TNFAIP3 mutation causing haploinsufficiency of A20 with a hemophagocytic lymphohistiocytosis phenotype: a report of two cases

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of multi-organ failure due to excessive immune activation. It can be presented at all ages but is most frequently reported in infants, occurring as a single or multiple recurring episodes. Fever, splenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, and hemophagocytosis constitute the criteria for diagnosis of HLH [1]. HLH can be sporadic or happen in the context of a genetic disorder. In both circumstances, infection is the most common trigger [2]. Some genes are found to be associated with familial HLH [3]. Moreover, diseases that alter the immunologic function, such as immunodeficiencies and autoinflammatory syndromes, can be potential causes of HLH [4].

A20 haploinsufficiency (HA20) is a newly introduced autosomal dominant autoinflammatory disorder [5]. TNF alpha-induced protein 3 (TNFAIP3) encodes protein A20, an essential negative regulator of nuclear factor–κB (NF-κB)-mediated inflammation. A20 is an E3-ligase that also possesses deubiquitinase activity [6]. HA20, due to reduced protein levels, leads to increased activation in response to tumor necrosis factor α (TNF-α) and toll-like receptor (TLR) stimulation. Heterozygous loss-of-function mutations in A20 cause HA20 and Behcet’s-like disease in children [7].

HA20 resembles Behcet’s disease in some symptoms; however, some features in specific individuals lead to an initial diagnosis of atypical systemic lupus erythematosus with central nervous system vasculitis, anterior uveitis, colon ulcers, and autoimmune lymphoproliferative syndrome (ALPS) phenotype [8, 9]. Features of the disease include recurrent oral, genital, and/or gastrointestinal (GI) ulcers, episodic fever, musculoskeletal symptoms, cutaneous lesions, and recurrent infections. Patients with TNFAIP3 mutation can develop autoantibodies. Fluctuating levels of low-titer autoantibodies, including antinuclear antibodies, anti-double-stranded DNA (dsDNA), anti-Sm/ribonucleoprotein (RNP), anticardiolipin, and lupus anticoagulant, were found in the largest reported cohort [10‐12]. Acute phase reactants tend to elevate with disease flares, but their levels are normal between flares, which is a characteristic of autoinflammatory syndromes [10].

The diagnosis of HA20 based on clinical symptoms is challenging due to various clinical manifestations in different patients. Therefore, the final diagnosis should be confirmed by genetic study and finding of TNFAIP3 mutation [10, 13, 14].

The scarcity of reported and published cases of HA20 and unknown presentations of the disease necessitate reporting the diagnosed cases of HA20 and sharing knowledge and experience in this area. Herein, we present a case of genetically confirmed HA20 with the primary presentation of HLH.

In this article, we have reported two cases of HA20 due to TNFAIP3 mutation that primarily manifested in early infancy with HLH. We explained the symptoms, laboratory evaluations, and treatments of these patients.

HLH is classified into familial (primary) and acquired (secondary) types [16]. Familial erythrophagocytic lymphohistiocytosis (FEL or FHL) is an autosomal recessive disease that in some patients is associated with decreased apoptosis triggering [17]. There are five subtypes of FHL: FHL type 1 is unknown in gene and its chromosomal location; FHL type 2 includes perforin gene (perforin 1 [PRF1], located at 10q21-22) mutation; FHL type 3 has the Munc 13-4 gene (unc-13 homolog D (UNC13D), located at 17q25) mutation; FHL type 4 includes mutations in STX11 (located at 6q24); and FHL type 5 has Munc 18-2 gene (syntaxin binding protein 2 [STXBP2], located at 19p13) mutation. Despite the name of FHL, the family history is often negative since the disease has recessive inheritance [18].

Many conditions can lead to the clinical manifestation of acquired HLH, including malignancies (leukemia, lymphoma, and other solid tumors), infections (viral, bacterial, or parasitic), and rheumatoid disorders [1]. Viral infections, especially Epstein-Barr virus (EBV), may trigger primary as well as secondary HLH [19]. Macrophage activation syndrome (MAS), which is defined as HLH associated with rheumatologic diseases, can be categorized as secondary HLH. Both of our patients met the European League Against Rheumatism (EULAR) Executive Committee and the American College of Rheumatology (ACR) criteria of MAS [20]. It should be noted that our first patient had anemia, yet the level of hemoglobin was 9.2 g/dl. The threshold for anemia in HLH criteria 2004 is 9 g/dl and it might be suggested that all five HLH criteria were not met in the first patient. Therefore, we suggest that the condition here be named as HLH/MAS.

High levels of IFN-γ are sufficient to induce many of the disease-associated hallmarks associated with autoinflammatory syndromes, including some of those associated with HLH. Some cytokines contribute to the pathogenesis of MAS, such as IFN-γ-producing CD8+ lymphocytes and TNF-α- and IL-6-producing macrophages [18, 21].

TNFAIP3 (OMIMI: 191163) on chromosome 6q23 encodes the A20 protein, which is a negative regulator of the TNF-nuclear factor-kB signaling pathway. Heterozygous mutation in TNFAIP3 leads to the autoinflammatory syndrome: familial Behcet’s-like disease [22]. This mutation has a vertical pattern in some families, consistent with autosomal dominant inheritance with a 50% probability of transfer from parents to their children.

In vitro experiments, in which mutant forms of the protein were overexpressed, did not lead to suppression of NF-κB activation. Cell types, which are affected by A20 deficiency in humans, include non-immune cells, such as skin fibroblasts and PBMC, monocyte-derived macrophages, and T cells, the latter of which can be induced to express excess pro-inflammatory cytokines, such as interleukin 9 (IL-9) and IL-17. Additionally, spontaneous NLR family pyrin domain containing 3 (NLRP3) inflammasome activity has been observed in cells from HA20 patients, expressing increased pro-IL-1β. As demonstrated by tumor necrosis factor receptor (TNFR) and IL-1R signaling blockade, the activity of these pathways is essential to maintain active disease [5].

Recurrent oral and genital aphthosis, GI ulcers, skin lesions, intermittent fever, ocular lesions, musculoskeletal involvement, vasculitis, neurologic symptoms, and recurrent infections are described in HA20 patients [23]. Although HA20 was first described by Zhou et al. as an autoinflammatory disorder, studies have found autoimmune-like features for the disease [22, 24]. Moreover, each patient seems to have a unique scenario. The three patients that Zhang et al. described in 2021, for instance, have their spectacular stories [25]. One patient presented with intermittent fever, diarrhea which was at times bloody, and monoarthritis which progressed into polyarthritis. The second patient manifested thoracic kyphosis, recurrent oral ulcers, and autoimmune hemolytic anemia. The third patient was referred for intermittent fever and perianal abscesses. The key to the diagnosis of many of the diagnosed cases of HA20 has been a genetic analysis in cases with obscure diagnoses. That is why reporting the cases of HA20 is mandatory for providing insights into diagnosis by summing up the presentations and laboratory evaluations.

It has been reported that several autoimmune and inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, Crohn’s disease, Behcet’s disease, and type 1 diabetes, are associated with TNFAIP3 polymorphisms [5, 16, 26‐28]. Our patients did not have any symptoms of Behcet’s-like or other autoimmune disorders in the first presentation and subsequent follow-ups, but their primary presentation was systemic inflammation. In addition, we did not find any positive autoantibodies, such as antinuclear antibodies, anti-dsDNA, anti-Sm/RNP, anticardiolipin, and lupus anticoagulant in our patients.

One of the unexpected presentations of HA20 is HLH. In case series of Li et al. [29], where four HA20 patients with various phenotypes are introduced, one patient is presented with macrophage activation syndrome (MAS), which is a subset of HLH. The patient had a fever, hyperferritinemia, hypertriglyceridemia, and hypofibrinogenemia. She had the c.259C > T (p.R87X) mutation in the TNFAIP3 gene (RS: NM_006290). This was the first report of HA20 patients presenting with MAS. Our patients also fulfilled the criteria for HLH. Since there are many etiologies for this catastrophic phenomenon, establishing the diagnosis at the time of presentation was impossible without genetic analysis.

Takagi et al. reported a case of a 7-month-old Japanese boy who developed a fever, skin rash, and hepatosplenomegaly with pancytopenia. The patient’s double negative T (DNT) cells were 5.1% of TCRαβ-positive cells. Immunological data and the patient’s phenotype supported the diagnosis of ALPS. In WES analysis, mutations associated with ALPS-1-like phenotypes were not identified, and de novo mutation of TNFAIP3 was detected [9]. Likewise this case, hepatosplenomegaly and pancytopenia in our patients were the primary presentations. Schulert et al. have also found a possible role for TNFAIP3 in immune dysregulation associated with HLH/MAS [30]. The patients introduced in literature with TNFAIP3 mutation and presentation of HLH/MAS are presented in Table 3.

A20 has a crucial role in downregulating NF-κB activity. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β1 (IL-1β1) impose their effects through the activity of NF-κB transcription factors. Failure to suppress NF-κB transcriptional activity results in chronic inflammation and cell death [6, 31]. Moreover, A20 is a negative regulator of NLRP3 inflammasome; deficiency of A20 triggers caspase-8-dependent enhancement of NLRP3 inflammasome activation [32‐34]. Therefore, haploinsufficiency of A20, through the mechanisms of NF-κB overactivation and NLRP3 inflammasome, results in the overproduction of proinflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α [14]. This state of cytokine storm is what we observe in the setting of HLH [35].

There is currently no standard treatment protocol for HA20. Nearly half of the patients were reported to respond well to colchicine, especially when used in combination with immunosuppressive or biological agents. Earlier initiation of biologic agents can possibly reduce the need for long-term glucocorticoid treatments [11, 14]. Therefore, the subsequent treatment plan for patients with such a specific mutation, after the initial control of inflammation, will be biological drugs, primarily anti-TNF and especially etanercept. Hematopoietic stem cell transplantation has been reported effective in patients with severe disease and cerebral vasculitis [10, 11]. Our first patient was receiving etoposide and oral corticosteroid before we got access to his genetic test, and then we prescribed etanercept as an anti-TNF drug. Unfortunately, his condition deteriorated before he had the time to begin the new therapy, and he passed away. In the case of our second patient, however, the genetic results were fortunately available just in time, and he received etanercept, which seems to have ceased the progression of the disease. We chose etanercept because it was more accessible in comparison with other biologics and since it was recommended in the literature, and continued it due to the favorable response in the patient.

The primary goal of treating secondary HLH is triggering the cause such as infection, malignancy, autoimmune disease, or autoinflammatory disorder. Moreover, it might be necessary to treat hyperinflammation apart from the etiology. Dexamethasone, etoposide, and cytokine inhibitors have been frequently used for that matter [15, 36]. The recombinant IL-1 receptor antagonist, anakinra [37], the anti-IL-6R antibody, tocilizumab [38], and the JAK inhibitor, ruxolitinib [36], are some of the biologic drugs that have been efficient in treating HLH/MAS. Considering the fact that TNFAIP3 mutation can be associated with HLH, and that HA20 can be presented with the symptoms of HLH/MAS, the biologic drugs seem to be the most reasonable choice for treatment of HA20 when HA20 is a possible trigger of HLH or is in differential diagnosis.

This report is a hypothesis for developing of the HLH phenotype in the presence of TNFAIP3 mutation. Our results provide a new perspective on the role of TNFAIP3 mutation in HLH phenotypes, but more extensive studies are required to confirm these preliminary results.