Profiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis

Abstract

Background: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific 'myocyte' cultures. Methods: From well-characterised MG patients we developed the 'myocyte' culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG). Results: OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p < 0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r > 0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10 - 6 ) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway. Conclusion: Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.