Autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase in patients with statin-associated autoimmune myopathy

Abstract

Objective: In addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing myopathy (IMNM), with autoantibodies that recognize ∼200-kd and ∼100-kd autoantigens. The purpose of this study was to identify these molecules to help clarify the disease mechanism and facilitate diagnosis.

Methods: The effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using sera from patients. The identity of the ∼100-kd autoantigen was confirmed by immunoprecipitation of in vitro-translated 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by enzyme-linked immunosorbent assay and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy.

Results: Statin exposure induced expression of the ∼200-kd/∼100-kd autoantigens in cultured cells. HMGCR was identified as the ∼100-kd autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ∼200-kd protein. In muscle biopsy tissues from anti-HMGCR-positive patients, HMGCR expression was up-regulated in cells expressing neural cell adhesion molecule, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients ages 50 years and older, 92.3% had taken statins. The prevalence of the rs4149056 C allele was not increased in patients with anti-HMGCR.

Conclusion: Statins up-regulate the expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy.

Figure 1

Expression of the 200 and 100 kDa autoantigens is up-regulated by statins (A) and the 100 kDa autoantigen is HMGCR (B). A: Radiolabeled lysates generated from HeLa cells treated in the absence (lane 1) or presence (lane 2) of 10 µM mevinolin for 24 hours were immunoprecipitated with patient serum 9190 as described in the Methods section. B: ³⁵S-methionine-labeled full-length IVTT HMGCR was immunoprecipitated using sera from anti-100/200 kDa positive patients (lanes 3–7; representative of 16 anti-200/100 positive serum samples tested), anti-100/200 kDa negative DM patients (lanes 8–10) or healthy controls (lanes 11–13). The input IVTT product is shown in lane 14. The data shown in A and B are representative of similar results obtained in at least 3 separate experiments.

Figure 2

Human anti-HMGCR antibodies immunoprecipitate full-length HMGCR and a piece corresponding to the C-terminus (aa 340–888). Immunoprecipitations were performed using 3 different ³⁵S-methionine labeled HMGCR products: full-length (lanes 4–8), C-terminus (lanes 9–13) and N-terminus (lanes 14–18). Sera 10009, 9190 and 8050 are all from anti-100/200 kDa patients, while 488 and 495 are normal controls. Lanes 1–3 show the input IVTT products, which, in each case, was 0.4 times the amount used for the immunoprecipitation. Data shown are representative of those obtained in 2–8 separate experiments.

Figure 3

Competition immunoprecipitations confirm that human anti-HMGCR antibodies detect the C-terminus (panel A) and that the 200 kDa protein is not recognized by a unique autoantibody (panel B). A: Sera 10,009 and 9190 were preincubated with the indicated amounts of unlabeled C-terminal HMGCR and subsequently used to immunoprecipitate full-length ³⁵S-methionine labeled HMGCR. B: Sera 9190 and 9176 were preincubated in the absence (lanes 11 & 13) or presence (lanes 12 & 14) of 300 ng unlabeled C-terminal HMGCR. They were subsequently added to radiolabeled lysates generated from HeLa cells treated with 10 µM mevinolin for 24 hours. The resulting immunoprecipitates were processed as described in the methods section. Identical data were obtained in 2 separate experiments using 4 (A) or 6 (B) different patient sera.

Figure 4

HMGCR expression is up-regulated in regenerating myofibers expressing NCAM. Muscle biopsies from anti-HMGCR positive (A, B, and C) and control subjects (D, E, and F) were co-stained with anti-NCAM (A and D; green), anti-HMGCR antibodies (B and E; red) and DAPI (blue) to stain nuclei. Overlay (C and F) demonstrates HMGCR and NCAM are frequently co-expressed at high levels in the same myofibers in anti-HMGCR positive biopsies (white arrows) but not control muscle tissue. To ensure comparability, images A–C and D–F were obtained using identical exposure settings for each channel. These results are representative of staining seen in 6 anti-HMGCR positive and 3 normal muscle biopsies. (20X objective).