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Page 160 Sulaiman et al. J Transl Genet Genom 2020;4:159-87 I https://doi.org/10.20517/jtgg.2020.27
[3,4]
and cancers may also be associated with mtDNA mutations . To date, there are about 350 causal genes for
[5]
mitochondrial diseases . Since mitochondria play a significant role in energy production via the oxidative
phosphorylation (OXPHOS) system and Krebs cycle, disruptions in their genome and protein functions may
affect various important cellular processes such as fatty acid metabolism, pyrimidine biosynthesis, calcium
[6]
homeostasis, cell signaling, beta-oxidation and heme biosynthesis .

Several factors are involved in determining disease onset, clinical symptoms, phenotypic variability, and
[7]
mitochondrial penetrance . One such factor is replicative or mitotic segregation that leads to several
conditions known as homoplasmy and heteroplasmy, threshold effect, clonal expansion, and the mtDNA
[7]
bottleneck . The ratio of wild type and mutant mtDNA is important in determining disease onset and
clinical symptoms. The vast majority of mtDNA mutations are present in the heteroplasmy condition, and
only some mtDNA mutations are homoplasmic and deleterious. Mitochondria are randomly segregated;
thus, there is a possibility that the daughter cells can shift from wild type to mutant and vice versa. A random
[8]
genetic drift results in clonal expansion, leading to acceleration of mtDNA mutation rate . Rapid segregation
of mammalian heteroplasmy with homoplasmy mtDNA between generations suggests that the mtDNA
bottleneck occurs during development. Subsequently, this condition leads to differences in heteroplasmy
[1]
levels in different mature oocytes of a woman .
[1,7]
mtDNA diseases have five unique characteristics, based on the mtDNA disease pedigree . First, mtDNA
mutations are inherited maternally. Second, mutations arise as a result of several factors, including lack
of histone protection, deficiency in DNA repair mechanisms, and increased levels of reactive oxygen
species (ROS) through OXPHOS activities in the mitochondria. Pathogenic mtDNA mutations comprise
rearrangement mutations and point mutations in genes affecting mitochondrial protein translation
and causing specific OXPHOS defects. Moreover, replicative segregation of mitochondria gives rise to
homoplasmy and heteroplasmy conditions. These conditions cause differential phenotypes in various
processes such as the transcription, translation, enzyme complex formation, respiratory complexes,
biochemical levels, and cellular phenotypes [1,6,7,9] . Third, the impact of mtDNA mutations is subject to the
amount of mitochondrial ATP production. In this case, tissues with the highest requirements for ATP can be
affected the most, such as the central nervous system (CNS). Fourth, mtDNA repairs occur synonymously
and replace mutations rapidly, about 5-10 times faster than nuclear OXPHOS genes due to mammalian
mtDNA genes evolving faster compared to a single copy of nDNA. Finally, aging could result in decreased
OXPHOS activities as well as mitochondrial dysfunction, which could be due to an accumulation of somatic
[10]
cell mtDNA mutations .

The complexity and multi-systemic involvement in mitochondrial diseases render early diagnosis difficult.
With ongoing advances in next-generation sequencing (NGS), the early diagnosis of mitochondrial diseases
becomes feasible, and accurate diagnosis can be made even before the symptoms occur. In this review,
we discuss the mitochondrial diseases, the challenges in their diagnosis, and future recommendations to
assist in the diagnosis. We also provide the details of several companies that offer NGS services to diagnose
mitochondrial diseases.

OVERVIEW OF mtDNA AND MITOCHONDRIAL DISEASES
mtDNA
[11]
Mitochondria have their own genome, which is known as mtDNA . The first complete sequence of the
[12]
human mitochondrial genome was published in 1981 . Following that, in 1999, the mtDNA sequence was
[13]
revised, and its final full genomic sequence was published . In humans, mtDNA spans about 16,500 bp
and consists of the heavy and light strands [12,13] . The heavy strand is rich in guanine bases and encodes 12
subunits of the oxidative phosphorylation (OXPHOS) system, two ribosomal RNAs (12S and 16S), and 14
tRNAs. The light strand encodes one subunit of OXPHOS and eight tRNAs. Altogether, mtDNA contains