Clinical and serological characteristics of 125 Dutch myositis patients

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1 J Neurol (2002) 249 : Steinkopff Verlag 2002 ORIGINAL COMMUNICATION G. J. D. Hengstman R. Brouwer W. T. M. Vree Egberts H. P. Seelig P. J. H. Jongen W. J. van Venrooij B. G. M. van Engelen Clinical and serological characteristics of 125 Dutch myositis patients Myositis specific autoantibodies aid in the differential diagnosis of the idiopathic inflammatory myopathies Received: 29 December 2000 Received in revised form: 24 April 2001 Accepted: 2 May 2001 G. J. D. Hengstman, MD ( ) P. J. H. Jongen, MD, PhD B. G. M. van Engelen, MD, PhD Neuromuscular Centre Nijmegen Institute of Neurology University Medical Centre Nijmegen PO Box HB Nijmegen, The Netherlands Tel.: / Fax:+31-24/ g.hengstman@czzoneu.azn.nl R. Brouwer W. T. M. Vree Egberts W. J. van Venrooij, PhD Department of Biochemistry University of Nijmegen Nijmegen, The Netherlands H. P. Seelig, MD Institute of Immunology and Molecular Genetics Karlsruhe, Germany Abstract The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of systemic diseases that include the familiar disease entities of dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM). A subset of patients has unique autoantibodies which are specific for IIM (myositis specific autoantibodies; MSAs). We studied the clinical and serological characteristics of IIM in 125 Dutch patients. Sera were analysed by immunoblotting, enzyme-linked immunosorbent assay, and immunoprecipitation. The most frequently encountered MSA was the anti-jo-1 autoantibody (20 %), followed by anti-trna His (6 %), anti-mi-2 (6 %), and anti- SRP (4 %). The presence of certain MSAs was clearly associated with specific clinical characteristics. Anti-Jo-1 and anti-trna His were associated with the anti-synthetase syndrome, anti-srp with PM with severe myalgia and arthralgia and a moderate response to immunosuppressive treatment. A novel finding was the presence of anti-mi-2, not only in DM, but also in PM. MSAs were frequently present in DM/PM sera, but were hardly ever detected in the sera of IBM patients. The few IBM patients with MSAs demonstrated a significant response to immunosuppressive treatment. It can be concluded that MSAs define specific clinical syndromes within the spectrum of IIM and that they can assist in the differential diagnosis and treatment plan of these enigmatic disorders by virtually excluding IBM by their presence, and by potentially identifying a subgroup of steroid-responsive IBM patients. Key words Myositis Autoantibody Serology Antibody Introduction The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of systemic diseases characterised by progressive proximal muscle weakness, elevated serum creatine kinase, characteristic electromyographic abnormalities, and inflammatory infiltrates in skeletal muscles [7,9].The three major categories of IIM are dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM) [7]. Various autoantibodies against nuclear and cytoplasmic antigens are found in up to 89 % of patients with IIM [20]. Most of these autoantibodies, referred to as myositis associated autoantibodies (MAAs), are not specific for IIM and are also frequently encountered in rheumatic disorders without the presence of myositis. A subset of patients has unique autoantibodies which are specific for IIM (table 1). Most of these myositis-specific autoantibodies (MSAs) bind to cytoplasmic ribonucleoproteins involved in the process of translation [8, 25]. The known autoantigens include five aminoacyl-transfer RNAs [5, 16, 17, 23], two transfer RNAs [4, 5], proteins of the signal recognition particle [19], and a 220 kda JON 577

2 70 Table 1 Characteristics of myositis specific autoantibodies according to the literature [4, 15, 18, 27] MSAAntigen Frequency Clinical characteristics anti-aminoacyl trna synthetase anti-jo-1 trna His synthetase 20% Anti-synthetase syndrome a anti-pl-7 trna Thr synthetase 2% Anti-synthetase syndrome anti-pl-12 trna Ala synthetase < 1% Anti-synthetase syndrome anti-ej trna Gly synthetase 3% Anti-synthetase syndrome anti-oj trna Ile synthetase 1% Anti-synthetase syndrome anti-trna anti-trna His anti-trna His 7% anti-trna Ala anti-trna Ala Anti-synthetase syndrome miscellaneous anti-srp SRP-complex 3% Severe PM with myalgia and cardiac involvement. Poor response anti-mi-2 unidentified protein 5% Classic mild DM with good response a Severe myositis with arthritis, Raynaud s phenomenon, interstitial lung disease and a moderate response to treatment. protein in a nuclear protein complex containing histone deacetylase and nucleosome remodelling activities [24, 27]. So far, epidemiological and clinical studies suggest that each type of MSA defines a unique IIM syndrome that differs from others in clinical manifestation, disease severity, response to immunosuppressive treatment, prognosis, epidemiological features, and immunoregulation [14, 15, 18]. Although our knowledge of MSAs has increased tremendously since their first description, there are still many questions concerning their clinical applicability that remain unanswered. First of all, while some of the MSA defined syndromes are based on solid grounds (e. g. anti-jo-1), others are highly questionable because they are based on a very small number of patients and the characteristics with which they are associated are very poorly defined (e. g. anti-srp). Secondly, different diagnostic criteria have been used in the different studies making comparison of data very difficult. Some studies used the Bohan and Peter criteria which do not distinguish between PM and IBM [2], while others used more recent criteria which do recognise IBM as a separate diagnosis [15]. Thirdly, the prevalence of MSAs in IIM may vary between geographical regions because of differences in genetic background and/or exposure to environmental factors between different populations [12]. Therefore, data from previous studies can not automatically be applied to the West-European population. In order to answer some of these questions we clinically evaluated, systematically and prospectively, 125 Dutch patients with IIM and subsequently tested their sera for the presence of MAAs and MSAs by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation. Patients and methods Patients and patient evaluation One hundred and twenty-five adult patients with IIM, seen between 1995 and 1997 at the Neuromuscular Centre Nijmegen of the University Medical Centre Nijmegen, were clinically and serologically evaluated. The study was approved by the Ethics Committee of the University Medical Centre Nijmegen and informed consent was obtained prior to inclusion. Patients were classified according to the classification scheme by Bohan and Peter, as modified by Cronin et al. [3, 6]. Diagnoses were made according to established criteria [7, 26]. The studied patient population consisted of 31 patients with DM, 41 with PM, 28 with IBM (14 definite IBM, 13 probable IBM, 1 possible IBM), 19 with myositis associated with another rheumatic disorder (CTM; 6 with DM, 13 with PM), 3 with myositis associated with malignancy (CAM; 1 with DM, 2 with PM), and 3 with a miscellaneous IIM diagnosis (including orbital myositis and granulomatous myositis). Clinical data were obtained by a standardised history taking and physical examination of the patients, and by reviewing their charts. Data were recorded in a standardised format and included: demographic data, symptoms and signs, presence of associated disorders, levels of muscle-associated enzymes at peak severity, results of EMG studies and muscle biopsies (including HLA-ABC and CD4/CD8 staining, and in some cases electromicroscopic studies), and therapeutic experiences. Treatment response was classified as complete (total recovery without clinical evidence of active disease), partial (evidence of clinical improvement short of a complete response), or none (no evidence of clinical improvement). Immunosuppressive treatment consisted most frequently of corticosteroids, azathioprine and/or methotrexate. Serological assays Sera were tested for the presence of following autoantibodies: anti-jo- 1, anti-trna His, anti-mi-2, anti-srp, anti-ro52, anti-ro60, anti-la, anti-pm/scl75, and anti-pm/scl100. Anti-Jo-1 and anti-trna His Anti-synthetase autoantibodies were detected by immunoprecipitation (IP) using HeLa cell S100 extract as described previously [4]. Anti-Jo-1 autoantibodies were distinguished from other anti-synthetase autoantibodies by performing additional immunoblotting and ELISA analyses as described previously [21]. All anti-jo-1 sera were positive in both IP and ELISA or immunoblot. In addition, anti-

3 71 synthetase positive sera were analysed for the presence of anti-trna autoantibodies by IP using total RNA from a deproteinised HeLa cell S100 extract as described previously [4]. Anti-Mi-2 The presence of anti-mi-2 autoantibodies was determined by ELISA. Four overlapping fragments (NT, aa 1 679; NM, aa ;M, aa ; CT, aa ), spanning the complete amino acid sequence of the Mi-2β autoantigen,were used.the cdnas of NT,M,and CT were synthesised essentially as described for fragment NM and ligated into an in house modified pex34 expression vector containing only 10 amino acids of the vectors MS2 polymerase fusion protein [22]. Proteins were expressed in transformed E. coli 537 cells and purified by Ni 2+ -affinity chromatography followed by preparative SDS- PAGE. With 0.2 µg/well of each of the proteins, medium binding microtitre plates (Greiner, Nürtingen, Germany) were coated as described [22]. Antibody screening was performed with 200 µl of diluted serum samples (1:100 in 20 mm Tris-HCl ph 7.4, 150 mm NaCl, 0.05 % NaN 3, 0.05 % Tween-20, 2.5 % bovine serum). After incubation (30 minutes, 37 0 C) and washing (3 times with PBS, 0.01 % Tween-20) plates were incubated (30 minutes, 37 0 C) with 200 µl/well of alkaline phosphatase-conjugated AffiniPure Fc-fragment specific goat antihuman IgG (Jackson Laboratories, West Baltimore Pike, USA) diluted 1:2000 in 20 mm Tris-HCl ph 7.4, 150 mm NaCl, 0.5 % BSA, 0.05 % Tween-20, 1 mm MgCl 2, 0.1 mm Zn-acetate, 0.05 % NaN 3.Bound antibodies were visualised with 38 mm disodiumparanitrophenyl-phosphate, 50 mm diethanolamine ph 9.8, 1 mm MgCl 2, 0.05 % NaN 3.The enzyme reaction was stopped by adding 50 µl 3N NaOH. ODs were measured bichromatically at 492 nm/620 nm (Titertek Multiscan MCC340 Mkll; Flow, Meckenheim, Germany). Tests were regarded positive at 2.5 fold ODs compared with that of three negative control sera. Positive and negative controls as well as patients sera were run in duplicate on every test plate. Anti-SRP Immunoprecipitations were performed as previously described [4]. Immunoprecipitated RNA recovered after phenol/chloroform extraction was spotted on Hybond N+ membranes (Amersham, United Kingdom). After drying and UV crosslinking, the dotblots were hybridised with an antisense RNA probe in (pre)hybridisation mix (6 SSC, 5 Denhardt s, 0.1 % SDS, 100 µg/ml sheared, denatured herring sperm DNA). After overnight incubation at 65 0 C, membranes were washed three times with 0.1 SSC/0.1 % SDS at 65 0 C and exposed to Kodak X-Omat AR imaging film (Eastman Kodak, Rochester, NY). Antisense RNA probes were in vitro transcribed in the presence of 32 P-UTP from linear templates. To detect anti-srp reactivity, a 7SL cdna/psp64 construct, kindly provided by dr. K. Strub (University of Geneva, Switzerland), was used. Antisense 7SL RNA probe was in vitro transcribed from the EcoRI linearised template by SP6 RNA polymerase. Statistical analysis Discontinuous grouped data were analysed by chi square frequency distribution. Fisher s exact test was used in cases of a predicted frequency 2. Continuous data were analysed using the Wilcoxon- Mann-Whitney test. A p-value < 0.05 was considered statistically significant. Patients whose data for a particular variable were not available were excluded from the analysis of that variable, and the number used for the calculation of percentages was adjusted accordingly. Results Myositis specific autoantibodies MSAs were detected in 32 % of the sera. The most frequently encountered MSA was the anti-jo-1 autoantibody which was present in 20 % of the patients.anti-mi- 2 autoantibodies were present in 6 %, anti-srp in 4 %, and anti-trna His autoantibodies in 6 % of the patients. Data from patients with anti-jo-1, anti-mi-2, or antitrna His autoantibodies and from patients without MSAs (MSA neg ) were statistically analysed. Diagnosis (figure 1,2) MSAs were hardly ever encountered in IBM (figure 1). Only three IBM patients had a MSA (1 with anti-jo-1, 2 with anti-mi-2). All three were atypical cases. The patient with the anti-jo-1 autoantibody was diagnosed with a definite IBM according to the criteria of Verschuuren et al. [26]. The patient improved significantly (clinically, chemically, and electrophysiologically) on corticosteroids. Of the two patients with anti-mi-2 autoantibodies, one had a probable IBM with Sjögren s Anti-Ro52, anti-ro60, anti-la Expression and purification of the Ro52, Ro60, and La autoantigens and corresponding ELISAs were performed as described previously [21]. Anti-PM/Scl The presence of anti-pm/scl75 and anti-pm/scl100 autoantibodies was determined by ELISA. For synthesis of the PM/Scl proteins cdnas from HEpG2 cell RNA (ATCC HB 8065) were constructed according to their sequence data (PM/Scl-75: Acc. No. M58460; PM/Scl-100: Acc. No. X66113) and processed essentially as described above for Mi-2 protein synthesis and purification. Each protein (0.4 µg/well of PM/Scl 75 and 0.2 µg/well of PM/Scl-100) was coated on microtitre plates. Coating, blocking, and assay performance followed the Mi-2 autoantibody protocols. Fig. 1 Prevalence of myositis-specific autoantibodies per IIM diagnosis. Numbers given in percent of patients.

4 72 Table 2 Clinical characteristics per serological group Characteristic Serological group Jo-1 trna His Mi-2 MSA neg Statistical significant n=25 n=8 n=7 n=87 difference Fig. 2 Prevalence of myositis-associated autoantibodies per IIM diagnosis. Numbers given in percent of patients. syndrome and the second had a probable IBM with a rheumatoid arthritis. Both demonstrated some improvement on immunosuppressive therapy. Anti-Jo-1 autoantibodies were more frequently seen in CTM than in DM or PM but this did not reach statistical significance. Anti-Mi-2 and anti-srp were seen in DM as well as in PM patients. Anti-tRNA His autoantibodies were seen significantly more frequent in CTM than in DM. With the exception of anti-trna His autoantibodies, which were only found in the presence of anti-jo-1 autoantibodies, no patient had two types of MSA. MSAs were not found in patients with a miscellaneous IIM diagnosis, and only in one patient with CAM (anti-srp). Demographic data (table 2) There were no differences found in the age of onset, disease duration, time interval between clinical onset and time of diagnosis and month or season of onset between the different MSA groups. The sex ratio (female:male) of the Mi-2 and trna His group tended to be lower than that of the Jo-1 and MSA neg group. Clinical characteristics (table 2) Patients with anti-jo-1 autoantibodies had significantly more often fevers, Raynaud s phenomenon, dry mouth/eyes, arthralgia, arthritis, myalgia, dyspnoea and rheumatic disorders than patients without MSAs. They also tended to have more frequent interstitial lung disease and a subacute disease onset. In general, patients with anti-trna His autoantibodies had characteristics comparable to those with just anti-jo-1 autoantibodies. demographic data age at onset (years) sex (F:M) 3.4:1 1.3:1 1.3:1 2.7:1 disease onset subacute Jo-1 vs MSA neg1 insidious Jo-1 vs MSA neg4 signs and symtoms fever Jo-1 vs MSA neg3 weigth loss Jo-1 vs Mi-2 4, MSA neg6 dry mouth/eyes Jo-1 vs MSA neg5 Mi-2 vs MSA neg3 Raynaud s Jo-1 vs MSA neg5 phenomenon arthralgia Jo-1 vs Mi-2 6, MSA neg7 myalgia Jo-1 vs MSA neg6 arthritis Jo-1 vs Mi-2 3, MSA neg7 trna his vs Mi-2 1, MSA neg7 atrophy Jo-1 vs MSA neg2 unexpected falling Jo-1 vs MSA neg4 dyspnoea Jo-1 vs Mi-2 2, MSA neg2 chest pain oedema Jo-1 vs MSA neg1 CTS trna his vs MSA neg1 associated disorders interstitial lung disease rheumatoid Jo-1 vs MSA neg4 disorders lab. investigations creatine kinase a normal EMG Jo-1 vs MSA neg4 trna his vs MSA neg5 treatment response complete partial none Numbers given in percent of patients. a Creatine kinase upon presentation in U/l. Adjusted p values: 1, p < 0.05; 2, p < 0.04; 3, p < 0.03; 4, p < 0.02; 5, p < 0.01; 6, p < 0.005; 7, p < They exhibited more often carpal tunnel syndrome (CTS) than patients without MSAs. Patients with anti- Mi-2 autoantibodies had more weight loss compared to patients with anti-jo-1 autoantibodies and significantly more often dry mouth/eyes and chest pain than patients without MSAs. In all anti-mi-2 positive patients the chest pain was atypical and there were no clinical signs of coronary heart disease or cardiomyopathy. Creatine kinase levels tended to be lower in the group without MSAs compared with those in the anti-jo-1, antitrna His, and anti-mi-2 group. The other muscle-associated enzymes exhibited a similar difference. A normal

5 73 EMG was significantly more frequently seen in patients with anti-jo-1 and anti-trna His autoantibodies than in patients without MSAs. There were no significant differences found in responses to immunosuppressive treatment between the different MSA groups. Patients with MSAs tended to have a slightly better response than patients without MSAs. Anti-SRP Autoantibodies to SRP were found in five patients, four with PM and 1 with DM. Four of the five patients were women,and one was diagnosed with breast cancer at the time of diagnosis of myositis. All patients had general signs of systemic disease, severe myalgia and arthralgia, a disease onset after the age of 40 years, creatine kinase levels between 5000 and 10,000 U/l, and a moderate response to immunosuppressive treatment. Cardiac involvement, investigated by clinical examination and laboratory investigations including electrocardiography and CKMB-ratios, was absent in all five. Two patients died within a year of diagnosis, one with pulmonary complications and one with the complications of her cancer. Myositis associated autoantibodies MAAs were found in all diagnostic groups, including IBM (figure 2). Anti-Ro52 autoantibodies occurred significantly more frequently in patients with anti-jo-1 or anti-trna His autoantibodies than in patients without MSAs (74 % and 62 % respectively versus 14 %). No associations were found between MAAs and specific demographic data or clinical characteristics. Discussion Table 3 Characteristics of myositis specific autoantibodies in 125 Dutch myositis patients MSAFrequency Clinical characteristics anti-jo-1 20 % Anti-synthetase syndrome a anti-trna His 6 % Anti-synthetase syndrome with carpal tunnel syndrome anti-srp 4 % Severe PM with myalgia and arthralgia. Moderate response anti-mi-2 6 % DM and PM with dry eyes/mouth and atypical chest pain a Myositis with arthritis, Raynaud s phenomenon, interstitial lung disease and a moderate response to treatment. The prevalence of MSAs and MAAs in this study was similar to those in other large series (table 3) [2, 10, 13, 15]. Clinical differences between patients with and without MSAs could in part be explained by the fact that almost all IBM patients, with their distinct clinical phenotype, were included in the latter group. This explained partially the virtual lack of symptoms of systemic disease, the lower levels of muscle-associated enzymes, and the worse response to treatment in the group without MSAs. The presence of the anti-jo-1 autoantibody was clearly associated with the anti-synthetase syndrome: Raynaud s phenomenon, arthritis, myalgia, and interstitial lung disease. Autoantibodies against trna His only occurred, as previously described, in the presence of anti-jo-1 autoantibodies [4].The cause of the generation of these autoantibodies and the reason for their association with anti-jo-1 autoantibodies are unclear but may involve epitope spreading [4]. The primary response may be directed against the protein component of the trna-trna synthetase complex, while prolonged exposure to the Jo- 1 antigen may induce an antibody response to additional portions of the antigen complex, including accessible sites of the trna molecule [4]. Patients with these autoantibodies had, with the exception of a higher prevalence of CTS, the same clinical characteristics as patients with just anti-jo-1 autoantibodies. Anti-Mi-2 autoantibodies were, unlike previous descriptions [15, 18], not associated with a classic DM and not with a good response to immunosuppression. In our patient population anti-mi-2 autoantibodies were seen in DM as well as in PM and were clinically associated with dry eyes/mouth (only two of the 14 patients met the diagnostic criteria for Sjögren s syndrome) and atypical chest pain without signs of cardiac involvement. The detection of anti-mi-2 autoantibodies in patients with PM might be explained by the increased sensitivity of the extended ELISA we used in this study compared to the techniques used by others. Autoantibodies to SRP were associated with PM with severe myalgia and arthralgia, and a moderate response to treatment. In contrast to other reports [15, 18] no cardiac abnormalities were found in this group. No associations were found between the presence of MAAs and specific clinical characteristics. Anti-Ro52 autoantibodies were, as previously described, clearly associated with the anti-jo-1 autoantibody [21]. It is not known what the role of anti-ro52 autoantibodies is in IIM and what causes their association with anti-jo-1. The overt association between the two does suggest that the immune response against Jo-1 and Ro52 might be coupled [21]. Recently, we showed that the presence of the most common MSA, the anti-jo-1 autoantibody, virtually excludes the diagnosis of IBM [11], which can be difficult to distinguish from DM and PM, even histologically [1]. This differential diagnosis is of clinical importance as

6 74 IBM, unlike DM and PM, lacks a consistent response to immunosuppressive treatment [7]. The results of the present study strengthen the observation that MSAs are hardly ever detected in IBM. For the anti-jo-1 autoantibody this was clearly statistically significant and for the other MSAs, because of the small number of patients exhibiting these antibodies, there was a clear trend. More importantly, the three IBM patients in this study with a MSA demonstrated a significant clinical response to immunosuppressive treatment.it is the experience of many clinicians that a small subgroup of IBM patients shows a partial response to immunosuppressive treatment. There are no specific characteristics to identify this subgroup. The partial response seen in the MSA positive IBM patients in this study raises the questions whether the presence of a MSA can aid in the identification of this subgroup. In summary, it can be concluded that MSAs are an important aid in the differential diagnosis and treatment plan of IIM by virtually excluding the diagnosis of IBM in the presence of the anti-jo-1 autoantibody and by questioning this diagnosis in the presence of other types of MSAs, and by warranting, by their presence, the start of immunosuppressive therapy. They further can define distinct clinical groups characterised by differences in extra-muscular complications, therapeutic responses, and prognosis. While some of these MSA associated syndromes are well defined (e. g. anti-jo-1 with the anti-synthetase syndrome) others remain uncertain and additional studies are required to clarify their clinical value (e. g. anti-srp, anti-mi-2). Awareness of these features will aid the clinician in the management of the patient with an idiopathic inflammatory myopathy. Besides their clinical usefullness, MSAs may aid in the unraveling of the pathogenesis of IIM. Their specificity for IIM, the presence of certain clinical characteristics and their association with certain HLA-genotypes indicate a close relationship between MSAs and the primary pathogenic process [15]. In the present study the clinical usefullness of MSAs was addressed. Further investigations, including HLA analysis, are currently being conducted in order to further elucidate the pathogenesis of IIM. Acknowledgements The authors thank B de Jong and E Nuy-Terwindt for their expert technical assistance and dr FHJ van den Hoogen (Department of Rheumatology, University Medical Centre Nijmegen) for patient referral. A special thanks to dr H Ehrfeld (Institute of Immunology and Molecular Genetics, Karlsruhe, Germany) for performing the anti-mi-2 and anti-pm/scl analysis. This study was financially supported by the Prinses Beatrix Fonds, grant and MW-NWO, grant References 1. 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