Incomplete penetrance of NOD2 C483W mutation underlining Blau syndrome

As a prototypic autoinflammatory granulomatous disease, dermatitis, polyarthritis, and uveitis are the classical triad of BS/EOS with rash being the first feature [1, 2, 15]. Usually painless and non-pruritic, non-caseating granulomas is the typical findings seen in skin biopsy of BS/EOS and our index case [5]. This is different from the caseating granulomas seen in CGD, ANCA-associated vasculitis and granuloma forming infections, such as tuberculosis, leprosy, atypical mycobacteria, or fungal infection [16]. Moreover, Crohn’s disease (CD) and sarcoidosis are also inflammatory syndromes characterized with non-caseating granuloma involving many organ systems [2, 5, 8]. In contrast to the simple granulomas seen in the biopsy specimen from patients with CD, the histopathogenical features of BS usually demonstrated polycyclic granulomas with large lymphocytic coronas and extensive emperipolesis of lymphocytes within multinucleated giant cells, accompany fibrinoid necrosis and fibrosis [17]. Classical sarcoidosis mostly affect young adults 30–50 years of age [8]. BS/EOS, on the other hands, are usually found in children harboring NOD2 mutation before the age of 5 with dominate extra-thoracic manifestations and less lymph node involvement [5].

Arthritis is a dominate feature seen in the proband and the most common manifestation presenting in over 90% of all BS/EOS patients [5, 18]. Although camptodactyly, the digital flexion deformity seen in half of BS/EOS cases, was not observed in the proband, extensive polyarthritis with “boggy” appearance involving wrists, knees, ankles and proximal interphalangeal joints in symmetry is compatible with most reported case diagnosed with BS/EOS [15, 18]. Similar to that in polyarticular JIA and enthesitis related arthritis, excess joint swelling and tenosynovitis with massive joint effusion can be seen in both large and small joints in symmetry [19]. However, the absent or subtle raise of acute phase reactants, lack of joint pain and joint destruction, and relatively well preserved range of motion in the large joints are more commonly observed in patients with BS/EOS and our proband [15]. Non-caseating granulomas in the synovial specimen are also in favor of BS/EOS [17, 20].

Uveitis is usually the latest feature presented in BS/EOS. It can lead to visual loss and is the most concerned morbidity of the disease [21]. Because BS/EOS associated uveitis mostly develops after a median disease duration of 12.1 years, it may not be observed early in the disease course, such as our proband [18]. Uveitis has been reported to affect up to 75% of the patients suffering BS/EOS and is predominately bilateral. Compared to the uveitis of JIA which is almost always anterior, panuveitis with typical multifocal choroidal scars is the most observed feature in BS/EOS uveitis [20, 21]. Optic disc abnormalities, band keratopathy, cataract, glaucoma, retinal vasculitis, and macular edema are ocular complications which have also been reported [20, 21]. Fortunately, the use of biologics, particularly TNF-α targeting monoclonal antibodies, have been shown to mitigate ocular inflammation and prevent blindness to a certain degree [8, 21]. Despite appropriate treatment, however, visual prognosis in Blau syndrome remains guarded.

While BS/EOS patients without NOD2 mutations were reported, mutations in the NOD2 gene is perhaps the most apparent difference distinguishing BS/EOS from other granulomatous conditions [5]. Unlike systemic sarcoidosis, JIA, CGD, ANCA-associated vasculitis and granuloma forming infections, NOD2 mutation is associated with chronic inflammatory disorders such as BS/EOS and CD [2, 4]. Accounting for approximal half of all reported BS/EOS, heterozygous missense mutations of R334W and R334Q are the most common disease causing mutations [10, 15, 18]. Recently, Maekawa et al. analyzed the crystal structure of NOD2 protein and revealed that most of the BS/EOS associated gain-of-function NOD2 mutations located in the NOD/NACHT domain interface surrounding the magnesium and adenosine triphosphate binding sites or concentrated on the helical domain 1 (HD1) [4, 6]. According to the structural analysis and its proinflammation nature, the BS/EOS associated mutations are likely to interfere NOD2 inner domain interactions and promote conformational changes to its bioactive form [4]. On the other hands, CD associated mutations are widely distributed throughout the protein [4]. Three variants within leucine rich repeat domains of the NOD2 gene, R702T, G908R and L1007fsinsC were identified as susceptibility loci associated with CD [2]. Unlike BS/EOS, these mutations have been postulated to disrupt the formation of oligomer or binding of its ligands, which result in defective NFκB activation, reduced α-defensin synthesis and altered intestinal microbiome [2, 4]. The C483W mutation discovered in our proband and her father, is located on the HD1 within the NOD/NACHT domain, clustered with the BS/EOS associated variants. According to Maekawa’s work [4], the positioning and molecular changes of C483W may potentially destabilized the closed form of NOD2 and promote conformational change from the inactive to the active form, leading to constitutive activation of the protein.

Physiologically, NOD2 protein directly recognizes intracellular bacterial fragments containing the MDP motif. Ligand interaction frees intra-molecular autoinhibitory conformation, leading to NOD2 oligomerization. Through caspase recruitment domain (CARD)-CARD interactions, subsequent activation of the NFκB and mitogen-activated protein kinase pathways results in the up-regulated transcriptions of pro-inflammatory and host defense genes [4, 6, 12, 22]. Specifically, many studies demonstrated that BS/EOS associated gain-of-function NOD2 mutations result in NFκB autoactivation and subsequently lead to overexpression of cytokines involved in the auto-inflammatory process [4, 6, 10, 15, 23, 24]. Impaired NOD2 activation to MDP resulted in mitigated NFκB signaling and absence of spontaneous proinflammatory cytokine production have also been reported by others [12, 24, 25].

NFκB autoactivation in BS/EOS have been demonstrated in various ways. An in vitro NFκB luciferase reporter system with overexpression of mutant NOD2 in the HEK293T cells, as demonstrated in Fig. 4B, is perhaps the most commonly performed test to functionally examine the impact of BS/EOS NOD2 mutations in real world practice [6, 10, 23]. However, due to unavoidable limitations of the artificial system in cell line experiments, these assays may not reflex the true physiologically alteration of the immune responses in BS/EOS. For example, HEK293T cell line may not contain the complete endogenous regulatory elements for NOD2 and the transfection of mutated NOD2 plasmid may likely mimic homozygotic NOD2 mutation. To elucidate the mechanisms of autoinflammation in patients with BS/EOS and to precisely evaluate the immune phenotypes, Takada et al. established a BS specific induced pluripotent stem cell (iPSC) line from a BS patient and applied the CRISPR-Cas9 system to correct the disease-associated NOD2 mutation [24]. Utilizing both an NFκB luciferase reporter assay and staining for intracellular NFκB p65, autoactivation of NFκB pathway was clearly demonstrated in human samples with mutant NOD2 [24]. However, to establish a BS specific iPSC line for each NOD2 mutation is not clinically practical considering its technical difficulty. Known that NOD2 is mainly expressed in hematopoietic lineage cells, particularly in monocytic cells [26], we gated on the CD11b⁺ PBMCs to evaluate their NFκB activity in subjects harboring wide type and mutant NOD2. Similar to Takada’s finding examine intracellular staining of NFκB p65 in NOD2-mutated immortalized proliferating myeloid cell lines with confocal microscope [24], the percentage of intracellular p-NFκB staining in the MDP treated CD11b⁺ cells were generally increased regardless of underlining NOD2 genotypes. The shifting of p-NFκB staining without MDP stimulation, however, was elevated only in confirmed BS control and the symptomatic proband harboring C483W NOD2 mutations. Extensive testing on other known BS/EOS NOD2 mutations is warranted to uphold this novel assay in assisting the diagnosis of BS/EOS functionally.

To date, E383K is the only confirmed NOD2 mutation underlining BS/EOS with incomplete penetrance [7, 10]. Due to the shortage of genetic material from elder family members of the proband, we were unable to confirmed the pattern of penetrance via classical segregation study. However, the fact that proband’s father harbors the same C483W NOD2 mutation but lacks clinical BS/EOS phenotype itself suggests that this NOD2 mutation was incompletely penetrated.

While the exact regulatory network of NOD2 and its impact in disease development requires further elucidation, the functional assays including the CD11b⁺ intracellular p-NFκB staining experiment and the plasma cytokine profile seem to closely associate with subjects’ clinical manifestations despite their NOD2 genotypes. Specifically, the percentage of p-NFκB⁺/CD11b⁺ cells and the plasma level of proinflammatory cytokines seem to be lower in proband’s father as compared to the proband and the positive BS control (Fig. 3C, D and E). Our immune system is made up with a complex and dynamic biological web of molecular, cellular, and organismal networks. The existence of promoting or protective factors may likely alter the development of the inflammation and clinical phenotype. For example, NOD2 interacts with receptor-interacting protein 2 as well as transforming growth factor β-activated kinase 1 and gene associated with retinoid-IFN-induced mortality 19 to mediate NFκB activation in cells [27]. Caspase-associated recruitment domain 12 negatively regulates NOD-protein-mediated NFκB activation through a NOD-NOD interaction and Erbin, a member of the PDZ domain-containing family, is known to inhibit NOD2 and capable of altering cytokine expression in response to MDP stimulation [27, 28]. Genetic and extrinsic factors interacting with this network of elements can potentially alter the immune responses and direct clinical manifestation beyond NOD2 mutation itself. Moreover, TNF-α, IFN-γ and toll-like receptor ligands were known priming triggers to promote NOD2 expression [24, 29, 30]. Environmental stimuli such as BCG vaccination or Propionibacterium acnes infection have also been reported to trigger the development of BS/EOS [8, 20, 24]. While the exact regulatory mechanism requires further elucidation, data from our p-NFκB intracellular staining assay and the level of plasma cytokines suggested that there likely exists other factors modulating the immune regulation in the asymptomatic subject harboring C483W NOD2 mutation.