State-of-the-art assays for myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease affecting 14-20 of every 100,000 people in the US,1 and 1-9 in 100,000 people in Europe. 2 The sad truth is that most of those afflicted go undiagnosed. MG causes severe muscle weakness, and significantly decreases quality of life. Diagnosis can be difficult, but state-of-theart disease biomarkers and targeted assays are available to increase the likelihood that a patient with MG will be diagnosed early, and can be treated appropriately. So how can these biomarkers, and use of the correct assays, help clinicians to monitor therapeutic efficacy and support better treatment outcomes for their patients?

Myasthenia gravis symptoms

In MG, the body’s own immune system produces antibodies that attack the skeletal muscles responsible for breathing and various other movements. This leads to patients experiencing muscle weakness, and prolonged rest is needed to recover. There are effective treatments for MG, 3 but they are only effective if diagnosis is made early enough.

In most cases of MG, autoantibodies target acetylcholine receptors, which are essential for transmitting electrical impulses through muscle tissue. These electrical signals stimulate the muscles to contract. Acetylcholine receptor autoantibodies (ARAbs) produced in MG patients prevent acetylcholine from binding to its receptor, which blocks normal muscle contractions.

Around 85 percent of people with MG have ARAbs, while the remaining 15 percent have so-called ‘seronegative’ MG. Despite having clear clinical symptoms, these patients don’t have detectable autoantibodies against the acetylcholine receptor. About half of these seronegative patients will instead have autoantibodies against a protein called muscle-specific tyrosine kinase (MuSK). MuSK plays an essential role in connecting the acetylcholine receptor to the muscles, making it important in MG. Patients with MuSK autoantibodies (MuSK-Abs) typically have a more severe form of the disease, with more debilitating symptoms.

ARAb and MuSK account for 85-90 percent of MG diagnoses, meaning that – even with good markers – there is no way to diagnose 10 percent of sufferers. One emerging marker that has shown diagnostic promise is low-density lipoprotein receptor-related protein 4 (LRP4). One study tested patients who were seronegative for ARAb and MuSK-Abs for the presence of antibodies against LRP4. They discovered that 12 out of the 13 patients tested were positive for LRP4, making this one of the most promising markers to help close the gap on people suffering with undiagnosed MG. 4

What makes myasthenia gravis diagnosis so complex?

ARAb and MuSK-Abs are the gold standard biomarkers for diagnosing MG. However, getting to that crucial early diagnosis is not simple. The first step is an assay for ARAbs, if MuSKAbs are not also measured right at the beginning. However, an ELISA for ARAbs is not sufficient, and is likely to yield a false positive result. To maximize the sensitivity and specificity of ARAb detection, a radioreceptor assay (RRA) is the preferred choice. The RRA is preferable because, unlike during an ELISA, the three-dimensional structure of the acetylcholine receptor is retained, which is essential for antibody recognition.

If the RRA test for ARAbs is positive, it aids in the diagnosis of MG. However, if the result is negative, it is important to consider the possibility of seronegative MG. To identify these cases, a follow-up test with a MuSK ELISA is necessary, as it can detect around 50 percent of these patients. Due to this reason, more and more laboratories directly involve the measurement of MuSK-Abs in the first line diagnostic test. The preferred test for this is a quantitative MuSK-Ab ELISA, which is highly accurate and reliable.

It also follows that if the test for MuSK is negative, then LRP4 could be the target. However, as there are no commercial tests currently available, laboratories currently have to develop in-house assays, which is now very time consuming under the In Vitro Diagnostic Regulation (IVDR). In such cases, it is probably best to collaborate with a commercial partner to get new tests registered under IVDR and commercially available as soon as possible.4

A quantitative MuSK-Ab assay enables therapeutic monitoring

Patients with MuSK-Abs not only have a more severe form of the disease, but the therapy they receive also tends to be more aggressive, commonly involving rituximab immunosuppression to achieve B-cell depletion. In the treatment of MG, the target reduction of ARAbs is 50 percent, whereas the clinical goal of treatment in MuSK-positive patients is complete elimination of the MuSK-Abs. Calculating when this has been achieved requires a highly sensitive and quantitative assay, rather than relying on qualitative results. Additionally, since therapy may require some time to take effect, clinicians can carefully monitor the efficacy of MG treatment by using a sensitive, quantitative assay. They can detect increases or decreases in MuSK-Ab levels, and modify both treatments and therapeutic decision making as needed. Thankfully, both ARAb and MuSK-Ab tests are commercially available under IVDR, and there will be a reliable supply going forward.

References

1. Myasthenia Gravis Foundation of America.

2. The Portal for Rare Diseases and Orphan Drugs.

3. National Institute of Neurological Disorders and Stroke. National Institutes of Health. Myasthenia Gravis.

4. Pevzner A, et al. Anti-LRP4 autoantibodies in AChR- and MuSKantibody-negative myasthenia gravis. J Neurol. 2012, 259 (3), 427-35.