We reviewed medical records of patients with myopathy, who were referred to Gangnam Severance Hospital between January 2002 and March 2022. Then we selected 99 patients with inflammatory myopathy (53 with polymyositis, 29 with IMNM, 9 with dermatomyositis, and 8 with inclusion body myositis). We also selected 63 healthy subjects and 36 patients with genetic myopathy. This study was approved by the Institutional Review Board of Gangnam Severance Hospital, Korea (approval number: 3-2020-0251). Written informed consent was obtained from all patients according to the protocol. In addition, the study was conducted following the Declaration of Helsinki for medical research involving human subjects.
Phenotype assessment
Clinical, laboratory, and pathologic data were obtained retrospectively through medical record review. Clinical information included assessments of age at symptom onset, disease duration, muscle impairments, dysphagia, myalgia, skin lesions, interstitial lung disease, cancers, and treatment regimens. Laboratory analysis included serum creatine kinase (CK) level. Physical disability was evaluated using the seven-level scale of the modified Rankin Scale (mRS) with 0 = no symptoms; 1 = symptoms but no disability for daily function; 2 = slight disability unable to carry out previous activities, but can function without assistance; 3 = moderate disability, requires some help but can walk without assistance; 4 = moderately severe, unable to walk without assistance and needs assistance for bodily care; 5 = severe disability, bedridden requiring constant nursing care; 6 = dead11.
Patients with inflammatory myopathy were classified according to the 2017 European League Against Rheumatism/American College of Rheumatology Classification (EULAR/ACR) criteria for adult inflammatory myopathies, the European Neuromuscular Centre (ENMC) 2017 criteria for immune mediated necrotizing myopathy, or the Griggs criteria for inclusion body myositis12-14.
Anti-HMGCR ELISA
Anti-HMGCR antibodies were measured in the sera of 99 patients with inflammatory myopathy, 63 healthy subjects, and 36 patients with genetic myopathy. The enzyme-linked immunosorbent assay (ELISA) plates coated with recombinant HMGCR were incubated with diluted serum. The assay was performed according to the manufacturer’s standard protocol for the QUANTA Lite assay (Inova Diagnostics Inc., San Diego, CA, USA)15. The cutoff was 20 units (U/ml).
Line blot immunoassay for 16 myositis-specific autoantibodies
We tested 16 myositis-specific autoantibodies (MSAs) against these antigens: Mi-2α, Mi-2β, TIF1γ, MDA5, NXP2, SAE1, Ku, PM-Scl100, PM-Scl75, Jo-, SRP, PL-7, PL-12, EJ, OJ, and Ro-52 in all patients with anti-HMGCR myopathy. These antibodies were tested by line blot immunoassay of Euroline Autoimmune Inflammatory Myopathies 16 Ag (Euroimmun, Lübeck, Germany) according to the standard protocol. Euroimmune recommends interpreting results based on signal intensity. Signal intensity greater than 25 in the EURO-Line Scan Flatbed scanner generated a medium to strong band and was considered a positive result for the particular antibody. Results are shown as + for the medium band, ++ for the strong band, +++ for the very strong band with an intensity comparable to the control band.
Histological examination
Muscle biopsy was performed in 10 patients (P1, P2, P3, P4, P5, P6, P7, P8, P12, and P14). All histological and histochemical staining was performed on frozen materials. Sections were cut in a cryostat at -23 to -25 ℃. Briefly, 10㎛-thick sections were prepared for histochemistry. All available slides were stained with hematoxylin and eosin, modified Gomori trichrome, and nicotinamide adenine dinucleotide–tetrazolium reductase and were evaluated by a pathologist.
Lower limb magnetic resonance imaging
Seven patients (P2, P4, P5, P6, P7, P10, and P16) underwent lower limb magnetic resonance imaging (MRI) of the pelvis, thigh, and calf muscles on one of the following three MRI machines: Two 1.5T systems (MAGNETOM Vision, Siemens, Erlangen, Germany or MAGNETOM Avanto, Siemens, Erlangen, Germany) and a 3.0T system (MAGNETOM Vida, Siemens, Erlangen, Germany). The imaging was done in the axial (field of view [FOV] 24–32 cm, slice thickness 10mm, slice gap 0.5–1 mm) and coronal planes (FOV 38–40 cm, slice thickness 4–5 mm, slice gap 0.5–1.0 mm). The protocol used for all patients was T1-weighted spin-echo (SE) (repetition time [TR] 570–650 milliseconds, echo time [TE] 14–20 milliseconds, 12 matrics) and short tau inversion recovery (STIR) -weighted SE (TR 3,090–4,900 milliseconds, TE 85–99 milliseconds, 12 matrics). The degree of fatty replacement was evaluated according to the Mercuri scale: grade 0 = normal appearance, grade 1 = traces of increased signal intensity, grade 2 = increased signal intensity with confluence in less than 50% of the muscle, grade 3 = increased signal intensity in more than 50% of the muscle, and grade 4 = increased signal intensity over the entire muscle16,17.
Statistical analysis
Fischer’s exact test was used to compare discrete variables. The Mann-Whitney test was used to compare age at symptom onset, disease duration, serum CK level, and titer of serum anti-HMGCR antibodies. Correlation studies between titers of anti-HMGCR antibodies and serum CK were performed using a Spearman’s correlation. We compared the mRS scale and the titers of anti-HMGCR antibody pre- and post- treatment using the Wilcoxon signed rank test. Differences were considered statistically significant at p- value ≤ 0.05. All statistical analyses were conducted using R software (version 4.2.0, www.r-project.org).