SMGr up - SM Journals | Open Access Journals | SM Online ......testing and diagnosis. However in routine clinical practice, patients with isolated or combined symptoms/signs consistent

1Mitochondrial Diseases and Therapy | www.smgebooks.comCopyright Kyriacou K.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited.

Gr upSMChallenges Associated with the Diagnosis of Mitochondrial Diseases: There is Still a Need

for a Detective’s Work

INTRODUCTIONMitochondria are essential double-membrane bound organelles responsible for housing

a number of important metabolic pathways in the cell. Although these pathways include fatty acid oxidation, the Kreb’s cycle, the respiratory chain, and part of the glucose metabolism, the term mitochondrial diseases was coined initially and is still used today, to describe defects in the respiratory chain.

Mitochondrial diseases are a group of clinically heterogeneous disorders that are caused primarily by gene defects affecting directly or indirectly the mitochondrial oxidative respiratory chain [1,2]. These diseases are characterized by extreme phenotypic diversity [3], making their diagnosis particularly challenging and necessitating the use of several laboratory techniques for confirmation [4,5]. Important diagnostic milestones that have greatly facilitated the diagnosis of mitochondrial diseases in chronological order, include the use of the modified Gomori Trichrome stain [6] and the cloning of the mitochondrial genome [7]. Currently the field is dominated by molecular studies, which are leading to the identification of a plethora of pathogenic mutations,

Th Kyriakides1,2, G Tanteles3, A Hadjisavvas2,4 and K Kyriacou2,4*1Department of Neuropathology, The Cyprus Institute of Neurology and Genetics, Cyprus2The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Cyprus3Department of Clinical Genetics, The Cyprus Institute of Neurology and Genetics, Cyprus4Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Cyprus

*Corresponding author: K Kyriacou, The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Cyprus, Email: [email protected]

Published Date: August 17, 2015


in an impressively expanding list of genes, encoded by both the Mitochondrial DNA (mtDNA) and the Nuclear DNA(nDNA) (http://www.mitomap.org). Taking into account the diagnostic challenges, including genetic and phenotypic heterogeneity and the large repertoire of laboratory tests necessary for accurate diagnosis, this chapter aims, to provide a practical and user friendly account to guide the non-specialist neurologist. The present chapter has been divided into the following four sections: historical background, contemporary diagnostic tools, diagnostic dilemmas and discussion with conclusion.

HISTORICAL BACKGROUNDThe first mitochondrial disease was described by Luft who upon examination of a muscle biopsy,

demonstrated the presence of variably sized mitochondria containing paracrystalline inclusions. During the next two decades many cases of mitochondrial diseases were described, with electron microscopy (EM) playing a very significant role in the diagnostic work up. The availability of this ultrastructural detail led to the use of Greek terms such as “pleoconial” myopathy to describe the presence of too many abnormal mitochondria [8] and “megaconial” myopathy, which referred to the presence, of greatly enlarged mitochondria [9].

In 1963 W. King Engel introduced the modified Gomori Trichrome stain [6], that demonstrated mitochondrial aggregates in muscle fibres, which appeared as irregular subsarcolemmal purplish patches and coined the term Ragged-Red Fibres (RRF). This was a major finding that linked together, abnormal mitochondrial proliferation with a histochemical stain and which could be used routinely during light microscopy (LM) on biopsies of skeletal muscle. Till today, the presence of RRFs represents the most easily recognizable pathognomonic feature of mitochondrial disease. In the early seventies biochemical assays were developed and applied systematically to detect defects in individual complexes of the respiratory chain, including complex III [10] and complex IV [11]. Subsequently DiMauro and co-workers [12] proposed a biochemical classification of mitochondrial diseases [13].

As described above, historically muscle histology, histochemistry, EM and biochemistry, played a pivotal role in advancing our understanding and ability to classify mitochondrial disorders. However, it is the molecular genetic studies that have revolutionized the diagnosis of these diseases and propelled mitochondrial disorders to the forefront of 21st century medical science. Following the cloning of the mtDNA [7] another landmark discovery, was the identification of large scale mtDNA deletions in patients with mitochondrial myopathies [14]. Subsequently numerous molecular genetic investigations led to the identification of a plethora of mutations in mtDNA (http://www.mitomap.org). These ranged from point mutations [15] in patients with Leber Hereditary Optic Neuropathy (LHON), to the more notable multiple mtDNA deletion mutations, which characterize Progressive External Opthalmoplegia (PEO) [16]. Consequently by the turn of the 21st century more than one hundred different mtDNA mutations were detected [17], although it is appreciated that these are nothing but the tip of the iceberg, since it is well known

http://www.mitomap.org


that 90% of mitochondrial proteins and 86% of the respiratory chain components are encoded by the nuclear genome. Indeed the first mutation in a nuclear gene, was detected in two sisters diagnosed with Leigh syndrome (LS) in 1995 [18], who carried a mutation in the flavoprotein subunit. Subsequently this was followed by the description of mutations in structural proteins in patients with isolated complex I deficiency [19] as well as in other components of the respiratory chain complex.

Currently the number of pathogenic mtDNA and nDNA mutations identified in patients with mitochondrial diseases amount to more than 300 [20]. A unique aspect of mitochondrial disorders is that in one cell there are two genomes, the mtDNA and the nDNA. This entails that there has to be strict mechanisms in place, for executing a very tight and stringent control on their regulation. This is effected via intergenomic communication and the first gene that was discovered that was a component of this pathway was TYMP. This gene encodes thymidine phosphorylase and when mutated, causes Mitochondrial Neurogastrointestinal Encephalopathy (MNGIE) [21]. It is anticipated that the availability of Next Generation Sequencing (NGS) platforms, which enable analysis of the whole genome at affordable costs, will escalate the rate of identification of new genes as well as mutations, responsible for causing mitochondrial diseases.

Another important aspect of mitochondria is that they are dynamic organelles, which are continuously reshuffled by the processes of fission and fusion, true to their bacterial origin. Mitochondria are capable of moving long distances, especially in the long axons of motor neurons, ensuring adequate energy distribution throughout the cell compartments [22]. Consequently disturbances in mitochondrial dynamics cause disease. Examples include some types of Charcot Marie Tooth neuropathies which may be caused by mutations in either the MEN2 gene [23] or in the GDAP1 gene [24].

CONTEMPORARY DIAGNOSTIC TOOLSMitochondrial diseases are characterized by extreme phenotypic diversity and currently

several laboratory techniques are required for their diagnosis. The majority of mitochondrial diseases are caused by defects in oxidative phosphorylation, which may give rise to a variable symptomatology, in multiple organs. Typically, organs that have a high demand for energy supply, such as the muscle and brain are frequently affected, leading also to the use of the term “mitochondrial encephalomyopathies”, to describe these diseases [25]. Mitochondrial diseases include several well characterized clinical syndromes such as CPEO (progressive external ophthalmoplegia), MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episode), MERRF (mitochondrial encephalopathy with ragged red fibres), LHOA (Leber’s hereditary optic atrophy) and MNGIE (mitochondrial neurogastrointestinal encephalomyopathy). However many patients, present with a non-syndromic or monosymptomatic phenotypes, which gives rise to diagnostic difficulties. Clinical heterogeneity relates to mitochondrial biology such as heteroplasmy, proliferation of mutated mitochondria, fission-fusion dynamics as well as threshold effects [20].


In the last decades in order to facilitate the diagnosis of mitochondrial disorders several diagnostic criteria were formulated [25,26]. These criteria are based on a multidisciplinary approach combining clinical, biochemical, histopathological, histochemical and genetic investigations. Emphasis is placed on morphological abnormalities as revealed by histopathological and histochemical methods. Electron microscopical observations are also included in these criteria, although their diagnostic importance needs to be placed in context. Towards this goal previous work by our group showed that EM could greatly contribute to the diagnosis of MEs in certain patients, especially in children [27,28].

DIAGNOSTIC DILEMMASClinical

The true incidence and prevalence of mitochondrial disorders remain unclear, but some studies estimated that the prevalence of inherited mitochondrial disorders is > 1 in 5000 births [29]. As already alluded to mitochondria diseases exhibit a highly variable clinical phenotype, fueled by the dual genetic control of the respiratory chain and the biological phenomena of mitotic segregation, heteroplasmy and threshold of mitDNA mutations [20]. Patients with mitochondrial diseases may present with multisystemic phenotypes or eponymous syndromes but a significant number do not. The clinician should be aware of the common well recognized syndromes (Table 1) which if present, either as complete or form fruste, greatly facilitate genetic testing and diagnosis. However in routine clinical practice, patients with isolated or combined symptoms/signs consistent with but not diagnostic of mitochondrial disease is not uncommon and the dilemma is then how to proceed (Table 2). As can be seen organs with high reliance on oxidative phosphorylation are commonly involved and multiple organ involvement makes mitochondrial disease more likely. It should be kept in mind that mendellian diseases, unrelated to the mitochondrial respiratory chain, may also cause multisystemic disorders so either genetic or tissue confirmation is preferable to confirm mitochondrial disease.


Table 1: Classification of mitochondrial disorders (based on) [40].

Clinical findings/major clinical syndromesPreferred sample initially tested Comments

Blood MuscleDefects of mtDNA maintenance caused by nDNA mutations – Mendelian disorders often characterized by mtDNA depletion or multiple mtDNA deletions.

Various clinical phenotypes including: AHS, MCHS, MEMSA, ANS, arPEO, adPEO.

POLG-related disorders [41] *** *

Mutations in the nuclear gene POLG. Affected muscle tissue may show mitochondrial depletion or multiple deletions.

Heterogeneous group of autosomal recessive disorders characterized by a severe reduction in mtDNA copy number. MDS may affect either a specific tissue (most commonly muscle or liver) or multiple organs (heart, brain and kidney) [41].

MDS [41] * ***Pathogenic variants in several nuclear genes: DGUOK, MPV17, RRM2B, SUCLA2, SUCLG1, TK2, C10orf2, SLC25A4, FBLX4, MGME1, and TYMP.

Examples of other disorders due to nDNA defects [40]Mutations in respiratory chain subunits (e.g. LS).Mutations in respiratory chain ancillary proteins (e.g. GRACILE, LS).Defects in mtRNA translation (e.g. SPG7, LBSL).Defects in MIM lipid milieu (e.g. Barth, Sengers, MEGDEL).Defects in mitochondrial dynamics (e.g. DOA, CMT2A, CMT4A).

Great number of nuclear DNA encoded genes. A more structured diagnostic approach is needed.

Disorders due to mtDNA defectsStroke-like episodes at age <40 years, seizures and/or dementia, RRF and/or lactic acidosis, diabetes mellitus, cardiomyopathy, bilateral deafness, pigmentary retinopathy, cerebellar ataxia.

MELAS [42] * ***

MT-TL1 mutations (account for ~80% of cases of MELAS). Other mtDNA genes: MT-ND5, MT-TC, MT-TK, MT-TV, MT-TF, MT-TQ, MT-TS1, MT-TS2, MT-TW, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND3, and MT-ND6.

Myoclonus, generalized epilepsy, cerebellar ataxia, RRF in muscle, myopathy, dementia, optic atrophy, SNHL, peripheral neuropathy, spasticity, multiple lipomata.

MERRF [43] *** ***

MT-TK (m.8344A>G, >80%; other mutations ~10%)Other mtDNA genes: MT-TF, MT-TL1, MT-TI, and MT-TP (<5%).

Subacute painless bilateral visual failure, male preponderance, dystonia, cardiac pre-excitation syndromes.

LHON [44] *** N/N

Mutations in the mitochondrial genes MT-ND1, MT-ND2, MT-ND4, MT-ND4L, MT-ND5, and MT-ND6, are known to cause LHON.(~90% of mutant alleles: m.11778G>A in MT-ND4, m.14484T>C in MT-ND6, m.3460G>A in MT-ND1).

Diabetes and deafness. Other: Short stature, arrhythmias, myopathy, kidney disease, macular dystrophy, strokes and gastrointestinal disease.

MIDD [45] * ***

In most cases, MIDD is caused by a point mutation (3243A>G) in MT-TL1. In rare cases, caused by mutations in MT-TE and MT-TK genes.

Sideroblastic anemia of childhood, pancytopenia, exocrine pancreatic failure, renal tubular defects.

PS [46] *** N/NSingle mtDNA deletion (varying lengths).In PS, mtDNA deletions are usually more abundant in blood than in other tissues.

PEO onset at age <20 years, pigmentary retinopathy, one of the following: CSF protein >1g/L, cerebellar ataxia, heart block. Other: bilateral deafness, myopathy, dysphagia, diabetes mellitus, hypoparathyroidism, dementia.

KSS [46] * ****

MtDNA deletion in 90% (~90% have a large-scale mtDNA deletion that is often undetectable in blood cells, necessitating examination of muscle). Many different mtDNA deletions have been associated with KSS, a common 4977 bp deletion known as m.8470_13446del4977 is encountered most frequently.

External ophthalmoplegia, bilateral ptosis, mild proximal myopathy.

PEO [47] * ****

MtDNA deletion in ~50% (confined to skeletal muscle). Maternally inherited PEOs caused by various mtDNA point mutations.Mendelian PEOs associated with multiple deletions of mtDNA caused by germline mutations in the following genes: SLC25A4, C10orf2, POLG (see above), TYMP and OPA1.


Subacute relapsing encephalopathy, cerebellar and brain stem signs, elevated lactate concentrations in blood and/or CSF, typically infantile onset, bilateral symmetric hypodensities in the basal ganglia on CT or bilateral symmetric hyperintense signal abnormality in the brain stem and/or basal ganglia on T2-weighted MRI, maternal history of neurologic disease or Leigh syndrome.

MILS [48] *** N/N

~10% either the MT-ATP6 m.8993T>G or m.8993T>C mutation~10%-20% have mutations in genes other than MT-ATP6.Mitochondrial DNA deletions (<5%) are detected in post-mitotic tissues such as muscle, but not in blood or other replicating cells.- In most, Leigh syndrome patients, the disease is not caused by a mtDNA mutation but represents an autosomal recessive or X-linked disorder of mitochondrial energy generation.

Late-childhood or adult-onset peripheral neuropathy, ataxia, pigmentary retinopathy, abnormal electroretinogram, sensorimotor neuropathy, MRI changes, SNHL, cardiac conduction defects.

NARP [48] *** N/N MT-ATP6 is the only mitochondrial gene in which mutations are known to cause NARP.

Usually moderate-to-profound hearing loss.

Non-syndromic

mitochondrial hearing loss

[49]

*** N/N

Pathogenic variants in either MT-RNR1 or MT-TS1. MT-RNR1 mutations can be associated with predisposition to AID and/or late-onset SNHL. Mutations MT-TS1 are usually associated with childhood onset SNHL. The m.7445A>G pathogenic variant in MT-TS1 is also associated with palmoplantar keratoderma.

adPEO: Autosomal Dominant Progressive External Ophthalmoplegia; AHS: Alpers-Huttenlocher Syndrome; AID: Aminoglycoside-Induced Deafness; ANS: Ataxia Neuropathy Spectrum Disorders; arPEO: Autosomal Recessive Progressive External Ophthalmoplegia; CMT: Charcot-Marie-Tooth Disease; CSF: Cerebrospinal Fluid; CT: Computed Tomography; DOA: Dominant Optic Atrophy; GRACILE: Growth Retardation, Aminoaciduria, Cholestasis, Iron Overload And Early Death; KSS: Kearns-Sayre Syndrome; LBSL: Leukoencephalopathy, Brainstem And Spinal Cord Involvement, Lactic Acidosis; LHON: Leber Hereditary Optic Neuropathy; LS: Leigh Syndrome; MCHS: Childhood Myocerebrohepatopathy Spectrum; MDS: Mitochondrial Depletion Syndromes; MEGDEL: 3-Methylglutaconic Aciduria With Sensorineural Deafness, Encephalopathy And Leigh-Like Syndrome; MELAS: Mitochondrial Encephalo-Myopathy, Lactic Acidosis, And Stroke-Like Episodes; MEMSA: Myoclonic Epilepsy Myopathy Sensory Ataxia; MERRF: Myoclonic Epilepsy With Ragged Red Fibers; MIDD: Maternally Inherited Diabetes and Deafness; MILS: Maternally Inherited Leigh Syndrome; MIM: Mitochondrial Inner Membrane; MRI: Magnetic Resonance Imaging; NARP: Neurogenic Muscle Weakness, Ataxia, And Retinitis Pigmentosa; N/N: Not Necessary; PEO: Progressive External Ophthalmoplegia; PS: Pearson Syndrome; RRF: Ragged-Red Fibers: SNHL: Sensorineural Hearing Loss; SPG7: Spastic Paraplegia 7;

*: Likelihood Of Detecting A Mutation In Certain Tissue; Major Clinical Findings In Bold.


Table 2: Clinical features consistent with ME.Skeletal muscle

Ptosis

Opthalmoplegia

Proximal weakness

Exercise intolerance (fatigue, myalgia)

Heart

Cardiomyopathy

Conductive abnormalities

Brain

Seizures, myoclonic

Ataxia

Dementia

Hemiparesis

Chorea

Dystonia

“Encephalopathy”

Migraine

Depression

Nonspecific Infantile presentation

Hypotonia

Failure to thrive

Neurodevelopmental regression

Renal

Fanconi syndrome

Liver

Hepatic dysfunction

Diabetes mellitus

Nerve

Neuropathy

Intestine

Gastrointestinal disturbance

Weight loss

Bone marrow

Neutropenia

Ear

Deafness

Eye

Optic neuropathy

Retinal degeneration

Cataracts


Among the clinical features progressive external opthalmoplegia, typically in the absence of diplopia, is highly suggestive of ME. This was present in 18/51 well characterized mitochondrial diseases from a tertiary center [30]. Early age of onset is perhaps more in favor of a nuclear defect since there is a more “ubiquitous” involvement of tissues while mitochondrial heteroplasmy in primary mtDNA defects, ensures variability in the age of onset with progressive worsening.

Although mitochondrial diseases are well known for their maternal inheritance, most cases are due to nuclear gene defects, commonly a result of autosomal recessive inheritance. Therefore parental consanguinity should be inquired for and particular attention paid to subtle features in the family history pertaining to table 2. The clinician should bear in mind that, for mtDNA based disease, heteroplasmy modulates phenotype making family history particularly challenging.

Laboratory Dilemmas

Studies aiming to determine the diagnostic value of laboratory methodologies suffer from several limitations including: bias in patient selection, inclusion only of well recognized mitochondrial syndromes whereas other studies, combine both children and adults [5]. In order to facilitate and streamline the diagnostic process several authors formulated various sets of diagnostic criteria [25,26] for the identification of mitochondrial diseases. All are based on a multidisciplinary approach combining clinical, laboratory, histopathological, histochemical, biochemical and molecular genetic findings.

Lactic Acid and Creatine Kinase

One of the most frequent laboratory findings in patients with mitochondrial diseases is elevated blood lactate. Impaired function in oxidative phosphorylation causes a reduction in the levels of DTP which causes upregulation of glycolysis. This results in an over production of pyruvate which has one of two metabolic fates. Excess pyruvate can either be transaminated to alanine, or reduced to form lactate thus causing an increase in the plasma levels. However several studies demonstrate that the incidence of increased plasma lactate varies between 20-50% in children with mitochondrial disease [31,32]. The current view is that the majority of children with mitochondrial diseases do not have lactic acidosis and when present, consideration must be given to the fact that is not necessarily a specific finding. In chronic PEO lactic acid is typically normal.

Overall lactic acidosis is elevated in the blood and/or cerebrospinal fluid depending on the tissue involved and the extent of involvement. In the Newcastle series blood lactic acid was elevated in only 4/19(21%) of patients presenting with skeletal muscle symptoms (proximal myopathy or ptosis/opthalmoplegia). Five out of the six with proximal muscle weakness (83%) and 1/13(8%) in those with ptosis /opthalmoplegia [30].

Cerebrospinal lactic acid tends to be elevated if there is prominent CNS involvement even in the presence of normal venous level. In the Newcastle series 16/19(84%) of cases with abnormal


CT scan had an elevated CSF lactate. In 9/12(75%) of the patients presenting with CNS symptoms there was elevation of CSF lactate [30].

Serum creatine kinase(CK) is at best mildly elevated in cases with muscle involvement except in situations where the genetic defect predisposes to myoglobinuria which is characterized by transient marked elevations. In the Newcastle series only 4/42(10%) patients was the CK mildly elevated [30].

Fibroblast Growth Factor 21

A new biomarker called fibroblast growth factor 21 (FGF-21), is said to have more than 90% sensitivity and specificity in identifying skeletal muscle involvement, from mitochondrial respiratory chain disease [33]. It should be noted that most of the patients in this study had syndromic presentations which were genetically confirmed. The real value of this marker remains to be proven in patients with isolated myopathic presentations or incomplete syndromes.

Electromyography

In the Newcastle series thirteen out of nineteen (68%) of patients presenting either with proximal myopathy and/or ptosis/opthalmoplegia had a myopathic EMG but the abnormalities were mainly mild and nonspecific.

Electroencephalography

In the Newcastle series 11/11 (100%) patients presented with a CNS symptoms and had an EEG that was abnormal.

Histopathology

Muscle biopsy remains the gold standard in the diagnosis of mitochondrial disease with clinical or subclinical involvement of muscle. It allows for safe harvesting of an adequate sample for histochemical, biochemical and genetic studies. The modified Gomori trichrome histological stain, enables the detection of RRFs, which is the most widely recognized pathognomic feature of mitochondrial disorders (Figure 1a). Muscle biopsies, should be snap frozen in isopentrane cooled by liquid nitrogen, and then studied with histological and histochemical methods. Histochemistry for succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) are particularly useful. RRFs which are considered the hallmark of mitochondrial diseases represent the sub-sarcolemmal accumulation of mitochondria; these can also be detected by SDH, and are usually described as Ragged Red Fibre equivalents (RRFes) (Figure 1b).

In both adults and children COX remains the most sensitive stain because three of its subunits are coded on mtDNA and thus vulnerable to large scale mtDNA deletions and mitochondrial tRNA or rRNA point mutations. COX histochemistry visually demonstrates heteroplasmy in muscle by showing normal, COX-deficient and COX negative fibres, all in one cross-section (Figure 1c). It should be noted that nuclear defects affecting mtDNA maintenance can also give a mosaic pattern in COX histochemistry.


Figure 1: Light micrographs of serial sections of frozen muscle obtained from the same adult patient, diagnosed with a mitochondrial myopathy (x200). (a)Stained with modified Gomori Trichrome, showing a ragged red fibre (RRF) (arrow) (b) Incubated in medium for Succinate

Dehydrogenase showing the same fibre characterized as ragged red equivalent (RRFe) (arrow) (c) Incubated in medium for Cytochrome Oxidase (COX), showing the same fibre which is COX

negative (arrow).

The most common pathologic finding of muscle specimens is the presence of a number of COX negative fibres. Usually these are more evident in muscle biopsies from adult patients. In children it is often difficult to detect RRFs, due to the fact that it takes time to develop the extensive mitochondrial aggregates seen by electron microscopy, and correspond to a COX negative fibre by light microscopy. We have demonstrated that in children a more frequent finding is a general reduction in the intensity of COX staining (Figure 2). For this reason we recommend the use of appropriately selected controls, in the diagnostic evaluation of paediatric biopsies, suspected of mitochondrial diseases in order not to miss subtle changes in COX staining intensity. Diffuse COX deficiency is suggestive of nuclear defects which may frequently present in infancy or childhood. SDH is entirely coded in the nucleus and therefore a good marker of mitochondrial proliferation which is driven by energy failure. A combined COX and SDH histochemistry protocol is particularly user friendly to identify abnormal fibers and visualize both COX negative fibres which appear blue, as well as COX deficient fibres, which appear light blue (Figure 3).

Figure 2: Sections of frozen muscle from two paediatric cases, incubated in medium for Cytochrome Oxidase (COX) (x200). (a) Control muscle showing normal intensity of staining in a chequerboard pattern (b) Muscle from patient with mitochondrial myopathy, showing a

generalized reduction in the intensity of muscle fibres.


Figure 3: Sections of frozen muscle from a paediatric case incubated with a combined COX followed by SDH stains. COX negative fibres stain blue (arrows), while COX-deficient fibres stain a lighter blue (arrowheads).

EM is very useful especially in children since in this age group one often detects enlarged and abnormal mitochondria (Figure 4a) as opposed to the situation in adults where the most frequent finding is subsarcolemmal mitochondrial aggregates (Figure 4b), with many mitochondria containing paracrystalline inclusions (Figure 4c).

Figure 4: Electron micrographs of muscle biopsies. (a) Paediatric case showing the presence of abnormal and large mitochondria (“megaconial” myopathy) (arrows) (x12,000) (b) Adult

cases showing the presence of subsarcolemmal mitochondrial aggregates (“pleoconial” myopathy) (arrows) (x4,000) (c) Same as above showing presence of crystalline inclusions

within mitochondria (arrows) (x15,000).

Indeed in order to gain a more accurate appreciation of the diagnostic role of EM we carried out a comparative morphological study on 33 cases of mitochondrial encephalomyopathy, including 14 adults, age range from 20-65 and 19 children ages 6 months to 18 years. The main aim of the study was to evaluate and compare the presence of mitochondrial abnormalities using the four routine morphological methods of Gomori Trichrome, COX and SDH histochemistry and EM. Our results confirmed that in both adults and children COX histochemistry appeared to be the most sensitive methods for detecting mitochondrial disorders [34]. In addition we noticed differences in the patterns of COX histochemistry between adults and children. The most common patterns


of COX histochemistry in children were either a diffuse reduction, usually without evidence of mitochondrial proliferation, (either on LM or on EM) or COX-deficient fibres usually with evidence of mitochondrial proliferation (on LM and/or on EM). In contrast the most common pattern of COX histochemistry in adults was COX deficient fibers usually with evidence of mitochondrial proliferation (on both LM and EM) [34].

In the Newcastle series 39/47(83%) had abnormal COX and 32/47 (68%) had abnormal SDH histochemistry. In 18/19(95%) patients presenting with skeletal muscle symptoms (proximal myopathy and/or ptosis/opthalmoplegia) COX histochemistry was abnormal [30].

Several issues need to be kept in mind when observing mitochondrial abnormalities in the muscle biopsy including the fact that subtle mitochondrial abnormalities are not uncommon in biopsies with non-specific myopathic appearance [35]. Secondly there are several primary muscle diseases that may be accompanied by secondary mitochondrial changes. Thirdly age per se increases the likelihood of finding such changes by coincidence. Therefore any mitochondrial abnormalities will need to be the predominant finding and “quantitively” important. There are specific diagnostic criteria, be it arbitrary, covering the quantitative aspects of light and EM in diagnosing MEs and these should be kept in mind when the results are being interpreted [26].

Biochemical Analysis

This is usually carried out on frozen muscle in parallel with morphological examination, or at times on cultured fibroblasts or other tissue. Biochemical assay of complexes I-IV is performed and an isolated complex deficiency is due to a single respiratory chain complex subunit gene defect either in the mtDNA or nDNA or due to a gene defect of an assembly protein pertaining to the specific complex. What is more commonly found is deficiency in multiple complexes which can have a variety of explanations, including point mutations or deletions of mtDNA genes involved in protein synthesis or mutations in nuclear genes regulating maintenance of mtDNA.

As with the histochemical markers the degree of variation from normal that is adequate proof for mitochondrial disease is a dilemma, since secondary effects on mitochondria from other primary myopathies need to be kept in mind. Again to the rescue there are proposed criteria, as for the morphological markers, which perhaps veer on the cautious side.

In the Newcastle series 35/43 (81%) patients had abnormal respiratory chain enzymes. Among patients presenting with skeletal muscle symptoms (proximal weakness, ptosis/progressive opthalmoplegia) 15/18(83%) had abnormal enzymes [30].

Genetic Analyses

DNA analysis is preferably performed on symptomatic tissue and skeletal muscle is very convenient. Guided by the clinical phenotype and particularly by the presence of specific syndromes, genetic analysis can be targeted (Table 1). The pattern of COX deficiency on muscle biopsy can also direct testing towards mtDNA or nuclear DNA, for detecting mutations in


mitochondrial maintenance genes. Southern blotting or quantitative real time PCR of mtDNA, for detecting single or multiple deletions, or mitDNA depletion, polymerase chain reaction - restriction fragment length polymorphism, (PCR-RFLP) for common mtDNA mutations, mtDNA sequencing and mito-nuclear exome sequencing can be performed, subject to accumulated clinical and laboratory data [36]. Traditionally PCR based assays are employed to detect a small number of specific mtDNA point mutations, which are common in specific mitochondrial syndromes such as MELAS and MERF. Southern blotting, or quantitative real time PCR are used for detecting large rearrangements and mtDNA depletion [37]. At the molecular level current practice encourages, the analysis of the entire mitDNA, in suspect cases, since Sanger sequencing is widely available and affordable, even in small scale laboratories [38]. For bigger laboratories the way forward is to use the Next-Generation Sequencing (NGS), platforms, which offer the advantage and capability to carry out in depth reads, which enable the detection of mosaism and heteroplasmy phenomena [36]. In addition the availability of the high throughput NGS technology facilitates the analysis, not only of the entire mitDNA but also of the entire exome. More in demand on NGS platforms, is the analysis of the entire mitDNA and nDNA, targeting the nuclear genes, which are most frequently associated with the mitochondrial disorders [39].

Brain Imaging

Clearly CT and MRI imaging, which are widely available, are particularly helpful and certain patterns such as signal alterations in the basal ganglia and brainstem or the cerebrum (crossing over arterial territories) are particularly helpful in confirming clinical or subclinical brain involvement. In the Newcastle series 13/15(86%) of patients with CNS presentation had an abnormal CT scan [30].

DISCUSSION AND CONCLUSIONSMitochondrial disorders consist of a wide spectrum of clinical phenotypes, which include

pure myopathies and multisystem disorders. The brain, the skeletal muscle and the kidney are particularly prone to pathologies, as a result of their high dependence on oxidative phosphorylation. In addition the dual genetic control of the respiratory chain, the biological phenomena of heteroplasmy, the threshold effect, mitotic segregation and mitochondrial fusion fission, greatly contribute to this phenotypic diversity. Furthermore the lack of a laboratory gold standard test, which can rule in or rule out a case of suspected ME, makes diagnosis of non-syndromic or atypical cases particularly challenging. In this context the aim of this chapter is to present a practical approach in diagnosing mitochondrial disease, based on clinical phenotype and routine laboratory investigations. Particular attention was devoted to diagnostic dilemmas which arise due to the lack of a golden standard diagnostic test and clinical heterogeneity.

We suggest the following diagnostic pathway;

Take a thorough history of presenting complaint and a family history bearing in mind the spectrum of phenotypes in Tables 1 and 2.


Carry out routine blood tests such as creatine kinase and blood lactic acid.

If a syndrome from Table 1 is suspected, then perform the recommended DNA test as per table 1, targeting known mutations.

If the patient does not fulfill a recognized syndrome then;

For predominantly skeletal muscle presentations (Table 2) carry out an EMG, a muscle biopsy for COX/SDH histochemistry, biochemical assay and DNA testing guided by COX histochemistry.

For predominantly brain presentation (Table 2) carry out brain imaging (CT/MRI), EEG and cerebrospinal fluid lactate/protein measurement and may proceed to a muscle biopsy as above.

Any combination of features from table 2 and high index of suspicion carry out a muscle biopsy as above.

If the muscle biopsy shows abnormal COX histochemistry;

If there is a mosaic pattern of COX deficiency then test for mtDNA mutation/deletion/depletion and if indicated, test nuclear genes concerned with mtDNA maintenance.

If there is a diffuse reduction in COX check for likely nuclear genes based on clinical phenotype.

Depending on clinical phenotype and availability mito/nuclear DNA exome sequencing may be performed.

In the absence of proof of a definitive pathogenic mutation either in the mtDNA or nDNA the criteria published by Bernier et al [26] may be used to facilitate a working diagnosis.

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SMGr up - SM Journals | Open Access Journals | SM Online ......testing and diagnosis. However in routine clinical practice, patients with isolated or combined symptoms/signs consistent