Identification of a novel NFKB2 mutation in a patient presenting with autoimmune cytopenia and generalized granulomatous lymphadenopathy

Common Variable Immunodeficiency (CVID) is a heterogenous clinical diagnosis characterized by hypogammaglobinemia and poor antibody response to specific antigens, leading to an increased susceptibility to infections. It is the most common symptomatic primary immunodeficiency, with an estimated prevalence of 1:10 000 to 1:100 000 depending on race and geographical region. While it is a clinical diagnosis, monogenic causes of CVID have been identified in up to 10% of CVID cases (Szczawinska-Poplonyk et al. 2021; Bogaert et al. 2016). One such monogenic cause is NFKB2 pathogenic variants.

Nuclear factor kappa B (NF-κB) proteins are transcription factors that reside in the cytoplasm until activated, upon which they migrate to the nucleus and bind to their gene targets. The NF-κB signal transduction pathway has important roles in inflammation and host immune responses (Hayden and Ghosh 2011). While NF-κB1 has broad immune functions, NF-κB2, a key part of the non-canonical NF-κB pathway, is more specialized to B cell development and maturation. The non-canonical pathway also has functions in secondary lymphoid organogenesis, thymic epithelial differentiation, and bone metabolism (Sun 2012; Hayden and Ghosh 2011).

NF-κB2 (also known as p100/p52) is encoded by the NFKB2 gene. Upon activation of its pathway by signals from cluster of differentiation 40 ligand (CD40L), lymphotoxin β receptor (LTβR), and B cell-activating factor receptor (BAFFR), NF-κB-inducing kinase (NIK) is activated, phosphorylating the homodimer IKKα/IKKα, which transfers the phosphate group to the C-terminal residues of p100. Subsequently, p100 is converted by proteasomal processing of its C-terminal into p52, the mature form of the NF-κB2 transcription factor. The p52 subunit can dimerize with other members of the NF-κB family, however, preferentially makes RelB/p52 dimers which are translocated to the nucleus where they regulate target gene expression (Pires et al. 2018; Sun 2012).

Immunodeficiency due to a heterozygous mutation in NF-κB2 was first described in 2013 as an early onset CVID associated with central adrenal insufficiency (adrenocorticotropin “ACTH” deficiency) (Chen et al. 2013), the so-called DAVID syndrome (deficit in anterior pituitary function and variable immune deficiency) (Quentien et al. 2012). Since then, other cases have been reported with and without anterior pituitary defects and with varying immune effects including CVID, autoimmunity, and combined immunodeficiency.

Here, we report the case of a patient who presented with autoimmune cytopenia and generalized granulomatous lymphadenopathy. Whole exome sequencing subsequently identified a novel causative missense variant in the NFKB2 gene.

Our patient is currently a 19-year-old male who first presented at the age of 2 with thrombocytopenia (Table 1), splenomegaly, and generalized lymphadenopathy. He was initially given the diagnosis of immune thrombocytopenia (ITP) and was treated with intravenous immunoglobulin replacement therapy (IVIG). Over the following year, he required 4 doses of IVIG and was thus diagnosed with chronic ITP. At the age of 3, he was referred to our Immunology clinic for assessment due to his persistent ITP and generalized lymphadenopathy. Other than several viral upper respiratory tract infections, he had no history of recurrent or severe infections. Immune evaluation revealed normal total immunoglobulin levels (Table 2), but given his recent IVIG, further humoral assessment was difficult. CD4+, CD8+ and CD19+ cell counts at the time were normal and a decrease in his total CD16+56+ cells was noted (Table 3). His lymphocyte proliferation responses to the mitogen phytohemagglutinin (PHA) were within normal range, although antigen recall responses (day 6) were low (<20 stimulation index) against Candida and Herpes zoster (Table 4). Screening for ALPS with double negative T cells was negative. He also underwent an assessment by Rheumatology at the same time and their work up was not suggestive of a rheumatologic disease.

In the following months, he continued to have recurring thrombocytopenia that was treated with anti-D and steroids. A reassessment of his humoral immunity was done 6 months post-IVIG and was normal.

At the age of 5, he developed autoimmune hemolytic anemia. He was known to be direct coomb’s positive since his initial presentation and was treated with oral steroids at the time. He continued to have recurrent thrombocytopenia and developed neutropenia which was thought to be autoimmune as well. A bone marrow biopsy at the time was normal and reactive.

One year later, at the age of 6, he had several episodes of ITP in a short duration, all treated with IVIG. Given the severity and persistence of his ITP, it was decided to start him on mycophenolate mofetil (MMF). A repeat bone marrow biopsy prior to the initiation of MMF was normal. After starting MMF, his cytopenia resolved and he had no recurrences of thrombocytopenia or hemolytic anemia.

After the initiation of MMF, he had a progressive decrease in his immunoglobulins and loss of specific antibodies to vaccine preventable infections (Table 2). He did not have any recurrent sinopulmonary infections. It was unclear at the time whether the hypogammaglobinemia was due to MMF treatment, GI losses, his underlying disease, or a combination of the above. Due to the persistent and significant decrease in his antibody levels he was started on regular IVIG at the age of 14, initially every 4 weeks then every 3 weeks due to persistently low IgG. He was transitioned to subcutaneous immunoglobulins at the age of 18.

Over the years he has developed multiple other complications summarized below:

Here we describe a novel pathogenic variant in NFKB2 in a patient who presented with persistent autoimmune thrombocytopenia, hemolytic anemia, and neutropenia, as well as granulomatous generalized lymphadenopathy, and splenomegaly. The patient also developed hypogammaglobinemia by the age of 6 years, although he was on immunosuppressive therapy at the time with MMF. Other clinical manifestations as noted above included protein losing enteropathy, delayed puberty, short stature, bilateral nephrocalcinosis, and asymptomatic interstitial lung infiltrates.

The variant we report at c.1700C>T is a missense variant targeting the ankyrin rich domain of p100, the precursor protein that is encoded by the NFKB2 gene (see Figure 1). It changes the amino acid sequence from alanine to valine at position 567. The variant is predicted by CADD and SIFT to be damaging. The cytosine residue at the c.1700 locus is also highly conserved in all vertebrate species. This variant has an allele frequency of 2.87 × 10⁻⁵ in gnomAD, and has been reported in ClinVar once as a variant of unknown significance.

In contrast to most patients with NFKB2 mutations, our patient presented initially with autoimmune cytopenia and generalized lymphadenopathy. While this presentation has been reported in other patients, most patients with NFKB2 mutations initially present with recurrent infections and variable immunodeficiency (Klemann et al. 2019). Four other patients with pathogenic variants in the same domain as our patient were found in the literature but their presentations were not similar (Klemann et al. 2019; Kuehn et al. 2017).

Although our patient’s hypogammaglobinemia did not develop until after starting MMF and the onset of his gastrointestinal complaints, he did show early signs of antibody deficiency with early loss of antibodies to pneumococcus post-vaccination and low isohemagglutinin titers prior to MMF initiation. The immunosuppressive therapy may have accelerated the progression of his antibody deficiency but is unlikely to be the sole cause of it.

Lymphadenopathy has been reported in multiple patients (Klemann et al. 2019) and granulomatous disease has been reported in some patients as well (Kuehn et al. 2017; Brue et al. 2014). Enteropathy and lymphocytic lung infiltrations are also known possible manifestations of this disease. (Klemann et al. 2019).

Many patients with NFKB2 mutations have shown T cell mediated autoimmunity in the form of alopecia and (or) trachyonychia which did not manifest in our patient. (Pérez Cavazos et al. 2021; Bienias et al. 2021; Klemann et al. 2019). Our patient also did not show any ACTH or other anterior pituitary hormonal deficiency which has been reported in other patients (Klemann et al. 2019) despite his delayed puberty which was likely constitutional.

Some patients have been reported with severe viral or opportunistic infections, lymphopenia, and a combined immune defect (Abraham et al. 2021; Pérez Cavazos et al. 2021; Bienias et al. 2021; Klemann et al. 2019). We also did not observe this in our patient, however, he did have a mild decrease in his T cell proliferation to mitogens compared to control on several occasions.

In summary, we present a novel pathogenic variant in NFKB2 in a patient who presented primarily with autoimmune cytopenia and generalized granulomatous lymphadenopathy.