Biological markers in Myasthenia gravis

In the project biological markers in myasthenia gravis we want to find muscle specific autoantibodies in patients with myasthenia gravis. Myasthenia gravis (MG) is an autoimmune disease in which the patients have fatigable muscle weakness. MG is very severe, the patients have problems moving, chewing, swallowing, and speaking, and as the weakness also affects muscles required for breathing, these patients can experience respiratory crisis and need to be ventilated artificially. MG is caused by autoantibodies that attack the junction between the nerve and the muscle and impair their communication, so that the signal for muscle contraction cannot pass to the muscle. In many patients the targets of these autoantibodies are known, but there are patients in whom the autoantibodies have not yet been identified. They have been classified as seronegativen myasthenia gravis (SNMG). Our hypothesis is that these patients have muscle specific autoantibodies that are yet unknown. To identify these, we will test if patients antibodies bind to muscle tissue from rodents and humans, and human and rodent mouse cell lines. If we find that autoantibodies bind to the muscle, we will identify which molecule exactly is bound, by lysing the cells and isolating the antibodies with their bound target, which will then be analysed by mass spectrometry. After identifying the target molecule that is attacked by the autoantibody, we will establish a specific diagnostic assay, which will allow for quick and specific screening for this particular autoantibody in other patients. This assay can also be used to characterize other properties of the autoantibody that could give information about the type of immune answer. The target molecule in SNMG patients is potentially very interesting as it might have an important function at the neuromuscular synapse. This function will be studied with standard biomolecular methods. We will also look at clinical and immunological aspects that may help us to understand how MG developed in the patients and how the antibody damages the neuromuscular synapse. By analysing the response to treatment of patients with specific autoantibodies, we will be able to improve treatment. Taken together, we want to identify autoantibodies in SNMG, which will help to select better therapies, and establish a new diagnostic assay that will also help to characterize properties of the autoantibody.

Many autoimmune diseases are caused by pathogenic autoantibodies that recognize and attack self-molecules in the body. In patients with myasthenia gravis (MG), autoantibodies targeting molecules at the neuromuscular junction with important function for neuromuscular signal transmission may cause severe fatigable muscle weakness. In project T996-B30, we searched for new autoantibodies in patients with MG. To this end we established a novel cell culture model of the muscle and the neuromuscular junction from muscle stem cells, and used patient serum derived antibodies to isolate their target molecules and identify these by mass spectrometry. Due to COVID-19 restrictions, which affected the cell culture experiments severely, the project was modified and expanded to include the study of antineuronal autoantibodies of IgG4 subclass, among these also antibodies in a sub form of MG (MuSK MG). IgG4 has a protective function in the healthy body, but in rare cases, IgG4 autoantibodies may cause autoimmune diseases. In project T996-B30, potential IgG4 autoimmune diseases were analyzed, classified and compared. We identified commonalities between the diseases, among these shared genetic risk factors, that suggest a common underlying immunopathogenesis. A histopathological and serological comparison led to the conclusion that IgG4-related diseases are not related to IgG4 autoimmune diseases and comprise a separate group of diseases. Most IgG4 autoimmune diseases are not well understood, and there is a lack of prognostic biomarkers and common treatment strategies for the patients. To address this lack of knowledge we investigated a cohort of longitudinal sera and CSF samples of patients with IgG4 autoimmune diseases, which we analyzed for antigen-specific IgG subclass profiles using flow cytometry that we then correlated with clinical parameters. We observed that autoantibody titers in the liquor cerebrospinalis could be valuable prognostic biomarkers. This analytical approach could improve diagnosis and prognosis of patients with IgG4 autoimmune diseases in the future. In addition to the gain of scientific knowledge, our project initiated several national and international collaborations which will ensure the study of IgG4 autoimmunity in the future.