Molecular Genetics and Metabolism 110 (2013) 3541

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Molecular Genetics and Metabolism

journal homepage: www.elsevier.com/locate/ymgme

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Diagnosis of mitochondrial myopathies

Margherita Milone a, Lee-Jun Wong b,
a
b

Department of Neurology, Neuromuscular Division, Mayo Clinic, Rochester, MN, USA

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

a r t i c l e

i n f o

Article history:
Received 23 June 2013
Received in revised form 10 July 2013
Accepted 10 July 2013
Available online 17 July 2013
Keywords:
Mitochondrial DNA
Mitochondrial myopathy
Progressive external ophthalmoplegia
Oxidative phosphorylation
NGS diagnosis of mitochondrial disorders
Molecular diagnosis

a b s t r a c t
Mitochondria are ubiquitous organelles and play crucial roles in vital functions, most importantly, the oxidative
phosphorylation and energy metabolism. Therefore, mitochondrial dysfunction can affect multiple tissues, with
muscle and nerve preferentially affected. Mitochondrial myopathy is a common clinical phenotype, which is
characterized by early fatigue and/or xed muscle weakness; rhabdomyolysis can seldom occur. Muscle biopsy
often identies signs of diseased mitochondria by morphological studies, while biochemical analysis may identify
respiratory chain deciencies. The clinical, morphological and biochemical data guide molecular analysis. Being
the mitochondrial function under the control of both mitochondrial DNA and nuclear DNA, the search for mitochondrial DNA mutations and mitochondrial DNA quantitation, may not be sufcient for the molecular diagnosis
of mitochondrial myopathies. Approximately 1500 nuclear genes can affect mitochondrial structure and function
and the targeting of such genes may be necessary to reach the diagnosis. The identication of causative molecular
defects in nuclear or mitochondrial genome leads to the denite diagnosis of mitochondrial myopathy.
2013 Elsevier Inc. All rights reserved.

Contents
1.
2.
3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clinical features of mitochondrial myopathies . . . . . . . . . . . . . . . . . . . . . .
Diagnosis of mitochondrial myopathies . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Non-invasive diagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Biochemical analysis in blood and urine . . . . . . . . . . . . . . . . .
3.1.2.
Electrodiagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Muscle imaging studies . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4.
Exercise test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Invasive diagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Morphological studies . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Biochemical studies
. . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Molecular studies of the mtDNA . . . . . . . . . . . . . . . . . . . . .
The ultimately denite molecular diagnosis: identication of causative mutations . . . . . .
4.1.
Mutations in mtDNA as primary defects . . . . . . . . . . . . . . . . . . . . . .
4.2.
Mutations in nuclear genes . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Mutations in nuclear genes causing secondary mtDNA defects . . . . . . .
4.2.2.
Mutations in nuclear genes as the primary cause of mitochondrial dysfunction
4.3.
Advantage of denite diagnosis . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2.
Genetic counseling . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3.
Prenatal diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ATP, adenosine triphosphate; CoQ10, coenzyme Q10; CK, creatine kinase; EMG, Electromyography; Pi, inorganic phosphate; mtDNA, mitochondrial DNA; nDNA, nuclear
DNA; (PCr), phosphocreatine; 31P-MRS, 31-phosphorous magnetic resonance spectroscopy; PEO, progressive external ophthalmoplegia; RRF, ragged-red bers.
Corresponding author at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, NAB 2015, Houston, TX 77030, USA.
E-mail address: ljwong@bcm.edu (L.-J. Wong).
1096-7192/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ymgme.2013.07.007

36

M. Milone, L.-J. Wong / Molecular Genetics and Metabolism 110 (2013) 3541

Conict of interest
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

1. Introduction
Muscle contraction and relaxation depend on energy derived from
the hydrolysis of adenosine triphosphate (ATP). Several biochemical
processes provide ATP, including oxidative phosphorylation, glycogen
and glucose metabolism, lipid metabolism, purine nucleotide cycle,
and creatine kinase (CK)-dependent reaction of phosphocreatine with
adenosine diphosphate [1]. Glycogen, glucose and free fatty acids
provide fuel for muscle energy metabolism [2] and oxidative phosphorylation is the principal method for the synthesis of ATP. Oxidative
phosphorylation occurs within the mitochondria, which, therefore,
plays a crucial role in energy metabolism.
Oxidative phosphorylation is accomplished through 5 multi-subunit
transmembrane complexes and 2 electron carriers, coenzyme Q10 and
cytochrome c, which transport electrons between complexes [3].
Thirteen subunits only of the complexes are encoded by mitochondrial
DNA (mtDNA), while the other subunits and the assembly factors are
nuclear DNA (nDNA)-encoded. Indeed, mtDNA contains only 37
genes, 24 encoding for the RNA apparatus (22 tRNA and 2 rRNA) and
13 for the subunits of the respiratory chain complexes I, III, IV and V.
Complex II is entirely encoded by nDNA. In addition to oxidative
phosphorylation, mitochondria play essential roles in other vital
functions, such as the modulation of calcium signaling, cellular redox
balance and apoptosis [4]. Several hundreds of nuclear genes are
required for the correct mitochondrial function. Through fusion and
ssion, mitochondria preserve their quality, efciency and cellular
distribution, warranting muscle cell integrity [5]. Failure to maintain
mitochondrial function results in failure to generate energy and
increased free-radical production, leading to disease [6]. Being the
mitochondrial function under the control of a dual genome, the maternally inherited mtDNA and the Mendelian inherited nDNA, mitochondrial diseases are potentially inherited with maternal, autosomal
dominant or recessive or X-linked modality.
2. Clinical features of mitochondrial myopathies
Mitochondria are ubiquitous organelles and therefore mitochondrial
dysfunction can affect multiple tissues. Mitochondrial myopathy is a
well-recognized feature of mitochondrial dysfunction. Mitochondrial
myopathy commonly manifests with exercise intolerance and premature fatigue. Muscle weakness occurs, but early fatigue is often out of
proportion to the degree of weakness. The myopathy may selectively
affect the extraocular muscles (progressive external ophthalmoplegia,
PEO), and/or extend to bulbar, limb and axial muscles. Limb muscle

Table 1
Mitochondrial genes that can result in isolated mitochondrial myopathy.
mtDNA
genes

Protein

Respiratory
chain complex

Phenotype

Reference

MTCYB

Cytochrome b

Complex III

[8,9]

MT-CO1

Cytochrome c oxidase
subunit I
Cytochrome c
oxidase subunit II
Cytochrome c
oxidase subunit III

Complex IV

Limb myopathy,
rhabdomyolysis
Limb myopathy,
rhabdomyolysis
Limb myopathy,
rhabdomyolysis
Limb myopathy,
rhabdomyolysis
Limb myopathy,
rhabdomyolysis

MT-CO2
MT-CO3
tRNAs

Complex IV
Complex IV

[10]
[11]

weakness is usually proximal but occasionally distal muscles are selectively involved and the clinical phenotype consists of distal myopathy
[7]. Recurrent rhabdomyolysis and myoglobinuria are rare in mitochondrial myopathy but have been described in sporadic cases of isolated
myopathy with mutations in mtDNA genes encoding cytochrome
b (MTCYB) of complex III [8,9], cytochrome c oxidase subunits I
(MT-CO1) [10], II (MT-CO2) [11] and III (MT-CO-III) of complex
[12], and tRNA [13] (Table 1). Resting lactic acidosis is often present
in these cases. The pathogenesis of rhabdomyolysis in mitochondrial
myopathies has remained indeterminate and there has been no
correlation between the severity of the oxidative defect and the
rhabdomyolysis [13]. Exercise-induced muscle contractures, typical
of glycolytic disorders, are not features of mitochondrial myopathies.
The myopathy can be the sole manifestation of mitochondrial
dysfunction or a facet of a multisystem disease (encephalopathy,
peripheral neuropathy, epilepsy, stroke-like events, gastrointestinal
dysmotility, diabetes, etc.) which increases the clinical suspicion
for a mitochondrial disease. For example, the complete clinical
spectrum of mitochondrial encephalomyopathy, lactic acidosis and
stroke-like events (MELAS) is often highly suggestive of a mitochondrial cytopathy, although matrilineal relatives of MELAS patients
may be oligosymptomatic and may lack the myopathy as well as
other clinical features. The clinical phenotypes are often genetically
heterogeneous, therefore, a MELAS-like presentation can be the
result of a mtDNA point mutation or of POLG mutations, or others.
Occasionally, unique phenotypes are highly suggestive of the causative gene, as in the case of the combined myopathy, lactic acidosis
and sideroblastic anemia due to YARS2 mutations [14].
Among the mitochondrial myopathies, it is of relevance to mention
the myopathic form of primary coenzyme Q10 (CoQ10) deciency
because patients improve with CoQ10 supplementation. CoQ10 is an
essential electron carrier from complexes I and II to complex III of the
mitochondrial respiratory chain and an antioxidant; mutations in
genes involved in its biosynthesis can result in a pure myopathy that
manifests with myalgia, muscle weakness, myoglobinuria, and hyperCKemia or multisystem disease [1517].

Table 2
Nuclear genes resulting in mitochondrial myopathy, in isolation or as part of multisystem
disease.
nDNA genes

Protein

mtDNA

Reference

POLG

mtDNA Pol, catalytic subunit

[73,77,78]

POLG2
C10ORF2
ANT1
OPA1
RRM2B
TK2

mtDNA Pol, accessory subunit

Mitochondrial helicase TWINKLE
Adenine nucleotide translocase 1
Optic atrophy 1
Ribonucleotide reductase p53R2
Thymidine kinase 2

DNA2
SUCLA2
EARS2

Nuclease/helicase
Succinate-CoA ligase, subunit
Mitochondrial glutamyl-tRNA
synthetase
Mitochondrial tyrosyl-tRNA
Electron transfer avoprotein
dehydrogenase
BCL-6 corepressor-like protein 1

Multiple deletions or
depletion
Multiple deletions
Multiple deletions
Multiple deletions
Multiple deletions
Multiple deletions
Multiple deletions or
depletion
Multiple deletions
Depletion
Normal

[27]
[94]
[95]

Normal

[14]
[96]

YARS2
ETFDH

[12]
BCORL1a
[13]

[19,90]
[21]
[18]
[20,91,92]
[23,93]
[22,2426]

Depletion

Pol, polymerase gamma.

a
Reported at the 2013 Mitochondrial Medicine meeting by A. Suomalainen.

M. Milone, L.-J. Wong / Molecular Genetics and Metabolism 110 (2013) 3541

Independently from the presence or absence of other tissue involvement, mitochondrial myopathies can be the result of primary mtDNA
mutations (point mutations or rearrangements) or nDNA mutations
(Table 2). Mutations arising postzygotically in the mtDNA of myogenic
stem cells manifest, as expected, with pure myopathy and therefore
can be detected only in muscle [10]. Isolated mitochondrial myopathies
due to nDNA mutations affecting mitochondrial function commonly
manifest as PEO and are accompanied by multiple mtDNA deletions in
muscle. To date, isolated PEO has been associated with mutations in
the catalytic and accessory subunits of the mtDNA polymerase gamma
(POLG and POLG2), mitochondrial helicase TWINKLE (C10ORF2),
adenine nucleotide translocase type 1 (ANT1), ribonucleotide reductase
p53R2 (RRM2B), and thymidine kinase 2 (TK2) [1823]. However,
isolated limb myopathy, even sparing extraocular muscles, can represent the sole clinical manifestation of nuclear gene defects affecting
mitochondrial function. Examples of such myopathies are those
described as a result of POLG and TK2 mutations, often, but not always,
accompanied by mtDNA depletion [7,2426], or secondary to DNA2
mutations [27].

37

myopathy patients compared to healthy controls. However, while the

specicity of the 31P-MRS ndings can be as high as 100%, its sensitivity
is low resulting in limited diagnostic strength of 31P-MRS [29].
3.1.4. Exercise test
A surrogate marker of oxidative phosphorylation integrity in muscle
is the level of O2 extraction from blood during exercise, measured as
systemic arteriovenous oxygen (a-vO2) difference, and can provide
in vivo information on mitochondrial function. The reduced capacity
to increase O2 extraction from blood is considered the hallmark of
myopathies with defects in oxidative phosphorylation [30]. As a consequence of this biochemical phenomenon, low levels of exercise lead to
marked tachycardia and dyspnea due to increased cardiac output and
ventilation trespassing the capacity of the muscle to use the increased
oxygen deliver occurring with the physiological responses.
3.2. Invasive diagnostic tests
Muscle biopsy provides tissue for morphological, biochemical and
molecular studies of mitochondrial myopathies.

3. Diagnosis of mitochondrial myopathies

3.1. Non-invasive diagnostic tests
3.1.1. Biochemical analysis in blood and urine
Resting and exercise-induced increases of blood lactate are an easy
screening tool for mitochondrial myopathy but lactic acidosis is not
always present. The blood lactate/pyruvate ratio is an indicator of the
cytosolic oxido-reduction state and may increase in inborn errors of
the mitochondrial respiratory chain [28]. CK values are normal or mildly
elevated, unless measured in the setting of rhabdomyolysis. In the latter
case, serum and urine myoglobin measurement might be informative.
3.1.2. Electrodiagnostic tests
Electromyography (EMG) may identify myopathic motor unit
potentials and early recruitment, which are nonspecic signs of myopathy, whereas the electrodiagnostic detection of a subclinical peripheral
neuropathy, when present, would provide a clue for a more systemic
disease and might indirectly suggest a mitochondrial disease. However,
EMG can also be normal in mitochondrial myopathy, especially when
the myopathy is restricted to the extraocular muscles.
3.1.3. Muscle imaging studies
31
-Phosphorous magnetic resonance spectroscopy (31P-MRS) is an
available technique to study mitochondrial function in vivo by measuring phosphocreatine (PCr) kinetics, ATP, and inorganic phosphate
(Pi) in muscle energy metabolism. 31P-MRS may show a lower resting
PCr/Pi ratio and lower leg muscle Pi recovery in mitochondrial

3.2.1. Morphological studies

Histological signs of mitochondrial dysfunction include ragged-red
bers (RRF) in Gomori trichrome stain and ragged-blue bers in succinate dehydrogenase stain, as a result of diseased mitochondrial aggregates in the subsarcolemmal regions of muscle bers. Frequently,
ragged-red bers are depleted of cytochrome c oxidase (complex IV)
by histoenzymatic reaction. However, mtDNA mutations in cytochrome
b, tRNA genes resulting in MELAS or mtDNA-encoded complex I
subunits are often associated with preserved cytochrome c oxidase
reactivity in RRF (Fig. 1) [9,3133]. In general, 2% of RRF in skeletal
muscle is a diagnostic criterion for mitochondrial myopathy in adults
but RRF may lack in children with mitochondrial cytopathy [34]. RRF,
ragged-blue-bers and cytochrome c oxidase negative bers should be
correlated with the coexisting pathological ndings, clinical phenotype
and clinical history, as they are not specic for an inherited mitochondrial myopathy. Indeed, RRF can develop with normal aging matching
the physiological decline of mitochondrial function and accumulation
of mtDNA deletions [35]. Histological evidence for mitochondrial
dysfunction can be associated with drug toxicity, such as antiretroviral
agents and statins [36], and with non-mitochondrial myopathies, such
as inammatory myopathies [37]. Increased number of lipid droplets
is present in the muscle biopsy of subjects with CoQ10 deciency.
Ultrastructural studies by electron microscopy provide little insights
into mitochondrial myopathy. Abnormal cristae and intracristal crystalloid inclusions can be observed not only in mitochondrial myopathies
but also in other myopathies and are a nonspecic indicator of defective
mitochondrial function [38].

Fig. 1. Muscle biopsy from a patient with MTCYB mutation. A classic ragged-red ber in trichrome stained section (A) shows preserved cytochrome c oxidase reactivity (B).

38

M. Milone, L.-J. Wong / Molecular Genetics and Metabolism 110 (2013) 3541

3.2.2. Biochemical studies

Since the major function of mitochondria is to generate energy currency, ATP, through electron transport chain (ETC) and oxidative phosphorylation, measurement of these functions in affected muscle tissue
will reect mitochondrial defects. Each complex can be assayed with
specic substrates. Isolated individual complex deciencies suggest defects in protein subunits or assembly factors of that particular complex
[3945]. Thus, molecular analysis can pinpoint down to a specic
group of genes. While combined deciencies in rotenone sensitive complexes I + III and complexes II + III may suggest defects in CoQ10, usually due to enzymes involved in CoQ10 biosynthetic pathways
[15,46,47]. The ATP synthesis rate can be measured with complex I or
complex II substrates, when coupled with oxygen consumption; the results will suggest the approximate site of defects. Due to supercomplex
interaction, defects in specic complex subunits or assembly genes may
also affect the activities of other complexes. This has been observed in
the case of defects in complex IV assembly showing reduced activities
of complexes I, I + III, or II + III in addition to complex IV [40,48,49].
Deciencies in respiratory chain function are usually tissue specic, particularly if the mutation is sporadic or somatic. Tissue specic expression depends on several factors, including genes (nuclear or
mitochondrial and which gene), mutations (mtDNA point mutation or
deletion), threshold, degree of heteroplasmy in the tissue, and penetrance [50]. Therefore, even muscle samples with proven mutations
and/or phenotype may not show deciency in mitochondrial functional
assays. Mitochondrial DNA defects, point mutations or large deletions,
are often accompanied by compensatory mitochondrial DNA overamplication or mitochondrial proliferation. Mitochondrial proliferation is indicated by an increased citrate synthetase (CS) activity. Therefore, the respiratory chain complex activities should be normalized to CS
in order to nd deciencies in complex activities. Bernier et al. recommended a b 30% or b 20% of normal complex activities to t a minor or
major criterion for the diagnosis of mitochondrial disorders [34]. In
fact, the complex activities of many muscle specimens with denite molecular diagnosis of mitochondrial disease may not t these stringent
criteria. Therefore, an apparently normal respiratory enzyme complex
activity does not exclude the diagnosis of mitochondrial disorders. A
denite diagnosis requires the identication of causative molecular
defects. Biochemical and functional studies can assist in the selection
of genes for analysis.
Except complex II (succinate dehydrogenase), all electron transport
chain complexes contain mitochondrial encoded subunits [51]. Thus, if
the molecular defects are in nuclear genes that affect mtDNA biosynthesis, transcription or translation, theoretically, they will affect all complexes that contain protein subunits encoded by nuclear genes.
Mitochondrial respiratory enzyme assay can serve as a screening test
to help in determining which group of genes to be sequenced [52,53].

3.2.3. Molecular studies of the mtDNA

Several molecular methods can also serve as screening tests to facilitate the diagnosis of mitochondrial disorders. MtDNA copy number
analysis by real time qPCR determines if the mtDNA content is depleted
or amplied. If the mtDNA depletion is observed, a group of genes responsible for mtDNA replication and maintenance of deoxynucleotide
pools can be analyzed [53,54]. MtDNA depletion syndromes are, in
general, recognizable autosomal recessive disorders caused by nuclear
gene defects [54]. The degree of mtDNA depletion in muscle specimens
(cut off: b 50% of matched control) of patients with myopathic or
encephalomyopathic form is usually not as obvious as it is in liver specimens of patients with hepatocerebral form [54]. MtDNA amplication
usually implies compensatory mechanism caused by mtDNA mutations
[55]. However, the genetic etiology of mtDNA over amplication can
be maternally inherited, autosomal recessive or dominant. MtDNA multiple deletions, detected only in the affected muscle tissue, not blood,
are usually secondary to nuclear gene defects [1823,56], although a

small amount of mtDNA deletions, as well as histological signs of mitochondrial dysfunction, normally accumulate with age.
Quantication of mtDNA point mutation mutant loads in various
tissues and family members is necessary for clinical correlation and
determination of inheritance [57,58]. A germ line mutation is expected
to be present in all types of tissue; however, a sporadic somatic mutation
is usually present in the affected tissue(s) only. In general, the higher the
mutant load, the more severe the affected status. However, the mutant
load in one tissue does not necessarily reect the mutant loads in other
tissues. Therefore, prenatal diagnosis of mtDNA mutations cannot be
accurately offered.
4. The ultimately denite molecular diagnosis: identication of
causative mutations
The purpose of molecular diagnosis is to identify the genetic cause of
the disease such that proper patient management, treatment, and
genetic counseling can be provided. For mitochondrial myopathies,
the genetic defects may be in the mitochondrial genome or the nuclear
genome. The diagnosis is challenging; not only both nuclear and mitochondrial genomes need to be considered, but also the detection of
different types of mutations including point mutations and large deletions. In addition, for heteroplasmic mtDNA mutations, the degree of
heteroplasmy needs to be quantied, and mtDNA deletions need to be
characterized regarding deletion breakpoints. Traditionally, point mutations are identied by Sanger sequencing, while large deletions are
detected by Southern blot analysis or target array comparative genome
hybridization [59,60]. In any case, a comprehensive analysis requires
the combined application of multiple methodologies. However, the
newly developed next generation high throughput sequencing technology allows simultaneous sequencing analysis of multiple genes and the
detection and quantication of point mutations and large deletions in
both genomes [6163].
4.1. Mutations in mtDNA as primary defects
When a maternally inherited mtDNA disorder is suspected, three
levels of mtDNA analysis can be performed. If the clinical features are
typical, specic mtDNA point mutations or a group of known common
mutations can be screened using various target mutation detection
methods [53,64]. If this is negative, and mtDNA disorders continue to
be hypothesized, then, the entire mitochondrial genome can be analyzed by Sanger sequencing [53,62]. However, Sanger sequencing has
a few limitations: (a) It does not detect large mtDNA deletions, regardless of single or multiple deletions, (b) heteroplasmy less than 20% may
not be detected, (c) it does not quantify mtDNA mutation heteroplasmy,
(d) it does not detect mtDNA deletion breakpoint sequences, and (e) the
sequence results may be skewed by the presence of SNPs and nuclear mitochondrial DNA homolog (NUMT). Next generation deep sequencing
overcomes these problems. By using one pair of back-to-back nonoverlapping primer, the circular double stranded mtDNA is amplied
as a single amplicon. This approach essentially eliminated the SNPs and
NUMT hurdles [6163]. Since every base of the 16.6 kb mitochondrial
genome is evenly covered, large deletions can be easily detected
[6163]. The deep coverage also facilitates the quantication of mtDNA
point mutation at low levels of heteroplasmy [61]. Multiple deletions
showed different coverage patterns compared to single deletions
[6163] that can be easily recognized. The deletion breakpoints can
also be determined [61].
4.2. Mutations in nuclear genes
The total number of nuclear candidate genes responsible for mitochondrial disorders is estimated to be around 1500 [6567]. Traditionally,
diagnosis is based on recognizable clinical syndromes followed by
sequencing analysis of known candidate genes one-by-one [53]. In

M. Milone, L.-J. Wong / Molecular Genetics and Metabolism 110 (2013) 3541

addition to clinical evaluation, biochemical and molecular screening

tests can assist with narrowing down the candidate genes to a smaller
number for sequencing analysis. For example, if a patient clinically has
Leigh disease, ETC analysis revealed complex IV deciency, and family
history suggests an autosomal recessive disorder, then genes encoding
protein subunits of complex IV and its complex assembly factors are
to be analyzed one-by-one starting from the most frequently mutated
gene [40,6871]. If the patient has mtDNA depletion in muscle,
based on clinical features, genes responsible for myopathic and/or
encephalomyopathic forms of mtDNA depletion syndrome should be
sequenced [54,72,73]. Due to the high genetic and clinical heterogeneity, very often, clinical phenotypes are not so recognizable. Therefore,
the list of candidate genes becomes too long and it is difcult to set
priority. In this case, a group of target genes related to mitochondrial
myopathy may be sequenced in parallel using the next generation
sequencing approach [62,7476].
4.2.1. Mutations in nuclear genes causing secondary mtDNA defects
Mutations in genes responsible for mtDNA biogenesis such as
DNA polymerase gamma (POLG) [73,77,78], or maintenance of
deoxynucleotide pools, such as deoxyguanosine kinase (DGUOK)
[7981] and thymidine kinase 2 (TK2) [22,25,26,82], are likely to affect
the copy number or integrity of mtDNA[1823,72,83]. Genes causing
autosomal dominant or recessive forms of myopathy, PEO and/or
hearing loss often show mtDNA multiple deletions. These include
POLG, POLG2, DNA helicase (TWINKLE), RRM2B, and OPA1 genes
[1823]. Therefore, the presence of mtDNA depletion and/or multiple
deletions suggests possible primary defects in nuclear genes. However, mtDNA multiple deletions associated mitochondrial myopathy
may be a multifactorial disorder that is correlated with aging [84].
4.2.2. Mutations in nuclear genes as the primary cause of
mitochondrial dysfunction
Although the number of proteins involving mitochondrial structure
and function is estimated to be about 1500 [6567], only about 200
have been identied to be responsible for a broad spectrum of mitochondrial disorders [52,85]. Molecular diagnosis can be performed at
different levels; single gene analysis by Sanger sequencing; target
gene analysis of a group of genes responsible for recognizable syndromes or particular biochemical/molecular characteristics; analysis of
genes known to be involved in mitochondrial structure and function;
and whole exome analysis [62].
There are two major methods for the enrichment of target genes;
multiplex PCR and capture by hybridization with probes. The enriched
coding sequences are analyzed by the next generation massively parallel
sequencing. Depending on the enrichment methods, depth of coverage,
chemistry of sequencing platform, degree of validation, analytical algorithms, variant conrmation and interpretation, the false negative, false
positive and the nal diagnostic positive rates vary from laboratory to
laboratory. This explains why testing 20,000 genes or 200 genes may
cost the same amount without much difference in positive detection
rates. In general, if clinical, biochemical, molecular and imaging studies
are consistent with mitochondrial myopathic disorders, panels of fully
validated target gene analysis with deep coverage may provide a more
denitive answer than a whole exome analysis.
4.3. Advantage of denite diagnosis
4.3.1. Treatment
Treatment of mitochondrial myopathy is supportive. A rigorous
review of the published studies on the treatment of mitochondrial diseases by drugs, diet modication, nutritional supplements and exercise,
identied no disease-modifying agent and showed no convincing evidence for supporting any intervention in mitochondrial disorders [86].
Study-drugs have included creatine, carnitine, CoQ10, dimethylglycine,
cysteine, dichloroacetate, L-arginine and lipoic acid. A trial with high-

39

dose CoQ10 and another with combined CoQ10, creatine monohydrate

and lipoic acid, showed biomarker improvement but no signicant clinical improvement. Peripheral neuropathy was the only serious adverse
effect of the long-term trial of dichloroacetate. A non-blinded study of Larginine vs. placebo showed a reduction in frequency and severity of
stroke-like events in MELAS. Randomized controlled clinical trials are
necessary to optimize the treatment of mitochondrial diseases. However, CoQ10 supplementation has shown benecial effects in primary
CoQ10 deciency linked to ETFDH mutations [46].
For patient care, it is important to mention drugs known to have mitochondrial toxicity, such as valproic acid [77,87] and anti-retrovirals
[88].
4.3.2. Genetic counseling
Sanger sequencing does not detect mtDNA heteroplasmy less than
20%. This limitation hinders accurate genetic counseling. Next generation
deep sequencing of the circular mitochondrial genome allows the detection of low heteroplasmic mtDNA mutations, accurate diagnosis and
counseling [61]. For example, an affected proband had 99.5% and 16%
of mtDNA mutation in her muscle and blood respectively, while her
mother did not carry the mutation in her blood [61]. Since this was
detected and determined by next generation deep sequencing, which
provides reliable quantitative heteroplasmy results, we can conclude
that the mutation is de novo in the proband, although germ line mosaicism cannot be ruled out. Should the heteroplasmy be determined by
Sanger sequencing or other less accurate methods, we would not be
able to conclude that the mother did not carry the mutation because
Sanger sequencing or other methods cannot detect low levels of
heteroplasmy. On the other hand, an affected woman with 37% mtDNA
mutation heteroplasmy in the muscle, but absolutely none in the blood,
has almost zero chances of passing the mutation to her children, because
the mutation is somatic in her muscle [61]. This was conrmed by her
unaffected sibling and daughter, who both did not carry mutation [61].
For autosomal recessive disorders, identication of the causative
mutations can help with prenatal and carrier diagnosis. However, as
discussed earlier, false negative rate varies among different laboratories.
Some laboratory offers fully validated, fully deeply covered panel
testing with zero false negative. In this case, even a negative result
provides a denitive conclusion that the patient most likely does not
carry mutations in any of the target genes captured and sequenced. A
denitive diagnosis should continue to be pursued by using other test
panels or whole exome sequencing.
4.3.3. Prenatal diagnosis
Since the mutant load in one tissue does not necessarily reect
the mutant loads in other tissues, prenatal diagnosis of mtDNA
mutations cannot be accurately offered. In the case of de novo or sporadic somatic mutations, the purpose of prenatal diagnosis is limited
to the re-conrmation of the sporadic situation and is of little value.
It is also a danger to provide pre-implantation genetic diagnosis
(PGD) for pregnant mother who carries mtDNA heteroplasmic mutations [89]. In this published PGD study, despite the implantation of the
blastocyst containing the lowest levels of mutation heteroplasmy, the
baby was born affected [89].
For severe autosomal recessive disorders, carrier status of parents
should be conrmed before prenatal diagnosis is considered. Prenatal
diagnosis for severe nuclear gene disorders is recommended.
Conict of interest
The authors have no conict of interest.
Acknowledgments
This work is partly supported by an MDA grant to LJW.

40

M. Milone, L.-J. Wong / Molecular Genetics and Metabolism 110 (2013) 3541

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