Original Contributions |

Autoantibodies to Lipoprotein-Related Protein 4 in Patients With Double-Seronegative Myasthenia Gravis

Bin Zhang, PhD; John S. Tzartos, MD, PhD; Maria Belimezi, PhD; Samia Ragheb, PhD; Beverly Bealmear, BS; Richard A. Lewis, MD; Wen-Cheng Xiong, PhD; Robert P. Lisak, MD; Socrates J. Tzartos, PhD; Lin Mei, PhD
Arch Neurol. 2012;69(4):445-451. doi:10.1001/archneurol.2011.2393.
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Objectives To determine whether patients with myasthenia gravis (MG) have serum antibodies to lipoprotein-related protein 4 (LRP4), a newly identified receptor for agrin that is essential for neuromuscular junction formation, and to establish whether such antibodies contribute to MG pathogenesis.

Design Serum samples from patients with MG with known status of serum antibodies to the acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) and serum samples from control subjects (healthy individuals and individuals with other diseases) were tested for antibodies to LRP4. Serum samples with such antibodies were tested to determine whether they had the ability to inhibit 2 different functions of LRP4 at the neuromuscular junction.

Setting Serum samples were collected at the Hellenic Pasteur Institute and Wayne State University. Samples were tested for LRP4 autoantibodies at Georgia Health Sciences University. Other immunoreactivities of the samples were tested at the Hellenic Pasteur Institute, Athens, Greece, or processed through University Laboratories of the Detroit Medical Center, Michigan.

Patients The study included 217 patients with MG, 76 patients with other neurologic or psychiatric diseases, and 45 healthy control subjects.

Results Anti-LRP4 antibodies were detected in 11 of 120 patients with MG without detectable anti-AChR or anti-MuSK antibodies (double seronegative) and in 1 of 36 patients without anti-AChR antibodies but with anti-MuSK antibodies, but they were not detected in any of the 61 patients with anti-AChR antibodies. No healthy control subjects and only 2 of the 76 control patients with neurologic disease had anti-LRP4 antibodies. Serum samples from patients with MG with anti-LRP4 antibodies were able to inhibit the LRP4-agrin interaction and/or alter AChR clustering in muscle cells.

Conclusions Anti-LRP4 antibodies were detected in the serum of approximately 9.2% of patients with double-seronegative MG. This frequency is intermediate compared with 2 recent studies showing anti-LRP4 antibodies in 2% and 50% of patients with double-seronegative MG from different geographic locations. Together, these observations indicate that LRP4 is another autoantigen in patients with MG, and anti-LRP4 autoantibodies may be pathogenic through different immunopathogenic processes.

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Figure 1. Preparation of ecto–lipoprotein-related protein 4 (LRP4). A, Structures of LRP4 and C-terminus–tagged ecto-LRP4. B, Preparation of ecto-LRP4 (arrow). Ecto-LRP4 was purified from transfected HEK293 cells by affinity chromatography and subjected to Western blotting by anti-Myc antibody.

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Figure 2. Enzyme-linked immunosorbent assays of anti–lipoprotein-related protein 4 autoantibodies. Mean (SD) optical density readings of normal human serum (control) were 0.31(0.22) (n = 45). The dotted line indicates the cutoff.

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Figure 3. Distribution of anti–lipoprotein-related protein 4 autoantibodies among patients with myasthenia gravis. Of 217 myasthenia gravis samples, 61 had anti–acetylcholine recepted (AChR) antibodies (AChR+), 36 had no anti-AChR antibodies (AChR−) but did have anti–muscle-specific kinase (MuSK) antibodies (MuSK+), and 120 were AChR− and had no anti-MuSK antibodies (MuSK−). The cutoff (the dotted line) was set as mean ± 4 SD.

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Figure 4. Recognition of full-length lipoprotein-related protein 4 (LRP4) by serum samples with anti-LRP4 antibodies. Lysates of LRP4-transfected HEK293 cells were incubated with serum samples with anti-LRP4 antibodies or normal human serum samples. Resulting immunocomplex and lysates (to indicate equal amounts of input) underwent Western blotting with anti-Myc antibody.

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Figure 5. Inhibition of agrin–lipoprotein-related protein 4 (LRP4) interaction by serum samples with anti-LRP4 antibodies. The interaction was tested by enzyme-linked immunosorbent assay in the presence of control serum samples or serum samples with anti-LRP4 antibodies. Data are shown as mean ± SD (n = 3). * P < .05 compared with normal human serum samples. MuSK indicates muscle-specific kinase.

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Figure 6. Serum samples with anti–lipoprotein-related protein 4 (LRP4) antibodies alter acetylcholine receptor (AChR) clustering in myotubes. A, Representative images. B, Quantitative data of basal AChR clusters. C, Quantitative data of induced AChR clusters. Data are shown as mean (SD). * P < .05 compared with control. MuSK indicates muscle-specific kinase.





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