Sarcoidosis is a systemic inflammatory disease with unknown etiology, but it can be clinically characterized by swelling of the bilateral hilar lymph nodes and histologically defined by the presence of noncaseating epithelioid cell granulomas. A special subtype called early-onset sarcoidosis (EOS; MIM no. 609464) occurs in children younger than 4 years of age and is characterized by a distinct triad of skin, joint, and eye disorders without apparent pulmonary involvement (1). An autosomal-dominant disease with clinical manifestations similar to those of EOS has been recognized as Blau syndrome (MIM no. 186580) (2, 3). The gene responsible for Blau syndrome has been mapped close to the inflammatory bowel disease 1 (IBD1) locus by linkage analysis (4), and later the nucleotide-binding oligomerization domain 2 gene (NOD2) was identified by Miceli-Richard et al to be responsible for Blau syndrome (5). In the study by Miceli-Richard et al, 2 European patients with EOS had no mutation in NOD2; therefore, it remained controversial whether Blau syndrome and EOS have the same etiology.
In 2004, we encountered a 27-year-old Japanese man with multiple lichenoid papules. He was almost blind, exhibited camptodactyly, and had a continuous low-grade fever. This case of sporadic systemic granulomatosis with clinical features of EOS showed the same NOD2 mutation, the arginine-to-tryptophan substitution at amino acid 334 (R334W), as that detected in Blau syndrome (6). Therefore, we expanded this report (6) and retrospectively examined cases of EOS in Japan and observed that 9 of 10 patients with EOS had NOD2 mutations (7). Until recently, other investigators have also confirmed that Blau syndrome and EOS are clinically and genetically identical across various ethnic groups (8–10).
NOD2 activates NF-κB after recognizing a signal from a bacterial cell wall component, muramyldipeptide, in the cytoplasm of monocytes, and thus can work as an intracellular sensor of bacteria (11, 12). NOD2 has a tripartite domain structure consisting of 2 amino-terminal domains (termed caspase activation and recruitment domains) that are composed of protein–protein interaction cassettes, 1 centrally located NOD, and carboxy-terminal leucine-rich repeats (LRRs) (13). Using assays of NF-κB activity, an impaired ligand-dependent response was demonstrated for 3 Crohn's disease–associated mutations located in NOD2 LRRs (14, 15), whereas enhanced ligand-independent NF-κB activity was demonstrated for NOD2 alleles associated with Blau syndrome and EOS (5, 7, 16). However, it remains unknown how increased basal NF-κB activity derived from gain-of-function mutations in NOD2 affects the pathogenesis of Blau syndrome/EOS and whether a genotype–phenotype correlation exists between the clinical manifestations or onset of Blau syndrome/EOS and NOD2 mutations.
Because Blau syndrome/EOS is so rare, very few reports are in the literature. Therefore, it was worthwhile to conduct a nationwide survey limited to patients with a specific ethnic background, such as Japanese patients. In this study, we precisely documented the clinical manifestations in a cohort of Japanese patients with Blau syndrome/EOS and NOD2 mutations, including 9 previously reported cases (7), and explored the genotype–phenotype correlation to the basal NF-κB activity associated with each mutation, especially focusing on the correlation of visual impairment with the most frequent mutations, R334W and R334Q.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Blau syndrome/EOS is a rare systemic granulomatosis that has been associated with NOD2. In this study, patients with Blau syndrome/EOS and NOD2 mutations were retrospectively recruited nationwide in Japan, to determine whether the NOD2 genotype and its functional abnormality predict the Blau syndrome/EOS clinical phenotype. This study is the first to investigate the correlation between the NOD2 genotype and its functional abnormality and the Blau syndrome/EOS clinical phenotype. Our findings suggest that NOD2 genotyping may help predict disease progression in patients with Blau syndrome/EOS, although the clinical severity of Blau syndrome/EOS was not clearly associated with basal NF-κB activity due to mutated NOD2 among the limited number of patients we studied.
The classic Blau syndrome/EOS symptom triad is skin rash, arthritis, and uveitis. Corresponding clinical manifestations include widespread erythematous papules, polyarthritis with boggy synovial swellings, and panuveitis (1, 9, 10, 23), which were also identified in the present study. Rose et al described 2 patients who also had 1 episode of erythema nodosum–like lesions during the course of the disease (9). In our cohort, 5 patients had erythema nodosum–like lesions, suggesting that this should be recognized as one of the skin manifestations associated with Blau syndrome/EOS.
In the current study, 55% of the patients had fever, which always accompanied at least 1 symptom of the classic triad. Arostegui et al also reported that 50% of their cohort had recurrent or persistent fever (10). These findings suggest that fever is one of the important symptoms of Blau syndrome/EOS and is the reason why Blau syndrome/EOS is misdiagnosed as systemic-onset juvenile idiopathic arthritis (JIA). In fact, patient 11 in our study (who had the R334W mutation) experienced persistent fever reaching 40°C and received aggressive immunosuppressive therapy, because systemic-onset JIA was initially diagnosed. This case alerts us to the possibility that patients with Blau syndrome/EOS can sometimes have fever, and that Blau syndrome/EOS can resemble systemic-onset JIA.
Bilateral hilar lymph nodes, which are often seen in adult sarcoidosis, are not observed in Blau syndrome/EOS, but this does not mean that pulmonary lesions do not occur in patients with Blau syndrome/EOS. In fact, 2 patients (patient 5 [with the R334Q mutation] and patient 10 [with the R334W mutation]) had pulmonary lesions; in particular, patient 10 had the first reported case of sporadic EOS in association with the NOD2 mutation (6). Another case of Blau syndrome/EOS with pulmonary lesions and interstitial pneumonitis, but not bilateral hilar lymph nodes, has also been reported (28). These findings suggest the importance of following up patients with Blau syndrome/EOS to check for not only the classic triad of symptoms but also other abnormalities, including pulmonary lesions.
Blau syndrome/EOS, which usually occurs in children younger than age 4 years, developed at 5 years and 8 years, respectively, in 2 patients in the present study (patient 2 [with the E383G mutation] and patient 17 [with the R334W mutation]). Because both of these patients had a family history of skin rash/arthritis/uveitis, they had been closely monitored by their parents as well as by their physicians. Therefore, it is unlikely that any symptoms that occurred when the patients were younger than 4 years of age were overlooked in these 2 cases. In the literature, there is 1 case of Blau syndrome in which skin rash, persistent fever, and camptodactyly started to develop at age 18 years (10). These findings indicate that the onset of Blau syndrome/EOS can be at age 5 years or older, and that disease onset in a patient younger than 4 years should not be considered requisite for a diagnosis of Blau syndrome/EOS.
In our cohort, the age at disease/symptom onset, organ involvement, and severity of Blau syndrome/EOS varied substantially even within affected families and between individuals with the same NOD2 mutation (e.g., R334W). In other genetic disorders, identical mutations have been associated with phenotypic variation in unrelated individuals, within a family, and even in monozygotic twins (29). Phenotypic variation in Blau syndrome/EOS has been reported in monozygotic twins; therefore, nongenetic factors such as environmental conditions and/or infectious agents might be involved in phenotypic variation (24). Interestingly, in 2 of our cases, BCG vaccination was an obvious triggering factor. In addition, a previous report noted that cutaneous lesions first arose after BCG vaccination in a patient with Blau syndrome/EOS (30). The BCG vaccine contains muramyldipeptide, a ligand for NOD-2 protein (11, 12), which is interesting from a pathophysiologic point of view. However, BCG vaccination did not always cause the onset of disease in patients with Blau syndrome/EOS, because most patients in our cohort were vaccinated with BCG according to the immunization protocol used in areas of Japan where the risk for tuberculosis was high. An unknown endogenous ligand for NOD-2 could influence disease onset and/or progression, similar to uric acid as an endogenous cryopyrin/NLRP3 ligand (31). The potential roles of endogenous ligands, pathogen-associated molecular patterns, and/or danger-associated molecular patterns in disease pathogenesis remain to be elucidated.
Although increased basal NF-κB activity due to mutated NOD2 has been proposed as an etiology of Blau syndrome/EOS, how such activity causes the characteristic symptoms remains unclear. We hypothesized that if increased basal NF-κB activity is the key to the pathophysiology of this disease, it should be related to disease severity or disease progression. Unfortunately, there was no clear correlation between basal NF-κB activity and the onset of disease/symptoms. However, patients with mutated NOD2 and low basal NF-κB activity tended to experience complications, e.g., arthritis and uveitis, at a later age. This finding raises the possibility that basal NF-κB activity may affect disease progression rather than disease onset. Given that NOD-2 protein signals through MAPK/ERK as well as the NF-κB pathway (32), the possibility cannot be excluded that the MAPK/ERK activation potential of each NOD2 genotype might also be correlated with disease severity or progression.
From the perspective of quality of life, the ocular manifestations of Blau syndrome/EOS require the closest attention (33). In a previous study, one-third of patients with Blau syndrome/EOS and NOD2 mutations had a poor or extremely poor visual outcome, and the progression of visual field loss was independent of the particular NOD2 mutant and was not associated with disease duration (9). In our cohort, however, patients with the R334W mutation experienced more visual impairment than did patients with the R334Q mutation, although 4 patients with the R334W mutation were from 2 families (patients 14 and 15 and patients 16 and 17, respectively). Therefore, familial genetic and environmental factors could easily influence the phenotype. Thus, in order not to favor our hypothesis, we excluded patients 15 and 17 from the analysis, and the trend was still evident. This observation was consistent with the findings of Rose et al (9), although those investigators did not address this issue. These findings suggest that NOD2 genotyping could help predict the course of eye disease in patients with Blau syndrome/EOS, especially those with the R334Q mutation or the R334W mutation.
The relationship between visual impairment and basal NF-κB activity also remains a matter for discussion. Our data showed that visual impairments were more severe in patients with the R334W mutation than in those with the R334Q mutation, which seems to be consistent with the hypothesis that higher basal NF-κB activity causes more severe disease or more disease progression. However, no ocular symptoms have developed during the 6 years since disease onset in patient 20 (with the C495Y mutation and the highest basal NF-κB activity in our cohort), although ocular symptoms developed in another patient with the same genotype (10). Also, in patient 2, who had the E383G mutation and the lowest basal NF-κB activity, severe visual impairment occurred when she was in her late twenties. These findings contradict our hypothesis that NOD2 genotypes with higher basal NF-κB activity are associated with severe disease. However, Blau syndrome/EOS was promptly diagnosed in patient 20 with the C495Y mutation, who luckily was under the care of the same pediatric rheumatologist who treated patient 19 (who had the N670K mutation) and was treated with systemic steroid therapy. Patient 2 (who had the E383G mutation) subsequently received inappropriate immunosuppressive therapy, because the patient refused steroid treatment. Furthermore, patient 10 (with the R334W mutation), who had no obvious systemic inflammatory findings and did not receive systemic steroid therapy, became blind at 20 years of age. These findings raise the possibility that the extent of visual impairment could be modified by therapy.
Finally, we were not able to prove a link between the clinical severity of Blau syndrome/EOS and basal NF-κB activity in the whole cohort, possibly because of the restricted number of patients and because of the differences in treatment among patients. Therefore, a prospective study involving a sufficient number of patients to allow analysis of each genotype–phenotype correlation would be required to test our hypothesis. Given that there is no standard treatment protocol for Blau syndrome/EOS, some predictors of disease progression, especially progression of visual impairment, would have great benefit for clinicians. We observed a difference in the development of visual impairment only between patients with the R334W mutation and those with the R334Q mutation, which provides a clue that predicts the development of visual impairment in patients with the R334W and R334Q mutations. We also believe that understanding the mechanisms of how NOD2 acts in disease pathogenesis should help in discovering therapeutic targets for the treatment of Blau syndrome/EOS.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Nishikomori had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Okafuji, Nishikomori, Heike, Miyachi, Nakahata.
Acquisition of data. Okafuji, Fujisawa, Saito, Yoshioka, Kawai, Sakai, Tanizaki.
Analysis and interpretation of data. Okafuji, Nishikomori.
Manuscript preparation. Okafuji, Nishikomori, Kanazawa, Kambe.
Statistical analysis. Yamazaki.