Gene expression profiling of orbital muscles in treatment-resistant ophthalmoplegic myasthenia gravis
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RESEARCH
Gene expression profiling of orbital muscles in treatment‑resistant ophthalmoplegic myasthenia gravis Tarin A. Europa1,2, Melissa Nel1,2 and Jeannine M. Heckmann1,2*
Abstract Background: Unbiased in silico approaches applied to genome-wide data prioritized putative functional gene variants associating with treatment-resistant ophthalmoplegic myasthenia gravis (OP-MG). Although altered expression of genes harbouring these variants, or associated pathways, were shown in patient-derived transdifferentiated-myocyte models, gene expression in orbital-derived muscle was required to test the validity of the predictions. Methods: We sampled orbicularis oculi muscle (OOM) and one paralysed extraocular muscle (EOM) from six individuals with OP-MG during blepharoptosis and re-alignment surgeries, respectively. For controls, the OOMs were sampled from four individuals without myasthenia undergoing surgery for non-muscle causes of ptosis, and one non-paralysed EOM. Using a qPCR array, expression of 120 genes was compared between OP-MG and control OOMs, profiling putative “OP-MG” genes, genes in related biological pathways and genes reported to be dysregulated in MG cases or experimental MG models, and in EOMs of cases with strabismus. Normalization was performed with two stable reference genes. Differential gene expression was compared between OP-MG and control samples using the ΔΔCT method. Co-expression was analysed by pairwise correlation of gene transcripts to infer expression networks. Results: Overall, transcript levels were similar in OOMs and EOMs (p = 0.72). In OOMs, significant downregulated expression of eight genes was observed in OP-MG cases compared with controls (> twofold; p ≤ 0.016), including TFAM, a mitochondrial transcription factor, and genes related to the following pathways: atrophy signalling; muscle regeneration and contraction; glycogen synthesis; and extracellular matrix remodelling. Several microRNAs, known to be highly expressed in EOMs, are predicted to regulate some of these genes. Co-expression analyses of genepairs suggested high interconnectedness of gene expression networks in OP-MG muscle, but not controls (r > 0.96, p 1 sample (Cq > 35) were excluded from these analyses (n = 5). Correlation plots using gene pairs with significant correlations (p 0.6 were excluded.
Results Six OP-MG muscle (1 EOM; 5 OOM) and five control muscle (1 EOM; 4 OOM) samples passed data quality analysis (Table 1). Differential gene expression in orbicularis oculi muscles
To assess differences in relative gene expression between 5 OP-MG and 4 control OOMs, data normalization was performed using the average values for RPLP0 and ACTN2 as reference genes (Additional file 1: Table S2). Figure 2 shows the genes that showed > twofold difference in gene expression levels between OP-MG vs control OOMs (FDR = 15%; p
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