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complement the DNA sequencing, can unravel hidden or deep intronic mutations which usually are missed
from interpretation og WES and WGS data [206] . An example of this RNA-seq approach was in the primary
muscle samples of the genetic myopathy patients, in which the RNA-seq was able to identify disease-causing
mutations in 21% of cases [207] . It is important to note that the DNA sequencing technique was unable to
detect the mutations in these patients [207] . However, the challenges of using RNA-seq are attributed to the
transcriptomic profiling issues, such as the batch effects, and the requirement of robust filtering pipelines to
confirm the results [180] . Nevertheless, the fact remains that the RNA-seq technique can detect mutations in
the patients who are not detected from WES and WGS sequencing.

PROTEOMICS
mtDNA encodes 13 proteins, including the mitochondrial respiratory chain proteins, ribosomal RNAs
and transfer RNAs. The remaining mitochondrial proteins, which include the TCA cycle components,
β-oxidation, protein transports, and the other respiratory chain subunits, are from nuclear DNA [208] .
Therefore, to characterize the proteome profile of mitochondrial diseases can be very challenging. Up until
now, the number of the mammalian mitochondrial proteins discovered is about 1,100 to 1,900, based on
the classifications in each database [209-213] . One of the earliest databases for the mitochondrial proteome
is MITOP, which was released in 1999 [210] and followed by the first comprehensive human mitochondrial
proteome database, the MitoProteome Project [211] . Currently, MitoProteome contains about 1,705 genes and
3,625 proteins that are associated with mitochondria [211] . After that, various databases with their analysis
tools have been released, including the MitoP2 [212] , MitoMiner [213] , and MitoCarta [209] databases.

An example of the mitochondrial dysfunction study using the proteome analysis is the identification
of C17orf89 (NDUFAF8) mutation in Leigh syndrome, in which mass spectrometry (MS) crosslinking
interactome analysis was able to show C17orf89/NDUFAF8 as a new candidate for the unresolved cases
of isolated complex I deficiency [214] . Another study of proteome profiling of the mitochondrial ribosomes
revealed that in the small ribosomal subunit, MRPS34 mutations were responsible for the destabilization of
the subunit and impaired monosome assembly in the fibroblasts of Leigh syndrome patients [215] . Importantly,
the findings [215] were after WES sequencing in those patients, indicating that proteome profiling could also
complement WES sequencing to improve the diagnostic detection of mitochondrial disease.

METABOLOMICS
Due to limited publications, the potential of metabolomics tools to diagnose mitochondrial disease is
uncertain. Lactate and pyruvate have been used as biomarkers for mitochondrial dysfunction, though these
biomarkers have low sensitivity and specificity [216] . One example is that the lactate stress test was used in the
diagnosis of mitochondrial myopathy. However, the sensitivity of the lactate stress test was 69%, but it can
complement the other clinical tests to confirm the diagnosis [217,218] . Advances in technologies allows for the
application of mass spectrometry-based metabolomics to profile thousands of small metabolites [180] . In a
study of the specific subgroup of the Leigh syndrome patients with mutations in the LRPPRC gene, analysis
of the blood and urine metabolites revealed that there were 45 distinct metabolites, including ketones,
lipids, kynurenine, lactate, and pyruvates [219] . These findings were important in highlighting the role of
metabolomics in unraveling the physiology of mitochondrial disease. However, whether these 45 signature
metabolites are specific to the subgroup of Leigh syndrome or applicable to all forms of mitochondrial
diseases is unknown. Therefore, further works are needed to confirm these findings, especially in a large
cohort, to establish the relationship and diagnostic capacity of the metabolomic approach in mitochondrial
disease.

FUNCTIONAL GENOMICS
Following the WES or WGS analysis, the presence of the rare variants or variants of unknown clinical
significance (VUS) is challenging to interpret for definitive mitochondrial disease diagnosis [220] . Functional