Pediatrics and Neonatology (2015) 56, 7e18

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INVITED REVIEW ARTICLE

Diagnostic Approach in Infants and Childrenwith Mitochondrial Diseases

Ching-Shiang Chi*

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

Received Mar 17, 2014; accepted Mar 27, 2014Available online 20 August 2014

Key Wordsdiagnosis;infants and children;mitochondrialdiseases;

Taiwan

* Department of Pediatrics, Tungs’ TE-mail address: chi-cs@hotmail.co

http://dx.doi.org/10.1016/j.pedneo.21875-9572/Copyright ª 2014, Taiwan

Mitochondrial diseases are a heterogeneous group of disorders affecting energy production inthe human body. The diagnosis of mitochondrial diseases represents a challenge to clinicians,especially for pediatric cases, which show enormous variation in clinical presentations, as wellas biochemical and genetic complexity.

Different consensus diagnostic criteria for mitochondrial diseases in infants and children areavailable. The lack of standardized diagnostic criteria poses difficulties in evaluating diag-nostic methodologies. Even though there are many diagnostic tools, none of them are sensitiveenough 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-basedmolecular diagnostic testing. The confirmation or exclusion of mitochondrial diseases remainsa 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 associatedphenotypes in infants and children with mitochondrial diseases, in addition to providing anoverview 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 rightsreserved.

aichung Metroharbor Hospital, 699, Taiwan Boulevard Section 8, Wuchi, Taichung, 435, Taiwan.m.

014.03.009Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.

8 C.-S. Chi

1. Introduction

Mitochondrial disease (MD) was first introduced in 1962,when a group of investigators, Luft et al1, described ayoung Swedish woman with severe nonthyroid origin hy-permetabolism. In 1963, Engel and Cunningham2 used amodification of the Gomori trichrome stain that allowed forthe detection of abnormal mitochondrial proliferation inmuscle as irregular purplish patches in fibers that weredubbed “ragged red fibers (RRFs)”. Since then, there hasbeen great interest in the diagnosis of mitochondrial dis-eases (MDs). The diagnosis of MD can be traced back to thepremolecular era, 1962e1988, when MDs were defined onthe basis of clinical examination, muscle biopsy, andbiochemical criteria. In the molecular era, the fullcomplexity of these disorders became evident.

MDs are a heterogeneous group of disorders affectingenergy production in the human body, which can occur witha probable frequency in preschool children (age < 6 years)of approximately 1 in 11,000,3 and the minimum birthprevalence for respiratory chain disorders with onset at anyage was estimated at 1 in 7634.4 MDs may present at anyage with a spectrum of symptoms and signs spanninga number of medical specialties. As cells with high energyrequirements, such as neurons, skeletal and cardiac

Glycolysis

Pyruvate LactateAlanine

Pyruvate

DIC

H+

H+ Pyruvate

Acetyl-CoAPDHC

Glucose

Amino acids

Amino acids

Citrate

α -Ketogluta

Succinyl-CoA

Succinate

Fumarate

Malate

Oxaloacetate

NADH

TCA cycle

CoQ CytbT

CoFAD FeS FeS FeSComplex II

FMN FeS FeS FeS FeSComplex I

CPT-II

Fatty –acyl-CoA

β -oxidationPC

Figure 1 Metabolic pathways in mitochondrion. ADP ZCACT Z carnitineeacylcarnitine translocase; CoQ Z coenzyme Q;Cytb Z cytochrome b; Cytc Z cytochrome c; DIC Z dicarboxylateprotein; FMNZ flavin mononucleotide; NADHZ nicotinamide adendehydrogenase complex; TCA Z tricarboxylic acid.

muscle, are particularly vulnerable to limited adenosinetriphosphate (ATP) supply, encephalopathy and myopathyare often prominent features in various mitochondrialphenotypes. Enzymes involved in mitochondrial energyproduction are coded by two genomes (mitochondrial andnuclear) in the organism. Due to their clinical, biochemical,and genetic complexity, MDs represent a challenge to cli-nicians, especially for pediatric cases, which show enor-mous variation in clinical presentations and course.

In Taiwan, the first clinical MD was reported in 1988,5

and the first mitochondrial DNA (mtDNA) mutation wasidentified in 1992.6 Many cases have since been reported inpediatric groups.7e47 Although MDs are being diagnosedmore frequently, clinical physicians still find diagnosis achallenge because clinical symptoms/signs evolve overtime and the number of genes known to be involved inmitochondrial energy production continues to increase.

The purpose of this study is to review the molecularclassification scheme and associated phenotypes in infantsand children with MDs, in addition to providing an over-view of the basic biochemical reactions and geneticcharacteristics in the mitochondrion, clinical manifesta-tions, and diagnostic methods. A diagnostic algorithm foridentifying MDs in pediatric neurology patients isproposed.

Outer m

itochondrial mem

brane

Inner mitochondrial m

embrane

Cytosol

Matrix

Mitochondrial DNA

Nuclear DNA

rate

Complex IV CytbK FeS Cytc1 Cytc Cyta Cyta3 O2

mplex V (ATPase 6,8)ATP synthase

ADP + Pi ATP

Complex III

Fatty acids

Fatty –acyl-CoA

CPT-I

CACT

Fatty –acyl-carnitine Carnitine

CarnitineFatty –acyl-carnitine

adenosine diphosphate; ATP Z adenosine triphosphate;CPT Z carnitine palmitoyltransferase; Cyta Z cytochrome a;carrier; FAD Z flavin adenine dinucleotide; FeS Z iron sulfur

ine dinucleotide; PCZ pyruvate carboxylase; PDHCZ pyruvate

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

Mitochondrial diseases (MDs) Type of mutation Mutated gene

mtDNA mutationsmtDNA rearrangementsChronic progressive external ophthalmoplegia (mtPEO) mtDNA deletion,

duplicationmtDNA

KearnseSayre syndrome (KSS) mtDNA deletion,duplication

mtDNA

Pearson’s syndrome (PS) mtDNA deletion,duplication

mtDNA

mtDNA point mutationMitochondrial encephalomyopathy, lactic acidosis, and

stroke-like episodes (MELAS)np 3243, np 3271,and others

mt-tRNALeu, mt-tRNAPhe,mt-tRNAVal, COXII, COXIII,ND1, ND5, ND6, rRNA

Myoclonic epilepsy with ragged-red fibers (MERRF) np 8344, np 8356,and others

mt-tRNALys

Leber’s hereditary optic neuropathy (LHON) np 11778, np 14484,and others

RCCI subunits

Leigh syndrome np 8993, np 10191,and others

ATPase 6

Neuropathy, ataxia, and retinitis pigmentosa (NARP) np 8993, and others ATPase6Maternal inherited Leigh syndrome (MILS) np 8993, and others ND1-6, COXIII, ATPase6, tRNAs

nDNA mutationsMutated RC subunitsNonsyndromic MDs with early-onset hypotonia,

cardiomyopathy, ataxia, psychomotor delaynDNA RCCI-subunits (NDUFS1,

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 inNDUFA11 or NDUFA1

Late-onset neurodegenerative disease nDNA RCCII (SDHA)Leigh syndrome (LS) nDNA RCCII (SDHA)Nonlethal phenotype of severe psychomotor retardation,

extrapyramidal signs, dystonia, athetosis and ataxia, mild axialhypotonia, and dementia

nDNA RCCIII (UQCRQ)

Severe infantile encephalomyopathy nDNA RCCIII (COX6B1)Mutated ancillary proteinsLeigh syndrome (LS) nDNA SURF1 encoding COX

assembly factorLeigh 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 PDHCLeigh syndrome (LS) nDNA E1-alpha-subunit of PDHCLeigh syndrome (LS), COX-deficient nDNA COX assembly factors

(SCO2, SCO1, COX10, COX15)Multisystem fatal infantile disorders, encephalomyopathy plus

cardiomyopathy, nephropathy, or hepatopathynDNA COX assembly factors

(SCO2, SCO1, COX10, COX15)Lethal infantile growth retardation, aminoaciduria, cholestasis,

iron overload, lactacidosis and early death (GRACILE) syndromenDNA RCCIII assembly protein

(BCS1L)Sensorineural hearing loss with pilli torli (Bjornstad syndrome) nDNA RCCIII assembly protein

(BCS1L)Congenital lactacidosis and fatal infantile multisystem disease,

involving brain, liver, heart, and musclenDNA RCCV assembly gene

(ATP12)Neonatal encephalo cardiomyopathy nDNA RCCV assembly gene (TMEM 70)

(continued on next page)

Mitochondrial diseases in infants and children 9

Table 1 (continued )

Mitochondrial diseases (MDs) Type of mutation Mutated gene

Defects of intergenomic signalingmtDNA breakage syndromes (large-scale mtDNA deletion)

Autosomal dominant PEO (adPEO) nDNA ANT1, PEO1, POLGAutosomal recessive PEO (arPEO) nDNA POLGSensory ataxic neuropathy, dysarthria, ophthalmoplegia (SANDO) nDNA POLGSpino-cerebellar ataxia and epilepsy with or without

ophthalmoplegia (SCAE)nDNA POLG

Infantile Aplers-Huttenlocher-syndrome (AHS) manifests asmyopathy, hepatopathy, epilepsy, migraine, intractable seizuresand mental retardation (hepatic poliodystrophy)

nDNA POLG

Mitochondrial neuro-gastrointestinal encephalomyopathy(MNGIE)

nDNA TYMP

MNGIE-like phenotype nDNA POLGmtDNA depletion syndromes (DPSs)Myopathic DPS

Muscle weakness, elevated creatine kinase, encephalopahy nDNA TK2adPEO, mild myopathy nDNA ANT1 (SLC25A4)Myopathy, renal tubulopathy nDNA RRM2BAHS, PEO, Myoclonic epilepsy myopathy sensory ataxia, ataxia

neuropathy spectrum, childhood myocerebrohepatopathyspectrum

nDNA POLG

Encephalomyopathic DPSLeigh-like syndrome with muscle hypotonia, lactic acidosis,

dystonic and moderate methylmalonic acidurianDNA SUCLA2,

Fatal infantile lactic acidosis, dysmorphism and methylmalonicaciduria with muscle and liver mtDNA depletion

nDNA SUCLG1

Hypotonia, developmental delay, and renal tubulopathy nDNA RRM2BHepato-cerebral DPS

Phenotypes as PLOG described above nDNA POLGAlpers-like phenotype nDNA PEO1Hepatic failure, hypoglycemia, muscle hypotonia, ataxia,

dystonia, or polyneuropathy, Navajo neurohepatopathynDNA MVP17

MNGIE nDNA TYMPHepatopathy, severe failure to thrive, lactic acidosis,

hypoglycemia, neurological symptomsnDNA DGUOK

Infantile-onset spinocerebellar ataxia, adPEO, myopathy,Parkinsonian features of rigidity tremor, and bradykinesia

nDNA C10orf2 (Twinkle)

Defective mitochondrial protein synthesis machineryMitochondrial myopathy and sideroblastic anemia (MLASA) nDNA PUS1Leukoencephalopathy with brainstem and spinal cord

involvement and lactacidosis (LBSL)-syndromenDNA DARS2

Pontocerebellar hypoplasia (PCH) with prenatal-onset,cerebellar/pontine atrophy/hypoplasia, microcephaly,neocortical atrophy and severe psychomotor impairment

nDNA RARS2

Agenesis of corpus callosum, dysmorphic features, fatal neonatallactic acidosis

nDNA MRPS16

ad intermediate Charcot-MarieeTooth neuropathy, type C nDNA YARS2Defects of the mitochondrial lipid milieu

Barth syndrome characterized by mitochondrial myopathy,hypertrophic or dilated cardiomyopathy, left ventricularhypertrabeculation/noncompaction, growth retardation,leukopenia and 3-methylglutaconic aciduria

nDNA TAZ

Sensorineural deafness, encephalopathy, Leigh-like syndrome,and 3-methylglutaconic aciduria

nDNA SERAC1

Sengers syndrome characterized by congenital cataracts,hypertrophic cardiomyopathy, skeletal myopathy, and lacticacidosis

nDNA AGK

10 C.-S. Chi

Table 1 (continued )

Mitochondrial diseases (MDs) Type of mutation Mutated gene

Coenzyme Q defectsNonsyndromic MDs: encephalomyopathy with recurrent

myoglobinuria, brain involvement and ragged red fibernDNA Primary CoQ deficiency

(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 machineryDeafness-dystonia syndrome (DDS): childhood-onset progressive

deafness, dystonia, spasticity, mental deterioration and blindnessnDNA DDP1/TIMM8A

X-linked sideroblastic anemia with ataxia (XLSA/A) nDNA ABCB7Congenital lactic acidosis, hypertrophic cardiomyopathy and

muscle hypotonianDNA SLC25A3

Defects in mitochondrial biogenesisDominant optic atrophy (ADOA) nDNA OPA1CharcoteMarieeTooth neuropathy-2A nDNA MFN2Severe infantile encephalopathy nDNA DLP1Hereditary spastic paraplegia nDNA KIF5A

ABCB7Z ATP-binding cassette, sub-family B (MDR/TAP), member 7; AGK Z acylglycerol kinase; ANT1Z adenine nucleotide translocase1 gene; C10orf2 Z chromosome 10 open reading frame 2; COX Z cytochrome c oxidase; DARS2 Z aspartyl-tRNA synthetase 2;DDP1Z deafness dystonia peptide 1; DGUOKZ deoxyguanosine kinase; DLP1Z dynamin-like protein-1; KIF5AZ kinesin family member5; 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-Sprotein; NDUFV Z NADH dehydrogenase (ubiquinone) flavoprotein; np Z nucleopeptide; OPA1 Z optic atrophy 1; PDHC Z pyruvatedehydrogenase 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-formingsuccinyl-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.

Mitochondrial diseases in infants and children 11

2. Biochemical reactions in the mitochondrion

The mitochondrion, an extranuclear organelle, is 0.5e1 mmin size. It consists of outer and inner membranes, anintermembranous space, and an inner matrix compartment.The matrix contains various enzymes, ribosomes, transferRNAs (tRNAs), and mtDNA molecules. Each cell containsmany mitochondria which are responsible for cellular ATPproduction by oxidative phosphorylation. Energy produc-tion of mitochondria comes from the metabolism of glucoseand fatty acids through a series of reactions (Figure 1).

Pyruvate, the end-product of aerobic glycolysis, isderived partly from blood-borne glucose but mainly fromendogenous glycogen. Once formed in the cell cytosol,pyruvate may be reduced to lactate, transaminated toalanine, or transported into the mitochondria where itundergoes oxidative decarboxylation to acetyl-coenzyme A

(acetyl-CoA) catalyzed by the pyruvate dehydrogenasecomplex (PDHC). Long-chain fatty acids, after being acti-vated to form fatty acyl-CoA in the cytosol, must betransferred across the inner mitochondrial membrane to beoxidized to acetyl-CoA. Acetyl-CoA then enters the citricacid cycle (Krebs cycle), which releases eight hydrogenmolecules and produces carbon dioxide and water throughoxidative phosphorylation. This process liberates energyalong the respiratory chain, which receives energy-richhydrogen atoms from nicotinamide adenine dinucleotide(NADH) or flavineadenine dinucleotide (FADH), producedmainly 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 energyproduction.

Mitochondrial respiratory chain (RC) complex is a uniquesystem in the cell coded by two genomes, known as the

12 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), andcytochrome c. Complexes I, III, and IV extrude protons fromthe mitochondrial matrix. Complex IV consumes oxygen toform water. Complex V couples ATP synthesis to protonreentry, which is powered by the electrochemicalgradient.48 Ultimately, the energy is stored as ATP. Theseassumptions are the basis for the biochemical evaluation ofmitochondrial RC enzymes’ activity.

The term “mitochondrial diseases” refers to a class ofdisorders 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 13genes encode subunits of the respiratory chain, and 22tRNAs and two ribosomal RNA genes (12S and 16S) translatemtDNA. Each cell contains many mitochondria with2e10 mtDNA molecules. The total mtDNA accounts forapproximately 0.5% of the DNA in a nucleated somatic cell.

The 13 mitochondrial protein-coding genes contribute tothe fourenzymecomponentsof theRCcomplexes required foroxidative phosphorylation: seven of them are subunits ofcomplex I (NADH-ubiquinoneoxidoreductase), oneofcomplexIII (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 thefactors involved in mtDNA replication, transcription, andtranslation, are encoded by nuclear DNA (nDNA).

Mitochondrial genetics differ from Mendelian genetics inseveral fundamental aspects. First, mtDNA is maternallyinherited because mitochondria derive solely from theoocyte. A normal cell has only a single mtDNA genotype andis therefore homoplasmic. However, if the genomesrepresent a mixture of a wild-type and a mutated-type, thecell genotype is heteroplasmic. The clinical pictures ofmitochondrial diseases are determined by the proportion ofnormal-to-mutated genomes in the mitochondria. Once theproportion exceeds a theoretical threshold, the biologicalbehavior of the cell will change. This minimum criticalamount differs from tissue to tissue and different tissuesmay be variously affected by different combinations and todifferent degrees. In addition, mitotic segregation of themitochondria influences the biological behavior governingthe stochastic redistribution of wild-type and mutated ge-nomes during mitochondrial and cell divisions. Thus, theconcepts of threshold effect and mitotic segregation pro-vide theoretical explanations for the variable phenotypeexpressions of the MDs.

The dual genetic control of the RC represents the ubiq-uitous nature of mitochondria. The mitochondrial metabolicpathway is under the control of not only the mitochondrial,but also the nuclear genomes, which are strictly coordinatedto ensure the correct functioning of the mitochondrial

machinery. To date, it has been established that 1500nuclear-encoded proteins are targeted to the mitochon-dria.49 Mutations in nDNA can affect: (1) components of RCsubunits; (2) mitochondrial ancillary proteins involved in RCcomplexes formation, turnover, and function; (3) inter-genomic signaling for mtDNA maintenance or expression; (4)biosynthetic enzymes for lipids or cofactors; (5) coenzymeQ; (6) mitochondrial trafficking or transport machinery; (7)mitochondrial biogenesis; or (8) apoptosis.50

A brief summary of published genetic classification ofMDs is shown in Table 1.49,50 Primary mtDNA mutationsmay exhibit a maternal inheritance, or Mendelian traitsthat present with multiple somatic mtDNA alterations.In the case of Mendelian disorders, autosomal recessive,autosomal dominant, and X-linked patterns have all beenobserved. Recent studies have demonstrated that approx-imately 10e15% of MDs are caused by mutations inmtDNA,48 and up to 25% of selected children with MDs arecaused by mutations in nDNA49; i.e., MDs in children resultmore often from nDNA mutations. In addition, it has beenreported that > 200 nuclear-encoded genes have beenlinked to human disease.49 Therefore, genetic diagnosis ofMDs in infants and children is a difficult task due to theheteroplasmy of mtDNA mutations and the large number ofnuclear genes involved in different tissues.

4. Clinical manifestations

MDs that result from diminishing ATP production cause clin-ical disorders. Several studies51e53 showed clinical featuresin patients with MDs which support the maxim “any tissueand any signs at any age”.54 However, infants and childrenwith MDs tend to have an acute onset of the clinical symp-toms and courses compared with adults with MDs, whotypically exhibit slow and/or progressive presentations. Ingeneral, the clinical spectrum is not limited to the neuro-muscular system; indeed, a number of nonneuromuscularorgans may exhibit symptoms and signs, such as the heart,eyes, ears, kidneys, endocrine glands, liver, bone marrow,and gastrointestinal tract.42 The clinical manifestations of103 pediatric patients with MDs in our case series, expandingdata from the previous report,42 are summarized in Table 2.The commonest clinical manifestation was symptoms andsigns of CNS (93/103; 90.3%). The CNS manifestations of MDswere variable, including seizures, developmental delay,altered level of consciousness, floppiness, spasticity, mentalretardation, sucking difficulty, involuntary movement,headache, apnea, external ocular motility limitation,tremor, apneustic respiration, dystonia, stroke, sudden in-fant death syndrome, and/or hypoventilation. The secondmost 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 systemincluded ptosis, retinitis pigmentosa, external oph-thalmoplegia, visual loss, nystagmus, exotropia, blurredvision, visual field defect, strabismus, corneal clouding, and/or optic nerve atrophy. The fourth most common clinicalfeature 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

Table 2 Clinical manifestations in 103 infants and chil-dren with mitochondrial diseases.

Involved organ system N Z 103

Central nervous system 93 (90.3)Ophthalmologic system 37 (35.9)Failure to thrive 37 (35.9)Cardiovascular system 26 (25.2)Gastrointestinal system 23 (22.3)Muscular system 22 (21.4)Hearing system 20 (19.4)Hepatic system 19 (18.4)Short stature 18 (17.5)Urologic system 9 (8.7)Endocrinologic system 8 (7.8)Peripheral nervous system 4 (3.9)Pancreas 2 (1.9)Hematologic system 1 (1.0)Psychic 1 (1.0)Pulmonary edema 1 (1.0)

Data are presented as n (%).

Mitochondrial diseases in infants and children 13

clinical features complicate the diagnosis of MDs and thepresentations may result in patients visiting numerous othersubspecialists and receiving various diagnoses.

There are two main clinical classifications of MDs: syn-dromic MDs and nonsyndromic MDs. Patients who presentedwith characteristic clinical phenotypes reported in theliterature were categorized as having specific mitochon-drial syndromes,55e68 including Leigh syndrome (LS),55,69

Alpers’ disease,57 lethal infantile mitochondrial disease(LIMM),59,60 Pearson’s syndrome (PS),58,70e72 KearnseSayresyndrome (KSS),56 mitochondrial encephalomyopathy, lac-tic acidosis, and stroke-like episodes (MELAS),64 myoclonicepilepsy with ragged-red fibers (MERRF),61,62 neuropathy,ataxia, and retinitis pigmentosa (NARP),67 mitochondrialneuro-gastrointestinal encephalomyopathy (MNGIE),65

chronic progressive external ophthalmoplegia (CPEO),63

Leber’s hereditary optic neuropathy (LHON),66 and Barthsyndrome (BTHS).68 The patients who fulfilled the diag-nostic criteria for MDs but not specific syndromes, wereclassified as having noncategorized or nonsyndromic MDs.42

Recently published studies42,51e53 show that pediatric pa-tients with nonsyndromic MDs are not uncommon, and theclinical manifestations are nonspecific.42,73

Specific mitochondrial syndromes are easy to diagnosebecause each of them has its own clinical phenotypes.However, it is not easy to diagnose nonsyndromic MDsduring the early stage of disease course due to the widevariety of clinical features. Thus, follow-up of clinicalsymptoms/signs in these patients is important.

5. Diagnostic approach of mitochondrialdiseases

5.1. Diagnostic criteria

Different consensus diagnostic criteria for MDs in childrenare available. Major and minor criteria are usually based onclinical, biochemical, pathologic, and molecular findings.

In 1996, Walker et al74 proposed diagnostic criteria foradults with respiratory chain disorders (Adult criteria; AC).In 2002, Bernier et al75 suggested a modified version of theAdult criteria (Modified Walker criteria or Modified AdultCriteria; MAC) in order to improve the sensitivity and expandits use to include pediatric patients as well as adults. Thediagnostic criteria of MDs were established on the basis ofassigning major or minor criteria for clinical, pathological,enzymatic, functional, molecular, and metabolic parame-ters. Mitochondrial encephalomyopathies were categorizedas definite, probable, or possible. A definite diagnosis isdefined as fulfillment of either of two major criteria or onemajor criterion plus two minor criteria. A probable diagnosisis defined as either one major criterion and one minor cri-terion or at least three minor criteria. A possible diagnosis isdefined as either a single major criterion or two minorcriteria, one of which must be clinical.

In 2002, Wolf et al76 proposed the consensus mitochon-drial diagnostic criteria (MDC) scoring system for infantsand children, which evaluates clinical features (I; maximumclinical score: 4 points) of muscle symptoms (IA; maximumscore: 2 points), CNS abnormalities (IB; maximal score: 2points), and multisystem involvement (IC; maximal score: 3points), adding metabolic abnormalities and neuroimaging(II; maximum addition score: 4 points). Histologic anomalies(III) could increase the score with 4 points, leading to amaximum score of 12 points. Scores of 8e12, 5e7, 2e4, and1 were defined as definite, probable, possible, or unlikelymitochondrial disorders, respectively. The MDC uses well-defined items (clinical, laboratory, pathologic, andbiochemical) for scoring and dividing criteria into twosubsets: general (clinical, metabolic, imaging, and patho-logic) and biochemical. This allows a critical and indepen-dent evaluation for general features and for results ofbiochemical investigations, before combining the two. Theadvantages of MDC criteria are that the general criteria(without the histochemical part) allow preclassification ofpatients before a muscle biopsy is performed. If a patientwith an undiagnosed disorder reaches the probable ordefinite general classification, a muscle biopsy may bestrongly recommended.

The different consensus diagnostic criteria for MDs inchildren described above show how difficult it can be todiagnose MDs; that is, the diagnosis of MDs with certaintyrequires analysis from multiple perspectives.

5.2. Diagnostic tools

5.2.1. Blood laboratory investigationLack of a clinically pathognomonic hallmark frequentlymakes laboratory investigations necessary to confirm thediagnosis.

For basic laboratory investigations, blood lactate is usedas a biochemical marker for the screening of MDs. Thedifferential diagnosis of lactic acidosis includes physiolog-ical anaerobic exercise, systemic diseases that increaseblood lactate levels, cerebral diseases that increase CSFlactate levels, metabolic diseases, poor collection tech-nique, or poor sample handling.77 However, blood lactatelevel is not always elevated in patients with MDs, and thus,normal serum lactic level does not exclude a mitochondrial

14 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 asingle blood lactate test.3,11,42 Lactic acidosis may be pro-portionate or disproportionate to the elevation of pyruvatebased on the associated effect of the biochemical defect onthe oxidationereduction potential. If the oxida-tionereduction potential is unaffected by the biochemicaldefect, the lactate and pyruvate elevations will be pro-portional and the lactate/pyruvate ratio will be normal. Bycontrast, if the oxidationereduction potential is disturbedby a primary defect involving the respiratory chain, thelactate values will be disproportionately elevated and thelactate/pyruvate ratio will be increased (> 25).

If available, CSF lactate, and pyruvate concentrationshould also be measured. It is useful to calculate a lactate-to-pyruvate ratio because an elevated ratio suggests thepossibility of a MD.

5.2.2. Metabolic surveyLactic acidosis can be caused not only by intramitochondrial(primary) disorders, but also by extramitochondrial (sec-ondary) disorders. The latter include defects of metabolicpathways for glycogen, gluconeogenesis, and organic acid-emia. Thus, a metabolic screening with measurement ofbasic 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 organicacids, is helpful for differential diagnosis of MDs.

5.2.3. Imaging studiesThe clinical diagnosis of MDs has been greatly improved byadvances in neuroimaging technology. Brain magnetic reso-nance imaging (MRI) is sensitive to these diseases, especiallyin clinically phenotypic mitochondrial syndromes, includingLS, MELAS, MNGIE, and KSS. In infants and children withnonsyndromic MDs, brain MRI can produce variable findings,from normal results to signal changes over the basal gangliaor brainstem.43 MR spectroscopy (MRS) for detecting theconcentration of a number of biochemical metabolitesin vivo has been reported to be a useful tool in conductingdifferential diagnoses and for monitoring of MDs.44,78

5.2.4. Tissue biopsy and MRC enzymatic assayA muscle biopsy using light microscopic and electron micro-scopic examinations is an auxiliary diagnostic method of MDs.Morphological examinations include the modified Gomoritrichrome staining for RRFs, ATPase staining for the assess-ment of myofibrillar integrity, muscle-type fiber predomi-nance and distribution, and cytochrome c oxidase andsuccinate dehydrogenase staining for oxidative enzymes.RRFs showing on themodified Gomori trichrome stain may benoted in aminority of patients, especially in childrenaged< 3years,51 but our previous study showed the presence of RRFwas not low in infants and children with MDs.42 Electronmicroscopicexaminationofmuscle cellsmayreveal abnormalmitochondrial configurations and/or subsarcolemmalabnormal mitochondrial accumulation in those patients.42 Inaddition to morphological examinations, mitochondrial RCcomplex enzyme analysis of biopsied muscle or skin fibro-blasts is helpful to confirm RC complex defects and provide a

clue for determining causative nDNA mutations. All patients,prior to undergoing a muscle biopsy, should have a carefulclinical and metabolic workup to detect the extent of organinvolvement and metabolic disturbance.

5.2.5. Mitochondrial genetic testingThe confirmation of the MD diagnosis by molecular methodsoften remains a challenge because of the large number ofknown genes involved in mitochondrial energy production,the complexity of the two genomes, and the heteroplasmicproportions of pathogenic mtDNA in a patient. A moleculardiagnostic approach for MDs has been proposed.79 If thephenotype is suggestive of syndromic MDs, screening ofcommon point mutations, common deletions in mtDNA orspecific nDNA mutations is the first step. In cases wherecharacteristic mitochondrial RC complex deficiencies andhistochemical abnormalities are observed, directsequencing of the specific causative nuclear genes can beperformed on white blood cell DNA. Measurement of mtDNAcontent in affected tissues, such as muscle, also allowsscreening for mtDNA depletion syndromes. In selectedcases testing negative, additional analyses can includewhole mtDNA genome screening for the detection of rareand novel point mutations. As new nDNA mutations areconstantly being discovered, clinicians may use next gen-eration sequencing (NGS) for molecular analysis. Knowncausative genes of MDs will improve genetic counseling.

5.3. Diagnostic algorithm

As the clinical manifestations of MDs are variable, diagnosisof MDs is much more difficult compared with other diseases.Therefore, the author suggests an evaluation process forclinical physicians to diagnose MDs in clinical practice(Figure 2). Diagnostic workups for suspected MDs are astepwise procedure. The first step comprises a comprehen-sive individual and family history, and clinical investigationsin neurology, ophthalmology, otology, endocrinology, car-diology, gastroenterology, nephrology, hematology, and soon. Important instrumental procedures include basicchemical investigations of serum and urine, electrophysio-logical investigations, and neuroimaging studies. Based onthe results, the probability for the presence of a MD can beassessed based on the Bernier diagnostic criteria.75 In thesecond step, clinicians need to decide whether an individualclinical phenotype conforms to any of the syndromic MDs oris characteristic of nonsyndromic MDs. When the presenta-tion is classic for specific mitochondrial syndromes, forexample, LS, MELAS, MERRF, LHON, or KSS, appropriatemtDNA studies should be obtained first. When the clinicalpicture is classic for a nDNA inherited syndrome and thegene or the linkage is known, for example, MNGIE, Alper’sdisease, some LS, or BTHS, proceed with genetic studies.When the clinical picture is nonsyndromic but biochemicalfindings are highly suggestive of a MD, clinicians can considerproceeding with muscle and/or tissue biopsies, and/ormeasurement of mitochondrial RC enzymatic assays. Byapplying this approach, clinicians can use diagnostic criteriato obtain a more accurate diagnosis. If the diagnosis is stilluncertain and difficult, it is necessary to follow up theclinical manifestations of the patients.

Does the infant or child have a mitochondrial disease?

• 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.

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.

Suspected nonsyndromic MDs

+

Genetic counseling

Suspected syndromic MDs

Common mtDNA mutations, Common mtDNA deletions, Specific nDNA mutations, or Depletion genes (Table 1)

Family member studies

Bernier diagnostic criteria75

Whole mtDNA sequencing, or NGS for other genes (Table 1)

Re-evaluate• Laboratory tests• Neuroimaging studies

+OGLST

Muscle and/or tissue biopsy

OGLST

−

+

Bernier diagnostic criteria75

−

NGS for other genes (Table 1)

+

Muscle and/or tissue biopsy

+Genetic counseling

Family member studies

-+

Common mtDNA mutations, Common mtDNA deletions, Specific nDNA mutations or Depletion genes (Table 1)

+

Genetic counseling

Family member studies

−

+ −

Follow-up • Neurological and

nonneurologic S/S• Clinical evolution

Re-evaluate• Laboratory tests• Neuroimaging studies

Follow-up • Neurological and

nonneurologic S/S• Clinical evolution

−

Muscle and/or tissue biopsy if indicated

Genetic counseling

Family member studies

Muscle and/or tissue biopsy if indicated

Figure 2 A diagnostic algorithm in infants and children with mitochondrial disease. ABG Z arterial blood gas; CBC Z completeblood cell count; CNS Z central nervous system; ECG Z electrocardiogram; EEG Z electroencephalography; MDs Z mitochondrialdiseases; 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.

Mitochondrial diseases in infants and children 15

5.4. Diagnostic dilemma

Although different consensus diagnostic criteria have beenproposed, an unresolved issue is the question of primaryversus secondary RC involvement. A growing number ofreports show RC involvement in other inborn errors ofmetabolism such as fatty acid oxidation disorder,thiamine-responsiveness megaloblastic anemia, andvarious neurodegenerative disorders of childhood.76

Careful interpretation of laboratory results is needed asenzyme deficiencies found on RC testing may be indicativeof the primary disease or be secondary to another primarypathological condition. The diagnosis of MDs shouldtherefore be conducted with care, especially in infantsand children.

6. Conclusion

The study of MDs is a challenging and rapidly evolving fieldof medicine. A single clinical feature or diagnostic criterionis rarely sufficient for the proper diagnosis of a MD. The

current approach to diagnosing and classifying MDs in-corporates clinical, biochemical, neuroradiological, patho-logic, and molecular information. The confirmation orexclusion of MDs is therefore a major challenge for clini-cians, especially in cases with nonspecific clinical pheno-types. Therefore, follow-up evaluations of clinicalsymptoms/signs and biochemical data are crucial.

Conflicts of interest

The author declares no conflicts of interest with respect tothe research, authorship, and/or publication of this article.

Acknowledgments

The author would like to thank Hsiu-Fen Lee, MD forcollection of clinical data and study design, Chi-Zen Tsai,MSC, Tzu-Chao Wang, MSC, Chia-Ju Li, MSC, and Tzu-YunHwang, MSC for molecular analysis, and Chia-Chi Hsu, MDand Liang-Hui Chen, MSC for assays of metabolic profiles.

16 C.-S. Chi

References

1. Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B. A case ofsevere hypermetabolism of nonthyroid origin with a defect inthe maintenance of mitochondrial respiratory control: acorrelated clinical, biochemical, and morphological study. JClin Invest 1962;41:1776e804.

2. Engel WK, Cunningham GG. Rapid examination of muscletissue. An improved trichrome method for freshefrozen bi-opsy sections. Neurology 1963;13:919e23.

3. Darin N, Oldfors A, Moslemi AR, Holme E, Tulinius M. Theincidence of mitochondrial encephalomyopathies in child-hood: clinical features and morphological, biochemical, andDNA abnormalities. Ann Neurol 2001;49:377e83.

4. Skladal D, Halliday J, Thorburn DR. Minimum birth prevalenceof mitochondrial respiratory chain disorders in children. Brain2003;126:1905e12.

5. Chi CS, Kao KP, Hsu NY, Lin E, Chen YC, Fu MC. KearnseSayresyndrome with proteinuria, glucosuria, copperuria and pro-lapse of the mitral valve: report of a case. Taiwan Yi Xue HuiZa Zhi 1988;87:95e100. [Article in Chinese].

6. Lee CC, Ko YM, Chen SS. Chronic progressive external oph-thalmoplegia with NADH-CoQ reductase deficiency: report ofa case. Zhonghua Yi Xue Za Zhi (Taipei) 1992;50:77e82.

7. Chen CH, Chi CS. Hypodensity in the basal ganglia demon-strated on CT brain scan studies in children. Chin Med J(Taipei) 1989;44:203e8.

8. Tsai ML, Hung KL, Chen TY. Subacute necrotizing encephalo-myelopathy (Leigh’s disease): report of a case. J Formos MedAssoc 1990;89:799e802.

9. Mak SC, Chi CS, Chen CH. Mitochondrial encephalomyopathypresenting with clinical Leigh’s disease: report of a case.Zhonghua Yi Xue Za Zhi (Taipei) 1991;47:54e8.

10. Lii YP, Chi CS, Mak SC, Chen CH. Myoclonic epilepsy withragged-red fibers: report of one case. Zhonghua Min Guo XiaoEr Ke Yi Xue Hui Za Zhi 1991;32:251e6.

11. Chi CS, Mak SC, Shian WJ, Chen CH. Oral glucose lactatestimulation test in mitochondrial disease. Pediatr Neurol1992;8:445e9.

12. Shian WJ, Chi CS, Mak SC. Intramyelin splitting in the spon-giform lesions of Leigh syndrome. Zhonghua Min Guo Xiao ErKe Yi Xue Hui Za Zhi 1993;34:308e13.

13. Mak SC, Chi CS, Chen CH, Shian WJ. Clinical manifestation ofmitochondrial diseases in children. Zhonghua Min Guo Xiao ErKe Yi Xue Hui Za Zhi 1993;34:247e56.

14. Wu TS, Lee CC, Lin JT, Hsu HH, Lin ST, Jong YJ, et al. Leighdisease (subacute necrotizing encephalomyelopathy): reportof one case. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi1993;34:301e7.

15. Chi CS, Mak SC, Shian WJ. Leigh syndrome with progressiveventriculomegaly. Pediatr Neurol 1994;10:244e6.

16. Lee ML, Chaou WT, Yang AD, Jong YJ, Tsai JL, Pang CY, et al.Mitochondrial myopathy, encephalopathy, lactic acidosis, andstrokelike episodes (MELAS): report of a sporadic case and re-view of the literature. Zhonghua Min Guo Xiao Er Ke Yi Xue HuiZa Zhi 1994;35:148e56.

17. Huang WY, Chi CS, Mak SC, Wu HM, Yang MT. Leigh syndromepresenting with dystonia: report of one case. Zhonghua MinGuo Xiao Er Ke Yi Xue Hui Za Zhi 1995;36:378e81.

18. Chiang LM, Jong YJ, Huang SC, Tsai JL, Pang CY, Lee HC,et al. Heteroplasmic mitochondrial DNA mutation in a patientwith mitochondrial myopathy, encephalopathy, lacticacidosis, and stroke-like episodes. J Formos Med Assoc 1995;94:42e7.

19. Shian WJ, Chi CS, Mak SC. Neuroimage in infants and chil-dren with mitochondrial disorders. Zhonghua Min Guo XiaoEr Ke Yi Xue Hui Za Zhi 1996;37:96e102.

20. Mak SC, Chi CS, Liu CY, Pang CY, Wei YH. Leigh syndromeassociated with mitochondrial DNA 8993 T –> G mutation andragged-red fibers. Pediatr Neurol 1996;15:72e5.

21. Lee WT, Wang PJ, Young C, Wang TR, Shen YZ. Cytochrome coxidase deficiency in fibroblasts of a patient with mitochon-drial encephalomyopathy. J Formos Med Assoc 1996;95:709e11.

22. Huang MY, Jong YJ, Tsai JL, Liu GC, Chiang CH, Pang CY, et al.Mitochondrial NADH-coenzyme Q reductase deficiency inLeigh’s disease. J Formos Med Assoc 1996;95:325e8.

23. Liu AM, Mak SC, Tsai CR, Chi CS. Childhood MELAS syndromepresenting with seizure and cortical blindness: a case report.Zhonghua Yi Xue Za Zhi (Taipei) 1998;61:730e5.

24. Mak SC, Chi CS, Tsai CR. Mitochondrial DNA 8993 T > C mu-tation presenting as juvenile Leigh syndrome with respiratoryfailure. J Child Neurol 1998;13:349e51.

25. Lin YC, Lee WT, Wang PJ, Shen YZ. Vocal cord paralysis andhypoventilation in a patient with suspected Leigh disease.Pediatr Neurol 1999;20:223e5.

26. Wang LC, Lee WT, Tsai WY, Tsau YK, Shen YZ. Mitochondrialcytopathy combined with Fanconi’s syndrome. Pediatr Neurol2000;22:403e6.

27. Liao SL, Huang SF, Lin JL, Lai SH, Chou YH, Kuo CY. Syndromeof mitochondrial myopathy of the heart and skeletal muscle,congenital cataract and lactic acidosis. Acta Paediatr Taiwan2003;44:360e4.

28. Chang CW, Chang CH, Peng ML. Leber’s hereditary optic neu-ropathy: a case report. Kaohsiung J Med Sci 2003;19:516e21.

29. Hung HL, Kao LY, Huang CC. Clinical features of Leber’s he-reditary optic neuropathy with the 11,778 mitochondrial DNAmutation in Taiwanese patients. Chang Gung Med J 2003;26:41e7.

30. Chang TM, Chi CS, Tsai CR, Lee HF, Li MC. Paralytic ileus inMELAS with phenotypic features of MNGIE. Pediatr Neurol2004;31:374e7.

31. Chou YJ, Ou CY, Hsu TY, Liou CW, Lee CF, Tso DJ, et al.Prenatal diagnosis of a fetus harboring an intermediate loadof the A3243G mtDNA mutation in a maternal carrier diag-nosed with MELAS syndrome. Prenat Diagn 2004;24:367e70.

32. Chung SH, Chen SC, Chen WJ, Lee CC. Symmetric basalganglia calcification in a 9-year-old child with MELAS.Neurology 2005;65:E19.

33. Lee HF, Lee HJ, Chi CS, Tsai CR, Chang P. Corneal clouding: aninfrequent ophthalmic manifestation of mitochondrial dis-ease. Pediatr Neurol 2006;34:464e6.

34. Lee HF, Lee HJ, Chi CS, Tsai CR, Chang TK, Wang CJ. Theneurological evolution of Pearson syndrome: case report andliterature review. Eur J Paediatr Neurol 2007;11:208e14.

35. Hung PC, Wang HS. A previously undescribed leukodystrophyin Leigh syndrome associated with T9176C mutation of themitochondrial ATPase 6 gene. Dev Med Child Neurol 2007;49:65e7.

36. Hung PC, Wang HS. Diffuse leukoencephalopathy: unusualsonographic finding in an infant with mitochondrial disease. JClin Ultrasound 2007;35:277e80.

37. Wang SB, Weng WC, Lee NC, Hwu WL, Fan PC, Lee WT. Mu-tation of mitochondrial DNA G13513A presenting with Leighsyndrome, WolffeParkinsoneWhite syndrome and cardiomy-opathy. Pediatr Neonatol 2008;49:145e9.

38. Chou HF, Liang WC, Zhang Q, Goto Y, Jong YJ. Clinical andgenetic features in a MELAS child with a 3271T>C mutation.Pediatr Neurol 2008;38:143e6.

39. Chen ST, Fan PC, Hwu WL, Wu MH. Fibrous dysplasia in a childwith mitochondrial A8344G mutation. J Child Neurol 2008;23:1447e50.

40. Wang W, Seak CJ, Liao SC, Chiu TF, Chen JC. Cardiac tam-ponade: a new complication in a patient with mitochondrial

Mitochondrial diseases in infants and children 17

myopathy, encephalopathy, lactic acidosis, and strokelikeepisodes. Am J Emerg Med 2008;26:382.e1e2.

41. Lee HF, Tsai CR, Chi CS, Lee HJ, Chen CC. Leigh syndrome:clinical and neuroimaging follow-up. Pediatr Neurol 2009;40:88e93.

42. Chi CS, Lee HF, Tsai CR, Lee HJ, Chen LH. Clinical manifes-tations in children with mitochondrial diseases. Pediatr Neu-rol 2010;43:183e9.

43. Chi CS, Lee HF, Tsai CR, Chen CC, Tung JN. Cranial magneticresonance imaging findings in children with nonsyndromicmitochondrial diseases. Pediatr Neurol 2011;44:171e6.

44. Chi CS, Lee HF, Tsai CR, Chen WS, Tung JN, Hung HC. Lactatepeak on brain MRS in children with syndromic mitochondrialdiseases. J Clin Med Assoc 2011;74:305e9.

45. Tsai JD, Liu CS, Tsao TF, Sheu JN. A novel mitochondrial DNA8597T > C mutation of Leigh syndrome: report of one case.Pediatr Neonatol 2012;53:60e2.

46. Lee IC, El-Hattab AW, Wang J, Li FY, Weng SW, Craigen WJ,et al. SURF1-associated Leigh syndrome: a case series andnovel mutations. Hum Mutat 2012;33:1192e200.

47. Liu HM, Tsai LP, Chien YH, Wu JH, Weng WC, Peng SF, et al. Anovel 3670-base pair mitochondrial DNA deletion resulting inmulti-systemic manifestations in a child. Pediatr Neonatol2012;53:264e8.

48. Debray FG, Lambert M, Mitchell GA. Disorders of mitochon-drial function. Curr Opin Pediatr 2008;20:471e82.

49. Goldstein AC, Bhatia P, Vento JM. Mitochondrial disease inchildhood: nuclear encoded. Neurotherapeutics 2013;10:212e26.

50. Finsterer J, Harbo HF, Baets J, Van Broeckhoven C, DiDonato S, Fontaine B, et al. EFNS guidelines on the moleculardiagnosis of mitochondrial disorders. Eur J Neurol 2009;16:1255e64.

51. Scaglia F, Towbin JA, Craigen WJ, Belmont JW, Smith EO,Neish SR, et al. Clinical spectrum, morbidity, and mortality in113 pediatric patients with mitochondrial disease. Pediatrics2004;114:925e31.

52. Garcıa-Cazorla A, De Lonlay P, Nassogne MC, Rustin P,Touati G, Saudubray JM. Long-term follow-up of neonatalmitochondrial cytopathies: a study of 57 patients. Pediatrics2005;116:1170e7.

53. Debray FG, Lambert M, Chevalier I, Robitaille Y, Decarie JC,Shoubridge EA, et al. Long-term outcome and clinical spec-trum of 73 pediatric patients with mitochondrial diseases.Pediatrics 2007;119:722e33.

54. Munnich A, Rotig A, Chretien D, Cormier V, Bourgeron T,Bonnefont JP, et al. Clinical presentation of mitochondrialdisorders in childhood. J Inherit Metab Dis 1996;19:521e7.

55. Leigh D. Subacute necrotizing encephalomyelopathy in aninfant. J Neurol Neurosurg Psychiatry 1951;14:216e21.

56. Kearns TP, Sayre GP. Retinitis pigmentosa, external oph-thalmoplegia, and complete heart block: unusual syndromewith histologic study in one of two cases. AMA Arch Oph-thalmol 1958;60:280e9.

57. Sandbank U, Lerman P. Progressive cerebral poliodystrophydAlpers’ disease. Disorganized giant neuronal mitochondria onelectron microscopy. J Neurol Neurosurg Psychiatry 1972;35:749e55.

58. Pearson HA, Lobel JS, Kocoshis SA, Naiman JL, Windmiller J,Lammi AT, et al. A new syndrome of refractory sideroblasticanemia with vacuolization of marrow precursors and exocrinepancreatic dysfunction. J Pediatr 1979;95:976e84.

59. DiMauro S, Mendell JR, Sahenk Z, Bachman D, Scarpa A,Scofield RM, et al. Fatal infantile mitochondrial myopathy andrenal dysfunction due to cytochromeec-oxidase deficiency.Neurology 1980;30:795e804.

60. DiMauro S, Nicholson JF, Hays AP, Eastwood AB,Papadimitriou A, Koenigsberger R, et al. Benign infantile

mitochondrial myopathy due to reversible cytochrome c oxi-dase deficiency. Ann Neurol 1983;14:226e34.

61. Fukuhara N, Tokiguchi S, Shirakawa K, Tsubaki T. Myoclonusepilepsy associated with ragged-red fibres (mitochondrialabnormalities): disease entity or a syndrome? Light- andelectron-microscopic studies of two cases and review of theliterature. J Neurol Sci 1980;47:117e33.

62. Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW,Wallace DC. Myoclonic epilepsy and ragged-red fiber disease(MERRF) is associated with a mitochondrial DNA tRNA(Lys)mutation. Cell 1990;61:931e7.

63. Mitsumoto H, Aprille JR, Wray SH, Nemni R, Bradley WG.Chronic progressive external ophthalmoplegia (CPEO): clin-ical, morphologic, and biochemical studies. Neurology 1983;33:452e61.

64. Pavlakis SG, Phillips PC, DiMauro S, De Vivo DC, Rowland LP.Mitochondrial myopathy, encephalopathy, lactic acidosis, andstrokelike episodes: a distinctive clinical syndrome. AnnNeurol 1984;16:481e8.

65. Bardosi A, Creutzfeldt W, DiMauro S, Felgenhauer K,Friede RL, Goebel HH, et al. Myo-, neuro-, gastrointestinalencephalopathy (MNGIE syndrome) due to partial defi-ciency of cytochrome-c-oxidase. A new mitochondrialmultisystem disorder. Acta Neuropathol 1987;74:248e58.

66. Vilkki J, Savontaus ML, Nikoskelainen EK. Genetic heteroge-neity in Leber hereditary optic neuroretinopathy revealed bymitochondrial DNA polymorphism. Am J Hum Genet 1989;45:206e11.

67. Makela-Bengs P, Suomalainen A, Majander A, Rapola J,Kalimo H, Nuutila A, et al. Correlation between the clinicalsymptoms and the proportion of mitochondrial DNA carryingthe 8993 point mutation in the NARP syndrome. Pediatr Res1995;37:634e9.

68. Barth PG, Scholte HR, Berden JA, Van der Klei-VanMoorsel JM, Luyt-Houwen IE, Van’t Veer-Korthof ET, et al.An X-linked mitochondrial disease affecting cardiac muscle,skeletal muscle, and neutrophil leucocytes. J Neurol Sci 1983;62:327e55.

69. Rahman S, Blok RB, Dahl HH, Danks DM, Kirby DM, Chow CW,et al. Leigh syndrome: clinical features and biochemical andDNA abnormalities. Ann Neurol 1996;39:343e51.

70. Rotig A, Cormier V, Blanche S, Bonnefont JP, Ledeist F,Romero N, et al. Pearson’s Marrow-Pancreas syndrome. Amultisystem mitochondrial disorder in infancy. J Clin Invest1990;86:1601e8.

71. Van den Ouweland JM, De Klerk JB, Van de Corput MP,Dirks RW, Raap AK, Scholte HR, et al. Characterization of anovel mitochondrial DNA deletion in a patient with a variantof the Pearson marrow-pancreas syndrome. Eur J Hum Genet2000;8:195e203.

72. Santorelli FM, Barmada MA, Pons R, Zhang LL, DiMauro S.Leigh-type neuropathology in Pearson syndrome associatedwith impaired ATP production and a novel mtDNA deletion.Neurology 1996;47:1320e3.

73. Kim JT, Lee YJ, Lee YM, Kang HC, Lee JS, Kim HD. Clinicalcharacteristics of patients with non-specific and non-categorized mitochondrial diseases. Acta Paediatr 2009;98:1825e9.

74. Walker UA, Collins S, Byrne E. Respiratory chain encephalo-myopathies: a diagnostic classification. Eur Neurol 1996;36:260e7.

75. Bernier FP, Boneh A, Dennett X, Chow CW, Cleary MA,Thorburn DR. Diagnostic criteria for respiratory chaindisorders in adults and children. Neurology 2002;59:1406e11.

76. Wolf NI, Smeitink JA. Mitochondrial disorders: a proposal forconsensus diagnostic criteria in infants and children.Neurology 2002;59:1402e5.

18 C.-S. Chi

77. Haas RH, Parikh S, Falk MJ, Saneto RP, Wolf NI, Darin N, et al.Mitochondrial disease: a practical approach for primary carephysicians. Pediatrics 2007;120:1326e33.

78. Dinopoulos A, Cecil KM, Schapiro MB, Papadimitriou A,Hadjigeorgiou GM, Wong B, et al. Brain MRI and proton MRS

findings in infants and children with respiratory chain defects.Neuropediatrics 2005;36:290e301.

79. Wong LJ, Scaglia F, Graham BH, Craigen WJ. Current molec-ular diagnostic algorithm for mitochondrial disorders. MolGenet Metab 2010;100:111e7.