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DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein-binding
protein with low pI) and Omi/HrtA2 are released into the cytoplasm [169-172] . The formation of the caspase-
activating complex or apoptosome composed of Cyto-C, Apaf-1, dATP and procaspase-9, thereby induce the
activation of the effector caspases required for apoptosis [173] .

Oxidative stress and mitochondrial dysfunction are associated with several neurodegenerative diseases
such as Alzheimer’s, Parkinson’s, Friedreich ataxia, and amyotrophic lateral sclerosis (ALS) [174] . The first
mtDNA disease was identified in 1988 in a patient with LHON, which is caused by mutations in genes
[71]
encoding OXPHOS subunit I, III, IV, and V . MTND4*LHON11778 is the most common cause of LHON,
[71]
representing about 40%-60% of all LHON cases . SOD1 gene mutations cause ALS with increased cellular
oxidative stress [175] . SOD1 gene encodes SOD1 protein, which acts as an antioxidant defense mechanism
for ROS detoxification. Mitochondrial dysfunction such as defective OXPHOS complexes, changes in
mitochondrial membrane potential (MMP), high level of oxidative stress, and decreased mitophagy
have been associated with idiopathic Parkinson’s disease (PD) [176] . Antony and colleagues showed that
mitochondria of idiopathic PD undergo morphological changes and increased resistance to depolarization.
The basal mitochondrial membrane potential (Ψm) of a skin biopsy from an idiopathic PD patient was
higher compared to healthy control. The mitochondrial morphology parameters such as node degree, mean
volume, skeleton size, perimeter, form factor, node count, erosion body count, endpoints, and mitochondria
count were reduced in idiopathic PD compared to control [177] . Previously, research on the pathogenesis of
PD has been focusing on oxidative stress, mitochondrial bioenergetics defects, and apoptosis mechanisms.
Currently, it has been postulated that the crosstalk dysregulation between mitochondria and endoplasmic
reticulum, as well as lysosome, may lead to mitochondrial dysfunction, including a bioenergetics defect,
abnormal protein aggregation, and finally neuronal cell death [178] .

MULTI-OMICS TECHNOLOGIES TO DIAGNOSE MITOCHONDRIAL DISEASE
The main problems in diagnosing mitochondrial disease are the heterogeneity of the individuals in their
clinical presentation and the incomplete understanding of the disease pathophysiology [1,179] . Advances in
molecular technologies, particularly the high-throughput omics that can characterize and identify many
targets in a single run [180] , have enabled a more in-depth and accurate diagnosis of mitochondrial diseases
and their causes. This section intends to summarize the roles of these omic technologies in mitochondrial
disease diagnosis.

Genomics
Genomics research for mitochondrial diseases started about 30 years ago when a report of the small
sequence of the mitochondrial genome was published [12,13] . Afterwards, various findings of the novel mtDNA
mutations associated with diseases were reported, including the early discoveries of mtDNA mutations in
[71]
ragged-red fiber myopathies [181] and LHON . During this time, it was clear that the mtDNA mutations were
not solely the main culprit, but that other nuclear DNA mutations may play a role in the disease, as in some
patients, the lack of mtDNA mutations were observed [182] . This situation was evident in OXPHOS deficiency
when a report of the succinate dehydrogenase-complex flavoprotein subunit A (SDHA) mutations in Leigh
syndrome (a disease caused by the deficiency of oxidative phosphorylation complex proteins) was published,
in which the SDHA gene mutations resulted in the deficiency of complex II protein in the patient [183] . Thus,
identification of mtDNA mutations and any related gene mutations is important for unraveling the disease-
causing or underlying cause of the mitochondrial disease.

In the early days, there was a limitation in detecting the mutations, as most of the disease-causing mutations
were identified using the single candidate gene sequencing techniques, and these methods were time-
and resource-consuming [184] . The most popular methods were Sanger sequencing and Affymetrix’s DNA
re-sequencing MitoChip [184] . To address the issue with heteroplasmy, several other methods were also