Biochimica et Biophysica Acta 1863 (2017) 1539–1555

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Biochimica et Biophysica Acta

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Review

Mitochondrial DNA maintenance defects

Ayman W. El-Hattab a, William J. Craigen b,⁎, Fernando Scaglia b
a
Division of Clinical Genetics and Metabolic Disorders, Pediatrics Department, Tawam Hospital, Al-Ain, United Arab Emirates
b
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

a r t i c l e i n f o a b s t r a c t

Article history: The maintenance of mitochondrial DNA (mtDNA) depends on a number of nuclear gene-encoded proteins in-
Received 15 December 2016 cluding a battery of enzymes forming the replisome needed to synthesize mtDNA. These enzymes need to be
Received in revised form 31 January 2017 in balanced quantities to function properly that is in part achieved by exchanging intramitochondrial contents
Accepted 14 February 2017
through mitochondrial fusion. In addition, mtDNA synthesis requires a balanced supply of nucleotides that is
Available online 16 February 2017
achieved by nucleotide recycling inside the mitochondria and import from the cytosol. Mitochondrial DNA main-
Keywords:
tenance defects (MDMDs) are a group of diseases caused by pathogenic variants in the nuclear genes involved in
mitochondrial diseases mtDNA maintenance resulting in impaired mtDNA synthesis leading to quantitative (mtDNA depletion) and
mitochondrial DNA (mtDNA) qualitative (multiple mtDNA deletions) defects in mtDNA. Defective mtDNA leads to organ dysfunction due to
mtDNA depletion syndromes insufficient mtDNA-encoded protein synthesis, resulting in an inadequate energy production to meet the
multiple mtDNA deletions needs of affected organs. MDMDs are inherited as autosomal recessive or dominant traits, and are associated
mitochondrial fusion with a broad phenotypic spectrum ranging from mild adult-onset ophthalmoplegia to severe infantile fatal he-
mtDNA replication patic failure. To date, pathogenic variants in 20 nuclear genes known to be crucial for mtDNA maintenance
have been linked to MDMDs, including genes encoding enzymes of mtDNA replication machinery (POLG,
POLG2, TWNK, TFAM, RNASEH1, MGME1, and DNA2), genes encoding proteins that function in maintaining a bal-
anced mitochondrial nucleotide pool (TK2, DGUOK, SUCLG1, SUCLA2, ABAT, RRM2B, TYMP, SLC25A4, AGK, and
MPV17), and genes encoding proteins involved in mitochondrial fusion (OPA1, MFN2, and FBXL4).
© 2017 Published by Elsevier B.V.

1. Introduction
Mitochondria are found in all nucleated human cells and generate
most of the cellular energy. Each nucleated human cell typically con-
tains several hundreds of cytoplasmic mitochondria depending on the
tissue energy needs. In addition to energy production, mitochondria
take part in a number of other processes including calcium homeostasis,
biosynthesis of heme and steroid hormones, apoptosis, and cell cycle
regulation via retrograde signaling [1,2]. Mitochondria have a character-
istic double membrane structure and are compartmentalized, with the
lipid and protein composition differing between the outer and inner
membranes and with functionally discrete intermembrane and matrix
spaces. The outer membrane is smooth and contains large amount of
porins which allow passage of small molecules, whereas the inner
membrane is highly folded into cristae, and is impermeable to most sol-
utes. The inner membrane large surface area accommodates the energy-
generating electron transport chain (ETC) complexes that generate ATP
via oxidative phosphorylation (OXPHOS), a process incorporating
⁎ Corresponding author at: Department of Molecular and Human Genetics, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
E-mail address: wcraigen@bcm.edu (W.J. Craigen).
electron transfer via complexes I-IV and ATP synthesis via complex V
[1,2]. Mitochondria are dynamic organelles that move, fuse, and divide
according to the needs of the cell. Therefore, mitochondria can be
regarded as a dynamic network rather than multiple isolated discrete
organelles. It has been estimated that 1500 proteins are needed for
the structure and function of normal mitochondria. Mitochondria are
under dual genome control. They contain their own DNA (mitochondri-
al DNA; mtDNA) that encodes a very small fraction of mitochondrial
proteins (13 total), and more than 99% of mitochondrial proteins are
encoded by nuclear genes, synthesized in cytoplasm, and imported
into mitochondria [1,2].
Mitochondrial diseases are a clinically and genetically heteroge-
neous group of disorders that result from dysfunction of the mitochon-
drial ETC due to pathogenic variants in mtDNA or nuclear genes
encoding mitochondrial proteins [3,4]. Impaired OXPHOS results in an
inability to generate adequate energy to meet the needs of different tis-
sues, particularly organs with high energy demands including the cen-
tral nervous system, cardiac and skeletal muscles, endocrine system,
liver, and renal system. Energy deficiency in various organs results in
multiorgan dysfunction leading to the variable manifestations observed
in mitochondrial diseases including cognitive impairment, epilepsy, car-
diac and skeletal myopathies, nephropathies, hepatopathies, and
endocrinopathies [3,4].

http://dx.doi.org/10.1016/j.bbadis.2017.02.017
0925-4439/© 2017 Published by Elsevier B.V.
1540 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
With the exception of early embryonic development, mtDNA is
continuously synthesized throughout the cell cycle, and cells require
adequate number of mtDNA for the production of mtDNA-encoded
subunits of ETC complexes and therefore for energy production. The
maintenance of mtDNA depends of on a number of nuclear gene-
encoded proteins that functions in mtDNA synthesis and the mainte-
nance of a balanced mitochondrial nucleotide pool [5,6]. Pathogenic
variants in these genes can result in impaired mtDNA synthesis leading
to quantitative (mtDNA depletion) and qualitative (multiple mtDNA
deletions) defects in mtDNA. Defective mtDNA results in insufficient
synthesis of the mtDNA-encoded ETC complex subunits, resulting in im-
paired OXPHOS and energy deficiency. Inadequate energy production to
meet the needs of affected organs results in organ dysfunction [5,6]. De-
fects in mtDNA maintenance have been classically viewed as two distinct
groups of diseases: mtDNA depletion syndromes and multiple mtDNA de-
letion syndromes, with the former typically presenting during infancy as
severe diseases whereas the latter usually exhibit milder diseases with
onset during adulthood. However, with the current understanding that
both mtDNA depletion and multiple mtDNA deletions have the same
pathomechanism, and with the notion that defects in many of the
mtDNA maintenance genes can result in both mtDNA depletion and mul-
tiple mtDNA deletions, it has become clearer that these two disease groups
represent the spectrum for a single disease group. Therefore, the term mi-
tochondrial DNA maintenance defects (MDMDs) has been used in this re-
view to represent this broader disease definition to replace the terms
mtDNA depletion syndromes and multiple mtDNA deletion syndromes.
In this review, we discuss the mechanism of mtDNA synthesis
and the major factors in its maintenance, the pathomechanism of
mtDNA depletion and multiple deletions, clinical features of the cur-
rently recognized MDMDs, and potential therapeutic options for these
diseases.
2. Mitochondrial DNA synthesis
A human cell can contain several thousand copies of mtDNA distribut-
ed within hundreds of mitochondria. MtDNA occurs as a double-stranded
supercoiled genome of 16,569 base pairs (~16.6 kb) that contains 37
genes including 13 protein-encoding ETC complex subunit genes, 22
transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. The two
mtDNA strands are named the heavy (H) and light (L) strands, with the
former rich in guanine nucleotides and latter rich in cytosines, and contain
a ~1.1 kb control region that harbors the H- and L-strand promoters and
the H-strand origin of replication (OH). The L-strand origin of replication
(OL) is located on the other side of the mtDNA circle within a cluster of
five tRNA genes [7]. MtDNA is organized in DNA-protein complexes called
nucleoids. Nucleoids have a uniform size and frequently contain a single
copy of mtDNA packed with a number of nucleoid-related proteins.
Some of the nucleoid proteins are multifunctional, with roles in both
mtDNA maintenance and intermediary metabolism. Nucleoids are an-
chored to the mitochondrial inner membrane on the matrix side. They
form autonomous replication units that can segregate independently
and are evenly distributed within the mitochondrial network [8,9].
A battery of enzymes forming the replisome are needed to
synthesize mtDNA during mtDNA replication. These enzymes need to
be in stoichiometrically balanced quantities to function properly. In
addition, a balanced supply of nucleotides is needed for accurate
functioning. Exchanging intramitochondrial contents through mitochon-
drial fusion allows the maintenance of a balanced proteome, including
the mtDNA synthesis enzymes, whereas maintaining balanced mitochon-
drial nucleotide pool is achieved by in organello recycling (salvage) of nu-
cleotides and import from the cytosol via specific transporters (Fig. 1).
2.1. The mitochondrial DNA replication machinery
MtDNA is synthesized during mtDNA replication by DNA polymer-
ase γ (pol γ) which is a heterotrimer composed of one 140-kDa catalytic
subunit (p140) encoded by POLG and a homodimeric processing sub-
unit composed of two p55 accessory proteins encoded by POLG2. The
p140 catalytic subunit harbors active sites for 5′-3′ DNA polymerase ac-
tivity and 3′-5′ exonuclease activity required for proofreading. The p55
homodimer binds asymmetrically to the catalytic subunit, where each
accessory monomer plays a distinct role in conferring high processivity
onto the holoenzyme. The proximal p55 increases the binding affinity to
DNA, while the distal p55 is important for the acceleration of nucleotide
incorporation [10–12]. Pol γ is one enzyme of the mtDNA replication
machinery and it functions in conjunction with a number of additional
proteins forming the mtDNA replisome, including Twinkle (encoded
by TWNK (previously designated as C10orf2)), mitochondrial topoisom-
erase I, mitochondrial RNA polymerase (mtRNAP), RNase H1 (encoded
by RNASEH1), and mitochondrial genome maintenance exonuclease 1
(MGME1; encoded by MGME1). Other proteins important for mtDNA
replication are mitochondrial single-stranded DNA binding protein
(mtSSB), DNA ligase III, DNA helicase/nuclease 2 (DNA2; encoded by
DNA2), and RNA and DNA flap endonuclease 1 (FEN1) [13].
Twinkle, which functions as a 5′-3′ DNA helicase, is required for dis-
ruption of the hydrogen bonds that hold the two DNA strands together,
causing mtDNA duplex denaturation and strand unwinding and separa-
tion. The mtSSB is required for stabilizing the single stands. In addition,
the mitochondrial topoisomerase I is required for mtDNA unwinding
ahead of the replication fork. The mtRNAP and the mitochondrial tran-
scription factor A (TFAM; encoded by TFAM), which are required for mi-
tochondrial transcription, are also needed for RNA primer formation to
initiate DNA replication by pol γ. Additionally, DNA primase-polymer-
ase (PrimPol), which contains both DNA primase and DNA polymerase
activities and is present in both nuclear and mitochondrial compart-
ments, can play an important role in re-priming DNA synthesis to rescue
a stalled replication fork [13].
Different mechanisms have been proposed for mtDNA replication,
with the asynchronous replication being the most accepted model. In
this mechanism, which is also called the strand displacement model,
two origins of replication direct the replisome to initiate continuous
DNA synthesis, but the initiation at both loci does not occur at the
same time. The replication of mtDNA is initiated by H-strand synthesis
at the OH. When H-strand synthesis reaches the OL (two-thirds of the
way around the mtDNA), the OL sequence is exposed and adopt a
stem-loop structure that is recognized by mtRNAP which initiates prim-
er synthesis, and L-strand synthesis is initiated from the OL and pro-
ceeds backward. Therefore, mtDNA is bidirectional but asynchronous.
MtSSB or RNA covers the parental H-strand, which is displaced during
mtDNA replication, during the prolonged interval between the initia-
tion of H-strand and O-strand synthesis [14–16].
The mtDNA replication needs to be terminated at OH and OL where
pol γ continues synthesis in the duplex region resulting in the displace-
ment of the RNA primers and the formation of a flap intermediate. Sub-
sequently, RNA primers are removed by the RNase H1. However, RNase
H1 alone is insufficient since this enzyme cannot remove the last two ri-
bonucleotides at the RNA-DNA junction. The remaining RNA and DNA in
the flap intermediate are removed by the nucleases DNA2, FEN1, and
MGME1. Once the flap is removed pol γ can extend or excise the end
to enable DNA ligase III to seal the nick [13,17,18] (Fig. 1).
2.2. Maintaining a balanced mitochondrial nucleotide pool
In addition to the mtDNA replisome enzymes, mtDNA synthesis re-
quires the availability of a balanced supply of the DNA building blocks
deoxyribonucleotide triphosphates (dNTPs). Nucleotides are synthe-
sized via either de novo pathways, which are cell cycle-regulated, there-
by operative only in S-phase cells and down-regulated in non-dividing
cells, or salvage pathways in which dNTPs are produced by utilizing
preexisting nucleosides. In contrast to nuclear DNA, which replicates
only during cell division, mtDNA synthesis is continuous throughout
the cell cycle and replicates independently of cell division [6]. In
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Fig. 1. A diagram showing the proteins that are involved in mtDNA maintenance and known to be associated with MDMDs: 1) Enzymes of mitochondrial nucleotide salvage pathway that
convert the deoxyribonucleosides (thymidine, deoxycytidine, deoxyguanosine, and deoxyadenosine) to deoxyribonucleotide monophosphates (dNMPs: thymidine monophosphate
(TMP), deoxycytidine monophosphate (dCMP), deoxyguanosine monophosphate (dGMP), and deoxyadenosine monophosphate (dAMP)), then to deoxyribonucleotide diphosphates
(dNDPs: thymidine diphosphate (TDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), and deoxyadenosine diphosphate (dADP)), then to
deoxyribonucleotide triphosphates (dNTPs: thymidine triphosphate (TTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxyadenosine
triphosphate (dATP)). Thymidine kinase 2 (TK2; encoded by TK2) and deoxyguanosine kinase (DGK; encoded by DGUOK) convert the deoxyribonucleosides to dNMPs. Nucleotide
monophosphate kinase (NMPK) converts dNMPs to dDMPs. Nucleotide diphosphate kinase (NDPK) converts dNMPs to dNTPs. NDPK forms complex with both succinyl-CoA ligase
(SUCL composed of an alpha subunit encoded by SUCLG1 and a beta subunit encoded by either SUCLA2 or SUCLG2) and gamma-aminobutyrate transaminase (GABAT; encoded by
ABAT). 2) Enzymes of cytosolic nucleotide metabolism: ribonucleotide reductase (RNR; composed of 2 catalytic subunits and two small subunits either R2 or p53-inducible small RNR
subunit (p53R2; encoded by RRM2B)) converts ribonucleotide diphosphates (NDPs) to dNDPs. Thymidine phosphorylase (TP; encoded by TYMP) converts thymidine to thymine. 3)
Proteins involved in mitochondrial nucleotide transport: adenine nucleotide translocator 1 (ANT1; encoded by SLC25A4), acylglycerol kinase (AGK; encoded by AGK), and MPV17
protein (encoded by MPV17). 4) Enzymes involved in mtDNA synthesis: Twinkle (encoded by TWNK) which is a DNA helicase that separates the DNA stands (presented as blue lines),
DNA polymerase γ (pol γ; consisting of catalytic subunit encoded by POLG and two accessory subunits encoded by POLG2) which needs RNA primer (presented as red lines) to initiate
DNA synthesis (presented as blue arrows), and mitochondrial transcription factor A (TFAM; encoded by TFAM) that is required for the generation RNA primer (presented as red lines).
5) Nucleases removing RNA primers and flap intermediate (presented as red and blue lines): RNase H1 (encoded by RNASEH1), DNA helicase/nuclease 2 (DNA2; encoded by DNA2),
and mitochondrial genome maintenance exonuclease 1 (MGME1; encoded by MGME1). 6) Proteins involved in mitochondrial fusion: mitofusin 1 (MFN1; encoded by MFN1),
mitofusin 2 (MFN2; encoded by MFN1), dynamin-related GTPase OPA1 (encoded by OPA1), and F-box and leucine-rich repeat 4 (FBXL4; encoded by FBXL4).

addition, nucleotides cannot be de novo synthesized in the mitochon-
dria, and the mitochondrial inner membrane is impermeable to charged
molecules, including nucleotides. Therefore, to support mtDNA synthe-
sis the mitochondrial dNTP pool needs to be maintained by a constant
supply of dNTPs from mitochondrial salvage pathways, where
preexisting deoxynucleosides are converted to dNTPs within the mito-
chondrial matrix, and by import of cytosolic dNTPs to mitochondrial
matrix via specific transporters [5,6,19].
The mitochondrial nucleotide salvage pathways include the mito-
chondrial thymidine kinase 2 (TK2; encoded by TK2) which plays an im-
portant role in the pyrimidine nucleotide salvage pathway. It mediates
the first, and rate limiting, step in the phosphorylation of pyrimidine
deoxyribonucleosides in the mitochondrial matrix as it converts
deoxycytidine and thymidine to deoxycytidine monophosphate
(dCMP) and thymidine monophosphate (TMP), respectively [20]. On
the other hand, the first step of the mitochondrial purine nucleoside sal-
vage pathway is mediated by the mitochondrial deoxyguanosine kinase
(DGK; encoded by DGUOK) which mediates the phosphorylation of
purine deoxyribonucleosides in the mitochondrial matrix. DGK
converts deoxyguanosine and deoxyadenosine to deoxyguanosine
monophosphate (dGMP) and deoxyadenosine monophosphate
(dAMP), respectively [21]. The resulting pyrimidine and purine deoxy-
ribonucleotide monophosphates (dNMPs) undergo two additional
phosphorylation steps: the nucleotide monophosphate kinase (NMPK)
1542 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
converts them to deoxyribonucleotide diphosphates (dDMPs), then the
nucleotide diphosphate kinase (NDPK) converts the dNMPs to deoxyri-
bonucleotide triphosphates (dNTPs). The mitochondrial NDPK
(encoded by NME4), the only mammalian NDPK with a mitochondrial
targeting leader sequence and confirmed mitochondrial localization, is
shown to form a complex with both succinyl CoA ligase (SUCL) and
gamma-aminobutyrate transaminase (GABAT). The physical interaction
among SUCL, GABAT, and NDPK and the formation of such complex is
essential for the function of the mitochondrial NDPK and the generation
of dNTPs. Therefore, both SUCL and GABAT are considered dual function
enzymes [22,23]. In addition to supporting the NDPK in dNTP synthesis,
SUCL is an enzyme of Krebs cycle that is composed of an alpha subunit,
encoded by SUCLG1 and a beta subunit, encoded by either SUCLA2 or
SUCLG2. It catalyzes the reversible conversion of succinyl-CoA and ADP
or GDP to succinate and ATP or GTP. When the alpha subunit forms a
heterodimer with SUCLA2-encoded beta subunits, the resulted SUCL en-
zyme utilizes ADP to generate ATP. However, when the alpha subunit
forms a heterodimer with the SUCLG2-encoded beta subunits, the
SUCL enzyme utilizes GDP to generate GTP. In addition to supporting
the NDPK in dNTP synthesis, GABAT (encoded by ABAT) catabolize the
principle inhibitory neurotransmitter GABA (gamma-aminobutyric
acid) in the mitochondrial matrix [22,23].
In addition to the mitochondrial nucleotide salvage pathways, the
mitochondrial dNTP pool relies in part on dNTPs imported from the cy-
tosol. Two cytosolic enzymes involved in nucleotide metabolism are im-
portant for maintaining a balanced mitochondrial dNTP pool.
Ribonucleotide reductase (RNR) is a cytosolic enzyme that catalyzes
the reduction of ribonucleotide diphosphates (NDPs) to dNDPs. There-
fore, RNR provides deoxyribonucleotides for nuclear and mitochondrial
DNA replication and repair. RNR is a heterotetramer consisting of two
catalytic large subunits (R1) and two small subunits, either R2 or
p53R2 (p53-inducible small RNR subunit). During S-phase of the cell
cycle, an R1/R2 complex is the main provider of deoxynucleotides. The
activity of the R1/R2 enzyme is tightly regulated by S-phase-specific
transcription and proteasome-mediated degradation of R2 in late mito-
sis. Therefore, post-mitotic cells are completely devoid of R2. However,
p53R2 is expressed in post-mitotic cells and R1/p53R2 synthesizes
deoxynucleotides in quiescent cells. Therefore, the p53R2 (encoded by
RRM2B) has an important function in the maintenance of dNTP pools
for mtDNA synthesis in post-mitotic cells [24]. Thymidine phosphory-
lase (TP; encoded by TYMP) is a cytosolic enzyme that catalyzes the con-
version of thymidine and deoxyuridine to thymine and uracil,
respectively. Therefore, TP is essential for the cytosolic nucleotide sal-
vage pathway [25].
Because the mitochondrial dNTP pool relies in part on dNTPs
imported from the cytosol, mitochondrial nucleotide transporters are
essential for importing cytosolic nucleotides to mitochondrial matrix
to maintain a balanced mitochondrial nucleotide pool. Adenine nucleo-
tide translocator (ANT) is a mitochondrial inner membrane protein that
forms a homodimeric gated channel that transports ADP inside and ATP
outside mitochondria. Different ANT isoforms exist. ANT1 (encoded by
SLC25A4) is expressed predominantly in post-mitotic cell types in skel-
etal muscle, brain, and cardiac muscle. ANT2 is expressed in proliferat-
ing tissues, whereas ANT3 is ubiquitously expressed [26,27]. ANT
plays a central role in cell bioenergetics by exporting ATP from mito-
chondrial matrix to cytosol and in regulating the ADP/ATP ratio in
mitochondrial OXPHOS. In addition, ANT has been suggested to partici-
pate in maintaining the mitochondrial nucleotide pool through
importing ADP which can be subsequently converted to dADP and
dATP [28]. It is important to mention that ANT is one of the most
abundant mitochondrial proteins and is bound to the inner membrane
lipids phosphatidic acid and cardiolipin [29]. These lipids are suggested
to be essential for proper inner membrane ANT assembly and stabiliza-
tion. The mitochondrial acylglycerol kinase (AGK; encoded by AGK),
which catalyzes the phosphorylation of diacylglycerol to phosphatidic
acid, has an essential function in membrane-lipid synthesis because
phosphatidic acid can take part in the synthesis of phospholipids includ-
ing cardiolipin. Therefore, AGK can play an indirect role in facilitating
nucleotide import through maintaining the lipid-membrane composi-
tion that is essential for the assembly and stabilization of ANT [30]. Fi-
nally, MPV17 protein (encoded by MPV17) is another mitochondrial
inner membrane protein that has been shown to be essential in main-
taining adequate dNTPs inside mitochondria. Therefore, it has been sug-
gested that MPV17 forms a channel in the mitochondrial inner
membrane that functions in importing dNTPs into the mitochondria
[31,32] (Fig. 1).
2.3. Maintaining a balanced mitochondrial proteome
Accurate stoichiometries of mitochondrial enzymes involved in
mtDNA synthesis are important for mtDNA maintenance. Mitochondria
maintain their balanced proteome through exchanging contents [33].
Mitochondria are mobile and dynamic organelles that continually un-
dergo fusion and division. In addition to their function in controlling mi-
tochondrial morphology, these dynamic processes allow mitochondria
to mix contents to maintain an even protein distribution among mito-
chondrial populations [33]. Mitochondrial fusion requires the coordi-
nated fusion of both the outer and inner membranes, and three large
GTPases are known to be important for this process. The mitofusin 1
(MFN1; encoded by MFN1) and mitofusin 2 (MFN2; encoded by
MFN2) are outer mitochondrial membrane proteins that are involved
in early steps of mitochondrial fusion. The dynamin-related GTPase,
OPA1 (encoded by OPA1), is an inner mitochondrial membrane that is
essential for the inner membrane fusion [33]. Mitofusins and OPA1 are
therefore essential for mtDNA maintenance as they mediate mitochon-
drial fusion which results in the exchange of intermitochondrial con-
tents and maintenance of a homogeneous and balanced pool of
mitochondrial proteins including the enzymes needed for mtDNA syn-
thesis. In addition, OPA1 encodes an alternatively spliced isoform,
OPA1-exon4b that is associated with the mitochondrial inner mem-
brane and directly interacts with the nucleoids, allowing their even dis-
tribution within the mitochondrial network. OPA1-exon4b also
promotes mtDNA replication potentially by interacting with and regu-
lating the replisome [34]. Finally, FBXL4 (F-box and leucine-rich repeat
4) is another protein that plays an important role in mtDNA mainte-
nance. Although the exact mechanism is unknown, it could be through
the regulation of mitochondrial dynamics. FBXL4 (encoded by FBXL4)
has recently been discovered to be a mitochondrial protein that resides
in the mitochondrial inter-membrane space. FBXL4 has a leucine-rich
repeat domain that is typically engaged in protein-protein interactions,
allowing FBXL4 to be present in a quaternary protein complex. FBXL4
has been found essential for the formation of a normal mitochondrial
network. Therefore, FBXL4 may play a role in the mitochondrial fusion
process via interacting and regulating other mitochondrial fusion pro-
teins [35–37] (Fig. 1).
3. Mitochondrial DNA maintenance defects
Mitochondrial DNA maintenance defects (MDMDs) are a group of
diseases caused by pathogenic variants in the nuclear genes
involved in mtDNA maintenance resulting in impaired mtDNA synthe-
sis leading to quantitative (mtDNA depletion) and qualitative (multiple
mtDNA deletions) defects in mtDNA. Therefore, MDMDs are character-
ized by mtDNA depletion and/or multiple mtDNA deletions in affected
organs. MDMDs are inherited in an autosomal recessive or dominant
manner and are associated with a broad phenotypic spectrum
ranging from mild adult-onset ophthalmoplegia to severe infantile
fatal hepatic failure. To date, pathogenic variants in 20 nuclear genes
known to be crucial for mtDNA maintenance have been linked to
MDMDs. These variants have been reported in: genes encoding en-
zymes of mtDNA replication machinery (mtDNA polymerization:
POLG, POLG2, TWNK and TFAM; and nucleases removing primers and
 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555 1543

Table 1
Clinical features of mitochondrial DNA maintenance defects due to abnormalities in mtDNA replication machinery.

Inh. Onset MtDNA Clinical manifestations

Defects in mitochondrial DNA polymerization

POLG-related Alpers-Huttenlocher syndrome AR Early childhood Depletion Encephalopathy, neuropathy, and hepatopathy
POLG-related MNGIE AR Infancy or childhood Depletion and multiple Gastrointestinal dysmotility, myopathy, and
deletions neuropathy
POLG-related MEMSA AR Young adulthood Multiple deletions Epilepsy, myopathy, neuropathy, and ataxia
POLG-related ANS AR Young adulthood Multiple deletions Ataxia, neuropathy, and encephalopathy
POLG-related ARPEO AR Adolescence or young Multiple deletions Ophthalmoplegia
adulthood
POLG-related ADPEO AD Adulthood Multiple deletions Ophthalmoplegia and myopathy
POLG2-related myopathic MDMD AD Infancy to adulthood Multiple deletions Myopathy and ophthalmoplegia
TWNK-related IOSCA AR Infancy Depletion Ataxia, encephalopathy, and neuropathy
TWNK-related hepatocerebral MDMD AR Neonatal period or infancy Depletion Encephalopathy and hepatopathy
TWNK-related ADPEO AD Early adulthood Multiple deletions Ophthalmoplegia and myopathy
TFAM-related hepatocerebral MDMD AR Neonatal period Depletion Hepatopathy

Defects in mitochondrial nucleases

RNASEH1-related encephalomyopathic AR Early adulthood Depletion and multiple Encephalopathy and myopathy
MDMD deletions
MGME1-related myopathic MDMD AR Childhood or early adulthood Depletion and multiple Myopathy
deletions
DNA2-related myopathic MDMD AD Childhood or early adulthood Multiple deletions Myopathy
DNA2-related Seckel syndrome AR Birth NA Dwarfism

Inh.: inheritance; AR: autosomal recessive; AD: autosomal dominant; MDMD: mitochondrial DNA maintenance defects; MNGIE: mitochondrial neurogastrointestinal encephalopathy;
MEMSA: myoclonic epilepsy-myopathy-sensory ataxia; ANS: ataxia-neuropathy spectrum; ARPEO: autosomal recessive progressive external ophthalmoplegia; ADPEO: autosomal dom-
inant progressive external ophthalmoplegia; IOSCA: infantile-onset spinocerebellar ataxia.

flap intermediates: RNASEH1, MGME1, and DNA2) (Table 1), genes
encoding proteins that function in maintaining a balanced mitochondri-
al nucleotide pool (mitochondrial salvage pathway: TK2, DGUOK,
SUCLG1, SUCLA2, and ABAT; cytosolic nucleotide metabolism RRM2B
and TYMP; and mitochondrial nucleotide import: SLC25A4, AGK, and
MPV17) (Table 2), and genes encoding proteins involved in mitochon-
drial dynamics (OPA1, MFN2, and FBXL4) (Table 3).
3.1. Defects in mitochondrial DNA polymerization: POLG, POLG2, TWNK
and TFAM
POLG, POLG2, and TWNK encode components of the mtDNA replica-
tion machinery that are responsible for mtDNA replication (see Section
2.1). Pathogenic variants in POLG result in a reduction in pol γ activity
leading to stalling at the replication fork [38]. Pathogenic variants in

Table 2
Clinical features of mitochondrial DNA maintenance defects due to abnormalities in maintaining a balanced mitochondrial nucleotide pool.

MDMD Inh. Onset MtDNA Clinical manifestations

Defects of mitochondrial nucleotide salvage pathway

TK2-related myopathic MDMD AR Infancy or childhood Depletion Myopathy
TK2-related ARPEO AR Mid-adulthood Multiple deletions Ophthalmoplegia and myopathy
DGUOK-related hepatocerebral Neonatal period, infancy, or Depletion Hepatopathy and encephalopathy
MDMD childhood
DGUOK-related myopathic MDMD AR Early or mid-adulthood Multiple deletions Myopathy
SUCLA2-related encephalomyopathic AR Infancy or early childhood Depletion Encephalopathy, myopathy, and elevated MMA
MDMA
SUCLG1-related encephalomyopathic AR Neonatal period or infancy Depletion Encephalopathy, myopathy, and elevated MMA
MDMA
ABAT-related encephalomyopathic AR Infancy Depletion Encephalopathy, myopathy, and elevated GABA
MDMD

Defects of cytosolic nucleotide metabolism

TYMP-related MNGIE AR Adolescence or early adulthood Depletion and multiple Gastrointestinal dysmotility, myopathy, encephalopathy, and
deletions neuropathy
RRM2B-related encephalomyopathic AR Neonatal period or infancy Depletion Encephalopathy and myopathy
MDMD
RRM2B-related MNGIE AR Early adulthood Depletion Gastrointestinal dysmotility, myopathy, encephalopathy, and
neuropathy
RRM2B-related ARPEO AR Childhood Multiple deletions Ophthalmoplegia and myopathy
RRM2B-related ADPEO AD Adulthood Multiple deletions Ophthalmoplegia and myopathy

Defects of mitochondrial nucleotide import

SLC25A4-related cardiomyopathic AR Early childhood Multiple deletions Cardiac and skeletal myopathy
MDMD
SLC25A4-related ADPEO AD Adulthood Multiple deletions Ophthalmoplegia and myopathy
AGK-related Sengers syndrome AR Neonatal period Depletion Cardiac and skeletal myopathy and cataract
MPV17-related Navajo AR Infancy or early childhood Depletion Neuropathy and hepatopathy
neurohepatopathy
MPV17-related hepatocerebral MDMD AR Neonatal period or infancy Depletion Encephalopathy and hepatopathy
MPV17-related neuromyopathic AR Adolescence or young adulthood Multiple deletions Neuropathy and myopathy
MDMD

Inh.: inheritance; AR: autosomal recessive; AD: autosomal dominant; MDMD: mitochondrial DNA maintenance defects; MNGIE: mitochondrial neurogastrointestinal encephalopathy;
ARPEO: autosomal recessive progressive external ophthalmoplegia; ADPEO: autosomal dominant progressive external ophthalmoplegia; MMA: methylmalonic acid; GABA: gamma-
aminobutyric acid.
1544 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
Table 3
Clinical features of mitochondrial DNA maintenance defects due to abnormalities in mitochondrial dynamics.
MDMD Inh. Onset
Defects in mitochondrial dynamics
OPA1-related ADOA AD Childhood or early adulthood
OPA1-related Behr syndrome AR Infancy or early childhood
OPA1-related encephalomyopathic MDMD AR Infancy
MFN2-related CMT2A AD Childhood or early adulthood
MFN2-related ADOA AD Early childhood
FBXL4-related encephalomyopathic MDMD AR Neonatal period or infancy
Inh.: inheritance; AR: autosomal recessive; AD: autosomal dominant; MDMD: mitochondrial DNA maintenance defects; ADOA: autosomal dominant optic atrophy; CMT2A: Charcot-
Marie-Tooth neuropathy type 2A.
POLG2 can result in decreased affinity and disturbed interaction be-
tween the p55 accessory subunit with the p140 catalytic subunit,
resulting in reduced pol γ processivity that can result in stalling of the
replication fork [12,39]. Pathogenic variants in POLG2 can have a domi-
nant negative effect, as the wild-type/mutant p55 heterodimer can im-
pair polγ function [40]. Pathogenic variants in TWNK impair the helicase
activity of Twinkle resulting in mtDNA replication stalling [41,42].
Stalling of mtDNA replication results in impaired mtDNA synthesis,
leading to mtDNA depletion and/or multiple mtDNA deletions. TFAM
encodes the mitochondrial transcription factor A that is essential for
mtDNA transcription. In addition, TFAM it is critical for mtDNA replica-
tion because of its role, along with mtRNAP, in producing the necessary
RNA primer for mtDNA replication. Furthermore, TFAM plays a role in
mtDNA compaction and organization in the nucleoid [43]. Pathogenic
variants in TFAM result in decreased number of nucleoids and impaired
mtDNA synthesis leading mtDNA depletion [44].
3.1.1. POLG-related mitochondrial DNA maintenance defects
POLG-related MDMDs constitute a continuum of overlapping pheno-
types with clinical onset spanning infantile period to late adulthood.
These disorders include: Alpers-Huttenlocher syndrome (AHS), POLG-
related mitochondrial neurogastrointestinal encephalopathy (MNGIE),
myoclonic epilepsy-myopathy-sensory ataxia (MEMSA), ataxia-neu-
ropathy spectrum (ANS), autosomal recessive progressive external
ophthalmoplegia (ARPEO), and autosomal dominant progressive exter-
nal ophthalmoplegia (ADPEO) [38] (Table 1).
3.1.1.1. Alpers-Huttenlocher syndrome. AHS is one of the most severe
phenotypes of POLG-related MDMDs. AHS is an autosomal recessive dis-
ease with a prevalence of ~1:50,000. It is characterized by a progressive
encephalopathy with psychomotor regression, epilepsy, and spasticity,
ataxia, neuropathy, and hepatopathy. Affected children are typically
healthy at birth and may develop normally until the onset of the disease,
which is usually between ages 2 and 4 years. Different types of epilepsy
occur in affected children including myoclonic, generalized, focal, status
epilepticus, and epilepsia partialis continua. The seizures can be initially
controlled by standard anticonvulsant medications in some children.
However, the seizures usually become increasingly refractory over
time. Affected individuals typically have areflexia and hypotonia early
in the disease and later develop spastic paraparesis evolving over
months to years. Ataxia, neuropathy, progressive spasticity, and loss of
cognitive function occur in all affected individuals. Other neurological
manifestations include movement disorders (myoclonus and
choreoathetosis), headaches typically associated with visual sensations,
stroke and stroke-like episodes, somnolence, irritability, parkinsonism,
nystagmus, cortical visual loss, retinopathy, and sensorineural hearing
impairment. Neurologic manifestations can worsen during infections
and with other physiologic stressors.
Cerebrospinal fluid (CSF) protein is usually elevated and neuro-
imaging typically shows generalized brain atrophy, gliosis, and
deep gray matter hyperintensities, particularly in the thalamus and
cerebellum. Affected individuals usually develop hepatopathy mani-
festing as coagulopathy, hypoalbuminemia, elevated transaminases,
MtDNA Clinical manifestations
Multiple deletions Optic atrophy
NA Optic atrophy, neuropathy, and spinocerebellar degeneration
Depletion Myopathy, encephalopathy, and optic atrophy
NA Axonal motor neuropathy
Multiple deletions Optic atrophy and neuropathy
Depletion Encephalopathy and myopathy
hyperammonemia, and fasting hypoglycemia. Within a few months,
the liver involvement can progress rapidly to end-stage liver failure.
Liver histology may demonstrate mitochondrial proliferation, bile
duct proliferation, fibrosis, cirrhosis, and steatosis. MtDNA depletion
is observed in clinically affected tissues including liver and muscle.
The progression of the disease is quite variable, and the typical life
expectancy being 3 months to 12 years from the onset of first symp-
toms [38,45–47].
3.1.1.2. POLG-related mitochondrial neurogastrointestinal encephalopathy
disease. MNGIE disease is characterized by progressive dysmotility of
the gastrointestinal tract, cachexia, ophthalmoplegia, ptosis,
leukoencephalopathy, sensorimotor neuropathy, myopathy, lactic aci-
dosis, and mtDNA depletion and multiple mtDNA deletions. The onset
of signs and symptoms is usually between the first and fifth decades.
It is primarily caused by biallelic pathogenic variants in TYMP [48]. To
date, nine individuals have been reported to have a MNGIE phenotype
caused by biallelic POLG pathogenic variants. These individuals present-
ed during infancy or childhood with gastrointestinal dysmotility,
ophthalmoplegia, cachexia, neuropathy, hypotonia, muscle weakness,
and neurosensory hearing impairment. Cytochrome c oxidase (COX)-
deficient fibers, ragged red fibers (RRF), decreased ETC complex I and
IV activities, and mtDNA depletion and multiple mtDNA deletions
were found in muscle tissue. However, leukoencephalopathy was ab-
sent upon neuroimaging in these individuals [49–53].
3.1.1.3. Myoclonic epilepsy-myopathy-sensory ataxia. MEMSA is an auto-
somal recessive disease that usually presents in young adulthood with
cerebellar ataxia, sensory neuropathy, and myopathy. Epilepsy, which
may be refractory to anticonvulsant medications, develops in later
years, often beginning focally and then spreading to become general-
ized. The myopathy can be distal or proximal, and it may present as ex-
ercise intolerance. Multiple mtDNA deletions are found in skeletal
muscle [38,47].
3.1.1.4. Ataxia-neuropathy spectrum. ANS is an autosomal recessive dis-
ease associated with multiple mtDNA deletions. Different clinical
phenotypes included in this spectrum are: mitochondrial recessive
ataxia syndrome (MIRAS), sensory ataxia-neuropathy-dysarthria-
ophthalmoplegia (SANDO), and spinocerebellar ataxia with epilepsy
(SCAE). ANS disorders typically present in young adulthood with
ataxia, neuropathy, and encephalopathy with seizures. The neuropa-
thy, which can be severe enough to contribute to ataxia, can be
motor, sensory, or mixed. Other frequent manifestations include mi-
graine headaches, vision impairment, myoclonus, hepatopathy, and
depression [38,47].
3.1.1.5. POLG-related autosomal recessive progressive external
ophthalmoplegia. POLG-related ARPEO is characterized by
ophthalmoplegia and ptosis with onset during adolescence or young
adulthood. It is typically not associated with systemic involvement.
However, other POLG-related MDMDs, including ANS, may present ini-
tially with ophthalmoplegia and develop other neurological
 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
manifestations over subsequent years. Therefore caution is needed
when making the diagnosis of POLG-related ARPEO. This disease is asso-
ciated with multiple mtDNA deletions [38,54].
3.1.1.6. POLG-related autosomal dominant progressive external
ophthalmoplegia. ADPEO is an adult-onset disease characterized by pro-
gressive ophthalmoplegia, ptosis, and multiple mtDNA deletions. Most
of the affected individuals exhibit generalized myopathy that can lead
to exercise intolerance and early fatigue. Other manifestations observed
in some individuals include ataxia, axonal neuropathy, sensorineural
hearing impairment, cataracts, depression, parkinsonism, and
hypogonadism. Less common features include gastrointestinal
dysmotility and cardiomyopathy [38,55].
3.1.2. POLG2-related mitochondrial DNA maintenance defect
Monallelic pathogenic variants in POLG2, which can have a dominant
negative effect, are associated with myopathic MDMD and multiple
mtDNA deletions [40]. POLG2-related myopathic MDMD is an autoso-
mal dominant disorder that has been reported in 12 individuals [12,
39,56,57]. The age of disease onset is variable and ranges from infancy
to adulthood. Common manifestations are proximal muscle weakness,
ophthalmoplegia, ptosis, exercise intolerance, and easy fatigability. In
addition, developmental delay, hypotonia, and growth failure have
been reported in affected children. Other less frequent manifestations
include muscle cramps, myalgia, peripheral neuropathy, ataxia, epilep-
sy, gastrointestinal dysmotility, hepatopathy, apnea, hypoventilation,
and respiratory failure. Elevated blood creatine phosphokinase (CPK)
and lactate, and elevated CSF protein have been described. Neuroimag-
ing may show cerebellar atrophy. COX-deficient fibers, RRF, and multi-
ple mtDNA deletions are observed in skeletal muscle [12,39,56,57]
(Table 1).
3.1.3. TWNK-related mitochondrial DNA maintenance defects
Biallelic pathogenic variants in TWNK can result in infantile-onset
spinocerebellar ataxia (IOSCA) or hepatocerebral MDMD, both of
which are associated with mtDNA depletion. ADPEO with multiple
mtDNA deletions can be caused by monoallelic TWNK pathogenic
variants that exhibit a dominant negative effect [58] (Table 1).
3.1.3.1. Infantile-onset spinocerebellar ataxia. IOSCA is a severe, progres-
sive autosomal recessive neurodegenerative disorder. Twenty four indi-
viduals with IOSCA have been reported to date. All affected individuals
with IOSCA have been identified in a genetically isolated population of
Finland. Affected children typically develop normally until one year of
age when the disease usually presents with ataxia, hypotonia,
hyporeflexia, and athetosis. By school age sensorineural hearing
impairment and ophthalmoplegia develop. By adolescence affected
individuals exhibit optic atrophy, sensory axonal neuropathy,
hypergonadotrophic hypogonadism in females, progressive pes cavus
foot deformity, scoliosis, and autonomic nervous system dysfunction
manifesting as, difficulty with urination, urinary incontinence,
obstipation, and increased perspiration. Later, epilepsy, psychiatric
symptoms, and migraine may develop. Epilepsy can have variable
types including myoclonic, tonic clonic, focal clonic, status epilepticus,
and epilepsia partialis continua. Neuroimaging shows atrophy of cortex,
cerebellum, and brain stem with T2 white matter hyperintensities in the
cerebellum. MtDNA depletion has been found in post-mortem brain tis-
sues [59–61].
3.1.3.2. TWNK-related hepatocerebral mitochondrial DNA maintenance de-
fect. Biallelic pathogenic variants in TWNK are a rare cause of
hepatocerebral MDMD, being reported in 8 children from 4 unrelated
families [62–65]. Affected children usually present in the neonatal or in-
fantile period with hepatopathy, lactic acidosis, psychomotor delay, and
hypotonia. Neurological manifestations are progressive and include
muscular atrophy, hyporeflexia, ataxia, sensory neuropathy, epilepsy,
1545
sensorineural hearing impairment, psychomotor regression, athetosis,
nystagmus, and ophthalmoplegia. Hepatopathy presents as hepatomeg-
aly, cholestasis, increased transaminases, coagulopathy, hypoalbumin-
emia, ascites, and liver failure. Feeding difficulties, recurrent vomiting,
failure to thrive, Fanconi tubulopathy, and fasting hypoglycemia can
be observed in affected infants. Plasma and CSF lactate are elevated.
Liver biopsy can show cirrhosis, cholestasis, and steatosis. Neuroimag-
ing can show T2 white matter hyperintensities and cerebellar atrophy.
ETC assay show reduced complex I, III, and IV activities, and mtDNA
depletion is evident in liver tissue. This disease has poor prognosis as
death occurs during infancy or early childhood in most affected children
[62–65].
3.1.3.3. TWNK-related autosomal dominant progressive external
ophthalmoplegia. ADPEO due to monoallelic pathogenic variants in
TWNK has been reported in more than 100 individuals [66]. Although
the disease typically starts in early adulthood, the age of onset varies
from 8 to 65 years with the mean being 42 years. Almost all affected in-
dividuals develop ptosis and ophthalmoplegia. Fatigue, exercise intoler-
ance, and proximal muscle weakness often occur. Other less frequent
manifestations are myalgia, dysphagia, cardiac involvement including
ventricular enlargement and arrhythmias, migraine, endocrinopathies
including diabetes and hypogonadism, cataracts, sensory axonal neu-
ropathy, depression, and sensorineural hearing impairment. COX-defi-
cient fibers, RRF, and multiple mtDNA deletions can be observed in
skeletal muscle [67]. Additionally, 9 individuals with TWNK-related
ADPEO developed late onset parkinsonism. The parkinsonism was de-
scribed as mild and stable, and generally manifests as bilateral postural
and rest tremor and mild rigidity of upper limbs and axial muscles typ-
ically responsive to L-Dopa [68].
3.1.4. TFAM-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in TFAM are associated with
hepatocerebral MDMD. TFAM-related hepatocerebral MDMD is an auto-
somal recessive disorder that has been recently described two siblings
with intrauterine growth restriction (IUGR) and neonatal presentation
of hypoglycemia, elevated transaminases, jaundice, and cholestasis.
Liver impairment progressed to liver failure with hypoalbuminemia, as-
cites, coagulopathy, and death in early infancy. Liver and muscle biopsy
were performed for one sibling. Liver histology revealed cirrhosis,
steatosis, and cholestasis. On electron microscopy proliferation of ab-
normally shaped mitochondria were observed in muscle and liver tis-
sue. MtDNA depletion was evident in both liver and skeletal muscle
tissues. ETC studies revealed reduction in complexes I, II, III and IV activ-
ities [44] (Table 1).
3.2. Defects in mitochondrial nucleases: RNASEH1, MGME1, and DNA2
RNASEH1, MGME1, and DNA2 encode components of the mtDNA rep-
lication machinery that are nucleases responsible for the removal RNA
primers and flap intermediates (see Section 2.1). Pathogenic variants
in RNASEH1 have been proposed to result in a reduced capacity to re-
move the RNA primers, leading to a slowing down and stalling of
mtDNA replication [69]. However, a subsequent study showed no evi-
dence of primer retention in human fibroblasts carrying a pathogenic
variant [70], therefore, further studies are needed before drawing final
conclusions about the exact effect of RNase H1 deficiency. Pathogenic
variants in MGME1 and DNA2 have been proposed to result in an inabil-
ity to remove flap intermediates, leading to improper mtDNA replica-
tion [13,17,71,72]. The impairment of removal of RNA primers and
flap intermediates are proposed to result in defective mtDNA synthesis,
leading to mtDNA depletion and multiple deletions.
3.2.1. RNASEH1-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in RNASEH1 can cause an
encephalomyopathic MDMD that is associated with mtDNA depletion
1546 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
and multiple mtDNA deletions. RNASEH1-related encephalomyopathic
MDMD is an autosomal recessive disease that has been reported in 6 in-
dividuals from 3 unrelated families. Affected individuals usually present
during early adulthood with ptosis, ophthalmoplegia, muscle weakness
and wasting, easy fatigability, exercise intolerance, ataxia, dysphagia,
cognitive impairment, pyramidal signs, and respiratory muscle weak-
ness presenting as dyspnea that can progress to respiratory failure,
resulting in the need for non-invasive ventilation in some. Other less
frequent manifestations include myalgia, muscle cramps, dysarthria,
dysphonia, elevated plasma lactate, and elevated CPK. Neuroimaging
can show cerebellar and cortical atrophy. RRF, COX-deficient, succinate
dehydrogenase (SDH)-positive fibers, abnormally shaped mitochondria
on electron microscopy, reduced ETC complex I and IV activities, mtDNA
depletion, and multiple mtDNA deletions can be found in muscle tissues
[69] (Table 1).
3.2.2. MGME1-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in MGME1 can cause a myopathic
MDMD which is associated with mtDNA depletion and multiple
mtDNA deletions. MGME1-related myopathic MDMD is an autosomal
recessive disease that has been reported in 6 individuals from 3 families.
Affected individuals usually present during childhood or early
adulthood with ptosis, ophthalmoplegia, easy fatigability, exercise
intolerance, progressive proximal muscular weakness, generalized
muscle wasting, underweight, and respiratory muscle weakness pre-
senting as dyspnea that can progress to respiratory failure resulting in
the need for non-invasive ventilation. Other common features are
kyphosis, facial weakness, nasal speech, intellectual disability, mi-
crocephaly, gastrointestinal manifestations (loss of appetite, nausea,
recurrent diarrhea, and flatulence), and ataxia. Less frequent fea-
tures were hyporeflexia, elevated CPK, dilated cardiomyopathy,
hypergonadotropic hypogonadism, and nephrolithiasis. Neuroimag-
ing can reveal cerebellar atrophy. RRF, COX-deficient fibers, reduced
ETC complex I and IV activities, multiple mtDNA deletions, and
mtDNA depletion are evident in skeletal muscle [71] (Table 1).
3.2.3. DNA2-related mitochondrial DNA maintenance defects
Monoallelic pathogenic variants in DNA2 are associated with
myopathic MDMD and multiple mtDNA deletions, whereas biallelic
pathogenic variants are associated with Seckel syndrome. DNA2-re-
lated myopathic MDMD is an autosomal dominant disease that has
been reported in 4 subjects from 3 different families. Affected indi-
viduals usually present during childhood or early adulthood with
ptosis, ophthalmoplegia, proximal muscle weakness, generalized
muscle atrophy, and easy fatigability. Myalgia, muscle cramps, ele-
vated CPK, and dyspnea can also occur in some. Type II fiber
hypotrophy, RRF, COX-deficient fibers, proliferation of abnormally
shaped mitochondria on electron microscopy, and multiple mtDNA
deletions are observed in skeletal muscle tissue [73]. Homozygous
pathogenic variants in DNA2 were identified in an uncle and niece
with dwarfism and distinctive facial features consistent with the
clinical phenotype of Seckel syndrome [74]. Although, the parents
of these individuals are heterozygous carriers of the DNA2 pathogen-
ic variants, they were healthy without any signs of myopathy or
ophthalmoplegia (Alkuraya FS: personal communication 09/2016).
One potential explanation is that the pathogenic variants causing
the dominant myopathic MDMD may have a dominant-negative ef-
fect, while loss-of-function pathogenic variants cause the recessive
Seckel syndrome and haploinsufficiency in carriers is not associated
with a disease phenotype. Indeed, the variant reported in Seckel syn-
drome causes loss-of-function [74]; however, mixing studies did not
support a dominant-negative effect for the pathogenic variants asso-
ciated with the myopathic MDMD [73], leaving the mechanism of
disease unresolved (Table 1).
3.3. Defects of mitochondrial nucleotide salvage pathway: TK2, DGUOK,
SUCLG1, SUCLA2, and ABAT
The mitochondrial nucleotide pool is tightly regulated and defects in
enzymes involved in mitochondrial or cytosolic nucleotide synthesis or
mitochondrial nucleotide transporters can affect the mitochondrial nu-
cleotide pool. Both a lack and overload of certain nucleosides can cause
impair mtDNA synthesis, leading to mtDNA depletion or multiple
mtDNA deletions [5,6,19].
TK2, DGUOK, SUCLA2, SUCLG1, and ABAT encode mitochondrial en-
zymes that are involved in nucleotide synthesis through the salvage
pathway, therefore they are essential in maintaining the mitochondrial
nucleotide pool (see Section 2.2). Defects in any of these enzymes result
in a depletion and imbalance of mitochondrial nucleotides leading to
impairment of mtDNA synthesis. SUCL and GABAT are dual function en-
zymes. Both are essential for the function of the mitochondrial NDPK;
therefore, SUCL deficiency (due to pathogenic variants in either
SUCLA2 or SUCLG1) or GABAT deficiency (due to pathogenic variants
in ABAT) results in impaired NDPK function leading to defective dNTP
synthesis. In addition, SUCL deficiency results in impaired conversion
of succinyl-CoA to succinate. Therefore, succinyl-CoA accumulates and
is converted to methylmalonyl-CoA by methylmalonyl-CoA mutase
leading to the accumulation of methylmalonic acid. On the other
hand, GABAT deficiency also results in impaired GABA catabolism lead-
ing to dysfunctional GABAergic system [22,23].
3.3.1. TK2-related mitochondrial DNA maintenance defects
Biallelic pathogenic variants in TK2 are associated with two pheno-
types: TK2-related myopathic MDMD, which has an early onset and is
associated with mtDNA depletion, and TK2-related ARPEO, which is
an adult-onset disease associated with multiple mtDNA deletions
(Table 2).
3.3.1.1. TK2-related myopathic mitochondrial DNA maintenance defect.
TK2-related myopathic MDMD is an autosomal recessive disorder. To
date, it has been reported in approximately 60 individuals. The clinical
presentation is variable, with a phenotypic continuum that ranges
from mild to severe. The most common presentation is the infantile or
childhood-onset isolated myopathy. Affected individuals typically
have unremarkable prenatal and perinatal histories, and normal initial
development. The onset of symptoms is usually during the first two
years of life when the affected children exhibit gradual-onset hypotonia,
followed by decreased physical activity, generalized fatigability, proxi-
mal muscle weakness, muscle atrophy, feeding difficulties, and loss of
previously acquired skills. However cognitive function is generally
spared. Bulbar weakness (dysarthria and dysphagia) can occur in
some affected children. Typically, the muscle weakness is rapidly pro-
gressive and can lead to respiratory failure and death within a few
years after disease onset. The commonest cause of death is pulmonary
infection. Less frequent manifestations include early-onset encephalop-
athy with severe muscle weakness and intractable epilepsy, myopathy
with liver involvement, a spinal muscular atrophy-like presentation,
adult-onset progressive myopathy, late-onset proximal muscle weak-
ness, PEO, and sensorineural hearing impairment. EMG (electromyogra-
phy) can show non-specific myopathic changes. CPK is typically
elevated. Fiber size variability, increased connective tissue, sarcoplasmic
vacuoles, RRF, COX-deficient and SDH-positive fibers, abnormally
shaped mitochondria on electron microscopy, and mtDNA depletion
are evident in skeletal muscle tissue. ETC assays usually shows low ac-
tivity of multiple complexes with complex I, III, and IV being the most
affected [75–77].
3.3.1.2. TK2-related autosomal recessive progressive external
ophthalmoplegia. TK2-related ARPEO has been reported in three individ-
uals from two families who presented in mid-adulthood with ptosis,
ophthalmoplegia, proximal muscle weakness and atrophy, facial
 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
weakness, and mild CPK elevations. One individual developed respirato-
ry failure required non-invasive ventilation. EMG showed myopathic
changes and muscle histology demonstrated fiber size variations, RRF,
COX-negative fibers, and SDH-positive fibers. Multiple mtDNA deletions
were evident in muscle tissue [78,79].
3.3.2. DGUOK-related mitochondrial DNA maintenance defects
DGK deficiency can result in early-onset hepatocerebral MDMD as-
sociated with mtDNA depletion or adult-onset myopathic MDMD asso-
ciated with multiple mtDNA deletions (Table 2).
3.3.2.1. DGUOK-related hepatocerebral mitochondrial DNA maintenance
defect. DGUOK-related hepatocerebral MDMD is an autosomal recessive
disorder. To date, it has been reported in over 100 individuals. It pre-
sents in two forms, a multiorgan disease with neonatal-onset and an
isolated hepatic disease that has an infantile- or childhood-onset. The
most comment form is the neonatal-onset multiorgan disease that usu-
ally presents in the first week of life with hypoglycemia and lactic acido-
sis. Subsequently, hepatic and neuromuscular manifestations appear in
all affected infants within weeks of birth. Neuromuscular manifesta-
tions include developmental regression, hypotonia, severe myopathy,
rotary nystagmus, and opsoclonus. Liver involvement includes jaundice,
cholestasis, hepatomegaly, and elevated transaminases. Hepatic dys-
function progresses in the majority of children causing neonatal- or in-
fantile-onset liver failure with coagulopathy, ascites, and edema. The
isolated hepatic disease form occurs in a minority of children who pres-
ent initially during infancy or childhood with jaundice, cholestasis, he-
patomegaly, and elevated transaminases. The onset of symptoms
occasionally occurs following a viral illness. Children with the isolated
hepatic disease form may exhibit mild hypotonia and proteinuria and
aminoaciduria indicating renal involvement. Other rare manifestations
include neonatal hemochromatosis with liver mtDNA depletion, and in-
fantile- or childhood-onset non-cirrhotic portal hypertension. The liver
disease in both forms is usually progressive and can lead to liver failure
in the majority of affected children. Liver failure is the most common
cause of death, and hepatocellular carcinoma is a potential
complication.
Elevated serum concentrations of tyrosine or phenylalanine on new-
born screening can be observed in the majority of affected newborns
with the multiorgan form of the disease. Serum concentrations of liver
transaminases, gammaglutamyltransferase (GGT), and conjugated
hyperbilirubinemia are typically elevated and indicate intrahepatic cho-
lestasis. Large number of affected infants exhibit elevated serum con-
centration of ferritin. Lactic acidosis and hypoglycemia are common
findings. ETC assays in liver usually demonstrate a combined deficiency
of complex I, III, and IV activities. Fibrosis, cirrhosis, cholestasis, and
giant cell hepatitis can be observed in liver biopsy. Liver electron mi-
croscopy can show an increase in the number of abnormally shaped mi-
tochondria. MtDNA depletion is evident in liver and muscle [80–83].
3.3.2.2. DGUOK-related myopathic mitochondrial DNA maintenance defect.
Biallelic pathogenic variants in DGUOK have been reported in 6 individ-
uals from 5 families with myopathic manifestations developing during
early or mid-adulthood. Common features are proximal muscle weak-
ness, ptosis, and ophthalmoplegia. Other variable features are muscle
cramps, myalgia, rhabdomyolysis, sensorineural deafness, dysphagia,
dysphonia, and neuropathy. CPK can be elevated and EMG can show
myopathic changes. Fiber size variability, RRF, COX-deficient fibers,
SDH-positive fibers, and multiple mtDNA deletions are evident in skel-
etal muscle tissue [84].
3.3.3. SUCLA2-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in SUCLA2 are associated with an
encephalomyopathic MDMD. SUCLA2-related encephalomyopathic
MDMD is an autosomal recessive disorder that, to date, has been report-
ed in 50 individuals. The median age of onset is two months, with a
1547
range from birth to six years. The vast majority of affected children pres-
ent during infancy with hypotonia and psychomotor retardation. Dysto-
nia, muscle atrophy, and sensorineural hearing impairment are
common. Less frequent neurologic manifestations include hypertonia,
choreoathetosis, ptosis, ophthalmoplegia, strabismus, and epilepsy (in-
cluding infantile spasms and generalized convulsions). Neuroimaging
typically shows basal ganglia hyperintensities, cerebral atrophy, and
leukoencephalopathy. Distinctive facial features including brachyceph-
aly, epicanthus, and upslanted palpebral fissures, have been reported.
Feeding difficulties and growth failure are common. Recurrent vomiting
and gastroesophageal reflux disease occasionally occur. Respiratory dis-
tress due to muscle weakness, obstructive sleep apnea, tracheomalacia,
and abnormal breathing has also been reported. Recurrent respiratory
infections, progressive kyphoscoliosis, contractures, hyperhidrosis, and
neonatal hypoglycemia occur occasionally. Rare manifestations include
anemia, dislocations of the hips and shoulders, irritability, and sleep
disturbance.
Methylmalonic acid (MMA) is modestly elevated in urine and blood.
Several other metabolites can be elevated in urine, including
methylcitrate, 3-methylglutaconic acid, 3-hydroxyisovaleric acid,
and Krebs cycle intermediates such as succinate, fumarate, and 2-
ketoglutarate. An acylcarnitine profile shows elevated C3
(propionylcarnitine); thus this condition can potentially be detected
by newborn screening. Plasma and CSF lactate levels are elevated in
most affected individuals. Increased fiber size variability, atrophic fibers,
intracellular lipid accumulation, COX-deficient fibers, structurally al-
tered mitochondria on electron microscopy, and mtDNA depletion are
evident in skeletal muscle tissue. ETC activity is abnormal in the major-
ity with the most common abnormalities being combined complex I and
IV deficiencies, combined complex I, III, and IV deficiencies, and isolated
complex IV deficiency. Life span is shortened, with median survival of
20 years. Approximately one third of affected individuals die during
childhood [85–87] (Table 2).
3.3.4. SUCLG1-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in SUCLG1 are associated with an
encephalomyopathic MDMD. SUCLG1-related encephalomyopathic
MDMD is an autosomal recessive disorder that, to date, has been report-
ed in 22 individuals. It can present from the prenatal period to 1 year of
age, with the majority presenting at birth. Some affected children have
prenatal manifestations including IUGR, oligohydramnios, abnormal
fetal heart rate, and congenital anomalies. The majority of affected
children demonstrate developmental delay, cognitive impairment, hy-
potonia, and muscle atrophy. Other less frequent neurological manifes-
tations include sensorineural hearing impairment, dystonia, hypertonia,
epilepsy, myoclonus, microcephaly, ptosis, and strabismus. Neuroimag-
ing typically shows basal ganglia hyperintensities, cerebral atrophy, and
leukoencephalopathy. Approximately half of affected individuals have
liver involvement manifesting as hepatomegaly, steatosis, and elevated
liver enzymes. One affected infant developed intermittent episodes of
liver failure. Failure to thrive, growth retardation, and feeding difficul-
ties, often necessitating tube feeding, occur commonly. Recurrent
vomiting and gastroesophageal reflux disease can be seen. Hypertrophic
cardiomyopathy and recurrent respiratory infections may also occur.
Respiratory distress due to muscle weakness, apnea, and abnormal
breathing has also been reported. One affected infant was reported to
have multiple congenital anomalies including cleft lip and palate, aortic
coarctation, patent ductus arteriosus (PDA), patent foramen ovale
(PFO), shortening of the left femur and both humeri, dilation of the
left renal collecting system, and an accessory left kidney. Other congen-
ital anomalies reported in single infant each are interrupted aortic arch,
polydactyly, and hypospadias. Given the small number of patients it is
unclear whether the structural birth defects are causally related. The
majority of affected children develop lactic acidosis that can be severe
and life-threatening. Five neonates were reported to present at birth
with severe metabolic acidosis and died during the neonatal period
1548 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
(fatal infantile lactic acidosis). Hypoglycemia was occasionally reported.
Other less frequent manifestations include hyperhidrosis, hypothermia,
sleeping disturbance, rhabdomyolysis, and joint contractures.
MMA is modestly elevated in urine and blood. Several other metab-
olites may be elevated in urine, including methylcitrate, 3-
methylglutaconic acid, 3-hydroxyisovaleric acid, and Krebs cycle inter-
mediates such as succinate, fumarate, and 2-ketoglutarate.
Acylcarnitine profiling can show elevated C3; thus, this condition can
similarly be potentially detected by newborn screening. Plasma and
CSF lactate levels are elevated in most affected individuals. Increased
fiber size variability, atrophic fibers, intracellular lipid accumulation,
RRF, COX-deficient fibers, structurally abnormal mitochondria on elec-
tron microscopy, and mtDNA depletion are evident in skeletal muscle
tissue. Abnormal ETC activities are present, with the most common ab-
normalities being combined complex I and IV deficiencies, combined
complex I, III, and IV deficiencies, and isolated complex IV deficiency.
Life span is shortened, with median survival of 20 months in affected
children. Two thirds of affected individuals are reported to have
died during neonatal period, infancy, or early childhood [85,88–90]
(Table 2).
3.3.5. ABAT-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in ABAT result in GABA-transaminase
(GABAT) deficiency. GABAT deficiency results in both the inhibition of
GABA metabolism and loss of mitochondrial NDPK activity in dNTP syn-
thesis. Therefore, GABA is elevated leading to dysfunctional GABAergic
system, and mitochondrial NDPK is impaired leading an
encephalomyopathic MDMD [22]. GABAT deficiency is extremely rare
with only 7 individuals from 5 families being reported since its initial
description in 1984. Affected children typically become symptomatic
during infancy with hypotonia, severe psychomotor retardation, lethar-
gy, hyperreflexia, and intractable seizures including infantile spasm and
myoclonic jerks. Variable features are a high-pitched cry, cortical visual
impairment, esotropia, dystonia, central hypoventilation, thermal
instability, accelerated height growth, obesity, and elevated growth hor-
mone. Neuroimaging can show T2 white matter signal hyperintensities,
thinning or agenesis of corpus callosum, generalized brain atrophy, and
brainstem and cerebellar atrophy. Magnetic resonance spectroscopy
(MRS) can demonstrate excess GABA in the brain. GABA,
homocarnosine, and beta-alanine can be elevated in blood and CSF.
GABAT activity is low in liver and leukocytes. Fiber size variation, in-
creased glycogen and lipid droplets, large mitochondrial of irregular
shape on electron microscopy, reduced ETC activity of complexes I, II,
III, and IV, and mtDNA depletion are evident in skeletal muscle tissue.
The disease is associated with refractory seizures and severe psychomo-
tor retardation. Half of affected children are reported to expire during
infancy or early childhood [22,91–94] (Table 2).
3.4. Defects of cytosolic nucleotide metabolism: TYMP and RRM2B
TYMP and RRM2B encode cytosolic enzymes that are involved in nu-
cleotide metabolism and are important for maintaining a balanced mi-
tochondrial nucleotide pool (see Section 2.2). Pathogenic variants in
TYMP and RRM2B result in the accumulation of cytosolic thymidine
and the reduction of cytosolic synthesis of deoxyribonucleosides, re-
spectively. Therefore, these defects result in an imbalance of the cytosol-
ic nucleotide pool. Because the mitochondrial nucleotide pool relies, in
part, on nucleotides imported from the cytosol, an imbalanced cytosolic
nucleotide pool can lead to an imbalanced mitochondrial nucleotide
pool that can impair mtDNA synthesis.
3.4.1. TYMP-related mitochondrial DNA maintenance defect
Biallelic pathogenic variants in TYMP are associated with TYMP-relat-
ed mitochondrial neurogastrointestinal encephalopathy disease
(MNGIE), which is an autosomal recessive disease with more than 120
affected individuals being reported to date. MNGIE is characterized by
gastrointestinal dysmotility and cachexia, ophthalmoplegia with ptosis,
peripheral neuropathy, myopathy, and leukoencephalopathy. The onset
of the disease is usually between the first and fifth decades, with the
mean age of onset being 18 years. Progressive gastrointestinal
dysmotility is caused mainly by enteric myopathy and develop in all af-
fected individuals. The manifestations of gastrointestinal dysmotility in-
clude gastroesophageal reflux, dysphagia, nausea, postprandial
vomiting, diarrhea, early satiety, and episodic abdominal pain and dis-
tention. Other common features are cachexia, weight loss, a thin body
habitus, reduced muscle mass, ophthalmoplegia, and ptosis. In addition,
all affected individuals develop peripheral neuropathy that is typically
demyelinating. However, about half of affected individuals also have
an axonal neuropathy. The neuropathic symptoms include paresthesias
occurring in a stocking-glove distribution and weakness that is usually
symmetric, initially distal, and affecting lower extremities more than
upper extremities. Foot drop than can be unilateral or bilateral and
clawed hand deformity can develop. Leukoencephalopathy is another
common feature; however, it is usually asymptomatic. Some individuals
may have intellectual disability, and dementia rarely occur later in the
course of the disease. Other less frequent manifestations include ane-
mia, hepatic cirrhosis with steatosis and increased transaminases,
hypogonadism, sensorineural hearing impairment, and diverticulitis.
CSF protein and plasma lactate can be elevated in affected individ-
uals, and plasma thymidine and deoxyuridine are universally high.
EMG and nerve conduction velocity testing can reveal myopathic
changes and decreased conduction velocity in motor and sensory
nerves. Neuroimaging usually show T2 white matter hyperintensities.
Mitochondrial proliferation, smooth muscle cell atrophy, and mtDNA
depletion can be found in the external layer of the muscularis propria
of the small intestine and the stomach. RRF, defects in single or multiple
ETC complexes, with the most common defect being in complex IV ac-
tivity, mMtDNA depletion, and multiple mtDNA deletions are evident
in skeletal muscle. The disease is progressive, with a poor prognosis
and the mean age of death being approximately 35 years [48,95].
(Table 2).
3.4.2. RRM2B-related mitochondrial DNA maintenance defects
Biallelic pathogenic variants in RRM2B are associated with the dis-
eases: RRM2B-related encephalomyopathic MDMD, RRM2B-related
MNGIE, and RRM2B-related ARPEO. Both the encephalomyopathic and
MNGIE diseases are associated with mtDNA depletion, whereas the
ARPEO is associated with multiple mtDNA deletions. Monoallelic patho-
genic variants can cause RRM2B-related ADPEO, which is associated
with multiple mtDNA deletions [96]. Pathogenic variants leading to
the recessive forms of the disease are predicted to be loss-of function,
while these associated with dominant inheritance are predicted to
have dominant negative effects [97,98] (Table 2).
3.4.2.1. RRM2B-related encephalomyopathic mitochondrial DNA mainte-
nance defect. RRM2B-related encephalomyopathic MDMD is the most
severe form of RRM2B-related disorders. It is an autosomal recessive
multisystem disease that, to date, has been reported in 15 individuals.
Affected children usually present during the first six months of life
with weakness, hypotonia, psychomotor delay, lactic acidosis, respira-
tory insufficiency, gastrointestinal dysmotility, failure to thrive,
nephrocalcinosis, and proximal renal tubulopathy. Seizures, sensori-
neural hearing loss, and microcephaly have also been reported. Neuro-
imaging can show cerebral atrophy and generalized central
hypomyelination. COX-deficient fibers, RRF, and mtDNA depletion are
evident in skeletal muscle tissue. ETC analysis typically shows deficien-
cies in complex I, III, IV, and V activities. The disease progresses rapidly,
leading to death in early childhood [96,99–101].
3.4.2.2. RRM2B-related mitochondrial neurogastrointestinal encephalopa-
thy. One individual has been reported with RRM2B-related MNGIE. She
was a 42 year old female who was in a good health until the age of
 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
30 years when she started to develop episodic vomiting due to gastroin-
testinal dysmotility, leading to significant weight loss. Beginning at
37 years of age, she had progressive ophthalmoplegia, ptosis, nystag-
mus, dysarthria, generalized muscle weakness unsteady gait, and loss
of deep tendon reflexes. Lactate levels were mildly elevated in plasma
and CSF. Nerve conduction studies showed demyelinating neuropathy.
Neuroimaging showed T2 hyperintensities in the basal ganglia and
periventricular and subcortical white matter. Fiber size variation, in-
creased endomysial connective tissue, RRF, SDH-positive fibers, and
mtDNA depletion were evident in muscle tissue [102].
3.4.2.3. RRM2B-related autosomal recessive progressive external
ophthalmoplegia. RRM2B-related ARPEO is a childhood disease with a
mean onset of seven years of age (range birth to 14 years). Five affected
children have been reported to date. Common features include
ophthalmoplegia, ptosis, proximal muscle weakness, fatigue bulbar dys-
function, facial weakness, ataxia, and sensorineural hearing impair-
ment. Less frequent features are endocrinopathies (hypogonadism and
hypoparathyroidism), cachexia, leukoencephalopathy, and learning dif-
ficulties. COX-deficient fibers, RRF, and multiple mtDNA deletions are
evident in skeletal muscle tissue [96,103].
3.4.2.4. RRM2B-related autosomal dominant progressive external
ophthalmoplegia. Monoallelic pathogenic variants in RRM2B have been
reported in 26 adults with ADPEO. The mean age of onset is 45 years
(range 15–70). Common features include ophthalmoplegia, ptosis,
proximal muscle weakness, ataxia, bulbar dysfunction, fatigue, and sen-
sorineural hearing impairment. Less frequent manifestations are hypo-
thyroidism, headache, facial weakness, ataxia, cognitive decline,
stroke-like episodes, cataracts, and pigmentary retinopathy. COX-defi-
cient fibers, RRF, and multiple mtDNA deletions are evident in skeletal
muscle tissue [96,103].
3.5. Defects of mitochondrial nucleotide import: SLC25A4, AGK, and MPV17
SLC25A4, AGK, and MPV17 encode proteins that play important roles
in importing nucleotides into mitochondria (see Section 2.2). Defects of
these proteins result in mitochondrial nucleotide deficiency and imbal-
ance leading to impaired mtDNA synthesis which results in mtDNA de-
pletion and multiple mtDNA deletions. Pathogenic variants in SLC25A4
result in a defective ANT1. As ANT1 is homodimer, monoallelic patho-
genic variants in SLC25A4 are expected to result in defective ANT1 di-
mers in two out of the three possible dimer combinations, while
biallelic pathogenic variants result in complete loss of ANT1 [28,104].
In addition to impaired OXPHOS and energy production, ANT1 deficien-
cy results in a shortage of ADP and its derivatives dADP and dATP. Defi-
ciency of dATP results in mitochondrial nucleotide pool imbalance [28,
105,106]. AGK deficiency results in a decrease in phosphatidic acid
and cardiolipin leading to a disturbed lipid-membrane composition
within the mitochondria. Because ANT1 is bound to the inner mem-
brane lipids phosphatidic acid and cardiolipin [29], and these lipids
are essential for proper inner-membrane ANT assembly and stabiliza-
tion, AGK deficiency can result in impaired ANT1 assembly and stability,
leading to a decrease in both the amount and activity of ANT1 in skeletal
muscle [107]. MPV17 deficiency results in decreased dNTPs in mito-
chondria, impaired mtDNA synthesis, and slowing of mtDNA replication
[31,32].
3.5.1. SLC25A4-related mitochondrial DNA maintenance defects
Biallelic pathogenic variants in SLC25A4 result in SLC25A4-related
cardiomyopathic MDMD, while monoallelic pathogenic variants cause
SLC25A4-related ADPEO, and both are associated with multiple mtDNA
deletions (Table 2).
3.5.1.1. SLC25A4-related cardiomyopathic mitochondrial DNA mainte-
nance defects. SLC25A4-related cardiomyopathic MDMD has been
1549
reported in two individuals who presented in early childhood with
easy fatigability, hypertrophic cardiomyopathy, and lactic acidosis. My-
algia, muscle wasting and weakness, elevated CPK, cataracts, obesity,
and cognitive impairment were each reported in one individual. EMG
showed myopathic changes, while RRF, COX-deficient fibers, accumula-
tion of abnormally shaped mitochondria on electron microscopy, re-
duced complex I, III, and IV activities, and multiple mtDNA deletions
were evident in skeletal muscle tissue [104,105].
3.5.1.2. SLC25A4-related autosomal dominant progressive external
ophthalmoplegia. To date, SLC25A4-related ADPEO has been reported in
10 unrelated families. Affected individuals present during adulthood,
i.e., the third to sixth decades, with ptosis and ophthalmoplegia. Easy fa-
tigability, exercise intolerance, muscle weakness, sensorineural hearing
impairment, and psychiatric illnesses have been reported in some indi-
viduals. EMG can show myopathic changes, while RRF, COX-deficient fi-
bers, accumulation of abnormally shaped mitochondria on electron
microscopy, and multiple mtDNA deletions occur in skeletal muscle tis-
sue [28,108–111].
3.5.2. Sengers syndrome
Sengers syndrome, first described in 1975, is an autosomal recessive
disease that is characterized by skeletal myopathy, hypertrophic cardio-
myopathy, lactic acidosis, congenital cataracts, and normal cognitive de-
velopment. The disease severity varies from a severe form that causes
death in infancy to a more benign form that allows for survival into
the fourth decade. Heart failure due to hypertrophic cardiomyopathy
is the typical cause of death in affected individuals. In 2002, a decrease
in both the amount and the activity of ANT1 in skeletal muscle was iden-
tified in families with Sengers syndrome; however at that time patho-
genic variants in SLC25A4 were excluded by linkage analysis [107]. In
2012, AGK was identified as the disease-causing gene for Sengers syn-
drome [30,112]. To date, 29 individuals from 27 families with AGK
biallelic pathogenic variants have been reported. The majority of affect-
ed individuals present in the neonatal period with cataracts, hypertro-
phic cardiomyopathy, and lactic acidosis. Some affected children may
present later in infancy or early childhood. About half of affected indi-
viduals had the severe form, with onset during the neonatal period
and an average survival of 4 months. Other common manifestations in-
clude muscle weakness, hypotonia, feeding difficulties, motor develop-
mental delay, easy fatigability, exercise intolerance, and respiratory
insufficiency. Other infrequent manifestations are nystagmus, esotropia,
recurrent headaches, stroke, seizures, failure to thrive, osteopenia, ar-
rhythmias, pulmonary hypertension, thrombocytopenia, and eosino-
philia. Fatty infiltration, RRF, COX-deficient fibers, combined
deficiency of ETC complexes I, II, III, and IV activities, or isolated deficien-
cies of complex I or IV activity, and mtDNA depletion occur in skeletal
muscle tissue [30,112,113] (Table 2).
3.5.3. MPV17-related mitochondrial DNA maintenance defects
Biallelic pathogenic variants in MPV17 are associated with three phe-
notypically overlapping MDMDs: MPV17-related hepatocerebral
MDMD and Navajo neurohepatopathy (NNH) are associated with
mtDNA depletion, whereas MPV17-related neuromyopathic MDMD is
associated with multiple mtDNA deletions (Table 2).
3.5.3.1. Navajo neurohepatopathy. NNH is an autosomal recessive disease
that is prevalent in the Navajo population of the southwestern United
States. It has an estimated incidence of 1 in 1600 live births in that pop-
ulation. The onset of symptoms is during infancy or early childhood.
NNH is characterized by progressive liver disease and sensorimotor
neuropathy manifested as distal weakness, areflexia, and loss of sensa-
tion that results in acral mutilation and corneal ulcerations. Children
with the classic NNH have striking neuropathic findings, with moderate
liver dysfunction reflected in elevated transaminases that can lead to
cirrhosis and liver failure. The non-classic infantile (onset before age
1550 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
6 months) and childhood (onset between 1 and 5 years) NNH pheno-
types are characterized by severe hepatic involvement and early death
from liver failure. Non-classic infantile NNH typically presents with lac-
tic acidosis, hypoglycemia, failure to thrive, jaundice, hepatomegaly,
and elevated transaminases. Liver dysfunction typically progresses to
liver failure and death during infancy or early childhood. Clinically ap-
parent neuropathy, which is typically absent at time of presentation, be-
comes evident over time, as these children demonstrate weakness,
motor delay, and areflexia during late infancy. Non-classic childhood
NNH typically presents in early childhood with an acute and aggressive
liver involvement that progresses to liver failure and death within
months. Neurological involvement is evident by the presence of motor
developmental delay and sensorimotor neuropathy.
All the NNH phenotypes are also characterized by T2 white matter
hyperintensities in neuroimaging, slow nerve conduction velocities,
and myelinated nerve fibers loss in nerve biopsies. Liver biopsy typically
shows fibrosis, cirrhosis, steatosis, and cholestasis. Reduced ETC com-
plex I, III, and IV activities, and mtDNA depletion are evident in liver tis-
sue. MPV17 sequencing reveals the founder homozygous p.Q50R
pathogenic variant in affected individuals [114,115].
3.5.3.2. MPV17-related hepatocerebral mitochondrial DNA maintenance
defect. MPV17-related hepatocerebral MDMD is an autosomal recessive
disease that has an infantile-onset. Affected children develop combined
hepatic, neurologic, and metabolic manifestations. To date, it has been
reported in about 50 individuals. All affected individuals exhibit
hepatic manifestations which include elevated transaminases,
hyperbilirubinemia, jaundice, cholestasis, coagulopathy, and hepato-
megaly. The liver disease is progressive and liver failure usually de-
velops during infancy or early childhood. Almost all affected
individuals exhibit neurologic manifestations, including hypotonia,
developmental delay, muscle weakness, and sensorimotor neuropathy,
which usually appear in early childhood as muscle wasting and
weakness, hyporeflexia, and loss of sensation in the hands and feet.
Other less common neurologic manifestations include ataxia, epilepsy,
microcephaly, dystonia, subdural hematomas, and cerebrovascular
infarctions. Poor feeding, failure to thrive, and metabolic derangements in-
cluding lactic acidosis and early-onset hypoglycemia are common. Other
infrequent manifestations include gastrointestinal dysmotility (cyclic
vomiting, gastroesophageal reflux, and diarrhea), endocrinopathies
(hypoparathyroidism and growth hormone deficiency), and renal
tubulopathy.
Neuroimaging can show cerebral and cerebellar atrophy and T2
white matter hyperintensities. Liver biopsy may show fatty infiltrate, fi-
brosis, cirrhosis, cholestasis and cirrhosis. Generalized fiber atrophy,
fatty infiltrate, and COX-deficient fibers are seen in skeletal muscle tis-
sue. MtDNA depletion is observed in liver and muscle tissues. ETC assays
usually reveal decreased multiple complexes (I, III, and IV) activities in
liver and skeletal muscle. Liver disease is typically progressive and
liver failure usually develops. Without liver transplantation death oc-
curs because of progressive liver failure during infancy or early child-
hood in the majority of affected children [116,117].
3.5.3.3. MPV17-related neuromyopathic mitochondrial DNA maintenance
defect. MPV17-related neuromyopathic MDMD has been reported in
two individuals who presented during adolescence or young adulthood
with sensorimotor neuropathy manifesting as distal muscle atrophy
and weakness, hyporeflexia, and distal numbness and sensory loss.
Myopathic features in one individual were exercise intolerance, proxi-
mal muscle weakness, ptosis, ophthalmoplegia, and elevated CPK;
whereas the other individual had proximal muscle weakness and diffi-
culty ambulating. Additional features, including sensorineural hearing
loss, gastrointestinal dysmotility, and parkinsonism, were observed in
one of the two affected individuals. Neuroimaging in one showed T2
white matter hyperintensities, while RRF, COX-deficient fibers, and
multiple mtDNA deletions were observed in skeletal muscle tissue
[118,119].
3.6. Defects in mitochondrial dynamics: OPA1, MFN2, and FBXL4
OPA1, MFN2, and FBXL4 encode proteins involved in mitochondrial
fusion (see Section 2.3). Pathogenic variants in OPA1 and MFN2 result
in impaired mitochondrial fusion leading to fragmented mitochondria
and impaired energy production. In addition, failure of fusion results
in impaired intramitochondrial content mixing leading to variations in
protein content within the mitochondrial populations. This heterogene-
ity in mitochondrial proteomes can result in an unbalance of mitochon-
drial proteins, including the enzymes needed for mtDNA synthesis, for
which precise stoichiometries are important for accurate and efficient
mtDNA synthesis. Therefore, defects in fusion can result in impaired
mtDNA synthesis leading to mtDNA depletion and multiple mtDNA de-
letions [33]. In addition, as OPA1-exon4b anchors the nucleoids to the
mitochondrial inner membranes, allowing for their even distribution
and promoting mtDNA replication, OPA1 defects can also result in the
inhibition of mtDNA replication and a distorted nucleoid distribution.
The disruption of the normal distribution of nucleoids in the mitochon-
drial network results in “clumpy” or aggregated nucleoids [34]. Defec-
tive FBXL4 results in dysfunctional mitochondrial respiration and
energy production, impaired mtDNA maintenance leading to mtDNA
depletion, and a disturbance of the mitochondrial network leading to
mitochondrial fragmentation and clumpy nucleoids [35–37].
3.6.1. OPA1-related mitochondrial DNA maintenance defects
Monoallelic pathogenic variants in OPA1, causing either
haploinsufficiency or a dominant negative effect, are associated with
OPA1-related autosomal dominant optic atrophy (ADOA). Biallelic path-
ogenic variants are associated with Behr syndrome and OPA1-related
encephalomyopathic MDMD (Table 3).
3.6.1.1. OPA1-related autosomal dominant optic atrophy. OPA1-related
ADOA is believed to be the most common cause of hereditary optic neu-
ropathy, with an estimated prevalence of 1:50,000. Affected individuals
usually present at a median age 5 years (between 4 and 6 years) with
decreased visual acuity. However, in some individuals visual acuity
may remain unaffected until early adulthood then symptoms appear
usually in the third decade. Visual acuity usually declines slowly with
age and the visual impairment is usually moderate, but ranges from le-
gally blind to mild visual impairment. The main feature of ADOP is optic
atrophy, which is bilateral, generally symmetric, and appears as tempo-
ral pallor of the optic disc implying the loss of central retinal ganglion
cells. The vision loss is typically progressive and irreversible, and occa-
sionally asymmetric. It is also associated with visual field defects and
color vision defects that can be either blue-yellow or red-green loss. Ad-
ditional extra-ophthalmologic abnormalities are observed in a minority
of affected individuals. Sensorineural hearing impairment that ranges
from severe and congenital to mild and subclinical is the most frequent
extraocular feature observed. Some individuals develop proximal my-
opathy, exercise intolerance, myalgia, muscle weakness, ataxia, axonal
sensorimotor neuropathy, ptosis, and ophthalmoplegia. These features
develop from the third decade of life onwards. RRF, COX-deficient fibers,
subsarcolemmal accumulation of abnormal mitochondria, and multiple
mtDNA deletions can be observed in skeletal muscle tissue. Penetrance
varies among different families and different pathogenic variants, and
can range from 40 to 100% [120–122].
3.6.1.2. Behr syndrome. Behr syndrome, which was first described in
1909, is characterized by the association of early-onset optic atrophy
with spinocerebellar degeneration resulting in ataxia, pyramidal signs,
sensory neuropathy, and developmental delay. Biallelic pathogenic var-
iants in OPA1 have been described in 7 children with Behr syndrome.
These children were identified during infancy or early childhood with
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optic atrophy, ataxia, and sensory axonal neuropathy. Other variable
features include gastrointestinal involvement (constipation, vomiting,
and dysphagia), developmental delay, nystagmus, deafness, and spas-
ticity. Neuroimaging typically shows cerebellar vermian atrophy, with
periventricular white matter changes in some affected children. Some
parents of these children who are heterozygous for the OPA1 pathogenic
variants demonstrated optic atrophy while others were asymptomatic,
consistent with the notion that ADOP has reduced penetrance [123–
125].
3.6.1.3. OPA1-related encephalomyopathic mitochondrial DNA mainte-
nance defect. Two sisters have been reported with homozygous OPA1
pathogenic variants and encephalomyopathic MDMD. By early infancy,
they exhibited developmental delay, muscle weakness and wasting,
poor feeding, failure to thrive, hypertrophic cardiomyopathy, hyperto-
nia and opisthotonic posturing, optic atrophy, sensorineural hearing im-
pairment, and lactic acidemia. Both infants died at 10 and 11 months
because of apneic episodes. ETC activities in muscle were reduced for
all complexes, with complex I and IV being the most affected and com-
plex II being relatively preserved. MtDNA depletion was also observed
in skeletal muscle tissue [126].
3.6.2. MFN2-related mitochondrial DNA maintenance defects
Monoallelic pathogenic variants in MFN2 results in two overlapping
phenotypes: Charcot-Marie-Tooth neuropathy type 2A (CMT2A) and
MFN2-related autosomal dominant optic atrophy (ADOA) (Table 3).
3.6.2.1. Charcot-Marie-Tooth neuropathy type 2A. With the overall preva-
lence of CMT2 being ~ 1:10,000 and CMT2A constituting ~ 10–30% of
CMT2, the incidence of CMT2A is estimated at 1–3:100,000 [127,128].
CMT2 is an axonal non-demyelinating peripheral neuropathy that has
an onset in the first or second decade in most affected individuals. It is
characterized by distal muscle weakness and atrophy, normal or near-
normal nerve conduction velocities, and mild sensory loss. Distal muscle
weakness and atrophy typically begin in the lower extremities and may
progress to involve the upper extremities. Foot drop or foot weakness
may be the initial presentation, and pes cavus can occur. Although the
motor signs of muscle weakness and atrophy are the predominant fea-
tures, sensory involvement including mild sensory loss in the feet and
absent tendon reflexes are common. Postural tremor is also common.
Affected individuals presenting before the age of 10 years usually have
a more severe disability than those with later onset. Individuals with
early onset may also have additional features including optic atrophy,
sensorineural hearing impairment, hoarse voice, proximal weakness,
scoliosis, and contractures. The disease has a progressive course, and
some individuals lose independent ambulation whereas most do not.
However, life span is typically not reduced. Neuroimaging can show
periventricular and subcortical white matter lesions in a minority of af-
fected individuals. Loss of myelinated nerve fibers especially large fi-
bers, mitochondrial abnormalities and, rarely, onion bulb structure can
be observed in nerve biopsies [128].
3.6.2.2. MFN2-related autosomal dominant optic atrophy. MFN2-related
ADOA was reported in 12 individuals from a large three generation fam-
ily. Optic atrophy typically starts during early childhood and presents as
vision impairment, scotomas, and color vision deficiency. Neuromuscu-
lar symptoms have an onset typically between 10 and 30 years of age,
and include sensorimotor axonal neuropathy, proximal and distal mus-
cle weakness, and hyporeflexia. Other variable features include ataxia,
pyramidal signs, sensorineural hearing impairment, cataracts, myalgia,
and cognitive impairment. Serum lactate and CPK can be elevated. Neu-
roimaging shows periventricular white matter abnormalities and cere-
bral atrophy in some affected individuals. COX-deficient fibers, RRF,
and multiple mtDNA deletions are evident in skeletal muscle tissue
[129].
1551
3.6.3. FBXL4-related encephalomyopathic mitochondrial DNA maintenance
defects
FBXL4-related encephalomyopathic MDMD is an autosomal reces-
sive disorder that to date has been reported in 40 individuals. Age of
onset ranges from birth to 2 years (mean 4 months). Affected children
commonly present with hypotonia, psychomotor delay, failure to thrive,
feeding difficulties, growth failure, and lactic acidosis. Less common
manifestations include cardiomyopathy (left ventricular non-compac-
tion or hypertrophic), seizures, microcephaly, sensorineural hearing im-
pairment, ataxia, dystonia, choreoathetosis, and hypoglycemia.
Infrequent manifestations include congenital cataracts, optic atrophy,
ptosis, stroke-like episodes, polyhydramnios, arrhythmias, pulmonary
hypertension, neutropenia, and lymphopenia. Dysmorphic features are
common and include prominent forehead, thick and diffuse eyebrows,
synophris, short upslanted palpebral fissures, a broad nasal root with
depressed nasal bridge, a smooth philtrum, and low-set malformed
ears. Neuroimaging can show T2 white matter hyperintensities, basal
ganglia and brainstem lesions, cerebral cysts, cerebral atrophy,
brainstem atrophy, and cerebellar hypoplasia. CPK and ammonia can
be elevated. COX-deficient fibers, decreased activities of all ETC en-
zymes, particularly I and IV, and mtDNA depletion occur commonly in
skeletal muscle tissue. More than a third of affected children die during
childhood. All long-term survivors develop severe psychomotor retar-
dation [35–37,130–134] (Table 3).
4. Management considerations
Although many of the MDMDs are severe disorders with a poor
prognosis, no curative therapy is available for any of these disorders.
The majority of MDMDs are multiorgan disorders, therefore, affected in-
dividuals should have a comprehensive evaluation to assess the degree
of involvement of different organs, including the hepatic, neuromuscu-
lar, gastrointestinal, renal, and cardiovascular systems. Management of
MDMDs should involve a multidisciplinary team that aims to provide
supportive care and symptomatic treatment for the complications asso-
ciated with these disorders.
4.1. Assessment of the disease extent
All MDMDs are associated with neuromuscular manifestations
hence a comprehensive neurologic examination including developmen-
tal and cognitive assessments are needed. The degree of neurological in-
volvement can be assessed using the following diagnostic modalities:
neuroimaging (mainly brain magnetic resonance imaging) to deter-
mine the degree of central nervous system involvement, nerve conduc-
tion velocity to assess the involvement of peripheral nervous system,
EMG to evaluate myopathy, electroencephalography when seizures
are suspected, and a swallowing study if bulbar signs are present. Thor-
ough hearing and ophthalmologic evaluations are also needed [5,38].
The degree of liver involvement in MDMDs with hepatopathy can be
evaluated by liver function tests, including albumin, liver transami-
nases, bilirubin, coagulation profile, ammonia, and fasting blood glu-
cose. A hepatic ultrasound can help in the assessment of liver size and
the presence of fibrosis or masses. Alpha fetoprotein (AFP) can be mea-
sured to screen for hepatocellular carcinoma and can be part the evalu-
ation [38,83].
Gastrointestinal evaluation for individuals with MNGIE disease de-
pends on their symptoms. In addition to gastrointestinal consultation,
these evaluation include abdominal imaging X-ray and computed to-
mography, esophagogastroduodenoscopy, upper gastrointestinal con-
trast radiography, sigmoidoscopy, liquid phase scintigraphy, small
bowel manometry, and antroduodenal manometry. These studies can
demonstrate gastroparesis, hypoperistalsis, reduced amplitude of
small bowel contractions, dilated duodenum, and diverticulosis [48].
Cardiac involvement can be assessed by echocardiogram and elec-
trocardiogram. Assessment of blood gases and pulmonary function
1552 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
tests are needed to assess for respiratory insufficiency in individuals
with myopathy. Sleep polysomnogram is helpful in evaluating central
or obstructive apnea or hypopnea. Swallowing assessment and nutri-
tional evaluation can be needed in individuals with feeding difficulty
and growth retardation. Urine analysis, amino acids, and electrolytes
can be performed to detect any renal tubulopathy.
4.2. Treatment of manifestations
Physical, occupational, and speech therapy can help maintaining
neurologic function as long as possible. Physical therapy and occupa-
tional therapy can help preserve mobility and prevent joint contrac-
tures. Antiepileptic medications are used to control seizures, however,
refractory epilepsy may be very difficult to control, and the use of
high-dose antiepileptic medications and treatment with multiple anti-
convulsant drugs are often needed to control refractory seizures. In ad-
dition to seizures, other non-epileptic movement, e.g. myoclonus can
occur in MDMDs. Benzodiazepines can be used to reduce the severity
of these abnormal movements and also help in managing seizures and
improving spasticity. Chorea and athetosis may also cause pain that
can be alleviated by muscle relaxants and pain medications, including
narcotics. Dopaminergic medication such as levodopa-carbidopa and
tetrabenazine can also be used to treat some movement disorders. Neu-
ropathic symptoms can be relieved by amitriptyline, nortriptyline, and
gabapentin. Ptosis blepharoplasty can be needed for cosmesis and
symptomatic relief, and cochlear implantation for sensorineural hearing
loss [38,48,96].
Failure to thrive and feeding difficulties may require consultation
with a gastroenterologist, nutritional support by an experienced dieti-
tian, occupational therapy to improve oromotor functions, and the use
of a nasogastric tube or gastrostomy tube feedings. Standard treatments
for liver failure can include, reduction in dietary protein, correction of
coagulopathy, frequent or continuous feeding to prevent hypoglycemia,
and treatment of hyperammonemia when present. Management of gas-
trointestinal dysmotility includes nutritional support using bolus feed-
ings, gastrostomy tube placement, and total parenteral nutrition, a
trial of domperidone for nausea and vomiting, antibiotic therapy for in-
testinal bacterial overgrowth which is a complication of dysmotility, ce-
liac plexus block which can help in reducing pain and increasing
gastrointestinal motility, and splanchnic nerve block which can help in
reducing abdominal pain [38,48].
Individuals with respiratory insufficiency may require aggressive
antibiotic treatment of chest infections, chest physiotherapy, and artifi-
cial ventilation that could include assisted nasal ventilation or intuba-
tion with the use of a tracheostomy and ventilator [75].
4.3. Nutritional modulation and cofactors
Formulas enriched with medium-chain triglycerides can provide
better nutritional support for children with cholestasis than formulas
containing mainly long-chain triglycerides. Avoidance of fasting by fre-
quent or continuous feeding can be required to prevent hypoglycemia.
In addition, the use of uncooked cornstarch can help in reducing symp-
tomatic hypoglycemia in children with hepatocerebral MDMDs. The use
of cornstarch may also slow the progression of the liver disease in
MPV17-related hepatocerebral MDMD [5,80,135,136].
CSF folate may be deficient in AHS. Therefore, it has been suggested
to test CSF folate and treat those with deficiency or apply empiric ther-
apy with folinic acid. Treatment with oral folinic acid results in increas-
ing CSF folate levels and improving the frequency of seizures, and
communicative abilities are reportedly improved [38,137]. Succinate
and ubiquinone were reported to slow the progression of liver dysfunc-
tion in children with MPV17-related MDMD [138]. Creatine
monohydrate, levocarnitine, B vitamins, coenzyme Q10, and antioxi-
dants, such as vitamin C, vitamin E, and alpha lipoic have been used as
mitochondrial supplements. These cofactors have been used in
MDMDs; however, there is very limited evidence for their effectiveness
[139,140].
4.4. Liver and stem cell transplantation
Although liver transplantation remains the only treatment option
for liver failure in hepatocerebral MDMD, liver transplantation in
mitochondrial hepatopathy is controversial, largely because of the
multiorgan involvement [141]. Liver transplantation is not recommend-
ed in children with AHS because liver transplantation does not change
the rapid progression of the neurological manifestations. On the other
hand, liver transplantation in adults with POLG-related MDMD who
have an acceptable quality of life may be beneficial [38]. Although sur-
vival after liver transplantation for DGUOK-related MDMD is lower
than survival after liver transplantation for other diseases, a significant
fraction of affected individuals survived more than five years despite
initial neurological abnormalities. Nevertheless, the decision to perform
liver transplantation for individuals with this disease remains difficult
because neurological manifestations may occur or worsen after liver
transplantation despite their absence before transplantation [142].
Liver transplantation outcomes for MPV17-related MDMD have not
been satisfactory, because approximately half of the children
transplanted died in the post-transplantation period because of
multiorgan failure and/or sepsis [143].
Allogeneic stem cell transplantation for TYMP-related MNGIE pro-
duces nearly complete biochemical correction of the thymidine and
deoxyuridine imbalances in blood and some clinical improvements.
However, because of the high morbidity and mortality, this procedure
is not generally recommended to treat individuals with TYMP-related
MNGIE [48].
4.5. Medications to avoid
Certain medications need to be avoided in MDMDs. Valproic acid,
which is commonly used for seizures, can inhibit mitochondrial respira-
tory chain and beta-oxidation. It is very important to avoid this medi-
cine in treating seizures in MDMDs, particularly POLG-related
disorders, because of the risk of precipitating and accelerating liver dis-
ease [144,145]. Metformin, which is used to treat diabetes, needs to be
avoided because of the theoretic risk of exacerbating metabolic acidosis
[146]. Chloramphenicol and tetracyclines should also be avoided be-
cause they inhibit mitochondrial protein synthesis. Prolonged use of
the antibiotic linezolid also needs to be avoided because of its associa-
tion with peripheral and optic neuropathy and lactic acidosis due to mi-
tochondrial toxicity. Zidovudine can interfere with mtDNA replication
and therefore its use carries the risk of inducing mitochondrial disease
[48,96].
5. Conclusions
MDMDs are a group of diseases caused by pathogenic variants in nu-
clear genes involved in mtDNA maintenance and are characterized by
mtDNA depletion and/or multiple mtDNA deletions in affected organs.
Defective mtDNA results in organ dysfunction due to insufficient syn-
thesis of ETC components resulting in inadequate energy production
to meet the needs of affected organs. MDMDs are associated with a
broad phenotypic spectrum. The nuclear genes that are known to be
crucial for mtDNA maintenance in which pathogenic variants have
been linked to MDMDs include: genes encoding enzymes of mtDNA
replication machinery (mtDNA polymerization: POLG, POLG2, TWNK
and TFAM; and nucleases removing flap intermediates: RNASEH1,
MGME1, and DNA2), genes encoding proteins that function in maintain-
ing a balanced mitochondrial nucleotide pool (mitochondrial salvage
pathway: TK2, DGUOK, SUCLG1, SUCLA2, and ABAT; cytosolic nucleotide
metabolism RRM2B and TYMP; and mitochondrial nucleotide import:
SLC25A4, AGK, and MPV17), and genes encoding proteins involved in
 A.W. El-Hattab et al. / Biochimica et Biophysica Acta 1863 (2017) 1539–1555
mitochondrial fusion (OPA1, MFN2, and FBXL4). No curative therapy is
available for any of these disorders, and treatment remains largely
symptomatic.
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