Leber’s hereditary optic neuropathy (LHON), an inherited mitochondrial disease, typically manifests as painless central visual loss (central scotoma). The visual loss can occur in just one eye or both. Mitochondrial mutations result in death of retinal ganglionic cells (RGCs). LHON is the most common mitochondrial diseases with an estimated prevalence from 1:30,000 to 1:54,000 worldwide.1–3 LHON is characterized by its incomplete penetrance with gender bias, affecting about 50% of male mutation carriers and about 10% of female carriers.4 Nearly 95% of LHON cases are caused by one of the three primary mitochondrial DNA (mtDNA) mutations: 3460G > A in MT-ND1, 11778G > A in MT-ND4 or 14484T > C in MT-ND6 subunit of complex I5–7. Recovery of the eyesight in LHON is minimal and clinical severity may depend on type of mutation. Prognosis of patients with 11778G > A mutation is the worst while 3460G > A and 14484T > C patients display a milder phenotype8,9.
In earlier studies, a mild to moderate reduction in complex I activity, and complex I dependent ATP synthesis was reported in LHON cybrids carrying either of the above three primary mutations10–13. However, LHON cybrids displayed severe ATP depletion and underwent apoptosis in galactose medium indicating bioenergetics insufficiency under OXPHOS stress condition14,15. Antioxidant defenses such as glutathione (GSH) content, activities of glutathione reductase (GR), glutathione peroxidase (GPX) or mitochondrial, superoxide dismutase 2 (SOD2) were reduced in LHON cybrids when growing in galactose medium or exposed to hydrogen peroxide (H2O2). These evidences show that cells carrying LHON mutation are vulnerable to oxidative and metabolic stress condition 14–16.
Therefore, increased reactive oxygen species (ROS) production and insufficient ATP production due to complex I defect have been regarded as major contributing factors to RGCs death in LHON. Like other neurons, RGCs demand the highest energy supply for firing action potential along the axon, especially in unmyelinated part17. Competent bioenergetics, along with functional antioxidant system are important to supply adequate ATP as well as prevent oxidative damage to mitochondrial DNAs, lipids and proteins under such conditions.
Compensatory increase in mitochondrial biogenesis of unaffected LHON carriers from European populations was proposed as an underlying factor for incomplete penetrance in 3460G > A and 11778G > A18,19. Additionally, mtDNA haplogroup backgrounds, 20,21 X-linked loci, 22,23 and potential nuclear modifier gene(s) explored in genome wide studies24–26 were reported to influence LHON penetrance though findings varied across populations probably owing to diverse nuclear background in different ethnicities. In addition, environmental factors, such as smoking27 and exposure to certain drugs28–30 may also trigger LHON expression.
With advances in high-throughput sequencing technologies, comparison of affected LHON patients and unaffected carriers using omics-wide approaches are of great help to identify disease modifying variants31. Monozygotic twins supposedly have identical genetic profiles but discrepancy between disease phenotypes may stem from small errors of DNA replication or variation in epigenetic predisposition32,33. Consequences of genetic or epigenetic variations could eventually reflect in gene expression, thus comparing transcriptomics of such discordant monozygotic twins will provide invaluable clues to exploration of disease modifying gene(s), associated signaling pathways and regulatory biological processes. We exploited an oxidative and metabolic insult culture in order to demonstrate a difference in cellular phenotype in 14484T > C mutation carrying monozygotic twins, one unaffected and one affected by LHON. We then created transcriptomic profiles using next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) on the Illumina platform. Differentially expressed genes were identified and explored to find potentially interplaying signaling pathways and biological processes, which might be beneficial in understanding LHON pathogenesis and application for therapeutic interventions.