Disorders associated with GOF mutations of NLRC4 are categorized into four groups: AIFEC, MAS, NOMID, and FCAS4. AIFEC is a chronic inflammatory disease presented with episodes of extreme acuity. Severe diarrhea which can start from the neonatal period is characteristic of AIFEC. MAS-like attacks can lead to intensive care unit (ICU) admission and death. Apart from the attacks, AIFEC is a chronic inflammatory disease. The patients display moderately elevated acute phase reactants and highly elevated serum IL-18 concentrations between flares and present various signs and symptoms such as those of the skin, hepatic, and nervous system [8, 12, 13]. All of our patients seem to fall into this category. The first patient was primarily diagnosed with MISN; however, the occurrence of the second attack, the history of recurrent fever in the mother and grandmother, and ultimately the WES results are very much compatible with NLRC4 inflammasomopathy and AIFEC. In light of the new findings, the first attack is now understood to be a MAS-like attack. Recent studies have found new associations with NLRC4 mutation, such as monogenic lupus [14].

A phenotype/genotype correlation for NLRC4 mutations has been suggested by Wang et al. It has been implied that the patients with mutations in the WHD domain presented with mild inflammatory symptoms of FCAS4, while those with mutations in the NBD and HD1 domains presented severe inflammation, AIFEC, and MAS [5]. On the other hand, Bardet et al. argue that NLRC4 GOF shows a poor phenotype/genotype correlation and mutations in NBD have been found in both severe and mild phenotypes [11]. Regarding our experience, our third patient had a mutation placed between CARD and NBD domain (exon 4), which has been reported for the first time. Our second patient had a mutation in HD1 domain, which is compatible with the hypothesis that mutations in the HD1 are associated with AIFEC phenotype. Remarkably, our first patient’s mutation is in the CARD domain, and to our knowledge, this is the first mutation found in the CARD domain of NLRC4 associated with autoinflammation (Fig. 2).

NLRC4 protein showing our reported mutations

Interestingly enough, somatic NLRC4 mosaicism can also be associated with autoinflammatory manifestations. Lonescu et al. introduced an adult female patient with recurrent episodes of fever, myalgia, arthralgia, diffuse abdominal pain, diarrhea, and systemic inflammation starting from the age of 47 years and responding to anti-IL-1 treatment. Postzygotic p.Ser171Phe NLRC4 variant was found in unfractionated blood [15]. Moreover, Wang et al. also reported a 69-year-old woman with recurrent rash and fever, and a skin biopsy in favor of vasculitis. They identified a somatic mutation in NLRC4 (p.His443Gln) with the highest mosaicism ratio in the patient’s monocytes. The patient responded well to anti-IL-6 treatment [16]. Both of these patients had elevated levels of serum IL-18. Kawasaki et al. introduced the first case of NLRC4 mutation with a presentation of NOMID. The patient was a male infant with a recurrent erythematous rash on the oral circumference, palm, and foot, growth restriction, sensorineural deafness, and brain atrophy. They found a heterozygous NLRC4 mutation, p. Thr177Ala, as a specific mutation in diseased induced pluripotent stem cells (iPSC) clones [10].

The variety in manifestations and severity spectrum mandates a variety of treatments. Most of the FCAS4 patients have been well controlled with non-steroid anti-inflammatory drugs (NSAIDs). Steroids, colchicine, and rarely anakinra have also been used in these patients depending on the severity of manifestations [5, 17]. Attacks of AIFEC and MAS have been treated with immunosuppressives and biologics, sometimes ineffectively and leading to death [8, 13, 18]. Our first and second patients had life-threatening flares of disease. We managed the first patient’s attack with methylprednisolone, and the second patient with prednisolone and cyclophosphamide due to CNS vasculitis. Afterward, we controlled their disease with adalimumab, and fortunately, they have not experienced any attacks since then (for 11 months and two years, respectively). Our third patient has not experienced a MAS-like attack, but he had been undergoing chronic diarrhea with high inflammatory markers. These manifestations have been controlled with prednisolone and azathioprine.

NLRC4 mutation is associated with increased levels of IL-1B and IL-18. The importance of IL-18 in the diagnosis of the disease has been well elucidated [7, 8]. Therefore, addressing IL-18 in treatment would be beneficial. Dramatic efficacy was found using recombinant IL-18 binding protein (BP) in one critically ill neonatal AIFEC patient whose disease was refractory to corticosteroids, cyclosporine, IL-1 inhibition, TNF-inhibition and integrin-inhibition [7]. This experience raises hope for curing severe cases of NLRC4 mutation inflammasomopathy. A clinical trial is being conducted on the efficacy, safety, and tolerability of MAS825, a bispecific IL-1β/IL-18 monoclonal antibody, in patients with monogenic IL-18-driven autoinflammatory diseases, including NLRC4-GOF [19]. However, serum analysis of IL-18 and treatment with the recombinant IL-18BP are not practical in resource-limited settings. This is also a limitation of our study. Recently, Bracaglia et al. reported the favorable outcome of a fecal microbiota transplant (FMT) in a patient with NLRC4 mutation with recurrent MAS attacks and persistent diarrhea. The patient had fever, enterocolitis, and recurrent HLH from the age of one month which were only partially controlled with immunomodulatory treatments, including high-dose glucocorticoids, cyclosporine, and high-dose anakinra. He developed multi-drug-resistant sepsis and recurrent intestinal obstructions. Finally, FMT was tried. With Anakinra and prednisolone being continued, the patient’s symptoms have not recurred in the past two years. IL-18 levels have remained high but decreased from more than 500,000 to 50,000 pg/ml [20].