Anti-MJ/NXP-2 autoantibody specificity in a cohort of adult Italian patients with polymyositis/ dermatomyositis

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Title:
Anti-MJ/NXP-2 autoantibody specificity in a cohort of adult Italian patients with polymyositis/ dermatomyositis
Series Title:
Arthritis Research & Therapy
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Book
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English
Creator:
Ceribelli, Angela
Fredi, Micaela
Taraborelli, Mara
Cavazzana, Ilaria
Franceschini, Franco
Quinzanini, Marzia
Tincani, Angela
Ross, Steven J.
Chan, Jason Y. F.
Pauley, Brad A.
Chan, Edward K. L.
Satoh, Minoru
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BioMed Central
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Abstract:
Introduction: Autoantibodies in patients with polymyositis/dermatomyositis (PM/DM) are associated with unique subsets, clinical course and outcome. Anti-MJ antibodies, which recognize the nuclear protein NXP-2/MORC3, are reported in ~25% of juvenile DM. Prevalence and clinical significance of anti-MJ antibodies in adult Italian PM/DM patients were studied. Methods: Sera from 58 consecutive adult Italian PM/DM patients were analyzed by immunoprecipitation of 35Slabeled K562 cells extract, ELISA (anti-MJ, Jo-1), Western blot and indirect immunofluorescence. Clinical associations were analyzed using information from medical charts. Results: Anti-MJ antibodies were the most prevalent specificity (17%) found mainly in DM (30%, 8 cases) vs 8% of PM (2 cases, P = 0.02). Comparing 10 anti-MJ (+) vs 48 anti-MJ (-) cases, DM was more common (P = 0.03), and age at onset was younger in anti-MJ (+) (P = 0.0006). In anti-MJ (+), heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were more frequent. None of them had heart or lung involvement, or malignancy. Myopathy in anti-MJ (+) patients responded well to therapy and none of them had elevated CPK at last visit (0% vs 25% in anti-MJ (-)). Only 60% of anti-MJ (+) showed immunofluorescent nuclear dots staining, despite PML localization of NXP-2/ MORC3. Conclusions: Anti-MJ antibodies are the most frequent specificity in our cohort of adult Italian PM/DM. Anti-MJ (+) were associated with young onset DM, calcinosis, no internal organ involvement and good response of myopathy to therapy. Anti-MJ reported in juvenile DM is also found in adult PM/DM, and could be a new useful biomarker.

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Abstract
Introduction
Autoantibodies in patients with polymyositis/dermatomyositis (PM/DM) are associated with unique subsets, clinical course and outcome. Anti-MJ antibodies, which recognize the nuclear protein NXP-2/MORC3, are reported in ~25% of juvenile DM. Prevalence and clinical significance of anti-MJ antibodies in adult Italian PM/DM patients were studied.
Methods
Sera from 58 consecutive adult Italian PM/DM patients were analyzed by immunoprecipitation of 35S-labeled K562 cells extract, ELISA (anti-MJ, Jo-1), Western blot and indirect immunofluorescence. Clinical associations were analyzed using information from medical charts.
Results
Anti-MJ antibodies were the most prevalent specificity (17%) found mainly in DM (30%, 8 cases) vs 8% of PM (2 cases, P = 0.02). Comparing 10 anti-MJ (+) vs 48 anti-MJ (-) cases, DM was more common (P = 0.03), and age at onset was younger in anti-MJ (+) (P = 0.0006). In anti-MJ (+), heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were more frequent. None of them had heart or lung involvement, or malignancy. Myopathy in anti-MJ (+) patients responded well to therapy and none of them had elevated CPK at last visit (0% vs 25% in anti-MJ (-)). Only 60% of anti-MJ (+) showed immunofluorescent nuclear dots staining, despite PML localization of NXP-2/MORC3.
Conclusions
Anti-MJ antibodies are the most frequent specificity in our cohort of adult Italian PM/DM. Anti-MJ (+) were associated with young onset DM, calcinosis, no internal organ involvement and good response of myopathy to therapy. Anti-MJ reported in juvenile DM is also found in adult PM/DM, and could be a new useful biomarker.
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Ceribelli, Angela
Fredi, Micaela
Taraborelli, Mara
Cavazzana, Ilaria
Franceschini, Franco
Quinzanini, Marzia
Tincani, Angela
Ross, Steven J
Chan, Jason YF
Pauley, Brad A
Chan, Edward KL
Satoh, Minoru
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Ceribelli et al.; licensee BioMed Central Ltd.
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Arthritis Research & Therapy. 2012 Apr 30;14(2):R97
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title p Anti-MJ/NXP-2 autoantibody specificity in a cohort of adult Italian patients with polymyositis/dermatomyositis
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au id A1 snm Ceribellifnm Angelainsr iid I1 email dott.ceribelli@libero.it
A2 FrediMicaelaI2 elmic83@libero.it
A3 TaraborelliMaramara.taraborelli@libero.it
A4 CavazzanaIlariailariacava@virgilio.it
A5 FranceschiniFrancofranceschini@bresciareumatologia.it
A6 QuinzaniniMarziamarziaquinz@libero.it
A7 TincaniAngelatincani@bresciareumatologia.it
A8 Rossmi JStevenstevenr966@gmail.com
A9 ChanYFJasonI3 jchan89@ufl.edu
A10 PauleyABradbrad_pollie@yahoo.com
A11 ChanKLEdwardechan@dental.ufl.edu
A12 ca yes SatohMinoruminoru.satoh@medicine.ufl.edu
insg
ins Department of Oral Biology, University of Florida, 1395 Center Drive, Gainesville, FL 32610-0424, USA
Rheumatology Unit, A.O. Spedali Civili, Piazzale Spedali Civili 1, 25123, Brescia, Italy
Division of Rheumatology and Clinical Immunology, Department of Medicine; Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 2
fpage R97
url http://arthritis-research.com/content/14/2/R97
xrefbib pubidlist pubid idtype doi 10.1186/ar3822pmpid 22546500
history rec date day 4month 12year 2012revrec 1232012acc 3042012pub 3042012
cpyrt 2012collab Ceribelli et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Autoantibodies in patients with polymyositis/dermatomyositis (PM/DM) are associated with unique subsets, clinical course and outcome. Anti-MJ antibodies, which recognize the nuclear protein NXP-2/MORC3, are reported in ~25% of juvenile DM. Prevalence and clinical significance of anti-MJ antibodies in adult Italian PM/DM patients were studied.
Methods
Sera from 58 consecutive adult Italian PM/DM patients were analyzed by immunoprecipitation of sup 35S-labeled K562 cells extract, ELISA (anti-MJ, Jo-1), Western blot and indirect immunofluorescence. Clinical associations were analyzed using information from medical charts.
Results
Anti-MJ antibodies were the most prevalent specificity (17%) found mainly in DM (30%, 8 cases) vs 8% of PM (2 cases, it P = 0.02). Comparing 10 anti-MJ (+) vs 48 anti-MJ (-) cases, DM was more common (P = 0.03), and age at onset was younger in anti-MJ (+) (P = 0.0006). In anti-MJ (+), heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were more frequent. None of them had heart or lung involvement, or malignancy. Myopathy in anti-MJ (+) patients responded well to therapy and none of them had elevated CPK at last visit (0% vs 25% in anti-MJ (-)). Only 60% of anti-MJ (+) showed immunofluorescent nuclear dots staining, despite PML localization of NXP-2/MORC3.
Conclusions
Anti-MJ antibodies are the most frequent specificity in our cohort of adult Italian PM/DM. Anti-MJ (+) were associated with young onset DM, calcinosis, no internal organ involvement and good response of myopathy to therapy. Anti-MJ reported in juvenile DM is also found in adult PM/DM, and could be a new useful biomarker.
bdy
Introduction
Myositis-specific autoantibodies (MSAs) are helpful in the diagnosis of polymyositis/dermatomyositis (PM/DM), in identifying distinct clinical subsets, and disease monitoring abbrgrp abbr bid B1 1B2 2B3 3. MSAs, including antibodies to aminoacyl-tRNA synthetases (Jo-1 is the most frequent), anti-p155/140, -CADM-140, -Mi-2, and -SRP are generally found in ~50% of adults with PM/DM 3. Since many patients do not have known MSAs, it is important to characterize additional autoantibodies in PM/DM, and to clarify their clinical significance. A new autoantibody, called anti-MJ, has been identified in juvenile DM (JDM) patients B4 4B5 5, and it was associated with severe muscle weakness, polyarthritis, joint contractures, and intestinal vasculitis 4. In a cohort of Argentine pediatric myositis patients, anti-MJ antibodies were the most prevalent specificity (25% of cases), associated with muscle contracture, atrophy and significant compromise of the functional status B6 6. The target of anti-MJ antibodies was identified as a nuclear protein called NXP-2 B7 7, which plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture 7. NXP-2 (also known as MORC3) B8 8B9 9 localizes in the PML (promyelocytic leukemia) nuclear bodies, where it recruits and activates p53 to induce cellular senescence 8B10 10. The presence of anti-MJ antibodies in adult patients with myositis has been described in an abstract analyzing a British cohort B11 11 and in a recent paper showing a possible link between anti-MJ antibodies and higher risk of malignancy in a Japanese cohort of adult PM/DM patients B12 12. It is also still unclear whether anti-MJ is found in adult PM/DM has same clinical significance as JDM. The aim of our study is to analyze the prevalence and clinical significance of anti-MJ antibodies in a cohort of adult Italian PM/DM patients.
Materials and methods
Patients
Fifty-eight consecutive adult Italian patients with PM/DM, who visited the Rheumatology Unit of Spedali Civili (Brescia, Italy) from 2009 to 2011, were studied. The diagnosis of PM/DM was based on Bohan and Peter criteria B13 13. Clinical information was obtained from medical records. The study was approved by the Institutional Review Board of the hospital. This study meets, and is in compliance with, all ethical standards of medicine, and informed consent was obtained from all patients in accordance with the Declaration of Helsinki.
Immunoprecipitation (IP)
Autoantibodies in sera were screened by IP using 35S-methionine-labeled K562 cell extract B14 14. Specificities of autoantibodies were determined using reference sera.
ELISA
Anti-MJ antibodies were also tested by antigen-capture ELISA B15 15. Briefly, a 96-well plate Immobilizer Amino (Nalge Nunc International, Rochester, NY, USA) was incubated with 50 μl/well of 2 μg/ml mouse monoclonal antibody (mAb) to MORC3 (MBL International, Woburn, MA, USA). Wells were then incubated with K562 cell extract, followed by 1:500 diluted sera, and probed with alkaline phosphatase conjugated mouse mAb anti-human Immunoglobulin G (1:2000 dilution) (Sigma, St. Louis, MO, USA). Anti-Ro52, -La B16 16B17 17, and -Jo-1 (Abazyme, Needham, MA, USA) antibodies were tested by ELISA using recombinant protein at 1:500 serum dilution. Optical density (OD) of the samples was converted into units using a standard curve created by a prototype positive serum 15.
Immunoprecipitation Western Blot (IP-Western)
Candidates for anti-MJ were selected based on immunoprecipitation of a 140 kDa protein that comigrates with MORC3 protein recognized by mAb. Identity of the 140 kDa proteins corresponding to MJ/MORC3/NXP-2 was verified by IP-Western 14. Cell extract from 107 K562 cells was immunoprecipitated by candidate sera. Proteins were fractionated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose filter. The filter was probed with 0.4 μg/ml of anti- MORC3 mouse mAb, followed by horseradish peroxidase ul (HRP) goat anti-mouse IgG (1: 7,500 dilution) (Southern Biotechnology, Birmingham, AL, USA) and developed using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Barrington, IL, USA).
Indirect immunofluorescence (IIF)
Immunofluorescent antinuclear/cytoplasmic antibodies (HEp-2 antinuclear antibody (ANA) slides) (INOVA Diagnostics, San Diego, CA, USA) were tested using a 1:80 dilution of human sera, or 4 μg/ml of anti-MORC3 mAb followed by DyLight 488 donkey IgG F(ab)'sub 2 anti-human IgG or goat anti-mouse IgG (1:100 dilution) (Jackson ImmunoResearch, West Grove, PA, USA). Double staining was also performed using rabbit anti-PML antiserum (1:300 dilution) B18 18 and Alexa 568 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA).
Statistical analyses
All parameters were analyzed by the Mann-Whitney test, or Fisher's exact test between groups using Prism version 5.0 d for Macintosh (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was accepted at P < 0.05.
Results
Demographic data
Fifty-eight patients (43 females, 15 males) with a diagnosis of PM (25 cases), DM (27 cases) or overlap syndrome (6 cases) were studied. In the whole cohort, the patients' age was 52 ± 16 (mean ± standard deviation (SD)) years, the age at disease onset was 43 ± 17.4 years, and the mean follow-up was 56 months (range 1 to 288 months).
Autoantibodies
Prevalence of myositis-related autoantibodies by IP in total PM/DM, DM, and PM is summarized (Table tblr tid T1 1). To our surprise, anti-MJ antibodies were the most prevalent specificity (10/58; 17%) in our PM/DM patients, followed by anti-Jo-1 (10%), -PM-Scl (10%), -U1RNP (7%), -p155/140 (5%), -SRP (5%), -EJ (3%), -Mi-2 (2%), and -OJ (2%). Anti-Ro and anti-Su were found in two cases (3%) each. In 22 patients (38%), no myositis-related autoantibodies were detected. In DM, no patient had anti-Jo-1 antibodies, while in PM it was the most common autoantibody specificity (0% vs 24%; P = 0.0087). Anti-MJ was found only in two cases in PM vs 30% in DM (P = 0.078). Thus, anti-MJ was common in DM, whereas anti-Jo-1 was found only in PM in our cohort. Candidates positive for anti-MJ were initially selected based on the IP of ~140 kD proteins compared with molecular weight markers and the mobility of other known autoantigens (Figure figr fid F1 1A). One of the known myositis autoantigen CADM140/MDA5, which migrates close to MJ, is not detectable in our IP system using K562 cells (Figure 1A, lane MDA5). IP of candidates were then run in SDS-PAGE along with anti-MORC3 mAb IP. If the mobility of a ~140 kD protein immunoprecipitated by the serum is considered same as that of the mAb to MORC3, sera were then tested by IP-Western to verify their identity as MJ. The 10 anti-MJ (+) were confirmed by 1) identical mobility of the 140 kDa protein with MORC3 recognized by mAb (Figure 1B); 2) IP- Western Blot (Figure 1C); and 3) positive results in antigen-capture ELISA (data not shown). None of anti-MJ (+) patients had other MSAs, consistent with previous observations 11, though the study in the Argentinian pediatric cohort reported 2/18 anti-MJ (+) cases with an additional autoantibody specificity 6. NXP-2/MORC3 localizes to nucleus and nuclear dots, known as PML bodies. Number and intensity of PML bodies stained by mAb appear to vary depending on cell type, cell cycle and other factors 10. In HEp-2 ANA slides, mAb to MORC3 stained nuclei in a fine speckled pattern with a few nuclear dots (Figure 1D, panel a). Six (three strong, three weak) of ten anti-MJ (+) sera showed PML bodies nuclear dots in indirect immunofluorescence, (Figure 1D, panels b to d), but PML bodies staining was not clear by other sera (Figure 1D, panel e), indicating that PML bodies immunofluorescence cannot be a good method for screening or confirmation of anti-MJ specificity.
tbl Table 1caption Autoantibody prevalence in a cohort of adult Italian patients with PM/DM.tblbdy cols 5
r
c
center
b Total
(n = 58)
DM
(n = 27)
PM
(n = 25)
Overlap syndrome
(n = 6)
cspan
hr
left
Myositis-related antibodies
Anti-MJ
17% (10)
30% (8)1
8% (2)1
0
Anti-Jo-1
10% (6)
02
24% (6)2
0
Anti-p155/140
5% (3)
7% (2)
0
17% (1)
(DM-SLE-Sjögren's)
Anti-SRP
5% (3)
0
8% (2)
17% (1)
(PM-RA)
Anti-EJ
3% (2)
4% (1)
4% (1)
0
Anti-Mi-2
2% (1)
5% (1)
0
0
Anti-OJ
2% (1)
0
4% (1)
0
Anti-PM/Scl
10%(6)
11% (3)
8% (2)
17% (1)
(DM-SSc)
Anti-U1RNP
7% (4)
4% (1)
4% (1)
33% (2)
(DM-SSc; PM-SLE)
Anti-Ro
3% (2)
0
8% (2)
0
Anti-Su
3% (2)
4% (1)
0
17% (1)
(PM-SSc)
Negative/unknown
38% (22)
41% (11)
40% (10)
17% (1)
(PM-SSc)
tblfn
1P = 0.078; 2P = 0.0087 by Fisher's exact test. DM, dermatomyositis; PM, polymyositis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. SSc, systemic sclerosis.
fig Figure 1Detection of anti-MJ antibodiestext
Detection of anti-MJ antibodies. A. Immunoprecipitation of anti-MJ and other autoantibodies that recognize proteins of similar molecular weight. 35S-methionine labeled K562 cell extract was immunoprecipitated by human sera and analyzed by 8% SDS-PAGE. Anti-MJ serum immunoprecipitates a ~140 kDa protein (arrow), which is different from the mobility of other known autoantigens. Reference sera for anti-MDA5, -MJ, -RNA helicase A (RHA), -p155/140, -Mi-2, OJ, and -RNA polymerases (RNAPs) are shown. NHS, normal human serum. B. Immunoprecipitation of anti-MJ positive sera. 35S-methionine labeled K562 cell extract was immunoprecipitated using mouse anti-MORC3 monoclonal antibody (lane mAb MORC3), human anti-MJ positive sera (lanes 1 to 10) or a normal human serum (NHS), and analyzed by 8% SDS-PAGE. MW, molecular weight marker. C. IP-Western blot of MJ. The identity of the 140 kDa protein as MJ/NXP-2/MORC3 was verified by IP-Western. The MJ protein is indicated with the arrow. MJ, anti-MJ reference serum, lanes 1 to 10, anti-MJ positive Italian samples. NHS, normal human serum. D. Immunofluorescence staining of anti-MJ positive sera. HEp-2 slides were stained with mouse anti-MORC3 monoclonal antibody (a), human anti-MJ (+) sera (b) to (e), or normal human serum (f). Serum dilution, 1:80.
graphic file ar3822-1
Clinical and laboratory features
Since anti-MJ antibodies were the most common in PM/DM (17%) or in DM (30%), clinical features of 10 anti-MJ (+) vs 48 anti-MJ (-) cases were compared (Table T2 2). Anti-MJ (+) patients had younger age of disease onset (25.5 ± 13.8 vs 46.1 ± 15.9 years, mean ± SD, P = 0.0006 by Mann-Whitney) and age at initial visit (37.6 ± 12.0 vs 54.6 ± 14.8 years, P = 0.0023 by Mann-Whitney) compared with anti-MJ (-). Two anti-MJ (+) patients had pediatric onset DM. DM is more common in anti-MJ (+) vs (-) (80% vs 40%, P = 0.03) and no overlap syndrome patients were found in the anti-MJ group (0% vs 13%). In anti-MJ (+) patients, heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were common, but none of them had heart involvement (0% vs 13%), interstitial lung disease (ILD) (0% vs 33%, P = 0.048), or malignancy (0% vs 8%). Myopathy in anti-MJ (+) patients responded well to therapy and elevated creatine phosphokinase (CPK) was not seen in any patient in the last visit (0% vs 25%). Thus, anti-MJ antibodies are associated with DM of young onset with heliotrope rash and calcinosis, without cardiac or lung involvement or overlapping features, and they respond well to therapy. Other clinical aspects, such as arthritis and Raynaud's phenomenon, were not significantly associated with anti-MJ antibodies (Table 2). Clinical characteristics of anti-MJ (+) DM compared to anti-MJ (-) DM were similar to those shown in Table 2, however, they did not reach statistical significance due to small numbers; heliotrope, 75% vs 47%; calcinosis 38% vs 21%; arthritis 25% vs 5%; heart involvement 0% vs 16%; and ILD 0% vs 32%.
Table 2Demographic, clinical and laboratory features in anti-MJ (+) and -MJ (-) patients.4
Anti-MJ (+)
n = 10
Anti-MJ (-)
n = 48
P
1
Demographic data
Male
40%
23%
Mean age, ys (± SD)
37.6 (± 12)
54.6 (± 14.8)
0.0023
DM/PM/overlap (%)
80/20/0
40/48/13
DM 0.03
Clinical and laboratory data
Heliotrope rash
60%
19%
0.01
Calcinosis
30%
8%
0.09
Facial erythema
60%
33%
ns
Gottron's papules
20%
19%
ns
Arthritis
20%
8%
ns
Raynaud's phenomenon
20%
27%
ns
Heart involvement
0%
13%
ns
Interstitial lung disease
0%
33%
0.048
Elevated CPK at last visit
0%
25%
ns
1P values are by Fisher's exact test except for mean age comparison by Mann-Whitney. CPK, creatine phosphokinase; DM/PM, dermatomyositis/polymyositis; ns, not significant; SD, standard deviation.
Discussion
Anti-MJ antibodies were originally described as an abstract in 1997 in a subset of patients with JDM, characterized by severe muscle involvement 4. Anti-MJ was reported in two juvenile DM studies 56, however, its detection in adult PM/DM was only in the form of an abstract 11 until recently, probably due to limited availability of the IP assay of this autoantibody 6. A recent study in an adult Japanese cohort of patients with inflammatory myopathy reported 1.6% (8/507) prevalence of anti-MJ antibodies 12. The most striking aspect observed in this cohort is that anti-MJ positive patients have higher prevalence of malignancy 12. Lack of malignancy in anti-MJ positive patients in our cohort may be related to their young age (average 37.6, range 24 to 56 with only 2/10 over age 50) compared to anti-MJ (+) patients with malignancy reported (ages 54 to 68) 12. This may be similar to a strong association of anti-p155/140 with malignancy in middle- to old-age DM but not in children or young adults B19 19B20 20.
Anti-MJ antibodies are the most prevalent specificity (30% in DM and 17% in PM/DM) in this Italian cohort, followed by other known MSAs, such as anti-Jo-1 and -Mi-2 (Table 1). The prevalence of anti-MJ in the present study is similar to two juvenile DM studies performed in Argentine 6 and UK/Ireland 5. However, our study is the first report on high prevalence of anti-MJ antibodies in a cohort of adult patients with PM/DM vs only 1.6% to 3% in PM/DM or 1.6% to 6% in DM in other reports 1112. This could be due to different ethnic background, techniques, or other reasons. Genetic and/or environmental factors within Caucasians may be important variables, since the prevalence of anti-MJ in American Caucasian adults is low B21 21. In fact, we have recently collected data on the different prevalence of autoantibodies in American Caucasian patients followed in our center, and only 3/73 (4%; all three DM cases) were anti-MJ (+) 21. Two additional anti-MJ (+) cases were identified in a male Hispanic and a male African American DM patient. All of the five American anti-MJ (+) cases have DM features similar to those identified in the Italian cohort. Significant differences within Caucasians in prevalence of scleroderma-related autoantibodies are also reported B22 22. In the same way, another unusual result from our study is the low prevalence of anti-Jo-1 antibodies in the Italian cohort (10%; 6 PM cases), and this is different from the American Caucasian cohort in which anti-Jo-1 was the most frequent specificity 21. It is not clear why these antibodies have such a different prevalence according to the cohorts studied, but also in this case we can hypothesize technical issues or factors involved in the disease pathogenesis.
The majority (8/10) of anti-MJ (+) patients has DM, consistent with data in previous cohorts 5612. Two of our patients with anti-MJ can be classified as juvenile onset DM, but even in the cases of adult onset, anti-MJ (+) patients have younger onset of DM, compared with anti-MJ (-) patients (Table 2). We were also able to identify specific clinical features in our anti-MJ (+) patients, who are characterized by severe skin disease and extensive calcinosis, in the absence of internal organ involvement. Myositis is usually well responsive to treatment, and CPK levels tend to normalize quickly after starting therapy (data not shown). These features are similar to those reported in a cohort of British JDM patients 5, but different from the results of another study in Argentinian juvenile myositis 6, even if the patients of this cohort were defined as 'Argentine Caucasian' due to the Spanish or Italian descent. This study shows that anti-MJ (+) Argentinian patients mainly had muscle contractures, atrophy and significant compromise of the functional status, which are not seen in our Italian cohort of adult PM/DM. The clinical expression of PM/DM associated with anti-MJ (+) antibodies is very peculiar when considering juvenile or adult onset of disease.
The target recognized by anti-MJ antibodies was identified as a protein called NXP-2 (also known as MORC3) 7, involved in transcriptional regulation and activation of the tumor suppressor p53, which prevents cell proliferation by inducing cellular senescence 810. Previous IIF studies showed that NXP-2/MORC3 localizes to both PML nuclear bodies and the nucleoplasm 10. In the IIF study of our cohort of adult Italian PM/DM, only 60% of anti-MJ (+) had nuclear dots consistent with PML bodies. One possible explanation for this partial positivity is that the MJ antigens in PML bodies are not well recognized by certain human autoantibodies, maybe due to denaturation by fixation of ANA slides, poor reactivity with post-translationally modified forms that accumulate in PML bodies 8, interference by other proteins, low titer of the antibodies, or other reasons. The link between NXP-2/MORC3, autoantibody production and disease development is not clear yet. The report of NXP-2 as a SUMO (small ubiquitin-like modifier) target with a possible role in SUMO-mediated transcriptional repression B23 23 is an interesting link with disease mechanisms, because antibodies to SUMO-1 activating enzyme (SAE) have been found in DM B24 24, and the p155 and Mi-2 antigens could also be involved in transcriptional regulation B25 25. Nevertheless, none of anti-MJ positive patients had anti-SAE antibodies that immunoprecipitate 90 kD and 40 kD proteins 24 (Figure 1B).
Conclusions
In summary, anti-MJ antibodies are the most prevalent specificity in our cohort of adult Italian PM/DM patients. This autoantibody is associated with specific clinical features in DM patients, similar to those identified in British patients with JDM, such as severe calcinosis, skin disease but no internal organ involvement 5. IIF cannot be considered as a screening or verification method to identify anti-MJ (+) samples, as only some anti-MJ (+) samples show a pattern consistent with PML nuclear bodies. Nevertheless, anti-MJ should be considered when PML body staining is seen in PM/DM patients. Further studies in various ethnicities and with larger cohorts of patients with anti-MJ antibodies are required to better understand clinical associations, etiopathogenesis and mechanisms of disease development.
Abbreviations
ANA: antinuclear antibody; CPK: creatine phosphokinase; ELISA: enzyme-linked immunosorbent assay; HRP: horseradish peroxidase; IgG: immunoglobulin G; IIF: indirect immunofluorescence; ILD: interstitial lung disease; IP: immunoprecipitation; JDM: juvenile dermatomyositis; kD: kiloDaltons; mAb: monoclonal antibody; MSAs: myositis-specific autoantibodies; OD: optical density; PM/DM: polymyositis/dermatomyositis; PML: promyelocitic leukemia; SAE: small ubiquitin-like modifier-1 activating enzyme; SD: standard deviation; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SUMO: small ubiquitin-like modifier.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AC, SJR, BAP, JYFC, EKLC, and MS carried out the immunoassays. AC, IC, FF, EKLC, and MS designed the study. MS performed the statistical analysis. AC, MF, MT, IC, FF, MQ, and AT enrolled patients for the study, collected information and maintained the database. AC, EKLC, and MS drafted the manuscript. All authors read and approved the final manuscript.
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ack
Acknowledgements
This work was supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
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adult dermatomyositisBetteridgeZEGunawardenaHChinoyHVenkovskyJAllardSAGordonPACooperRGMcHughNJArthritis Rheum200960S304Anti-NXP2 autoantibodies in adult patients with idiopathic inflammatory myopathies: possible association with malignancyIchimuraYMatsushitaTHamaguchiYKajiKHasegawaMTaninoYInokoshiYKawaiKKanekuraTHabuchiMIgarashiASogameRHashimotoTKogaTNishinoAIshiguroNSugimotoNAokiRAndoNAbeTKandaTKuwanaMTakeharaKFujimotoMAnn Rheum Dis2012717101310.1136/annrheumdis-2011-20069722258483Computer-assisted analysis of 153 patients with polymyositis and dermatomyositisBohanAPeterJBBowmanRLPearsonCMMedicine (Baltimore)197756255286Autoantibodies to RNA helicase A: A new serological marker of early lupusYamasakiYNarainSYoshidaHHernandezLBarkerTHahnPCSobelESRichardsHBChanEKLReevesWHSatohMArthritis Rheum20075659660410.1002/art.2232917265494Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune 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(Oxford)200847324328Myositis-specific anti-155/140 autoantibodies target transcriptional intermediary factor 1 family proteinsFujimotoMHamaguchiYKajiKMatsushitaTIchimuraYKoderaMIshiguroNUeda-HayakawaIAsanoYOgawaFFujikawaKMiyagiTMabuchiEHiroseKAkimotoNHattaNTsutsuiKHigashiAIgarashiASeishimaMHasegawaMTakeharaKArthritis Rheum20126451352210.1002/art.3340321987216Distinctive pattern of myositis-specific autoantibody production between American Caucasian and Italian patients with polymyositis/dermatomyositisCeribelliALYCavazzanaIFranceschiniFRossSJChanJYFPauleyBASobelESReevesWHChanEKLetal Arthritis Rheum201163S232Anti-Th/To are common antinucleolar autoantibodies in Italian patients with sclerodermaCeribelliACavazzanaIFranceschiniFAiroPTincaniACattaneoRPauleyBAChanEKSatohMJ Rheumatol2010372071207510.3899/jrheum.10031620682663NXP-2 association with SUMO-2 depends on lysines required for transcriptional repressionRosendorffASakakibaraSLuSKieffEXuanYDiBaccoAShiYGillGProc Natl Acad Sci USA20061035308531310.1073/pnas.0601066103145935116567619Identification of a novel autoantibody directed against small ubiquitin-like modifier activating enzyme in dermatomyositisBetteridgeZGunawardenaHNorthJSlinnJMcHughNArthritis Rheum2007563132313710.1002/art.2286217763420Newly identified autoantibodies: relationship to idiopathic inflammatory myopathy subsets and pathogenesisGunawardenaHBetteridgeZEMcHughNJCurr Opin Rheumatol20082067568010.1097/BOR.0b013e328313bff418946327



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RESEARCHARTICLE OpenAccessAnti-MJ/NXP-2autoantibodyspecificityina cohortofadultItalianpatientswithpolymyositis/ dermatomyositisAngelaCeribelli1,MicaelaFredi2,MaraTaraborelli2,IlariaCavazzana2,FrancoFranceschini2,MarziaQuinzanini2, AngelaTincani2,StevenJRoss1,JasonYFChan3,BradAPauley1,EdwardKLChan1andMinoruSatoh3*AbstractIntroduction: Autoantibodiesinpatientswithpolymyositis/dermatomyositis(PM/DM)areassociatedwithunique subsets,clinicalcourseandoutcome.Anti-MJantibodies,whichrecognizethenuclearproteinNXP-2/MORC3,are reportedin~25%ofjuvenileDM.Prevalenceandclinicalsignificanceofanti-MJantibodiesinadultItalianPM/DM patientswerestudied. Methods: Serafrom58consecutiveadultItalianPM/DMpatientswereanalyzedbyimmunoprecipitationof35SlabeledK562cellsextract,ELISA(anti-MJ,Jo-1),Westernblotandindirectimmunofluorescence.Clinicalassociations wereanalyzedusinginformationfrommedicalcharts. Results: Anti-MJantibodieswerethemostprevalentspecificity(17%)foundmainlyinDM(30%,8cases)vs8%of PM(2cases, P =0.02).Comparing10anti-MJ(+)vs48anti-MJ(-)cases,DMwasmorecommon( P =0.03),and ageatonsetwasyoungerinanti-MJ(+)( P =0.0006).Inanti-MJ(+),heliotroperash( P =0.01)andcalcinosis( P = 0.09)weremorefrequent.Noneofthemhadheartorlunginvolvement,ormalignancy.Myopathyinanti-MJ(+) patientsrespondedwelltotherapyandnoneofthemhadelevatedCPKatlastvisit(0%vs25%inanti-MJ(-)). Only60%ofanti-MJ(+)showedimmunofluorescentnucleardotsstaining,despitePMLlocalizationofNXP-2/ MORC3. Conclusions: Anti-MJantibodiesarethemostfrequentspecificityinourcohortofadultItalianPM/DM.Anti-MJ(+) wereassociatedwithyoungonsetDM,calcinosis,nointernalorganinvolvementandgoodresponseofmyopathy totherapy.Anti-MJreportedinjuvenileDMisalsofoundinadultPM/DM,andcouldbeanewusefulbiomarker.IntroductionMyositis-specificautoantibodies(MSAs)arehelpfulin thediagnosisofpolymyosit is/dermatomyositis(PM/ DM),inidentifyingdistinctclinicalsubsets,anddisease monitoring[1-3].MSAs,includingantibodiestoaminoacyl-tRNAsynthetases(Jo-1isthemostfrequent), anti-p155/140,-CADM-140,-Mi-2,and-SRParegenerallyfoundin~50%ofadultswithPM/DM[3].Since manypatientsdonothaveknownMSAs,itisimportant tocharacterizeadditionalautoantibodiesinPM/DM, andtoclarifytheirclinicalsignificance.Anew autoantibody,calledanti-MJ,hasbeenidentifiedinjuvenileDM(JDM)patients[4,5],anditwasassociatedwith severemuscleweakness,polyarthritis,jointcontractures, andintestinalvasculitis[4].InacohortofArgentine pediatricmyositispatients,anti-MJantibodieswerethe mostprevalentspecificity(25%ofcases),associatedwith musclecontracture,atrophyandsignificantcompromise ofthefunctionalstatus[6].Thetargetofanti-MJantibodieswasidentifiedasanuclearproteincalledNXP-2 [7],whichplaysimportantrolesindiversenuclearfunctions,includingRNAmetabolismandmaintenanceof nucleararchitecture[7].NXP-2(alsoknownas MORC3)[8,9]localizesinthePML(promyelocyticleukemia)nuclearbodies,whereitrecruitsandactivates p53toinducecellularsenescence[8,10].Thepresence *Correspondence:minoru.satoh@medicine.ufl.edu3DivisionofRheumatologyandClinicalImmunology,Departmentof Medicine;DepartmentofPathology,Immunology,andLaboratoryMedicine, UniversityofFlorida,1600SWArcherRoad,Gainesville,FL32610-0221,USA FulllistofauthorinformationisavailableattheendofthearticleCeribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 2012Ceribellietal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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ofanti-MJantibodiesinadultpatientswithmyositishas beendescribedinanabstractanalyzingaBritishcohort [11]andinarecentpapershowingapossiblelink betweenanti-MJantibodiesandhigherriskofmalignancyinaJapanesecohortofadultPM/DMpatients [12].Itisalsostillunclea rwhetheranti-MJisfoundin adultPM/DMhassameclinicalsignificanceasJDM. Theaimofourstudyistoanalyzetheprevalenceand clinicalsignificanceofanti-MJantibodiesinacohortof adultItalianPM/DMpatients.MaterialsandmethodsPatientsFifty-eightconsecutiveadultItalianpatientswithPM/ DM,whovisitedtheRheumatologyUnitofSpedali Civili(Brescia,Italy)from2009to2011,werestudied. ThediagnosisofPM/DMwasbasedonBohanand Petercriteria[13].Clinicalinformationwasobtained frommedicalrecords.Thestudywasapprovedbythe InstitutionalReviewBoardofthehospital.Thisstudy meets,andisincompliancewith,allethicalstandardsof medicine,andinformedconsentwasobtainedfromall patientsinaccordancewiththeDeclarationofHelsinki.Immunoprecipitation(IP)AutoantibodiesinserawerescreenedbyIPusing35Smethionine-labeledK562cell extract[14].Specificities ofautoantibodiesweredeterminedusingreferencesera.ELISAAnti-MJantibodieswerealsotestedbyantigen-capture ELISA[15].Briefly,a96-wellplateImmobilizerAmino (NalgeNuncInternational,Rochester,NY,USA)was incubatedwith50 l/wellof2 g/mlmousemonoclonal antibody(mAb)toMORC3(MBLInternational, Woburn,MA,USA).Wellswerethenincubatedwith K562cellextract,followedby1:500dilutedsera,and probedwithalkalinephosphataseconjugatedmouse mAbanti-humanImmunoglob ulinG(1:2000dilution) (Sigma,St.Louis,MO,USA).Anti-Ro52,-La[16,17], and-Jo-1(Abazyme,Needham,MA,USA)antibodies weretestedbyELISAusingrecombinantproteinat 1:500serumdilution.Opticaldensity(OD)ofthesampleswasconvertedintounitsusingastandardcurve createdbyaprototypepositiveserum[15].Immunoprecipitation-WesternBlot(IP-Western)Candidatesforanti-MJwereselectedbasedonimmunoprecipitationofa140kDaproteinthatcomigrateswith MORC3proteinrecognizedbymAb.Identityofthe140 kDaproteinscorrespondingtoMJ/MORC3/NXP-2was verifiedbyIP-Western[14].Cellextractfrom107K562 cellswasimmunoprecipitatedbycandidatesera.Proteinswerefractionatedby8%sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE)and transferredtoanitrocellulosefilter.Thefilterwas probedwith0.4 g/mlofanti-MORC3mousemAb, followedbyhorseradishperoxidase (HRP)goatantimouseIgG(1:7,500dilution)(SouthernBiotechnology, Birmingham,AL,USA)anddevelopedusingSuperSignal WestFemtochemiluminescentsubstrate(Thermo Scientific,Barrington,IL,USA).Indirectimmunofluorescence(IIF)Immunofluorescentantinucl ear/cytoplasmicantibodies (HEp-2antinuclearantibody(ANA)slides)(INOVA Diagnostics,SanDiego,CA,USA)weretestedusinga 1:80dilutionofhumansera,or4 g/mlofanti-MORC3 mAbfollowedbyDyLight 488donkeyIgGF(ab) 2antihumanIgGorgoatanti-mouseIgG(1:100dilution) (JacksonImmunoResearch,WestGrove,PA,USA). DoublestainingwasalsoperformedusingrabbitantiPMLantiserum(1:300dilution)[18]andAlexa568goat anti-rabbitIgG(Invitrogen,Carlsbad,CA,USA).StatisticalanalysesAllparameterswereanalyzedbytheMann-Whitney test,orFisher sexacttestbetweengroupsusingPrism version5.0dforMacintosh(GraphPadSoftware,Inc., LaJolla,CA,USA).Statisticalsignificancewasaccepted at P <0.05.ResultsDemographicdataFifty-eightpatients(43females,15males)withadiagnosisofPM(25cases),DM(27cases)oroverlapsyndrome(6cases)werestudied.Inthewholecohort,the patients agewas5216(meanstandarddeviation (SD))years,theageatdiseaseonsetwas4317.4years, andthemeanfollow-upwas56months(range1to288 months).AutoantibodiesPrevalenceofmyositis-relatedautoantibodiesbyIPin totalPM/DM,DM,andPMissummarized(Table1). Tooursurprise,anti-MJantibodieswerethemostprevalentspecificity(10/58;17%)inourPM/DMpatients, followedbyanti-Jo-1(10%),-PM-Scl(10%),-U1RNP (7%),-p155/140(5%),-SRP(5%),-EJ(3%),-Mi-2(2%), and-OJ(2%).Anti-Roandanti-Suwerefoundintwo cases(3%)each.In22patients(38%),nomyositisrelatedautoantibodiesweredetected.InDM,nopatient hadanti-Jo-1antibodies,whileinPMitwasthemost commonautoantibodyspecificity(0%vs24%; P = 0.0087).Anti-MJwasfoundonlyintwocasesinPMvs 30%inDM( P =0.078).Thus,anti-MJwascommonin DM,whereasanti-Jo-1wasfoundonlyinPMinour cohort.Candidatespositiv eforanti-MJwereinitiallyCeribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 Page2of6

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selectedbasedontheIPof~140kDproteinscompared withmolecularweightmarkersandthemobilityof otherknownautoantigens(Figure1A).Oneofthe knownmyositisautoantigenCADM140/MDA5,which migratesclosetoMJ,isnotdetectableinourIPsystem usingK562cells(Figure1A,laneMDA5).IPofcandidateswerethenruninSDS-PAGEalongwithantiMORC3mAbIP.Ifthemobilityofa~140kDprotein immunoprecipitatedbytheserumisconsideredsameas thatofthemAbtoMORC3,serawerethentestedby IP-WesterntoverifytheiridentityasMJ.The10antiMJ(+)wereconfirmedby1)identicalmobilityofthe 140kDaproteinwithMORC3recognizedbymAb(Figure1B);2)IP-WesternBlot(Figure1C);and3)positive resultsinantigen-captureELISA(datanotshown). Noneofanti-MJ(+)patientshadotherMSAs,consistentwithpreviousobservations[11],thoughthestudy intheArgentinianpediatriccohortreported2/18antiMJ(+)caseswithanadditional autoantibodyspecificity [6].NXP-2/MORC3localizestonucleusandnuclear dots,knownasPMLbodies.Numberandintensityof PMLbodiesstainedbymAbappeartovarydepending oncelltype,cellcycleandotherfactors[10].InHEp-2 ANAslides,mAbtoMORC3stainednucleiinafine speckledpatternwithafewnucleardots(Figure1D, panela).Six(threestrong,threeweak)oftenanti-MJ (+)serashowedPMLbodiesnucleardotsinindirect immunofluorescence,(Figure1D,panelsbtod),but PMLbodiesstainingwasnotclearbyothersera(Figure 1D,panele),indicatingthatPMLbodiesimmunofluorescencecannotbeagoodmethodforscreeningorconfirmationofanti-MJspecificity.ClinicalandlaboratoryfeaturesSinceanti-MJantibodieswerethemostcommonin PM/DM(17%)orinDM(30%),clinicalfeaturesof10 anti-MJ(+)vs48anti-MJ(-)caseswerecompared (Table2).Anti-MJ(+)patientshadyoungerageofdiseaseonset(25.513.8vs46.115.9years,meanSD, P =0.0006byMann-Whitney)andageatinitialvisit (37.612.0vs54.614.8years, P =0.0023byMannWhitney)comparedwithanti-MJ(-).Twoanti-MJ(+) patientshadpediatriconsetDM.DMismorecommon inanti-MJ(+)vs(-)(80%vs40%, P =0.03)andno overlapsyndromepatientswerefoundintheanti-MJ group(0%vs13%).Inanti-MJ(+)patients,heliotrope rash( P =0.01)andcalcinosis( P =0.09)werecommon, butnoneofthemhadheartinvolvement(0%vs13%), interstitiallungdisease(ILD)(0%vs33%, P =0.048),or malignancy(0%vs8%).Myopathyinanti-MJ(+) patientsrespondedwelltotherapyandelevatedcreatine phosphokinase(CPK)wasnotseeninanypatientinthe lastvisit(0%vs25%).Thus,anti-MJantibodiesareassociatedwithDMofyoungonsetwithheliotroperashand calcinosis,withoutcardiacorlunginvolvementoroverlappingfeatures,andthey respondwelltotherapy. Otherclinicalaspects,suchasarthritisandRaynaud s phenomenon,werenotsignificantlyassociatedwith anti-MJantibodies(Table2).Clinicalcharacteristicsof anti-MJ(+)DMcomparedtoanti-MJ(-)DMweresimilartothoseshowninTable2,however,theydidnot reachstatisticalsignificanceduetosmallnumbers; heliotrope,75%vs47%;calcinosis38%vs21%;arthritis 25%vs5%;heartinvolvement0%vs16%;andILD0% vs32%.DiscussionAnti-MJantibodieswereoriginallydescribedasan abstractin1997inasubsetofpatientswithJDM, characterizedbyseveremuscleinvolvement[4].AntiMJwasreportedintwojuvenileDMstudies[5,6], however,itsdetectioninadultPM/DMwasonlyin theformofanabstract[11]untilrecently,probably duetolimitedavailabilityoftheIPassayofthisautoantibody[6].ArecentstudyinanadultJapanese cohortofpatientswithinflammatorymyopathy reported1.6%(8/507)prevalenceofanti-MJantibodies Table1Autoantibodyprevalenceinacohortofadult ItalianpatientswithPM/DM.Total ( n = 58) DM ( n = 27) PM ( n = 25) Overlap syndrome ( n =6) Myositis-related antibodies Anti-MJ17% (10) 30%(8)18%(2)10 Anti-Jo-110%(6)0224%(6)20 Anti-p155/1405%(3)7%(2)017%(1) (DM-SLESjgren s) Anti-SRP5%(3)08%(2)17%(1) (PM-RA) Anti-EJ3%(2)4%(1)4%(1)0 Anti-Mi-22%(1)5%(1)00 Anti-OJ2%(1)04%(1)0 Anti-PM/Scl10%(6)11%(3)8%(2)17%(1) (DM-SSc) Anti-U1RNP7%(4)4%(1)4%(1)33%(2) (DM-SSc;PMSLE) Anti-Ro3%(2)08%(2)0 Anti-Su3%(2)4%(1)017%(1) (PM-SSc) Negative/unknown 38% (22) 41% (11) 40% (10) 17%(1) (PM-SSc)1P =0.078;2P =0.0087byFisher sexacttest.DM,dermatomyositis;PM, polymyositis;RA,rheumatoidarthritis;SLE,systemiclupuserythematosus.SSc, systemicsclerosis.Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 Page3of6

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[12].Themoststrikingasp ectobservedinthiscohort isthatanti-MJpositivepatientshavehigherprevalence ofmalignancy[12].Lackofmalignancyinanti-MJ positivepatientsinourcohortmayberelatedtotheir youngage(average37.6,range24to56withonly2/10 overage50)comparedtoanti-MJ(+)patientswith malignancyreported(ages54to68)[12].Thismaybe similartoastrongassociationofanti-p155/140with malignancyinmiddle-toold-ageDMbutnotinchildrenoryoungadults[19,20]. Anti-MJantibodiesarethemostprevalentspecificity (30%inDMand17%inPM/DM)inthisItaliancohort, followedbyotherknownMSAs,suchasanti-Jo-1and -Mi-2(Table1).Theprevalenceofanti-MJinthepresentstudyissimilartotwojuvenileDMstudiesperformedinArgentine[6]andUK/Ireland[5].However, ourstudyisthefirstreportonhighprevalenceofantiMJantibodiesinacohortofadultpatientswithPM/ DMvsonly1.6%to3%inPM/DMor1.6%to6%in DMinotherreports[11,12].Thiscouldbeduetodifferentethnicbackground,t echniques,orotherreasons. Geneticand/orenvironmentalfactorswithinCaucasians maybeimportantvariables,sincetheprevalenceofantiMJinAmericanCaucasianadultsislow[21].Infact,we haverecentlycollecteddataonthedifferentprevalence ofautoantibodiesinAmericanCaucasianpatientsfollowedinourcenter,andonly3/73(4%;allthreeDM cases)wereanti-MJ(+)[21].Twoadditionalanti-MJ(+) caseswereidentifiedinamaleHispanicandamale AfricanAmericanDMpatient.AllofthefiveAmerican anti-MJ(+)caseshaveDMfeaturessimilartothose identifiedintheItaliancoho rt.Significantdifferences withinCaucasiansinprevalen ceofscleroderma-related autoantibodiesarealsoreported[22].Inthesameway, Figure1 Detectionofanti-MJantibodies A.Immunoprecipitationofanti-MJandotherautoantibodiesthatrecognizeproteinsof similarmolecularweight .35S-methioninelabeledK562cellextractwasimmunoprecipitatedbyhumanseraandanalyzedby8%SDS-PAGE. Anti-MJserumimmunoprecipitatesa~140kDaprotein(arrow),whichisdifferentfromthemobilityofotherknownautoantigens.Referencesera foranti-MDA5,-MJ,-RNAhelicaseA(RHA),-p155/140,-Mi-2,OJ,and-RNApolymerases(RNAPs)areshown.NHS,normalhumanserum. B. Immunoprecipitationofanti-MJpositivesera .35S-methioninelabeledK562cellextractwasimmunoprecipitatedusingmouseanti-MORC3 monoclonalantibody(lanemAbMORC3),humananti-MJpositivesera(lanes1to10)oranormalhumanserum(NHS),andanalyzedby8% SDS-PAGE.MW,molecularweightmarker. C.IP-WesternblotofMJ .Theidentityofthe140kDaproteinasMJ/NXP-2/MORC3wasverifiedbyIPWestern.TheMJproteinisindicatedwiththearrow.MJ,anti-MJreferenceserum,lanes1to10,anti-MJpositiveItaliansamples.NHS,normal humanserum. D.Immunofluorescencestainingofanti-MJpositivesera .HEp-2slideswerestainedwithmouseanti-MORC3monoclonal antibody (a) ,humananti-MJ(+)sera (b) to (e) ,ornormalhumanserum (f) .Serumdilution,1:80. Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 Page4of6

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anotherunusualresultfromourstudyisthelowprevalenceofanti-Jo-1antibodiesintheItaliancohort(10%; 6PMcases),andthisisdifferentfromtheAmerican Caucasiancohortinwhichanti-Jo-1wasthemostfrequentspecificity[21].Itisnotclearwhytheseantibodieshavesuchadifferentprevalenceaccordingtothe cohortsstudied,butalsointhiscasewecanhypothesize technicalissuesorfactorsinvolvedinthedisease pathogenesis. Themajority(8/10)ofanti-MJ(+)patientshasDM, consistentwithdatainpreviouscohorts[5,6,12].Twoof ourpatientswithanti-MJcanbeclassifiedasjuvenile onsetDM,buteveninthecasesofadultonset,anti-MJ (+)patientshaveyoungeronsetofDM,comparedwith anti-MJ(-)patients(Table2).Wewerealsoableto identifyspecificclinical featuresinouranti-MJ(+) patients,whoarecharacterizedbysevereskindisease andextensivecalcinosis,intheabsenceofinternal organinvolvement.Myositisisusuallywellresponsive totreatment,andCPKlevelstendtonormalizequickly afterstartingtherapy(datanotshown).Thesefeatures aresimilartothosereportedinacohortofBritishJDM patients[5],butdifferentfromtheresultsofanother studyinArgentinianjuvenilemyositis[6],evenifthe patientsofthiscohortweredefinedas ArgentineCaucasian duetotheSpanishorItaliandescent.Thisstudy showsthatanti-MJ(+)Argentinianpatientsmainlyhad musclecontractures,atrophyandsignificantcompromiseofthefunctionalstatus,whicharenotseeninour ItaliancohortofadultPM/DM.Theclinicalexpression ofPM/DMassociatedwithanti-MJ(+)antibodiesis verypeculiarwhenconsideringjuvenileoradultonset ofdisease. Thetargetrecognizedbyanti-MJantibodieswasidentifiedasaproteincalledNXP-2(alsoknownas MORC3)[7],involvedintran scriptionalregulationand activationofthetumorsuppressorp53,whichprevents cellproliferationbyinducingcellularsenescence[8,10]. PreviousIIFstudiesshowedthatNXP-2/MORC3localizestobothPMLnuclearbodiesandthenucleoplasm [10].IntheIIFstudyofourcohortofadultItalianPM/ DM,only60%ofanti-MJ(+)hadnucleardotsconsistentwithPMLbodies.Onepossibleexplanationforthis partialpositivityisthattheMJantigensinPMLbodies arenotwellrecognizedbycertainhumanautoantibodies,maybeduetodenaturationbyfixationofANA slides,poorreactivitywithpo st-translationallymodified formsthataccumulateinPMLbodies[8],interference byotherproteins,lowtiteroftheantibodies,orother reasons.ThelinkbetweenNXP-2/MORC3,autoantibodyproductionanddiseasedevelopmentisnotclear yet.ThereportofNXP-2asaSUMO(smallubiquitinlikemodifier)targetwithapossibleroleinSUMOmediatedtranscriptionalrepression[23]isaninteresting linkwithdiseasemechanisms,becauseantibodiesto SUMO-1activatingenzyme(SAE)havebeenfoundin DM[24],andthep155andMi-2antigenscouldalsobe involvedintranscriptionalregulation[25].Nevertheless, noneofanti-MJpositivepatientshadanti-SAEantibodiesthatimmunoprecipitate90kDand40kDproteins [24](Figure1B).ConclusionsInsummary,anti-MJantibodiesarethemostprevalent specificityinourcohor tofadultItalianPM/DM patients.Thisautoantibodyisassociatedwithspecific clinicalfeaturesinDMpatients,similartothoseidentifiedinBritishpatientswithJDM,suchasseverecalcinosis,skindiseasebutnointernalorganinvolvement[5]. IIFcannotbeconsideredasascreeningorverification methodtoidentifyanti-MJ(+)samples,asonlysome anti-MJ(+)samplesshowapatternconsistentwith PMLnuclearbodies.Nevertheless,anti-MJshouldbe consideredwhenPMLbodystainingisseeninPM/DM patients.Furtherstudiesinv ariousethnicitiesandwith largercohortsofpatientswithanti-MJantibodiesare requiredtobetterunderstandclinicalassociations,etiopathogenesisandmechanismsofdiseasedevelopment.Abbreviations ANA:antinuclearantibody;CPK:creatinephosphokinase;ELISA:enzymelinkedimmunosorbentassay;HRP:horseradishperoxidase;IgG: immunoglobulinG;IIF:indirectimmunofluorescence;ILD:interstitiallung disease;IP:immunoprecipitation;JDM:juveniledermatomyositis;kD: kiloDaltons;mAb:monoclonalantibody;MSAs:myositis-specific autoantibodies;OD:opticaldensity;PM/DM:polymyositis/dermatomyositis; Table2Demographic,clinicalandlaboratoryfeaturesin anti-MJ(+)and-MJ(-)patients.Anti-MJ(+) n =10 Anti-MJ(-) n =48 P1Demographicdata Male40%23% Meanage,ys(SD)37.6(12)54.6(14.8)0.0023 DM/PM/overlap(%)80/20/040/48/13DM0.03 Clinicalandlaboratorydata Heliotroperash60%19%0.01 Calcinosis30%8%0.09 Facialerythema60%33%ns Gottron spapules20%19%ns Arthritis20%8%ns Raynaud sphenomenon20%27%ns Heartinvolvement0%13%ns Interstitiallungdisease0%33%0.048 ElevatedCPKatlastvisit0%25%ns1P valuesarebyFisher sexacttestexceptformeanagecomparisonbyMannWhitney.CPK,creatinephosphokinase;DM/PM,dermatomyositis/polymyositis; ns,notsignificant;SD,standarddeviation.Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 Page5of6

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PML:promyelociticleukemia;SAE:smallubiquitin-likemodifier-1activating enzyme;SD:standarddeviation;SDS-PAGE:sodiumdodecylsulfatepolyacrylamidegelelectrophoresis;SUMO:smallubiquitin-likemodifier. Acknowledgements ThisworkwassupportedinpartbyagrantfromtheLupusResearch InstituteandtheNationalInstitutesofHealthgrantAI47859.Publicationof thisarticlewasfundedinpartbytheUniversityofFloridaOpen-Access PublishingFund. Authordetails1DepartmentofOralBiology,UniversityofFlorida,1395CenterDrive, Gainesville,FL32610-0424,USA.2RheumatologyUnit,A.O.SpedaliCivili, PiazzaleSpedaliCivili1,25123,Brescia,Italy.3DivisionofRheumatologyand ClinicalImmunology,DepartmentofMedicine;DepartmentofPathology, Immunology,andLaboratoryMedicine,UniversityofFlorida,1600SWArcher Road,Gainesville,FL32610-0221,USA. Authors contributions AC,SJR,BAP,JYFC,EKLC,andMScarriedouttheimmunoassays.AC,IC,FF, EKLC,andMSdesignedthestudy.MSperformedthestatisticalanalysis.AC, MF,MT,IC,FF,MQ,andATenrolledpatientsforthestudy,collected informationandmaintainedthedatabase.AC,EKLC,andMSdraftedthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:4December2012Revised:12March2012 Accepted:30April2012Published:30April2012 References1.TargoffIN: Laboratorytestinginthediagnosisandmanagementof idiopathicinflammatorymyopathies. RheumDisClinNorthAm 2002, 28 :859-890,viii. 2.NakashimaR,MimoriT: Clinicalandpathophysiologicalsignificanceof myositis-specificandmyositis-associatedautoantibodies. IntJClin Rheumatol 2010, 5 :523-536. 3.ZongM,LundbergIE: Pathogenesis,classificationandtreatmentof inflammatorymyopathies. NatRevRheumatol 2011, 7 :297-306. 4.OddisCV,FertigN,GoelA,EspadaG,ConfaloneGregorianM,Maldonado CoccoJA,LondinoAV: Clinicalandserologicalcharacterizationofthe anti-MJantibodyinchildhoodmyositis. ArthritisRheum 1997, 40 :S139. 5.GunawardenaH,WedderburnLR,ChinoyH,BetteridgeZE,NorthJ, OllierWE,CooperRG,OddisCV,RamananAV,DavidsonJE,McHughNJ: Autoantibodiestoa140-kdproteininjuveniledermatomyositisare associatedwithcalcinosis. ArthritisRheum 2009, 60 :1807-1814. 6.EspadaG,MaldonadoCoccoJA,FertigN,OddisCV: Clinicalandserologic characterizationofanArgentinepediatricmyositiscohort:identification ofanovelautoantibody(anti-MJ)toa142-kDaprotein. JRheumatol 2009, 36 :2547-2551. 7.TargoffIN,TrieuEP,Levy-NetoM,FertigN,OddisCV: Serawith autoantibodiestotheMJantigenreactwithNXP2. ArthritisRheum 2007, 56 :S787. 8.MimuraY,TakahashiK,KawataK,AkazawaT,InoueN: Two-step colocalizationofMORC3withPMLnuclearbodies. JCellSci 2010, 123 :2014-2024. 9.KimuraY,SakaiF,NakanoO,KisakiO,SugimotoH,SawamuraT,SadanoH, OsumiT: ThenewlyidentifiedhumannuclearproteinNXP-2possesses threedistinctdomains,thenuclearmatrix-binding,RNA-binding,and coiled-coildomains. JBiolChem 2002, 277 :20611-20617. 10.TakahashiK,YoshidaN,MurakamiN,KawataK,IshizakiH,TanakaOkamotoM,MiyoshiJ,ZinnAR,ShimeH,InoueN: Dynamicregulationof p53subnuclearlocalizationandsenescencebyMORC3. MolBiolCell 2007, 18 :1701-1709. 11.BetteridgeZE,GunawardenaH,ChinoyH,VenkovskyJ,AllardSA, GordonPA,CooperRG,McHughNJ: Autoantibodiestothep140 autoantigenNXP-2inadultdermatomyositis. ArthritisRheum 2009, 60 : S304. 12.IchimuraY,MatsushitaT,HamaguchiY,KajiK,HasegawaM,TaninoY, InokoshiY,KawaiK,KanekuraT,HabuchiM,IgarashiA,SogameR, HashimotoT,KogaT,NishinoA,IshiguroN,SugimotoN,AokiR,AndoN, AbeT,KandaT,KuwanaM,TakeharaK,FujimotoM: Anti-NXP2 autoantibodiesinadultpatientswithidiopathicinflammatory myopathies:possibleassociationwithmalignancy. AnnRheumDis 2012, 71 :710-13. 13.BohanA,PeterJB,BowmanRL,PearsonCM: Computer-assistedanalysisof 153patientswithpolymyositisanddermatomyositis. Medicine(Baltimore) 1977, 56 :255-286. 14.YamasakiY,NarainS,YoshidaH,HernandezL,BarkerT,HahnPC,SobelES, RichardsHB,ChanEKL,ReevesWH,SatohM: AutoantibodiestoRNA helicaseA:Anewserologicalmarkerofearlylupus. ArthritisRheum 2007, 56 :596-604. 15.YamasakiY,NarainS,HernandezL,BarkerT,IkedaK,SegalMS,RichardsHB, ChanEK,ReevesWH,SatohM: Autoantibodiesagainstthereplication proteinAcomplexinsystemiclupuserythematosusandother autoimmunediseases. ArthritisResTher 2006, 8 :R111-120. 16.TsengC-E,ChanEKL,MirandaE,GrossM,DiDonatoF,BuyonJP: The52kDproteinasatargetofintermolecularspreadingoftheimmune responsetocomponentsoftheSS-A/Ro-SS-B/Lacomplex. ArthritisRheum 1997, 40 :936-944. 17.ChanEK,HamelJC,BuyonJP,TanEM: Moleculardefinitionandsequence motifsofthe52-kDcomponentofhumanSS-A/Roautoantigen. JClin Invest 1991, 87 :68-76. 18.IshovAM,SotnikovAG,NegorevD,VladimirovaOV,NeffN,KamitaniT, YehET,StraussJF,MaulGG: PMLiscriticalforND10formationand recruitsthePML-interactingproteindaxxtothisnuclearstructurewhen modifiedbySUMO-1. JCellBiol 1999, 147 :221-234. 19.GunawardenaH,WedderburnLR,NorthJ,BetteridgeZ,DunphyJ, ChinoyH,DavidsonJE,CooperRG,McHughNJ: Clinicalassociationsof autoantibodiestoap155/140kDadoubletproteininjuvenile dermatomyositis. Rheumatology(Oxford) 2008, 47 :324-328. 20.FujimotoM,HamaguchiY,KajiK,MatsushitaT,IchimuraY,KoderaM, IshiguroN,Ueda-HayakawaI,AsanoY,OgawaF,FujikawaK,MiyagiT, MabuchiE,HiroseK,AkimotoN,HattaN,TsutsuiK,HigashiA,IgarashiA, SeishimaM,HasegawaM,TakeharaK: Myositis-specificanti-155/140 autoantibodiestargettranscriptionalintermediaryfactor1family proteins. ArthritisRheum 2012, 64 :513-522. 21.CeribelliALY,CavazzanaI,FranceschiniF,RossSJ,ChanJYF,PauleyBA, SobelES,ReevesWH,ChanEKL, etal : DistinctivepatternofmyositisspecificautoantibodyproductionbetweenAmericanCaucasianand Italianpatientswithpolymyositis/dermatomyositis. ArthritisRheum 2011, 63 :S232. 22.CeribelliA,CavazzanaI,FranceschiniF,AiroP,TincaniA,CattaneoR, PauleyBA,ChanEK,SatohM: Anti-Th/Toarecommonantinucleolar autoantibodiesinItalianpatientswithscleroderma. JRheumatol 2010, 37 :2071-2075. 23.RosendorffA,SakakibaraS,LuS,KieffE,XuanY,DiBaccoA,ShiY,GillG: NXP-2associationwithSUMO-2dependsonlysinesrequiredfor transcriptionalrepression. ProcNatlAcadSciUSA 2006, 103 :5308-5313. 24.BetteridgeZ,GunawardenaH,NorthJ,SlinnJ,McHughN: Identificationof anovelautoantibodydirectedagainstsmallubiquitin-likemodifier activatingenzymeindermatomyositis. ArthritisRheum 2007, 56 :3132-3137. 25.GunawardenaH,BetteridgeZE,McHughNJ: Newlyidentified autoantibodies:relationshiptoidiopathicinflammatorymyopathy subsetsandpathogenesis. CurrOpinRheumatol 2008, 20 :675-680.doi:10.1186/ar3822 Citethisarticleas: Ceribelli etal .: Anti-MJ/NXP-2autoantibody specificityinacohortofadultItalianpatientswithpolymyositis/ dermatomyositis. ArthritisResearch&Therapy 2012 14 :R97.Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R97 http://arthritis-research.com/content/14/2/R97 Page6of6


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui ar3822
ji 1478-6354
fm
dochead Research article
bibl
title p Anti-MJ/NXP-2 autoantibody specificity in a cohort of adult Italian patients with polymyositis/dermatomyositis
aug
au id A1 snm Ceribellifnm Angelainsr iid I1 email dott.ceribelli@libero.it
A2 FrediMicaelaI2 elmic83@libero.it
A3 TaraborelliMaramara.taraborelli@libero.it
A4 CavazzanaIlariailariacava@virgilio.it
A5 FranceschiniFrancofranceschini@bresciareumatologia.it
A6 QuinzaniniMarziamarziaquinz@libero.it
A7 TincaniAngelatincani@bresciareumatologia.it
A8 Rossmi JStevenstevenr966@gmail.com
A9 ChanYFJasonI3 jchan89@ufl.edu
A10 PauleyABradbrad_pollie@yahoo.com
A11 ChanKLEdwardechan@dental.ufl.edu
A12 ca yes SatohMinoruminoru.satoh@medicine.ufl.edu
insg
ins Department of Oral Biology, University of Florida, 1395 Center Drive, Gainesville, FL 32610-0424, USA
Rheumatology Unit, A.O. Spedali Civili, Piazzale Spedali Civili 1, 25123, Brescia, Italy
Division of Rheumatology and Clinical Immunology, Department of Medicine; Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 2
fpage R97
url http://arthritis-research.com/content/14/2/R97
xrefbib pubidlist pubid idtype doi 10.1186/ar3822pmpid 22546500
history rec date day 4month 12year 2012revrec 1232012acc 3042012pub 3042012
cpyrt 2012collab Ceribelli et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Autoantibodies in patients with polymyositis/dermatomyositis (PM/DM) are associated with unique subsets, clinical course and outcome. Anti-MJ antibodies, which recognize the nuclear protein NXP-2/MORC3, are reported in ~25% of juvenile DM. Prevalence and clinical significance of anti-MJ antibodies in adult Italian PM/DM patients were studied.
Methods
Sera from 58 consecutive adult Italian PM/DM patients were analyzed by immunoprecipitation of sup 35S-labeled K562 cells extract, ELISA (anti-MJ, Jo-1), Western blot and indirect immunofluorescence. Clinical associations were analyzed using information from medical charts.
Results
Anti-MJ antibodies were the most prevalent specificity (17%) found mainly in DM (30%, 8 cases) vs 8% of PM (2 cases, it P = 0.02). Comparing 10 anti-MJ (+) vs 48 anti-MJ (-) cases, DM was more common (P = 0.03), and age at onset was younger in anti-MJ (+) (P = 0.0006). In anti-MJ (+), heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were more frequent. None of them had heart or lung involvement, or malignancy. Myopathy in anti-MJ (+) patients responded well to therapy and none of them had elevated CPK at last visit (0% vs 25% in anti-MJ (-)). Only 60% of anti-MJ (+) showed immunofluorescent nuclear dots staining, despite PML localization of NXP-2/MORC3.
Conclusions
Anti-MJ antibodies are the most frequent specificity in our cohort of adult Italian PM/DM. Anti-MJ (+) were associated with young onset DM, calcinosis, no internal organ involvement and good response of myopathy to therapy. Anti-MJ reported in juvenile DM is also found in adult PM/DM, and could be a new useful biomarker.
bdy
Introduction
Myositis-specific autoantibodies (MSAs) are helpful in the diagnosis of polymyositis/dermatomyositis (PM/DM), in identifying distinct clinical subsets, and disease monitoring abbrgrp abbr bid B1 1B2 2B3 3. MSAs, including antibodies to aminoacyl-tRNA synthetases (Jo-1 is the most frequent), anti-p155/140, -CADM-140, -Mi-2, and -SRP are generally found in ~50% of adults with PM/DM 3. Since many patients do not have known MSAs, it is important to characterize additional autoantibodies in PM/DM, and to clarify their clinical significance. A new autoantibody, called anti-MJ, has been identified in juvenile DM (JDM) patients B4 4B5 5, and it was associated with severe muscle weakness, polyarthritis, joint contractures, and intestinal vasculitis 4. In a cohort of Argentine pediatric myositis patients, anti-MJ antibodies were the most prevalent specificity (25% of cases), associated with muscle contracture, atrophy and significant compromise of the functional status B6 6. The target of anti-MJ antibodies was identified as a nuclear protein called NXP-2 B7 7, which plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture 7. NXP-2 (also known as MORC3) B8 8B9 9 localizes in the PML (promyelocytic leukemia) nuclear bodies, where it recruits and activates p53 to induce cellular senescence 8B10 10. The presence of anti-MJ antibodies in adult patients with myositis has been described in an abstract analyzing a British cohort B11 11 and in a recent paper showing a possible link between anti-MJ antibodies and higher risk of malignancy in a Japanese cohort of adult PM/DM patients B12 12. It is also still unclear whether anti-MJ is found in adult PM/DM has same clinical significance as JDM. The aim of our study is to analyze the prevalence and clinical significance of anti-MJ antibodies in a cohort of adult Italian PM/DM patients.
Materials and methods
Patients
Fifty-eight consecutive adult Italian patients with PM/DM, who visited the Rheumatology Unit of Spedali Civili (Brescia, Italy) from 2009 to 2011, were studied. The diagnosis of PM/DM was based on Bohan and Peter criteria B13 13. Clinical information was obtained from medical records. The study was approved by the Institutional Review Board of the hospital. This study meets, and is in compliance with, all ethical standards of medicine, and informed consent was obtained from all patients in accordance with the Declaration of Helsinki.
Immunoprecipitation (IP)
Autoantibodies in sera were screened by IP using 35S-methionine-labeled K562 cell extract B14 14. Specificities of autoantibodies were determined using reference sera.
ELISA
Anti-MJ antibodies were also tested by antigen-capture ELISA B15 15. Briefly, a 96-well plate Immobilizer Amino (Nalge Nunc International, Rochester, NY, USA) was incubated with 50 μl/well of 2 μg/ml mouse monoclonal antibody (mAb) to MORC3 (MBL International, Woburn, MA, USA). Wells were then incubated with K562 cell extract, followed by 1:500 diluted sera, and probed with alkaline phosphatase conjugated mouse mAb anti-human Immunoglobulin G (1:2000 dilution) (Sigma, St. Louis, MO, USA). Anti-Ro52, -La B16 16B17 17, and -Jo-1 (Abazyme, Needham, MA, USA) antibodies were tested by ELISA using recombinant protein at 1:500 serum dilution. Optical density (OD) of the samples was converted into units using a standard curve created by a prototype positive serum 15.
Immunoprecipitation Western Blot (IP-Western)
Candidates for anti-MJ were selected based on immunoprecipitation of a 140 kDa protein that comigrates with MORC3 protein recognized by mAb. Identity of the 140 kDa proteins corresponding to MJ/MORC3/NXP-2 was verified by IP-Western 14. Cell extract from 107 K562 cells was immunoprecipitated by candidate sera. Proteins were fractionated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose filter. The filter was probed with 0.4 μg/ml of anti- MORC3 mouse mAb, followed by horseradish peroxidase ul (HRP) goat anti-mouse IgG (1: 7,500 dilution) (Southern Biotechnology, Birmingham, AL, USA) and developed using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Barrington, IL, USA).
Indirect immunofluorescence (IIF)
Immunofluorescent antinuclear/cytoplasmic antibodies (HEp-2 antinuclear antibody (ANA) slides) (INOVA Diagnostics, San Diego, CA, USA) were tested using a 1:80 dilution of human sera, or 4 μg/ml of anti-MORC3 mAb followed by DyLight 488 donkey IgG F(ab)'sub 2 anti-human IgG or goat anti-mouse IgG (1:100 dilution) (Jackson ImmunoResearch, West Grove, PA, USA). Double staining was also performed using rabbit anti-PML antiserum (1:300 dilution) B18 18 and Alexa 568 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA).
Statistical analyses
All parameters were analyzed by the Mann-Whitney test, or Fisher's exact test between groups using Prism version 5.0 d for Macintosh (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was accepted at P < 0.05.
Results
Demographic data
Fifty-eight patients (43 females, 15 males) with a diagnosis of PM (25 cases), DM (27 cases) or overlap syndrome (6 cases) were studied. In the whole cohort, the patients' age was 52 ± 16 (mean ± standard deviation (SD)) years, the age at disease onset was 43 ± 17.4 years, and the mean follow-up was 56 months (range 1 to 288 months).
Autoantibodies
Prevalence of myositis-related autoantibodies by IP in total PM/DM, DM, and PM is summarized (Table tblr tid T1 1). To our surprise, anti-MJ antibodies were the most prevalent specificity (10/58; 17%) in our PM/DM patients, followed by anti-Jo-1 (10%), -PM-Scl (10%), -U1RNP (7%), -p155/140 (5%), -SRP (5%), -EJ (3%), -Mi-2 (2%), and -OJ (2%). Anti-Ro and anti-Su were found in two cases (3%) each. In 22 patients (38%), no myositis-related autoantibodies were detected. In DM, no patient had anti-Jo-1 antibodies, while in PM it was the most common autoantibody specificity (0% vs 24%; P = 0.0087). Anti-MJ was found only in two cases in PM vs 30% in DM (P = 0.078). Thus, anti-MJ was common in DM, whereas anti-Jo-1 was found only in PM in our cohort. Candidates positive for anti-MJ were initially selected based on the IP of ~140 kD proteins compared with molecular weight markers and the mobility of other known autoantigens (Figure figr fid F1 1A). One of the known myositis autoantigen CADM140/MDA5, which migrates close to MJ, is not detectable in our IP system using K562 cells (Figure 1A, lane MDA5). IP of candidates were then run in SDS-PAGE along with anti-MORC3 mAb IP. If the mobility of a ~140 kD protein immunoprecipitated by the serum is considered same as that of the mAb to MORC3, sera were then tested by IP-Western to verify their identity as MJ. The 10 anti-MJ (+) were confirmed by 1) identical mobility of the 140 kDa protein with MORC3 recognized by mAb (Figure 1B); 2) IP- Western Blot (Figure 1C); and 3) positive results in antigen-capture ELISA (data not shown). None of anti-MJ (+) patients had other MSAs, consistent with previous observations 11, though the study in the Argentinian pediatric cohort reported 2/18 anti-MJ (+) cases with an additional autoantibody specificity 6. NXP-2/MORC3 localizes to nucleus and nuclear dots, known as PML bodies. Number and intensity of PML bodies stained by mAb appear to vary depending on cell type, cell cycle and other factors 10. In HEp-2 ANA slides, mAb to MORC3 stained nuclei in a fine speckled pattern with a few nuclear dots (Figure 1D, panel a). Six (three strong, three weak) of ten anti-MJ (+) sera showed PML bodies nuclear dots in indirect immunofluorescence, (Figure 1D, panels b to d), but PML bodies staining was not clear by other sera (Figure 1D, panel e), indicating that PML bodies immunofluorescence cannot be a good method for screening or confirmation of anti-MJ specificity.
tbl Table 1caption Autoantibody prevalence in a cohort of adult Italian patients with PM/DM.tblbdy cols 5
r
c
center
b Total
(n = 58)
DM
(n = 27)
PM
(n = 25)
Overlap syndrome
(n = 6)
cspan
hr
left
Myositis-related antibodies
Anti-MJ
17% (10)
30% (8)1
8% (2)1
0
Anti-Jo-1
10% (6)
02
24% (6)2
0
Anti-p155/140
5% (3)
7% (2)
0
17% (1)
(DM-SLE-Sjögren's)
Anti-SRP
5% (3)
0
8% (2)
17% (1)
(PM-RA)
Anti-EJ
3% (2)
4% (1)
4% (1)
0
Anti-Mi-2
2% (1)
5% (1)
0
0
Anti-OJ
2% (1)
0
4% (1)
0
Anti-PM/Scl
10%(6)
11% (3)
8% (2)
17% (1)
(DM-SSc)
Anti-U1RNP
7% (4)
4% (1)
4% (1)
33% (2)
(DM-SSc; PM-SLE)
Anti-Ro
3% (2)
0
8% (2)
0
Anti-Su
3% (2)
4% (1)
0
17% (1)
(PM-SSc)
Negative/unknown
38% (22)
41% (11)
40% (10)
17% (1)
(PM-SSc)
tblfn
1P = 0.078; 2P = 0.0087 by Fisher's exact test. DM, dermatomyositis; PM, polymyositis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. SSc, systemic sclerosis.
fig Figure 1Detection of anti-MJ antibodiestext
Detection of anti-MJ antibodies. A. Immunoprecipitation of anti-MJ and other autoantibodies that recognize proteins of similar molecular weight. 35S-methionine labeled K562 cell extract was immunoprecipitated by human sera and analyzed by 8% SDS-PAGE. Anti-MJ serum immunoprecipitates a ~140 kDa protein (arrow), which is different from the mobility of other known autoantigens. Reference sera for anti-MDA5, -MJ, -RNA helicase A (RHA), -p155/140, -Mi-2, OJ, and -RNA polymerases (RNAPs) are shown. NHS, normal human serum. B. Immunoprecipitation of anti-MJ positive sera. 35S-methionine labeled K562 cell extract was immunoprecipitated using mouse anti-MORC3 monoclonal antibody (lane mAb MORC3), human anti-MJ positive sera (lanes 1 to 10) or a normal human serum (NHS), and analyzed by 8% SDS-PAGE. MW, molecular weight marker. C. IP-Western blot of MJ. The identity of the 140 kDa protein as MJ/NXP-2/MORC3 was verified by IP-Western. The MJ protein is indicated with the arrow. MJ, anti-MJ reference serum, lanes 1 to 10, anti-MJ positive Italian samples. NHS, normal human serum. D. Immunofluorescence staining of anti-MJ positive sera. HEp-2 slides were stained with mouse anti-MORC3 monoclonal antibody (a), human anti-MJ (+) sera (b) to (e), or normal human serum (f). Serum dilution, 1:80.
graphic file ar3822-1
Clinical and laboratory features
Since anti-MJ antibodies were the most common in PM/DM (17%) or in DM (30%), clinical features of 10 anti-MJ (+) vs 48 anti-MJ (-) cases were compared (Table T2 2). Anti-MJ (+) patients had younger age of disease onset (25.5 ± 13.8 vs 46.1 ± 15.9 years, mean ± SD, P = 0.0006 by Mann-Whitney) and age at initial visit (37.6 ± 12.0 vs 54.6 ± 14.8 years, P = 0.0023 by Mann-Whitney) compared with anti-MJ (-). Two anti-MJ (+) patients had pediatric onset DM. DM is more common in anti-MJ (+) vs (-) (80% vs 40%, P = 0.03) and no overlap syndrome patients were found in the anti-MJ group (0% vs 13%). In anti-MJ (+) patients, heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were common, but none of them had heart involvement (0% vs 13%), interstitial lung disease (ILD) (0% vs 33%, P = 0.048), or malignancy (0% vs 8%). Myopathy in anti-MJ (+) patients responded well to therapy and elevated creatine phosphokinase (CPK) was not seen in any patient in the last visit (0% vs 25%). Thus, anti-MJ antibodies are associated with DM of young onset with heliotrope rash and calcinosis, without cardiac or lung involvement or overlapping features, and they respond well to therapy. Other clinical aspects, such as arthritis and Raynaud's phenomenon, were not significantly associated with anti-MJ antibodies (Table 2). Clinical characteristics of anti-MJ (+) DM compared to anti-MJ (-) DM were similar to those shown in Table 2, however, they did not reach statistical significance due to small numbers; heliotrope, 75% vs 47%; calcinosis 38% vs 21%; arthritis 25% vs 5%; heart involvement 0% vs 16%; and ILD 0% vs 32%.
Table 2Demographic, clinical and laboratory features in anti-MJ (+) and -MJ (-) patients.4
Anti-MJ (+)
n = 10
Anti-MJ (-)
n = 48
P
1
Demographic data
Male
40%
23%
Mean age, ys (± SD)
37.6 (± 12)
54.6 (± 14.8)
0.0023
DM/PM/overlap (%)
80/20/0
40/48/13
DM 0.03
Clinical and laboratory data
Heliotrope rash
60%
19%
0.01
Calcinosis
30%
8%
0.09
Facial erythema
60%
33%
ns
Gottron's papules
20%
19%
ns
Arthritis
20%
8%
ns
Raynaud's phenomenon
20%
27%
ns
Heart involvement
0%
13%
ns
Interstitial lung disease
0%
33%
0.048
Elevated CPK at last visit
0%
25%
ns
1P values are by Fisher's exact test except for mean age comparison by Mann-Whitney. CPK, creatine phosphokinase; DM/PM, dermatomyositis/polymyositis; ns, not significant; SD, standard deviation.
Discussion
Anti-MJ antibodies were originally described as an abstract in 1997 in a subset of patients with JDM, characterized by severe muscle involvement 4. Anti-MJ was reported in two juvenile DM studies 56, however, its detection in adult PM/DM was only in the form of an abstract 11 until recently, probably due to limited availability of the IP assay of this autoantibody 6. A recent study in an adult Japanese cohort of patients with inflammatory myopathy reported 1.6% (8/507) prevalence of anti-MJ antibodies 12. The most striking aspect observed in this cohort is that anti-MJ positive patients have higher prevalence of malignancy 12. Lack of malignancy in anti-MJ positive patients in our cohort may be related to their young age (average 37.6, range 24 to 56 with only 2/10 over age 50) compared to anti-MJ (+) patients with malignancy reported (ages 54 to 68) 12. This may be similar to a strong association of anti-p155/140 with malignancy in middle- to old-age DM but not in children or young adults B19 19B20 20.
Anti-MJ antibodies are the most prevalent specificity (30% in DM and 17% in PM/DM) in this Italian cohort, followed by other known MSAs, such as anti-Jo-1 and -Mi-2 (Table 1). The prevalence of anti-MJ in the present study is similar to two juvenile DM studies performed in Argentine 6 and UK/Ireland 5. However, our study is the first report on high prevalence of anti-MJ antibodies in a cohort of adult patients with PM/DM vs only 1.6% to 3% in PM/DM or 1.6% to 6% in DM in other reports 1112. This could be due to different ethnic background, techniques, or other reasons. Genetic and/or environmental factors within Caucasians may be important variables, since the prevalence of anti-MJ in American Caucasian adults is low B21 21. In fact, we have recently collected data on the different prevalence of autoantibodies in American Caucasian patients followed in our center, and only 3/73 (4%; all three DM cases) were anti-MJ (+) 21. Two additional anti-MJ (+) cases were identified in a male Hispanic and a male African American DM patient. All of the five American anti-MJ (+) cases have DM features similar to those identified in the Italian cohort. Significant differences within Caucasians in prevalence of scleroderma-related autoantibodies are also reported B22 22. In the same way, another unusual result from our study is the low prevalence of anti-Jo-1 antibodies in the Italian cohort (10%; 6 PM cases), and this is different from the American Caucasian cohort in which anti-Jo-1 was the most frequent specificity 21. It is not clear why these antibodies have such a different prevalence according to the cohorts studied, but also in this case we can hypothesize technical issues or factors involved in the disease pathogenesis.
The majority (8/10) of anti-MJ (+) patients has DM, consistent with data in previous cohorts 5612. Two of our patients with anti-MJ can be classified as juvenile onset DM, but even in the cases of adult onset, anti-MJ (+) patients have younger onset of DM, compared with anti-MJ (-) patients (Table 2). We were also able to identify specific clinical features in our anti-MJ (+) patients, who are characterized by severe skin disease and extensive calcinosis, in the absence of internal organ involvement. Myositis is usually well responsive to treatment, and CPK levels tend to normalize quickly after starting therapy (data not shown). These features are similar to those reported in a cohort of British JDM patients 5, but different from the results of another study in Argentinian juvenile myositis 6, even if the patients of this cohort were defined as 'Argentine Caucasian' due to the Spanish or Italian descent. This study shows that anti-MJ (+) Argentinian patients mainly had muscle contractures, atrophy and significant compromise of the functional status, which are not seen in our Italian cohort of adult PM/DM. The clinical expression of PM/DM associated with anti-MJ (+) antibodies is very peculiar when considering juvenile or adult onset of disease.
The target recognized by anti-MJ antibodies was identified as a protein called NXP-2 (also known as MORC3) 7, involved in transcriptional regulation and activation of the tumor suppressor p53, which prevents cell proliferation by inducing cellular senescence 810. Previous IIF studies showed that NXP-2/MORC3 localizes to both PML nuclear bodies and the nucleoplasm 10. In the IIF study of our cohort of adult Italian PM/DM, only 60% of anti-MJ (+) had nuclear dots consistent with PML bodies. One possible explanation for this partial positivity is that the MJ antigens in PML bodies are not well recognized by certain human autoantibodies, maybe due to denaturation by fixation of ANA slides, poor reactivity with post-translationally modified forms that accumulate in PML bodies 8, interference by other proteins, low titer of the antibodies, or other reasons. The link between NXP-2/MORC3, autoantibody production and disease development is not clear yet. The report of NXP-2 as a SUMO (small ubiquitin-like modifier) target with a possible role in SUMO-mediated transcriptional repression B23 23 is an interesting link with disease mechanisms, because antibodies to SUMO-1 activating enzyme (SAE) have been found in DM B24 24, and the p155 and Mi-2 antigens could also be involved in transcriptional regulation B25 25. Nevertheless, none of anti-MJ positive patients had anti-SAE antibodies that immunoprecipitate 90 kD and 40 kD proteins 24 (Figure 1B).
Conclusions
In summary, anti-MJ antibodies are the most prevalent specificity in our cohort of adult Italian PM/DM patients. This autoantibody is associated with specific clinical features in DM patients, similar to those identified in British patients with JDM, such as severe calcinosis, skin disease but no internal organ involvement 5. IIF cannot be considered as a screening or verification method to identify anti-MJ (+) samples, as only some anti-MJ (+) samples show a pattern consistent with PML nuclear bodies. Nevertheless, anti-MJ should be considered when PML body staining is seen in PM/DM patients. Further studies in various ethnicities and with larger cohorts of patients with anti-MJ antibodies are required to better understand clinical associations, etiopathogenesis and mechanisms of disease development.
Abbreviations
ANA: antinuclear antibody; CPK: creatine phosphokinase; ELISA: enzyme-linked immunosorbent assay; HRP: horseradish peroxidase; IgG: immunoglobulin G; IIF: indirect immunofluorescence; ILD: interstitial lung disease; IP: immunoprecipitation; JDM: juvenile dermatomyositis; kD: kiloDaltons; mAb: monoclonal antibody; MSAs: myositis-specific autoantibodies; OD: optical density; PM/DM: polymyositis/dermatomyositis; PML: promyelocitic leukemia; SAE: small ubiquitin-like modifier-1 activating enzyme; SD: standard deviation; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SUMO: small ubiquitin-like modifier.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AC, SJR, BAP, JYFC, EKLC, and MS carried out the immunoassays. AC, IC, FF, EKLC, and MS designed the study. MS performed the statistical analysis. AC, MF, MT, IC, FF, MQ, and AT enrolled patients for the study, collected information and maintained the database. AC, EKLC, and MS drafted the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
This work was supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
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