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patients with a single large mtDNA deletion, none of the patients had a polyneuropathy [115] . However, other
than the presence or absence of peripheral neuropathy, the clinical phenotype of CPEO is similar between
single and multiple deletions in mtDNA [113] . There has recently been a patient who has a single large 5-kb
mtDNA deletion caused by a nuclear-encoded gene involved in mitochondrial DNA replication, SSBP1 [105] .
This gene’s product binds and protects single-stranded DNA during mtDNA replication. Phenotypically,
this child presented with bone marrow failure and infantile anemia, similar to Pearson syndrome; and short
stature, ptosis, ophthalmoplegia, retinal dystrophy, sensorineural hearing loss, and multiple endocrine
deficiencies, similar to Kearns-Sayre syndrome. He also developed metabolic strokes. A dominant variant
in SSBP1 has been reported to induce optic atrophy and foveopathy [116] .

Mitochondrial DNA replication and maintenance
The replication of mtDNA is continuous throughout the cell cycle and even in those cells not undergoing
active replication. The apparatus needed for replication is exclusively encoded by nuclear genes. The
requirement of multiple copies of mtDNA per mitochondrion and numerous mitochondria per cell
underscore the importance of mechanisms of ongoing mtDNA integrity and stability. The constant
synthesis of mtDNA requires a balanced pool of precursor nucleotides ready for incorporation by the
replisome machinery into new DNA molecules [117] . The precursor nucleotide pools are provided by salvage
pathways and import from the cytosol by specific transporters. Single copies of mtDNA are packaged into
nucleoprotein complexes, named nucleoids within the IMM. The precise location of mtDNA replication
is thought to occur within the nucleoid complex, as, in experiments with purification of necleoids,
POLG, Twinkle, and mtSSB are found [118] . Each nucleoid complex has multiple copies of mitochondrial
transcription factor A (TFAM), which is involved in mtDNA compaction and responsible for replication
and transcription within mitochondria [119] . When TFAM is at high concentrations, replication and
transcription is blocked, but, when compaction is mild, both processes can continue. Enriched nucleoid
preparations also contain components of transcription, RNA helicases, RNA-binding proteins, quality
control proteases, RNA processing proteins, and a subset of mitochondrial ribosomal proteins [120] . Newly
replicated nucleoids are likely coupled to mitochondrial fission at ER contact sites [121] . Mitochondrion
[41]
constantly undergo fusion and fission, termed mitochondrial dynamics . The central role of nucleoid
function, in addition to mtDNA replication and mitochondrial transcription and translation, is still being
defined. However, the importance of the integrity of the nucleoid can be demonstrated when deficiencies
of mtDNA replication and editing, repair, fusion, fission, and alterations in the balance within nucleotide
pools result in mtDNA depletion, multiple mtDNA deletions (and single large deletion), and age specific
mtDNA point mutations producing disease [Tables 3 and 4].

Mitochondrial DNA replication apparatus
The center piece of mtDNA replication revolves around a specialized mtDNA polymerase, POL gamma.
The polymerase is a heterotrimer composed of one 140-kDa catalytic subunit encoded by POLG (also
reported in the literature as POLG1 and POLGA) and a homodimeric processing subunit composed of
two p55 accessory proteins encoded by POLG2. POL gamma is responsible for the replication of mtDNA,
proofreading, and repair of replication mistakes [122] . The replisome consists of POL gamma, one POLG
subunit and two POLG2 subunits, helicase Twinkle (encoded by TWNK formerly named C10orf2),
mitochondrial topoisomerase I, mitochondrial RNA polymerase, RNase H1 (encoded by RNASEH1), and
mitochondrial genome maintenance exonuclease 1 (MGME1 encoded MGME1). Other proteins involved
in mtDNA replication are mitochondrial single-stranded DNA binding protein 1 (mtSSBP1), DNA ligase
III, DNA helicase/nuclease 2 (DNA2 encoded by DNA2), and RNA and DNA flap endonuclease (FEN1) [123] .
POLG-related disease depends on the location of the pathological variant within the gene and likely
unknown environmental and epigenetic factors. The intermixing of gene variants and other factors largely
determine the clinical presentation and course of disease from the severe infantile onset of hepatocerebral
[33]
disorder, Alpers-Huttenlocher syndrome, to adult onset CPEO . The most severe spectrum of POLG