care Dexamethasone; rituximab − 
Allogeneic HCT − − − 
Current age 12 y 4 y Deceased 3 y Deceased 24 y 16 y 28 y Deceased 4 y 
Family history Yes Yes No No (No) No No Yes Yes Yes 

HCT indicates hematopoietic cell transplantation;CNS, central nervous system; EBV, Epstein-Barr virus; CMV, cytomegalo virus; NA, not applicable; and NT, not tested.

*

Patients 2 and 8 are maternally related.

Patients 5 and 10 are maternally related.

As determined by Western blotting and/or intracellular flow cytometry.

§

Tested while on immunosuppressive therapy.

Maternally related to patient 10 who was asymptomatic at the time of diagnosis.

Patient was diagnosed and treated with allogeneic HCT before being symptomatic.

These clinical phenotypes suggest that XIAP deficiency is predominantly associated with FHLH phenotypes, and not XLP phenotypes. The phenotypic differences may be the result of differences in the molecular basis of each disease. SAP, consisting almost entirely of an SH2 domain, functions as an intracellular adaptor molecule involved in SLAM family signaling.11  XIAP is an inhibitor of apoptosis family member, consisting of 3 BIR regions and a C-terminal RING domain, best known for its caspase-inhibitory and antiapoptotic properties, and is also involved in several signaling pathways (nuclear factor-κB, c-Jun N-terminal kinase 1 transforming growth factor-β, Nucleotide-binding Oligomerization Domain Protein) and possesses E-3 ubiquitination function.12-20 

In patients with SAP deficiency, HLH may develop because of absence of iNKT cells,21  defective granule-mediated cytotoxic lymphocyte cytotoxicity,11  and defective T-cell reactivation-induced cell death (RICD).22  It is unknown why XIAP deficiency confers HLH susceptibility, and it appears unlikely that there is overlap with the defects associated with SAP deficiency. We previously reported that iNKT cells are numerically normal in many of the patients in this cohort, indicating this is unlikely to be a pathologic basis for disease.23  We also studied NK-cell function, which was observed to be normal by Rigaud et al.1  We observed decreased NK function using standard chromium release assays that use PBMCs as the NK-cell source, but this is likely an artifact because of patient NK lymphopenia (7 of 10 patients), which results in effector cell dilution. We therefore evaluated NK-cell degranulation via measurement of surface CD107a up-regulation after K562 cell exposure and found this to be normal (supplemental Figure 1). We lastly studied RICD and found that T cells from patients with XIAP deficiency have increased susceptibility to RICD, confirming the observation made by Rigaud et al,1  which contrasts sharply with the defective RICD observed in SAP deficiency (supplemental Figure 2A). However, in contrast to the findings by Riguad et al,1  we observed normal T-cell susceptibility to Fas-mediated apoptosis (supplemental Figure 2B). This is in line with the observation of normal Fas-mediated apoptosis in XIAP-deficient mice24  and the finding that human peripheral blood lymphocyte Fas-mediated apoptosis is not increased by treatment with XIAP inhibitors,25,26  suggesting that XIAP is specifically important for TCR-mediated T-cell survival in the context of immune responses.

In conclusion, we have demonstrated that the clinical phenotypes of XIAP deficiency are not consistent with the spectrum of abnormalities observed in XLP. XIAP deficiency is associated with FHLH phenotypes in the patients presented here, and also in patients reported in the literature (Table 2). In addition, lymphocyte defects associated with SAP deficiency are not observed in many patients with XIAP deficiency (Table 2). We conclude that XIAP deficiency is a unique primary immunodeficiency that is best classified as X-linked FHLH.

Table 2

Summary of clinical presentations and laboratory findings of interest among patients with XIAP deficiency reported to date

Rigaud et al1 Zhao et al6 Marsh et al (current study)
No. of patients 12 10 
Patients with HLH 11 (92%) 9 (90%) 
Median patient age at presentation, y (range) 3 (0.5-20) 1.7 0.3 (birth to 8) 
Patients with recurrent HLH or HLH-like illness 6 (50%) 6 (60%) 
Patients with Epstein-Barr virus infection-associated HLH 8 (67%) 3 (30%) 
Patients with hypogammaglobulinema 4 (33%) 2 (20%) 
Patients with lymphoma 
NK cell function Normal Not Tested Normal 
Peripheral blood iNKT cell populations Decreased Not Tested Normal 
T-cell Fas-mediated apoptosis Increased Not Tested Normal 
T-cell restimulation-induced cell death Increased Not Tested Increased 
Rigaud et al1 Zhao et al6 Marsh et al (current study)
No. of patients 12 10 
Patients with HLH 11 (92%) 9 (90%) 
Median patient age at presentation, y (range) 3 (0.5-20) 1.7 0.3 (birth to 8) 
Patients with recurrent HLH or HLH-like illness 6 (50%) 6 (60%) 
Patients with Epstein-Barr virus infection-associated HLH 8 (67%) 3 (30%) 
Patients with hypogammaglobulinema 4 (33%) 2 (20%) 
Patients with lymphoma 
NK cell function Normal Not Tested Normal 
Peripheral blood iNKT cell populations Decreased Not Tested Normal 
T-cell Fas-mediated apoptosis Increased Not Tested Normal 
T-cell restimulation-induced cell death Increased Not Tested Increased 

HLH indicates hemophagocytic lymphohistiocytosis; and NK, natural killer.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank the patients and their families for their support of our work and Colin Duckett for critical reading of the manuscript.

This work was supported by the Histiocytosis Association of America and National Institutes of Health (R03 1R03AI079797-01).

National Institutes of Health

Contribution: R.A.M. designed the study, collected patient data, performed experiments, and wrote the manuscript; L.M., B.J.K., R.M., and B.M. contributed to the collection of data and writing of the manuscript; M.B.J. and J.J.B. edited the manuscript; K.Z. performed the BIRC4 analysis and edited the manuscript; and A.H.F. designed the study and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Rebecca A. Marsh, Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail: [email protected].

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