Clinical significance of anti-NOR90 antibodies in systemic sclerosis and idiopathic interstitial pneumonia.

OBJECTIVE: Anti-NOR90 antibodies are usually found in patients with SSc; however, their clinical relevance remains obscure. We developed an ELISA for measuring them to investigate the clinical features of patients with anti-NOR90 antibodies. METHODS: Serum samples from 1252 patients with various conditions from Nagoya University Hospital and 244 patients with idiopathic interstitial pneumonia (IIP) from Tosei General Hospital were included. Anti-NOR90 antibodies were assayed by an ELISA using the recombinant protein produced by in vitro transcription/translation. RESULTS: Five (0.4%) patients in the Nagoya University Hospital cohort had anti-NOR90 antibodies. One patient with diffuse cutaneous SSc, three with limited cutaneous SSc, and one with Raynaud's disease were positive for anti-NOR90 antibodies. Anti-NOR90 antibodies were found more frequently in patients with systemic scleroderma-spectrum disorders (SSDs) than without SSDs (5/316 vs 0/936, P <0.00101) and were found more frequently in patients with SSc than without SSc (4/249 vs 0/528, P <0.0104) in the systemic autoimmune rheumatic diseases cohort. Three of the four anti-NOR90-positive SSc patients had interstitial lung disease (ILD), and two of those four had cancer. Three (1.2%) patients in the Tosei General Hospital cohort had anti-NOR90 antibodies. All three of the anti-NOR90-positive IIP patients had gastrointestinal tract involvement, and two of those three had cancer or skin lesions observed in SSc. CONCLUSIONS: Although anti-NOR90 antibodies are rarely found in clinics, our ELISA is useful for their detection. Further studies are needed to confirm the association of anti-NOR90 antibodies with ILD and cancer in SSc and IIP patients.

Anti-Zo antibodies in Japanese myositis patients detected by a newly developed ELISA.

OBJECTIVES: The myositis-specific autoantibodies that characterise certain forms of idiopathic inflammatory myopathy (IIM) are useful for diagnosing dermatomyositis (DM) / polymyositis (PM) and predicting its prognosis. The autoantibody to phenylalanyl-tRNA synthetase (anti-Zo) has been identified as a disease marker antibody for anti-synthetase syndrome only in a UK cohort. Here we aim to establish an ELISA for the measurement of anti-Zo and to characterise the clinical features of Japanese patients who have this autoantibody. METHODS: Anti-Zo was investigated by immunoprecipitation with recombinant phenylalanyl-tRNA synthetase α/ß proteins. The results were confirmed by immunoprecipitation-Western blotting with cell extract. Sera from patients with DM/PM (n=224) were screened by an ELISA with the recombinant proteins. Medical records were retrospectively reviewed to obtain detailed information on the clinical phenotypes of the anti-Zo-positive patients. RESULTS: Only two male patients were confirmed to have anti-Zo. Both patients had fever, myopathy, interstitial lung disease, and mechanic's hands, and these clinical features are consistent with those of anti-synthetase syndrome. Another patient's serum showed a higher level than the cut-off value for anti-phenylalanyl-tRNA synthetase α by our in-house ELISA, but was judged to be negative for anti-Zo by immunoprecipitation-Western blotting. CONCLUSIONS: This is the first report of anti-Zo-positive IIM patients from Asia. Although Japanese patients with anti-Zo have a clinical phenotype similar to that of Caucasian patients, further large cohort studies are necessary to confirm the frequency of anti-Zo in Japanese IIM patients. Our newly developed ELISA should be validated for sensitivity and specificity in large cohorts.

Immune recognition of lysyl-tRNA synthetase and isoleucyl-tRNA synthetase by anti-OJ antibody-positive sera.

OBJECTIVE: Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are useful for identifying a clinical subset of patients with idiopathic inflammatory myopathies (IIMs). Anti-OJ antibodies, which recognize multi-enzyme synthetase complexes including isoleucyl-tRNA synthetase (IARS) and lysyl-tRNA synthetase (KARS), are among the anti-ARS antibodies. Although testing antibodies to other ARSs have been used clinically, no validated immunoassays for detecting anti-OJ antibodies are available. We aimed to establish an anti-OJ ELISA. METHODS: Serum samples were collected from 279 patients with IIMs and 22 patients with idiopathic interstitial pneumonia. Sixty-four of the samples that had been confirmed to be negative for anti-OJ by standard immunoprecipitation were used as the negative control, and 12 anti-OJ-positive reference sera were used as the positive control. Antibodies to IARS and KARS were assayed by ELISA using biotinylated recombinant proteins generated by in vitro transcription/translation. RESULTS: The anti-OJ-positive sera strongly reacted with the KARS and IARS recombinant proteins in ELISA. Although all 12 reference sera were positive in the anti-KARS ELISA, 4 of the 64 anti-OJ-negative sera were also weakly positive. The sensitivity and the specificity were 100% and 93.8%, respectively. Since our anti-KARS ELISA performed well, showing a high agreement with the results for immunoprecipitation (Cohen's κ > 0.8), the remaining 237 samples were also tested. Thirteen anti-KARS-positive sera were newly found by ELISA, all of which were anti-OJ positive by immunoprecipitation. CONCLUSION: Immunoassays for detecting anti-OJ antibodies using KARS and IARS recombinant proteins were developed. Our ELISAs performed well, with very high agreement of the results by immunoprecipitation and can be applied to the first reliable, easy-to-use measurement assays for anti-OJ antibodies.

2019 Diagnostic criteria for mixed connective tissue disease (MCTD): From the Japan research committee of the ministry of health, labor, and welfare for systemic autoimmune diseases.

OBJECTIVE: To update and revise the diagnostic criteria for mixed connective tissue disease (MCTD) issued by the Japan Research Committee of the Ministry of Health, Labor, and Welfare (MHLW), a round table discussion by experts from rheumatology, dermatology, and pediatric medicine was conducted in multiple occasions. METHODS: The definition of MCTD, and items included in the diagnostic criteria were generated by consensus method and evaluation using clinical data of typical and borderline cases of MCTD, by applying to the diagnostic criteria for MCTD proposed in 1996 and 2004 by the Research Committee of MHLW. RESULTS: To the end, all committee members reached consensus. Then, the criteria were assessed in an independent validation cohort and tested against preexisting criteria. The revised criteria facilitate an understanding of the overall picture of this disease by describing the concept of MCTD, common manifestations, immunological manifestation and characteristic organ involvement. Conditions with characteristic organ involvement include pulmonary arterial hypertension, aseptic meningitis and trigeminal neuropathy. Even if the overlapping manifestations are absent, MCTD can be diagnosed based on the presence of the characteristic organ involvement. Furthermore, the criteria were validated for applicability in actual clinical cases, and public comments were solicited from the Japan College of Rheumatology and other associated societies. CONCLUSION: After being reviewed through public comments, the revised diagnostic criteria have been finalized.

Splicing variant of WDFY4 augments MDA5 signalling and the risk of clinically amyopathic dermatomyositis.

OBJECTIVES: Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare autoimmune diseases in which both genetic and environmental factors play important roles. To identify genetic factors of IIM including polymyositis, dermatomyositis (DM) and clinically amyopathic DM (CADM), we performed the first genome-wide association study for IIM in an Asian population. METHODS: We genotyped and tested 496 819 single nucleotide polymorphism for association using 576 patients with IIM and 6270 control subjects. We also examined the causal mechanism of disease-associated variants by in silico analyses using publicly available data sets as well as by in in vitro analyses using reporter assays and apoptosis assays. RESULTS: We identified a variant in WDFY4 that was significantly associated with CADM (rs7919656; OR=3.87; P=1.5×10-8). This variant had a cis-splicing quantitative trait locus (QTL) effect for a truncated WDFY4isoform (tr-WDFY4), with higher expression in the risk allele. Transexpression QTL analysis of this variant showed a positive correlation with the expression of NF-κB associated genes. Furthermore, we demonstrated that both WDFY4 and tr-WDFY4 interacted with pattern recognition receptors such as TLR3, TLR4, TLR9 and MDA5 and augmented the NF-κB activation by these receptors. WDFY4 isoforms also enhanced MDA5-induced apoptosis to a greater extent in the tr-WDFY4-transfected cells. CONCLUSIONS: As CADM is characterised by the appearance of anti-MDA5 autoantibodies and severe lung inflammation, the WDFY4 variant may play a critical role in the pathogenesis of CADM.

Strong correlation between cancer progression and anti-transcription intermediary factor 1γ antibodies in dermatomyositis patients.

OBJECTIVES: Transcription intermediary factor 1γ (ΤΙF1γ) protein is known as a tumour suppressor that promotes cellular differentiation. Autoantibodies to ΤΙF1γ have a strong clinical association with cancers associated with dermatomyositis (DM). This study aims to identify the clinical characteristics of cancers in anti-ΤΙF1γ antibody-positive adult patients with DM. METHODS: This retrospective analysis covered 160 adult DM patients who visited Nagoya University Hospital or collaborating medical centres between 2003 and 2016. Anti-TIF1γ antibody and other myositis-specific autoantibodies were detected by ELISA. Based on a review of medical charts, the cancers were staged according to the TNM Classification of Malignant Tumours of the Union for International Cancer Control and were divided into the two groups of "advanced" or "non-advanced" according to the stage classification. RESULTS: Forty-one of the 160 (26%) patients had cancer. The incidence was significantly higher in the anti-TIF1γ-positive patients than in the anti-TIF1γ-negative patients (23/34=68% vs. 18/126=14%, p<1x10-6). Anti-TIF1γ-positive patients with cancer were found more frequently in the "advanced" group than in the "non-advanced" group (21/23=91% vs. 9/18=50%, p<0.0046). The intervals between DM diagnosis and cancer diagnosis were significantly shorter in the anti-TIF1γ-positive patients than in the anti-TIF1γ-negative patients (p=0.047). CONCLUSIONS: Not only did anti-TIF1γ antibodies correlate strongly with malignancy in DM patients, but cancers were also significantly more advanced in anti-TIF1γ-positive DM patients than in anti-TIF1γ-negative patients. Cancers in such cases were very frequently found close to the time of the DM diagnosis.

Two novel anti-aminoacyl tRNA synthetase antibodies: Autoantibodies against cysteinyl-tRNA synthetase and valyl-tRNA synthetase.

Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are useful for identifying a clinical subset of patients with inflammatory myopathies. Since the myositis of anti-ARS-positive patients is characterized by a unique set of non-myopathic manifestations, including interstitial lung disease, mechanic's hands, and arthralgia, the patients are classified as having anti-synthetase syndrome. Autoantibodies have been identified to eight kinds of ARSs. Of the other 12 ARSs, eight are components of the "OJ" multi-synthetase complex. Autoantibodies to the four remaining ARSs (CysARS, ValARS, SerARS, and TrpARS) have not been reported to be present in patients with inflammatory myopathies. In this study, we first screened samples from more than 300 Japanese patients majorly consisting of those with dermatomyositis (DM) by our established in-house ELISA to find autoantibodies against the four ARSs described above. Since sera from two DM patients specifically reacted to CysARS or ValARS, we determined their reactivities by immunoprecipitation (IP) with the corresponding recombinant proteins and IP-Western blotting with cellular extract. One patient had several features found in anti-synthetase syndrome, but the other did not. The clinical differences among the various anti-ARS antibodies should be explored in a future work.

A case with overlapping features of IgG4-related autoimmune pancreatitis, Sjögren's syndrome and anti-aminoacyl-tRNA synthetase syndrome.

A 55-year-old man who had been diagnosed with autoimmune pancreatitis five years earlier was referred to our department because of finger swelling, finger stiffness and the presence of interstitial lung disease (ILD). The patient was diagnosed with Sjögren's syndrome according to the pathological findings of minor salivary glands and positive anti-SS-A antibodies. Later, at age 58, he was hospitalised due to the exacerbation of the ILD. Serum IgG4 level was checked and was found to be elevated (417 mg/dL). After the introduction of cyclosporine in addition to the prednisolone, at age 60, the ILD disease activity stabilised. However, at age 62, fever, myalgia and mechanic's hands appeared. His serum creatine kinase level was high, and magnetic resonance imaging showed inflammatory findings of muscle. In-house ELISA clarified that his serum carried anti-PL-7 antibody among anti-aminoacyl-tRNA synthetase antibodies. This is a unique case who had overlapping features of IgG4-related autoimmune pancreatitis, Sjögren's syndrome and anti-synthetase syndrome. Although the aetiology of the complications in this patient is obscure, autoimmunity might have played a significant role in the disease conditions and prognosis of the present case with IgG4-related disease.