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Rare Disease Research Insights : GNE Myopathy

A new cellular model of GNE Myopathy reveals disease mechanism and holds promise for testing potential therapies.


Jason Doles & Team at their lab Those of us living with GNE myopathy constantly pray for a treatment to emerge that could stop the relentless grip this disease has on our bodies. The failure of sialic acid supplementation therapy in the phase 3 Ultragenyx trial was a big disappointment for us. Gene replacement therapies, although highly promising, are in nascent stages of development, have serious safety issues, and are going to be formidably priced. Alternative approaches, especially therapies based on small-molecule drugs, are hence our urgent need. These therapies are typically easier and relatively inexpensive to produce and test. It is also possible to repurpose already known small molecule drugs for our benefit if we know what to look for; that is, if we know how GNE Myopathy causes so much muscle damage we could try and reverse this process using small molecule drugs.

A new study (Ref. 1) holds out such a promise for GNE myopathy. For the first time this study clearly points in the direction of the cellular processes that are damaged in GNE Myopathy muscle cells, and provides a much-needed cell-based model system to test out drugs that could reverse this damage. This work was done by Dr. Jason Doles’ lab at the Indiana University School of Medicine, USA, in collaboration with Dr. Margherita Milone’s lab at the Department of Neurology, Mayo Clinic, USA.


A brief introduction to GNE Myopathy (GNEM) GNEM is a muscle-wasting disease that typically manifests in adulthood; most commonly beginning with weakness of lower limbs, gradually progressing to upper limbs and other skeletal muscles. The eventual result is loss of ambulation and extreme disability. It is a genetic disorder caused by mutations in the GNE gene. This gene codes for a protein needed to synthesize a sugar called sialic acid (SA) which is required for normal functioning of all cells and tissues in our body. So, the question arises that if SA is needed by all cells of the body, why do mutations in the GNE gene affect only skeletal muscles and not our other organs? This is one of many baffling questions about the mechanism by which GNE mutations cause this disease. Finding answers to these questions is extremely important as they hold the key for discovering potential treatments. The need for model systems to study the disease To understand the mechanism of any biological process one typically needs to apply harsh procedures. Some of these allow us to see what happens inside a cell or tissue when a gene is mutated. Sometimes we may use chemicals to stop, or speed up a process that may be linked to the system we are studying. Such manipulations allow us to infer the underlying mechanisms. Obviously, one cannot apply these invasive methods on humans directly. Hence the need for model systems (lab-maintained animals or cells) that are designed to replicate the human disease sufficiently closely.

Dr. Jason Doles Prevailing model systems for GNEM The most favoured lab animal to generate disease models is the mouse. The first successful mouse model for GNEM was made in 2007 in the lab of Dr. Nishino at the National Institute of Neuroscience, Japan (2). This transgenic mouse (that is, carrying the genes of another organism) carried a copy of the mutated human GNE gene and could replicate key features of the human disease, including presence of rimmed vacuoles in muscle cells, reduced sialylation and reduced motor performance. However, this model was difficult to generate and maintain. Other GNEM labs, notably those in Israel and USA, generated knock-in mouse models in which both copies of the mouse GNE gene were mutated like the human GNEM mutation (3-5). These models are easier to generate but the mice showed prominent kidney (rather than muscle) defect. Apart from mouse, the Israel group of Dr. Mitrani-Rosenbaum developed a knockdown zebrafish model of GNEM in which the gene is not mutated but its expression is brought down (6). This displayed muscle defects like in human disease, but the model has not been further analyzed. To sum up, a fully satisfactory animal model of GNEM is still not available. A Cellular Model of GNEM In this backdrop the Doles’ lab decided to adopt a different approach, namely to use induced pluripotent stem cells (iPSCs) to generate a cellular model of GNEM. For this they obtained tissue samples from two GNEM patients. Using standard methods they re-programmed the patient cells to get iPSCs. The reprogramming process can itself sometimes cause genetic variations. Hence the selected iPSC clones were sequenced to confirm the presence of the expected GNEM mutations. Next, they confirmed that the iPSCs were functional, that is, they were indeed pluripotent and could be induced to differentiate into desired cell types.

These iPSCs were now made to differentiate into muscle cells (myotubes) in a three-step process. In the first step, iPSCs were converted to muscle stem cells (or satellite cells). These were grown to expand their numbers, and in step 2 they were differentiated into myoblasts. Further, in step 3 the myoblasts were allowed to fuse to form myotubes (Figure 1).

Figure 1 - Diagram to illustrate how iPSCs differentiate into satellite cells that are committed to form muscle. These further form myoblasts (single nucleus in each cell) and finally, myotubes (multiple nuclei in each cell). (Adapted from Schmitt et al. NPJ Regen Med. 2022) Myotubes generated from patient iPSCs showed the features of GNEM GNE Myopathy goes by various names, one of them being hereditary inclusion body Myopathy (HIBM). As this name suggests, muscle cells from GNEM patients when viewed under the microscope show presence of abnormal structures called inclusion bodies which contain aggregates of defective proteins. One of the proteins in these aggregates is TDP-43. When myotubes from patient iPSCs were examined, they did, indeed contain inclusion bodies with TDP-43. Thus, the lab-generated myofibers from GNEM patient iPSCs recapitulated the defect seen in muscle biopsies. Differentiation into normal muscle is impaired in GNEM iPSCs The three-stage process in which iPSCs from GNEM and normal (control) samples were differentiated into myotubes was studied closely. Proteins that are important for muscle differentiation, like myogenic factor 5 (Myf5), myogenin (MyoG), and myoblast determination protein (MyoD) were monitored in the three stages. Till stage 2 (myoblast) the GNEM samples did not markedly differ from control samples in the expression of proteins needed for muscle differentiation. However, the patterns deviated markedly in stage 3 (myotube). There was almost one-third reduction in the number of myotubes formed from GNEM samples compared with the control samples. Many of the proteins normally abundant in muscle cells (e.g. myosin heavy chain 1) were present in very low amounts in myotubes obtained from GNEM iPSCs. Thus, GNE mutation impaired the ability of muscle stem cells to differentiate into mature muscle fibers. The maximum impairment was seen in the last stage of conversion of myoblasts to myotubes.

Dr. Margherita Milone Possible factors impairing the production of mature muscle fibers in GNEM There could be several factors contributing to the observed defect. The data from this study suggest that the most important factor causing this impairment could be defective autophagy. Autophagy is the process by which a cell clears up its junk and renews itself. In the life of a cell many defective components tend to accumulate, which are continually degraded and recycled through the controlled process of autophagy. Defects in this process can lead to accumulation of junk, and could hinder normal cellular functioning; hence leading to disease. If autophagy defect was indeed responsible for the inability of GNEM iPSCs to differentiate into normal muscle fibers, one could reverse it by using chemicals that induce autophagy and overcome the defect. A candidate drug, SB203580, known to induce autophagy, did indeed substantially improve the capacity of GNEM iPSCs to differentiate into muscle fibers. In Summary This study provides a cellular model of GNE Myopathy that recapitulates the inclusion body feature of GNEM muscle. In comparison to animal models, cellular models are easier to generate directly from patient-derived samples in a noninvasive way. In addition, cellular models are simpler to maintain and study in the lab.


This study provides evidence that the inability of GNEM stem cells to differentiate into mature muscle fibres could be linked to defective autophagy. Therapies that reverse the autophagic defect could potentially boost muscle regeneration in GNE Myopathy. The cellular model could be used to test new drugs, or to study the efficacy of therapeutic molecules in GNE Myopathy. References 1. Schmitt et al. NPJ Regen Med. 2022, doi: 10.1038/s41536-022-00238-3.

2. Malicdan et al. Hum. Mol. Genet. 2007, doi:10.1093/hmg/ddm220

3. Galeano et al. J. Clin. Invest. 2007, doi:10.1172/JCI30954

4. Ito et al. PLoS One 2012, doi:10.1371/journal.pone.0029873

5. Sela et al. Neuromol. Med. 2013, DOI 10.1007/s12017-012-8209-7

6. Daya et al. Hum Mol. Genet. 2014, doi:10.1093/hmg/ddu045

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