Juvenile idiopathic inflammatory myopathies with anti‐3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase antibodies in a Chinese cohort

Abstract Aims To characterize the clinical and histopathological characteristics and treatment outcomes of juvenile idiopathic inflammatory myopathies (JIIMs) with anti‐3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) antibodies in a Chinese cohort. Methods We detected anti‐HMGCR antibodies in a series of Chinese JIIM by ELISA and indirect immunofluorescence assay on HEK293 cells, and summarized the clinical findings of these anti‐HMGCR antibody‐positive patients. Results Of 32 JIIM patients, 5 (15.63%) were found to be anti‐HMGCR antibody‐positive. The disease duration was 1.20 ± 0.45 months. Statin exposure was not found. Four patients had skin lesions, while typical pathological features of dermatomyositis such as perifascicular atrophy or myxovirus resistance protein A expression were not found. The mean creatine kinase level was 16771.60 U/L. Among the four patients who received long‐term (10.46 ± 1.42 years) follow‐up, three exhibited favorable outcomes with prednisone and additional immunosuppressants. Conclusions Our study indicates that anti‐HMGCR antibodies may not be rare in Chinese JIIM. These anti‐HMGCR‐positive JIIMs were characterized by acute onset, substantially elevated creatine kinase level, and skin lesions without perifascicular changes in muscle pathology. The treatment outcome is generally favorable with the combination of steroid and immunosuppressant.


| INTRODUC TI ON
Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune disorders, which are characterized by proximal muscle weakness and elevated creatine kinase (CK) levels. 1 Patients with onset age of first symptom ≤18 years are regarded as juvenile idiopathic inflammatory myopathy (JIIM). 2 Autoantibodies recognizing 3-hydroxy-3-methylglutaryl-coe nzyme A reductase (HMGCR), a target of statin medications, are typically associated with high CK levels and pathological features of necrotizing autoimmune myopathy (NAM) in adult IIM. 3,4 Clinical and pathological features of anti-HMGCR-positive JIIM have not yet been well characterized in the literature. [5][6][7][8][9][10][11] It has been previously reported that the prevalence of anti-HMGCR autoantibodies in JIIM varies markedly from 0.77% to 14.52%. [9][10][11][12] Additionally, the disease duration of anti-HMGCR-positive JIIM patients could also vary substantially. For instance, in two series studies with Japanese cohorts, one showed that more than half of the patients with anti-HMGCR antibody had disease duration of more than 2 years, and the other study showed that all 5 anti-HMGCR-positive JIIM had over 3 years of disease duration and 4 of these 5 patients even had disease course longer than 10 years of mimicking muscular dystrophy (MD), 8,11 while two separate studies of Chinese and American cohorts reported 60% and 100% anti-HMGCR-positive JIIM showed acute onset, respectively. 5,10 Consistently, most previous research showed extremely high serum CK level (≥ 8000 U/L) in anti-HMGCR-positive JIIM patients, with a record of over 20000 U/L in a study with European patients. 5,7,9,11,[13][14][15][16][17] However, it is noted that a Japanese study reported that the CK level of anti-HMGCR-positive JIIM was merely about 2000 U/L. 8 While multi-immunotherapy has been reported to be a common treatment method for anti-HMGCRpositive JIIM, 9,10 a few recent cases showed that intravenous immunoglobulin (IVIG) could also lead to dramatic clinical responses. In some cases, no medication was required. 14,15,17 Indeed, these inconsistent reports in the literature have led to confusion for devising appropriate treatments for this condition, calling for more clinical research.
In this study, we explored the prevalence of anti-HMGCR autoantibodies among 32 JIIM patients and summarized the clinical, serological, and histopathological characteristics and treatment outcomes of 5 anti-HMGCR-positive patients to improve the diagnosis and treatment of this disease.

| Participants
Clinical data were collected from 227 patients with IIM in the Department of Neurology at Qilu Hospital of Shandong University from April 2005 to September 2019. The diagnosis and classification of IIM were based on the criteria proposed by the European Neuromuscular Centre. 18 Among them, 32 were defined as JIIM patients whose onset age of first symptom was ≤18 years. 2 All the patients were Chinese in origin and assessed by two IIM experts. We reviewed their clinical manifestations, laboratory findings, muscle pathological features, treatment regimens, and outcomes. In order to establish an optimized in-house cutoff for anti-HMGCR antibody detection and to compare the clinical sample, we also studied a healthy control (HC) group of 100 age-and sex-matched individuals who received a routine health examination and for whom other diseases were excluded. Serum samples were collected from all IIM patients and HCs, and stored at −80°C until measurements.
In regard to clinical assessments, "chronic disease duration" was defined as a duration over 12 months from the disease onset to first examination. 19 Muscle strength was evaluated by the ordinal sixpoint (0-5) manual muscle testing (MMT) scale; asymmetric muscle weakness was defined as no less than 1 grade measured by MMT between two sides of the same muscle group or the same joint activity. 20 Treatment outcomes were graded as follows: no improvement, mild improvement (1 grade improvement in at least one muscle group, persistently requiring assistance in daily activities), moderate improvement (>1 grade in multiple muscle groups, occasionally requiring assistance in daily activities), marked improvement (only mild weakness without functional impairment), or return to baseline (no symptoms or signs of muscle weakness); a favorable outcome was defined as marked improvement or return to baseline. 21 The study was performed in accordance with the Declaration of Helsinki. All patients or their guardian gave their written informed consent, which was approved by the Ethics Committee of Qilu Hospital (Qingdao), Shandong University, China (KYLL-qdql2020019).

| Myositis-specific antibody (MSA) detection
Anti-HMCGR antibodies were measured in 227 patients with IIM and 100 HCs with QUANTA Lite ® HMGCR Kit (Inova Diagnostics), following the manufacturer's instructions. 22 We defined and

| Muscle biopsy
Muscle biopsies were taken from the biceps brachii, the deltoid,

| Statistical analysis
Qualitative variables were expressed as percentages and absolute frequencies, while quantitative features were reported as mean and standard deviation (SD) values. The Shapiro-Wilk normality test was used for checking the assumption of normal distribution.
The Mann-Whitney U test or Student's t test was applied to continuous data. Categorical variables were analyzed by Fisher's exact test as predicted frequency <5. Statistical significance was defined as p < 0.05. All analyses were performed using SPSS 22.0 (IBM Corp.).

| Detection of anti-HMGCR antibody in JIIM patient
Anti-HMGCR antibodies were detected in 5 (15.63%) of 32 patients with JIIM, and all these 5 patients were anti-HMGCR-positive by confirmatory IIFA ( Figure 1A). As anti-HMGCR antibodies were found in 16 adult IIM patients, the frequency of anti-HMGCR antibodies did not show significant difference between adult IIM and JIIM patients (p = 0.189). Moreover, the titer of anti-HMGCR antibody also did not show significant difference between these two groups (p = 0.275) ( Figure 1B).

| Clinical features in anti-HMGCR-positive JIIM patients
The clinical features of the 5 JIIM patients with anti-HMGCR antibody are summarized in Table 1. All these 5 patients were female with age at onset ranging from 4 to 15 years (mean ± SD, 10.20 ± 4.55 years). The duration from disease onset to the first visit was all within 2 months (mean ± SD, 1.20 ± 0.45 months).
Notably, none of these 5 patients had prior exposure to statin or statin-containing foods (including oyster, mushrooms, red yeast rice, or pu-erh tea). Skin lesions were seen in four patients. Specifically, two patients had Gottron's sign, which was the typical rash of DM, 1 with hypopigmentation in the forearms and one with malar rash.
Besides, all five patients experienced proximal limb weakness, two patients with neck weakness, and one patient with asymmetric muscle weakness. Dysphagia was seen in one patient and myalgia in two patients. Except for skin lesions, no extramuscular manifestations such as malignancy, interstitial lung disease (ILD), cardiac involvement, rheumatic disease, or Raynaud's phenomenon were found in these five patients.

| Treatment and outcomes
In the present study, among 5 anti-HMGCR-positive JIIM, 4 patients were followed up for more than 3 years (mean ±SD, 10.46±1.42 years). The detailed drug therapy and treatment outcomes of these 5 anti-HMGCR-positive patients are summarized in Table 2. One patient with marked improvement was not followed up after 30 months of treatment with oral prednisone. With the 4 patients who undergo a long-time follow-up, they were all initially treated with oral prednisone (1 mg/kg/day) and received additional immunotherapies including IVIG, cyclophosphamide, methotrexate, and azathioprine subsequently. All 4 patients suffered from relapse during taper or discontinuation of the drugs. While only one patient was drug-free at the last follow-up, they all showed improvement of muscle strength and decrease in CK level. In addition, 3 (75.00%) of these 4 patients had favorable outcomes. In our JIIM cohort, the clinical features of anti-HMGCRantibody-positive patients were distinct. Firstly, they were predominantly females, which is similar to a previous Chinese study. 5 Importantly, unlike many reported cases with chronic progressive disease onset mimicking MD, 8,11,14,17 patients in our cohort generally showed acute disease duration less than 2 months, indicating that anti-HMGCR-positive JIIM should be considered in pediatric patients with acute disease duration. In addition, the prevalence of skin rashes in Chinese JIIM patients with anti-HMGCR antibodies was more than 60%, which was in line with the statistics reported in America (about 80%) but considerably higher than that reported in Japan (about 20%). 5,8,10,11 Furthermore, our study suggested that non-typical DM skin rashes can be common in anti-HMGCRpositive JIIM patients, and careful assessments of skin condition should be performed. With regard to the laboratory examination of JIIM patients with anti-HMGCR antibody, the serum CK level was extremely high, consistent with previous studies. 5,7,9,11,17 Moreover, our patients with anti-HMGCR antibody did A limitation of our present study is that anti-HMGCR antibody was not tested by the gold standard immunoprecipitation. 7,26 However, the high concordance between the commercial ELISA and

CO N FLI C T S O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
YH and KS contributed equally to this work. YH contributed to study design, evaluation of the patients, data acquisition, interpretation and analysis, and manuscript drafting. KS contributed to MSA testing and manuscript revising. BZ, TD, QW, YZ, CY, YY, and XM contributed to data acquisition. WL contributed to study design, result interpretation, and manuscript revising. All authors approved the version of the article to be published.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data relevant to the study are either included in the article or will be shared at the request of any qualified investigator. Kai Shao https://orcid.org/0000-0003-1563-5722