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Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis
Andrea Stoccoro1, Adam R Smith2, Lorena Mosca3
1Department of Translational Research and of New Surgical and Medical Technologies, Lab. of Medical Genetics, University of Pisa, Medical School, Via Roma 55, 56126, Pisa, Italy.
Background:
Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms.
Results:
In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females.
Conclusions:
Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms.
Insights
Epigenetic changes in mitochondrial DNA (mtDNA) are linked to amyotrophic lateral sclerosis (ALS). This study found lower mtDNA D-loop methylation in sporadic ALS patients, suggesting a compensatory mechanism for mitochondrial dysfunction.
Area of Science:
- Neuroscience
- Genetics
- Epigenetics
Background:
- Mitochondrial dysregulation and epigenetic alterations are implicated in neurodegenerative diseases like ALS.
- Previous studies noted reduced mtDNA D-loop methylation in SOD1 carriers, but its role in sporadic ALS remained unexplored.
- Sporadic ALS accounts for most cases, highlighting the need to investigate its underlying mechanisms.
Purpose of the Study:
- To investigate D-loop methylation levels and mtDNA copy number in sporadic ALS patients.
- To compare these levels with those in healthy controls and familial ALS patients.
- To explore the relationship between mtDNA methylation and copy number in ALS.
Main Methods:
- Pyrosequencing analysis of D-loop methylation levels in peripheral white blood cells.
- Quantitative analysis of mtDNA copy number in peripheral white blood cells.
- Comparison between 36 sporadic ALS patients, 51 controls, and 27 familial ALS patients (SOD1 or C9orf72 mutations).
Main Results:
- ALS patients exhibited significantly lower D-loop methylation levels compared to controls.
- An inverse correlation was found between D-loop methylation and mtDNA copy number.
- Lower D-loop methylation was observed in sporadic ALS and SOD1-mutant ALS, but not in C9orf72-ALS patients.
Conclusions:
- Altered D-loop methylation is present in sporadic ALS.
- mtDNA methylation and replication are inversely correlated and may act as compensatory mechanisms.
- These findings suggest potential therapeutic targets for sporadic and SOD1-related ALS.
