The possession of exon 2 or exon 3 variants in the MEFV gene promotes inflammasome activation in Japanese patients with familial Mediterranean fever with a heterozygous exon 10 mutation.

OBJECTIVES: The modification and pathogenesis of MEFV exon 2 or 3 variants in familial Mediterranean fever (FMF) remains unclear. We compared the clinical and laboratory characteristics between the coexistence and noncoexistence of MEFV exon 2 or 3 variants in patients with FMF that had a heterozygous MEFV exon 10 mutation. METHODS: We excluded patients with FMF that had two MEFV exon 10 mutations in one or more alleles and/or MEFV mutations in exons other than in exons 2, 3, or 10. Finally, we reviewed 131 Japanese patients with FMF that had a heterozygous MEFV exon 10 mutation, and they were divided into the groups with and without MEFV exon 2 or 3 variants of 97 and 34, respectively. RESULTS: All patients with MEFV exon 2 variants had either E148Q and/or L110P variants, none of patients had exon 3 variants. In the univariate analysis, the group with variants had significantly earlier onset, a higher percentage of thoracic pain with febrile attacks, a higher frequency of attack, and a higher IL-18 level at remission compared to the group without variants (all, p<0.05). Importantly, multivariate analyses showed that the coexistence of MEFV exon 2 variants was independently and significantly associated with earlier onset of FMF and thoracic pain (both, p<0.05). CONCLUSIONS: Our results suggested that coexistence of MEFV exon 2 variants have additional effects on manifestations of FMF with MEFV exon 10 mutations. Our findings highlighted the modifications and pathogenesis of such MEFV variants in FMF.

Next-generation sequencing of the whole MEFV gene in Japanese patients with familial Mediterranean fever: a case-control association study.

OBJECTIVES: We aimed to identify the whole nucleotide sequence of the Mediterranean fever (MEFV) gene in familial Mediterranean fever (FMF) and reveal novel single nucleotide variants (SNVs) associated with the susceptibility of FMF. METHODS: SeqCap capturing technique followed by Illumina next-generation sequencing have been used to assess two hundred SNVs in the whole region of MEFV in 266 Japanese patients with FMF and 288 ethnically matched controls. We performed an association analysis using these SNVs to identify genetic variants that predispose to FMF. RESULTS: We identified the two most significant SNVs [rs28940578; M694I in exon 10, odds ratio (OR) = 153, p=2.47×10-21 and rs3743930; E148Q in exon 2, OR = 1.65, p<0.0005]. Stratified analysis identified rs28940578 as a risk allele in typical FMF. Haplotype AG, defined by rs401298 and rs28940578, was the most significant and prevalent among patients with typical FMF compared with controls (22.4% vs. 0%, respectively; OR = 137, p=1.44×10-31). Haplotype GTC, defined by rs11466018, rs224231, and rs401877, was the most significant among patients with typical FMF without the rs28940578 mutation compared with controls (15.9% vs. 6%, respectively; OR = 12.4, p=0.004). CONCLUSIONS: rs28940578 is associated with the highest risk in typical FMF cases. This is consistent with results from previous studies in Japan. We found a novel MEFV gene haplotype that confers susceptibility of FMF among typical FMF without the rs28940578 mutation. There were no relevant SNVs identified in MEFV among the atypical FMF group.

KN3014, a piperidine-containing small compound, inhibits auto-secretion of IL-1ß from PBMCs in a patient with Muckle-Wells syndrome.

NLRP3, an intracellular pattern recognition receptor, recognizes numerous pathogens and/or its own damage-associated molecules, and forms complexes with the adaptor protein ASC. These complexes constitute the NLRP3 inflammasome, a platform for processing interleukin (IL)-1ß and/or IL-18. Several NLRP3 mutations result in constitutive activation of the NLRP3 inflammasome, causing cryopyrin-associated periodic syndrome (CAPS). To the best of our knowledge, small compounds that specifically inhibit inflammasome activation through the pyrin domain (PYD) have not yet been developed. This study describes an attempt to develop small compounds targeting the NLRP3 inflammasome. A core chemical library of 9,600 chemicals was screened against reconstituted NLRP3 inflammasome in a cell-free system with an amplified luminescence proximity homogeneous assay and a cell-based assay by human peripheral blood mononuclear cells (PBMCs). Inflammasome activation was evaluated by ASC-speck formation in human PBMCs, accompanied by IL-1ß secretion and processing, and by using IL-1ß-based dual operating luciferase (IDOL) mice. The activity of these compounds was evaluated clinically using PBMCs from a patient with Muckle-Wells syndrome (MWS), a type of CAPS, with an R260W mutation in NLRP3. Screening identified KN3014, a piperidine-containing compound targeting the interaction between NLRP3 and ASC through the PYD. KN3014 reduced ASC-speck formation in human PBMCs, luminescence from IDOL mice, and auto-secretion of IL-1ß by PBMCs from the patient with MWS. These findings suggest that KN3014 may be an attractive candidate for treatment of MWS, as well as other NLRP3 inflammasomopathies.

[AN ADULT CASE OF PERIODIC FEVER, APHTHOUS STOMATITIS, PHARYNGITIS, AND ADENITIS (PFAPA) SYNDROME DEVELOPED IN CHILDHOOD AND DIAGNOSED AFTER PROGRESS MORE THAN 30 YEARS].

We report an adult case of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome, who had a tonsillectomy at 10 years old and relapsed later. An early 40's-year-old man had been suffering from recurrent fever attack once in 1-2 months during childhood. He was accompanied by fever which was persist for several days, aphthous stomatitis, tongued tonsillitis with moss, pharyngitis, and submandibular lymphadenitis with tenderness. He was not doing well during fare-up. At the time of admission, CRP level was 12.5mg/dl and the remarkably increased expression of CD64 on neutrophils was found. Bacterial infections and collagen diseases were excluded by the several examinations. We suspected PFAPA syndrome, and treated with cimetidine, but cimetidine was not effective. At the time of flare up, administration of prednisolone was remarkably effective. We diagnosed PFAPA syndrome on the basis of clinical courses. Genetic analysis of responsible gene of familial Mediterranean fever, MEFV showed E148Q heterozygous mutation in exon 2.Since an adult case of PFAPA syndrome is likely to be made misunderstanding for infectious recurrent pharyngitis, it is important to note that we should consider PFAPA syndrome as a differential diagnosis when we meet with the adult patient of recurrent fever.

Clinical features and new diagnostic criteria for the syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis.

AIM: The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a common inflammatory disease that presents with periodic fever. We aimed to establish more specific diagnostic criteria for PFAPA based on the clinical characteristics of PFAPA patients in our directory. METHOD: The clinical, laboratory, genetic, and family history details of 257 Japanese PFAPA patients treated at our and other affiliated hospitals between April 2000 and April 2018 were analyzed along with quantitative measurements of the number of CD64 molecules on neutrophils, and the levels of serum inflammatory cytokines. The sensitivity and specificity of the criteria were calculated for several diseases. RESULTS: Because recurrent fevers were crucial findings, they were defined as the required criterion. Tonsillitis/pharyngitis with white moss were important accompanying signs. Other symptoms associated with febrile episodes were cervical lymphadenitis with tenderness, aphthous stomatitis, sore throat, vomiting, and headache but not cough. A total of 159 (62%) patients had a family history of recurrent fevers, indicating autosomal dominant inheritance. C-reactive protein levels were extremely elevated during febrile attacks but normal in attack-free periods. Serum immunoglobulin D levels were high in 72 of the 199 tested patients. Oral glucocorticoid and cimetidine were extremely effective in all and 51.6% of the patients, respectively. We defined the above as supportive criteria. These criteria were sensitive and specific enough to distinguish PFAPA from other recurrent fever diseases. Raised serum interferon-γ levels and remarkable CD64 expression on neutrophils during flare-ups were recognized, indicating they contributed to diagnosis. CONCLUSION: Our new criteria are useful for diagnosing PFAPA.

Mosaicism of an ELANE Mutation in an Asymptomatic Mother.

PURPOSE: We report normal neutrophil count in a mother, who carries the same ELANE mutation as her daughter with severe congenital neutropenia. We hypothesized that the mother possessed wild- and mutant-type clones and the wild-type clones could generate neutrophils, whereas the mutant clones could not. METHODS: We confirmed mutant variant ratio by sequence signals and measured the frequency of the mutant allele by subcloning in various cell types. We established the ELANE-mutated and non-mutated induced pluripotent stem cells (iPSCs) from the mother's T cells and compared granulopoiesis between these iPSCs. RESULTS: In the sequence analysis of isolated peripheral blood (PB), nail and hair, the mutant variant was detected in approximately 40-60% of lymphocytes, monocytes, hematopoietic progenitor cells, and hair as well as in a small percentage of nail, but in none of the neutrophils. In the subcloning analysis of extracted DNA from CD3+ and CD34+ cells, the mutant allele was identified in 37.5% and 38.1%, respectively. We reprogrammed the mother's PB cells and established the ELANE-mutated and non-mutated iPSCs. Granulopoiesis from mutated iPSCs revealed little sensitivity to granulocyte colony-stimulating factor in comparison with non-mutated iPSCs. CONCLUSIONS: These observations strongly suggest that mutant-carrying neutrophils did not appear in the mother's PB because mutated clones could not differentiate into neutrophils. The mother's normal hematological phenotype could be explained by the perseverance of normal, non-mutated granulopoiesis.

Musculoskeletal manifestations occur predominantly in patients with later-onset familial Mediterranean fever: Data from a multicenter, prospective national cohort study in Japan.

BACKGROUND: We showed previously that Japanese individuals with familial Mediterranean fever (FMF) have a more atypical phenotype compared to endemic areas. The clinical differences between young-onset FMF (YOFMF), adult-onset FMF (AOFMF), and late-onset FMF (LOFMF) in Japan are unclear. METHODS: We enrolled 395 consecutive patients. We defined YOFMF, AOFMF, and LOFMF as the onset of FMF at < 20, 20-39, and ≥ 40 years of age, respectively. We compared clinical manifestations and MEFV mutations patterns among these groups. RESULTS: Median ages at onset were YOFMF 12.5 years (n = 182), AOFMF 28 years (n = 115), and LOFMF 51 years (n = 90). A family history, MEFV mutations in exon 10, and more than two MEFV mutations were significantly more frequent in the earlier-onset groups (p < 0.01, p < 0.0001, and p < 0.001, respectively). In the accompanying manifestations, thoracic and abdominal pain were significantly more frequent in the earlier-onset groups (p < 0.01 and p < 0.0001, respectively), whereas arthritis and myalgia were significantly more frequent in the later-onset groups (p < 0.0001 and p < 0.01, respectively). The multiple logistic regression analysis revealed that the presence of MEFV exon 10 mutations and earlier onset were significantly associated with serositis, whereas the absence of MEFV exon 10 mutations, later onset, and the presence of erysipelas-like erythema were significantly associated with musculoskeletal manifestations. There was no significant between-group difference in the responsiveness to colchicine. CONCLUSIONS: Our results indicate that the later-onset FMF patients had a lower percentage of MEFV mutations in exon 10 and predominantly presented arthritis and myalgia. It is important to distinguish their FMF from other inflammatory diseases.

Heterozygous Mutations in OAS1 Cause Infantile-Onset Pulmonary Alveolar Proteinosis with Hypogammaglobulinemia.

Pulmonary alveolar proteinosis (PAP) is characterized by accumulation of a surfactant-like substance in alveolar spaces and hypoxemic respiratory failure. Genetic PAP (GPAP) is caused by mutations in genes encoding surfactant proteins or genes encoding a surfactant phospholipid transporter in alveolar type II epithelial cells. GPAP is also caused by mutations in genes whose products are implicated in surfactant catabolism in alveolar macrophages (AMs). We performed whole-exome sequence analysis in a family affected by infantile-onset PAP with hypogammaglobulinemia without causative mutations in genes associated with PAP: SFTPB, SFTPC, ABCA3, CSF2RA, CSF2RB, and GATA2. We identified a heterozygous missense variation in OAS1, encoding 2,'5'-oligoadenylate synthetase 1 (OAS1) in three affected siblings, but not in unaffected family members. Deep sequence analysis with next-generation sequencing indicated 3.81% mosaicism of this variant in DNA from their mother's peripheral blood leukocytes, suggesting that PAP observed in this family could be inherited as an autosomal-dominant trait from the mother. We identified two additional de novo heterozygous missense variations of OAS1 in two unrelated simplex individuals also manifesting infantile-onset PAP with hypogammaglobulinemia. PAP in the two simplex individuals resolved after hematopoietic stem cell transplantation, indicating that OAS1 dysfunction is associated with impaired surfactant catabolism due to the defects in AMs.

MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase γ pathway.

Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.

Analysis of biomarker serum levels in IVIG and infliximab refractory Kawasaki disease patients.

Infliximab (IFX) is effective for treatment of refractory Kawasaki disease (KD). However, the precise mechanisms and biomarkers for IFX efficacy are unknown. We tried to evaluate the effect and response to IFX therapy by measuring serum cytokine levels. Twenty-nine children with KD who had been resistant to two courses of high-dose intravenous immunoglobulin were enrolled and treated with IFX. Plasma samples were analyzed for cytokines before and after IFX administration. Serum levels of interleukin-6, granulocyte colony-stimulating factor (G-CSF), interferon-gamma-induced monokine, interferon-gamma inducible protein 10 (IP-10), monocyte chemotactic protein 1, and soluble tumor necrosis factor-alpha receptor (sTNFR) 1 and 2 were significantly elevated before IFX treatment, but promptly decreased after the administration. The pre-treatment G-CSF and sTNFR1 levels in non-responders to IFX were significantly higher than in responders, who were defined as patients who defervesce (< 37.5 °C). After IFX administration, elevated cytokines declined to normal ranges in responders, but in non-responsive group, G-CSF and sTNFR1 remained elevated without failing to normal levels. IFX treatment significantly reduced the levels of serum cytokines, chemokines, and sTNFRs in refractory KD. G-CSF and sTNFR1 may be indicators predictive of poor response to IFX.

Hematopoietic stem cell transplantation for pulmonary alveolar proteinosis associated with primary immunodeficiency disease.

Pulmonary alveolar proteinosis (PAP) is a rare disorder that is characterized by the excessive accumulation of surfactant-like materials in the alveoli, leading to hypoxemic respiratory failure. We describe two Japanese infants with PAP associated with hypogammaglobulinemia and monocytopenia. These patients may have underlying primary immunodeficiency (PID) and were successfully treated with allogeneic hematopoietic stem cell transplantation (HSCT). This report indicates that allogeneic HSCT may provide a curative treatment for PAP associated with PID.

Acute Myeloid Leukemia in a Patient With X-linked Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is a defect in the differentiation and function of T cells. An increased malignancy risk, mainly lymphatic malignancy, has been described in patients with SCID. We report a patient with X-linked SCID who developed acute myeloid leukemia, derived from the recipient with somatic NRAS mutation 4 months after cord blood transplantation (CBT). Loss of heterozygosity phenomenon of the recipient at 6q14 locus was observed at 2 months post-CBT and progressed to 6q deletion (6q-) chromosome abnormality. Somatic NRAS mutation was detected at 3 months post-CBT. Thus, 6q- and NRAS mutation were strongly associated with the leukemic transformation in our patient.

Early diagnosis of early-onset sarcoidosis: a case report with functional analysis and review of the literature.

This study examined the pathogenesis of early-onset sarcoidosis (EOS) in a patient with a rare NOD2 mutation and surveyed the literature to identify the hallmark features for early diagnosis. An infant girl suffering from prolonged fever and skin rash of multiple pinkish papules and subsequent erythema nodosum was referred to our institution. Skin biopsy and DNA sequencing were performed along with cytokine profiling of the patient's serum and stimulated mononuclear cells. NF-κB activation was analyzed using transfected cells. Multiple non-caseating granuloma inclusions were recognized in biopsy specimens obtained from the patient's rash. DNA sequencing revealed a very rare heterozygous Met513Thr (M513T) mutation in NOD2. Mononuclear cells produced a low amount of IL-1ß upon stimulation as compared with normal control cells. Mutated NOD2 transfection enhanced NF-κB activation. We suspected that the M513T mutation in NOD2 decreased IL-1ß production and enhanced NF-κB activation, which was likely responsible for the patient's granuloma involvement. A comprehensive review of the literature on 30 cases of sporadic type of EOS revealed that all patients had cutaneous manifestations, with all but one displaying granulation. A majority of EOS patients have R334W/Q. But about half of sporadic EOS had NOD2 mutations other than R334W/Q, as in the present case. Accordingly, skin rash with granuloma formation and specific NOD2 mutations may represent early diagnostic hallmarks of EOS in infants with persistent inflammation.

Familial Mediterranean fever with P369S/R408Q exon3 variant in pyrin presenting as symptoms of PFAPA.

Familial Mediterranean fever (FMF) can be classified into typical and incomplete/atypical types. Periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) syndrome-like symptoms have been found in atypical type carrying P369S-R408Q mutations in the responsible gene MEFV. A 28-year-old female with recurrent fever and her young sisters and mother, all of whom had tonsillectomy for tonsillitis, carried heterozygous alterations involving E148Q/P369S/R408Q. A diagnosis of atypical FMF, MEFV exon3 variants with PFAPA syndrome-like symptoms, was made.

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

Familial Mediterranean fever is no longer a rare disease in Japan.

BACKGROUND: The aim of this study was to evaluate the clinical manifestations and prevalence of familial Mediterranean fever (FMF) in Japanese patients with unexplained fever and rheumatic manifestations. METHODS: We enrolled 601 patients with unexplained fever or suspected FMF throughout Japan between 2009 and 2015. Patients were divided into three groups according to Tel Hashomer criteria: sure FMF, probable FMF, and non-FMF patients, including definitive rheumatic diseases. Mutation detection in exons 1, 2, 3, and 10 of the FMF gene MEFV was performed by direct sequencing. RESULTS: A total of 192 patients (31.9 %) were diagnosed with FMF according to FMF diagnostic criteria. These could be divided into sure FMF (56.3 %, n = 108) and probable FMF (43.7 %, n = 84) patients. Fever, abdominal symptoms, and thoracic symptoms were significantly more common in FMF than non-FMF patients. Among FMF patients, 26 (13.5 %) had concomitant rheumatic diseases. Most FMF patients (94.3 %, 181/192) carried at least one MEFV mutation. Allele frequencies of M694I (13.5 % vs 0 %) and E148Q (39.1 % vs 24.8 %) mutations were significantly higher in FMF compared with healthy subjects. Allele frequencies of common MEFV mutations in FMF patients were M694I (13.5 %), P369S (8.6 %), R408Q (8.1 %), G304R (2.9 %), R202Q (4.4 %), E148Q (39.1 %), L110P (11.7 %), and E84K (3.1 %). Patients with a sure FMF phenotype had a higher frequency of MEFV exon 10 mutation (M694I) and a lower frequency of MEFV exon 3 mutations (P369S, R408Q) compared with those with a probable FMF phenotype. CONCLUSION: The high prevalence of FMF in Japanese patients with unexplained fever was confirmed in the present study. FMF should be suspected in cases of unexplained fever or non-specific rheumatic manifestations, and mutational analysis of MEFV could be useful to predict the clinical phenotypes of FMF in Japan.

Novel heterozygous C243Y A20/TNFAIP3 gene mutation is responsible for chronic inflammation in autosomal-dominant Behçet's disease.

OBJECTIVE: Although Behçet's disease (BD) is a chronic inflammatory disorder of uncertain aetiology, the existence of familial BD with autosomal-dominant traits suggests that a responsibility gene (or genes) exists. We investigated a Japanese family with a history of BD to search for pathogenic mutations underlying the biological mechanisms of BD. METHODS: 6 patients over 4 generations who had suffered from frequent oral ulcers, genital ulcers and erythaema nodosum-like lesions in the skin were assessed. Whole-exome sequencing was performed on genomic DNA, and cytokine production was determined from stimulated mononuclear cells. Inflammatory cytokine secretion and Nod2-mediated NF-κB activation were analysed using the transfected cells. RESULTS: By whole-exome sequencing, we identified a common heterozygous missense mutation in A20/TNFAIP3, a gene known to regulate NF-κB signalling, for which all affected family members carried a heterozygous C243Y mutation in the ovarian tumour domain. Mononuclear cells obtained from the proband and his mother produced large amounts of interleukin 1ß, IL-6 and tumour necrosis factor α (TNF-a) on stimulation as compared with those from normal controls. Although inflammatory cytokine secretion was suppressed by wild-type transfected cells, it was suppressed to a much lesser extent by mutated C243Y A20/TNFAIP3-transfected cells. In addition, impaired suppression of Nod2-mediated NF-κB activation by C243Y A20/TNFAIP3 was observed. CONCLUSIONS: A C243Y mutation in A20/TNFAIP3 was likely responsible for increased production of human inflammatory cytokines by reduced suppression of NF-κB activation, and may have accounted for the autosomal-dominant Mendelian mode of BD transmission in this family.

Multiple Serum Cytokine Profiling to Identify Combinational Diagnostic Biomarkers in Attacks of Familial Mediterranean Fever.

The precise cytokine networks in the serum of individuals with familial Mediterranean fever (FMF) that are associated with its pathogenesis have been unknown. Here, we attempted to identify specific biomarkers to diagnose or assess disease activity in FMF patients. We measured serum levels of 45 cytokines in 75 FMF patients and 40 age-matched controls by multisuspension cytokine array. FMF in "attack" or "remission" was classified by Japan College of Rheumatology-certified rheumatologists according to the Tel Hashomer criteria. Cytokines were ranked by their importance by a multivariate classification algorithm. We performed a logistic regression analysis to determine specific biomarkers for discriminating FMF patients in attack. To identify specific molecular networks, we performed a cluster analysis of each cytokine. Twenty-nine of the 45 cytokines were available for further analyses. Eight cytokines' serum levels were significantly elevated in the FMF attack versus healthy control group. Nine cytokines were increased in FMF attack compared to FMF remission. Multivariate classification algorithms followed by a logistic regression analysis revealed that the combined measurement of IL-6, IL-18, and IL-17 distinguished FMF patients in attack from the controls with the highest accuracy (sensitivity 89.2%, specificity 100%, and accuracy 95.5%). Among the FMF patients, the combined measurement of IL-6, G-CSF, IL-10, and IL-12p40 discriminated febrile attack periods from remission periods with the highest accuracy (sensitivity 75.0%, specificity 87.9%, and accuracy 84.0%). Our data identified combinational diagnostic biomarkers in FMF patients based on the measurement of multiple cytokines. These findings help to improve the diagnostic performance of FMF in daily practice and extend our understanding of the activation of the inflammasome leading to enhanced cytokine networks.