Diagnostic approach in infants and children with mitochondrial diseases.

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Pediatrics and Neonatology (2014) xx, 1e12

Available online at www.sciencedirect.com

ScienceDirect 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 diagnosis; infants and children; mitochondrial diseases; Taiwan

Mitochondrial diseases are a heterogeneous group of disorders affecting energy production in the human body. The diagnosis of mitochondrial diseases represents a challenge to clinicians, especially for pediatric cases, which show enormous variation in clinical presentations, as well as biochemical and genetic complexity. Different consensus diagnostic criteria for mitochondrial diseases in infants and children are available. The lack of standardized diagnostic criteria poses difficulties in evaluating diagnostic 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 clinical, 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. Therefore, 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 mitochondrial 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: [email protected]. 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.

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

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C.-S. Chi

1. Introduction

particularly vulnerable to limited adenosine triphosphate (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, especially 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 overview of the basic biochemical reactions and genetic characteristics in the mitochondrion, clinical manifestations, and diagnostic methods. A diagnostic algorithm for identifying MDs in pediatric neurology patients is proposed.

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 hypermetabolism. 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 diseases (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 estimated 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

Glucose

Fatty acids

Glycolysis Pyruvate

Alanine H+

Amino acids

Lactate

Pyruvate

CPT-II

CACT Matrix

Fatty –acyl-carnitine Carnitine Fatty –acyl-CoA

H+ Pyruvate

Acetyl-CoA

β -oxidation

Citrate

Oxaloacetate

Mitochondrial DNA

TCA cycle α -Ketoglutarate Malate Succinyl-CoA Fumarate

NADH

Outer mitochondrial membrane

PDHC

Inner mitochondrial membrane

PC

Cytosol

CPT-I Fatty –acyl-carnitine Carnitine

Amino acids

DIC

Nuclear DNA

Fatty –acyl-CoA

Succinate

Complex III

Complex I FMN

FeS

FeS

Complex II FAD FeS

FeS FeS

FeS FeS

CoQ

CytbT

CytbK

Complex IV

FeS

Cytc1

Cytc

Cyta

Cyta3

O2

Complex V (ATPase 6,8) 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. 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

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Mitochondrial diseases in infants and children Table 1

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

Mitochondrial diseases (MDs) mtDNA mutations mtDNA rearrangements Chronic progressive external ophthalmoplegia (mtPEO) KearnseSayre syndrome (KSS) Pearson’s syndrome (PS) mtDNA point mutation Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) Myoclonic epilepsy with ragged-red fibers (MERRF) Leber’s hereditary optic neuropathy (LHON) Leigh syndrome Neuropathy, ataxia, and retinitis pigmentosa (NARP) Maternal inherited Leigh syndrome (MILS) nDNA mutations Mutated RC subunits Nonsyndromic MDs with early-onset hypotonia, cardiomyopathy, ataxia, psychomotor delay

Type of mutation

Mutated gene

mtDNA deletion, duplication mtDNA deletion, duplication mtDNA deletion, duplication

mtDNA

np 3243, np 3271, and others

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

np 8344, np 8356, and others np 11778, np 14484, and others np 8993, np 10191, and others np 8993, and others np 8993, and others

nDNA

ATPase 6 ATPase6 ND1-6, COXIII, ATPase6, tRNAs

nDNA

RCCIII (COX6B1)

nDNA

SURF1 encoding COX assembly factor RCCI assembly factors (NDUFA12L) RCCI assembly factors (NDUFAF1, C6ORF66) E1-alpha-subunit of PDHC E1-alpha-subunit of PDHC COX assembly factors (SCO2, SCO1, COX10, COX15) COX assembly factors (SCO2, SCO1, COX10, COX15) RCCIII assembly protein (BCS1L) RCCIII assembly protein (BCS1L) RCCV assembly gene (ATP12) RCCV assembly gene (TMEM 70)

Encephalocardiomyopathy with severe lactic acidosis

nDNA

Leigh syndrome (LS) manifest as leukoencephalopathy

nDNA

Leigh syndrome (LS) manifest as cardio-encephalo-myopathy

nDNA

Leigh syndrome (LS) Leigh syndrome (LS) Leigh syndrome (LS), COX-deficient

X chromosome nDNA nDNA

Congenital lactacidosis and fatal infantile multisystem disease, involving brain, liver, heart, and muscle Neonatal encephalo cardiomyopathy

RCCI subunits

nDNA nDNA nDNA

nDNA

Multisystem fatal infantile disorders, encephalomyopathy plus cardiomyopathy, nephropathy, or hepatopathy Lethal infantile growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis and early death (GRACILE) syndrome Sensorineural hearing loss with pilli torli (Bjornstad syndrome)

mtDNA

RCCI-subunits (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS8, NDUFV1, NDUFV2) or assembly factors RCCI-subunits (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS8, NDUFV1, NDUFV2) or assembly factors RCCI splicing mutations in NDUFA11 or NDUFA1 RCCII (SDHA) RCCII (SDHA) RCCIII (UQCRQ)

Leigh syndrome (LS)

Late-onset neurodegenerative disease Leigh syndrome (LS) Nonlethal phenotype of severe psychomotor retardation, extrapyramidal signs, dystonia, athetosis and ataxia, mild axial hypotonia, and dementia Severe infantile encephalomyopathy Mutated ancillary proteins Leigh syndrome (LS)

mtDNA

nDNA nDNA nDNA nDNA nDNA

(continued on next page)


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

Type of mutation

Mutated gene

nDNA nDNA nDNA nDNA

ANT1, PEO1, POLG POLG POLG POLG

nDNA

POLG

nDNA

TYMP

nDNA

POLG

nDNA nDNA nDNA nDNA

TK2 ANT1 (SLC25A4) RRM2B POLG

nDNA

SUCLA2,

nDNA

SUCLG1

nDNA

RRM2B

nDNA nDNA nDNA

POLG PEO1 MVP17

nDNA nDNA

TYMP DGUOK

nDNA

C10orf2 (Twinkle)

nDNA nDNA

PUS1 DARS2

nDNA

RARS2

nDNA

MRPS16

nDNA

YARS2

nDNA

TAZ

nDNA

SERAC1

nDNA

AGK


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Mitochondrial diseases in infants and children

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Table 1 (continued ) Mitochondrial diseases (MDs) Coenzyme Q defects Nonsyndromic MDs: encephalomyopathy with recurrent myoglobinuria, brain involvement and ragged red fiber

Type of mutation

Mutated gene

nDNA

Primary CoQ deficiency (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Primary CoQ deficiency (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Primary CoQ deficiency (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Secondary CoQ deficiency (APTX) Secondary CoQ deficiency (ETFDH) Secondary CoQ deficiency (BRAF)

Severe infantile multisystem MDs

nDNA

Leigh syndrome (LS)

nDNA

Cerebellar ataxia

nDNA

Pure myopathy

nDNA

Cardio-facio-cutaneous syndrome

nDNA

Defects of mitochondrial transport machinery Deafness-dystonia syndrome (DDS): childhood-onset progressive deafness, dystonia, spasticity, mental deterioration and blindness X-linked sideroblastic anemia with ataxia (XLSA/A) Congenital lactic acidosis, hypertrophic cardiomyopathy and muscle hypotonia Defects in mitochondrial biogenesis Dominant optic atrophy (ADOA) CharcoteMarieeTooth neuropathy-2A Severe infantile encephalopathy Hereditary spastic paraplegia

nDNA

DDP1/TIMM8A

nDNA nDNA

ABCB7 SLC25A3

nDNA nDNA nDNA nDNA

OPA1 MFN2 DLP1 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 phosphorylase; YARS2 Z tyrosyl-tRNA synthetase 2.

2. Biochemical reactions in the mitochondrion 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 production 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

(acetyl-CoA) catalyzed by the pyruvate dehydrogenase complex (PDHC). Long-chain fatty acids, after being activated 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 respiratory 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 powerhouse system of the cell, where the energy production occurs through the oxidative phosphorylation pathway. The mitochondrial RC system is located in the mitochondrial inner membrane, organized in five enzymatic complexes (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 mitochondrial RC, where most cellular ATP is generated.

3. Mitochondrial genetics The human mitochondrial genome is a 16,569-bp doublestranded 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 complex IV (cytochrome c oxidase), and two of complex V (Hþtranslocating ATP synthase). All of the subunits of complex II (succinateeubiquinone oxidoreductase), the remaining subunits 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 genomes during mitochondrial and cell divisions. Thus, the concepts of threshold effect and mitotic segregation provide theoretical explanations for the variable phenotype expressions of the MDs. The dual genetic control of the RC represents the ubiquitous 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

C.-S. Chi machinery. To date, it has been established that 1500 nuclear-encoded proteins are targeted to the mitochondria.49 Mutations in nDNA can affect: (1) components of RC subunits; (2) mitochondrial ancillary proteins involved in RC complexes formation, turnover, and function; (3) intergenomic 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, autosomal 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 mutations 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 clinical 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 symptoms 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 neuromuscular 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 infant death syndrome, and/or hypoventilation. The second most common clinical features were ophthalmologic problems (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 ophthalmoplegia, 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 cardiomyopathy, arrhythmia, mitral valve prolapse, hypertension, and/or dilated cardiomyopathy. Various symptoms and


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Table 2 Clinical manifestations in 103 infants and children with mitochondrial diseases. Involved organ system

N Z 103

Central nervous system Ophthalmologic system Failure to thrive Cardiovascular system Gastrointestinal system Muscular system Hearing system Hepatic system Short stature Urologic system Endocrinologic system Peripheral nervous system Pancreas Hematologic system Psychic Pulmonary edema

93 37 37 26 23 22 20 19 18 9 8 4 2 1 1 1

(90.3) (35.9) (35.9) (25.2) (22.3) (21.4) (19.4) (18.4) (17.5) (8.7) (7.8) (3.9) (1.9) (1.0) (1.0) (1.0)

Data are presented as n (%).

clinical features complicate the diagnosis of MDs and the presentations may result in patients visiting numerous other subspecialists and receiving various diagnoses. There are two main clinical classifications of MDs: syndromic MDs and nonsyndromic MDs. Patients who presented with characteristic clinical phenotypes reported in the literature were categorized as having specific mitochondrial syndromes,55e68 including Leigh syndrome (LS),55,69 Alpers’ disease,57 lethal infantile mitochondrial disease (LIMM),59,60 Pearson’s syndrome (PS),58,70e72 KearnseSayre syndrome (KSS),56 mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS),64 myoclonic epilepsy with ragged-red fibers (MERRF),61,62 neuropathy, ataxia, and retinitis pigmentosa (NARP),67 mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE),65 chronic progressive external ophthalmoplegia (CPEO),63 Leber’s hereditary optic neuropathy (LHON),66 and Barth syndrome (BTHS).68 The patients who fulfilled the diagnostic criteria for MDs but not specific syndromes, were classified as having noncategorized or nonsyndromic MDs.42 Recently published studies42,51e53 show that pediatric patients with nonsyndromic MDs are not uncommon, and the clinical manifestations are nonspecific.42,73 Specific mitochondrial syndromes are easy to diagnose because each of them has its own clinical phenotypes. However, it is not easy to diagnose nonsyndromic MDs during the early stage of disease course due to the wide variety of clinical features. Thus, follow-up of clinical symptoms/signs in these patients is important.

5. Diagnostic approach of mitochondrial diseases 5.1. Diagnostic criteria Different consensus diagnostic criteria for MDs in children are available. Major and minor criteria are usually based on clinical, biochemical, pathologic, and molecular findings.

In 1996, Walker et al74 proposed diagnostic criteria for adults with respiratory chain disorders (Adult criteria; AC). In 2002, Bernier et al75 suggested a modified version of the Adult criteria (Modified Walker criteria or Modified Adult Criteria; MAC) in order to improve the sensitivity and expand its use to include pediatric patients as well as adults. The diagnostic criteria of MDs were established on the basis of assigning major or minor criteria for clinical, pathological, enzymatic, functional, molecular, and metabolic parameters. Mitochondrial encephalomyopathies were categorized as definite, probable, or possible. A definite diagnosis is defined as fulfillment of either of two major criteria or one major criterion plus two minor criteria. A probable diagnosis is defined as either one major criterion and one minor criterion or at least three minor criteria. A possible diagnosis is defined as either a single major criterion or two minor criteria, one of which must be clinical. In 2002, Wolf et al76 proposed the consensus mitochondrial diagnostic criteria (MDC) scoring system for infants and children, which evaluates clinical features (I; maximum clinical score: 4 points) of muscle symptoms (IA; maximum score: 2 points), CNS abnormalities (IB; maximal score: 2 points), and multisystem involvement (IC; maximal score: 3 points), adding metabolic abnormalities and neuroimaging (II; maximum addition score: 4 points). Histologic anomalies (III) could increase the score with 4 points, leading to a maximum score of 12 points. Scores of 8e12, 5e7, 2e4, and 1 were defined as definite, probable, possible, or unlikely mitochondrial disorders, respectively. The MDC uses welldefined items (clinical, laboratory, pathologic, and biochemical) for scoring and dividing criteria into two subsets: general (clinical, metabolic, imaging, and pathologic) and biochemical. This allows a critical and independent evaluation for general features and for results of biochemical investigations, prior to combining the two. The advantages of MDC criteria are that the general criteria (without the histochemical part) allow preclassification of patients prior to when a muscle biopsy is performed. If a patient with an undiagnosed disorder reaches the probable or definite general classification, a muscle biopsy may be strongly recommended. The different consensus diagnostic criteria for MDs in children described above show how difficult it can be to diagnose MDs; that is, the diagnosis of MDs with certainty requires analysis from multiple perspectives.

5.2. Diagnostic tools 5.2.1. Blood laboratory investigation Lack of a clinically pathognomonic hallmark frequently makes laboratory investigations necessary to confirm the diagnosis. For basic laboratory investigations, blood lactate is used as a biochemical marker for the screening of MDs. The differential diagnosis of lactic acidosis includes physiological anaerobic exercise, systemic diseases that increase blood lactate levels, cerebral diseases that increase CSF lactate levels, metabolic diseases, poor collection technique, or poor sample handling.77 However, blood lactate level is not always elevated in patients with MDs, and thus, normal serum lactic level does not exclude a mitochondrial


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8 disorder.42,51 A glucose challenge test followed by successive blood lactate examinations, oral glucose lactate stimulation test (OGLST), is a better screening method than a single blood lactate test.3,11,42 Lactic acidosis may be proportionate or disproportionate to the elevation of pyruvate based on the associated effect of the biochemical defect on the oxidationereduction potential. If the oxidationereduction potential is unaffected by the biochemical defect, the lactate and pyruvate elevations will be proportional 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 lactateto-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 (secondary) disorders. The latter include defects of metabolic pathways for glycogen, gluconeogenesis, and organic acidemia. 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 resonance 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 microscopic examinations is an auxiliary diagnostic method of MDs. Morphological examinations include the modified Gomori trichrome staining for RRFs, ATPase staining for the assessment of myofibrillar integrity, muscle-type fiber predominance 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 fibroblasts is helpful to confirm RC complex defects and provide a

C.-S. Chi 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 generation 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 comprehensive individual and family history, and clinical investigations in neurology, ophthalmology, otology, endocrinology, cardiology, gastroenterology, nephrology, hematology, and so on. Important instrumental procedures include basic chemical investigations of serum and urine, electrophysiological 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 presentation 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.


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

+

OGLST + Muscle and/or tissue biopsy

−

-

+ Genetic counseling

OGLST +

Family member studies

−

−

Muscle and/or tissue biopsy +

Bernier diagnostic criteria75 Whole mtDNA sequencing, or NGS for other genes (Table 1) + Genetic counseling Family member studies

− Follow-up • Neurological and nonneurologic S/S • Clinical evolution Re-evaluate • Laboratory tests • Neuroimaging studies Muscle and/or tissue biopsy if indicated

Bernier diagnostic criteria75 Common mtDNA mutations, Common mtDNA deletions, Specific nDNA mutations or Depletion genes (Table 1) +

−

Genetic counseling

NGS for other genes (Table 1)


+ Genetic counseling

Follow-up • Neurological and nonneurologic S/S • Clinical evolution Re-evaluate • Laboratory tests • Neuroimaging 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 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.

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

current approach to diagnosing and classifying MDs incorporates clinical, biochemical, neuroradiological, pathologic, and molecular information. The confirmation or exclusion of MDs is therefore a major challenge for clinicians, especially in cases with nonspecific clinical phenotypes. 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 The author would like to thank Hsiu-Fen Lee, MD for collection of clinical data and study design, Chi-Zen Tsai, MSC, Tzu-Chao Wang, MSC, Chia-Ju Li, MSC, and Tzu-Yun Hwang, MSC for molecular analysis, and Chia-Chi Hsu, MD and Liang-Hui Chen, MSC for assays of metabolic profiles.


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