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Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
The Mitochondrial Epigenome: An Unexplored Avenue to Explain Unexplained Myopathies?
Archibold Mposhi1,2, Lin Liang1, Kevin P Mennega1,3
1Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
Abstract:
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.
Insights
Investigating mitochondrial DNA (mtDNA) methylation in unexplained myopathies revealed enhanced methylation in patient muscle tissue. This suggests mtDNA methylation may serve as a biomarker for diagnosing mitochondrial diseases when genetic causes are unknown.
Area of Science:
- Mitochondrial Biology and Genetics
- Epigenetics
- Neuromuscular Disorders
Background:
- Mitochondrial diseases arise from mutations in mitochondrial DNA (mtDNA) or nuclear genes, but some myopathies lack identified genetic causes.
- Mitochondrial DNA (mtDNA) methylation has been linked to various pathologies, suggesting a potential role in disease mechanisms.
- Investigating epigenetic modifications like mtDNA methylation may uncover novel diagnostic markers for unexplained mitochondrial myopathies.
Purpose of the Study:
- To determine if mitochondrial DNA (mtDNA) methylation is associated with impaired mitochondrial function in patients with unexplained myopathies.
- To explore the potential of mtDNA methylation patterns as diagnostic biomarkers for myopathies of unknown genetic origin.
- To investigate the correlation of mtDNA methylation between muscle tissue and skin fibroblasts in myopathy patients.
Main Methods:
- Pyrosequencing was used to analyze mitochondrial DNA (mtDNA) methylation at specific cytosines in the Cytochrome B (CYTB) gene and D-loop region in muscle biopsies from myopathy patients and healthy controls.
- Mass spectrometry was employed to validate pyrosequencing results and assess total methylated cytosines, while also identifying nuclear DNA (nDNA) contamination.
- Gene expression analysis of solute carrier family 25A26 (SLC25A26) and mtDNA copy number quantification were performed on fibroblast samples.
Main Results:
- Enhanced mitochondrial DNA (mtDNA) methylation was observed in the Cytochrome B (CYTB) gene of muscle biopsies from myopathy patients compared to controls.
- A correlation in mtDNA methylation patterns was found between muscle tissue and skin fibroblasts within individual myopathy patients.
- Myopathy fibroblasts showed increased expression of SLC25A26 and elevated mtDNA copy numbers, though no disease-associated mtDNA methylation differences were found in fibroblasts.
Conclusions:
- Mitochondrial DNA (mtDNA) methylation patterns, particularly in the CYTB gene, may serve as potential molecular biomarkers for diagnosing unexplained myopathies.
- Analyzing the mitochondrial genome beyond sequence variations, including epigenetic modifications, offers new diagnostic avenues for mitochondrial diseases.
- Further research is needed to refine mtDNA enrichment techniques and fully elucidate the role of mtDNA methylation in the pathogenesis of myopathies.
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