SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

Objective

Autoantibodies against melanoma differentiation–associated protein 5 (MDA-5) have been described in several Asian dermatomyositis (DM) cohorts, often associated with amyopathic DM and rapidly progressive interstitial lung disease (ILD). A recent study of a DM cohort seen at a US dermatology clinic reports that MDA-5 autoantibodies are associated with a unique cutaneous phenotype. Given the widening spectrum of clinical findings, we evaluated the clinical features of anti–MDA-5–positive patients seen at a US myositis referral center.

Methods

One hundred sixty DM patients were screened for MDA-5 autoantibodies by immunoprecipitation and antibody titers were analyzed in longitudinal serum samples. Anti–MDA-5–positive patients were evaluated for the presence of additional myositis autoantibodies. Patient clinical characteristics were compared by retrospective chart review.

Results

MDA-5 was targeted in 11 (6.9%) of 160 patients with DM. Of these, 9 presented with a symmetric polyarthropathy, 6 demonstrated overt clinical myopathy, and 8 had ILD. Eight anti–MDA-5–positive patients exhibited the clinical attributes of the antisynthetase syndrome in the absence of Jo-1 or other antisynthetase autoantibodies. MDA-5 autoantibody titers did not correlate with clinical course.

Conclusion

MDA-5 autoantibodies are found in DM patients presenting with a symmetric polyarthritis, clinically similar to rheumatoid arthritis. These patients often have features of the antisynthetase syndrome, but in the absence of antisynthetase autoantibodies. Most anti–MDA-5–positive patients had overt clinical myopathy and ILD. The latter, while occasionally severe, typically resolved with immunosuppressive therapy. In this cohort, the MDA-5 phenotype is frequently a clinical mimic of the antisynthetase syndrome and is not associated with rapidly progressive ILD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

Dermatomyositis (DM) is a systemic autoimmune disease that affects muscle, lungs, and skin to varying extents in different patients. Like many other systemic autoimmune diseases, patients with DM frequently have specific autoantibodies that are strongly associated with distinct clinical phenotypes, making autoantibodies useful for disease diagnosis and prognosis ([1]). For example, autoantibodies that recognize Mi-2 are associated with a more severe cutaneous form of DM that responds favorably to therapy ([2-4]), whereas antibodies against the aminoacyl–transfer RNA synthetases (aaRS) are associated with a clinical phenotype termed the “antisynthetase syndrome,” consisting of myopathy, fever, interstitial lung disease (ILD), Raynaud's phenomenon, nonerosive arthritis, and mechanics hands ([4-6]).

Autoantibodies against the interferon (IFN)–inducible antigen melanoma differentiation–associated protein 5 (MDA-5) have recently been described in 10–20% of Japanese DM patients. Anti–MDA-5 antibody–positive patients had predominantly amyopathic DM and a high risk for ILD, including rapidly progressive ILD, which was frequently fatal (7–11; for review, see ref.[12]). To date, only one US cohort of patients with DM has been systematically evaluated with regard to the prevalence of MDA-5 autoantibodies and the associated clinical attributes ([13]). These patients were drawn from an academic dermatology practice and, similar to the Japanese study, 13% of DM patients had MDA-5 autoantibodies. Consistent with previous reports, these patients were more likely to be amyopathic and have ILD. In addition, they demonstrated a characteristic cutaneous phenotype consisting of skin ulceration, tender palmar papules, or both.

We sought to determine the prevalence of MDA-5 autoantibodies in a cohort of 160 DM patients evaluated at a US tertiary myositis specialty referral center, and to define the clinical features of these patients. In this cohort, anti–MDA-5 antibody–positive patients often demonstrated hallmark features of the antisynthetase syndrome in the absence of transfer RNA (tRNA) synthetase autoantibodies. ILD was less severe than previously reported ([7-11]), and was absent completely in some patients over years of followup. Therefore, anti–MDA-5–associated myositis should be strongly considered when a patient with features of the antisynthetase syndrome is negative for antisynthetase antibodies.

Box 1. Significance & Innovations

  • Individuals with melanoma differentiation–associated protein 5 (MDA-5) autoantibodies typically present with a symmetric inflammatory polyarthropathy, which was frequently clinically indistinguishable from rheumatoid arthritis. A majority of MDA-5 antibody–positive patients had a clinical myopathy, and interstitial lung disease (ILD), when present, typically resolved with treatment.
  • A majority of MDA-5 antibody–positive patients exhibited ≥3 clinical features of the antisynthetase syndrome, with many demonstrating all 6 clinical features (fever, nonerosive arthritis, myopathy, Raynaud's phenomenon, mechanics hands, and ILD).
  • Jo-1 antibodies, which are strongly associated with the antisynthetase syndrome, were not detected in any MDA-5 autoantibody–positive patients. However, Ro 52 antibodies, which are frequently found in anti–Jo-1–positive patients, were detected in 3 (27%) of 11 of anti–MDA-5 autoantibody–positive patients.
  • MDA-5 antibody titers did not vary significantly over time, nor did they track with clinical course.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

Patients and sera.

One hundred sixty consecutive patients with a Bohan and Peter diagnosis of definite or probable DM ([14, 15]) or a diagnosis of amyopathic or hypomyopathic DM by Sontheimer's criteria ([16]) were evaluated. Patients underwent routine clinical care at the Johns Hopkins Myositis Center between 2006 and 2012 and provided serum samples for research, which were stored at −80°C. Normal sera from 32 donors were also used for this study. Informed consent was obtained from all subjects, and all samples were obtained under the auspices of Johns Hopkins Medicine Institutional Review Board–approved protocols. Clinical information was retrospectively retrieved from all patients by medical record review.

Assessment of muscle disease.

Muscle disease was evaluated clinically by strength assessment using the Medical Research Council 5-point scale in addition to electrophysiologic testing, radiographic assessment by muscle magnetic resonance imaging (MRI), and laboratory testing for serum muscle enzymes, and when deemed clinically appropriate, by muscle biopsy. All patients with longitudinal followup were subsequently reassessed by the same physician.

Assessment of ILD.

Patients with MDA-5 autoantibodies were evaluated for the presence of radiographic findings consistent with ILD, including radiologist-documented ground-glass opacities, reticulation, or honeycombing ([17]). Pulmonary function testing (PFT), when available, was reviewed for the presence of restriction (forced vital capacity [FVC] <80% predicted in the absence of obstruction) and decreased diffusing capacity for carbon monoxide (DLCO; <80% predicted). Patients were considered to have ILD if defining features on computed tomography (CT) were present.

Immunoprecipitation (IP) using 35S-methionine–labeled proteins generated by in vitro transcription/translation (IVTT).

Complementary DNAs encoding full-length human MDA-5, Mi-2, NXP-2, and Ro 60 were used to generate 35S-methionine–labeled proteins by IVTT, per the manufacturer's protocol (Promega). IPs using these products were performed as described ([13]) and the immunoprecipitates were electrophoresed on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels and visualized by fluorography. For densitometry, radiograph films were scanned using an AGFA Arcus II scanner and densities were quantified using Bio-Rad Quantity One software.

IP from radiolabeled HeLa cell extracts.

HeLa cells were cultured using standard protocols. In some cases, cells were treated with purified leukocyte IFNα (Sigma; 1,000 units/ml) for 22 hours before beginning the radiolabeling protocol. HeLa cells were radiolabeled for 2 hours with 35S-methionine/cysteine; where appropriate, IFNα was also added to the radiolabeling incubations (bringing the total treatment time with IFNα to 24 hours). Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS) and precleared with immobilized protein A agarose (Thermo Scientific). IPs were performed by adding 1 μl of patient serum to the lysate (1 hour at 4°C), followed by protein A agarose (20 minutes, 4°C), electrophoresis on 10% SDS-polyacrylamide gels, and visualization of radiolabeled immunoprecipitates by fluorography. An important difference between IPs performed using IVTT proteins and radiolabeled cell lysates is that the latter detects all antibody specificities present in the patient serum (e.g., antisynthetases, MDA-5, Ro 52, etc.), whereas the former reads out antibodies only against the single input protein.

Enzyme-linked immunosorbent assay (ELISA).

Ro 52 and Jo-1 antibodies were assessed by ELISA using commercially available kits (Inova Diagnostics), according to the manufacturer's protocol.

Statistical analysis.

Chi-square (and, where appropriate, Fisher's exact test) and independent 2-sample t-tests were used for comparisons of frequencies. P values of 0.05 or less were considered statistically significant. Each clinical feature was tested separately with the statistical methods mentioned such that the correction for multiple comparisons was not performed. Comparison of MDA-5–positive cohorts was performed using a 1-sample test of proportion.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

Determination of MDA-5 autoantibody prevalence and association with known myositis autoantigens.

In this cohort, patients were referred with a confirmed or suspected diagnosis of myositis and presented with muscle weakness, rash, and/or ILD. The demographics of the 160 DM patients at the time of presentation are shown in Table 1. To determine MDA-5 autoantibody prevalence, we performed IP of IVTT-generated proteins using patient sera. IP of IVTT-radiolabeled proteins is a sensitive method to determine the presence of defined autoantibodies ([13, 18-20]), since autoantibody detection by immunoblotting typically underestimates actual antibody frequency ([21]). MDA-5 antibodies were detected in 11 (6.9%) of 160 patients with DM and in 0 of 32 control sera (all anti–MDA-5 antibody–positive sera [DM 1–11], 2 representative anti–MDA-5 antibody–negative sera [DM 12 and 13], and 2 controls are shown in Figure 1A). Of note, these antibodies were high titer in 9 of 11 sera, whereas the levels in the remaining 2 (DM 4 and DM 8) were low. Longer autoradiographic exposures (results not shown) demonstrated clearly that the levels of IVTT 35S-methionine–MDA-5 immunoprecipitated by these 2 sera were above the background obtained using control sera or anti–MDA-5 antibody–negative sera.

Table 1. Demographic characteristics of dermatomyositis patients (n = 160).
 Value
Sex, no. (%) 
Male42 (26.3)
Female118 (73.8)
Race, no. (%) 
White132 (82.5)
American Indian1 (0.6)
Hispanic or Latino3 (1.9)
Pacific Islander0 (0)
Asian5 (3.1)
African American19 (11.9)
Age at diagnosis, mean ± SD years44.7 ± 15.9
Disease duration, median (range)8 months (1 month to 28.8 years)
image

Figure 1. Detection of melanoma differentiation–associated protein 5 (MDA-5) antibodies using 2 different immunoprecipitation (IP) methods. A, IP using 35S-methionine–labeled MDA-5 generated by in vitro transcription/translation (IVTT). Sera from 160 dermatomyositis (DM) patients and 32 controls were assayed by IVTT IP. All 11 anti–MDA-5 antibody–positive sera (DM 1–11) are shown, as well as 2 representative anti–MDA-5 antibody–negative DM sera (DM 12 and 13) and 2 normal controls (C 1 and 2). On longer autoradiographic exposures, the signals detected by DM 4 and 8 were robust and well above negative controls (results not shown). B, Untreated (lanes denoted “−”) and interferon α (IFNα)–treated (lanes denoted “+”) HeLa cells were radiolabeled and cell lysates were immunoprecipitated with 4 different anti–MDA-5 antibody–positive sera. Representative results from DM 8, 9, 10, and 11 are shown. The open triangle shows MDA-5 and the solid triangle shows Ro 52.

Download figure to PowerPoint

To define whether MDA-5 autoantibodies occur in association with other prominent myositis autoantibodies, we screened anti–MDA-5 antibody–positive patient sera for antibodies against Jo-1 and Ro 52 by ELISA and Mi-2, NXP-2, and Ro 60 by IP of appropriate IVTT products. Jo-1 antibodies, which are strongly associated with the antisynthetase syndrome, were not detected in any of the MDA-5 autoantibody–positive patients, whereas Ro 52 antibodies were detected in 3 (27.3%) of 11 patients. Antibodies against Mi-2, which is found in 10–20% of DM patients ([4, 6]), were absent in all anti–MDA-5 antibody–positive patients, as were Ro 60 antibodies. Low titers of anti–NXP-2 antibodies ([22]) were detected in 1 patient.

As an alternate method to assess whether other antibody specificities are detected in the sera of anti–MDA-5 antibody–positive patients, we immunoprecipitated lysates of radiolabeled HeLa cells (Figure 1B, lanes denoted “−”). This assay is useful for identifying antibody specificities present in patient sera ([23]). Interestingly, the majority of MDA-5 antibody–positive sera were monospecific, with only 1 patient (DM 10) demonstrating another antibody specificity (∼82 kDa) that was detected in untreated HeLa lysates. Given the association of antisynthetase antibodies with ILD ([4-6]), we tested whether this antigen was an aaRS by immunoprecipitating lysates of radiolabeled HeLa cells with reference sera from patients with confirmed tRNA synthetase autoantibodies and comparing the migration patterns of DM 10 and these standards. The unidentified 82 kDa antigen did not comigrate with any of the aaRS references (results not shown).

Since MDA-5 is an IFN-inducible protein that senses double-stranded RNA (dsRNA) in the cytoplasm of cells ([24, 25]), we investigated whether antibodies against other IFN-inducible proteins were found in the anti–MDA-5 antibody–positive sera. Sera from anti–MDA-5 antibody–positive patients were screened by IP of lysates from IFNα-treated, radiolabeled HeLa cells, and the profiles were compared to those obtained in the absence of IFNα treatment. MDA-5 autoantibodies were not detected in the absence of IFNα treatment (Figure 1B, lanes denoted “−”), but were readily detected in lysates of IFNα-treated cells (Figure 1B, lanes denoted “+”). Similar to MDA-5, Ro 52 antibodies were undetectable in the absence of IFNα treatment, but were detected when patient sera were screened against lysates of IFNα-treated cells (Figure 1B). No additional IFN-induced specificities were seen in the MDA-5 antibody–positive patients. Immunoblots performed on unlabeled lysates incubated in the absence or presence of IFNα using a rabbit polyclonal anti–MDA-5 antibody confirmed robust biochemical levels of MDA-5 only in the latter lysates (results not shown). This likely explains our inability to readily detect these antibodies using untreated radiolabeled HeLa lysates as source material for the IPs. Therefore, MDA-5 autoantibodies are found in the absence of antibodies against aaRS, but can occur in association with Ro 52 autoantibodies.

Clinical characteristics of MDA-5 autoantibody–positive patients.

The first symptom in the majority of anti–MDA-5 antibody–positive patients was the appearance of the characteristic DM rash ([14, 15]). Palmar papules and/or a livedoid appearance were noted in 2 of the anti–MDA-5 antibody–positive patients (these data were unavailable in 1 patient) and ulcerations (mouth, nails, fingers, knuckles, thighs, and/or buttocks) were present in 9 (Table 2). Interestingly, 8 anti–MDA-5 antibody–positive patients had a phenotype similar to that of the antisynthetase syndrome with ≥3 clinical features of this syndrome (Table 2). Three patients had all 6 clinical features of this syndrome. Calcinosis was present in 3 patients, with 1 having calcinosis universalis in the absence of other features of myositis with the exception of having a heliotrope rash (Table 2). Two patients presented with elevated liver function tests out of proportion to muscle enzyme elevation in the absence of use of hepatotoxic therapy; however, liver biopsy, when performed, revealed no specific abnormalities, and frequently, liver function test elevations normalized over time.

Table 2. Clinical features of anti–melanoma differentiation–associated protein 5 antibody–positive DM patients*
Patient IDILDWeaknessHigh CKHigh aldolaseArthritisFeverRPCalcinosisUlcerationsMouth painMechanics handsGottron's signHeliotropePalmar erythemaMedicationsa
  1. DM = dermatomyositis; ID = identification; ILD = interstitial lung disease; CK = creatine kinase; RP = Raynaud's phenomenon; Y = yes; U = unknown; N = no; MTX = methotrexate; pred. = prednisone; MMF = mycophenolate mofetil; N/A = not available; HCQ = hydroxychloroquine; AZA = azathioprine; IVIG = intravenous immunoglobulin; RTX = rituximab.

  2. a

    Medication use is indicated for the entire course of followup.

DM 1YYYUYYYNYYYYYNMTX, pred., MMF
DM 2YYYYYNNNYN/ANYYUMTX, pred., HCQ
DM 3YNNNYYNNNNYYNNMTX, pred., AZA
DM 4NYNYYYNNYNNYYNPred., HCQ
DM 5YNYYNNNNNYYYYNPred., MMF, HCQ
DM 6YYNYYYYYYYYYYYMTX, MMF, pred., AZA
DM 7NNNNYNYNYNYYYNMTX, IVIG, HCQ
DM 8NNNNNNNYYNYYYNMMF, pred., IVIG, MTX
DM 9YYNNYNYNYYYYYYPred., MMF, RTX, AZA, IVIG
DM 10YNNYYNNYYNYYYNPred., MMF, IVIG, MTX
DM 11YYYYYYYNYYYYNNMMF, pred., MTX, AZA, tacrolimus
Total positive, %72.754.536.46081.845.545.527.381.85081.810081.820 

We compared the demographics and clinical features of the 11 anti–MDA-5 antibody–positive patients and 149 anti–MDA-5 antibody–negative patients (Table 3). There were no statistically significant differences in sex, race, age at diagnosis, or disease duration. Although some clinical features (e.g., Gottron's sign, Raynaud's phenomenon) did not differ, there were several significant clinical differences between these groups. Anti–MDA-5 antibody–positive patients exhibited an increased prevalence of mechanics hands (81.8% versus 19.0%; P < 0.001), inflammatory arthritis (81.8% versus 26.7%; P < 0.001), and fever (45.5% versus 16.4%; P = 0.017). The arthritis observed in the anti–MDA-5–positive patients was frequently symmetric, present in the small joints of the hands, and associated with morning stiffness, clinically similar to rheumatoid arthritis (RA). These patients had undergone laboratory testing for rheumatoid factor and anti–cyclic citrullinated peptide (anti-CCP) antibodies due to suspicion of RA. Of these, 1 patient had an elevated rheumatoid factor and 1 had anti-CCP antibodies. Only 1 of these patients fulfilled the 2010 American College of Rheumatology diagnostic criteria for RA ([26]). A small erosion on the radial site of the second metacarpal head was seen in 1 patient (DM 10), but only by hand MRI. Conventional radiographs did not demonstrate erosive disease in any of the patients with arthritis.

Table 3. Demographics and clinical characteristics of anti–MDA-5–positive and anti–MDA-5–negative dermatomyositis patients*
 Anti–MDA-5 positive (n = 11)Anti–MDA-5 negative (n = 149)P
  1. Values are the number (percentage) unless otherwise indicated. Clinical data were not available for every anti–melanoma differentiation–associated protein 5 (anti–MDA-5)–negative patient.

  2. a

    N = 148.

  3. b

    N = 146.

  4. c

    N = 145.

  5. d

    N = 147.

Demographics   
Sex  0.94
Male3 (27.3)39 (26.2) 
Female8 (72.7)110 (73.8) 
Race  0.24
White8 (72.7)124 (83.2) 
African American2 (18.2)17 (11.4) 
Asian0 (0)6 (4.0) 
Other1 (9.1)2 (1.3) 
Age at diagnosis, mean years41.444.90.48
Disease duration, median months24.526.30.9
Clinical features   
Gottron's papules/sign11 (100)111 (75)a0.055
Heliotrope rash9 (81.8)71 (48.0)a0.03
Weakness6 (54.5)138 (93.2)a< 0.001
Fever5 (45.5)24 (16.4)b0.017
Inflammatory arthropathy9 (81.8)39 (26.7)b< 0.001
Raynaud's phenomenon5 (45.5)44 (30.3)c0.3
Mechanics hands9 (81.8)28 (19.0)d< 0.001
Interstitial lung disease8 (72.7)17 (11.4)< 0.001
Calcinosis3 (27.3)18 (12.1)0.15

ILD was enriched in anti–MDA-5 antibody–positive patients (72.7% versus 11.4%; P < 0.001), with 8 of 11 patients having evidence of ILD by radiographic criteria (Tables 2 and 3). Of the 8 patients with radiographic evidence of lung involvement on chest CT scan, 6 had reticular changes, 1 had ground-glass opacities only, and 1 had atelectasis. The initial PFT of the cohort of anti–MDA-5 patients is shown in Figure 2A. The median value for FVC was 89.7% predicted, total lung capacity was 87.7% predicted, and DLCO was 70.7% predicted. Serial PFTs were available on 6 patients. Of those with ILD, the majority stabilized or improved with regard to their lung disease once treated with immunosuppressive medications (Figures 2B–E). Only 2 patients to date have demonstrated progressive ILD on immunosuppressive therapy. Both of these patients were found to have Ro 52 autoantibodies. Figure 2F shows a patient representative of this declining course. One additional anti–MDA-5 antibody–positive patient had Ro 52 autoantibodies, but did not have evidence of ILD.

image

Figure 2. Pulmonary function testing (PFT) in the anti–melanoma differentiation–associated protein 5 (anti–MDA-5)–positive cohort. A, Results of initial PFT (where available) performed at or prior to the first clinic visit for all 11 anti–MDA-5–positive patients. Forced vital capacity (FVC), total lung capacity (TLC), and diffusing capacity for carbon monoxide (DLCO) for each individual are shown as the percent predicted. The median value for each test is shown by the solid black bar. BF, Longitudinal PFTs for 5 anti–MDA-5–positive patients demonstrating B, lung function improvement, C and D, a stable pattern within the normal range, E, a stable pattern in the moderate to severely reduced range, and F, declining lung function. The x-axis shows months of followup. DM = dermatomyositis.

Download figure to PowerPoint

Anti–MDA-5 antibody–positive patients demonstrated less weakness (54.5% versus 93.2%; P < 0.001) than the rest of the cohort; however, these patients demonstrated overt clinical myopathy. Muscle enzymes were elevated in 7 of 11 patients, with 3 of these patients exhibiting an isolated elevated aldolase level with a normal creatine kinase value (Table 2). Of those with elevated muscle enzymes, 6 were clinically weak. Five patients with elevated muscle enzymes had a STIR sequence bilateral thigh muscle MRI performed and 1 had a deltoid MRI. Four MRIs demonstrated muscle edema of which 2 were normal, but they were performed >2 years after the initial disease presentation. Electromyogram (EMG) studies were performed on 9 of 11 patients: 4 patients had an irritable myopathy, 3 had a nonirritable myopathy, and 2 who were characterized as amyopathic showed no evidence of myopathy by EMG testing.

Correlation between MDA-5 autoantibody titers and disease activity.

Longitudinal serum samples were available from 6 of the 11 anti–MDA-5 antibody–positive patients. These ranged from 2–5 samples over a followup period of 1–5 years. To determine whether MDA-5 autoantibody levels fluctuated and if they correlated with disease activity, the samples were assayed by IP of 35S-methionine–labeled MDA-5 generated by IVTT. All IPs using sera from the same patient were electrophoresed on a single gel for the purposes of comparison. The images were scanned and density values were calculated for each time point and plotted on an arbitrary scale of anti–MDA-5 units (Figure 3). In general, patients with low-titer MDA-5 autoantibodies had less severe disease. For each patient tested, MDA-5 autoantibody titers did not vary much over the entire course of followup. In contrast, disease course, as determined by a composite score of rash, muscle weakness, ILD, number of immunosuppressive medications, and prednisone dose, generally improved over time. Although all patients received immunosuppressive therapy, the treatments varied, with azathioprine and mycophenolate being the most common (Table 2). Nine of the 11 patients had clear clinical resolution of their symptoms (rash, myositis, and/or lung disease) enabling a decrease in dose or cessation of prednisone, although length of therapy was variable. All 11 patients were able to reduce their steroid dose by ≥50% by the time of their last visit, with 4 patients taking no prednisone at all. Three of those 4 were also taking no other immunosuppressive medications at the time of their last visit.

image

Figure 3. Longitudinal analysis of melanoma differentiation–associated protein 5 (MDA5) autoantibody titers. Anti–MDA5 antibody–positive patients with multiple banked serum samples were screened for MDA5 autoantibodies by in vitro transcription/translation immunoprecipitation. Data were scanned and density values are shown as arbitrary units. Antibody titers at each visit are shown. The x-axis shows the date of sampling. DM = dermatomyositis.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

Phenotype is closely linked with autoantibody status among patients with the idiopathic inflammatory myopathies, including DM ([1]). It is therefore important that as new DM-specific antibodies are described, the associated phenotype(s) need to be carefully defined. Reports of MDA-5 autoantibodies, found in 10–20% of Japanese DM patients, have defined an association with clinically amyopathic disease and a high incidence of ILD; the latter was often rapidly progressive and fatal ([7-11]). Additional reports have confirmed the association with rapidly progressive ILD, but have shown that these antibodies occur in patients with myopathic disease ([27]). More recently, MDA-5 autoantibodies were identified in 13% of patients in a DM cohort seen at the Stanford University Dermatology Clinic ([13]). The association with an increased prevalence of ILD and amyopathic disease was confirmed in this US cohort. Prominent skin manifestations, including ulceration and tender palmar papules, were also noted in these patients ([13]).

Our study of predominantly white (8 [72.7%] of 11) anti–MDA-5 antibody–positive DM patients from a US tertiary myositis referral center contrasts in some regard with previously reported anti–MDA-5 phenotypes ascertained in Asian cohorts, but is similar in many aspects to the report from a dermatology cohort in the US ([7, 10, 13]). The frequency of MDA-5 autoantibodies in our cohort was lower (6.9% versus 13% [13]; P = 0.212); this difference is noteworthy because it exists even when identical methodologies were used for assaying anti–MDA-5 antibodies. Compared to the Japanese cohorts, our patients more often had clinical myopathy (6 [54.5%] of 11), which required a potent immunosuppressive combination treatment regimen (Table 2). Additionally, ILD was generally mild in our cohort of anti–MDA-5 antibody–positive patients, and even those with a relatively fulminant clinical course of ILD were often able to attain sustained clinical remission. Various factors may contribute to these discrepancies, including ethnic differences and subspecialty referral bias. It is conceivable, for example, that amyopathic DM patients are more likely to be evaluated by a dermatologist, given the prominent cutaneous features of the disease, and not referred on for further evaluation at a rheumatology specialty center. Conversely, DM patients presenting with prominent arthritis will likely be evaluated by a rheumatologist rather than a dermatologist, accounting for the potential differences in the clinical spectrum. Additionally, a treatment bias may exist, owing to earlier immunomodulatory therapies (e.g., methotrexate, mycophenolate, or rituximab) in patients with symptomatic polyarthritis and/or myositis, leading to a better prognosis of concomitant ILD.

In our experience, there is a broad spectrum of clinical disease associated with MDA-5 antibodies. Many patients were initially labeled as a rheumatologic “overlap” phenotype owing to their impressive synovitis in combination with Raynaud's phenomenon and myositis. Several were incorrectly diagnosed with RA upon presentation of their disease when arthritis was a prominent component. Our patients expressed many of the defining features of the antisynthetase syndrome, including arthritis, fevers, Raynaud's phenomenon, mechanics hands, and ILD, in the absence of antibodies against aaRS. These patients may be clinically indistinguishable from patients with the antisynthetase syndrome. There is even heterogeneity of clinical features and outcomes among antisynthetase patients, with anti–Jo-1 patients reported to have more severe myositis, joint impairment, and increased risk of cancer, whereas anti–PL-7/PL-12 antibody–positive patients have earlier and more severe ILD and gastrointestinal complications ([28]). It is unclear why autoantibodies directed against distinct cellular proteins (e.g., tRNA synthetases and MDA-5) can manifest in similar phenotypes. Perhaps the antibodies read out pathways that drive pathogenesis (e.g., MDA-5 and Ro 52 are both IFN-induced proteins). Defining the expression of tRNA synthetases and MDA-5 in lung tissue from patients with ILD may provide important clues into the connection between these seemingly unrelated pathways, and are a priority for future studies.

Interestingly, 27% of the anti–MDA-5 antibody–positive patients in our cohort also had Ro 52 autoantibodies; a similar percentage was reported in the study by Fiorentino et al (of note, Jo-1 antibodies were not found in any of the anti–MDA-5 antibody–positive sera in the study by Fiorentino et al, consistent with our current findings) ([13]). Both Ro 52 and MDA-5 are cytoplasmic proteins that are highly induced by IFN. MDA-5 is a member of the retinoic acid–inducible gene 1 (RIG-1)–like receptor family of proteins, which sense viral dsRNA structures in the cytoplasm of cells and induce the production of type I IFNs through activation of IFN regulatory factor 3 (IRF-3) ([24]). Recent studies have shown that RIG-1–like receptor family members RIG-1 and MDA-5 can also be activated by ubiquitin-induced oligomerization to produce type I IFN ([29]). For RIG-1, this process is mediated via the E3 ubiquitin ligase TRIM25 ([30]). Ro 52 (TRIM21) is a member of the TRIM family of proteins, and has documented E3 ubiquitin ligase activity ([31]). Ro 52 has been shown to regulate IFN signaling through interactions with IRF-3 ([32]). Perhaps interactions between Ro 52 and MDA-5 induce the formation of novel complexes that are particularly immunogenic ([33]), possibly explaining the coexistence of these antibodies in a subset of DM patients.

The prognosis and associated clinical features for patients with anti–MDA-5 antibodies have been variable in the medical literature, ranging from those developing rapidly progressive ILD associated with a high mortality to those with more cutaneous features (ulceration, etc.), both with attenuated muscle disease. In our cohort, anti–MDA-5 antibody–positive patients often presented with an inflammatory arthritis that was clinically similar in pattern to RA. MDA-5 autoantibodies should be added to the differential diagnosis when the antisynthetase hallmark features are present in addition to the classic DM rash, especially when antisynthetase autoantibodies are not detected. We have found that those patients with a relatively fulminant clinical course with regard to ILD, myositis, and cutaneous arthritis were often able to obtain sustained clinical remission, in some cases even after discontinuation of immunosuppression. In our experience, although ILD is an associated feature, patients may be able to have complete resolution of their pulmonary disease, suggesting that in at least some anti–MDA-5 antibody–positive patients, the prognosis may be more favorable than both the antisynthetase syndrome and the previous reports of anti–MDA-5 antibody–positive patients.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Casciola-Rosen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Hall, Casciola-Rosen, Danoff, Christopher-Stine.

Acquisition of data. Hall, Casciola-Rosen, Samedy, Werner, Owoyemi, Christopher-Stine.

Analysis and interpretation of data. Hall, Casciola-Rosen, Danoff, Christopher-Stine.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES

The authors would like to thank Po-Han Chen, ScM, from the Johns Hopkins Biostatistics, Epidemiology and Data Management Core, Johns Hopkins Bayview Medical Center, for assistance with the statistical analyses.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. ACKNOWLEDGMENT
  9. REFERENCES
  • 1
    Gunawardena H, Betteridge ZE, McHugh NJ.Myositis-specific autoantibodies: their clinical and pathogenic significance in disease expression.Rheumatology (Oxford)2009;48:60712.
  • 2
    Targoff IN, Reichlin M.The association between Mi-2 antibodies and dermatomyositis.Arthritis Rheum1985;28:796803.
  • 3
    Ghirardello A, Zampieri S, Iaccarino L, Tarricone E, Bendo R, Gambari PF, et al.Anti-Mi-2 antibodies.Autoimmunity2005;38:7983.
  • 4
    Mammen AL.Autoimmune myopathies: autoantibodies, phenotypes and pathogenesis.Nat Rev Neurol2011;7:34354.
  • 5
    Nishikai M, Reichlin M.Heterogeneity of precipitating antibodies in polymyositis and dermatomyositis: characterization of the Jo-1 antibody system.Arthritis Rheum1980;23:8818.
  • 6
    Targoff IN.Laboratory testing in the diagnosis and management of idiopathic inflammatory myopathies.Rheum Dis Clin North Am2002;28:85990.
  • 7
    Sato S, Hirakata M, Kuwana M, Suwa A, Inada S, Mimori T, et al.Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis.Arthritis Rheum2005;52:15716.
  • 8
    Sato S, Hoshino K, Satoh T, Fujita T, Kawakami Y, Fujita T, et al.RNA helicase encoded by melanoma differentiation–associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: association with rapidly progressive interstitial lung disease.Arthritis Rheum2009;60:2193200.
  • 9
    Nakashima R, Imura Y, Kobayashi S, Yukawa N, Yoshifuji H, Nojima T, et al.The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific autoantigen identified by the anti-CADM-140 antibody.Rheumatology (Oxford)2010;49:43340.
  • 10
    Hamaguchi Y, Kuwana M, Hoshino K, Hasegawa M, Kaji K, Matsushita T, et al.Clinical correlations with dermatomyositis-specific autoantibodies in adult Japanese patients with dermatomyositis: a multicenter cross-sectional study.Arch Dermatol2011;147:3918.
  • 11
    Koga T, Fujikawa K, Horai Y, Okada A, Kawashiri SY, Iwamoto N, et al.The diagnostic utility of anti-melanoma differentiation-associated gene 5 antibody testing for predicting the prognosis of Japanese patients with DM.Rheumatology (Oxford)2012;51:127884.
  • 12
    Chaisson NF, Paik J, Orbai AM, Casciola-Rosen L, Fiorentino D, Danoff S, et al.A novel dermato-pulmonary syndrome associated with MDA-5 antibodies: report of 2 cases and review of the literature.Medicine (Baltimore)2012;91:2208.
  • 13
    Fiorentino D, Chung L, Zwerner J, Rosen A, Casciola-Rosen L.The mucocutaneous and systemic phenotype of dermatomyositis patients with antibodies to MDA5 (CADM-140): a retrospective study.J Am Acad Dermatol2011;65:2534.
  • 14
    Bohan A, Peter JB.Polymyositis and dermatomyositis (first of two parts).N Engl J Med1975;292:3447.
  • 15
    Bohan A, Peter JB.Polymyositis and dermatomyositis (second of two parts).N Engl J Med1975;292:4037.
  • 16
    Sontheimer RD.Dermatomyositis: an overview of recent progress with emphasis on dermatologic aspects.Dermatol Clin2002;20:387408.
  • 17
    American Thoracic Society, European Respiratory Society.American Thoracic Society/European Respiratory Society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias: this joint statement of the American Thoracic Society (ATS) and the European Respiratory Society (ERS) was adopted by the ATS Board of Directors, June 2001 and by the ERS Executive Committee, June 2001.Am J Respir Crit Care Med2002;165:277304.
  • 18
    Ulanet DB, Wigley FM, Gelber AC, Rosen A.Autoantibodies against B23, a nucleolar phosphoprotein, occur in scleroderma and are associated with pulmonary hypertension.Arthritis Rheum2003;49:8592.
  • 19
    Pillemer SR, Casciola-Rosen L, Baum BJ, Rosen A, Gelber AC.Centromere protein C is a target of autoantibodies in Sjögren's syndrome and is uniformly associated with antibodies to Ro and La.J Rheumatol2004;31:11215.
  • 20
    Mammen AL, Chung T, Christopher-Stine L, Rosen P, Rosen A, Doering KR, et al.Autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase in patients with statin-associated autoimmune myopathy.Arthritis Rheum2011;63:71321.
  • 21
    Casciola-Rosen LA, Pluta AF, Plotz PH, Cox AE, Morris S, Wigley FM, et al.The DNA mismatch repair enzyme PMS1 is a myositis-specific autoantigen.Arthritis Rheum2001;44:38996.
  • 22
    Ichimura Y, Matsushita T, Hamaguchi Y, Kaji K, Hasegawa M, Tanino Y, et al.Anti-NXP2 autoantibodies in adult patients with idiopathic inflammatory myopathies: possible association with malignancy.Ann Rheum Dis2012;71:7103.
  • 23
    Christopher-Stine L, Casciola-Rosen LA, Hong G, Chung T, Corse AM, Mammen AL.A novel autoantibody recognizing 200-kd and 100-kd proteins is associated with an immune-mediated necrotizing myopathy.Arthritis Rheum2010;62:275766.
  • 24
    Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, Miyagishi M, Taira K, et al.Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity.J Immunol2005;175:28518.
  • 25
    Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature2006;441:1015.
  • 26
    Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO III, et al.2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative.Arthritis Rheum2010;62:256981.
  • 27
    Kang EH, Nakashima R, Mimori T, Kim J, Lee YJ, Lee EB, et al.Myositis autoantibodies in Korean patients with inflammatory myositis: anti-140-kDa polypeptide antibody is primarily associated with rapidly progressive interstitial lung disease independent of clinically amyopathic dermatomyositis.BMC Musculoskelet Disord2010;11:223.
  • 28
    Marie I, Josse S, Decaux O, Dominique S, Diot E, Landron C, et al.Comparison of long-term outcome between anti-Jo1- and anti-PL7/PL12 positive patients with antisynthetase syndrome.Autoimmun Rev2012;11:73945.
  • 29
    Jiang X, Kinch LN, Brautigam CA, Chen X, Du F, Grishin NV, et al.Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response.Immunity2012;36:95973.
  • 30
    Gack MU, Shin YC, Joo CH, Urano T, Liang C, Sun L, et al.TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity.Nature2007;446:91620.
  • 31
    Wada K, Kamitani T.Autoantigen Ro52 is an E3 ubiquitin ligase.Biochem Biophys Res Commun2006;339:41521.
  • 32
    Higgs R, Ni Gabhann J, Ben Larbi N, Breen EP, Fitzgerald KA, Jefferies CA.The E3 ubiquitin ligase Ro52 negatively regulates IFN-β production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.J Immunol2008;181:17806.
  • 33
    Rosen A, Casciola-Rosen L, Ahearn J.Novel packages of viral and self-antigens are generated during apoptosis.J Exp Med1995;181:155761.