SAP and XIAP deficiency in hemophagocytic lymphohistiocytosis


  • Xi Yang,

    1. Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
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  • Toshio Miyawaki,

    1. Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
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  • Hirokazu Kanegane

    Corresponding author
    1. Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
      Hirokazu Kanegane, MD PhD, Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. Email:
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Hirokazu Kanegane, MD PhD, Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. Email:


Hemophagocytic lymphohistiocytosis (HLH) is a multisystem inflammatory disorder due to cytokine overproduction from excessively activated lymphocytes and macrophages. HLH has been divided into two subgroups: primary HLH and secondary HLH. Primary HLH includes PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, SH2D1A and XIAP gene mutations; and secondary HLH is associated with infections, malignancies and autoimmune diseases. Among primary HLH-related genes, SH2D1A and XIAP are genetically responsible for X-linked lymphoproliferative syndrome (XLP) due to signaling-lymphocytic-activation-molecule-associated protein (SAP) and XIAP deficiencies, respectively. XLP is characterized by extreme vulnerability to Epstein–Barr virus infection. The major clinical manifestations of XLP consist of HLH (60%), lymphoproliferative disorder (30%) and dysgammaglobulinemia (30%). Analysis of clinical phenotypes of XLP patients suggests that XLP predominantly shows familial HLH phenotypes, whereas some XLP patients present sporadic HLH. For many decades, clinicians and investigators have been concerned with possible XLP in young boys presenting with Epstein–Barr-virus-associated HLH. This review aims to describe the new knowledge about XLP and to draw the attention of the pediatrician to XLP, which should be differentiated from other forms of HLH.

Hemophagocytic lymphohistiocytosis (HLH) was first described in 1952 as an autosomal recessive immune dysregulatory disorder of childhood, called “familial hemophagocytic reticulosis.”1 As HLH patients have high mortality, we suppose that many HLH patients have been underdiagnosed. Allen et al.2 estimated that the incidence of HLH is one case per 3000 inpatients in tertiary care pediatric hospitals. On the basis of its etiological factors, HLH is divided into two different conditions: primary and secondary HLH.3 In primary HLH, patients have a family history or documented genetic mutations that are associated with the development of HLH. Mutations in genes including PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, SH2D1A and XIAP have been linked to this disease. Genotype and phenotype correlations observed in patients with PRF1, UNC13D, STX11, STXBP2 and RAB27A, and hypomorphic mutations might be associated with later-onset HLH.4,5 Secondary HLH encompasses a number of categories that include designations such as infection-associated HLH, autoimmune-associated HLH or macrophage-activation syndrome, and malignancy-associated HLH.6

X-linked lymphoproliferative syndrome (XLP) is a rare inherited immunodeficiency estimated to affect approximately one in 1 000 000 males, although it may be underdiagnosed.7 A gene responsible for XLP was first identified by using positional cloning and functional cloning approaches in 1998.8–10 It is located in the region of Xq25, designated SH2D1A/signaling-lymphocytic-activation-molecule (SLAM)-associated protein (SAP). The SH2D1A gene encodes a 128-amino-acid protein consisting of a 5-amino-acid N-terminal sequence, an SH2 domain and a 25-amino-acid C-terminal tail. In 2006, a second causative gene of XLP was found, XIAP/BIRC4, which encodes X-linked inhibitor of apoptosis (XIAP) protein.11XIAP is located close to SH2D1A on the X-chromosome (Fig. 1), and consists of six coding exons, producing an anti-apoptotic molecule that belongs to the inhibitor of apoptosis (IAP) family.12–14 XLP is now divided into two types: SAP deficiency (XLP-1), caused by mutations in the SH2D1A gene, and XIAP deficiency (XLP-2), due to mutations in the XIAP gene. While HLH can be diagnosed according to clinical criteria, XLP is definitively diagnosed by gene and protein analyses. Flow cytometry is useful for rapid screening of several primary immunodeficiencies, including XLP (Fig. 2). However, it is notable that there are a few patients who cannot be diagnosed only by protein analysis.15–18 Needless to say, gene sequencing of SH2D1A and XIAP is the gold standard method for the identification of XLP disease. There are various types of mutations, including large and small deletions, nonsense mutation, missense mutation and splicing anomaly (Fig. 3). Arg55stop mutation was observed in four of 21 Japanese families with XLP-1, and it is a hotspot mutation in SH2D1A.

Figure 1.

Physical map of SH2DA and XIAP in Xq25. The physical position of each gene is indicated according to NCBI Homo sapiens map. SH2D1A and XIAP genes are located quite close together.

Figure 2.

Flow cytometric analysis in patients with SAP and XIAP deficiency. (a) SAP expression by flow cytometry. The patient showed decreased expression of SAP protein in CD56+ NK cells.15 The gray and black areas indicate control and SAP antibodies (KST-3). (b) XIAP expression by flow cytometry. The patient showed decreased expression of XIAP protein in lymphocytes.16 The gray and black areas indicate control and XIAP antibodies (clone 48).

Figure 3.

SH2D1A mutations in Japanese patients with XLP-1. Various types of SH2D1A mutations were identified in Japanese patients with XLP-1. Parentheses indicate the number of the XLP family.

As the clinical features of secondary HLH and XLP are basically similar, and often triggered by an infection, especially that of Epstein–Barr virus (EBV), it maybe difficult to distinguish these two disease conditions.19 In this report, we describe the characteristics of SAP and XIAP deficiency, and explain how we can identify XLP among HLH patients.

Pathophysiology of SAP and XIAP deficiency

The central pathophysiological abnormality of HLH is cytokine dysfunction, resulting in uncontrolled accumulation of activated T lymphocytes and histiocytes in many organs. Lykens et al.20 have recently demonstrated this condition with an animal model. Defective NK cell function is also associated with decreased amounts of perforin, which may also be important in regulating T cell function.21

SAP is a key regulator of normal immune function in T, natural killer (NK) and NKT cells, and possibly in B cells as well.10,22,23 SAP-deficient patients have been found to exhibit impaired development of memory B cells. This may explain the lower levels of immunoglobulin (Ig) secreted by B cells from XLP patients in vitro compared with those from normal donors because Ig production in such assays is dominated by memory B cells, rather than naïve ones.24,25 SAP is required for the functions of SLAM receptors by its capacity to recruit the Src-related protein tyrosine kinase Fyn, allowing SLAM receptors, including NTB-A, 2B4, CD84, Ly-9, CD84 and CRACC, to transduce tyrosine phosphorylation signals.26,27 In T cells, the SLAM-SAP-Fyn signaling regulates cytokine production by activating two pathways: one is SH2-containing inositol phosphatase (SHIP), which suppresses production of interferon (IFN)-γ;28 and the other consists of protein kinase C (PKC)θ, Bcl10 and nuclear factor κB (NF-κB), which increases expression of the transcription factor GATA-3 and optimal production of IL-4 by anti-CD3-stimulated CD4+ T cells29 (Fig. 4a). In NK cells, the 2B4-SAP-Fyn module triggers a pathway involving SHIP, Vav-2, c-Cbl and phospholipase Cγ1 that activates cell cytotoxicity.30 In the absence of SAP, 2B4 may deliver negative signals to NK cells by recruiting molecules with inhibitory functions, such as SHIP, SHP-1, SHP-2 and Csk. The co-activation effect of 2B4 has also been studied in detail in EBV-specific cytolytic T lymphocytes (CTL)31 (Fig. 4b). NTB-A appears to play a similar role to 2B4 on NK cells, in that cross-linking of NTB-A results in enhanced cytotoxicity and cytokine secretion.32,33 The ability of NTB-A to bind protein tyrosine phosphatases requires clarification because NTB-A can bind SHP-2, but not SHP-1, in primary human NK cells32 (Fig. 4c). Studies of human NK cells and CD8+ T cells from XLP-1 patients have shown that, in the absence of SAP, the activating function of SLAM-R, 2B4 and NTB-A shifts toward the inhibition of cell-mediated cytotoxicity to EBV-infected B cells, which develop the clinical signs of EBV-HLH.34,35

Figure 4.

Defective SLAM receptor functions in the pathophysiology of SAP deficiency. (a) Signaling pathways downstream of SLAM in T cells. In T cells, SAP and its SLAM-SAP-Fyn signaling module regulate cytokine production by activating two pathways: one is SHIP, and the other involves PKCθ, Bcl10 and NF-κB. (b) Signaling pathways downstream of 2B4 in NK and CTL cells. In NK and CTL cells, a 2B4-SAP-Fyn module triggers a pathway involving at least EAT-2 and Csk, which activates cell cytotoxicity. (c) Signaling pathways downstream of NTB-A in NK cells. In NK cells, NTB-A appears to play a similar role to the 2B4 pathway, which can bind SHP-2, but not SHP-1.

XIAP gene encodes a 497-amino-acid anti-apoptotic molecule that belongs to the IAP, which are composed of three baculovirus IAP repeat (BIR) domains and a C-terminal RING domain with E3 ubiquitin ligase activity (Fig. 5).36 Unlike SAP, XIAP is ubiquitously expressed, and it can be detected in all hematopoietic cells.37 The main function ascribed to XIAP is the suppression of apoptosis through its direct interaction with caspases via its BIR domains, which contain a conserved sequence of cysteines and histidine that coordinate an atom of zinc and are primarily involved in mediating protein–protein interactions.38 BIR2 and its N-terminal linker region inhibit caspase-3 and caspase-7, whereas BIR3 inhibits caspase-9.39 The BIR regions of XIAP can also interact with noncaspase proteins, such as RIP2 and TAB1.37 However, BIR regions are also involved in transforming growth factor-β receptor and Notch signaling, as well as pathways that participate in c-JunN-terminal kinase (JNK) and NF-κB activation, although the functional implications of these interactions remain unclear.40,41 Inhibition of caspases by XIAP can be relieved by second mitochondria-derived activator of caspases (Smac), Omi/HtrA2 and apoptosis-related protein in the TGF-β signaling pathway (ARTS), which are released by the mitochondria after proapoptotic stimuli. The RING domain possesses E3 ubiquitin ligase function, although knowledge regarding the specific functions of XIAP's RING domain is limited.42 The pathophysiology of HLH in XIAP-deficient patients is not fully understood and currently seems to be consistent with the paradigm of HLH resulting from defects in the cytotoxic pathway.43 The SH2D1A and XIAP genes are located close together on the same chromosome, and they may interact with each other.

Figure 5.

Protein interactions of XIAP. XIAP is a member of IAPs, and plays a role in cell death signaling pathways. XIAP protein contains three baculovirus IAP repeats (BIR), which can bind directly to caspases-3, -7 and -9, thereby inhibiting their proteolytic activity. However, the correlation of impaired XIAP function and clinical phenotypes has not been well resolved.

EBV-associated HLH and SAP and XIAP deficiency

EBV infects the majority of the adult population worldwide and persists in B cells throughout the lifetime in normal individuals, usually without causing disease. After the interaction of the viral surface glycoproteins with the CD21 receptor, EBV entry into B cells is mediated by HLA class II and other co-receptors.44 The mechanism of T cell infection by EBV in HLH remains unclear, but one hypothesis is that, in certain situations, CD8+ T cells express CD21, which can mediate EBV infection. Although T cells do not express the glycoprotein, they contain mRNA for CD21.44,45 EBV-specific CTL cells are required to regulate infected cells, so that memory cells can be produced.46 HLH patients display strongly uncontrolled expansion of antigen-specific effector T cells, which secrete high levels of IFN-γ, further activating macrophages.43 In EBV-HLH, it has been shown that the virus predominantly infects CD8+ T lymphocytes (occasionally CD4+ or both)47 (Fig. 6). It is worth mentioning that EBV infects B cells, but EBV infection into B cells may be delayed in patients with EBV-HLH.47

Figure 6.

Pathophysiology of hemophagocytic lymphohistiocytosis (HLH). Patients with HLH display uncontrolled strong expansion of antigen-specific effector T cells, which secrete high levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ, further activating macrophages. Then activated lymphocytes and macrophages infiltrate various organs, resulting in massive tissue necrosis, organ failure and hemophagocytosis.

It has been reported that the immune responses to other viruses are apparently normal in SAP-deficient patients. This finding suggests that SAP and SLAM receptors are preferentially involved in anti-EBV immune responses.22 While EBV predominantly infects CD8+ T lymphocytes in Japanese patients with HLH, it mostly infects CD19+B cells in XLP patients.48 This information could provide a method for distinguishing XLP patients from sporadic EBV-HLH patients. The persistence of EBV-infected B cells could serve as a stimulus for continued T cell activation and Th1-type cytokine production, which would lead to the secondary activation of macrophages in SAP deficiency.22

The excess of lymphocyte apoptosis in XIAP-deficient patients might account for the abnormal immune response to EBV.36 It is difficult to understand how a defect in an XIAP mainly involved in anti-apoptotic pathways leads to lymphoproliferative disorders, such as HLH. Xiap-deficient mice have been reported with no obvious phenotype, and these mice had not been triggered with a virus.49 To understand this phenotype more clearly, more studies should be carried out in the future. Recently, a report suggested that IL-2-inducible T cell kinase (ITK) deficiency causes a syndrome that leads to a fatally inadequate immune response to EBV,50 which prompted us to think of more candidate genes for XLP-like phenotype.

Invariant NKT (iNKT) cells coexpressing T-cell receptor (TCR) Vα24 and Vβ11 chains in humans recognize a conserved family of glycolipids and α-galactosylceramide. These iNK T cells are associated with autoimmune disease, tumor surveillance and infections. SAP-deficient humans and mice show deficient iNKT cells. Patients with XIAP deficiency also show a low number of iNKT cells. Although Xiap-deficient mice show a normal number of iNKT cells, deficient expression of iNKT cells might be associated with anti-EBV immune response in the common pathway in SAP and XIAP deficiency. In addition, patients with ITK deficiency, who exhibit susceptibility to EBV infection, also show iNKT cell deficiency. Although there are very low numbers of iNKT cells in human circulation, the immune response of iNKT cells to EBV infection should be clarified in the future.

Clinical features of XLP patients

The most common clinical features of HLH are fever, hepatosplenomegaly and cytopenias. Other findings include hypertriglyceridemia, coagulopathy with hypofibrinogenemia, liver dysfunction, elevated levels of ferritin and serum transaminases, and neurological symptoms. In addition, lymphadenopathy, skin rash, jaundice, and edema have also been reported.3 EBV is one of the most frequent pathogens found in HLH patients, and approximately half of all infection-associated HLH cases involve EBV.51 Most cases of EBV-HLH are sporadic, and a few cases may exhibit the first presentation of XLP.52 Despite increasing insights into this disease, we believe that HLH remains a syndrome defined and diagnosed by a unique pattern of clinical findings according to the HLH2004 guidelines (Table 1).3

Table 1.  Diagnostic guidelines for HLH
The diagnosis of HLH is established by fulfilling 1 or 2 of the following criteria:
  • Adapted from Henter et al.3 HLH, hemophagocytic lymphohistiocytosis.

1. A molecular diagnosis consistent with HLH (e.g. PRF mutations, SH2D1A mutations)
2. Having 5 out of 8 of the following:
 a. Fever
 b. Splenomegaly
 c. Cytopenia (affecting > 2 cell lineages)
  Hemoglobin (<9g/dL or <10g/dL for infants < 4 weeks of age)
  Platelets < 100,000/µL
  Neutrophils < 1,000/µL
 d. Hypertriglyceridemia (>265 mg/dL) and/or hypofibrinogenemia (<150 mg/dL)
 e. Hemophagocytosis in the bone marrow, spleen or lymph nodes without evidence of malignancy
 f. Low or absent NK cell cytotoxicity
 g. Hyperferritinemia (>500µg/L)
 h. Elevated soluble CD25 (e.g. soluble IL-2 receptor >2,400 U/mL)

Since SAP deficiency was first identified in XLP patients in 1998, its clinical features have been widely observed. In SAP deficiency, HLH, malignant B-cell lymphoma and progressive dysgammaglobulinemia occur quite freqently.53 Other rare clinical findings, including vasculitis, aplastic anemia and pulmonary lymphoid granulomatosis, have been found in SAP deficiency.54,55 XIAP deficiency has been recently recognized, and reports on its clinical features are limited. Malignant B-cell lymphoma has not been reported in XIAP deficiency until now, but other major phenotypes have already been reported. Although the clinical features in these two types of XLP patients show a small difference, it should be stressed that the most common feature in both types is HLH, especially EBV-HLH.

The incidences of HLH in XLP are 39.6–55% in SAP deficiency and 67–90% in XIAP deficiency, and EBV-HLH in HLH with SAP deficiency is 78.8–92%, while that with XIAP deficiency is 30–84% (Table 2).16,56–59 Importantly, concerning the prognosis of HLH seen in XLP, it has been reported that more frequent neurological involvements and fatal outcomes were observed in SAP deficiency compared with those in XIAP deficiency.56 While SAP-deficient CD8+ T and NK cells exhibited defective cytotoxicity responses in mice and humans, NK-cell and T-cell cytotoxic responses appear to be somewhat preserved in XIAP-deficient patients.60 This may account for the lower severity of the HLH in XIAP deficiency. HLH relapses seem to be more common in XIAP deficiency than in SAP deficiency in survivors.16,56,61 The presence of a family history is also very important in XLP. Recently, XIAP deficiency has been defined as more readily fitting the definition of familial HLH.57 The manifestations in HLH can be found even in XLP patients who have never encountered EBV.56 We propose that XLP should be suspected in HLH patients having at least three conditions as above: one is familial HLH with EBV infection; the other two are sporadic HLH with or without EBV infection. Patients with SAP deficiency show more clinically severe HLH with elevated levels of serum IgA and IgM. Clinical features of both types of XLP patients are summarized in Table 2.

Table 2.  Comparison of clinical features in SAP- and XIAP-deficient patients
 SAP deficiency (XLP-1)XIAP deficiency (XLP-2)
Booth, et al.59Pachlopnik-Schmid, et al.56Kanegane, et al.58Marsh, et al.57Pachlopnik-Schmid, et al.56Yang, et al.16
  1. EBV, Epstein–Barr virus; HLH, hemophagocytic lymphohistiocytosis.

Number of patients91333310309
Family history16.5%69.7%52%40%83.3%66.7%
Recurrent HLHunknown29%Unknown60%61%83%
EBV-associated HLH78.8%92%89%30%84%67%

Patients with XLP often develop more than one phenotype over time, but not HLH. It is also noted that other manifestations can be present, even in some XLP patients who have never encountered EBV.56 Splenomegaly and dysgammaglobulinemia are similarly seen in both SAP deficiency and XIAP deficiency.16,56,57 In addition, recurrent splenomegaly occurs more frequently in XIAP deficiency than in SAP deficiency, and it may occur even in the absence of systemic HLH.56 XIAP-deficient patients recover from hypogammaglobulinemia. In contrast, hypogammaglobulinemia in SAP-deficient patients persist, resulting in recurrent infections leading to a poor prognosis.56

Although SAP and XIAP deficiencies share some clinical features, they display specific manifestations. Lymphoproliferative disorders, such as lymphomas, have thus far only been described in SAP deficiency. Some studies have demonstrated that XIAP protein is a potent target for the treatment of cancer based on the anti-apoptotic function of XIAP.62 Therefore, it seems that the absence of XIAP protects patients from cancer, explaining why XIAP-deficient patients do not develop lymphoma. Some XIAP-deficient patients develop chronic colitis, which is a very rare manifestation in SAP-deficient patients. In this regard, XIAP has been shown to be involved in the activation of NOD2, the gene of which is accepted to be key to susceptibility to Crohn's disease.63

Treatment and prognosis of XLP

The initiation of immune chemotherapy according to the HLH-94 protocol has dramatically improved the outcome of children with HLH.64 In most cases, disease activity is stabilized with dexamethasone, etoposide/VP16 and cyclosporine A, being the backbone of the HLH-2004 protocol. In EBV-HLH, early immunochemotherapy results in high response rates, so patients with reactive HLH associated with an infectious organism, except leishmaniasis, should start specific therapy because pathogen-specific therapy cannot stabilize the disease activity by itself.46 It has been reported that only 60–70% of patients achieve recovery with treatment of the underlying infection alone.65

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for XLP-1 patients. A recent report has suggested that the mortality in untransplanted patients was lower than expected.59 It fact, it was found that all the untransplanted patients with XLP-1 in Japan died (Fig. 7).58 The outcomes of myeloablative conditioning and reduced-intensity conditioning (RIC) seem to be similar, and RIC is recommended to avoid regimen-related toxicity. Patients with XIAP deficiency may also be cured by allogeneic HSCT, although the outcome of HSCT in XIAP deficiency has not been reported. Patients with SAP deficiency have been successfully treated with B-cell-directed therapy using an anti-CD20 mAb (rituximab).66 The main target cells of EBV are B cells in SAP and XIAP deficiency, and thus B-cell target therapy could be a viable therapeutic option in the initial stage of EBV-HLH in both XLP-1 and XLP-2 patients.48

Figure 7.

Outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in Japanese patients with XLP-1. Survival curve of the transplanted and untransplanted XLP-1 patients in Japan. The outcome of the transplanted patients is significantly better than that of the untransplanted patients. inline image, Transplanted; inline image, untransplanted.


We have described the recent advances in understanding of the pathogenesis and clinical features of SAP and XIAP deficiency. It may still be difficult to distinguish XLP from HLH. However, EBV-target cells in XLP and sporadic EBV-HLH are different, and this information is also useful regarding the choice of treatment. EBV-HLH associated with SAP deficiency may be more severe and fatal with increased levels of serum IgA and IgM, whereas HLH sometimes occurs frequently in XIAP deficiency. When patients presenting HLH are presumed to have XLP, we recommend flow cytometric analysis or Western blot of SAP and XIAP expression followed by gene analysis. Allogeneic HSCT with RIC regime is recommended in patients with SAP deficiency. The pathogenesis of XLP, especially XIAP deficiency, is not fully understood, and extensive studies are required in the future.


This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and grants from the Ministry of Health, Labour, and Welfare of Japan. We thank Dr Sylvain Latour for critical discussion, and are grateful to many doctors for providing us with samples and patient data.