Immune-Mediated Rippling Muscle Disease Associated With Thymoma and Anti-MURC/Cavin-4 Autoantibodies

Case Report

A 40-year-old man presented in February 2018 with myalgias and rippling waves of muscle contraction in the pectoralis major and deltoid muscles (Figure 1A; Videos 1 and 2). Surface EMG (EMG) showed electrical silence during muscle rippling. Needle EMG studies revealed fibrillation potentials and pseudomyotonic discharges in deltoid muscles. The serum creatine kinase (CK) level was increased at 1,890 IU/L (normal value <200 IU/L). Anti-Titin antibodies were positive without anti-Lgi1, anti-Caspr2, anti-AChR, anti-MuSK, and anti–myositis-specific or -associated antibodies. Molecular investigations revealed no mutation in the CAV3 and PTRF/CAVIN1 genes. Whole-body fluorodeoxyglucose-PET showed a hypermetabolic mediastinal left paracardiac tumor in contact with the lower part of the thymus (Figure 1A.c).

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Figure 1 Rippling Patient (RIP) Clinical, Radiologic Features and Deltoid Muscle Biopsy vs Control (CT)

Rippling (A.a) Voluntary contraction and active stretching induced rippling phenomenon, which traversed the pectoralis major and deltoid muscles in a wave-like manner. (A.b) Quick and firm percussion of biceps muscle induced muscle mounding (see black arrow). (A.c) Whole-body fluorodeoxyglucose-PET showed a hypermetabolic mediastinal left paracardiac tumor (white arrow) in contact with the lower part of the thymus. (B) Hematoxylin-phloxine-saffron (HPS) staining and immunohistochemistry analyses. (B.a) HPS analysis revealed peri- and endomysial inflammatory infiltrates in the Rippling (RIP) muscle patient. (B.b) Increased expression of peri- and intrafascicular HLA-I in the RIP muscle patient. (B.c) Upregulated expression of peri- and intrafascicular HLA-DR in the RIP muscle patient. (B.d) Increased expression of peri- and intrafascicular CD56 in the RIP muscle patient. (B.e) Caveolin-3 expression in control (CT) muscle. (B.f) Caveolin-3 expression in the RIP muscle patient. Note the strong decrease of Caveolin-3 staining in RIP patient muscle. (B.g) Dysferlin expression in control (CT) muscle. (B.h) Dysferlin expression in RIP patient muscle. Note the strong decrease of staining and distribution perturbation of dysferlin in RIP patient muscle. Magnification ×100.

Hematoxylin-phloxine-saffron staining performed on the patient's deltoid muscle biopsy revealed rare perifascicular and endomysial necrotic fibers associated with few pericapillary perimysial and endomysial inflammatory infiltrates (Figure 1B.a). The inflammatory infiltrates were composed mostly of CD20-positive B cells, few T cells (more CD4 than CD8-positive cells), and CD68-positive macrophages. Immunohistochemistry (IHC) investigations identified peri- and intrafascicular HLA-I and HLA-DR upregulated expression associated with positive CD56-regenerating fibers (Figure 1B.b–B.d). Complementary IHC on frozen muscle sections did not reveal any positive signal for C3, C1q, IgG, -A, and -M, and free light chain kappa and lambda. The terminal membrane attack complex C5b-9 was observed as rare endomysial punctiform deposits without endomysial capillaries stainings (data not shown). Overall, this set of data appeared evocative of an overlap myositis pattern.¹⁰ Of interest, a significative decreased sarcolemmal expression of Caveolin-3 was observed in a mosaic-like pattern associated with disturbed localization of its interacting partner dysferlin (Figure 1B.e–B.h).

Electron microscopy (EM) approaches revealed as well a reduction of caveolae formation at the sarcolemma level at the expense of an accumulation of vesicular and tubular structures beneath the sarcolemma (Figure 2) as commonly found in inherited caveolinopathies.¹ The patient underwent surgical resection of an encapsulated type B2 thymoma of the World Health Organization (WHO) classification and received steroids during 4 months with only partial improvement of his symptoms and persistently elevated CK level (593 IU/L). Azathioprine was then added with progressive disappearance of rippling phenomenon within few months. The serum CK level decreased at 291 IU/L.

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Figure 2 Electron Microscopy Analyses of Control (CT) and Rippling Patient Skeletal Muscles

CT individual and RIP-thymoma associated rippling patient. Note the presence of caveolae structures in CT (white arrows), which are mainly absent from the RIP patient at the expense of vesicular and tubular structures that accumulate beneath the sarcolemma (white arrowheads and black arrows, respectively). Scale bars 100 and 200 nm, respectively.

We next investigated the presence in the patient serum (PS) of specific antibodies targeting skeletal muscle antigens by Western blotting on human myoblasts (MBs) and myotubes (MTs). A major 55-kDa immunoreactive band was detected with PS in MT extract (Figure 3A). Conversely, when probed with the posttreatment patient serum, a drastic signal decrease of the 55-kDa band was observed (Figure 3A), thus suggesting strong autoantibody titer reduction after tumor resection and immunotherapy. No signal was observed with healthy control serum (Figure 3, A and B). These data were confirmed by immunofluorescence microscopy (Figure 3B).

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Figure 3 iRMD Associated With Thymoma and MURC/Cavin-4 Autoantibodies

(A) Representative WB of myoblast (MB; lane 1) and myotube (MT; lane 2) protein extracts from the immortalized Y711i myoblast cell line or HEK293T cells (lane 3) and probed with anti-Myosin Heavy Chain (MHC) (marker of differentiated MT), patient serum (PS), healthy control serum (HCS) as negative control, patient serum after tumor ablation and immunotherapy (TPS for treated patient serum) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as loading control. (B) Representative immunofluorescence of Y711i MT probed with HCS, PS, or TPS (red signal). Note the presence of a strong signal distributed near the sarcolemma when PS was used as a primary antibody (see white arrows). A drastic signal decrease was observed when MT extracts were probed with TPS. No specific signal was observed in MT when probed with HCS. The scale bar is 20 μm. (C) WB validation of the 55 kDa as MURC/cavin-4 immunoreactive band. WB on untransfected HEK293T cells (NT) or transfected with human Flag-tagged Cavin-1, -2, -3, or -4 expressing constructs and probed with the anti-Flag antibody, PS, commercial anti-MURC/Cavin-4 and anti-Cavin-1 antibodies, or GAPDH antibodies as loading control. Note the specific detection of MURC/Cavin-4 by PS. (D) Representative immunofluorescence images of MT probed with the commercial anti-MURC/Cavin-4 antibody. Note the presence of a perfect yellow merge colocalization signal between commercial anti-MURC/Cavin-4 and PS. The scale bar is 10 μm. (E) IHC of control individual (CT) and rippling patient (RIP) skeletal muscle sections probed with the commercial anti-MURC/Cavin-4. Note the decrease of MURC/Cavin-4 expression in a mosaic-like pattern in the thymoma rippling affected patient (see black arrows; ×20 magnification).

Immunoprecipitation-MS–based proteomic approaches on MTs using PS revealed that MURC/Cavin-4 and Cavin-1 were the most specifically enriched proteins (data not shown). We validated PS specificity for MURC/Cavin-4 by WB on HEK293T cells expressing Flag-tagged Cavin-1-4 (Figure 3C) and immunofluorescence colabeling in human MT using PS and commercial anti-MURC/Cavin-4 (Figure 3D). Isotype antibody determination was performed by WB using anti-IgG1, 2, 3, or 4 secondary antibodies. Anti-MURC/cavin-4 autoantibodies were recognized by anti-IgG1, 2, and 3 but not by anti-IgG4 (data not shown). IHC experiments performed on patient muscle biopsy using commercial anti-MURC/Cavin-4 antibody revealed a decrease of subsarcolemmal MURC/Cavin-4 staining in a mosaic-like pattern (Figure 3E), thus suggesting that MURC/Cavin-4 autoantibodies may induce antibody-mediated protein depletion.

To determine whether the thymoma itself could express MURC/Cavin-4 autoantigen that could lead to the induction of MURC/Cavin-4 autoantibodies by resident immune cells, the patient thymoma section was analyzed by IHC using a commercial anti-MURC/Cavin-4. We failed to detect MURC/Cavin-4 signal in the section through this approach, whereas a strong sarcolemmal signal was concomitantly observed in control healthy individual muscle tissue (eFigure 1A, links.lww.com/NXI/A786). Western blotting investigations performed on the non-iRMD B2 thymoma sample compared with the iRMD B2 thymoma and using the commercial anti-MURC/Cavin-4 confirmed the absence of the 55-kDa MURC/Cavin-4 band in both thymomas (eFigure 1, links.lww.com/NXI/A786). However, a faint MURC/Cavin-4 immunoreactive band around 43 kDa (see red arrow) was specifically detected in both iRMD thymoma and MT extracts but absent from the non-iRMD thymoma sample (eFigure 1B, links.lww.com/NXI/A786). This may suggest a possible weak expression of a MURC/Cavin-4 distinct isoform in the iRMD patient thymoma (see Discussion).