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Diagnostic utility of anti-Ro52 detection in systemic autoimmunity
  1. Manish Dugar1,
  2. Sally Cox1,
  3. Vidya Limaye1,
  4. Tom Paul Gordon2,
  5. Peter John Roberts-Thomson2
  1. 1Royal Adelaide Hospital, Adelaide, Australia
  2. 2Flinders Medical Centre, Adelaide, Australia
  1. Correspondence to Dr Manish Dugar, Department of Rheumatology, Level 4 Eleanor Harrald Building, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5042, Australia; manish.dugar{at}health.sa.gov.au

Abstract

Objectives To determine the prevalence and diagnostic utility of monospecific anti-Ro52 (defined as an immune response against Ro-52 antigen in the absence of reactivity to Ro-60 antigen) reactivity in selected autoimmune diseases.

Study design Stored diagnostic non-consecutive serum samples obtained from patients with systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), systemic sclerosis, idiopathic inflammatory myopathies (IIM), rheumatoid arthritis, primary biliary cirrhosis and mixed essential cryoglobulinaemia were analysed by line immunoassay to detect the presence of anti-Ro52 and other autoantibodies.

Results Monospecific anti-Ro52 reactivity was found in 51 (12.7%) of the 402 samples tested. Anti-Ro52 was the most common serological marker in patients with IIM (35/147, 23.8%) and co-occurred with anti-Jo1 (10/18, 55.6%; p=0.02). The prevalence of anti-Ro52 reactivity was significantly more than anti-Ro60 reactivity in patients with IIM, systemic sclerosis, primary biliary cirrhosis, mixed essential cryoglobulinemia and pSS. The mean signal intensity of anti-Ro52 reactivity was significantly higher in pSS than SLE and associated with rheumatoid factor positivity. The mean signal intensity of anti-Ro52 correlated with anti-Ro60 and anti-La in pSS and SLE.

Conclusions Monospecific anti-Ro52 reactivity is not disease specific but may be of importance in patients with IIM. Furthermore, as anti-Ro52 reactivity is more prevalent than anti-Ro60 reactivity in certain autoimmune conditions, specific testing for their distinction in clinical practice is recommended.

  • Ro52
  • Ro52 antigen
  • autoimmune diseases
  • myositis
  • diagnostic tests
  • rheumatology

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Introduction

Autoantibodies to Ro-52 are not disease specific and are prevalent in many systemic autoimmune diseases including systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS).1–9 The diagnostic utility of ‘monospecific’ or ‘isolated’ anti-Ro52 autoantibodies (anti-Ro52 reactivity without concomitant anti-Ro60 reactivity) is controversial1 2 and has been shown to have low diagnostic value in evaluation of autoimmune diseases.3 Recent evidence for their clinical relevance comes from a study in patients with idiopathic inflammatory myopathies (IIM), where anti-Ro52 autoantibodies were the most common immune marker detected and associated with a favourable response to prednisone.5 The Ro-52 antigen is a member of the RING/Bbox/coiled-coil tripartite motif protein and induces cellular events through ubiquitination.10 11 In contrast, the Ro-60 antigen binds to misfolded non-coding RNAs and tags them for degradation.12 It has been proposed that detection of single reactivity to Ro-60 and Ro-52 is desirable as they represent two distinct functional autoantibody systems.12

The prevalence of anti-Ro52 autoantibodies without anti-Ro60 has been reported to be 1% of all antinuclear antibody (ANA) positive sera.1 Since anti-Ro52 autoantibodies are not disease specific, we sought to determine their prevalence in various selected autoimmune diseases, as opposed to testing serial laboratory samples sent for detection of antibodies to extractable nuclear antigens (ENAs), which has been reported to be of limited clinical value.2 The aims of our study were to determine the prevalence and diagnostic utility of detecting monospecific anti-Ro52 (defined as an immune response against Ro-52 antigen in the absence of reactivity to Ro-60 antigen) reactivity in selected systemic autoimmune diseases by the Euroline ANA profile line immunoassay and its correlations with other serological markers.

Methods

Design

This cross-sectional observational study was conducted at the Flinders Medical Centre, Adelaide, South Australia, during the period September to December 2007. Stored non-consecutive diagnostic serum samples (n=402) obtained from patients with SLE (n=67), pSS (n=40), systemic sclerosis (SSc, n=106), IIM (n=147), rheumatoid arthritis (RA, n=19), primary biliary cirrhosis (PBC, n=14), mixed essential cryoglobulinaemia (MEC, n=9), and controls (n=15, patients with insect venom allergy but otherwise healthy and with no autoimmune diseases) were analysed by line immunoassay (LIA, Euroline ANA profile assay, Euroimmun AG, Luebeck, Germany) to detect the presence of autoantibodies against different ENAs as described below, with a focus on anti-Ro52 autoantibodies. The study patients were attending the private and public hospital rheumatology clinics in the region and their diagnosis was confirmed by review of medical records and laboratory results in accordance with the current classification criteria being used.13–19 All patients with IIM had a positive muscle biopsy and were further classified as polymyositis (PM), dermatomyositis and inclusion body myositis based on their clinical features, histopathology and investigations.16 The stored serum samples were part of ongoing research projects investigating the epidemiology, clinical features and serology of systemic autoimmune diseases for which ethics approval has been obtained from the Clinical Ethics Committee, Flinders Medical Centre, Adelaide, Australia.

Laboratory tests

The Euroline assay is a qualitative in vitro assay containing immunoblot strips coated with parallel highly purified native and recombinant antigens. This assay can detect IgG autoantibodies to 15 antigens: nRNP, Sm, Ro-60, Ro-52, La, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosomes, histones, ribosomal P-protein, and AMA-M2. The assay was performed according to the recommendations of the manufacturer. The immunoblot strips placed on a work sheet were scanned and evaluated digitally using the EUROline scan. The results were recorded as negative to strong positive based on the numerical value of the signal intensity (SI) displayed. A comparison between the SI of anti-Ro52 reactivity on the LIA and Ro-52 enzyme linked immunosorbent assay (ELISA) (Quanta Lite SS-A 52 ELISA, INOVA Diagnostics Inc, San Diego, California, USA) was performed in 175 non-randomly selected serum samples. In addition, Euroline myositis antigen profile LIA which detects IgG antibodies against Mi-2, Ku, Pm-Scl, Jo-1, PL-7, PL-12 and Ro-52 was performed as per the manufacturer's recommendations on all sera of patients with IIM. Rheumatoid factor (RF) was measured by rate nephelometry (IMMAGE Immunochemistry Systems, Beckman Coulter, Fullerton, California, USA) and a value <20 IU/ml was considered negative.

Data analysis

Data were entered on an Excel worksheet and analysed for prevalence of anti-Ro52 and anti-Ro60 reactivity. To ascertain the diagnostic utility of detecting ‘monospecific’ anti-Ro52 reactivity, the prevalence of anti-Ro52 and anti-Ro60 reactivity, the SI of anti-Ro52 reactivity in different autoimmune diseases studied and correlation of SI of anti-Ro52 reactivity with other serological markers (anti-Ro60 and anti-La reactivity) was examined. The difference in prevalence of anti-Ro52 and anti-Ro60 reactivity was assessed by an identity binomial generalised estimating equation model using SAS statistical package version 9.2 and considered statistically significant if p<0.05. Graph Pad Prism 5.0 software was used to analyse and compare the SI of anti-Ro52 reactivity in SLE and pSS by Mann–Whitney–Wilcoxon non-parametric test and the SI of anti-Ro52 reactivity with other serological markers (anti-Ro60 and anti-La reactivity) in pSS and SLE by the Pearson's correlation test. The measure of agreement between categorical data of anti-Ro52 reactivity in 175 samples tested with LIA and ELISA was evaluated through κ statistics.

Results

Prevalence of anti-Ro52 reactivity

Monospecific anti-Ro52 reactivity was found in 51 (12.7%) of the 402 samples tested. Anti-Ro52 autoantibodies were detected in all disease groups but none in the controls tested (n=15). The prevalence of anti-Ro52 reactivity (table 1) was varied, ranging from 97.5% in pSS to 14.1% in SSc. The prevalence of monospecific anti-Ro52 reactivity (table 1) ranged from 5.9% in SLE to 35.7% in PBC. Patients with triple reactivity (autoantibodies to Ro-60, Ro-52 and La) most frequently had a diagnosis of pSS (64.9%) followed by SLE (32.4%); double reactivity (autoantibodies to Ro-60 and Ro-52) had a diagnosis of SLE (44.9%), IIM (24.5%) and pSS (22.4%); and single reactivity to Ro-60 had a diagnosis of SLE (100%). There were no patients with autoantibodies directed to Ro-60 and La or La alone. Twelve IIM patients with anti-Ro60 reactivity also had reactivity to Ro-52 (table 1). The prevalence of anti-Ro52 reactivity was significantly higher than anti-Ro60 reactivity in patients with IIM (p<0.001), SSc (p<0.001), PBC (p=0.005), MEC (p=0.03) and pSS (p=0.03), but not in patients with SLE (p=0.06) and RA (p=0.13).

Table 1

Autoantibody profile as detected by Euroline ANA profile line immunoassay in different autoimmune conditions

Autoantibodies in patients with IIM

The autoantibody profile of 147 patients with IIM as detected by the Euroline myositis antigen profile LIA is summarised in table 2 and anti-Ro52 was the most common autoantibody against ENA identified in these patients (35/147, 23.8%). Autoantibodies against Jo-1 were detected in 18 patients with IIM and the concurrence with Ro-52 was seen in 10 patients (10/18, 55.6%; p=0.02). Anti-Ro52 was the only autoantibody against ENAs detected in 15 patients with IIM by both Euroline ANA profile and myositis antigen profile LIAs. Furthermore, Ro-52 was the only ENA detected by Euroline ANA profile LIA in three patients with pSS, PBC and MEC, two with RA and one with SLE.

Table 2

Distribution of myositis specific and myositis associated autoantibodies in patients with idiopathic inflammatory myopathies as detected by Euroline myositis antigen and antinuclear antibody (ANA)* profile line immunoassay

SI of anti-Ro52 reactivity

The mean SI of anti-Ro52 on the LIA was significantly higher in pSS compared with SLE (p<0.0001, figure 1). The mean anti-Ro52 SI was significantly higher in patients with pSS who were RF positive (p=0.02, figure 2). The anti-Ro52 SI correlated significantly with anti-Ro60 (p<0.0001, figure 3) and anti-La (p<0.0001, figure 4) levels in patients with pSS and SLE.

Figure 1

Anti-Ro52 signal intensity in different autoimmune disorders as measured by line immunoassay. IIM, idiopathic inflammatory myositis; MEC, mixed essential cryoglobulinaemia; PBC, primary biliary cirrhosis; pSS, primary Sjögren's syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.

Figure 2

Rheumatoid factor positive patients with primary Sjögren's syndrome (pSS) have higher anti-Ro52 signal intensity (p=0.02). RF, rheumatoid factor.

Figure 3

Anti-Ro52 signal intensity correlates with anti-Ro60 in primary Sjögren's syndrome and systemic lupus erythematosus as measured by line immunoassay.

Figure 4

Anti-Ro52 signal intensity correlates with anti-La in primary Sjögren's syndrome and systemic lupus erythematosus as measured by line immunoassay.

Comparison between LIA and ELISA to detect anti-Ro52 reactivity

There was a strong concordance in detection of anti-Ro52 reactivity by Euroline ANA profile LIA and ELISA in the 175 serum samples tested with a κ value of 0.82 (95% confidence interval 0.73 to 0.90, discordant samples=16/175. 9.1%) suggesting almost perfect agreement.

Discussion

Specific testing for anti-Ro52 reactivity in patients with well defined systemic autoimmune conditions as described in our study has not been described earlier. In our hands, the LIA appeared to be a simple and reliable method to detect anti-Ro52 reactivity. The prevalence of monospecific anti-Ro52 reactivity (12.7%) in our selected patient cohort was considerably higher than the previously reported figures of 0.5%2 and 1%1 in serial laboratory samples tested, possibly reflecting selection bias. Triple reactivity to Ro52, Ro60 and La was more commonly found in pSS than SLE as previously reported,8 which may be explained by the higher specificity of anti-La for pSS.

Anti-Ro52 reactivity has been reported to be the most commonly detected myositis associated autoantibody.5 9 Almost one-quarter of our patients with IIM had antibodies to Ro-52. All patients with anti-Ro60 reactivity in IIM had anti-Ro52 reactivity as well, confirming previous reports that the most common anti-Ro specificity in IIM is anti-Ro52.5 9 The prevalence of anti-Ro52 reactivity was significantly higher than anti-Ro60 reactivity (p<0.001) in our patients with IIM, supporting a role for specific testing for anti-Ro52 in this condition. The presence of anti-Ro52 reactivity may thus serve as an additional or isolated serum immune marker in patients with autoimmune conditions. However, further studies are needed to investigate fully the prognostic role of anti-Ro52 detection. The concurrence of antibodies against Jo-1 and Ro-52 was also confirmed in our study as previously reported.4 5 7 9 The concurrence of these autoantibodies is difficult to explain in the absence of demonstrable molecular cross-reactivity or mimicry between these antigens,7 20 but possible co-expression in apoptosis or inflamed tissues20 and a coupling effect (pairing of autoantibody generation for Jo-1 and Ro-52 antigens) during generation of autoimmunity may explain their concordance.

The mean SI of anti-Ro52 reactivity was significantly higher in patients with pSS who were RF positive. The Ro-52 antigen (also called TRIM21) binds to the Fc region of human IgG isotypes 1, 2 and 4 through its carboxy terminal B30.2 domain.21 Viral Fc receptors22 and RF21 (anti-IgG autoantibodies) bind to the same Fc region of human IgG. The significance of this common feature is unknown but it is plausible there is an immunopathogenic relationship between Ro-52 and RF, which generates immune complexes and autoantibodies in predisposed individuals. In our study, stronger anti-Ro52 responses were linked to stronger anti-Ro60 and anti-La responses. This is consistent with an intermolecular epitope spreading to physically linked Ro52/Ro60/La complexes in pSS and SLE.23

The limitations of our study include the cross-sectional design and use of stored non-consecutive diagnostic serum samples.

In summary, we have shown that anti-Ro52 positivity is the more common anti-Ro specificity in certain autoimmune conditions, especially IIM. The presence of anti-Ro52 reactivity in the absence of anti-Ro60 reactivity points to the Ro-52 antigen being immunologically distinct from Ro-60 antigen; therefore, their distinction in clinical practice is recommended. The clinical, prognostic and therapeutic utility of detecting anti-Ro52 reactivity is yet to be established; however, diagnostic utility of anti-Ro52 reactivity as an immune marker looks promising in patients with IIM.

Main messages

  1. The presence of autoantibodies to Ro-52 is not disease-specific.

  2. Anti-Ro52 reactivity is the most common immune marker detected in patients with idiopathic inflammatory myopathy.

  3. The distinction between anti-Ro52 and anti-Ro60 reactivity in clinical practice is recommended.

Current research question

To assess the diagnostic utility of anti-Ro52 detection in systemic autoimmune diseases.

Acknowledgments

We thank Paul Hakendorf and Thomas Sullivan for statistical analysis. INOVA Diagnostics Inc. gifted the two Ro-52 ELISA kits.

References

Footnotes

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the Clinical Ethics Committee, Flinders Medical Centre, Adelaide, Australia.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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