Advances in diagnosis of mitochondrial dysfunction and primary mitochondrial disease

Mitochondria are critical cellular organelles engaged in energy production, diverse intermediary metabolic functions and signaling. Inherited genetic disorders directly affecting mitochondrial structure and/or function, known collectively as primary mitochondrial diseases, are among the most complex and heterogeneous inherited conditions, involving dual-genome origin, multisystem manifestations and widely variable clinical severity. These diseases can follow different inheritance patterns and are caused by pathogenic variants in nearly 400 genes encoded by either the mitochondrial or nuclear genome, which commonly impair proteins or RNAs in distinct molecular pathways, resulting in defective energy production by disrupting oxidative phosphorylation. Mitochondrial diseases are challenging to accurately diagnose because of their extensive clinical variability and common features with other metabolic and neuromuscular disorders, as well as limitations in existing diagnostic tools. However, advances in molecular genetics, imaging and systems biology have transformed the diagnostic landscape, enabling more comprehensive and integrative approaches. This Review provides an overview of mitochondrial physiology and outlines the current state of diagnostic strategies, ranging from conventional biochemical assessments and tissue-based analyses to next-generation genome-wide sequencing and the emerging omics technologies. We discuss how combining classical and modern methods can improve diagnostic accuracy and inform clinical decision making. Additionally, we highlight the need for continued refinement of diagnostic frameworks to better support personalized management and future therapeutic development in mitochondrial medicine.