Laboratory testing in the diagnosis and management of idiopathic inflammatory myopathies

Rheum Dis Clin N Am 28 (2002) 859 – 890

Laboratory testing in the diagnosis and management of idiopathic inf lammatory myopathies Ira N. Targoff, MD * Department of Medicine, Veterans Affairs Medical Center, Oklahoma Medical Research Foundation and University of Oklahoma Health Sciences Center, 825 NE 13th Street, Oklahoma City, OK 73104, USA

Laboratory testing commonly used to assess the idiopathic inflammatory myopathies (IIMs) can be divided into three categories: (1) measurement of serum activities or concentrations of muscle-derived factors—such as enzymes, myoglobin, and other molecules—in order to assess muscle injury; (2) immunologic tests that detect markers of the disease process, including serum autoantibodies that have been associated with myositis; and (3) general laboratory tests that are used to assess the patient’s general status and medical condition. The laboratory assessment of muscle-derived factors that reflect muscle injury, and the determination of serum autoantibodies, play valuable roles in the diagnosis and management of the IIM. Enzyme elevations do not correlate with disease activity in all patients, however, and they must be interpreted within the clinical context. Autoantibodies can identify disease subsets with distinctive patterns of clinical manifestations, genetics, responses to therapy and prognosis, but diseasespecific autoantibodies are present in only half of patients with IIM. Recent studies have defined additional myositis autoantibodies that may improve our capacity to diagnose and manage the IIMs. The laboratory is a crucial component in the evaluation of patients with suspected IIM. Elevations of muscle enzyme activities in the serum represent one of the criteria of Bohan and Peter [87] and are included in nearly all criteria sets proposed. Recent criteria sets have also included detection of specific autoantibodies as tools in patient diagnosis. None of these tests is necessary or sufficient for diagnosis of IIM. Laboratory tests commonly used to assess IIM, including those described below, are essential tools for the diagnosis and management of myositis. Recent

* E-mail address: [email protected] 0889-857X/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 0 8 8 9 - 8 5 7 X ( 0 2 ) 0 0 0 3 2 - 7

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studies suggest that detection of additional immunologic parameters may be clinically useful in the future. In addition, laboratory tests can be helpful in the exclusion of certain other conditions that arise in the differential diagnosis of muscle pain or weakness. This article focuses on laboratory testing in the context of the measurement of serum activities or concentrations of muscle-derived factors—such as enzymes, myoglobin, and other molecules—in order to assess muscle injury, and immunologic testing that detects markers of the disease process, including serum autoantibodies that have been associated with myositis. Table 1 provides a summary of laboratory tests currently used in diagnosis and management of IIM.

Muscle-derived factors Measurement of serum activities of enzymes released from muscle have been used for many years, and remain the most widely used laboratory tests for muscle injury [1]. Myoglobin is detectable in serum in most patients with active IIM and may be a more sensitive measure of active myositis, but because it is not as readily available, it is assessed less frequently than enzyme activities. Troponin measurements have become more widely available in recent years, but primarily are used for assessment of cardiac muscle injury. Urinary creatine levels were used in the past to assess myositis, but are rarely used now because of the cumbersome nature of urine collections, difficulties in interpretation, and lack of availability. Enzymes The elevations of serum activities of muscle-derived enzymes observed in patients with the IIM reflect the presence of muscle injury, and help differentiate IIM from conditions that primarily involve atrophy, such as steroid myopathy [2]. Release of muscle enzymes in these conditions presumably occurs from necrotic or injured muscle fibers. The enzymes are usually measured biochemically, by detecting their enzyme activity, which means that their measurement will vary from laboratory to laboratory and can be affected by inhibitors of their biochemical activity [3]. Creatine kinase Creatine kinase catalyzes the transfer of a high-energy phosphate from ATP to creatine to form creatine phosphate (and ADP), as well as the reverse process. Thus, creatine phosphate serves as a storage form of high energy phosphate, and provides a source of energy for regenerating ATP during the high energy demands of muscle exertion, and serves as a shuttle to move energy from mitochondria to the sarcoplasm (the ‘‘creatine –creatine phosphate energy shuttle’’) [4]. Creatine kinase has traditionally been considered the most useful serum enzyme for diagnosis and assessment of adult patients with IIM. This is primarily

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Table 1 Laboratory tests in idiopathic inflammatory myopathies Total CK

> 90% in adult PM 70 – 90% in DM 80% in IBM

CK – MB

50%

Aldolase

LDH, AST, ALT

Serum myoglobin Urine myoglobin Troponin

Cardiac Tn-I: < 5% Cardiac Tn-T: 40% Skeletal TN-I: high

Carbonic anhydrase III 24-hr urinary creatine

Erythrocyte sedimentation rate Factor VIII-related antigen Neopterin Immunoglobulins

50%

Rheumatoid factor Antinuclear antibody test Defined specific antibodies

8% 60 – 80%

1) Lower frequency of elevations in elderly than in young adults 2) Average level lower in IBM (< 1000) than in PM or DM 3) Can vary with disease activity in individuals, can predict flares or treatment responses. 4) Should not be used by itself for assessing disease activity; Does not correlate well with strength in studies; Disease can be active despite normal CK 5) Elevation not specific for myositis (see Table 2) 6) Consider ethnic group, muscle mass, individual variation, and other conditions in assessing significance of elevations. Fraction of CK – MB can be increased in IIM (50%) from muscle source; can be " without cardiac involvement, or normal with cardiac Useful to help assess muscle, especially if " aldolase and normal CK, but aldolase is less muscle-specific than CK. Can be useful in assessing activity, especially in children or when CK normal. Wider distribution makes elevation difficult to interpret. Elevation of ALT can occur in IIM, and elevations can be confused with liver injury in unrecognized IIM. Elevated in most IIM patients, can vary with disease activity. Can cause positive chemical test for blood without RBCs. Renal injury rare. 1) Cardiac Troponin I is usually not elevated in IIM, even if CK-MB is elevated. 2) Cardiac Troponin T can increase with active myositis without cardiac involvement; believed to arise from skeletal muscle 3) Skeletal Troponin I correlates with CK and may be a useful marker Skeletal muscle specific form can be increased in active IIM Increased creatine/[creatine + creatinine] in muscle disease; can increase in atrophy as well as damage; thus it is less specific Not usually used to assess myositis activity Elevated in a subset of active juvenile DM May correlate with activity in juvenile DM Can be increased but if deficient, consider echovirus infection Appears to be associated with anti-synthetases Nuclear more common than cytoplasmic patterns See Table 3

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because of the relative specificity of CK – MM for skeletal and cardiac muscle. Additionally, among the enzymes that are commonly measured, CK is the most sensitive to muscle injury; in mild muscle injury, CK is occasionally the only enzyme elevated. Also, in muscle injury, the degree of elevation of CK is often greater than that seen with other enzymes. The mean increase of CK in adult patients with IIM is approximately tenfold the upper limit of normal, but it may be elevated more than 100 times. The normal range for CK varies, depending on the relative amount of muscle mass present, among different populations. It is higher for men than for women and is higher for African-American than for white patients [5,6]. The normal CK level may be lower in patients taking corticosteroids [7] or in patients with connective tissue diseases or rheumatoid arthritis [8,9], possibly because of the specific inflammatory processes in those diseases. In a recent study, elevations of CK in patients with overlap syndromes of lupus and IIM had comparable CK elevations to those with IIM alone [10]. Most adults with active myositis (80 – 90% of patients with polymyositis [PM] or dermatomyositis [DM]) have CK levels above normal when first evaluated; 95% have CK levels above normal at some time during their disease course. Nonetheless, a small proportion of patients with IIM have active myositis with persistently normal CK levels. This seems to be more frequent during later disease exacerbations than during the initial presentation of myositis. In very advanced disease, reduced muscle mass may lead to lower than expected CK, but it is possible for patients with advanced disease to have increases in CK levels during exacerbation. Inhibitors of the enzymatic measurement of CK may also account for this finding in some patients [3]. Changes in CK within the normal range may be significant, particularly if that range is not adjusted for the patient’s gender or ethnic group. Thus, an increase from the lower to the higher portion of the range may reflect disease recurrence, although some increase may occur with steroid tapering alone. In patients with DM there is a somewhat higher frequency of normal CK at presentation compared with PM. This is, in part, because some patients with DM present with a rash before the onset of myositis; diagnosis of DM is usually easier than PM in the absence of CK increases (enzyme elevations would be required to diagnose ‘‘definite’’ PM by traditional criteria). The patients with DM who develop rash without clinically evident myositis, even after repeated evaluations over time (amyopathic DM), must be distinguished from the small proportion of adult patients who have normal CK despite having myositis. It was suggested that myositis without CK elevation is a sign of poor prognosis [11]. This was disputed in subsequent studies [1,12] but was observed in some series of patients with DM with interstitial lung disease, several of which were from Japan [13 –17]. Creatine kinase is usually lower in patients with inclusion body myositis (IBM) compared with those who have PM or DM. Among 35 patients described by Felice and North [18], the mean CK at presentation was 444 U/L (upper normal is 269). Only three patients had CK >1000, and six had normal levels. Serum levels of

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CK activity were lower in patients with PM who were resistant to treatment and had symptoms that resembled IBM [19]. In a related finding, Pautas et al [20] observed that normal CK is more common in elderly patients with PM or DM (40%), compared with younger patients (5%), and elderly patients usually have a more chronic course. Clinical experience suggests a general correlation of serum CK activity with myositis activity, over time, in patients with IIM [21,22]. Several recent studies were unable to show a correlation with strength or with functional measures of disease activity [23,24]; CK cannot be used by itself to assess disease activity [25,26]. There was no association of CK with a mechanical strength measure or with aerobic exercise capacity, although the latter two are correlated [27]. Although CK cannot be relied upon as an absolute guide, and many factors can affect it, CK activities can be very useful in patient management. In particular, increases of CK in apparently stable patients can frequently predict a disease exacerbation, often preceding it by about 6 weeks. Conversely, CK usually decreases before strength recovers during treatment, often by 3 to 4 weeks. Steroids may lower serum CK activities, however, without adequate suppression of disease and recovery of strength. Persistent elevation of CK is often a sign of persistent inflammatory activity, and should lead to caution in tapering of treatment. Nonetheless, some patients will have persistent elevations of CK, usually after a substantial decrease from its peak, without evidence of continuing muscle inflammation. When serum CK activities increase during the disease course in a patient with myositis who had been previously normalized with treatment, it is important to exclude other potential causes before attributing this to recurrent myositis. Creatine kinase is elevated in patients with conditions that lead to muscle necrosis, such as rhabdomyolysis, muscular dystrophies, hypothyroidism, and many druginduced myopathies (including cholesterol-lowering agent myopathy), as summarized in Table 2. Creatine kinase is not usually elevated in patients who have conditions that lead to muscle atrophy, such as steroid myopathy or denervation, although elevations have been found in patients amyotrophic lateral sclerosis. Serum CK activities can increase with seizures or malignant hyperthermia, as well as excessive or unaccustomed exercise [28,29]. Creatine kinase can be elevated for a number of reasons other than muscle injury, such as diabetic nephrotic syndrome with edema [30], or from drugs that interfere with elimination. Intramuscular injections, electromyography, muscle biopsy, or other surgery can also cause transient CK elevations. Creatine kinase is mainly present in muscle and the brain. The enzyme exists as a dimer of M or B forms, with the MM isoenzyme predominant in skeletal muscle, and the BB isoenzyme predominant in smooth muscle and brain. The MB isoenzyme represents a small proportion (< 5%) of skeletal muscle total CK, but a higher proportion (20 –25%) of total cardiac muscle CK. It is also increased in regenerating skeletal muscle compared with mature skeletal muscle. Thus, MB isoenzymes elevations can be seen in patients with IIM as a result of myocarditis, but they more commonly represent regenerating or immature skeletal muscle.

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Table 2 Causes of elevated serum creatine kinase levels (other than idiopathic inflammatory myopathies) 1) Muscle trauma a) Muscle injury b) Needle stick c) Electromyogram d) Surgery e) Convulsions, delirium tremens 2) Diseases affecting muscle a) Infectious myositis b) Metabolic or mitochondrial myopathies c) Muscular dystrophy d) Myocardial infarction e) Rhabdomyolysis f) Amyotrophic lateral sclerosis 3) Drug/toxin-induced myopathy a) Lipid-lowering agents, especially HMG-CoA-reductase inhibitors b) Alcoholic myopathy c) Drugs of abuse (eg, cocaine, amphetamines, phencyclidine,) d) Malignant hyperthermia and neuroleptic malignant syndrome e) Other medications: Zidovudine, colchicine, chloroquine, Ipecac, others 4) Drug-induced myositis a) D-penicillamine b) Interferon 5) Drug-induced CK elevation Inhibition of excretion (barbiturates, morphine, diazepam) 6) Endocrine and metabolic abnormalities a) Hypothyroidism b) Hypokalemia c) Hyperosmolar state or ketoacidosis d) Diabetic nephrotic syndrome with edema e) Renal failure 7) Elevation of CK – BB a) CNS disease b) Tumors (GI, bronchial, other) 8) Elevation without disease a) Strenuous, prolonged, and/or unaccustomed exercise b) Ethnic group (black > white) c) Increased muscle mass

Such elevations may occur in over half of patients with PM or dermatomyositis [21]. Elevations are usually less severe than those seen in patients with myocardial infarction, but can sometimes be over 20% of total CK [31]. Cardiac involvement can occur in the absence of CK –MB elevation. Macro CK refers to a form of enzyme that is larger relectrophorectically than the standard CK. Two forms exist, macro CK type 1 and type 2. Macro CK type 1, a complex of an antibody with CK, can occur in patients with myositis [32]. Macro CK type 2 is usually derived from mitochondria and may be a marker of malignancy [33].

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Other muscle enzymes Other commonly measured serum enzyme activities that are elevated after muscle injury (eg, ALT, AST, LDH, aldolase) are useful for monitoring disease activity in patients who have normal CK levels, despite having active myositis. Most patients have at least one serum enzyme elevation at some point during their disease course [12]. Aldolase is sometimes considered an alternative muscle marker enzyme, but because it is more widely distributed in tissues than CK, it can increase as a result of liver injury or other conditions [34]. It may be of more use late in the course of patients with juvenile DM; aldolase remains elevated when CK may have returned to normal [1]. In juvenile patients with DM, physician global disease activity correlated better with LDH than any other enzyme [26]. The LDH-5 isoenzyme is the predominant form in skeletal muscle, whereas the LDH-1 isoenzyme is the predominant form in the heart. Like CK-MB, however, the LDH-1 isoenzyme activity may be elevated in patients with IIM without cardiac involvement [35]. With isolated elevation of LDH, unrecognized malignancy or hemolysis should be considered. The transaminases, AST and ALT, can be elevated in patients with muscle injury and vary with disease activity. Aspartate aminotransferase is present in higher amounts than ALT [36] and may be more useful in monitoring disease activity, especially in patients with juvenile DM [26]. Alanine aminotransferase may also be elevated in patients with muscle injury [37], including those with IIM. This can lead to a misdiagnosis of liver disease if the muscle source is not recognized [38]. It can also complicate monitoring of treatment with methotrexate, particularly if disease activity is not adequately suppressed. Serial measurements and comparisons of ratios of CK, ALT, and LDH activities can often help clarify the clinical meaning of these enzyme abnormalities. Carbonic anhydrase III is found exclusively in skeletal muscle, and can increase in patients with active myositis [39 – 41]. Although it has potential advantages over conventionally measured enzymes, it is not as readily available. Other muscle factors Myoglobin Myoglobin can be a useful serum marker of muscle damage. It has similar specificity for skeletal and cardiac muscle, and is elevated at least as frequently as CK in the serum of patients with IIM who have active myositis [35]. It varies with disease activity [22], sometimes predicting exacerbation; serial measures can be useful. An advantage of myoglobin is that it is detected by a nonenzymatic immunologic reaction that does not rely upon an enzymatic activity [42], but it remains less readily available than CK. Troponin Troponin, a structural component of muscle thin filaments, is also released during muscle injury. It is composed of three proteins (troponins C, I, and T) [43]. The cardiac form of troponin I (the component that interacts with actomyosin

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Table 3 Autoantibodies in polymyositis and dermatomyositis Antigen

MW (kDa)

Tests

IIFa

Freq (%)

ID;IPP; WB;EIA

Cyto

1) 18 – 20 HLA-DQA1 * 0501 Myo (95); ILD (80); or * 0401 c Arth (60); RP (60); 2) 6 MH (70); Fever

ID;IPP;EIA

Cyto

<3

(Similar to Jo-1)

(Similar to Jo-1)

ID;IPP

Cyto

<3

(Similar to Jo-1)

IPP IPP

Cyto

<3 <2

(Similar to Jo-1)

(Similar to Jo-1 except lower freq myo, esp. Japan) (Similar to Jo-1)

(Similar to Jo-1)

(Similar to Jo-1)

Myositis-specific autoantibodies (established) Antisynthetases autoantibodies Jo-1 1) Histidyl-tRNA 50 (dimer) synthetase 2) TRNAhis (direct reaction) PL-7 Threonyl-tRNA 80 synthetase PL-12 1) Alanyl-tRNA 110 synthetase 2) TRNAala OJ 1) Isoleucyl-tRNA 150 synthetase 2) Multienzyme complex 170, 130, 75 EJ Glycyl-tRNA synthetase 75

IPP;WB

Cyto

<2

KS

IPP

Cyto

<1

IPP; WB

Cyto

4–5

Asparaginyl-tRNA 65 synthetase Other established myositis-specific autoantibodies SRP Signal recognition 54, 72, particle ?other

WB;AA1

HLA

Clinicalb

<1

(Similar but less myo, esp. Japan) DR5

PM, Possible " in cardiac and distal involvement

Comments

1) PM > DM 2) Adult > juvenile 3) anti-RNA always with antienzyme More DM than with anti – Jo-1 Anti-tRNA usually with antinezyme Ile-RS usual main antigen; leu-RS, lys-RS can be seen with ile-RS More DM than with anti – Jo-1 Very few cases; most with ILD a) May be severe, acute, resistant; b) Biopsy may show less inflammation

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Name

Mi-2

NuRD helicases 1) Mi-2a (CHD3) 2) Mi-2b (CHD4)

240 ID;IPP; 1) 208 – 226 WB;ElA 2) 218

UIRNP

U1 small nuclear ribonucleoprotein NonU1-snRNPs 1) U2RNP 2) U5RNP 3) U4/6RNP Ku DNA binding compex Other myositis-associated autoantobidies Ro/SSA Ro60

56kd Fer Mas KJ

70,32A, C25 1) A0, B00 2) 200

5 – 14

DR7

DP (PM only when EIA used)

All sera react with both forms but not by all tests

ID; IPP; WB; ElA

NS/NO

a) 5 – 10 b) 3 – 5

DR3

Myo(75); SSc(75); Arth; MH

a) Myo may be more responsive; b) SSc usually limited-cutaneous

ID; IPP; WB; ElA IPP; WB

NS

5 – 10

Myo; SSc; SLE

NS

1) Rare 2) Rare 3) Rare 1 (in US)

1) Myo; SSc 2) Myo; ?SSc 3) SSc, Myo SSc, SLE; Myo

70,80

ID; IPP; WB NS

60

Cyto/NU 10

Ro52

52

ID;IPP; ElA;WB ElA; WB

Ribonucleoprotein component Elongation factor 1a tRNAsel & protein Translation factor

56

WB

Nu

62 – 87

All forms esp. JDM

IPP IPP ID; WB

Cyto Cyto Cyto

<1 <2 <1

Uncertain myo assoc Alcohol; hepatitis Myo;ILD

48 45 120

Sjo¨gren’s

25

1) Anti-La can also be seen; 2) Both forms associated with antisynthetases

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Myositis-associated autoantibodies (established) Overlap syndrome associated autoantibodies PM-Scl Exosome proteins a) 100 kDa b) 75 kDa c) Other

NS

867

868

Table 3 (continued ) Name

Antigen

Se

Unidentified Endothelial cell

MW (kDa)

Test

IIF

120

IPP

Nu

140 155

IPP;WB IPP;WB

Nu Nu

95

IPP;WB

Nu

Proteasome aC9

EIA;WB

Nuclear pore

IIF

Freq

7.5 4 4 17.5 15.5

5 36 62

Clinical

Comments

Very few cases reported

Different antienzymes can occur together or separately [50]

JDM DM

[51] a) May be increased in amyopathic DM

Similar to 155kd a) All subgroups b) ILD may be"

b) Subset has anti-Se [52] Similar to 155kd [53] [54] 58% in SLE and frequent in other autoimmune disease [55] Seen in 2/30 French-Canadians [56]

Abbreviations: Arth, arthritis; cyto, cytoplasmic pattern; EIA, enzyme immunoassay; Freq, frequency; ID, immunodiffusion; IIF, indirect immunofluorescence; ILD, interstitial lung disease; IPP, immunoprecipitation; kDa, kilodaltons (kd used in antigen names); MH, mechanic’s hands; MW, molecular weight; Myo, myositis; NO, nucleolar; NS, nuclear speckled; Nu, nuclear pattern; RP, Raynaud’s phenomenon; SSc, systemic sclerosis; WB, Western immunoblotting. a IIF pattern is that caused by the antibody indicated; individual sera may have other antibodies causing other patterns. Thus, a cytoplasmic pattern may suggest an antisynthetase but a nuclear pattern does not exclude. b Numbers in parentheses are percents roughly estimated from available studies and experience. Numbers in brackets are references. c In Japanese patients, antisynthetases were associated with HLA-DQA * 0102 and * 0103.

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New myositis autoantibodies PMS1 related DNA mismatch repair enzymes: PMS1 PMS2 MLH1 MJ Unidentified 155kd Unidentified 155kd protein

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ATPase) is unlike CK-MB; it is not expressed in regenerating skeletal muscle, fetal skeletal muscle, or in muscle from patients with Duchenne’s muscular dystrophy or PM [44]. It is rarely elevated in patients with PM or DM [21,45] and could be used as a more specific marker of cardiac muscle injury. Its ability to detect cardiac involvement in patients with IIM has not been investigated; its elevation in a single case was attributed to other factors [45]. Cardiac troponin T (the component that interacts with tropomyosin) is frequently elevated in patients with myositis (41% for T versus 2.5% for I), possibly because of its production in regenerating skeletal muscle [21,46]. Skeletal troponin I correlates well with CK; its potential use as a marker of muscle injury needs to be validated [45]. Cardiac troponin I does not correlate with CK or other measures of skeletal muscle injury. Creatine Creatine is normally taken up by the muscle and used in energy storage as discussed earlier. It is broken down to creatinine. Many conditions that affect muscle can lead to defects in creatine uptake and retention, which results in an increase in its excretion [35]. Creatine excretion is usually measured in a 24-hour urine sample, and is expressed as [creatine: (creatine + creatine)]  100 with the normal range being < 6%. Although creatine is elevated in most patients with myositis, it is less specific than CK because it can increase in patients with diseases associated with muscle atrophy, such as neuropathies or steroid myopathy. In patients with IIM, muscle atrophy can lead to persistent elevations of creatine after disease suppression.

Autoantibodies The autoantibodies that have been most extensively studied in patients with IIM are antinuclear or anticytoplasmic autoantibodies that react with essential cellular proteins that are present in all cells, rather than the antibodies that react with muscle-specific antigens. The reason why these autoantibodies are produced, and their role in tissue injury remain uncertain and are the subjects of continuing research; the clinical associations of the autoantibodies can assist the clinician in patient diagnosis and classification. The autoantibodies were reviewed previously in this series [47] with extensive reference to previous findings; this section will summarize and focus on new developments. Antinuclear antibodies (ANAs) The ANA test by indirect immunofluorescence is positive in 50% to 80% of patients with IIM [48,49]. Antinuclear antibodies are primarily associated with patients with PM or DM, but the frequency in patients with IBM, approximately 20%, is higher than in the normal population. In patients with PM or DM, the frequency is highest in those who have myositis overlap syndromes with other connective tissue diseases, and is lowest in patients with malignancy-associated

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myositis. The ANA alone can be a valuable diagnostic clue, particularly in high titer, in differentiating PM or DM from dystrophies and nonautoimmune myopathies. It is not specific enough to allow firm conclusions and thus supplant conventional diagnostic tests. In patients with IIM, the frequency of positive autoantibody screening tests, such as ANAs, remains higher than the frequency of autoantibodies with defined antigen specificities, although new autoantibodies continue to be described. Overall, nuclear speckled ANA patterns are the most common type seen in patients with IIM, but 10% to 15% of patients with PM – DM will have pure cytoplasmic patterns without nuclear staining by indirect immunofluorescence. Autoantibodies to defined antigens The defined autoantibodies in patients with PM and DM have been divided into ‘‘myositis-specific autoantibodies’’ (in which most patients have myositis, ‘‘MSAs’’), and ‘‘myositis-associated autoantibodies’’ (which are frequently found in patients without myositis, ‘‘MAAs’’) (Table 3). These terms are relative and with exceptions, but in general, myositis is the primary manifestation of the syndromes associated with MSAs; this is not the case with most MSAs. Nevertheless, certain MAAs can be as useful for evaluation of some individual patients with myositis as MSAs; some, such as anti – PM-Scl, can be nearly as valuable for diagnosis and classification. About half of patients with PM or DM have defined autoantibodies, but even the most common individual established antibodies are found in less than a quarter of patients with IIM (except possibly anti-56 kDa). Each MSA, and many MAAs, have characteristic clinical associations (Table 3) [48,57]. Some distinctive muscular features of different MSA-defined groups have been noted [58]; many clinical associations of the MSAs relate to extramuscular manifestations, such as interstitial lung disease [59] or DM rash [60]. In general, individual patients tend to have only a single MSA (MSAs are mutually exclusive), but one or more MAAs may occur with MSAs, or independently [61]. Recent studies [57] have shown that patients with MSAs may be more likely than patients without antibodies to have associated MAAs [61 –63]; certain MAAs, such as anti-Ro, are more likely to be associated with MSAs than others, (eg, anti-U1RNP) [61,63]. As a group, MSAs are more likely to occur in patients with PM compared with DM [61]. This is because of the overwhelming effect of anti –Jo-1, which is more common than all other MSAs taken together in adults, and is more frequently associated with patients with PM compared with DM. The MSA anti– Mi-2 [64] is more common in patients with DM. Recent studies have defined new autoantibodies that have stronger associations with DM compared with PM [51,52], although the specificity of these for myositis has not been extensively studied. As a group, MAAs are more likely to be associated with PM compared with DM, but DM is at least as common as PM in patients with anti – PM-Scl. The clinical associations and the tendency of mutual exclusivity of MSAs and certain MAAs, such as anti –PM-Scl, have allowed the definition of clinical subgroups of PM and DM, that may differ

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from the clinically defined subgroups, and can be complementary to them [48,65,66]. Although rare instances of coexistence of MSAs was previously reported [67], Brouwer et al [68] described sera with more than one MSA more frequently than had been previous reported. These sera represented a small proportion of MSA-positive sera ( < 4%), but enough to represent a meaningful difference from previous findings. This seems to be due in part to differences in the technique used in that study for the detection of anti –Mi-2 antibodies. Because the antibodies are so infrequent in the general population, the expected rate of coexistence in an individual patient by chance would be very low; occurrence in even a small proportion may affect hypotheses regarding the mechanisms behind the development of the autoantibodies. Love et al [48] did not find MSAs in 26 patients with IBM. Few other studies have investigated MSAs in patients with IBM, although the experience of this investigator is consistent with Love et al’s original findings. In contrast, Brouwer et al [68] found 7 MSAs (antisynthetase, anti-SRP, anti –Mi-2) among 38 IBM sera, using a combination of highly sensitive tests. Although rare cases of anti – Jo-1 have been reported in patients with IBM [69], Brouwer et al supported their report with sophisticated, but different, detection methods used to define these reactivities. These findings raise important questions about the relationship of IBM, generally thought to be distinctive and in which autoimmunity is of uncertain significance, with PM and DM, generally felt to be autoimmune-mediated. At least one patient with anti –Jo-1 antibody with IBM responded unexpectedly well to corticosteroid treatment; this suggests that the antibody reflected clinical and possibly pathogenetic differences from other IBM patients [70]. The higher frequency of antibodies noted in patients with IBM may relate to differences between patient populations, or, more likely, to differences in techniques for antibody detection. Despite these exceptions, MSAs are much more common in patients with PM/DM than in other conditions, including IBM, and generally remain useful for distinguishing these conditions. Using traditional immunoprecipitation and enzyme inhibition methods, it was noted that anti – Jo-1 autoantibodies do not usually occur in patients with IBM [71]; we and others have not found MSAs in patients with muscular dystrophies or other myopathies. Myositis-specific autoantibodies also usually absent from patients with malignancy-associated myositis. When evaluating patients, MSAs can strongly support and increase the confidence in clinical impressions, but must be interpreted in light of the total clinical picture. The type of tests employed should be taken into account. It is possible (see later discussion) that the high specificity of MSAs that was noted in previous studies depended on reactivity in tests that required either higher titers or reaction with particular epitopes. MSAs: antisynthetases Anti – Jo-1, the prototype antisynthetase, is usually the most common of the established MSAs in most populations of patients with IIM. Anti –Jo-1 was found in 18% of patients with IIM in two large, recent studies [61,68], and in 20% of

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patients with IIM in another recent study [72]. Although the data are limited, there is increasing evidence that the frequency of MSAs differs in different ethnogeographic populations. For example, the frequency of anti – Jo-1 was surprisingly low in a French-Canadian population of patients with IIM (0 of 30 patients), although only eight had primary PM [56]. Anti–Jo-1 and other antisynthetases were not as frequent (3%) in Meso-Americans (from Mexico and Guatemala), another group with a lower frequency of PM [73]. In 84 Polish patients with IIM, as in most other populations, anti – Jo-1 was the most frequent MSA [74], but unlike most studies it was found in only 10.7% of patients, and was much less frequent than anti– PM-Scl (23.8%). There was a trend toward a higher frequency of anti – Jo-1 in Japanese patients (6 of 21 patients, 29% ) [61]. Anti –Jo-1 is usually the most common autoantibody in the group of patients clinically classified as having primary PM (adult PM without overlap or malignancy). Arnett et al [61] found anti – Jo-1 in 27% of this group, compared with 7% of patients with DM, a statistically significant difference ( P = 0.009). The difference found by Brouwer et al was less striking; anti– Jo-1 was found in 22% of patients with PM and 16% of patients with DM. The proportion of juvenile patients with IIM with anti– Jo-1 is much smaller, although several cases have been noted [75,76]. When anti – Jo-1 is present, juvenile patients with IIM may show a clinical picture similar to adults with the antibody. Although connective tissue disease features are common in patients with anti – Jo-1, and they may clinically resemble patients with mixed connective tissue disease, they seldom satisfy criteria for scleroderma or systemic lupus. Only 8% of the patients with connective tissue disease overlap studied by Love et al [48] had antisynthetases, compared with 33% of the patients with PM. Anti –Jo-1 reacts with histidyl-tRNA synthetase, the enzyme that catalyzes the formation of histidyl-tRNA, (ie, the covalent attachment of the amino acid histidine to its cognate tRNA, ‘‘tRNAhis’’). There is a separate, immunologically and enzymatically distinct aminoacyl-tRNA synthetase enzyme for each of the amino acids. A high degree of accuracy in the attachment of each amino acid to its appropriate tRNA is required for proper translation of the messenger RNA and protein synthesis; each of the aminoacyl-tRNA synthetases must uniquely identify the amino acid, as well as its cognate tRNA. Thus, antisynthetase antibodies generally react specifically with a single synthetase. Antibodies to five other aminoacyl-tRNA synthetases have been described in patients with IIM. The most recently described reacts with asparaginyl-tRNA synthetase [77]. Although an individual usually has antibodies to only one of these, exceptions to this mutual exclusion have been noted [67]. An additional exception exists for anti-OJ. The synthetases can be divided into two classes based on structural features; all antisynthetases, other than anti-OJ, react with class II synthetases, most of which exist free (uncomplexed) in the cytoplasm. Anti-OJ reacts with isoleucyl-tRNA synthetase, a class I synthetase that exists as part of a multi-enzyme synthetase complex [78]. Some sera with anti-OJ react with other components of the complex [79]. Although isoleucyl-tRNA synthetase is the main antigen for most anti-OJ sera, it is possible that a patients who have

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this very rare autoantibody react primarily with lysyl-tRNA synthetase [67,79] or possibly other components. It is unknown whether antibodies ever occur that react with the remaining aminoacyl-tRNA synthetases that have not been described as antigens. It is clear that anti –Jo-1 is found much more frequently than other antisynthetases in most populations studied. Brouwer et al [68] found that anti – Jo-1 was six times more common than all other antisynthetases combined. Arnett et al [61] found that anti – Jo-1 was approximately twice as common as the other synthetases combined. The difference between these reports is probably a result of the high frequency of anti – PL-12 in patients in the latter study and the inclusion of patients with antisynthetase who did not have myositis [80]. Although the relative frequency of different non– Jo-1 antisynthetases is difficult to judge because of their low overall frequency, there seem to be differences between populations. In white patients with myositis, anti – PL-12 and anti – PL-7 are more common than anti-EJ, anti-OJ, and anti-KS; anti-EJ may be more common in Asian populations compared with white populations [81,82]. In general, however, it is not understood why antibodies to histidyl-tRNA synthetase are much more common than antibodies to other synthetases. Known immunogenetic associations with anti – Jo-1 may contribute to this [48,83]. This same observation in ethnically different populations [61], as well as the recent generation of antibodies to histidyl-tRNA synthetase in a murine model in which upregulation of MHC class I alone induced a form of myositis [84], suggest that immunogenetics may not fully explain this. The clinical associations of anti – Jo-1 autoantibodies have been extensively studied, and differ significantly in several aspects compared with patients with IIM who do not have antisynthetases [48]. Despite the low frequency of other antisynthetases, it is clear that there are strong clinical similarities in patients with either anti – Jo-1 or other antisynthetase autoantibodies [59,85]. The clinical picture seen in association with all antisynthetase autoantibodies has been referred to as the antisynthetase syndrome. There may be a higher frequency of DM in persons with non – Jo-1 antisynthetases. Although there are distinctive features to the group overall, including the prominence of lung involvement and severity of myositis, individual patients may resemble those who have anti – PM-Scl or antiU1RNP, or possibly other antibodies [86]. Thus, the syndrome cannot be diagnosed in the absence of the antibody. Clinically, the myositis of patients with antisynthetases generally resembles that of patients with PM or DM who do not have antisynthetases. The myositis usually shows subacute onset of proximal muscle weakness, and satisfies the diagnostic criteria of Bohan and Peter [87,163]. The myositis usually responds as expected to corticosteroids and immunosuppressive agents. In one study, however, as treatment was withdrawn, myositis in patients with antisynthetase recurred more frequently (60%) than in patients without antibodies (20%) [48]. One report described a flare in a patient with anti – Jo-1 after 7 years of remission [88]. There were also suggestions that, in a retrospective study, patients with antisynthetase responded differently to some immunosuppressive agents [89]. The prognosis for patients

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with antisynthetase was worse than that of patients without antibodies, although this was not necessarily due to the myositis itself [48]. One recent study suggested more fundamental differences in the pathogenesis of myositis of patients with anti –Jo-1 antibodies. Previous reports found that the pathogenesis of DM and PM were quite different, with a predominance of a complement-mediated vasculopathy in DM, and a cell-mediated attack on muscle fibers in PM (as well as in IBM). This implies that PM and DM are fundamentally different diseases, and that antisynthetases could occur in at least two distinct forms of IIM. In most studies, however, the PM was defined clinically (absence of rash), and some of these patients have ‘‘DM’’ histology. Mozaffar et al [58] recently compared the histology of 11 patients with anti –Jo-1 antibodies to patients with other IIM; they found a frequent pattern that differed from patients who had PM or DM. Perifascicular atrophy was found in all 11 patients with myositis; this is usually associated with DM and was found in all 8 patients who had definite DM. Perimysial inflammation was also found in all 11 patients with myositis; this was more frequent than in patients with DM or PM. Perivascular and endomysial inflammation were less frequent than in patients with DM or PM. Despite this pattern, usually considered to be related to the vasculopathy of DM, patients with anti – Jo-1 antibodies did not show the marked drop in capillary density that patients with DM did (capillary index of 0.88 in patients with anti – Jo-1 was significantly higher than the 0.55 in patients with DM, and similar to the 0.95 for patients with PM). All 11 biopsies from patients with anti – Jo-1 antibodies showed a distinctive feature, perimysial connective tissue fragmentation, which the investigators associated with fasciitis. Further study is needed to confirm these results, and we have seen some apparent exceptions to this pattern. The findings of Mozaffar et al [58] suggest that the pathogenesis of myositis in patients who have anti – Jo-1 antibodies may be different from that in other forms of IIM. Several extramuscular clinical features have been found to be more frequent in patients with antisynthetases than in other patients with IIM or in other patients with PM/DM. The most important of these is interstitial lung disease (ILD), because of its high frequency in patients with antisynthetases and its potential impact on prognosis and disability. The ILD of patients with antisynthetase does not have any unique features, and can vary in severity, rate of progression, and responsiveness. It can present fulminantly as acute respiratory distress syndrome [90,91], although this is not the most frequent manner of presentation. Douglas et al [92] found that the ILD associated with PM and DM was usually nonspecific interstitial pneumonia (NSIP). Of 70 patients with ILD, NSIP was seen in 18 of 22 who had biopsies. Nonspecific interstitial pneumonia is a pathologic form that has a better response to prednisone as well as a better overall prognosis compared with idiopathic pulmonary fibrosis. The ILD associated with PM and DM showed a survival curve that resembled that of NSIP. Thirty-eight percent of the 50 patients who were tested had anti –Jo-1; these patients had survival rates that were similar to other patients with PM\DM who had ILD. Other autoantibodies were not discussed. There were several reported cases of another histologic pattern, bronchiolitis obliterans organizing pneumonia (BOOP), in patients with either

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anti – Jo-1 or other antisynthetases [80,93 – 95]. BOOP is usually more responsive to treatment than idiopathic pulmonary fibrosis or usual interstitial pneumonia. These findings suggest that the ILD in with patients with antisynthetase autoantibodies may be more responsive to treatment, and suggest that a trial of corticosteroids or immunosuppressive agents would be worthwhile. Interstitial lung disease in patients with antisynthetase can be severe [90]. Nevertheless, the prognosis was poorer for patients with antisynthetases compared with patients with IIM who had no antibodies [48], which is probably related in great part to the morbidity and mortality caused by ILD. Other studies found a higher proportion of anti – Jo-1 antibodies in patients with PM\DM who had ILD; Grau et al [96] found anti – Jo-1 in 75% of patients with DM who had ILD compared with 3% of patients with DM who did not have ILD. Patients may present with the ILD rather than myositis. Some patients may have subclinical myositis, which may be revealed by the antisynthetase, or myositis may never occur. In some cases, suppression of myositis, which may have otherwise appeared, was caused by treatment of the ILD. Available data suggest that ILD without myositis is more common in patients with anti– PL-12 (anti-alaRS) [61,80], and anti-KS [77] autoantibodies compared with patients with anti – Jo-1 autoantibodies. In preliminary studies from Japan, this pattern was striking; only 13% of patients with anti-PL-12, and no patients with anti-KS, had clinical evidence of myositis, but all had ILD [97]. Interstitial lung disease without clinical myositis was also noted in patients with anti-EJ in Japan [97], and with patients with anti-OJ in the United States [80]. Inflammatory polyarthritis is another characteristic feature of the ‘‘antisynthetase syndrome.’’ Love et al [48] found arthritis in 94% of patients with antisynthetases, compared with 34% of patients without antibodies. Patients may present with the arthritis [98,99] or develop it later. Arthritis is rarely the predominant feature of the syndrome [100]; this may be due to suppression of other features by treatments such as methotrexate [101]. The arthritis is usually nonerosive but can lead to finger deformity and subluxations in one third of patients [102]. Occasional cases with erosive disease have been attributed to an overlap syndrome [103] and the arthritis can be associated with calcinosis in the fingers [104,105]. The cutaneous feature known as ‘‘mechanic’s hands’’, characterized by hyperkeratosis with cracking on the edges and sides of the fingers, was found in 71% of patients with antisynthetase. It is much less common in patients without antibodies. Mechanic’s hands is not specific for patients with antisynthetases and has been found in some patients with other antibodies, such as anti – PM-Scl [106] and anti – Mi-2. Histologically, ‘‘mechanic’s hands’’ resembles the rash of DM [107], but is not usually considered a DM-defining manifestation and may occur in patients considered to have PM [108]. Raynaud’s phenomenon is also increased in patients with antisynthetases (62% versus 26% in patients without MSA) [48]. It usually is not severe enough to cause ulceration, but can be significant for some patients. Patients with antisynthetases also have more fevers during flares of disease (87% of patients with antisynthetases compared with 23% of patients without MSA) [48].

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Extensive studies have been done to characterize the epitopes on the enzyme that are targeted by anti– Jo-1 [110,111], and, to a lesser extent, other antisynthetases [109,112]. Shared predominant epitopes have been identified, including one important epitope in the N-terminal portion of hisRS [111]; they are not exclusive epitopes, with patients showing multiple epitopes and polyclonal reactivity. This supports the concept that the response is driven by the synthetase antigens [110], but the initiating factor has not been determined. No clinical associations with different epitope reactivities have been noted for antisynthetase autoantibodies. Antisynthetases in patients with myositis are frequently recognized by their ability to specifically immunoprecipitate tRNAs for the antigenic synthetase [79,113,114]. It was originally found that the ability of anti – Jo-1 to immunoprecipitate tRNAhis was lost if the protein was removed [115]. This was thought to indicate exclusive reaction with the enzyme protein, with the tRNA precipitated indirectly due to its affinity for the enzyme. Studies found this to be the case with other antisynthetases except for anti –PL-12 (anti-alaRS). Most anti-PL-12 sera also react with a subset of tRNAala [116,117], with separate antibodies to the tRNA and to the enzyme. A recent study [118], however, found that one third of anti –Jo-1 sera actually do have antibodies that react directly with tRNAhis. Such antibodies were only found in sera with antibodies to the enzyme. Unlike anti-tRNAala, antitRNAhis did not seem to react with the anticodon loop, and required magnesium for stabilization of the epitope; this indicated that it was conformational, possibly accounting for previous difficulties in detection. Similar studies of anti-U1RNA in anti-U1RNP sera suggested that the antibodies varied with disease activity; this raised the possibility that the titers of these antibodies may be of use in patient management, a possibility that requires further study. The frequency of anti-tRNA antibodies in sera with other antisynthetases has not been comparably studied. The presence of antisynthetases is strongly associated with HLA-DQA1 *0501 or *0401 [61]. This was not seen in Japanese patients, who had an increased frequency of HLA-DQA*0102 and *0103. Previous studies had noted an increase in HLA-DR3 in white patients with the anti –Jo-1 antibody [48]. One study suggested a variation in titer of anti –Jo-1 with disease activity [119], but the clinical value of following titers has not been established. In most patients, the antibody remains present throughout the course; suppression of disease activity can be achieved despite its persistence. Disappearance of the antibody is a good prognostic sign [119]. The clinical value of antisynthetase antibodies lies in diagnosis because of their high disease specificity [120], and in predicting clinical manifestations and prognosis, because of their subgroup associations. Although some non– Jo-1 antisynthetases might be considered less myositis-specific, they demonstrate strong specificity for the antisynthetase syndrome. Patients almost always have myositis, ILD, or both during their course. Patients who have antisynthetases detected by a reliable test, with other evidence of a myopathy compatible with PM or DM (typical weakness, enzymes, and electromyography), probably do not require a biopsy [120]. If clinical features of IBM exist (insidious onset, slow progression, lack of response to treatment, distal weakness), biopsy would be indicated.

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Testing for anti –Jo-1 is readily available, usually by enzyme immunoassay (ELISA), RNA immunoprecipitation, or Ouchterlony immunodiffusion. Detection of the antibody by two different laboratories or methods improves reliability. Enzyme immunoassay tests generally show a higher false positive rate; confirmation of positive results by other standard methods is helpful [121]. Positive ELISA in low titer, with a negative confirmatory test, may be the result of a higher sensitivity of the ELISA, or a false positive result, but any ELISA result that cannot be confirmed by other methods should be regarded as suspect [100]. Antisynthetase testing should be particularly useful when clinical features of the syndrome are present, or when other studies, such as a cytoplasmic pattern by ANA (indirect immunofluorescence) testing suggest its presence. MSAs: anti-signal recognition particle (SRP) Signal recognition particle is a ribonucleoprotein complex of six proteins with the 7SL RNA. It functions in assisting the translocation of nascent polypeptides from the cytoplasm to the endoplasmic reticulum. The 54-kDa protein of SRP binds to a nascent protein near the ribosome. SRP also binds to corresponding docking proteins on the endoplasmic reticulum. Studies in U.S. patients suggested that anti-SRP antibodies most frequently bound to the 54 kDa protein [122]. Japanese patients show a different pattern, with more patients binding the 72 and 9 kDa proteins, although binding to the 54 kDa protein is still frequent [123]. No clinical associations with the targeting of different SRP proteins have been identified. Patient sera that contain anti-SRP antibodies immunoprecipitate the 7SL RNA. This can be used in detection of the antibodies; sera do not seem to bind the RNA directly. Indirect immunofluorescence usually shows a cytoplasmic pattern, but it is not detectable with all sera, and this method cannot specifically identify the antibody. ELISA [122] and immunoprecipitation have been used to detect the antibody, and recently a dot blot was used in which immunoprecipitated RNAs were tested with a 7SL RNA antisense probe [68]. The original studies of anti-SRP [48,122,124] using immunoprecipitation (with analysis of both RNA and protein) noted the almost exclusive association with clinical PM rather than DM. Recently, Brouwer et al [68] found PM in 14 of 20 patients with anti-SRP. Although PM remained the clinical group most commonly associated with anti-SRP, the presence of anti-SRP in five patients with DM and one patient with IBM is a significant departure from previous observations. This difference may relate to differences in patient populations, classification criteria, antibody detection techniques, or other factors. The clinical group defined by anti-SRP antibodies differs from patients with antisynthetase antibodies, and also from the group of patients with IIM without antibodies [122]. The extramuscular features associated with antisynthetases, including ILD, arthritis, Raynaud’s, fevers, and mechanic’s hands, were not significantly increased in patients with anti-SRP compared with those without antobodies who had IIM. A higher frequency of cardiac involvement was reported in patients with anti-SRP in some [48,122], but not all studies [121]. An increase in

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distal weakness has also been noted [48]. Muscle biopsies from patients with antiSRP often show myopathy with necrosis but little or no inflammation [123,125]. A seasonal predominance in onset of myositis in patients with anti-SRP was noted, being greater in and around November. Myositis in patients with anti – Jo-1 has a more frequent onset in the Spring [126]; this raises the possibility that different environmental factors are involved in the pathogenesis of myositis in these serologic groups. Love et al [48] noted a high frequency of severe disease of acute onset, among their six patients with anti-SRP. Furthermore, these patients had myositis that was relatively resistant to treatment, with a reduced rate of complete responses, and an increase in flares with taper. Subsequently, other patients with anti-SRP who had myositis that was difficult to treat were described [127]. The survival rate of patients with anti-SRP who have PM was lower in the study of Love et al; patients with anti-SRP had the worst prognosis of any IIM clinical or serologic groups with a 5-year survival of about 25%. As pointed out by Hengstman et al [121], however, only a small number of patients were evaluated, and the survival rate of the group with anti-SRP has not been fully established. Hengstman et al did confirm the low rate of complete remission and frequent need for long-term, highdose immunosuppressive agents. Unlike patients with IBM, most patients with anti-SRP do respond partially to treatment, which should be instituted. This combination of distinctive features associated with anti-SRP raises the possibility that anti-SRP defines a unique myopathy in some or all cases, but further study is needed. MSAs: anti – Mi-2 Anti –Mi-2 was among the first autoantibodies to be associated with myositis, but the antigen was characterized only recently. Anti –Mi-2 antibodies immunoprecipitate a complex containing at least eight proteins; the major antigenic protein is the largest, which migrates at 240 kDa [128]. There are two forms of the Mi-2 autoantigen, which have been labeled Mi-2a and Mi-2b [129]. These forms are 75% identical, but are clearly different proteins [130 –133]. By immunoprecipitation, both forms seem to react with all sera that are positive for anti – Mi-2. Both forms have motifs consistent with a helicase function [132,133], including the DEAD/H sequence, and an ATPase area. Mi-2a and Mi-2b have been more formally designated ‘‘CHD3’’ and ‘‘CHD4’’ respectively, as part of the CHD family of proteins so called because they contain a ‘‘chromo’’ domain (chromatin organization modifier), the ‘‘helicase’’ domain, and a ‘‘DNA-binding’’ domain [133]. Mi-2b is part of a protein complex that includes histone deacetylases and other proteins [134,135]; this probably accounts for the immunoprecipitation results. This complex has been labeled ‘‘NuRD’’ [135], for ‘‘nucleosome remodeling deacetylase’’, and it functions in chromosomally mediated regulation of transcription. This method of transcriptional control, in which modification of the structure of the nucleosomes of chromatin controls access to DNA, is involved in a number of cellular processes. Two major mechanisms for altering chromatin structure are

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known: active, ATP-dependent nucleosome remodeling, and acetylation of histones, which affects their binding. NuRD complex exhibits both activities, the latter through histone deacetylase enzymes and the former through Mi-2. Recombinant Mi-2 demonstrates the ATP-dependent nucleosome remodeling function of NuRD [136]. Initial studies, that used immunodiffusion, and later immunoprecipitation, showed a very strong association of anti – Mi-2 with myositis, and almost all of the patients had DM [48,60,61,64,74,76]. Until recently, it was the only antibody predominantly associated with DM. It was found in adults and children, and is the most common MSA in children, although the MAA, anti– PM-Scl, may be higher in some populations. The skin manifestations were sometimes prominent with typical Gottron’s papules and heliotrope rash, as well as the ‘‘V’’ sign (a rash at the base of the neck/upper chest) and ‘‘shawl’’ sign (a rash over the upper back and shoulder) involvement [48]. Although a rare patient with amyopathic DM had anti – Mi-2, the frequency appears to be lower than in patients with classical DM. Recently, an ELISA that used overlapping fragments spanning the sequence of Mi-2b was studied [68]. This highly sensitive ELISA detected a relatively high frequency of anti –Mi-2 (14% of European myositis patients), compared with the frequency seen in Polish patients (4.9%) [74] or in patients in U.S. studies that used immunodiffusion or immunoprecipitation (5 – 8%) [48,64]. The clinical subgroup associated with anti – Mi-2 antibodies as detected by the ELISA is different from that found using other techniques, with a higher rate of PM and even IBM. A major epitope on the NM fragment (‘‘N-terminal/middle’’) corresponded to that used in a previous study [132] in which the ELISA showed good correlation with other measures of anti – Mi-2 and relatively high disease specificity in extensive testing. Reaction with other fragments appears to be less DM-specific, and data that demonstrate disease specificity for reaction with these fragments are not available [137]. Thus, the ELISA detected the original anti – Mi-2-defined group plus additional patients, who appeared to be a different clinical group and whose antibody generally had different epitope specificity. This emphasizes that reported clinical associations can be significantly affected by the techniques used for antibody detection and raises the need for more standardization in this field. Like antisynthetase autoantibodies, strong immunogenetic associations have been noted with anti –Mi-2 autoantibodies. There is a strong association of anti – Mi-2 with HLA-DR7 [48,60]. Although anti – Mi-2 has been found in most populations studied, there is some variation in its frequency. A general trend toward increased DM was found in lower latitudes of Europe compared with northern areas [138]; a comparable trend was found for anti –Mi-2 and other MSAs [68]. In Mexico and Guatemala, there was a marked increase in the proportion of myositis patients with anti – Mi-2 (39% compared with 6% of white myositis patients in the United States), but fewer patients with antisynthetases [73]. Anti – Mi-2 can be a useful adjunct in the diagnosis of certain patients because of its myositis specificity. Some patients with anti – Mi-2 have severe or recurrent disease, but as a group with a low frequency of ILD, the prognosis appears to be better than for patients with antisynthetases [48].

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MAAs: anti – PM-Scl Anti – PM-Scl autoantibodies were initially identified by immunodiffusion [139], and were associated with a nucleolar and nuclear staining pattern by indirect immunofluorescence. Immunoprecipitation with serum positive for anti – PM-Scl antibodies results in a characteristic group of protein bands [39] that include: (1) a 100 kDa protein that is the main antigen reacting with almost all patient sera [140]; (2) a protein that migrates at 70 to 75 kDa that reacts with at least half of anti – PM-Scl sera [141]; and (3) at least nine other bands of 20 to 40 kDa, some of which may react with a small proportion of sera [142]. The sequence of the 100 kDa protein has homology to a yeast exonuclease Rrp6p involved in 5.8S ribosomal RNA processing [143]. Further study revealed that other PM-Scl proteins corresponded to other components of the yeast ‘‘exosome’’, a large complex of 30-50 exonucleases involved in RNA processing [144,145]. Several studies have analyzed the epitopes bound by anti –PM-Scl antibodies, and predominant epitopes have been noted [146,147]; thus far, no clinical or pathogenetic associations with differing epitope reactivity have been demonstrated. Anti –PM-Scl is almost as common in children with IIM as in adults [148]. There is a strong genetic association with HLA-DR3 [149]. It is most common in whites and is uncommon in blacks and Japanese. Anti – PM-Scl antibodies are most often associated with a characteristic overlap syndrome with features of systemic sclerosis (scleroderma) and PM or DM, that has been referred to as ‘‘scleromyositis’’ [66,74,149]. Arthritis is often a prominent part of the syndrome. The scleroderma usually shows limited cutaneous involvement. The myositis is often mild and responds well to treatment [74]. A typical DM rash may be seen, as well as mechanic’s hands, and calcinosis [66]. Raynaud’s and interstitial lung disease can be seen, as may occur in patients with either scleroderma or IIM; occasionally other organ involvement is similar to that seen in patients with systemic sclerosis. Approximately 75% of patients with anti –PM-Scl have signs of myositis [66], some of whom ( < 25% of total) do not show signs of scleroderma. Others have myositis that resolves easily with treatment, whereas manifestations of scleroderma remain. A small proportion may show neither myositis nor scleroderma, with only arthritis, or other symptoms [150]. Because some patients do not show myositis, the antibody is not considered ‘‘myositisspecific.’’ There is relative specificity for features of the overlap syndrome; in the proper clinical setting anti – PM-Scl autoantibodies can have comparable usefulness in diagnosis to the MSAs. Additionally, there is relative mutual exclusivity with MSAs (as well as scleroderma-specific antibodies), that allows them to define a subgroup, in contrast to some of the other MAAs. MAAs: antibodies to snRNPs Patients with anti-U1RNP, especially those without anti-Sm who have antibodies to the 70K protein, often have an undifferentiated rheumatic syndrome. This has been referred to as mixed connective tissue disease (MCTD), with nonspecific features associated with lupus, scleroderma, or PM, such as Raynaud’s phenomenon, arthritis, and esophageal dysmotility [65]. The frequency of myositis in

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patients with anti-U1RNP or MCTD varies from 16% to 79%, apparently due to differing criteria for inclusion and other factors [151]. Interstitial lung disease may occur, so that individual patients may resemble those with antisynthetases [86]. It has been suggested that the myositis may be milder than in patients with PM without overlap, but the severity is variable and myositis may be significant [10]. Anti-U1RNP was seen in 16% of patients in the series studied by Arnett et al [61], but at lower frequency in whites (4%) than in blacks (26%) or other groups. Consistent with this, Brouwer et al [68] found anti-U1RNP in only 6% of patients with IIM. Arnett et al [61] found anti-U1RNP in 60% of patients with myositis in SLE. Anti-Sm was also seen in 7% of Arnett et al’s group, including some who did not fulfill criteria for SLE. Anti-U1RNP is less likely to occur in patients with MSA (6%) compared with patients without MSA (23%) patients, and thus seems to define a subgroup. Patients may also have autoantibodies that react specifically with proteins unique to a single Sm snRNP, in the absence of anti-Sm. These are rare antibodies, much less common than anti-U1RNP or anti-Sm. Very few patients have been described, but most of those reported had myositis, alone, or as part of overlap syndromes. Specific anti-U2RNP [152], anti-U4/U6RNP [153], and anti-U5RNP [154], were described in occasional patients. Patients with anti-U2RNP may also show anti-U1RNP; this may be due to cross-reaction between proteins specific to each snRNP [154], but isolated anti-U4/6RNP and anti-U5RNP have been seen. All patients in a preliminary study of anti-U5RNP had myositis [155]. MAAs: antibodies to Ro/SSA Anti-Ro/SSA and anti-La/SSB have been thought to occur in a small percentage of patients with myositis; Arnett et al [61] found them in 17% and 6%, respectively, with an increased frequency (37% for Ro) in patients with antisynthetases or antiSRP. Anti-Ro60 and anti-La were infrequent in Brouwer et al’s study (4% and 5%) [68]. Recent reports showed a surprisingly high frequency of anti-Ro52 (25%), often without anti-Ro60, in patients with myositis [62,63,68]. There was an even higher frequency of anti-Ro52 in patients with anti –Jo-1 (58%) [63]; this was later shown to be true for other antisynthetases and anti– PM-Scl [62]. Anti-Ro52 was the most common MAA [68]. Anti-Ro52 may therefore have particular value in the diagnosis of myositis; this is tempered by its lack of disease specificity and the fact that many of the patients have other, disease-specific antibodies. MAAs: anti-Ku Anti-Ku autoantibodies react with 70 and 80 kDa proteins, which can be associated with a large, 350 kDa protein (DNA-dependent protein kinase). They bind to free ends of DNA, and are involved in DNA repair. In the United States, anti-Ku is most often found in patients with SLE or scleroderma; in Japanese, and occasional U.S. patients, it is associated with an overlap syndrome of myositis and scleroderma, sometimes with SLE [81,156]. Arnett et al [61] found anti-Ku in 19% of Japanese patients with myositis, 3% of blacks, 12% of MexicanAmericans, but not in any white patients with IIM. Anti-Ku was associated with

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DPB1* 0501, which is common in Japanese but rare in whites; this may account for this difference in frequency [156]. MAAs: anti-56kd An autoantibody was described in 1987 that reacted with a 56 kDa component of large nuclear ribonucleoproteins [157]. Almost all of the patients with the antibody had myositis. It was remarkable for its high frequency in patients with IIM [158,159]; it was found in up to 86% of adults, including 75% of patients with cancer-associated myositis, a group that others found to have only 31% positive ANAs [48]. The highest frequency (92%) was in patients with juvenile DM [159]. The antibody titer varied with disease activity, which, along with its higher sensitivity, could make it particularly valuable clinically; its high frequency suggests it may have a role in myositis pathogenesis. The high frequency also suggests that it crossed MSA/MAA-defined groups, although this was not directly shown. Difficulty with detection has hampered the more widespread study and clinical use of this antibody. In a recent preliminary study, the antibody was found in 62% of patients with juvenile DM, more often in those with DQA1* 0501 [160]. New antibodies Several new autoantibodies have been recently identified in patients with myositis, some of which have been described only in preliminary reports. Of particular note is a possible new MSA, anti-PMS1, and new autoantibodies in DM. Certain autoantibodies that are frequent in several autoimmune diseases, including antiendothelial cell autoantibodies [54] and autoantibodies to the 20S proteasome aC9 subunit [55], are prevalent in patients with myositis (Table 3). Antibodies that are reactive with the nuclear pore complex were found in 2 of 30 French- Canadian patients with myositis, but the disease specificity, and frequency in other populations, is unknown. PMS1 A small proportion (7.5%) of myositis sera was recently discovered to have autoantibodies to the DNA mismatch repair enzyme PMS1 [50]. None of 94 controls had these antibodies. Two other mismatch repair enzymes, PMS2 and MLH1, were tested; 3 sera were found to react with one or both of these enzymes, including one that also reacted with PMS1. One anti-PMS1 serum also had anti – Mi-2. There were not enough patients to draw further conclusions about the clinical associations, but the investigators considered anti-PMS1 to be myositis specific. Three of six patients had clinical and histologic DM. New DM antibodies Oddis et al described antibodies to a 140kd protein, called MJ, in 14 of 80 patients with IIM (17.5%); this is a relatively high frequency for myositis autoantibodies [51,161]. Of particular note was that 13 of the 14 patients had DM. It was more common in patients with juvenile DM, and was the most common

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autoantibody detected in a cohort from Argentina. A separate antibody that reacted with a 155kd protein was identified in 31 patients with myositis, [52]; 29 patients with antibodies to the 155 kDa protein had DM, including 9 adults and 20 children. Anti-155kd was particularly common in patients with amyopathic DM, a group that had a low frequency of MSAs and MAAs [53]. About one third of patients with anti-155kd also had antibodies to a 95 kDa protein labeled ‘‘Se’’. Usefulness of autoantibody testing Autoantibody testing can be used as a tool to assist in the diagnosis of PM and DM. It is not usually used for monitoring disease activity, although it is reasonable to repeat testing in unusual circumstances, (eg, to see if the antibody has disappeared). It was suggested [117] that the Bohan and Peter diagnostic criteria for PM and DM [85] be modified to include MSAs as an additional criterion. The criteria include weakness, muscle enzyme elevation, the DM rash, and characteristic findings by EMG and by biopsy. The requirement of meeting three criteria for probable disease and four criteria for definite disease would be maintained. An MSA could substitute for any other criterion, but could not, by itself, establish the diagnosis. Tanimoto et al [162] suggested adding anti –Jo-1 as a criterion. Certain MAAs, particularly anti –PM-Scl and anti-U1RNP, can be very useful in assessment of individual patients. ANAs and unidentified autoantibodies can suggest an autoimmune origin, but are not specific enough to allow conclusions.

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