Mitochondria are important cellular organelles, primarily responsible for generating energy required for biological functions. Dysfunctional mitochondria result in abnormalities in ATP generation, cell metabolism, cell function, and promote ageing and diseases. Separate from nuclear DNA (nDNA), mitochondria possess their own genome: mitochondrial DNA (mtDNA). For nDNA, chemical modifications (methylation) play an important role in gene expression regulation. In recent decades, methylation on mtDNA has also gained increasing attention, given its potential role as a biomarker in various diseases. However, its low levels and technical difficulties question the existence of mtDNA methylation.
One of the main concerns in the mtDNA methylation measurements is nDNA contamination, especially for liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. In this project if Lin Liang, we developed an LC-MS/MS-based method for quantifying mtDNA methylation. For this, we used mtDNA isolated from the upper aqueous phase of TRIzol isolation, which efficiently reduced nDNA contamination. This time and cost-effective isolation method allows us to assess mtDNA gene expression and mtDNA methylation in one TRIzol preparation. With this method, we assessed mtDNA methylation in the context of diseases with known mitochondrial dysfunction. In mitochondrial myopathy, we detected an increase in mtDNA methylation compared to healthy controls. In aging-related lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), we found modest changes in mtDNA gene expression and no profound effect of the disease status on mtDNA methylation.
Together, this thesis contributes to understanding the chances and challenges of mtDNA methylation measurement, providing a methodology foundation for future investigation of mtDNA methylation.
