Mutations in either mitochondrial DNA (mtDNA) or nuclear genes that encode mitochondrial proteins may lead to dysfunctional mitochondria, giving rise to mitochondrial diseases. Some mitochondrial myopathies, however, present without a known underlying cause. Interestingly, methylation of mtDNA has been associated with various clinical pathologies. The present study set out to assess whether mtDNA methylation could explain impaired mitochondrial function in patients diagnosed with myopathy without known underlying genetic mutations. Enhanced mtDNA methylation was indicated by pyrosequencing for muscle biopsies of 14 myopathy patients compared to four healthy controls, at selected cytosines in the Cytochrome B (CYTB) gene, but not within the displacement loop (D-loop) region. The mtDNA methylation patterns of the four healthy muscle biopsies were highly consistent and showed intriguing tissue-specific differences at particular cytosines with control skin fibroblasts cultured in vitro. Within individual myopathy patients, the overall mtDNA methylation pattern correlated well between muscle and skin fibroblasts. Despite this correlation, a pilot analysis of four myopathy and five healthy fibroblast samples did not reveal a disease-associated difference in mtDNA methylation. We did, however, detect increased expression of solute carrier family 25A26 (SLC25A26), encoding the importer of S-adenosylmethionine, together with enhanced mtDNA copy numbers in myopathy fibroblasts compared to healthy controls. To confirm that pyrosequencing indeed reflected DNA methylation and not bisulfite accessibility, mass spectrometry was employed. Although no myopathy-related differences in total amount of methylated cytosines were detected at this stage, a significant contribution of contaminating nuclear DNA (nDNA) was revealed, and steps to improve enrichment for mtDNA are reported. In conclusion, in this explorative study we show that analyzing the mitochondrial genome beyond its sequence opens novel avenues to identify potential molecular biomarkers assisting in the diagnosis of unexplained myopathies.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by a constant accumulation of lipids in the liver. This hepatic lipotoxicity is associated with a dysregulation of the first step in lipid catabolism, known as beta oxidation, which occurs in the mitochondrial matrix. Eventually, this dysregulation will lead to mitochondrial dysfunction. To evaluate the possible involvement of mitochondrial DNA methylation in this lipid metabolic dysfunction, we investigated the functional metabolic effects of mitochondrial overexpression of CpG (MSssI) and GpC (MCviPI) DNA methyltransferases in relation to gene expression and (mito)epigenetic signatures. Overall, the results show that mitochondrial GpC and, to a lesser extent, CpG methylation increase bile acid metabolic gene expression, inducing the onset of cholestasis through mito-nuclear epigenetic reprogramming. Moreover, both increase the expression of metabolic nuclear receptors and thereby induce basal overactivation of mitochondrial respiration. The latter promotes mitochondrial swelling, favoring lipid accumulation and metabolic-stress-induced mitophagy and autophagy stress responses. In conclusion, both mitochondrial GpC and CpG methylation create a metabolically challenging environment that induces mitochondrial dysfunction, which may contribute to the progression of MASLD.

Life and social adversities experienced before adulthood are decisive drivers of later life health inequalities, which hinge on immune, endocrine and epigenetic changes that reflect an individual’s interaction with the environment. Since monozygotic (MZ) twins share the same genetic background and perinatal environment, they provide a robust approach to identify environmental influences on biological features while controlling genetic confounders. We screened MZ and dizygotic (DZ) twin pairs (aged of 17 to 32 years) from the German TwinLife study for recent and time-distant adversities in their psychosocial environment (PSE). Here, we present three statistical approaches we developed to quantify adverse experiences in 739 twin pairs (349 MZ, 390 DZ). We calculated divergence scores based on score differences between each twin pair. By combining these approaches, we identified adversity-divergent twins (144 MZ, 173 DZ), suggesting that in approximately 41% - 44% of twin pairs, there was a clear divergence in their adversarial experiences and how they interpreted their PSE. Variance modelling suggested that 43% of the adversity experienced by the twins was due to their unique environment, while 23% was from their shared environment while 33% of the variance was explained by genetic factors.

Introduction: Research has shown that social and physical stressors of early-life adversity (ELA) can negatively affect long-term health trajectories. Despite differences in types of ELA exposure, previous studies have identified common health-related outcomes in adults who had experienced less favourable conditions during developmentally sensitive periods. This meta-analysis investigates the potential role of DNA methylation in mediating these adverse health trajectories by identifying common biological signatures across cohorts with distinct adversity exposures and environmental backgrounds. Materials and Methods: DNA methylation data from previously published studies was used to perform a meta-analysis on 227 individuals across three cohorts. These include the EpiPath cohort consisting of adults who were exposed early institutional care, ImmunoTwin cohort consisting of adversity discordant monozygotic twin pairs and lastly a cohort of young children exposed to early institutional care. Results: DNA methylation analysis across the three cohorts revealed differential methylation at CpG loci associated with 15 genes common to all cohorts. These genes are involved in neuronal development, chromatin remodeling and metabolism. Pathway enrichment analysis of the combined dataset showed potential associations with oxytocin signalling, regulation of nervous system development, and calcium signalling in relation to the later-life phenotype of the adversity exposed individuals. In addition, a poly-epigenetic score was developed by identifying a subset of 200 differentially methylated CpG sites through PLS-DA analysis with the combined beta matrix of these cohorts. Conclusion: This study highlights the long-term impact of adversity by identifying common DNA methylation signatures of negative life experiences across three cohorts. The development of a poly-epigenetic score represents the first steps towards identifying group differences by combining weighted methylation values for CpG sites of interest. This method illustrates the potential to track changes in individuals across long-term studies that may benefit research in lifelong health outcomes.

Introduction: Early-life adversity (ELA) encompasses a range of environmental stressors, including physical, emotional, and social challenges that can affect health during the critical early developmental period. Extensive research has linked ELA to negative long-term health outcomes, yet the underlying biological mechanisms remain poorly understood. The current study investigates how early institutional care changes the epigenetic landscape in young adults. The study also provides insights into role of DNA methylation as a potential mediator for disease susceptibility and altered health trajectories. Materials and Methods: DNA was extracted from blood samples obtained from 111 individuals (71 Controls; 40 ELA) who were part of the EpiPath cohort. DNA methylation was measured using the Infinium Methylation EPIC v2.0 BeadChip. Results: 3,785 differentially methylated CpG loci were identified in the ELA group in comparison with the control group (FDR <0.05). Pathway enrichment analysis highlighted biological processes involved in metabolic regulation, stress response, and neurodevelopment, with novel pathways such as GTPase-mediated signalling, efferocytosis and glucuronosyltransferase emerging as potential drivers of the ELA phenotype. A subset of 28 CpG loci was used to develop an epigenetic signature, which showed a significant association with the development of chronic diseases in ELA-exposed individuals. Conclusion: This study reinforces Barker’s concept of sensitive periods and it underscores the enduring impact of ELA in shaping long-term health outcomes. The persistence of DNA methylation patterns decades after exposure to ELA, and their clear association with the resultant phenotype confirms that stable epigenetic imprints play a potential role in long-term disease risk and resilience.