Abstract
Objectives:
To define the clinical features of anti-Ku-positive myositis patients and to determine the reliability of the Euroline assay to detect anti-Ku autoantibodies.
Methods:
Serum samples were screened for anti-Ku autoantibodies by Euroline and positive samples were confirmed by ELISA. The prevalence and severity of clinical features at onset and during follow-up in patients with anti-Ku-positive myositis were compared to those with dermatomyositis, immune-mediated necrotizing myopathy (IMNM), the antisynthetase syndrome (AS), inclusion body myositis (IBM), anti-U1-RNP-positive myositis, and anti-PM/Scl-positive myositis.
Results:
72 (2.9%) of 2475 samples were anti-Ku positive by Euroline using the manufacturer’s recommended cutoff of >15. Just 17 (23.6%) of these were confirmed by ELISA and considered anti-Ku-positive for the analysis. Comparators included 169 IMNM, 168 AS, 387 IBM, 20 anti-U1-RNP-positive, and 47 anti-PM/Scl-positive patients. Muscle weakness was a presenting feature in 38% of anti-Ku-positive patients; 81% developed weakness during follow-up. Anti-Ku-positive patients had increased distal weakness compared to the non-IBM comparators. Interstitial lung disease (ILD) was present in 19% of anti-Ku-positive patients at the first visit and eventually developed in 56% of them. Throughout the course of disease, Gottron’s papules and/or heliotrope rashes were less common in anti-Ku-positive patients (19%) compared to those with dermatomyositis (94%) or anti-PM/Scl-positive myositis (89%). Anti-Ku-positive patients never developed calcinosis.
Conclusions:
The phenotype of anti-Ku positive myositis is distinguished by distal weakness, frequent ILD, infrequent rash, and no calcinosis. When used according to the current manufacturer’s instructions, the Euroline assay has a high false-positive rate for anti-Ku autoantibodies.
SEARCH TERMS: myositis, ELISA, EUROLINE, anti-Ku
INTRODUCTION
The inflammatory myopathies (IM) are a heterogeneous family of systemic autoimmune diseases. In addition to skeletal muscle, the lungs, skin, and joints may also be affected.1 Importantly, recognized autoantibodies are present in at least two-thirds of patients with IM. Myositis-specific autoantibodies (MSAs) are only rarely present only in patients without IM and include those recognizing Mi2, tRNA synthetases (e.g., Jo1), NXP2, TIF1γ, MDA, SRP, and HMGCR.2 In contrast, myositis-associated autoantibodies (MAA), which include anti-PM/Scl and anti-U1-RNP, are found not only in patients with myositis, but also in those with other autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and rheumatoid arthritis (RA).2
Importantly, most MSAs and MAAs are associated with distinct clinical phenotypes. For example, we have previously shown that anti-Mi2-positive dermatomyositis (DM) patients have more severe muscle disease than other DM patients3, anti-PM/Scl-positive myositis patients have a unique pattern of weakness with arm abductors weaker than hip flexors4, and anti-U1-RNP-positive myositis patients may develop glomerulonephritis or pericarditis.5
Anti-Ku autoantibodies are MAAs recognizing a 70–80 kDa protein that binds double-stranded DNA.6,7 These autoantibodies have been described in patients with several autoimmune diseases including SLE, SSc, mixed connective tissue disease (MCTD),8–19 Sjögren’s syndrome (SS),18,20,21 and RA.9,15,21
Although anti-Ku autoantibodies have been described in patients with myositis20,22–24, the phenotype of anti-Ku-positive myositis patients remains poorly described. Consequently, the objective of this study was to define the phenotype of anti-Ku-positive myositis patients and to compare the clinical features of anti-Ku-positive myositis patients to other types of IM in a large cohort of myositis patients. We also aimed to evaluate the utility of the widely used Euroline assay to test for anti-Ku autoantibodies.
MATERIALS AND METHODS
Patients:
Patients enrolled in the Johns Hopkins Myositis Center Longitudinal Cohort study with suspicion of an inflammatory muscle disease between 2006 and 2016 that were positive for anti-Ku autoantibodies by both line blot (EUROLINE myositis profile) and confirmed by enzyme-linked immunosorbent assay (ELISA; see below) were included in the study. We included as comparators all those myositis patients that had inclusion body myositis (IBM) according to Lloyd-Greenberg criteria25 or were positive for autoantibodies recognizing Mi2, NXP2, TIF1γ, MDA5, Jo1, PL-7, PL- 12, SRP, HMGCR, PM/Scl, or U1RNP by at least by 2 different techniques from among the following: ELISA, immunoprecipitation of in vitro transcribed and translated protein, line blotting (EUROLINE myositis profile), and immunoprecipitation from S35-labeled HeLa cell lysates (S35 IP). Comparators were included in the DM group if they had autoantibodies recognizing Mi2, NXP2, TIF1γ, or MDA5. Alternatively, patients were classified as having antisynthetase syndrome (AS) if they had autoantibodies against Jo-1, PL-7, or PL- 12. Patients were included in the immune-mediated necrotizing myopathy (IMNM) group if they tested positive for anti-SRP or anti-HMGCR autoantibodies. Anti-Ro-52 autoantibodies were detected by Euroline line blot both in patients and comparator groups. Patients confirmed positive for anti-Ku as well as those positive for anti-PM/Scl and anti-U1RNP were classified as SLE or SSc if they fulfilled the 1997 American College of Rheumatology (ACR) classification criteria for lupus26 or the 2013 classification criteria for SSc from the ACR/European Union League Against Rheumatism (ACR/EULAR)27 respectively. The 2017 EULAR/ACR criteria for inflammatory myopathy28 were also used to classify the anti-Ku-positive patients.
Muscle strength was evaluated by the examining physician using the Medical Research Council scale. This scale was transformed to Kendall′s 0–10 scale for analysis as previously described.29 Serial strength measurements for each patient were made by the same physician. For analyses, right- and left-side measurements for arm abduction and hip flexion strength were combined and the average was used for calculations (possible range 0–10).
The presence or absence of clinical signs and symptoms at disease onset was established retrospectively at the first patient visit based on a review of prior patient records and patient recollection. Interstitial lung disease (ILD) at the onset of the disease (often diagnosed before the first visit to the Myositis Center) was assessed by retrospective chart review. During the follow-up period, ILD was defined through a multidisciplinary approach as recommended by the American Thoracic Society.30 All patients with suspicion of pulmonary hypertension (PH) (compatible clinical and echocardiographic features) underwent a right heart catheterization. If the mean pulmonary arterial pressure PAPm was ≥ 25 mmHg at rest, the patients were considered as having PH.31
Skin manifestations specific for DM i.e., heliotrope rash or Gottron’s sign, symptoms of esophageal involvement (reflux and dysphagia), antisynthetase syndrome-associated clinical features mechanics hands, Raynaud’s phenomenon, arthralgia, arthritis, and fever), and SSc-specific skin involvement (i.e., sclerodactyly, telangiectasias, subcutaneous edema, puffy hands, or fingertip ulcers), were assessed prospectively. Glomerulonephritis was assessed by kidney biopsy and pericardial effusion by echocardiography.
The treatment at each visit was obtained retrospectively by chart review. Treatment decisions were made individually by each treating physician.
Line blot assay (Euroline):
We performed the Euroline line blot assay (EUROLINE Myositis Antigens Profile 4, EuroImmun, Lübeck, Germany) by using the first available serum of each patient, which generally corresponded to the first visit. This assay detects autoantibodies against Ku, Mi2a, Mi2b, TIF1g, MDA5, NXP2, SAE1, PMScl100, PMScl75, Jo1, SRP, PL7, PL12, EJ, OJ, and Ro52 autoantigens. Euroline strips were processed using the EUROBlotOne device. A signal intensity of 15 units was set as the cutoff for positivity as recommended by the vendor through their automated interpretation software (EUROLineScan). Of note, the methodology for the selection of the cutoff is not specified in the kit. Supplementary information about the diagnostic performance and validation of the test is available in the Supplementary Materials.
ELISA testing:
To confirm the positivity of the patients that tested positive for anti-Ku by Euroline, we performed an ELISA using the same sera. Ninety-six-well microtiter plates were coated with 100 ng per well of commercial recombinant heterodimer Ku70–80 (Sino Biological), incubated overnight at 4°C, washed, and blocked with 300 ul of 5% BSA-TBST at 37°C for one hour. After washing the wells, 1uL of each human sera was diluted 1:100 in 1%BSA-TBST, added, and incubated at 37°C for one hour. After washing, 100 ul per well of horseradish peroxidase-labeled (HRP) anti-human antibody diluted 1:10,000 with 1% BSA-PBS-T were added and incubated at 37°C for 40 min in the darkness. For color development, 100 ul per well of SureBlue peroxidase reagent (KPL) was added after 6 washes. The reaction was stopped by adding 100 uL of 1N HCl per well. We determined the absorbances at 450 nm and normalized the results to an arbitrary positive anti-Ku patient that was included in every ELISA plate. Sera from 192 healthy comparators enrolled at the NIH Clinical Center were tested using this ELISA, resulting in a mean absorbance of 0.09 with an SD of 0. 07. The cutoff for a negative anti-Ku autoantibody titer was subsequently defined as the mean absorbance value plus 2 SDs of the healthy control participants (0.23).
Standard protocol approvals:
The study received the approval of the Johns Hopkins University School of Medicine and National Institutes of Health Institutional Review Boards.
Statistical analysis:
Dichotomous variables were expressed as percentages and absolute frequencies, and continuous features were reported as means and standard deviations (SD). Pairwise comparisons for categorical variables between groups were made using the chi-square test or Fisher’s exact test, as appropriate. The Studentś t-test was used to compare continuous variables among groups. CK, a highly positively skewed variable, was expressed as median, first, and third quartile for descriptive purposes. All statistical analyses were performed using Stata/MP 14.1. A 2-sided p-value of 0.05 or less was considered significant with no correction for multiple comparisons.
RESULTS
Patients
From among 2475 patients enrolled in the Johns Hopkins Myositis Center Longitudinal Study, 72 were anti-Ku positive by Euroline using the manufacturer’s recommended cutoff of >15. Among these, only 17 (23%) were positive by ELISA. Of note, most (n=11, 65%) samples verified by ELISA had Euroline values over 150 (Supplementary Figure 1). Of note, 26 samples that were anti-Ku-positive by Euroline were also tested by immunodiffusion and immunoprecipitation at the Oklahoma Medical Research Foundation (OMRF) clinical laboratory. Only 5 of these were positive for anti-Ku autoantibodies at OMRF and each of these was also positive by ELISA. Taken together, these results confirm that the Euroline test has a high false-positive rate for anti-Ku autoantibodies and that the ELISA can be used to identify the true-positives.
The patients who were anti-Ku positive by Euroline and negative by ELISA constituted a very heterogeneous group, including 15 with DM, 12 with IBM, 16 with PM, and 11 who did not fulfill myositis criteria. Among these false anti-Ku-positive patients, we found 11 with MSA (4 with anti-HMGCR, 1 with anti-Jo1, 1 with anti-MDA5, 1 with anti-PL12, and 4 with anti-SRP autoantibodies).
Comparator groups were also obtained from patients enrolled at the Johns Hopkins Myositis Center and included those with MSAs (202 with DM, 168 with AS, and 169 with IMNM) and MAAs recognizing PM/Scl (47 patients) or U1-RNP (20 patients), and IBM (387 patients). Among the anti-Ku positive patients, 2 tested positive for anti-U1RNP, 1 tested positive for anti-Jo1, and 1 tested positive for anti-TIF1 gamma autoantibodies. For the purposes of this analysis, these patients were included only in the anti-Ku group. Among the anti-Ku positive subjects, 12 (71%) fulfilled the 2017 ACR/EULR criteria for inflammatory myopathy, one (6%) fulfilled the 2013 ACR/EULAR criteria for SSc, and one fulfilled the 2013 SLICC SLE criteria for lupus. In contrast, among the patients with anti-PM/Scl or anti-U1RNP autoantibodies, 96% and 70% met the criteria for myositis, 30%, and 35% met the criteria for SSc, respectively, and 50% of the subjects with anti-U1RNP autoantibodies fulfilled SLE criteria (Supplementary Table 1).
The general features of the anti-Ku-positive patients and the comparator groups are shown in Table 1. Anti-Ku-positive patients were less likely to be white than those with DM, IBM, or anti-PM/Scl autoantibodies. Those with anti-Ku autoantibodies were younger than those with IBM but older than those with AS, anti-PM/Scl autoantibodies, or anti-U1-RNP autoantibodies. Only one anti-Ku-positive patient had cancer-associated myositis, defined as cancer starting within 3 years of the myositis onset, which was similar to the prevalence of cancer in the other groups. Three patients from the anti-Ku positive group died during the follow-up period but only one died of complications related to the anti-Ku syndrome (worsening of ILD). Only patients with anti-U1-RNP autoantibodies had a higher prevalence of co-existing anti-Ro52 autoantibodies than those with anti-Ku autoantibodies. Compared to non-IBM myositis patients, anti-Ku-positive patients tended to be prescribed fewer steroids and other immunosuppressive agents (Table 1).
Table 1.
General features of anti-Ku patients compared with the myositis groups, the anti-U1RNP positive, and the anti-PM/Scl positive.
| Anti-Ku (n=17) |
DM (n=202) |
AS (n=168) |
IBM (n=387) |
IMNM (n=169) |
Anti-U1RNP (n=20) |
Anti-PM/SCl (n=47) |
Total (n=1010) |
|
|---|---|---|---|---|---|---|---|---|
| Female sex | 71% (12) | 73% (148) | 71% (119) | 36% (139)** | 63% (106) | 85% (17) | 77% (36) | 57% (577) |
| Race | ||||||||
| White | 47% (8) | 75% (151)* | 59% (99) | 82% (319)** | 67% (114) | 40% (8) | 89% (42)*** | 73% (741) |
| Black | 29% (5) | 13% (27) | 30% (51) | 10% (37)* | 24% (41) | 55% (11) | 6% (3)* | 17% (175) |
| Other races | 24% (4) | 12% (24) | 11% (18) | 8% (31)* | 8% (14) | 5% (1) | 4% (2)* | 9% (94) |
| Age of onset (years) | 54.4 (17.2) | 47.1 (15.4) | 46.1 (13.1)* | 64.9 (9.8)*** | 50.2 (15.5) | 37.8 (18.1)** | 42.2 (14.1)** | 54.0 (15.9) |
| Cancer associated myositis | 6% (1) | 9% (19) | 2% (4) | 15% (57) | 4% (7) | 0% (0) | 0% (0) | 9% (88) |
| Death during follow-up | 18% (3) | 4% (8)* | 8% (14) | 7% (29) | 3% (5)* | 0% (0) | 0% (0)* | 6% (59) |
| Time of follow-up (years) | 7.5 (4.9) | 4.3 (3.6)*** | 4.6 (3.9)** | 2.2 (2.9)*** | 4.0 (3.8)*** | 5.9 (4.6) | 6.3 (4.5) | 3.7 (3.8) |
| Number of visits per participant | 11.7 (9.1) | 9.6 (7.2) | 9.9 (7.0) | 3.9 (4.4)*** | 9.1 (8.9) | 21.2 (19.8) | 11.9 (8.0) | 7.7 (7.9) |
| Anti-Ro52 | 12% (2) | 21% (43) | 21% (43) | 15% (58) | 22% (37) | 65% (13)** | 30% (14) | 30% (302) |
| Irritable EMG | 25% (4) | 53% (98)* | 41% (61) | 79% (192)*** | 76% (121)*** | 41% (7) | 41% (17) | 62% (500) |
| Treatment # | ||||||||
| Corticosteroids | 41% (7) | 84% (169)*** | 94% (158)*** | 32% (125) | 74% (125)** | 85% (17)** | 89% (42)*** | 64% (643) |
| Azathioprine | 24% (4) | 29% (59) | 53% (89)* | 5% (21)* | 25% (43) | 35% (7) | 40% (19) | 24% (242) |
| Methotrexate | 29% (5) | 53% (108) | 42% (70) | 18% (69) | 51% (87) | 40% (8) | 34% (16) | 36% (363) |
| Mycophenolate | 18% (3) | 37% (74) | 41% (69) | 5% (18) | 20% (33) | 40% (8) | 60% (28)** | 23% (233) |
| IVIG | 18% (3) | 48% (97)* | 36% (61) | 8% (32) | 41% (70) | 30% (6) | 30% (14) | 28% (283) |
| Rituximab | 6% (1) | 16% (33) | 23% (38) | 0% (0)* | 24% (40) | 35% (7)* | 15% (7) | 12% (126) |
p < 0.05,
p < 0.01,
p < 0.001.
Cumulative treatments at the end of the follow-up.
Abbreviations: DM = dermatomyositis; AS = antisynthetase syndrome; IBM = inclusion body myositis; IMNM = immune-mediated necrotizing myopathy; anti_RPN = anti ribonucleoprotein; anti_PM/Scl = anti-PM/SCl complex; IVIG = IV immunoglobulin; SSc = scleroderma; EMG= electromyography, Dichotomous variables expressed as percentage (count) and continuous variables as mean (SD). Bivariate comparisons of continuous variables were made using the Student t-test while bivariate comparisons of dichotomous variables were made either using χ2 test or Fisher exact test, as appropriate. Each of the clinical groups was compared to the sample of anti-Ku-positive patients.
Muscle involvement
Prominent distal weakness in patients with anti-Ku autoantibodies
At disease onset, only 38% of anti-Ku-positive patients had muscle weakness, which was similar to the other groups except for IBM and IMNM patients, who had a significantly higher prevalence of weakness at disease onset (96% and 88%, respectively) (Table 2). Although the majority (81%) of those with anti-Ku autoantibodies developed weakness during the follow-up period, weakness continued to be more prevalent in the IBM and IMNM groups (Table 3). With regard to proximal muscle weakness severity, arm abductors were similarly weak in anti-Ku-positive patients and all the other groups except those with AS, who had stronger arm abductors (Table 4). Similarly, the severity of hip flexor weakness was similar in the anti-Ku positive patients and others except for AS and anti-PM/Scl patients, who were stronger (Table 4).
Table 2.
Clinical features of the anti-Ku patients compared with the myositis groups, the anti-U1RNP, and the anti-PM/SCl at the onset of the disease.
| Anti-Ku (n=17) |
DM (n=202) |
AS (n=168) |
IBM (n=387) |
IMNM (n=169) |
Anti-U1RNP (n=20) |
Anti-PM/SCl (n=47) |
Total (n=1010) |
|
|---|---|---|---|---|---|---|---|---|
| Muscle involvement | ||||||||
| Muscle weakness | 38% (6) | 50% (101) | 56% (94) | 96% (372)*** | 88% (149)*** | 32% (6) | 34% (16) | 74% (744) |
| Skin involvement | ||||||||
| DM-specific skin involvement | 6% (1) | 68% (138)*** | 11% (18) | 0% (0)* | 2% (4) | 16% (3) | 32% (15) | 18% (179) |
| Sclerodactyly | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 9% (4) | 0% (4) |
| Raynaud’s phenomenon | 25% (4) | 5% (10)* | 14% (24) | 0% (0)*** | 7% (11)* | 16% (3) | 28% (13) | 6% (65) |
| Telangiectasias | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) |
| Ulcers | 6% (1) | 2% (5) | 0% (0) | 0% (0)* | 0% (0) | 0% (0) | 0% (0) | 1% (6) |
| Carpal tunnel | 0% (0) | 0% (1) | 2% (4) | 0% (0) | 1% (1) | 0% (0) | 9% (4) | 1% (10) |
| Livedo reticularis | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 5% (1) | 0% (0) | 0% (1) |
| Mechanics hands | 0% (0) | 4% (8) | 15% (26) | 0% (0) | 1% (1) | 0% (0) | 19% (9) | 4% (44) |
| Calcinosis | 0% (0) | 2% (4) | 2% (4) | 0% (0) | 0% (0) | 0% (0) | 2% (1) | 1% (9) |
| Subcutaneous edema | 12% (2) | 7% (14) | 7% (12) | 0% (0)** | 2% (3) | 11% (2) | 17% (8) | 4% (41) |
| Puffy hands | 6% (1) | 1% (3) | 1% (1) | 0% (0)* | 0% (0) | 5% (1) | 9% (4) | 1% (10) |
| Lung involvement | ||||||||
| Interstitial lung disease | 19% (3) | 4% (9)* | 49% (82)* | 0% (0)*** | 1% (2)** | 5% (1) | 11% (5) | 10% (102) |
| Pulmonary hypertension | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) |
| Esophageal involvement | ||||||||
| Gastroesophageal reflux disease | 0% (0) | 0% (0) | 7% (12) | 0% (1) | 1% (1) | 11% (2) | 9% (4) | 2% (20) |
| Dysphagia | 0% (0) | 10% (20) | 9% (15) | 28% (110)** | 10% (17) | 16% (3) | 11% (5) | 17% (170) |
| Joint involvement | ||||||||
| Arthritis | 19% (3) | 6% (13) | 20% (34) | 2% (6)** | 1% (1)** | 16% (3) | 17% (8) | 7% (68) |
| Arthralgia | 25% (4) | 17% (34) | 43% (73) | 0% (1)*** | 5% (9)* | 37% (7) | 28% (13) | 14% (141) |
| Systemic involvement | ||||||||
| Fever | 0% (0) | 6% (13) | 10% (16) | 0% (0) | 2% (3) | 21% (4) | 2% (1) | 4% (37) |
| Sicca syndrome | 0% (0) | 1% (2) | 2% (3) | 0% (0) | 0% (0) | 0% (0) | 4% (2) | 1% (7) |
| Pericardial effusion | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 5% (1) | 0% (0) | 0% (1) |
| Glomerulonephritis | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) | 0% (0) |
p < 0.05,
p < 0.01,
p < 0.001.
Abbreviations: DM = dermatomyositis; AS = antisynthetase syndrome; IBM = inclusion body myositis; IMNM = immune-mediated necrotizing myopathy; Anti-U1RNP= anti-U1 ribonucleoprotein; anti-PM/SCl = anti-PM/SCl complex. Values are % (n). Chi-squared or Fisher exact tests were used to compare each of the clinical groups with anti-Ku-positive patients. The clinical features at the onset of the disease are based on retrospective reports from the patients.
Table 3.
Cumulative clinical features of the anti-Ku positive patients compared with the myositis groups, the anti-U1RNP, and the anti-PM/SCl.
| Anti-Ku (n=17) |
DM (n=202) |
AS (n=168) |
IBM (n=387) |
IMNM (n=169) |
Anti_U1RNP (n=20) |
Anti PM/Scl (n=47) |
Total (n=1010) |
|
|---|---|---|---|---|---|---|---|---|
| Muscle involvement | ||||||||
| Muscle weakness | 81% (13) | 90% (181) | 93% (156) | 99% (385)*** | 97% (164)* | 84% (16) | 94% (44) | 95% (959) |
| Skin involvement | ||||||||
| DM-specific skin involvement | 19% (3) | 94% (190)*** | 57% (96)** | 0% (1)*** | 8% (14) | 47% (9) | 89% (42)*** | 35% (355) |
| Sclerodactyly | 19% (3) | 1% (3)** | 10% (17) | 0% (0)*** | 0% (0)*** | 16% (3) | 57% (27)** | 5% (53) |
| Raynaud’s phenomenon | 56% (9) | 25% (50)* | 38% (64) | 0% (1)*** | 16% (27)*** | 79% (15) | 81% (38) | 20% (204) |
| Telangiectasias | 19% (3) | 18% (36) | 15% (26) | 0% (0)*** | 7% (11) | 11% (2) | 60% (28)** | 11% (106) |
| Ulcers | 6% (1) | 12% (25) | 5% (8) | 0% (0)* | 0% (0) | 5% (1) | 6% (3) | 4% (38) |
| Carpal tunnel | 0% (0) | 7% (15) | 15% (25) | 0% (0) | 8% (13) | 11% (2) | 13% (6) | 6% (61) |
| Livedo reticularis | 6% (1) | 10% (20) | 7% (12) | 0% (0)* | 3% (5) | 16% (3) | 11% (5) | 5% (46) |
| Mechanics hands | 38% (6) | 27% (55) | 57% (96) | 0% (0)*** | 4% (7)*** | 42% (8) | 74% (35)** | 21% (207) |
| Calcinosis | 0% (0) | 22% (45)* | 7% (12) | 0% (0) | 1% (1) | 26% (5)* | 36% (17)** | 8% (80) |
| Subcutaneous edema | 25% (4) | 22% (45) | 26% (43) | 0% (0)*** | 6% (10)* | 32% (6) | 45% (21) | 13% (129) |
| Puffy hands | 6% (1) | 7% (15) | 8% (13) | 0% (0)* | 0% (0) | 21% (4) | 38% (18)* | 5% (51) |
| Lung involvement | ||||||||
| Interstitial lung disease | 56% (9) | 13% (26)*** | 80% (135)* | 0% (0)*** | 6% (10)*** | 42% (8) | 62% (29) | 22% (217) |
| Pulmonary hypertension | 12% (2) | 2% (5) | 16% (27) | 0% (0)** | 1% (1)* | 26% (5) | 11% (5) | 4% (45) |
| Esophageal involvement | ||||||||
| Gastroesophageal reflux disease | 38% (6) | 31% (62) | 29% (49) | 1% (2)*** | 25% (43) | 42% (8) | 57% (27) | 20% (197) |
| Dysphagia | 56% (9) | 53% (108) | 38% (64) | 39% (152) | 45% (76) | 58% (11) | 55% (26) | 44% (446) |
| Joint involvement | ||||||||
| Arthritis | 38% (6) | 23% (46) | 57% (95) | 2% (8)*** | 5% (9)*** | 53% (10) | 53% (25) | 20% (199) |
| Arthralgia | 62% (10) | 54% (109) | 63% (106) | 1% (2)*** | 33% (56)* | 63% (12) | 81% (38) | 33% (333) |
| Systemic involvement | ||||||||
| Fever | 6% (1) | 17% (34) | 23% (38) | 0% (0)* | 7% (12) | 32% (6) | 9% (4) | 9% (95) |
| Sicca syndrome | 19% (3) | 25% (50) | 36% (60) | 0% (0)*** | 15% (25) | 16% (3) | 53% (25)* | 16% (166) |
| Pericardial effusion | 12% (2) | 0% (0)** | 1% (1)* | 0% (0)** | 0% (0)** | 32% (6) | 0% (0) | 1% (9) |
| Glomerulonephritis | 6% (1) | 0% (0) | 1% (1) | 0% (0)* | 0% (0) | 16% (3) | 0% (0) | 0% (5) |
p < 0.05,
p < 0.01,
p < 0.001.
Abbreviations: DM = dermatomyositis; AS = antisynthetase syndrome; IBM = inclusion body myositis; IMNM = immune-mediated necrotizing myopathy; Anti-U1RNP= anti-U1 ribonucleoprotein; anti-PM/SCl = = anti-PM/SCl complex. Values are % (n). Chi-squared or Fisher exact tests were used to compare each of the clinical groups with anti-Ku-positive patients. The clinical features at the onset of the disease are based on retrospective reports from the patients.
Table 4.
The pattern of weakness at the first visit of the anti-Ku positive patients compared to the myositis groups; the anti-U1RNP, and the anti-PM/SCl.
| Anti-Ku (n=17) |
DM (n=202) |
AS (n=168) |
IBM (n=387) |
IMNM (n=169) |
Anti-U1RNP (n=20) |
Anti-PM/SCl (n=47) |
Total (n=1010) |
|
|---|---|---|---|---|---|---|---|---|
| Neck flexors | 10.0 (0.0) | 8.8 (2.2) | 9.5 (1.3) | 7.7 (2.3) | 8.7 (1.9) | 10.0 (0.0) | 9.5 (0.8) | 8.9 (1.9) |
| Neck extensors | 10.0 (0.0) | 9.8 (0.7) | 9.7 (0.8) | 10.0 (0.0) | 9.8 (0.9) | 10.0 (0.0) | 9.7 (0.7) | 9.8 (0.7) |
| Arm abductors | 8.4 (2.4) | 8.4 (2.4) | 9.3 (1.4)* | 9.1 (1.8) | 8.0 (2.1) | 8.9 (1.8) | 8.2 (2.5) | 8.7 (2.0) |
| Elbow flexors | 8.2 (3.3) | 9.1 (1.5) | 9.7 (0.6)*** | 8.9 (1.7) | 8.8 (1.9) | 9.6 (1.0) | 9.1 (1.7) | 9.1 (1.6) |
| Elbow extensors | 8.0 (3.0) | 8.9 (1.6) | 9.6 (0.9)*** | 8.1 (1.7) | 8.6 (1.9) | 9.6 (1.0) | 9.0 (1.7) | 8.6 (1.7) |
| Wrist flexors | 8.9 (2.8) | 9.7 (0.7)** | 9.9 (0.4)** | 8.1 (2.0) | 9.7 (0.9)* | 9.8 (0.7) | 9.7 (1.0) | 9.0 (1.7) |
| Wrist extensors | 8.9 (3.0) | 9.7 (0.9) | 9.9 (0.4)** | 9.1 (1.9) | 9.8 (0.8)* | 9.8 (0.7) | 9.6 (1.1) | 9.4 (1.5) |
| Finger flexors | 8.5 (3.2) | 9.8 (0.6)*** | 9.8 (0.6)** | 5.7 (3.1)* | 9.6 (0.9)* | 9.8 (0.7) | 9.3 (1.7) | 7.9 (2.9) |
| Finger extensors | 8.2 (3.5) | 9.7 (1.0)** | 9.8 (0.8)** | 8.8 (2.0) | 9.8 (0.7)*** | 9.7 (0.8) | 9.2 (2.0) | 9.3 (1.7) |
| Hip flexors | 7.0 (3.9) | 8.1 (2.5) | 8.8 (1.8)*** | 7.6 (2.6) | 5.8 (3.1) | 8.6 (2.2) | 8.8 (2.0)* | 7.7 (2.7) |
| Hip extensors | 9.7 (0.8) | 9.5 (1.5) | 9.8 (0.7) | 9.7 (1.2) | 8.8 (2.5) | 9.7 (0.8) | 9.6 (1.5) | 9.5 (1.5) |
| Knee flexors | 8.3 (2.9) | 9.8 (0.9)*** | 9.9 (0.4)*** | 9.2 (1.6) | 8.9 (2.0) | 9.9 (0.4)* | 9.7 (0.7)** | 9.4 (1.5) |
| Knee extensors | 8.9 (1.2) | 9.7 (1.0)* | 9.8 (0.5)*** | 6.8 (3.1) | 9.1 (2.0) | 9.4 (2.0) | 9.8 (0.5)** | 8.3 (2.7) |
| Ankle flexors | 8.4 (3.1) | 9.8 (1.0)*** | 9.9 (0.3)*** | 8.2 (2.6) | 9.7 (1.1)** | 10.0 (0.0)* | 10.0 (0.2)** | 9.1 (2.1) |
| Ankle extensors | 8.6 (2.9) | 9.9 (0.5)*** | 10.0 (0.0)*** | 9.3 (2.0) | 9.7 (1.2)* | 10.0 (0.0)* | 10.0 (0.2)** | 9.6 (1.5) |
p < 0.05,
p < 0.01,
p < 0.001.
Abbreviations: DM = dermatomyositis; AS = antisynthetase syndrome; IBM: inclusion body myositis; IMNM = immune-mediated necrotizing myopathy; anti-U1RNP= anti-U1 ribonucleoprotein, anti-PM/SCl = anti-PM/SCl complex. Strength values(Kendall scale) expressed as mean (SD) and bivariate comparisons made using Student’s t-test.
Of note, anti-Ku-positive patients presented with more severe distal weakness than all of the non-IBM comparator groups (Table 4). Specifically, ankle extensors and ankle dorsiflexors were weaker in anti-Ku-positive patients compared to those with DM, AS, IMNM, or either of the other MAAs. Similarly, those with anti-Ku autoantibodies had weaker wrist flexors, wrist extensors, finger flexors, and finger extensors than those with DM, AS, or IMNM (Table 4). We did not identify significant contractures of sensory-motor neuropathy to explain this finding. Unlike IBM, weakness in finger extensors was similar to that of finger flexors in anti-Ku patients.
Mean maximum CK levels were no different between those with anti-Ku autoantibodies and the other groups except for DM patients, who had lower CK levels, and IMNM patients, who had higher CK levels (Table 5). Anti-Ku-positive patients were less likely to have an irritable myopathy on EMG than those with DM, IBM, or IMNM. Among the four anti-Ku-positive patients who had muscle biopsies available for review, three had histopathological features consistent with a necrotizing myopathy and the fourth had regenerating myofibers but no necrotic or actively degenerating muscle cells. Two of the patients had mild perivascular inflammation but none had endomysial inflammation or perifascicular atrophy.
Table 5.
Disease activity during follow-up.
| Anti-Ku (n=17) |
DM (n=202) |
AS (n=168) |
IBM (n=387) |
IMNM (n=169) |
Anti-U1RNP (n=20) |
Anti-PM/SCl (n=47) |
Total (n=1010) |
|
|---|---|---|---|---|---|---|---|---|
| Mean hip flexor strength | 7.5 (3.3) | 8.7 (1.9)* | 9.1 (1.4)*** | 7.5 (2.5) | 6.4 (2.9) | 8.8 (1.9) | 9.2 (1.5)** | 8.0 (2.4) |
| Hip flexors strength at last visit | 9.2 (1.0) | 9.0 (2.0) | 9.3 (1.4) | 7.3 (2.7)* | 6.7 (3.6)* | 9.8 (0.4) | 9.1 (1.8) | 8.1 (2.7) |
| Mean arm abductor strength | 8.4 (2.2) | 9.0 (1.6) | 9.4 (1.0)*** | 9.0 (1.8) | 8.4 (2.0) | 9.4 (0.7) | 8.6 (2.4) | 9.0 (1.7) |
| Arm abductors strength at last visit | 9.2 (1.7) | 9.2 (1.9) | 9.5 (1.1) | 8.8 (2.2) | 8.5 (2.5) | 9.7 (0.6) | 8.6 (2.5) | 9.0 (2.1) |
| Median CK | 493 (207–872) | 121 (70–328)* | 282 (108–1020) | 426 (218–703) | 1387 (501–2874)** | 237 (135–696) | 138 (78–509) | 362 (136–868) |
| Maximum CK | 567 (328–1361) | 806 (139–3513) | 1439 (330–6000) | 543 (270–977) | 5372 (2274–10000)*** | 760 (507–4166) | 1149 (247–3000) | 948 (314–3600) |
| Mean aldolase | 9.5 (5.7) | 9.6 (7.4) | 45.7 (296.0) | 11.6 (9.5) | 34.0 (47.0) | 20.1 (19.0) | 13.8 (11.9) | 21.8 (133.5) |
| Maximum aldolase | 14.5 (12.5) | 13.0 (14.7) | 131.0 (1177.7) | 14.3 (30.8) | 55.8 (70.3)* | 36.1 (41.0) | 23.3 (22.2) | 44.8 (524.8) |
p < 0.05,
p < 0.01,
p < 0.001.
Abbreviations: DM = dermatomyositis; AS = antisynthetase syndrome; IBM = inclusion body myositis; IMNM = immune-mediated necrotizing myopathy; anti-U1RNP = anti-U1 ribonucleoprotein; anti-PM/SCl = anti-PM/SCl complex;; CK = creatine kinase
Strength and aldolase values expressed as mean (SD) and CK as median (Q1–Q3). Bivariate comparisons made using Student’s t-test for strength and Wilcoxon rank-sum test for CK. Each of the clinical groups was compared to the sample of anti-Ku-positive patients
Extramuscular involvement
Skin.
Among anti-Ku-positive patients, 19% developed DM-specific skin features (i.e., Gottron’s or heliotrope rashes) at some time during the follow-up period, which was lower than the prevalence of these rashes in those with DM (94%), AS (57%), or anti-PM/Scl autoantibodies. Only anti-PM/Scl-positive patients were more likely to have mechanic’s hands than anti-Ku patients (74% vs. 38%). No patient with anti-Ku autoantibodies developed calcinosis during the follow-up period; in contrast, 22% of DM, 26% of anti-U1-RNP-positive, and 36% of anti-PM/Scl-positive patients had calcinosis, Sclerodactyly, a feature typically associated with SSc, was present in 19% of anti-Ku patients; this was increased compared to those with DM (1%) and decreased compared to those with anti-PM/Scl.
Lung, heart, and kidney.
Although only 19% of anti-Ku-positive patients presented with ILD, 56% eventually developed this clinical feature. DM and IMNM patients developed ILD less frequently (13% and 6%, respectively), and only the patients in the AS group had significantly more ILD than those with anti-Ku autoantibodies 80% vs 56%, p <0.05). Pulmonary hypertension, pericardial effusion, and glomerulonephritis were virtually absent at disease presentation in all patient groups. Among anti-Ku-positive patients, two (12%) eventually developed pericardial effusion, compared to 0% of DM and only 1% of AS patients. Similarly, only small numbers of anti-Ku positive patients developed pulmonary hypertension (12%) or glomerulonephritis (6%), which was similar to the prevalence of these clinical features in those with DM, AS, IMNM, and both of the other MAA groups.
Association of CK values with ILD.
Since a prior report suggested that anti-Ku-positive patients with high CK levels were more likely to develop ILD19, we analyzed the association of CK levels with ILD in our cohort. Among 17 anti-Ku-positive patients, 16 (94%) had available CK levels. Seventy-five percent of them (n=12) were found to have high levels of this muscle enzyme, and in this group, 67% (n=8) suffered also from ILD. Fifty percent (n=2) of the anti-Ku patients with normal levels of CK had also ILD, but the prevalence of ILD between those with high CK levels and the ones with normal CK was not statistically significant (p= 0.6).
Dysphagia.
Although dysphagia was not a presenting feature in any anti-Ku-positive patient, 56% had difficulty swallowing at some time during the follow-up period. The prevalence of dysphagia was higher at disease onset among those with IBM (28%) but was similar among all groups during the course of the disease but not significantly different than in the other groups during the course of the disease.
Joint involvement.
Arthritis was a presenting feature in 19% and eventually developed in 38% of anti-Ku-positive patients. The prevalence of arthritis was similar at disease onset and during follow-up in the other groups, except for those with IBM and IMNM, who had low rates of arthritis even during the follow-up period (1% and 5%, respectively).
Arthralgias were relatively common at disease onset (25%) and during follow-up (62%) in those with anti-Ku autoantibodies; those with IBM (1%) and IMNM (33%) had a lower prevalence of arthralgias during the course of the disease.
Raynaud’s phenomenon was present in 56% of anti-Ku patients compared to just 25% of DM and 16% of IMNM patients.
DISCUSSION
In this study, we demonstrated that anti-Ku-positive myositis patients have a unique clinical phenotype that distinguishes them not only from patients with IBM, DM, IMNM, and ASyS but also from myositis patients with other MAAs, including anti-U1RNP and anti-PM/Scl autoantibodies. First, anti-Ku-positive myositis have an unusual pattern of weakness characterized by distal muscle involvement that is more severe than in any of the non-IBM comparator groups especially in the absence of any sensori-motor nerve involvement. Second, more than half of patients with anti-Ku autoantibodies eventually develop ILD, which distinguishes them from those with DM, IMNM, or IBM. Third, unlike Spielmann et al.19 we did not find a significantly higher prevalence of ILD in those anti-Ku patients with high CK levels compared to those with normal CK levels. Fourth, in contrast to those with DM or anti-PM/Scl autoantibodies, Gottron’s papules and heliotrope rash are uncommon in anti-Ku-positive myositis patients. And finally, unlike DM or myositis patients with the other MAAs, anti-Ku-positive patients do not develop calcinosis. Thus, the anti-Ku-positive patients are the only autoantibody-defined myositis group characterized by distal weakness, a high risk of ILD, infrequent rash, and absence of calcinosis.
We found that extramuscular manifestations commonly develop in myositis patients with anti-Ku autoantibodies. These include arthralgia (62%), Raynaud’s phenomenon (56%), interstitial lung disease (56%), mechanic’s hands 38%, and arthritis (38%). Among all the comparator groups in this study, only those with anti-PM/Scl autoantibodies had more extramuscular involvement. Despite the prevalence of extramuscular disease manifestations, very few anti-Ku-positive patients fulfilled the criteria for SSc or SLE. By comparison, nearly one-third of anti-PM/Scl-positive and over half of anti-U1RNP-positive myositis patients had “overlap myositis” with concomitant lupus or scleroderma.
Although only 4 anti-Ku positive patients had a muscle biopsy available for review, it was notable that 3 (75%) of these revealed typical features of a necrotizing myopathy. Necrotizing muscle biopsies were less common in those with anti-PM/Scl autoantibodies (24%; p=0.049), underscoring that the different MAAs may be associated not only with different clinical manifestations but also with different histological features.
Although commercial line blot assays are a convenient and popular method to test for autoantibodies in patients with myositis, they may have false positive and negative issues for certain autoantibody specificities.20,32–36 In the current study, we found that the Euroline line blot assay had a striking high false-positive rate for anti-Ku autoantibodies using the manufacturer’s cutoff value; only 23% of Euroline-positive samples were confirmed positive by ELISA. The high false-positive rate of Euroline for anti-Ku autoantibodies was further confirmed by immunodiffusion and immunoprecipitation assays in a representative subset of the samples. Importantly, most (65%) of the samples validated by ELISA had Euroline values over 150 (Supplementary Figure 1), underscoring the need to individualize cutoffs for the Euroline assay for each autoantibody to improve its performance.
This study has several limitations. First, our analyses are based on signs and symptoms that were documented from the beginning of the study in 2006, prior to the development of some activity and damage tools now available. Second, patients included in this study presented to a multidisciplinary myositis center. Thus, the results could be biased towards including patients with active muscle and lung disease. Third, given the small number of anti-Ku–positive patients included and the low number of muscle biopsies available, this study may have been underpowered to detect differences in some key features. Future studies including larger numbers of patients could reveal additional differences between anti-Ku-positive myositis patients and those with other types of myositis.
These limitations notwithstanding, we show here that anti-Ku-positive patients have a distinctive phenotype characterized by distal weakness and frequent ILD with minimal cutaneous manifestations. Recognizing that the different MAAs define different clinical phenotypes, we propose that “the anti-Ku syndrome” should be recognized, along with the “anti-PM/Scl syndrome” and “anti-U1RNP syndrome”, as distinct types of myositis in future myositis classification schemes.
Supplementary Material
FUNDING:
This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (AR-041203). We are also grateful to Dr. Peter Buck, whose generous support made this work possible. IPF’s research was supported by a Fellowship from the Myositis Association. JJP’s research was supported by an award from the National Institutes of Health (K23-AR073927). We also want to acknowledge the Hopkins Rheumatic Diseases Research Core center, which is funded by the National Institutes of Health and the National Institute of Arthritis, Musculoskeletal and Skin Diseases grant P30-AR070254.
Footnotes
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Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
CONFLICT OF INTERESTS: No potential conflict of interest was reported by the author
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