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provided by Elsevier - Publisher Connector Pediatrics and Neonatology (2015) 56,7e18

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INVITED REVIEW ARTICLE Diagnostic Approach in Infants and Children with Mitochondrial Ching-Shiang Chi*

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

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

Key Words Mitochondrial diseases are a heterogeneous group of disorders affecting production in diagnosis; the . The diagnosis of mitochondrial diseases represents a challenge to clinicians, infants and children; especially for pediatric cases, which show enormous variation in clinical presentations, as well mitochondrial as biochemical and genetic complexity. diseases; Different consensus diagnostic criteria for mitochondrial diseases in infants and children are Taiwan available. The lack of standardized diagnostic criteria poses difficulties in evaluating diag- nostic methodologies. Even though there are many diagnostic tools, none of them are sensitive enough to make a confirmative diagnosis without being used in combination with other tools. The current approach to diagnosing and classifying mitochondrial diseases incorporates clin- ical, biochemical, neuroradiological findings, and histological criteria, as well as DNA-based molecular diagnostic testing. The confirmation or exclusion of mitochondrial diseases remains a challenge in clinical practice, especially in cases with nonspecific clinical phenotypes. There- fore, follow-up evolution of clinical symptoms/signs and biochemical data is crucial. The purpose of this study is to review the molecular classification scheme and associated phenotypes in infants and children with mitochondrial diseases, in addition to providing an overview of the basic biochemical reactions and genetic characteristics in the , clinical manifestations, and diagnostic methods. A diagnostic algorithm for identifying mito- chondrial disorders in pediatric 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. 8 C.-S. Chi

1. Introduction muscle, are particularly vulnerable to limited (ATP) supply, and Mitochondrial (MD) was first introduced in 1962, are often prominent features in various mitochondrial when a group of investigators, Luft et al1, described a phenotypes. involved in mitochondrial energy young Swedish woman with severe nonthyroid origin hy- production are coded by two (mitochondrial and permetabolism. In 1963, Engel and Cunningham2 used a nuclear) in the organism. Due to their clinical, biochemical, modification of the Gomori trichrome stain that allowed for and genetic complexity, MDs represent a challenge to cli- the detection of abnormal mitochondrial proliferation in nicians, especially for pediatric cases, which show enor- muscle as irregular purplish patches in fibers that were mous variation in clinical presentations and course. 5 dubbed “ragged red fibers (RRFs)”. Since then, there has In Taiwan, the first clinical MD was reported in 1988, been great interest in the diagnosis of mitochondrial dis- and the first mitochondrial DNA (mtDNA) was 6 eases (MDs). The diagnosis of MD can be traced back to the identified in 1992. Many cases have since been reported in 7e47 premolecular era, 1962e1988, when MDs were defined on pediatric groups. Although MDs are being diagnosed the basis of clinical examination, muscle biopsy, and more frequently, clinical physicians still find diagnosis a biochemical criteria. In the molecular era, the full challenge because clinical symptoms/signs evolve over complexity of these disorders became evident. time and the number of genes known to be involved in MDs are a heterogeneous group of disorders affecting mitochondrial energy production continues to increase. energy production in the human body, which can occur with The purpose of this study is to review the molecular a probable frequency in preschool children (age < 6 years) classification scheme and associated phenotypes in infants of approximately 1 in 11,000,3 and the minimum birth and children with MDs, in addition to providing an over- prevalence for respiratory chain disorders with onset at any view of the basic biochemical reactions and genetic age was estimated at 1 in 7634.4 MDs may present at any characteristics in the mitochondrion, clinical manifesta- age with a spectrum of symptoms and signs spanning tions, and diagnostic methods. A diagnostic algorithm for a number of medical specialties. As cells with high energy identifying MDs in pediatric neurology patients is requirements, such as neurons, skeletal and cardiac proposed.

Glucose Fatty acids Glycolysis Nuclear Fatty –acyl-CoA DNA Pyruvate Lactate Amino acids Cytosol CPT-I H+ Pyruvate Amino acids Fatty –acyl-carnitine Carnitine DIC CPT-II CACT Matrix Fatty –acyl-carnitine Carnitine H+ Pyruvate Fatty –acyl-CoA Inner mitochondrial membrane Outer mitochondrial membrane

PC PDHC Acetyl-CoA β -oxidation Citrate Oxaloacetate TCA cycle Mitochondrial DNA α -Ketoglutarate Malate Succinyl-CoA Fumarate NADH Succinate

Complex I Complex III Complex IV

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

ADP + Pi ATP

Figure 1 Metabolic pathways in mitochondrion. ADP Z adenosine diphosphate; ATP Z adenosine triphosphate; CACT Z carnitineeacylcarnitine translocase; CoQ Z coenzyme Q; CPT Z carnitine palmitoyltransferase; Cyta Z cytochrome a; Cytb Z cytochrome b; Cytc Z cytochrome c; DIC Z dicarboxylate carrier; FAD Z flavin adenine dinucleotide; FeS Z iron sulfur protein; FMN Z flavin mononucleotide; NADH Z adenine dinucleotide; PC Z pyruvate carboxylase; PDHC Z complex; TCA Z tricarboxylic acid. Mitochondrial diseases in infants and children 9

Table 1 A brief summary of published genetic classification of mitochondrial diseases.49,50 Mitochondrial diseases (MDs) Type of mutation Mutated gene mtDNA mtDNA rearrangements Chronic progressive external ophthalmoplegia (mtPEO) mtDNA deletion, mtDNA duplication KearnseSayre syndrome (KSS) mtDNA deletion, mtDNA duplication Pearson’s syndrome (PS) mtDNA deletion, mtDNA duplication mtDNA Mitochondrial encephalomyopathy, , and np 3243, np 3271, mt-tRNALeu, mt-tRNAPhe, -like episodes (MELAS) and others mt-tRNAVal, COXII, COXIII, ND1, ND5, ND6, rRNA Myoclonic with ragged-red fibers (MERRF) np 8344, np 8356, mt-tRNALys and others Leber’s hereditary optic neuropathy (LHON) np 11778, np 14484, RCCI subunits and others np 8993, np 10191, ATPase 6 and others Neuropathy, , and retinitis pigmentosa (NARP) np 8993, and others ATPase6 Maternal inherited Leigh syndrome (MILS) np 8993, and others ND1-6, COXIII, ATPase6, tRNAs nDNA mutations Mutated RC subunits Nonsyndromic MDs with early-onset , nDNA RCCI-subunits (NDUFS1, cardiomyopathy, ataxia, psychomotor delay NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS8, NDUFV1, NDUFV2) or assembly factors Leigh syndrome (LS) nDNA RCCI-subunits (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS8, NDUFV1, NDUFV2) or assembly factors Encephalocardiomyopathy with severe lactic acidosis nDNA RCCI splicing mutations in NDUFA11 or NDUFA1 Late-onset neurodegenerative disease nDNA RCCII (SDHA) Leigh syndrome (LS) nDNA RCCII (SDHA) Nonlethal phenotype of severe psychomotor retardation, nDNA RCCIII (UQCRQ) extrapyramidal signs, , and ataxia, mild axial hypotonia, and Severe infantile encephalomyopathy nDNA RCCIII (COX6B1) Mutated ancillary proteins Leigh syndrome (LS) nDNA SURF1 encoding COX assembly factor Leigh syndrome (LS) manifest as leukoencephalopathy nDNA RCCI assembly factors (NDUFA12L) Leigh syndrome (LS) manifest as cardio-encephalo-myopathy nDNA RCCI assembly factors (NDUFAF1, C6ORF66) Leigh syndrome (LS) E1-alpha-subunit of PDHC Leigh syndrome (LS) nDNA E1-alpha-subunit of PDHC Leigh syndrome (LS), COX-deficient nDNA COX assembly factors (SCO2, SCO1, COX10, COX15) Multisystem fatal infantile disorders, encephalomyopathy plus nDNA COX assembly factors cardiomyopathy, nephropathy, or hepatopathy (SCO2, SCO1, COX10, COX15) Lethal infantile growth retardation, aminoaciduria, cholestasis, nDNA RCCIII assembly protein iron overload, lactacidosis and early death (GRACILE) syndrome (BCS1L) Sensorineural hearing loss with pilli torli (Bjornstad syndrome) nDNA RCCIII assembly protein (BCS1L) Congenital lactacidosis and fatal infantile multisystem disease, nDNA RCCV assembly gene involving brain, liver, , and muscle (ATP12) Neonatal encephalo cardiomyopathy nDNA RCCV assembly gene (TMEM 70) (continued on next page) 10 C.-S. Chi

Table 1 (continued) Mitochondrial diseases (MDs) Type of mutation Mutated gene Defects of intergenomic signaling mtDNA breakage syndromes (large-scale mtDNA deletion) Autosomal dominant PEO (adPEO) nDNA ANT1, PEO1, POLG Autosomal recessive PEO (arPEO) nDNA POLG Sensory ataxic neuropathy, dysarthria, ophthalmoplegia (SANDO) nDNA POLG Spino-cerebellar ataxia and epilepsy with or without nDNA POLG ophthalmoplegia (SCAE) Infantile Aplers-Huttenlocher-syndrome (AHS) manifests as nDNA POLG myopathy, hepatopathy, epilepsy, , intractable and mental retardation (hepatic poliodystrophy) Mitochondrial neuro-gastrointestinal encephalomyopathy nDNA TYMP (MNGIE) MNGIE-like phenotype nDNA POLG mtDNA depletion syndromes (DPSs) Myopathic DPS Muscle weakness, elevated creatine kinase, encephalopahy nDNA TK2 adPEO, mild myopathy nDNA ANT1 (SLC25A4) Myopathy, renal tubulopathy nDNA RRM2B AHS, PEO, myopathy sensory ataxia, ataxia nDNA POLG neuropathy spectrum, childhood myocerebrohepatopathy spectrum Encephalomyopathic DPS Leigh-like syndrome with muscle hypotonia, lactic acidosis, nDNA SUCLA2, dystonic and moderate methylmalonic aciduria Fatal infantile lactic acidosis, dysmorphism and methylmalonic nDNA SUCLG1 aciduria with muscle and liver mtDNA depletion Hypotonia, developmental delay, and renal tubulopathy nDNA RRM2B Hepato-cerebral DPS Phenotypes as PLOG described above nDNA POLG Alpers-like phenotype nDNA PEO1 Hepatic failure, , muscle hypotonia, ataxia, nDNA MVP17 dystonia, or polyneuropathy, Navajo neurohepatopathy MNGIE nDNA TYMP Hepatopathy, severe , lactic acidosis, nDNA DGUOK hypoglycemia, neurological symptoms Infantile-onset , adPEO, myopathy, nDNA C10orf2 (Twinkle) Parkinsonian features of rigidity , and bradykinesia Defective mitochondrial protein synthesis machinery and sideroblastic (MLASA) nDNA PUS1 Leukoencephalopathy with and nDNA DARS2 involvement and lactacidosis (LBSL)-syndrome Pontocerebellar hypoplasia (PCH) with prenatal-onset, nDNA RARS2 cerebellar/pontine atrophy/hypoplasia, , neocortical atrophy and severe psychomotor impairment Agenesis of corpus callosum, dysmorphic features, fatal neonatal nDNA MRPS16 lactic acidosis ad intermediate Charcot-MarieeTooth neuropathy, type C nDNA YARS2 Defects of the mitochondrial lipid milieu Barth syndrome characterized by mitochondrial myopathy, nDNA TAZ hypertrophic or , left ventricular hypertrabeculation/noncompaction, growth retardation, leukopenia and 3-methylglutaconic aciduria Sensorineural deafness, encephalopathy, Leigh-like syndrome, nDNA SERAC1 and 3-methylglutaconic aciduria Sengers syndrome characterized by congenital cataracts, nDNA AGK hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis Mitochondrial diseases in infants and children 11

Table 1 (continued) Mitochondrial diseases (MDs) Type of mutation Mutated gene Coenzyme Q defects Nonsyndromic MDs: encephalomyopathy with recurrent nDNA Primary CoQ deficiency myoglobinuria, brain involvement and ragged red fiber (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Severe infantile multisystem MDs nDNA Primary CoQ deficiency (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Leigh syndrome (LS) nDNA Primary CoQ deficiency (COQ2, COQ8, ADCK3, CABC1, PDSS1, PDSS2) Cerebellar ataxia nDNA Secondary CoQ deficiency (APTX) Pure myopathy nDNA Secondary CoQ deficiency (ETFDH) Cardio-facio-cutaneous syndrome nDNA Secondary CoQ deficiency (BRAF) Defects of mitochondrial transport machinery Deafness-dystonia syndrome (DDS): childhood-onset progressive nDNA DDP1/TIMM8A deafness, dystonia, spasticity, mental deterioration and blindness X-linked with ataxia (XLSA/A) nDNA ABCB7 Congenital lactic acidosis, hypertrophic cardiomyopathy and nDNA SLC25A3 muscle hypotonia Defects in mitochondrial biogenesis Dominant optic atrophy (ADOA) nDNA OPA1 CharcoteMarieeTooth neuropathy-2A nDNA MFN2 Severe infantile encephalopathy nDNA DLP1 Hereditary spastic paraplegia nDNA KIF5A ABCB7 Z ATP-binding cassette, sub-family B (MDR/TAP), member 7; AGK Z acylglycerol kinase; ANT1 Z adenine nucleotide translocase 1 gene; C10orf2 Z open reading frame 2; COX Z ; 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 complex, subunit A; SERAC1 Z serine active site containing 1; SLC25A3 Z solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3; SUCLA2 Z b-subunit of the adenosine diphosphate-forming succinyl-CoA-ligase; SUCLG1 Z alpha-subunit of GDP-forming succinyl-CoA-ligase; TAZ Z tafazzin; TK Z thymidine-kinase; TMEM70 Z transmembrane protein 70; TYMP Z thymidine phosphory- lase; YARS2 Z tyrosyl-tRNA synthetase 2.

2. Biochemical reactions in the mitochondrion (acetyl-CoA) catalyzed by the pyruvate dehydrogenase complex (PDHC). Long-chain fatty acids, after being acti- The mitochondrion, an extranuclear , is 0.5e1 mm vated to form fatty acyl-CoA in the cytosol, must be in size. It consists of outer and inner membranes, an transferred across the inner mitochondrial membrane to be intermembranous space, and an inner matrix compartment. oxidized to acetyl-CoA. Acetyl-CoA then enters the citric The matrix contains various enzymes, ribosomes, transfer acid cycle (Krebs cycle), which releases eight hydrogen RNAs (tRNAs), and mtDNA molecules. Each contains molecules and produces carbon dioxide and water through many mitochondria which are responsible for cellular ATP oxidative phosphorylation. This process liberates energy production by oxidative phosphorylation. Energy produc- along the respiratory chain, which receives energy-rich tion of mitochondria comes from the metabolism of hydrogen atoms from nicotinamide adenine dinucleotide e and fatty acids through a series of reactions (Figure 1). (NADH) or flavin adenine dinucleotide (FADH), produced Pyruvate, the end-product of aerobic glycolysis, is mainly in the Krebs cycle and from oxidation. derived partly from -borne glucose but mainly from Electrons from the hydrogen are passed between respira- endogenous . Once formed in the cell cytosol, tory complexes in the chain and result in energy pyruvate may be reduced to lactate, transaminated to production. alanine, or transported into the mitochondria where it Mitochondrial respiratory chain (RC) complex is a unique undergoes oxidative decarboxylation to acetyl-coenzyme A system in the cell coded by two genomes, known as the 12 C.-S. Chi powerhouse system of the cell, where the energy produc- machinery. To date, it has been established that 1500 tion occurs through the oxidative phosphorylation pathway. nuclear-encoded proteins are targeted to the mitochon- The mitochondrial RC system is located in the mitochon- dria.49 Mutations in nDNA can affect: (1) components of RC drial inner membrane, organized in five enzymatic com- subunits; (2) mitochondrial ancillary proteins involved in RC plexes (IeV), ubiquinone (or ), and complexes formation, turnover, and function; (3) inter- cytochrome c. Complexes I, III, and IV extrude protons from genomic signaling for mtDNA maintenance or expression; (4) the mitochondrial matrix. Complex IV consumes oxygen to biosynthetic enzymes for lipids or cofactors; (5) coenzyme form water. Complex V couples ATP synthesis to proton Q; (6) mitochondrial trafficking or transport machinery; (7) reentry, which is powered by the electrochemical mitochondrial biogenesis; or (8) apoptosis.50 gradient.48 Ultimately, the energy is stored as ATP. These A brief summary of published genetic classification of assumptions are the basis for the biochemical evaluation of MDs is shown in Table 1.49,50 Primary mtDNA mutations mitochondrial RC enzymes’ activity. may exhibit a maternal inheritance, or Mendelian traits The term “mitochondrial diseases” refers to a class of that present with multiple somatic mtDNA alterations. disorders characterized by an impairment of the mito- In the case of Mendelian disorders, autosomal recessive, chondrial RC, where most cellular ATP is generated. autosomal dominant, and X-linked patterns have all been observed. Recent studies have demonstrated that approx- imately 10e15% of MDs are caused by mutations in 3. Mitochondrial genetics mtDNA,48 and up to 25% of selected children with MDs are caused by mutations in nDNA49; i.e., MDs in children result The human mitochondrial is a 16,569-bp double- more often from nDNA mutations. In addition, it has been stranded DNA circle that contains 37 genes, of which 13 reported that > 200 nuclear-encoded genes have been genes encode subunits of the respiratory chain, and 22 linked to human disease.49 Therefore, genetic diagnosis of tRNAs and two ribosomal RNA genes (12S and 16S) translate MDs in infants and children is a difficult task due to the mtDNA. Each cell contains many mitochondria with of mtDNA mutations and the large number of 2e10 mtDNA molecules. The total mtDNA accounts for nuclear genes involved in different tissues. approximately 0.5% of the DNA in a nucleated somatic cell. The 13 mitochondrial protein-coding genes contribute to the four components of the RC complexes required for 4. Clinical manifestations oxidative phosphorylation: seven of them are subunits of complex I (NADH-ubiquinone oxidoreductase), one of complex MDs that result from diminishing ATP production cause clin- III (ubiquinolecytochrome c oxidoreductase), three of com- ical disorders. Several studies51e53 showed clinical features plex IV (cytochrome c oxidase), and two of complex V (Hþ- in patients with MDs which support the maxim “any tissue translocating ATP synthase). All of the subunits of complex II and any signs at any age”.54 However, infants and children (succinateeubiquinone oxidoreductase), the remaining sub- with MDs tend to have an acute onset of the clinical symp- unitsoftheothermitochondrialRCcomplexesaswellasthe toms and courses compared with adults with MDs, who factors involved in mtDNA replication, transcription, and typically exhibit slow and/or progressive presentations. In translation, are encoded by nuclear DNA (nDNA). general, the clinical spectrum is not limited to the neuro- Mitochondrial genetics differ from Mendelian genetics in ; indeed, a number of nonneuromuscular several fundamental aspects. First, mtDNA is maternally organs may exhibit symptoms and signs, such as the heart, inherited because mitochondria derive solely from the , ears, kidneys, endocrine glands, liver, bone marrow, oocyte. A normal cell has only a single mtDNA genotype and and gastrointestinal tract.42 The clinical manifestations of is therefore homoplasmic. However, if the genomes 103 pediatric patients with MDs in our case series, expanding represent a mixture of a wild-type and a mutated-type, the data from the previous report,42 are summarized in Table 2. cell genotype is heteroplasmic. The clinical pictures of The commonest clinical manifestation was symptoms and mitochondrial diseases are determined by the proportion of signs of CNS (93/103; 90.3%). The CNS manifestations of MDs normal-to-mutated genomes in the mitochondria. Once the were variable, including seizures, developmental delay, proportion exceeds a theoretical threshold, the biological altered level of consciousness, floppiness, spasticity, mental behavior of the cell will change. This minimum critical retardation, sucking difficulty, involuntary movement, amount differs from tissue to tissue and different tissues , apnea, external ocular motility limitation, may be variously affected by different combinations and to tremor, apneustic respiration, dystonia, stroke, sudden in- different degrees. In addition, mitotic segregation of the fant death syndrome, and/or hypoventilation. The second mitochondria influences the biological behavior governing most common clinical features were ophthalmologic prob- the stochastic redistribution of wild-type and mutated ge- lems (37/103; 35.9%) and failure to thrive (37/103; 35.9%). nomes during mitochondrial and cell divisions. Thus, the The symptoms and signs of the ophthalmologic system concepts of threshold effect and mitotic segregation pro- included ptosis, retinitis pigmentosa, external oph- vide theoretical explanations for the variable phenotype thalmoplegia, visual loss, , exotropia, blurred expressions of the MDs. vision, visual field defect, strabismus, corneal clouding, and/ The dual genetic control of the RC represents the ubiq- or optic atrophy. The fourth most common clinical uitous nature of mitochondria. The mitochondrial metabolic feature was cardiovascular system dysfunction (26/103; pathway is under the control of not only the mitochondrial, 25.2%), including pericardial effusion, hypertrophic cardio- but also the nuclear genomes, which are strictly coordinated myopathy, arrhythmia, mitral valve prolapse, , to ensure the correct functioning of the mitochondrial and/or dilated cardiomyopathy. Various symptoms and Mitochondrial diseases in infants and children 13

In 1996, Walker et al74 proposed diagnostic criteria for Table 2 Clinical manifestations in 103 infants and chil- adults with respiratory chain disorders (Adult criteria; AC). dren with mitochondrial diseases. In 2002, Bernier et al75 suggested a modified version of the Involved organ system N Z 103 Adult criteria (Modified Walker criteria or Modified Adult Central 93 (90.3) Criteria; MAC) in order to improve the sensitivity and expand Ophthalmologic system 37 (35.9) its use to include pediatric patients as well as adults. The Failure to thrive 37 (35.9) diagnostic criteria of MDs were established on the basis of Cardiovascular system 26 (25.2) assigning major or minor criteria for clinical, pathological, Gastrointestinal system 23 (22.3) enzymatic, functional, molecular, and metabolic parame- Muscular system 22 (21.4) ters. Mitochondrial encephalomyopathies were categorized Hearing system 20 (19.4) as definite, probable, or possible. A definite diagnosis is Hepatic system 19 (18.4) defined as fulfillment of either of two major criteria or one Short stature 18 (17.5) major criterion plus two minor criteria. A probable diagnosis Urologic system 9 (8.7) is defined as either one major criterion and one minor cri- Endocrinologic system 8 (7.8) terion or at least three minor criteria. A possible diagnosis is Peripheral nervous system 4 (3.9) defined as either a single major criterion or two minor 2 (1.9) criteria, one of which must be clinical. 76 Hematologic system 1 (1.0) In 2002, Wolf et al proposed the consensus mitochon- Psychic 1 (1.0) drial diagnostic criteria (MDC) scoring system for infants Pulmonary edema 1 (1.0) and children, which evaluates clinical features (I; maximum clinical score: 4 points) of muscle symptoms (IA; maximum Data are presented as n (%). score: 2 points), CNS abnormalities (IB; maximal score: 2 points), and multisystem involvement (IC; maximal score: 3 clinical features complicate the diagnosis of MDs and the points), adding metabolic abnormalities and neuroimaging presentations may result in patients visiting numerous other (II; maximum addition score: 4 points). Histologic anomalies subspecialists and receiving various diagnoses. (III) could increase the score with 4 points, leading to a There are two main clinical classifications of MDs: syn- maximum score of 12 points. Scores of 8e12, 5e7, 2e4, and dromic MDs and nonsyndromic MDs. Patients who presented 1 were defined as definite, probable, possible, or unlikely with characteristic clinical phenotypes reported in the mitochondrial disorders, respectively. The MDC uses well- literature were categorized as having specific mitochon- defined items (clinical, laboratory, pathologic, and e drial syndromes,55 68 including Leigh syndrome (LS),55,69 biochemical) for scoring and dividing criteria into two Alpers’ disease,57 lethal infantile subsets: general (clinical, metabolic, imaging, and patho- e (LIMM),59,60 Pearson’s syndrome (PS),58,70 72 KearnseSayre logic) and biochemical. This allows a critical and indepen- syndrome (KSS),56 mitochondrial encephalomyopathy, lac- dent evaluation for general features and for results of tic acidosis, and stroke-like episodes (MELAS),64 myoclonic biochemical investigations, before combining the two. The epilepsy with ragged-red fibers (MERRF),61,62 neuropathy, advantages of MDC criteria are that the general criteria ataxia, and retinitis pigmentosa (NARP),67 mitochondrial (without the histochemical part) allow preclassification of neuro-gastrointestinal encephalomyopathy (MNGIE),65 patients before a muscle biopsy is performed. If a patient chronic progressive external ophthalmoplegia (CPEO),63 with an undiagnosed disorder reaches the probable or Leber’s hereditary optic neuropathy (LHON),66 and Barth definite general classification, a muscle biopsy may be syndrome (BTHS).68 The patients who fulfilled the diag- strongly recommended. nostic criteria for MDs but not specific syndromes, were The different consensus diagnostic criteria for MDs in classified as having noncategorized or nonsyndromic MDs.42 children described above show how difficult it can be to e Recently published studies42,51 53 show that pediatric pa- diagnose MDs; that is, the diagnosis of MDs with certainty tients with nonsyndromic MDs are not uncommon, and the requires analysis from multiple perspectives. clinical manifestations are nonspecific.42,73 Specific mitochondrial syndromes are easy to diagnose 5.2. Diagnostic tools 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 5.2.1. Blood laboratory investigation variety of clinical features. Thus, follow-up of clinical Lack of a clinically pathognomonic hallmark frequently symptoms/signs in these patients is important. makes laboratory investigations necessary to confirm the diagnosis. 5. Diagnostic approach of mitochondrial For basic laboratory investigations, blood lactate is used diseases as a biochemical marker for the screening of MDs. The differential diagnosis of lactic acidosis includes physiolog- ical anaerobic exercise, systemic diseases that increase 5.1. Diagnostic criteria blood lactate levels, cerebral diseases that increase CSF lactate levels, metabolic diseases, poor collection tech- Different consensus diagnostic criteria for MDs in children nique, or poor sample handling.77 However, blood lactate are available. Major and minor criteria are usually based on level is not always elevated in patients with MDs, and thus, clinical, biochemical, pathologic, and molecular findings. normal serum lactic level does not exclude a mitochondrial 14 C.-S. Chi disorder.42,51 A glucose challenge test followed by succes- clue for determining causative nDNA mutations. All patients, sive blood lactate examinations, oral glucose lactate stim- prior to undergoing a muscle biopsy, should have a careful ulation test (OGLST), is a better screening method than a clinical and metabolic workup to detect the extent of organ single blood lactate test.3,11,42 Lactic acidosis may be pro- involvement and metabolic disturbance. portionate or disproportionate to the elevation of pyruvate based on the associated effect of the biochemical defect on 5.2.5. Mitochondrial genetic testing e the oxidation reduction potential. If the oxida- The confirmation of the MD diagnosis by molecular methods e tion reduction potential is unaffected by the biochemical often remains a challenge because of the large number of defect, the lactate and pyruvate elevations will be pro- known genes involved in mitochondrial energy production, portional and the lactate/pyruvate ratio will be normal. By the complexity of the two genomes, and the heteroplasmic e contrast, if the oxidation reduction potential is disturbed proportions of pathogenic mtDNA in a patient. A molecular by a primary defect involving the respiratory chain, the diagnostic approach for MDs has been proposed.79 If the lactate values will be disproportionately elevated and the phenotype is suggestive of syndromic MDs, screening of > lactate/pyruvate ratio will be increased ( 25). common point mutations, common deletions in mtDNA or If available, CSF lactate, and pyruvate concentration specific nDNA mutations is the first step. In cases where should also be measured. It is useful to calculate a lactate- characteristic mitochondrial RC complex deficiencies and to-pyruvate ratio because an elevated ratio suggests the histochemical abnormalities are observed, direct possibility of a MD. sequencing of the specific causative nuclear genes can be performed on white blood cell DNA. Measurement of mtDNA 5.2.2. Metabolic survey content in affected tissues, such as muscle, also allows Lactic acidosis can be caused not only by intramitochondrial screening for mtDNA depletion syndromes. In selected (primary) disorders, but also by extramitochondrial (sec- cases testing negative, additional analyses can include ondary) disorders. The latter include defects of metabolic whole mtDNA genome screening for the detection of rare pathways for glycogen, gluconeogenesis, and organic acid- and novel point mutations. As new nDNA mutations are emia. Thus, a metabolic screening with measurement of constantly being discovered, clinicians may use next gen- basic blood and urine analytes such as plasma ketone body eration sequencing (NGS) for molecular analysis. Known (3-OH butyrate/acetoacetate) ratio, serum transaminase, causative genes of MDs will improve genetic counseling. plasma quantitation, tandem mass spectrometry (MS/MS), plasma acylcarnitine profile, and urinary organic acids, is helpful for differential diagnosis of MDs. 5.3. Diagnostic algorithm

5.2.3. Imaging studies As the clinical manifestations of MDs are variable, diagnosis The clinical diagnosis of MDs has been greatly improved by of MDs is much more difficult compared with other diseases. advances in neuroimaging technology. Brain magnetic reso- Therefore, the author suggests an evaluation process for nance imaging (MRI) is sensitive to these diseases, especially clinical physicians to diagnose MDs in clinical practice in clinically phenotypic mitochondrial syndromes, including (Figure 2). Diagnostic workups for suspected MDs are a LS, MELAS, MNGIE, and KSS. In infants and children with stepwise procedure. The first step comprises a comprehen- nonsyndromic MDs, brain MRI can produce variable findings, sive individual and family history, and clinical investigations from normal results to signal changes over the in neurology, ophthalmology, otology, endocrinology, car- or brainstem.43 MR spectroscopy (MRS) for detecting the diology, gastroenterology, nephrology, hematology, and so concentration of a number of biochemical metabolites on. Important instrumental procedures include basic in vivo has been reported to be a useful tool in conducting chemical investigations of serum and urine, electrophysio- differential diagnoses and for monitoring of MDs.44,78 logical investigations, and neuroimaging studies. Based on the results, the probability for the presence of a MD can be 5.2.4. Tissue biopsy and MRC enzymatic assay assessed based on the Bernier diagnostic criteria.75 In the A muscle biopsy using light microscopic and electron micro- second step, clinicians need to decide whether an individual scopic examinations is an auxiliary diagnostic method of MDs. clinical phenotype conforms to any of the syndromic MDs or Morphological examinations include the modified Gomori is characteristic of nonsyndromic MDs. When the presenta- trichrome staining for RRFs, ATPase staining for the assess- tion is classic for specific mitochondrial syndromes, for ment of myofibrillar integrity, muscle-type fiber predomi- example, LS, MELAS, MERRF, LHON, or KSS, appropriate nance and distribution, and cytochrome c oxidase and mtDNA studies should be obtained first. When the clinical succinate dehydrogenase staining for oxidative enzymes. picture is classic for a nDNA inherited syndrome and the RRFs showing on the modified Gomori trichrome stain may be gene or the linkage is known, for example, MNGIE, Alper’s noted in a minority of patients, especially in children aged < 3 disease, some LS, or BTHS, proceed with genetic studies. years,51 but our previous study showed the presence of RRF When the clinical picture is nonsyndromic but biochemical was not low in infants and children with MDs.42 Electron findings are highly suggestive of a MD, clinicians can consider microscopic examination of muscle cells may reveal abnormal proceeding with muscle and/or tissue biopsies, and/or mitochondrial configurations and/or subsarcolemmal measurement of mitochondrial RC enzymatic assays. By abnormal mitochondrial accumulation in those patients.42 In applying this approach, clinicians can use diagnostic criteria addition to morphological examinations, mitochondrial RC to obtain a more accurate diagnosis. If the diagnosis is still complex enzyme analysis of biopsied muscle or skin fibro- uncertain and difficult, it is necessary to follow up the blasts is helpful to confirm RC complex defects and provide a clinical manifestations of the patients. Mitochondrial diseases in infants and children 15

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 , optic 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 , etc.

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

Suspected syndromic MDs Suspected nonsyndromic MDs

Common mtDNA mutations, OGLST Common mtDNA deletions, + − Specific nDNA mutations, or Depletion genes (Table 1) + − Muscle and/or tissue biopsy + - Genetic counseling OGLST 75 + − Bernier diagnostic criteria Follow-up • Neurological and Family member nonneurologic S/S studies Muscle and/or tissue biopsy Common mtDNA mutations, • Clinical evolution + − Common mtDNA deletions, Specific nDNA mutations or 75 Re-evaluate Bernier diagnostic criteria Follow-up Depletion genes (Table 1) • Laboratory tests • Neurological and + − • Neuroimaging studies nonneurologic S/S Whole mtDNA sequencing, or • Clinical evolution NGS for other genes (Table 1) Genetic counseling NGS for other + genes (Table 1) Muscle and/or tissue Re-evaluate biopsy if indicated Genetic counseling Family member + • Laboratory tests studies Genetic counseling • Neuroimaging studies Family member studies Family member Muscle and/or tissue studies biopsy if indicated

Figure 2 A diagnostic algorithm in infants and children with mitochondrial disease. ABG Z arterial blood gas; CBC Z complete blood cell count; CNS Z ; 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 current approach to diagnosing and classifying MDs in- corporates clinical, biochemical, neuroradiological, patho- Although different consensus diagnostic criteria have been logic, and molecular information. The confirmation or proposed, an unresolved issue is the question of primary exclusion of MDs is therefore a major challenge for clini- versus secondary RC involvement. A growing number of cians, especially in cases with nonspecific clinical pheno- reports show RC involvement in other inborn errors of types. Therefore, follow-up evaluations of clinical metabolism such as fatty acid oxidation disorder, symptoms/signs and biochemical data are crucial. -responsiveness megaloblastic anemia, and 76 various neurodegenerative disorders of childhood. Conflicts of interest Careful interpretation of laboratory results is needed as enzyme deficiencies found on RC testing may be indicative The author declares no conflicts of interest with respect to of the primary disease or be secondary to another primary the research, authorship, and/or publication of this article. pathological condition. The diagnosis of MDs should therefore be conducted with care, especially in infants and children. Acknowledgments

6. Conclusion The author would like to thank Hsiu-Fen Lee, MD for collection of clinical data and study design, Chi-Zen Tsai, The study of MDs is a challenging and rapidly evolving field MSC, Tzu-Chao Wang, MSC, Chia-Ju Li, MSC, and Tzu-Yun of medicine. A single clinical feature or diagnostic criterion Hwang, MSC for molecular analysis, and Chia-Chi Hsu, MD is rarely sufficient for the proper diagnosis of a MD. The and Liang-Hui Chen, MSC for assays of metabolic profiles. 16 C.-S. Chi

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