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Pediatrics and Neonatology (2014) xx, 1e12

Available online at www.sciencedirect.com

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journal homepage: http://www.pediatr-neonatol.com

INVITED REVIEW ARTICLE

Diagnostic Approach in Infants and Children

with Mitochondrial Diseases
Ching-Shiang Chi*

Department of Pediatrics, Tungs’ Taichung Metroharbor Hospital, Taichung, Taiwan

Received Mar 17, 2014; accepted Mar 27, 2014

Key Words Mitochondrial diseases are a heterogeneous group of disorders affecting energy production in
diagnosis; the human body. The diagnosis of mitochondrial diseases represents a challenge to clinicians,
infants and children; especially for pediatric cases, which show enormous variation in clinical presentations, as well
mitochondrial as biochemical and genetic complexity.
diseases; Different consensus diagnostic criteria for mitochondrial diseases in infants and children are
Taiwan available. The lack of standardized diagnostic criteria poses difficulties in evaluating diag-
nostic methodologies. Even though there are many diagnostic tools, none of them are sensitive
enough to make a confirmative diagnosis without being used in combination with other tools.
The current approach to diagnosing and classifying mitochondrial diseases incorporates clin-
ical, biochemical, neuroradiological findings, and histological criteria, as well as DNA-based
molecular diagnostic testing. The confirmation or exclusion of mitochondrial diseases remains
a challenge in clinical practice, especially in cases with nonspecific clinical phenotypes. There-
fore, follow-up evolution of clinical symptoms/signs and biochemical data is crucial.
The purpose of this study is to review the molecular classification scheme and associated
phenotypes in infants and children with mitochondrial diseases, in addition to providing an
overview of the basic biochemical reactions and genetic characteristics in the mitochondrion,
clinical manifestations, and diagnostic methods. A diagnostic algorithm for identifying mito-
chondrial disorders in pediatric neurology patients is proposed.
Copyright ª 2014, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights
reserved.

* Department of Pediatrics, Tungs’ Taichung Metroharbor Hospital, 699, Taiwan Boulevard Section 8, Wuchi, Taichung, 435, Taiwan.
E-mail address: chi-cs@hotmail.com.

http://dx.doi.org/10.1016/j.pedneo.2014.03.009
1875-9572/Copyright ª 2014, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.

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2 C.-S. Chi

1. Introduction particularly vulnerable to limited adenosine triphosphate

Mitochondrial disease (MD) was first introduced in 1962,
when a group of investigators, Luft et al1, described a
young Swedish woman with severe nonthyroid origin hy-
permetabolism. In 1963, Engel and Cunningham2 used a
modification of the Gomori trichrome stain that allowed for
the detection of abnormal mitochondrial proliferation in
muscle as irregular purplish patches in fibers that were
dubbed “ragged red fibers (RRFs)”. Since then, there has
been great interest in the diagnosis of mitochondrial dis-
eases (MDs). The diagnosis of MD can be traced back to the
premolecular era, 1962e1988, when MDs were defined on
the basis of clinical examination, muscle biopsy, and
biochemical criteria. In the molecular era, the full
complexity of these disorders became evident.
MDs are a heterogeneous group of disorders affecting
energy production in the human body, which can occur with
a probable frequency in preschool children (age < 6 years)
of w 1 in 11,000,3 and the minimum birth prevalence for
respiratory chain disorders with onset at any age was esti-
mated at 1 in 7634.4 MDs may present at any age with a
spectrum of symptoms and signs spanning a number of
medical specialties. As cells with high energy requirements,
such as neurons, skeletal and cardiac muscle, are
(ATP) supply, encephalopathy and myopathy are often
prominent features in various mitochondrial phenotypes.
Enzymes involved in mitochondrial energy production are
coded by two genomes (mitochondrial and nuclear) in the
organism. Due to their clinical, biochemical, and genetic
complexity, MDs represent a challenge to clinicians, espe-
cially for pediatric cases, which show enormous variation in
clinical presentations and course.
In Taiwan, the first clinical MD was reported in 1988,5
and the first mitochondrial DNA (mtDNA) mutation was
identified in 1992.6 Many cases have since been reported in
pediatric groups.7e47 Although MDs are being diagnosed
more frequently, clinical physicians still find diagnosis a
challenge because clinical symptoms/signs evolve over
time and the number of genes known to be involved in
mitochondrial energy production continues to increase.
The purpose of this study is to review the molecular
classification scheme and associated phenotypes in infants
and children with MDs, in addition to providing an over-
view of the basic biochemical reactions and genetic
characteristics in the mitochondrion, clinical manifesta-
tions, and diagnostic methods. A diagnostic algorithm for
identifying MDs in pediatric neurology patients is
proposed.

Glucose
Fatty acids
Glycolysis Nuclear
Fatty –acyl-CoA DNA
Alanine Pyruvate Lactate Amino acids Cytosol

CPT-I
H+ Pyruvate Amino acids
Fatty –acyl-carnitine Carnitine
DIC CPT-II CACT
Matrix
H+ Pyruvate Fatty –acyl-carnitine Carnitine
Fatty –acyl-CoA

Inner mitochondrial membrane

Outer mitochondrial membrane
PC PDHC Acetyl-CoA β -oxidation

Citrate
Oxaloacetate
TCA cycle Mitochondrial
DNA
α -Ketoglutarate
Malate
Succinyl-CoA
Fumarate NADH
Succinate

Complex I Complex III Complex IV

FMN FeS FeS FeS FeS CoQ CytbT CytbK FeS Cytc1 Cytc Cyta Cyta3 O2
Complex II Complex V (ATPase 6,8)
FAD FeS FeS FeS ATP synthase

ADP + Pi ATP

Figure 1 Metabolic pathways in mitochondrion. ADP Z adenosine diphosphate; ATP Z adenosine triphosphate;
CACT Z carnitineeacylcarnitine translocase; CoQ Z coenzyme Q; CPT Z carnitine palmitoyltransferase; Cyta Z cytochrome a;
Cytb Z cytochrome b; Cytc Z cytochrome c; DIC Z dicarboxylate carrier; FAD Z flavin adenine dinucleotide; FeS Z iron sulfur
protein; FMN Z flavin mononucleotide; NADH Z nicotinamide adenine dinucleotide; PC Z pyruvate carboxylase; PDHC Z pyruvate
dehydrogenase complex; TCA Z tricarboxylic acid.

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Mitochondrial diseases in infants and children 3

Table 1 A brief summary of published genetic classification of mitochondrial diseases.49,50

Mitochondrial diseases (MDs) Type of mutation Mutated gene
mtDNA mutations
mtDNA rearrangements
Chronic progressive external ophthalmoplegia (mtPEO) mtDNA deletion, mtDNA
duplication
KearnseSayre syndrome (KSS) mtDNA deletion, mtDNA
duplication
Pearson’s syndrome (PS) mtDNA deletion, mtDNA
duplication
mtDNA point mutation
Mitochondrial encephalomyopathy, lactic acidosis, and np 3243, np 3271, mt-tRNALeu, mt-tRNAPhe,
stroke-like episodes (MELAS) and others mt-tRNAVal, COXII, COXIII,
ND1, ND5, ND6, rRNA
Myoclonic epilepsy with ragged-red fibers (MERRF) np 8344, np 8356, mt-tRNALys
and others
Leber’s hereditary optic neuropathy (LHON) np 11778, np 14484, RCCI subunits
and others
Leigh syndrome np 8993, np 10191, ATPase 6
and others
Neuropathy, ataxia, and retinitis pigmentosa (NARP) np 8993, and others ATPase6
Maternal inherited Leigh syndrome (MILS) np 8993, and others ND1-6, COXIII, ATPase6, tRNAs
nDNA mutations
Mutated RC subunits
Nonsyndromic MDs with early-onset hypotonia, nDNA RCCI-subunits (NDUFS1,
cardiomyopathy, ataxia, psychomotor delay NDUFS2, NDUFS3, NDUFS4,
NDUFS6, NDUFS8, NDUFV1,
NDUFV2) or assembly factors
Leigh syndrome (LS) nDNA RCCI-subunits (NDUFS1,
NDUFS2, NDUFS3, NDUFS4,
NDUFS6, NDUFS8, NDUFV1,
NDUFV2) or assembly factors
Encephalocardiomyopathy with severe lactic acidosis nDNA RCCI splicing mutations in
NDUFA11 or NDUFA1
Late-onset neurodegenerative disease nDNA RCCII (SDHA)
Leigh syndrome (LS) nDNA RCCII (SDHA)
Nonlethal phenotype of severe psychomotor retardation, nDNA RCCIII (UQCRQ)
extrapyramidal signs, dystonia, athetosis and ataxia, mild axial
hypotonia, and dementia
Severe infantile encephalomyopathy nDNA RCCIII (COX6B1)
Mutated ancillary proteins
Leigh syndrome (LS) nDNA SURF1 encoding COX
assembly factor
Leigh syndrome (LS) manifest as leukoencephalopathy nDNA RCCI assembly factors
(NDUFA12L)
Leigh syndrome (LS) manifest as cardio-encephalo-myopathy nDNA RCCI assembly factors
(NDUFAF1, C6ORF66)
Leigh syndrome (LS) X chromosome E1-alpha-subunit of PDHC
Leigh syndrome (LS) nDNA E1-alpha-subunit of PDHC
Leigh syndrome (LS), COX-deficient nDNA COX assembly factors
(SCO2, SCO1, COX10, COX15)
Multisystem fatal infantile disorders, encephalomyopathy plus nDNA COX assembly factors
cardiomyopathy, nephropathy, or hepatopathy (SCO2, SCO1, COX10, COX15)
Lethal infantile growth retardation, aminoaciduria, cholestasis, nDNA RCCIII assembly protein
iron overload, lactacidosis and early death (GRACILE) syndrome (BCS1L)
Sensorineural hearing loss with pilli torli (Bjornstad syndrome) nDNA RCCIII assembly protein
(BCS1L)
Congenital lactacidosis and fatal infantile multisystem disease, nDNA RCCV assembly gene
involving brain, liver, heart, and muscle (ATP12)
Neonatal encephalo cardiomyopathy nDNA RCCV assembly gene (TMEM 70)
(continued on next page)

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4 C.-S. Chi

Table 1 (continued )
Mitochondrial diseases (MDs) Type of mutation Mutated gene
Defects of intergenomic signaling
mtDNA breakage syndromes (large-scale mtDNA deletion)
Autosomal dominant PEO (adPEO) nDNA ANT1, PEO1, POLG
Autosomal recessive PEO (arPEO) nDNA POLG
Sensory ataxic neuropathy, dysarthria, ophthalmoplegia (SANDO) nDNA POLG
Spino-cerebellar ataxia and epilepsy with or without nDNA POLG
ophthalmoplegia (SCAE)
Infantile Aplers-Huttenlocher-syndrome (AHS) manifests as nDNA POLG
myopathy, hepatopathy, epilepsy, migraine, intractable seizures
and mental retardation (hepatic poliodystrophy)
Mitochondrial neuro-gastrointestinal encephalomyopathy nDNA TYMP
(MNGIE)
MNGIE-like phenotype nDNA POLG
mtDNA depletion syndromes (DPSs)
Myopathic DPS
Muscle weakness, elevated creatine kinase, encephalopahy nDNA TK2
adPEO, mild myopathy nDNA ANT1 (SLC25A4)
Myopathy, renal tubulopathy nDNA RRM2B
AHS, PEO, Myoclonic epilepsy myopathy sensory ataxia, ataxia nDNA POLG
neuropathy spectrum, childhood myocerebrohepatopathy
spectrum
Encephalomyopathic DPS
Leigh-like syndrome with muscle hypotonia, lactic acidosis, nDNA SUCLA2,
dystonic and moderate methylmalonic aciduria
Fatal infantile lactic acidosis, dysmorphism and methylmalonic nDNA SUCLG1
aciduria with muscle and liver mtDNA depletion
Hypotonia, developmental delay, and renal tubulopathy nDNA RRM2B
Hepato-cerebral DPS
Phenotypes as PLOG described above nDNA POLG
Alpers-like phenotype nDNA PEO1
Hepatic failure, hypoglycemia, muscle hypotonia, ataxia, nDNA MVP17
dystonia, or polyneuropathy, Navajo neurohepatopathy
MNGIE nDNA TYMP
Hepatopathy, severe failure to thrive, lactic acidosis, nDNA DGUOK
hypoglycemia, neurological symptoms
Infantile-onset spinocerebellar ataxia, adPEO, myopathy, nDNA C10orf2 (Twinkle)
Parkinsonian features of rigidity tremor, and bradykinesia
Defective mitochondrial protein synthesis machinery
Mitochondrial myopathy and sideroblastic anemia (MLASA) nDNA PUS1
Leukoencephalopathy with brainstem and spinal cord nDNA DARS2
involvement and lactacidosis (LBSL)-syndrome
Pontocerebellar hypoplasia (PCH) with prenatal-onset, nDNA RARS2
cerebellar/pontine atrophy/hypoplasia, microcephaly,
neocortical atrophy and severe psychomotor impairment
Agenesis of corpus callosum, dysmorphic features, fatal neonatal nDNA MRPS16
lactic acidosis
ad intermediate Charcot-MarieeTooth neuropathy, type C nDNA YARS2
Defects of the mitochondrial lipid milieu
Barth syndrome characterized by mitochondrial myopathy, nDNA TAZ
hypertrophic or dilated cardiomyopathy, left ventricular
hypertrabeculation/noncompaction, growth retardation,
leukopenia and 3-methylglutaconic aciduria
Sensorineural deafness, encephalopathy, Leigh-like syndrome, nDNA SERAC1
and 3-methylglutaconic aciduria
Sengers syndrome characterized by congenital cataracts, nDNA AGK
hypertrophic cardiomyopathy, skeletal myopathy, and lactic
acidosis

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Mitochondrial diseases in infants and children 5

Table 1 (continued )
Mitochondrial diseases (MDs) Type of mutation Mutated gene
Coenzyme Q defects
Nonsyndromic MDs: encephalomyopathy with recurrent nDNA Primary CoQ deficiency
myoglobinuria, brain involvement and ragged red fiber (COQ2, COQ8, ADCK3,
CABC1, PDSS1, PDSS2)
Severe infantile multisystem MDs nDNA Primary CoQ deficiency
(COQ2, COQ8, ADCK3,
CABC1, PDSS1, PDSS2)
Leigh syndrome (LS) nDNA Primary CoQ deficiency
(COQ2, COQ8, ADCK3,
CABC1, PDSS1, PDSS2)
Cerebellar ataxia nDNA Secondary CoQ deficiency
(APTX)
Pure myopathy nDNA Secondary CoQ deficiency
(ETFDH)
Cardio-facio-cutaneous syndrome nDNA Secondary CoQ deficiency
(BRAF)
Defects of mitochondrial transport machinery
Deafness-dystonia syndrome (DDS): childhood-onset progressive nDNA DDP1/TIMM8A
deafness, dystonia, spasticity, mental deterioration and blindness
X-linked sideroblastic anemia with ataxia (XLSA/A) nDNA ABCB7
Congenital lactic acidosis, hypertrophic cardiomyopathy and nDNA SLC25A3
muscle hypotonia
Defects in mitochondrial biogenesis
Dominant optic atrophy (ADOA) nDNA OPA1
CharcoteMarieeTooth neuropathy-2A nDNA MFN2
Severe infantile encephalopathy nDNA DLP1
Hereditary spastic paraplegia nDNA KIF5A
ABCB7 Z ATP-binding cassette, sub-family B (MDR/TAP), member 7; AGK Z acylglycerol kinase; ANT1 Z adenine nucleotide translocase
1 gene; C10orf2 Z chromosome 10 open reading frame 2; COX Z cytochrome c oxidase; DARS2 Z aspartyl-tRNA synthetase 2;
DDP1 Z deafness dystonia peptide 1; DGUOK Z deoxyguanosine kinase; DLP1 Z dynamin-like protein-1; KIF5A Z kinesin family member
5; MFN2 Z mitofusin 2; MPV17 Z mitochondrial inner membrane protein; MRPS16 Z mitochondrial ribosomal protein S16;
mtDNA Z mitochondrial DNA; nDNA Z nuclear DNA; ND Z NADH dehydrogenase; NDUFS Z NADH dehydrogenase (ubiquinone) Fe-S
protein; NDUFV Z NADH dehydrogenase (ubiquinone) flavoprotein; np Z nucleopeptide; OPA1 Z optic atrophy 1; PDHC Z pyruvate
dehydrogenase complex; PDSS Z prenyl (decaprenyl) diphosphate synthase; PEO1 Z C10orf2 encoding a mitochondrial helicase
(Twinkle); POLG gene Z polymerase-gamma gene; PUS1 Z pseudouridylate synthetase-1; RARS2 Z arginyl-tRNA synthetase 2;
RCC Z respiratory chain complex; RRM2B Z p53-dependent ribonucleotide reductase; SDHA Z succinate dehydrogenase complex,
subunit A; SERAC1 Z serine active site containing 1; SLC25A3 Z solute carrier family 25 (mitochondrial carrier; phosphate carrier),
member 3; SUCLA2 Z b-subunit of the adenosine diphosphate-forming succinyl-CoA-ligase; SUCLG1 Z alpha-subunit of GDP-forming
succinyl-CoA-ligase; TAZ Z tafazzin; TK Z thymidine-kinase; TMEM70 Z transmembrane protein 70; TYMP Z thymidine phosphory-
lase; YARS2 Z tyrosyl-tRNA synthetase 2.

2. Biochemical reactions in the mitochondrion (acetyl-CoA) catalyzed by the pyruvate dehydrogenase

The mitochondrion, an extranuclear organelle, is 0.5e1 mm
in size. It consists of outer and inner membranes, an
intermembranous space, and an inner matrix compartment.
The matrix contains various enzymes, ribosomes, transfer
RNAs (tRNAs), and mtDNA molecules. Each cell contains
many mitochondria which are responsible for cellular ATP
production by oxidative phosphorylation. Energy produc-
tion of mitochondria comes from the metabolism of glucose
and fatty acids through a series of reactions (Figure 1).
Pyruvate, the end-product of aerobic glycolysis, is
derived partly from blood-borne glucose but mainly from
endogenous glycogen. Once formed in the cell cytosol,
pyruvate may be reduced to lactate, transaminated to
alanine, or transported into the mitochondria where it
undergoes oxidative decarboxylation to acetyl-coenzyme A
complex (PDHC). Long-chain fatty acids, after being acti-
vated to form fatty acyl-CoA in the cytosol, must be
transferred across the inner mitochondrial membrane to be
oxidized to acetyl-CoA. Acetyl-CoA then enters the citric
acid cycle (Krebs cycle), which releases eight hydrogen
molecules and produces carbon dioxide and water through
oxidative phosphorylation. This process liberates energy
along the respiratory chain, which receives energy-rich
hydrogen atoms from nicotinamide adenine dinucleotide
(NADH) or flavineadenine dinucleotide (FADH), produced
mainly in the Krebs cycle and from fatty acid oxidation.
Electrons from the hydrogen are passed between respira-
tory complexes in the chain and result in energy
production.
Mitochondrial respiratory chain (RC) complex is a unique
system in the cell coded by two genomes, known as the

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6 C.-S. Chi
powerhouse system of the cell, where the energy produc-
tion occurs through the oxidative phosphorylation pathway.
The mitochondrial RC system is located in the mitochon-
drial inner membrane, organized in five enzymatic com-
plexes (IeV), ubiquinone (or coenzyme Q10), and
cytochrome c. Complexes I, III, and IV extrude protons from
the mitochondrial matrix. Complex IV consumes oxygen to
form water. Complex V couples ATP synthesis to proton
reentry, which is powered by the electrochemical
gradient.48 Ultimately, the energy is stored as ATP. These
assumptions are the basis for the biochemical evaluation of
mitochondrial RC enzymes’ activity.
The term “mitochondrial diseases” refers to a class of
disorders characterized by an impairment of the mito-
chondrial RC, where most cellular ATP is generated.
3. Mitochondrial genetics
The human mitochondrial genome is a 16,569-bp double-
stranded DNA circle that contains 37 genes, of which 13
genes encode subunits of the respiratory chain, and 22
tRNAs and two ribosomal RNA genes (12S and 16S) translate
mtDNA. Each cell contains many mitochondria with
2e10 mtDNA molecules. The total mtDNA accounts for w
0.5% of the DNA in a nucleated somatic cell.
The 13 mitochondrial protein-coding genes contribute to
the four enzyme components of the RC complexes required for
oxidative phosphorylation: seven of them are subunits of
complex I (NADH-ubiquinone oxidoreductase), one of complex
III (ubiquinolecytochrome c oxidoreductase), three of com-
plex IV (cytochrome c oxidase), and two of complex V (Hþ-
translocating ATP synthase). All of the subunits of complex II
(succinateeubiquinone oxidoreductase), the remaining sub-
units of the other mitochondrial RC complexes as well as the
factors involved in mtDNA replication, transcription, and
translation, are encoded by nuclear DNA (nDNA).
Mitochondrial genetics differ from Mendelian genetics in
several fundamental aspects. First, mtDNA is maternally
inherited because mitochondria derive solely from the
oocyte. A normal cell has only a single mtDNA genotype and
is therefore homoplasmic. However, if the genomes
represent a mixture of a wild-type and a mutated-type, the
cell genotype is heteroplasmic. The clinical pictures of
mitochondrial diseases are determined by the proportion of
normal-to-mutated genomes in the mitochondria. Once the
proportion exceeds a theoretical threshold, the biological
behavior of the cell will change. This minimum critical
amount differs from tissue to tissue and different tissues
may be variously affected by different combinations and to
different degrees. In addition, mitotic segregation of the
mitochondria influences the biological behavior governing
the stochastic redistribution of wild-type and mutated ge-
nomes during mitochondrial and cell divisions. Thus, the
concepts of threshold effect and mitotic segregation pro-
vide theoretical explanations for the variable phenotype
expressions of the MDs.
The dual genetic control of the RC represents the ubiq-
uitous nature of mitochondria. The mitochondrial metabolic
pathway is under the control of not only the mitochondrial,
but also the nuclear genomes, which are strictly coordinated
to ensure the correct functioning of the mitochondrial
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machinery. To date, it has been established that 1500
nuclear-encoded proteins are targeted to the mitochon-
dria.49 Mutations in nDNA can affect: (1) components of RC
subunits; (2) mitochondrial ancillary proteins involved in RC
complexes formation, turnover, and function; (3) inter-
genomic signaling for mtDNA maintenance or expression; (4)
biosynthetic enzymes for lipids or cofactors; (5) coenzyme
Q; (6) mitochondrial trafficking or transport machinery; (7)
mitochondrial biogenesis; or (8) apoptosis.50
A brief summary of published genetic classification of
MDs is shown in Table 1.49,50 Primary mtDNA mutations may
exhibit a maternal inheritance, or Mendelian traits that
present with multiple somatic mtDNA alterations. In the
case of Mendelian disorders, autosomal recessive, auto-
somal dominant, and X-linked patterns have all been
observed. Recent studies have demonstrated that w
10e15% of MDs are caused by mutations in mtDNA,48 and up
to 25% of selected children with MDs are caused by muta-
tions in nDNA49; i.e., MDs in children result more often from
nDNA mutations. In addition, it has been reported that
> 200 nuclear-encoded genes have been linked to human
disease.49 Therefore, genetic diagnosis of MDs in infants
and children is a difficult task due to the heteroplasmy of
mtDNA mutations and the large number of nuclear genes
involved in different tissues.
4. Clinical manifestations
MDs that result from diminishing ATP production cause clin-
ical disorders. Several studies51e53 showed clinical features
in patients with MDs which support the maxim “any tissue
and any signs at any age”.54 However, infants and children
with MDs tend to have an acute onset of the clinical symp-
toms and courses compared with adults with MDs, who
typically exhibit slow and/or progressive presentations. In
general, the clinical spectrum is not limited to the neuro-
muscular system; indeed, a number of nonneuromuscular
organs may exhibit symptoms and signs, such as the heart,
eyes, ears, kidneys, endocrine glands, liver, bone marrow,
and gastrointestinal tract.42 The clinical manifestations of
103 pediatric patients with MDs in our case series, expanding
data from the previous report,42 are summarized in Table 2.
The commonest clinical manifestation was symptoms and
signs of CNS (93/103; 90.3%). The CNS manifestations of MDs
were variable, including seizures, developmental delay,
altered level of consciousness, floppiness, spasticity, mental
retardation, sucking difficulty, involuntary movement,
headache, apnea, external ocular motility limitation,
tremor, apneustic respiration, dystonia, stroke, sudden in-
fant death syndrome, and/or hypoventilation. The second
most common clinical features were ophthalmologic prob-
lems (37/103; 35.9%) and failure to thrive (37/103; 35.9%).
The symptoms and signs of the ophthalmologic system
included ptosis, retinitis pigmentosa, external oph-
thalmoplegia, visual loss, nystagmus, exotropia, blurred
vision, visual field defect, strabismus, corneal clouding, and/
or optic nerve atrophy. The fourth most common clinical
feature was cardiovascular system dysfunction (26/103;
25.2%), including pericardial effusion, hypertrophic cardio-
myopathy, arrhythmia, mitral valve prolapse, hypertension,
and/or dilated cardiomyopathy. Various symptoms and
 + MODEL
Mitochondrial diseases in infants and children 7

In 1996, Walker et al74 proposed diagnostic criteria for

Table 2 Clinical manifestations in 103 infants and chil-
adults with respiratory chain disorders (Adult criteria; AC).
dren with mitochondrial diseases.
In 2002, Bernier et al75 suggested a modified version of the
Involved organ system N Z 103 Adult criteria (Modified Walker criteria or Modified Adult
Central nervous system 93 (90.3) Criteria; MAC) in order to improve the sensitivity and expand
Ophthalmologic system 37 (35.9) its use to include pediatric patients as well as adults. The
Failure to thrive 37 (35.9) diagnostic criteria of MDs were established on the basis of
Cardiovascular system 26 (25.2) assigning major or minor criteria for clinical, pathological,
Gastrointestinal system 23 (22.3) enzymatic, functional, molecular, and metabolic parame-
Muscular system 22 (21.4) ters. Mitochondrial encephalomyopathies were categorized
Hearing system 20 (19.4) as definite, probable, or possible. A definite diagnosis is
Hepatic system 19 (18.4) defined as fulfillment of either of two major criteria or one
Short stature 18 (17.5) major criterion plus two minor criteria. A probable diagnosis
Urologic system 9 (8.7) is defined as either one major criterion and one minor cri-
Endocrinologic system 8 (7.8) terion or at least three minor criteria. A possible diagnosis is
Peripheral nervous system 4 (3.9) defined as either a single major criterion or two minor
Pancreas 2 (1.9) criteria, one of which must be clinical.
Hematologic system 1 (1.0) In 2002, Wolf et al76 proposed the consensus mitochon-
Psychic 1 (1.0) drial diagnostic criteria (MDC) scoring system for infants
Pulmonary edema 1 (1.0) and children, which evaluates clinical features (I; maximum
clinical score: 4 points) of muscle symptoms (IA; maximum
Data are presented as n (%).
score: 2 points), CNS abnormalities (IB; maximal score: 2
points), and multisystem involvement (IC; maximal score: 3
clinical features complicate the diagnosis of MDs and the points), adding metabolic abnormalities and neuroimaging
presentations may result in patients visiting numerous other (II; maximum addition score: 4 points). Histologic anomalies
subspecialists and receiving various diagnoses. (III) could increase the score with 4 points, leading to a
There are two main clinical classifications of MDs: syn- maximum score of 12 points. Scores of 8e12, 5e7, 2e4, and
dromic MDs and nonsyndromic MDs. Patients who presented 1 were defined as definite, probable, possible, or unlikely
with characteristic clinical phenotypes reported in the mitochondrial disorders, respectively. The MDC uses well-
literature were categorized as having specific mitochon- defined items (clinical, laboratory, pathologic, and
drial syndromes,55e68 including Leigh syndrome (LS),55,69 biochemical) for scoring and dividing criteria into two
Alpers’ disease,57 lethal infantile mitochondrial disease subsets: general (clinical, metabolic, imaging, and patho-
(LIMM),59,60 Pearson’s syndrome (PS),58,70e72 KearnseSayre logic) and biochemical. This allows a critical and indepen-
syndrome (KSS),56 mitochondrial encephalomyopathy, lac- dent evaluation for general features and for results of
tic acidosis, and stroke-like episodes (MELAS),64 myoclonic biochemical investigations, prior to combining the two. The
epilepsy with ragged-red fibers (MERRF),61,62 neuropathy, advantages of MDC criteria are that the general criteria
ataxia, and retinitis pigmentosa (NARP),67 mitochondrial (without the histochemical part) allow preclassification of
neuro-gastrointestinal encephalomyopathy (MNGIE),65 patients prior to when a muscle biopsy is performed. If a
chronic progressive external ophthalmoplegia (CPEO),63 patient with an undiagnosed disorder reaches the probable
Leber’s hereditary optic neuropathy (LHON),66 and Barth or definite general classification, a muscle biopsy may be
syndrome (BTHS).68 The patients who fulfilled the diag- strongly recommended.
nostic criteria for MDs but not specific syndromes, were The different consensus diagnostic criteria for MDs in
classified as having noncategorized or nonsyndromic MDs.42 children described above show how difficult it can be to
Recently published studies42,51e53 show that pediatric pa- diagnose MDs; that is, the diagnosis of MDs with certainty
tients with nonsyndromic MDs are not uncommon, and the requires analysis from multiple perspectives.
clinical manifestations are nonspecific.42,73
Specific mitochondrial syndromes are easy to diagnose
because each of them has its own clinical phenotypes.
5.2. Diagnostic tools
However, it is not easy to diagnose nonsyndromic MDs
during the early stage of disease course due to the wide 5.2.1. Blood laboratory investigation
variety of clinical features. Thus, follow-up of clinical Lack of a clinically pathognomonic hallmark frequently
symptoms/signs in these patients is important. makes laboratory investigations necessary to confirm the
diagnosis.
5. Diagnostic approach of mitochondrial For basic laboratory investigations, blood lactate is used
as a biochemical marker for the screening of MDs. The
diseases differential diagnosis of lactic acidosis includes physiolog-
ical anaerobic exercise, systemic diseases that increase
5.1. Diagnostic criteria blood lactate levels, cerebral diseases that increase CSF
lactate levels, metabolic diseases, poor collection tech-
Different consensus diagnostic criteria for MDs in children nique, or poor sample handling.77 However, blood lactate
are available. Major and minor criteria are usually based on level is not always elevated in patients with MDs, and thus,
clinical, biochemical, pathologic, and molecular findings. normal serum lactic level does not exclude a mitochondrial

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 + MODEL
8 C.-S. Chi
disorder.42,51 A glucose challenge test followed by succes-
sive blood lactate examinations, oral glucose lactate stim-
ulation test (OGLST), is a better screening method than a
single blood lactate test.3,11,42 Lactic acidosis may be pro-
portionate or disproportionate to the elevation of pyruvate
based on the associated effect of the biochemical defect on
the oxidationereduction potential. If the oxida-
tionereduction potential is unaffected by the biochemical
defect, the lactate and pyruvate elevations will be pro-
portional and the lactate/pyruvate ratio will be normal. By
contrast, if the oxidationereduction potential is disturbed
by a primary defect involving the respiratory chain, the
lactate values will be disproportionately elevated and the
lactate/pyruvate ratio will be increased (> 25).
If available, CSF lactate, and pyruvate concentration
should also be measured. It is useful to calculate a lactate-
to-pyruvate ratio because an elevated ratio suggests the
possibility of a MD.
5.2.2. Metabolic survey
Lactic acidosis can be caused not only by intramitochondrial
(primary) disorders, but also by extramitochondrial (sec-
ondary) disorders. The latter include defects of metabolic
pathways for glycogen, gluconeogenesis, and organic acid-
emia. Thus, a metabolic screening with measurement of
basic blood and urine analytes such as plasma ketone body
(3-OH butyrate/acetoacetate) ratio, serum transaminase,
plasma amino acid quantitation, tandem mass spectrometry
(MS/MS), plasma acylcarnitine profile, and urinary organic
acids, is helpful for differential diagnosis of MDs.
5.2.3. Imaging studies
The clinical diagnosis of MDs has been greatly improved by
advances in neuroimaging technology. Brain magnetic reso-
nance imaging (MRI) is sensitive to these diseases, especially
in clinically phenotypic mitochondrial syndromes, including
LS, MELAS, MNGIE, and KSS. In infants and children with
nonsyndromic MDs, brain MRI can produce variable findings,
from normal results to signal changes over the basal ganglia
or brainstem.43 MR spectroscopy (MRS) for detecting the
concentration of a number of biochemical metabolites
in vivo has been reported to be a useful tool in conducting
differential diagnoses and for monitoring of MDs.44,78
5.2.4. Tissue biopsy and MRC enzymatic assay
A muscle biopsy using light microscopic and electron micro-
scopic examinations is an auxiliary diagnostic method of MDs.
Morphological examinations include the modified Gomori
trichrome staining for RRFs, ATPase staining for the assess-
ment of myofibrillar integrity, muscle-type fiber predomi-
nance and distribution, and cytochrome c oxidase and
succinate dehydrogenase staining for oxidative enzymes.
RRFs showing on the modified Gomori trichrome stain may be
noted in a minority of patients, especially in children aged < 3
years,51 but our previous study showed the presence of RRF
was not low in infants and children with MDs.42 Electron
microscopic examination of muscle cells may reveal abnormal
mitochondrial configurations and/or subsarcolemmal
abnormal mitochondrial accumulation in those patients.42 In
addition to morphological examinations, mitochondrial RC
complex enzyme analysis of biopsied muscle or skin fibro-
blasts is helpful to confirm RC complex defects and provide a
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Neonatology (2014), http://dx.doi.org/10.1016/j.pedneo.2014.03.009
clue for determining causative nDNA mutations. All patients,
prior to undergoing a muscle biopsy, should have a careful
clinical and metabolic workup to detect the extent of organ
involvement and metabolic disturbance.
5.2.5. Mitochondrial genetic testing
The confirmation of the MD diagnosis by molecular methods
often remains a challenge because of the large number of
known genes involved in mitochondrial energy production,
the complexity of the two genomes, and the heteroplasmic
proportions of pathogenic mtDNA in a patient. A molecular
diagnostic approach for MDs has been proposed.79 If the
phenotype is suggestive of syndromic MDs, screening of
common point mutations, common deletions in mtDNA or
specific nDNA mutations is the first step. In cases where
characteristic mitochondrial RC complex deficiencies and
histochemical abnormalities are observed, direct
sequencing of the specific causative nuclear genes can be
performed on white blood cell DNA. Measurement of mtDNA
content in affected tissues, such as muscle, also allows
screening for mtDNA depletion syndromes. In selected
cases testing negative, additional analyses can include
whole mtDNA genome screening for the detection of rare
and novel point mutations. As new nDNA mutations are
constantly being discovered, clinicians may use next gen-
eration sequencing (NGS) for molecular analysis. Known
causative genes of MDs will improve genetic counseling.
5.3. Diagnostic algorithm
As the clinical manifestations of MDs are variable, diagnosis
of MDs is much more difficult compared with other diseases.
Therefore, the author suggests an evaluation process for
clinical physicians to diagnose MDs in clinical practice
(Figure 2). Diagnostic workups for suspected MDs are a
stepwise procedure. The first step comprises a comprehen-
sive individual and family history, and clinical investigations
in neurology, ophthalmology, otology, endocrinology, car-
diology, gastroenterology, nephrology, hematology, and so
on. Important instrumental procedures include basic
chemical investigations of serum and urine, electrophysio-
logical investigations, and neuroimaging studies. Based on
the results, the probability for the presence of a MD can be
assessed based on the Bernier diagnostic criteria.75 In the
second step, clinicians need to decide whether an individual
clinical phenotype conforms to any of the syndromic MDs or
is characteristic of nonsyndromic MDs. When the presenta-
tion is classic for specific mitochondrial syndromes, for
example, LS, MELAS, MERRF, LHON, or KSS, appropriate
mtDNA studies should be obtained first. When the clinical
picture is classic for a nDNA inherited syndrome and the
gene or the linkage is known, for example, MNGIE, Alper’s
disease, some LS, or BTHS, proceed with genetic studies.
When the clinical picture is nonsyndromic but biochemical
findings are highly suggestive of a MD, clinicians can consider
proceeding with muscle and/or tissue biopsies, and/or
measurement of mitochondrial RC enzymatic assays. By
applying this approach, clinicians can use diagnostic criteria
to obtain a more accurate diagnosis. If the diagnosis is still
uncertain and difficult, it is necessary to follow up the
clinical manifestations of the patients.
 + MODEL
Mitochondrial diseases in infants and children 9

Does the infant or child have a mitochondrial disease?

History taking, physical examinations, and neurological examinations

• Variable clinical manifestations, including unexplained multiorgan disorders
• CNS symptoms and signs, including refractory seizures with a progressive encephalopathy or encephalomyopathy, etc.
• “Red flag” of extra-CNS symptoms and signs, including short stature, neurosensory hearing loss, progressive external ophthalmoplegia,
pigmentary retinopathy, optic neuropathy, peripheral neuropathy, cardiomyopathy, cardiac arrhythmia, hepatopathy, renal tubular acidosis, etc.
• Family history of unexplained developmental delay, epileptic seizures, or early death, etc.
• Soft signs in maternal relatives, including short stature, migraine, deafness, and diabetes, etc.

• Laboratory tests: CBC, electrolytes, ABG, blood sugar, blood ketone, ammonia, lactate, Ms/Ms assay, amino acids assays, UOA assay, etc.
• Specific examinations: Hearing test, ECG, EEG, echocardiography, fundus examination, NCV, evoked potentials, etc.
• Neuroimaging studies: Brain MRI and MRS, etc.

Suspected syndromic MDs Suspected nonsyndromic MDs

Common mtDNA mutations, OGLST

Common mtDNA deletions, + −
Specific nDNA mutations, or
Depletion genes (Table 1)
+ − Muscle and/or tissue biopsy
+ -
Genetic counseling OGLST
+ − Bernier diagnostic criteria75 Follow-up
• Neurological and
Family member
Muscle and/or tissue biopsy nonneurologic S/S
studies Common mtDNA mutations, • Clinical evolution
+ − Common mtDNA deletions,
Specific nDNA mutations or
Bernier diagnostic criteria75 Depletion genes (Table 1) Re-evaluate
Follow-up
• Laboratory tests
• Neurological and + − • Neuroimaging studies
nonneurologic S/S
Whole mtDNA sequencing, or
• Clinical evolution
NGS for other genes (Table 1) Genetic counseling NGS for other
+ genes (Table 1) Muscle and/or tissue
Re-evaluate biopsy if indicated
Genetic counseling Family member +
• Laboratory tests
studies Genetic counseling
• Neuroimaging studies
Family member
studies Family member
Muscle and/or tissue
studies
biopsy if indicated

Figure 2 A diagnostic algorithm in infants and children with mitochondrial disease. ABG Z arterial blood gas; CBC Z complete
blood cell count; CNS Z central nervous system; ECG Z electrocardiogram; EEG Z electroencephalography; MDs Z mitochondrial
diseases; MRI Z magnetic resonance imaging; MRS Z magnetic resonance spectroscopy; Ms/Ms Z tandem mass spectrometry;
mtDNA Z mitochondrial DNA; NCV Z nerve conduction velocity; nDNA Z nuclear DNA; NGS Z next generation sequencing;
OGLST Z oral glucose lactate stimulation test; ; S/S Z symptoms and signs; UOA Z urinary organic acids.

5.4. Diagnostic dilemma
Although different consensus diagnostic criteria have been
proposed, an unresolved issue is the question of primary
versus secondary RC involvement. A growing number of
reports show RC involvement in other inborn errors of
metabolism such as fatty acid oxidation disorder,
thiamine-responsiveness megaloblastic anemia, and
various neurodegenerative disorders of childhood.76
Careful interpretation of laboratory results is needed as
enzyme deficiencies found on RC testing may be indicative
of the primary disease or be secondary to another primary
pathological condition. The diagnosis of MDs should
therefore be conducted with care, especially in infants
and children.
current approach to diagnosing and classifying MDs in-
corporates clinical, biochemical, neuroradiological, patho-
logic, and molecular information. The confirmation or
exclusion of MDs is therefore a major challenge for clini-
cians, especially in cases with nonspecific clinical pheno-
types. Therefore, follow-up evaluations of clinical
symptoms/signs and biochemical data are crucial.
Conflicts of interest
The author declared no conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Acknowledgments

6. Conclusion The author would like to thank Hsiu-Fen Lee, MD for

collection of clinical data and study design, Chi-Zen Tsai,
The study of MDs is a challenging and rapidly evolving field MSC, Tzu-Chao Wang, MSC, Chia-Ju Li, MSC, and Tzu-Yun
of medicine. A single clinical feature or diagnostic criterion Hwang, MSC for molecular analysis, and Chia-Chi Hsu, MD
is rarely sufficient for the proper diagnosis of a MD. The and Liang-Hui Chen, MSC for assays of metabolic profiles.

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Neonatology (2014), http://dx.doi.org/10.1016/j.pedneo.2014.03.009
 + MODEL
10 C.-S. Chi

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Please cite this article in press as: Chi C-S, Diagnostic Approach in Infants and Children with Mitochondrial Diseases, Pediatrics and
Neonatology (2014), http://dx.doi.org/10.1016/j.pedneo.2014.03.009