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Mitochondrial diseases
Handbook of Clinical Neurology, Vol. 145 (3rd series) Neuropathology G.G. Kovacs and I. Alafuzoff, Editors http://dx.doi.org/10.1016/B978-0-12-802395-2.00010-9 Copyright © 2018 Elsevier B.V. All rights reserved
Chapter 10
Mitochondrial diseases 1
MARIA J. MOLNAR1* AND GABOR G. KOVACS2 Institute of Genomic Medicine, Rare Disorders, Semmelweis University, Budapest, Hungary 2
Institute of Neurology, Medical University of Vienna, Vienna, Austria
Abstract Mitochondrial disorders represent a major challenge in medicine. Most of the mitochondrial proteins are encoded by the nuclear DNA (nDNA), whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction. The disease usually affects multiple organs in varying locations and severity; however, there are some forms which affect a single organ. The diagnosis of mitochondrial disorders is based on clinical examination, biochemical and histopathologic examinations, functional studies, and molecular genetic testing. Neuropathologic alterations of the muscle are variable and can range from striking abnormalities, such as cytochrome oxidase-negative and ragged red fibers, to nonspecific or minimal changes. Neuropathologic alterations in the brain show common features in disorders with different genetic background. These are characterized by various degrees of vacuolation in the white and gray matter, regional neurodegeneration with reactive astrogliosis, loss of oligodendrocytes, presence of macrophages and microgliosis, capillary proliferation, and mineralization of vessel walls. The advent of molecular genetics, the discovery of biomarkers and new sequencing platforms to perform targeted exome and whole-genome sequencing have changed traditional approaches to diagnose mitochondrial diseases.
INTRODUCTION Mitochondrial disorders represent a major challenge in medicine. The first patient with mitochondrial disease was described in 1962; the patient had hypermetabolism and normal thyroid function (Luft et al., 1962). Most of the mitochondrial proteins are encoded by nuclear DNA (nDNA), whereas a very small fraction is encoded by mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction. This leads to a wide range of cellular alterations, such as excessive reactive oxygen species production, abnormal calcium homeostasis, dysregulated apoptosis, and insufficient energy generation in various organs, especially those with high energy demand. The disease usually affects multiple organs in varying locations and severity; however, there are some forms which only affect a single organ (e.g., the eyes in
Leber hereditary optic neuropathy (LHON)). The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathologic alterations, functional studies, and molecular genetic testing. However, with the advent of molecular genetics, the discovery of improved biomarkers and new sequencing platforms to perform targeted exome and whole-genome sequencing, traditional approaches to diagnose mitochondrial disease are now changing.
PREVALENCE Combined epidemiologic data on childhood and adult mitochondrial disease suggest that the prevalence is at least 1 in 5,000 (20 per 100,000) (Schafer et al., 2004; Gorman et al., 2015). The prevalence of nuclear mitochondrial disease is 2.9 per 100,000 adults (Schafer
*Correspondence to: Maria Judit Molnar, MD, PhD, DSc, Institute of Genomic Medicine and Rare Disorders, Semmelweis University, T€omőstr. 25–29, 1083 Budapest, Hungary. E-mail: [email protected]
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et al., 2004; Gorman et al., 2015). Gorman et al. (2015) calculated the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (1 in 4,300), as one of the commonest adult forms of inherited neurologic disorders. A prospective American study estimated the prevalence as 1:200 to harbor a pathogen mutation at risk of developing mitochondrial disease (Saneto and Sedensky, 2013). The prevalence of the specific mtDNA mutation m.3243A > G, as the most prevalent pathogenic mtDNA point mutation in the north-east of England (Gorman et al., 2015), is 7.8/100,000. The prevalence of LHON, the most prevalent mtDNA disorder overall, for affected individuals is 3.65 per 100,000; carrier frequency of the three common LHON mutations is 4.42 per 100,000, with evident familial clustering (m.11778G > A: 12 pedigrees, m.3460G > A: 5 pedigrees, m.14484 T > C: single pedigree). Myoclonus epilepsy with ragged red fibers (MERRF) related to the m.8344A > G mutation remains a rare form of mitochondrial disease (0.7/100,000) in the north-east of England. To date, 14 nuclear encoded genes, TYMP (thymidine phosphorylase), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4), POLG, PEO1, OPA1, POLG2, RRM2B, TK2 (thymidine kinase 2), DGUOK (deoxyguanosine kinase), MPV17 (mitochondrial inner membrane protein), MGME1 (mitochondrial genome maintenance exonuclease), DNA2 (DNA replication helicase/nuclease 2), SPG7, and AFG3L2 (AFG3-like AAA ATPase 2) have been reported to be associated with ophthalmoparesis and multiple mtDNA deletions in muscle of adult-onset chronic progressive external ophthalmoplegia (CPEO) patients. Notably, mutations in SPG7 (4/100,000) and PEO1 (3/100,000), followed by OPA1 (1/100,000) and RRM2B (0.9/ 100,000), have emerged as major causes of adult mitochondrial disease. Recessive mutations in POLG (0.6/100,000) are also emerging as an important cause of mitochondrial disease in the north-east of England (Gorman et al., 2015).
diabetes, deafness, visual, heart, liver, and kidney problems, stroke, and migraine have been described (Calvo et al., 2006). The disorders are caused either by mutations of the maternally inherited mitochondrial genome, or by nDNA mutations. Today more than 200 different diseasecausing mutations of mtDNA are known and due to the increased knowledge about nuclear genetics during the last few years, further at least 100 nuclear gene mutations have been described. In 70–85% of mitochondrial diseases nuclear mutations are responsible for the disease. Owing to the unequal distribution of mitochondria in the different tissues and the coexistence of mutant and wild-type mtDNA in these organelles, these disorders may present with a huge variety of symptoms, even related to the same mutation (Molnar, 2009). Children often have severe disease associated with nDNA mutation, causing respiratory chain defect and crashing mitochondrial integrity. In addition to their fundamental role in cellular energy metabolism, mitochondria seem to contribute to the pathogenesis of many degenerative diseases, aging, and cancer.
MANIFESTATION OF MITOCHONDRIAL DISEASES
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A variety of organs can be affected by mitochondrial dysfunction. The most oxidative tissues (brain, muscle, retina, cochlea, liver, and kidney) are the most vulnerable to OXPHOS defects (Shoubridge and Molnar, 2003; DiMauro and Hirano, 2005). In patients with mitochondrial disorders, most commonly the symptoms of epilepsy, ataxia, delayed motor and mental development, failure to thrive, signs of neurodegenerative diseases,
GENETICS AND SYNDROMES mtDNA-related mitochondrial disorders mtDNA is inherited mostly maternally; the nuclear genes are inherited autosomal-dominantly, recessively, or in an X-linked manner. The most important characteristics of mtDNA are as follows (Shoubridge and Molnar, 2003): ● ● ●
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The mitochondrial genome is a 16,569-bp long circular DNA. It consists of 37 genes that encode 13 proteins, 22 tRNAs, and 2 rRNAs. The mitochondrial genome is not able to independently produce all of the proteins needed for functionality; thus, mitochondria rely heavily on imported nuclear gene products. In one mitochondrion there are multiple copies of its mtDNA. A cell contains several thousand copies of its mitochondrial genome (polyplasmy). Wild-type and mutated mtDNA are present at the same time in a cell (heteroplasmy). To develop a clinical sign a certain amount of mutated mtDNA has to be present in a tissue (threshold effect).
Hundreds of pathogenic mtDNA mutations have been reported in the literature (for example, a database of reported mtDNA mutations can be found at www. mitomap.org). The most common mitochondrial disease due to mtDNA mutations is LHON with homoplasmic mtDNA mutations G11778A (69%), G3460A (13%), T14484C (14%) and other substitutions (Remenyi
MITOCHONDRIAL DISEASES et al., 2015). Men are involved more than women. Painless visual loss begins in young adulthood with optic atrophy. The next prevalent phenotype is mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndromes (MELAS) with external ophthalmoplegia, diabetes mellitus, hearing loss, early-onset stroke-like symptoms, migraine, and cognitive dysfunction due to mutations in the mitochondrial tRNA Leu gene (A3243G 80%, T3271C 7%, A3260G and A3252G 5–5%). Further common mtDNA-related syndromes are the MERRF, neuropathy, ataxia, retinitis pigmentosa (NARP), and Kearns–Sayre syndrome. MERRF evolves due to mtDNA tRNA Lys mutations as A8344G (80%), T8356C, G8363A, and G8361A (10%), causing myoclonus epilepsy, ataxia, dementia, neuropathy, myopathy, multiple lipomas in “collar” distribution, hearing loss, declining cognitive function, and neuropathy (Cohen, 2013). The A8993C or T substitution may result in the NARP syndrome or maternally inherited Leigh disease. Bilateral basal ganglia lesions, psychomotor retardation, seizures, movement disorders, and lactic acidosis characterize Leigh disease. Kearns–Sayre syndrome is the result of the mtDNA “common” large-scale deletion. Clinical symptoms include CPEO, retinal pigmentary abnormalities, cardiac conduction abnormalities, proximal and bulbar weakness, and sometimes ataxia. The ratio of heteroplasmy in many cases is very high in patients with mtDNA disorders. Usually the higher ratio of heteroplasmy is associated with more severe clinical phenotype. It is important to know that identical pathogenic mtDNA mutations may cause disease at different ages and different clinical syndromes. Even within the same family carrying the same mutation, there is extreme phenotypic variability (Crimmins et al., 1993). The m.A3243G mutation may result in lethal disease in childhood but we know patients with milder phenotypes such as maternally inherited diabetes and deafness or deafness (Remenyi et al., 2015). It is presumably due to mechanisms such as the different tissue distribution of mtDNA mutations, the threshold effects of the tissue, environmental stressors, and underlying nuclear genetic backgrounds.
nDNA-related mitochondrial disorders nDNA encodes 1,000 mitochondrial proteins, OXPHOS proteins (74) and factors, forming the respiratory chain complexes or their assembly factors; mitochondrial membrane proteins and transporters and proteins responsible for networking; or proteins involved in mtDNA maintenance (Wong, 2010). Defects in nDNA genes can be inherited in an autosomal or X-linked manner. In recent years next-generation sequencing
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technologies have been revolutionizing diagnostic screening for rare primary mitochondrial disorders, particularly those caused by nuclear gene defects. Wholeexome sequencing expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine (Legati et al., 2016). As far as we know, presently more than 200 nuclearencoded genes are implicated in mitochondrial diseases (Table 10.1). Every year about 10 new disease-causing genes are discovered (Calvo et al., 2012). There are five groups of nDNA mutations causing diseases: 1. Mutations in genes encoding respiratory chain proteins. Most frequently these alterations result in Leigh syndrome. 2. Defects of mitochondrial dynamics: fusion genes are in the inner membrane such as OPA1 (optic atrophy 1) or in the outer membrane, like MFN2 (mitofusin2). Their mutations cause optic atrophy and Charcot–Marie–Tooth disease type 2A, respectively. The fission gene is DLPA1 (dynamin-like protein1). 3. Defects of mitochondrial protein synthesis: e.g., mutations in genes of tRNA-modifying enzymes (PUS1, TRMU), elongation factors (TUFM, TSFM), or mitochondrial aminoacyl tRNA synthetases (RARS2, DARS2, YARS2). 4. Defects in lipid milieu/transporter carriers of the inner mitochondrial membrane, like TAZ gene (taffazin transacylase) catalyzing cardiolipin maturation, or DDP1 (TIMM8A), causing Mohr–Tranebjaerg syndrome. 5. Defects of mtDNA maintenance (see the next section).
DEFECTS OF MTDNA MAINTENANCE Cross-talk between nuclear and mitochondrial genomes is crucial for mitochondrial biogenesis and function, and the two genomes are probably subjected to coevolutionary processes. The defect of intergenomic signaling can affect mtDNA quantitatively (mtDNA depletion) and qualitatively (multiple mtDNA deletion). Nuclear genes responsible for the integrity of the mtDNA can be classified as follows: genes responsible (1) for mitochondrial DNA replication and repair (POLG, Twinkle); (2) for mitochondrion biogenesis and for the maintenance of nucleotide pool (SLC25A3, SLC25A4, TYMP, DGUOK, TK2, RRM2B, MVP17, SUCLAA, SUCLG1); and (3) for mitochondrial translation (PUS1, TRMU, LRPPRC, TUFM, TSFM and GFM1, MRPS16 and MRPS22, RARS2, DARS2, and YARS2). The most frequent gene, affected in intergenomic communication disturbances is POLG (polymerase gamma). Mutations in
Table 10.1 Phenotype–genotype correlation in some mitochondrial disorders due to nDNA mutations Phenotype Respiratory complex disorders Childhood encephalopathy Cardiomyopathy and encephalopathy with complex I deficiency Encephalopathy, hypertrophic cardiomyopathy Multisystemic complex I deficiency Leigh syndrome with complex 1 deficiency
Gene
NDUFS1 NDUFS2 NDUFS2, NDUFV2 NDUFS4 NDUFS1, NDUFS4, NDUFS4, NDUFS7, NDUFS8, NDUFV1, SDHAF1 NDUFV1 SDHB, SDHC, SDHD, and SDHAF2 L SDHA BCSL1 COX10 COX15, SCO2 SCO1 ETHE-1
Leigh syndrome with complex 2 deficiency Leukodsytrophy, myoclonic epilepsy Hereditary paraganglioma, pheochromocytoma Optic atrophy and ataxia (complex II deficiency GRACILE syndrome Encephalomyopathy, renal tubulopathy Infantile cardioencephalomyopathy Hepatoketoacidotic coma Ethylmalonic encephalopathy Disorders of mitochondrial protein synthesis Leukoencephalopathy DARS2 Myopathy, lactic acidosis, sideroblastic anaemia YARS2 Disorders of mtDNA maintenance (disorders associated with mtDNA deletion or depletion) CPEO, myopathy POLG1, POLG2, Twinkle, RRM2Bm SLC25A4 IOSCA POLG1 Alpers–Huttenlocher syndrome POLG1 Ataxia, neuropathy syndromes POLG1, Twinkle,OPA1 MIRAS POLG1 MNGIE TP SMA-like myopathy TK Hypotonia, movement disorder, and/or Leigh SUCLA2 syndrome with methylmalonic aciduria Hepatoencephalopathy DGUOK Defects of mitochondrial dynamics Charcot–Marie–Tooth II MNF2 Hereditary spastic paraplegia KIF5A Optic atrophy OPA1, OPA2 Defects of the lipid milieu Barth syndrome Tafazzin Other nDNA encoded mitochondrial disorders Dilatated cardiomyopathy with ataxia (DCMA) DNAJC19 Epilepsy, episodic ataxia, encephalopathy Pyruvate dehydrogenase Encephalopathy, hepatomegaly HMC-CoA-lyase Epilepsy, encephalopathy HMGCS2 Friedreich ataxia FXN Hepatopathy, hypotonia, failure to thrive DGUOK Hereditary spastic paraplegia SPG7 Hypocarnitinemia, hypolysinemia DCAR Menkes disease, occipital horn syndrome ATP7A Microcephaly SCL25A19 Mohr–Tranebjaerg syndrome DDP1/TIMM8A Myopathy, retinopathy, hepatomegaly HADHA Wolfram syndrome WFS1
GRACILE, growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis, and early death; IOSCA, infantile-onset spinocerebellar ataxia; MIRAS, mitochondrial recessive ataxia syndrome; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; SMA, spinal muscular atrophy.
MITOCHONDRIAL DISEASES the POLG gene, which encodes for the catalytic subunit of the mitochondrial DNA polymerase (pol-g), have been related to a wide variety of autosomal disorders, inherited both in a dominant and recessive fashion (Milone and Massie, 2010). POLG mutation may result in secondary mtDNA multiple deletions or depletions. The most common phenotype is CPEO. The bestknown autosomal-recessive inherited disorders are the sensory-ataxic neuropathy and dysarthria with external ophthalmoplegia (SANDO), Alpers-Huttenlocher syndrome, a severe pediatric hepatoencephalopathy, mitochondrial recessive ataxia syndrome, and myoclonic epilepsy myopathy sensory ataxia. Mutations in Twinkle and ANT1 genes provoke multiplex deletion with autosomal-dominant CPEO phenotype. Proper balance of the mitochondrial deoxynucleotide pools is essential for the maintenance of mtDNA copy number. Defects in these genes lead to depletion of mtDNA. The mitochondrial depletion syndrome may manifest as myopathic, hepatocerebral, and encephalomyopathic forms. The myopathic form usually occurs in the first year of life with feeding difficulty, failure to thrive, hypotonia, muscle weakness, and occasionally CPEO. It is due to mutations mostly in ANT1 (responsible for ADP/ATP balance), TK2 (in charge of pyrimidine biosynthesis), or RRM2B, and POLG. The hepatocerebral form is frequently due to mutated DGUOK (in charge of purin biosynthesis), POLG, or Twinkle. A peculiar form of hepatocerebral mitochondrial depletion syndrome is the Alpers–Huttenlocher syndrome, an early-onset, fatal disease, characterized by hepatic failure and intractable seizures. In these cases, due to POLG single-nucleotide polymorphism severe valproate hepatotoxicity is observed (Delarue et al., 2000). The encephalomyopathy usually is due to mutated SUCLA and RRM2B genes and characterized by infantile onset of hypotonia with severe psychomotor retardation, high lactate in blood, progressive neurologic deterioration, a hyperkinetic-dystonic movement disorder, external ophthalmoplegia, deafness, generalized seizures, and variable renal tubular dysfunction. Brain magnetic resonance imaging could be suggestive of Leigh syndrome (Spinazzola and Zeviani, 2007). The second large group of the mitochondrial integrity disorders are the mtDNA deletion syndromes. The main clinical manifestations associated with multiple deletions are: 1.
CPEO (autosomal-dominant or recessive) (Filosto et al., 2003; Luoma et al., 2004). Additional symptoms may include cataracts, hearing loss, sensory axonal neuropathy, ataxia, depression, and parkinsonism.
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2. SANDO is an autosomal-recessive systemic disorder characterized mainly by adult-onset sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (Spinazzola and Zeviani, 2007). 3. Mitochondrial neurogastrointestinal encephalomyopathy, an autosomal-recessive disorder clinically characterized by young age at onset, gastrointestinal dysmotility (often pseudo-obstruction), cachexia, diffuse leukoencephalopathy, peripheral neuropathy, and early death. mDNA abnormalities can include depletion, multiple deletions, and point mutations (Hirano et al., 1994). 4. Spinocerebellar ataxia – epilepsy syndrome disorder is similar to SANDO but with a higher frequency of migraine headaches and seizures (Winterthun et al., 2005).
NEUROPATHOLOGIC CHANGES IN MITOCHONDRIAL DISORDERS Muscle Muscle biopsy is a very common diagnostic procedure in the diagnosis of the mitochondrial disorders. The pathologic changes of the muscle are variable and can range from striking abnormalities, highly evocative of a mitochondrial disease, to nonspecific or minimal. The absence of pathologic changes does not exclude a mitochondrial problem. The two most valuable tools in pathologic assessment of muscle biopsy for suspected mitochondrial myopathy are the succinate dehydrogenase (SDH) enzyme histochemical reaction and the cytochrome oxidase (COX) reaction, which can be performed together (Fig. 10.1). The modified G€om€ori trichrome reaction is also useful, but less specific and less sensitive because only well-developed ragged red fibers with prominent red stain throughout the fiber, and particularly at the periphery, reflecting the presence of structurally abnormal mitochondria (Fig. 10.1). Using hematoxylin and eosin staining these fibers have a granular, basophilic appearance and react intensely for SDH and NADH-TR (Fig. 10.1). Modification of the SDH reaction by the addition of phenazine methosulfate is useful because it suppresses the reactivity of normal mitochondria. It shows both ragged red fibers and ragged red equivalents. The fibers that are positive with the modified SDH have been called ragged blue fibers. Further pathologic features in mitochondrial myopathies include variation in fiber size, with atrophy but little or no hypertrophy, an increase in internal nuclei, necrosis and fiber regeneration in cases with myoglobinuria. In conditions in which there is associated peripheral nerve involvement there may be associated fiber-type grouping and the presence of angular atrophic fibers.
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Fig. 10.1. Myopathologic alterations in mitochondrial disorders. (A) (Cytochrome oxidase (COX) staining. (B) Succinate dehydrogenase (SDH) staining. (C) Combined COX/SDH staining: the strongly blue fibers indicate loss of COX staining. (D) Hematoxylin and eosin staining showing fibers with basophilic rim representing ragged-red fibers. (E) Combined COX/SDH staining reveals a ragged-blue fiber. (F) Typical ragged-red fiber as seen in Gomori-trichrome staining (the same fiber as shown in E).
The ultrastructural abnormalities of mitochondria include an alteration in shape, increase in size, disruption and/or distortion of the cristae, and paracrystalline or osmiophilic inclusions (Fig. 10.2). Electron microscopy may reveal varying degrees of myofibrillar loss and disruption and an increase in intracellular lipid, which can often be detected with oil red O or Sudan black staining as well. It must be emphasized that ragged red and COXnegative fibers are not specific for mitochondrial myopathies and may be a secondary feature in some muscular dystrophies and inflammatory myopathies, in particular inclusion body myositis. As the number of COXnegative fibers increases with age the observation of 2-3 COX-negative fibers in a sample of an elderly patient should not be interpreted as significant.
Brain Neuropathologic alterations in the brain show common features in disorders with different genetic backgrounds. The distribution of neuropathologic alterations is usually characteristic for a syndrome (Table 10.2). Various
degrees of vacuolation in the white and gray matter, regional neurodegeneration with reactive astrogliosis, loss of oligodendrocytes, presence of macrophages and microgliosis, capillary proliferation, and mineralization of vessel walls can be seen in most of those, which are associated with central nervous system symptoms (Fig. 10.3). Neurons may be preserved in the vacuolated gray-matter areas, giving the appearance of pseudonecrosis. A recent study suggested an intramyelinic nature of white and gray-matter vacuolation and presence of intraoligodendroglial vacuoles (Szalardy et al., 2016). It has been suggested that oligodendrocytes are important players of the pathogenesis of central nervous system lesioning (Szalardy et al., 2016). The affection of myelin is supported also by the observation of syndromes with hypomyelination in the brainstem and spinal cord (Taft et al., 2013). There are rare reports of multiple sclerosis-like lesioning with selective demyelination and prominent inflammatory infiltrates in the brain in LHON (Kovacs et al., 2005), which, on the other hand highlights the role of mitochondria in the pathogenesis of multiple sclerosis.
MITOCHONDRIAL DISEASES
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Fig. 10.2. (A–D) Representative electron microscopy images of paracrystalline mitochondrial inclusions. Table 10.2 Characteristic neuropathologic findings in the brain in mitochondrial disorders with brain involvement Neuropathologic feature
Syndrome
Remark
Vacuolation of the WM
KSS MDS; LS, MERRF, LHON, MELAS AHS KSS
Prominent and characteristic in cerebral and cerebellar WM Variably present in cerebral WM
LS
Characteristic feature in subcortical areas (i.e., thalamus) and brainstem but not in the cortex Only mild degree, mostly in cortex Focal or missing Not seen Characteristic feature in the cortex (i.e., striate) and mild in subcortical areas Not seen Associates with spongy degeneration of the GM Characteristically seen with GM lesions Seen in the cerebral and cerebellar cortex In the hepatocerebral form Purkinje cell loss Retinal ganglion cells Focal/mild or not seen
Spongy degeneration of the GM
MELAS MERRF LHON AHS
Neuron loss/astrogliosis
Capillary proliferation/ prominence Other
MDS KSS MELAS, MERRF, LS AHS MDS LHON KSS, LHON, MERRF, AHS MELAS, LS MELAS LS
Usually not involved Present mostly in the brainstem
Characteristically seen Cortical laminar necrosis: infarct-like lesions “Vasculonecrotic” lesions
AHS, Alpers–Huttenlocher syndrome; GM, gray matter; KSS, Kearns–Sayre syndrome; LS, Leigh syndrome; LHON, Leber hereditary optic neuropathy; MDS, mitochondrial depletion syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndromes; MERRF, myoclonus epilepsy with ragged-red fibers; MDS, mitochondrial depletion syndrome; WM, white matter.
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Fig. 10.3. Neuropathologic alterations in the brain in mitochondrial disorders. (A and B) Spongy vacuolation in the white matter in Kearns–Sayre syndrome. (C) Capillary proliferation in the basal ganglia (left side of image) in Leigh syndrome associated with spongy degeneration of the thalamus (D), the pontine tegmentum (E), and vacuolation of the white and gray matter of the spinal cord (F).
DIAGNOSTIC PRINCIPLES During the diagnostic evaluation of suspected mitochondrial disease, the following strategy is recommended (Shoubridge and Molnar, 2003). The most important laboratory parameters are resting serum lactate and pyruvate, which are frequently increased. The ratio of lactate/pyruvate is increased in many cases. Serum creatine kinase levels are either normal or slightly elevated. Serum lactate increases during slight exercise in mitochondrial patients and 30 minutes after the exercise will not decline to the baseline. The use of a biomarker such as serum of fibroblast growth factor 21 levels is a promising new approach in the diagnostic pathway for patients with mitochondrial disease (Suomalainen et al., 2011). Electromyogram is normal, neurogenic or myogenic, or not specific. The muscle biopsy mostly displays the characteristic ragged red or ragged blue fiber pathology. The ragged red fibers usually do not have COX activity. Ultrastructural analysis of the muscle reveals aberrant and enlarged mitochondria, usually with paracrystalline inclusions or abnormally organized cristae. Biochemical investigations detect the reduced activity of the affected
enzyme. Searching for mtDNA mutation genetic testing is recommended on postmitotic tissue (e.g., muscle specimen). In the routine diagnostic for the mtDNA mutation hotspots, mtDNA deletion and depletion are screened. In many cases the whole mtDNA is sequenced. Mutations in the nuclear genes were searched based on the clinical and imaging phenotype for many years. In the near future the targeted panel sequencing of these nuclear genes and whole-exome analysis by next-generation sequencing will improve the diagnostics of mitochondrial disorders due to nuclear gene mutations.
REFERENCES Calvo S, Jain M, Xie X et al. (2006). Systematic identification of human mitochondrial disease genes through integrative genomics. Nat Genet 38: 576–582. Calvo SE, Compton AG, Hershman SG et al. (2012). Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci Transl Med 4: 118. Cohen BH (2013). Neuromuscular and systemic presentations in adults: diagnoses beyond MERRF and MELAS. Neurotherapeutics 10: 227–242.
MITOCHONDRIAL DISEASES Crimmins D, Morris JG, Walker GL et al. (1993). Mitochondrial encephalomyopathy: variable clinical expression within a single kindred. J Neurol Neurosurg Psychiatry 56: 900–905. Delarue A, Paut O, Guys J et al. (2000). Inappropriate liver transplantation in a child with Alpers–Huttenlocher syndrome misdiagnosed as valproate-induced acute liver failure. Pediatr Transplant 4: 67–71. DiMauro S, Hirano M (2005). Mitochondrial encephalomyopathies: an update. Neuromusc Disord 15: 276–286. Filosto M, Mancuso M, Nishigaki Y et al. (2003). Clinical and genetic heterogeneity in progressive external ophthalmoplegia due to mutations in polymerase-gamma. Arch Neurol 60: 1279–1284. Gorman GS, Schaefer AM, Ng Y et al. (2015). Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann Neurol 77(5): 753–759. Hirano M, Silvestri G, Blake DM et al. (1994). Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): clinical, biochemical, and genetic features of an autosomal recessive mitochondrial disorder. Neurology 44: 721–727. Kovacs GG, H€oftberger R, Majtenyi K et al. (2005). Neuropathology of white matter disease in Leber’s hereditary optic neuropathy. Brain 128: 35–41. Legati A, Reyes A, Nasca A et al. (2016). New genes and pathomechanisms in mitochondrial disorders unraveled by NGS technologies. Biochim Biophys Acta 1857: 1326–1335. Luft R, Ikkos D, Palmieri G et al. (1962). A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 41: 1776–1804. Luoma P, Melberg A, Rinne JO et al. (2004). Parkinsonism, premature menopause, and mitochondrial DNA polymerase-gamma mutations: clinical and molecular genetic study. Lancet 364: 875–882. Milone M, Massie R (2010). Polymerase gamma 1 mutations: clinical correlations. Neurologist 16: 84–91.
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Molnar MJ (2009). Encyclopedia of Molecula Mechanisms of Disease In: F Lang (Ed.), Springer-Verlag Berlin Heidelberg. Remenyi V, Inczedy-Farkas G, Komlosi K et al. (2015). Retrospective assessment of the most common mitochondrial DNA mutations in a large Hungarian cohort of suspect mitochondrial cases. Mitochondrial DNA 4: 572–578. Saneto RP, Sedensky MM (2013). Mitochondrial disease in childhood: mtDNA encoded. Neurotherapeutics 10: 199–211. Schaefer AM, Taylor RW, Turnbull DM et al. (2004). The epidemiology of mitochondrial disorders—past, present and future. Biochim et Biophys Acta - Bioenergetics. 1659 (2–3): 115–120. Shoubridge EA, Molnar MJ (2003). Oxidative phosphorylation defects. In: G Karpati (Ed.), Structural and molecular basis of skeletal muscle disease. ISN Neuropath Press, Basel, pp. 202–213. Spinazzola A, Zeviani M (2007). Disorders of nuclearmitochondrial intergenomic communication. Biosci Rep 27: 39–51. Suomalainen A, Elo JM, Pietilainen KH et al. (2011). FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study. Lancet Neurol 10: 806–881. Szalardy L, Molnar M, Torok R et al. (2016). Histopathological comparison of Kearns-Sayre syndrome and PGC-1a-deficient mice suggests a novel concept for vacuole formation in mitochondrial encephalopathy. Folia Neuropathol 54: 9–22. Taft RJ, Vanderver A, Leventer RJ et al. (2013). Mutations in DARS cause hypomyelination with brain stem and spinal cord involvement and leg spasticity. Am J Hum Genet 92: 774–780. Winterthun S, Ferrari G, He L et al. (2005). Autosomal recessive mitochondrial ataxic syndrome due to mitochondrial polymerase-gamma mutations. Neurology 64: 1204–1208. Wong CL-J (2010). Molecular genetics of mitochondrial disorders. Dev Disabil Res Rev 16: 154–162.
Chapter 10
Mitochondrial diseases 1
MARIA J. MOLNAR1* AND GABOR G. KOVACS2 Institute of Genomic Medicine, Rare Disorders, Semmelweis University, Budapest, Hungary 2
Institute of Neurology, Medical University of Vienna, Vienna, Austria
Abstract Mitochondrial disorders represent a major challenge in medicine. Most of the mitochondrial proteins are encoded by the nuclear DNA (nDNA), whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction. The disease usually affects multiple organs in varying locations and severity; however, there are some forms which affect a single organ. The diagnosis of mitochondrial disorders is based on clinical examination, biochemical and histopathologic examinations, functional studies, and molecular genetic testing. Neuropathologic alterations of the muscle are variable and can range from striking abnormalities, such as cytochrome oxidase-negative and ragged red fibers, to nonspecific or minimal changes. Neuropathologic alterations in the brain show common features in disorders with different genetic background. These are characterized by various degrees of vacuolation in the white and gray matter, regional neurodegeneration with reactive astrogliosis, loss of oligodendrocytes, presence of macrophages and microgliosis, capillary proliferation, and mineralization of vessel walls. The advent of molecular genetics, the discovery of biomarkers and new sequencing platforms to perform targeted exome and whole-genome sequencing have changed traditional approaches to diagnose mitochondrial diseases.
INTRODUCTION Mitochondrial disorders represent a major challenge in medicine. The first patient with mitochondrial disease was described in 1962; the patient had hypermetabolism and normal thyroid function (Luft et al., 1962). Most of the mitochondrial proteins are encoded by nuclear DNA (nDNA), whereas a very small fraction is encoded by mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction. This leads to a wide range of cellular alterations, such as excessive reactive oxygen species production, abnormal calcium homeostasis, dysregulated apoptosis, and insufficient energy generation in various organs, especially those with high energy demand. The disease usually affects multiple organs in varying locations and severity; however, there are some forms which only affect a single organ (e.g., the eyes in
Leber hereditary optic neuropathy (LHON)). The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathologic alterations, functional studies, and molecular genetic testing. However, with the advent of molecular genetics, the discovery of improved biomarkers and new sequencing platforms to perform targeted exome and whole-genome sequencing, traditional approaches to diagnose mitochondrial disease are now changing.
PREVALENCE Combined epidemiologic data on childhood and adult mitochondrial disease suggest that the prevalence is at least 1 in 5,000 (20 per 100,000) (Schafer et al., 2004; Gorman et al., 2015). The prevalence of nuclear mitochondrial disease is 2.9 per 100,000 adults (Schafer
*Correspondence to: Maria Judit Molnar, MD, PhD, DSc, Institute of Genomic Medicine and Rare Disorders, Semmelweis University, T€omőstr. 25–29, 1083 Budapest, Hungary. E-mail: [email protected]
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et al., 2004; Gorman et al., 2015). Gorman et al. (2015) calculated the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (1 in 4,300), as one of the commonest adult forms of inherited neurologic disorders. A prospective American study estimated the prevalence as 1:200 to harbor a pathogen mutation at risk of developing mitochondrial disease (Saneto and Sedensky, 2013). The prevalence of the specific mtDNA mutation m.3243A > G, as the most prevalent pathogenic mtDNA point mutation in the north-east of England (Gorman et al., 2015), is 7.8/100,000. The prevalence of LHON, the most prevalent mtDNA disorder overall, for affected individuals is 3.65 per 100,000; carrier frequency of the three common LHON mutations is 4.42 per 100,000, with evident familial clustering (m.11778G > A: 12 pedigrees, m.3460G > A: 5 pedigrees, m.14484 T > C: single pedigree). Myoclonus epilepsy with ragged red fibers (MERRF) related to the m.8344A > G mutation remains a rare form of mitochondrial disease (0.7/100,000) in the north-east of England. To date, 14 nuclear encoded genes, TYMP (thymidine phosphorylase), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4), POLG, PEO1, OPA1, POLG2, RRM2B, TK2 (thymidine kinase 2), DGUOK (deoxyguanosine kinase), MPV17 (mitochondrial inner membrane protein), MGME1 (mitochondrial genome maintenance exonuclease), DNA2 (DNA replication helicase/nuclease 2), SPG7, and AFG3L2 (AFG3-like AAA ATPase 2) have been reported to be associated with ophthalmoparesis and multiple mtDNA deletions in muscle of adult-onset chronic progressive external ophthalmoplegia (CPEO) patients. Notably, mutations in SPG7 (4/100,000) and PEO1 (3/100,000), followed by OPA1 (1/100,000) and RRM2B (0.9/ 100,000), have emerged as major causes of adult mitochondrial disease. Recessive mutations in POLG (0.6/100,000) are also emerging as an important cause of mitochondrial disease in the north-east of England (Gorman et al., 2015).
diabetes, deafness, visual, heart, liver, and kidney problems, stroke, and migraine have been described (Calvo et al., 2006). The disorders are caused either by mutations of the maternally inherited mitochondrial genome, or by nDNA mutations. Today more than 200 different diseasecausing mutations of mtDNA are known and due to the increased knowledge about nuclear genetics during the last few years, further at least 100 nuclear gene mutations have been described. In 70–85% of mitochondrial diseases nuclear mutations are responsible for the disease. Owing to the unequal distribution of mitochondria in the different tissues and the coexistence of mutant and wild-type mtDNA in these organelles, these disorders may present with a huge variety of symptoms, even related to the same mutation (Molnar, 2009). Children often have severe disease associated with nDNA mutation, causing respiratory chain defect and crashing mitochondrial integrity. In addition to their fundamental role in cellular energy metabolism, mitochondria seem to contribute to the pathogenesis of many degenerative diseases, aging, and cancer.
MANIFESTATION OF MITOCHONDRIAL DISEASES
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A variety of organs can be affected by mitochondrial dysfunction. The most oxidative tissues (brain, muscle, retina, cochlea, liver, and kidney) are the most vulnerable to OXPHOS defects (Shoubridge and Molnar, 2003; DiMauro and Hirano, 2005). In patients with mitochondrial disorders, most commonly the symptoms of epilepsy, ataxia, delayed motor and mental development, failure to thrive, signs of neurodegenerative diseases,
GENETICS AND SYNDROMES mtDNA-related mitochondrial disorders mtDNA is inherited mostly maternally; the nuclear genes are inherited autosomal-dominantly, recessively, or in an X-linked manner. The most important characteristics of mtDNA are as follows (Shoubridge and Molnar, 2003): ● ● ●
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The mitochondrial genome is a 16,569-bp long circular DNA. It consists of 37 genes that encode 13 proteins, 22 tRNAs, and 2 rRNAs. The mitochondrial genome is not able to independently produce all of the proteins needed for functionality; thus, mitochondria rely heavily on imported nuclear gene products. In one mitochondrion there are multiple copies of its mtDNA. A cell contains several thousand copies of its mitochondrial genome (polyplasmy). Wild-type and mutated mtDNA are present at the same time in a cell (heteroplasmy). To develop a clinical sign a certain amount of mutated mtDNA has to be present in a tissue (threshold effect).
Hundreds of pathogenic mtDNA mutations have been reported in the literature (for example, a database of reported mtDNA mutations can be found at www. mitomap.org). The most common mitochondrial disease due to mtDNA mutations is LHON with homoplasmic mtDNA mutations G11778A (69%), G3460A (13%), T14484C (14%) and other substitutions (Remenyi
MITOCHONDRIAL DISEASES et al., 2015). Men are involved more than women. Painless visual loss begins in young adulthood with optic atrophy. The next prevalent phenotype is mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndromes (MELAS) with external ophthalmoplegia, diabetes mellitus, hearing loss, early-onset stroke-like symptoms, migraine, and cognitive dysfunction due to mutations in the mitochondrial tRNA Leu gene (A3243G 80%, T3271C 7%, A3260G and A3252G 5–5%). Further common mtDNA-related syndromes are the MERRF, neuropathy, ataxia, retinitis pigmentosa (NARP), and Kearns–Sayre syndrome. MERRF evolves due to mtDNA tRNA Lys mutations as A8344G (80%), T8356C, G8363A, and G8361A (10%), causing myoclonus epilepsy, ataxia, dementia, neuropathy, myopathy, multiple lipomas in “collar” distribution, hearing loss, declining cognitive function, and neuropathy (Cohen, 2013). The A8993C or T substitution may result in the NARP syndrome or maternally inherited Leigh disease. Bilateral basal ganglia lesions, psychomotor retardation, seizures, movement disorders, and lactic acidosis characterize Leigh disease. Kearns–Sayre syndrome is the result of the mtDNA “common” large-scale deletion. Clinical symptoms include CPEO, retinal pigmentary abnormalities, cardiac conduction abnormalities, proximal and bulbar weakness, and sometimes ataxia. The ratio of heteroplasmy in many cases is very high in patients with mtDNA disorders. Usually the higher ratio of heteroplasmy is associated with more severe clinical phenotype. It is important to know that identical pathogenic mtDNA mutations may cause disease at different ages and different clinical syndromes. Even within the same family carrying the same mutation, there is extreme phenotypic variability (Crimmins et al., 1993). The m.A3243G mutation may result in lethal disease in childhood but we know patients with milder phenotypes such as maternally inherited diabetes and deafness or deafness (Remenyi et al., 2015). It is presumably due to mechanisms such as the different tissue distribution of mtDNA mutations, the threshold effects of the tissue, environmental stressors, and underlying nuclear genetic backgrounds.
nDNA-related mitochondrial disorders nDNA encodes 1,000 mitochondrial proteins, OXPHOS proteins (74) and factors, forming the respiratory chain complexes or their assembly factors; mitochondrial membrane proteins and transporters and proteins responsible for networking; or proteins involved in mtDNA maintenance (Wong, 2010). Defects in nDNA genes can be inherited in an autosomal or X-linked manner. In recent years next-generation sequencing
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technologies have been revolutionizing diagnostic screening for rare primary mitochondrial disorders, particularly those caused by nuclear gene defects. Wholeexome sequencing expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine (Legati et al., 2016). As far as we know, presently more than 200 nuclearencoded genes are implicated in mitochondrial diseases (Table 10.1). Every year about 10 new disease-causing genes are discovered (Calvo et al., 2012). There are five groups of nDNA mutations causing diseases: 1. Mutations in genes encoding respiratory chain proteins. Most frequently these alterations result in Leigh syndrome. 2. Defects of mitochondrial dynamics: fusion genes are in the inner membrane such as OPA1 (optic atrophy 1) or in the outer membrane, like MFN2 (mitofusin2). Their mutations cause optic atrophy and Charcot–Marie–Tooth disease type 2A, respectively. The fission gene is DLPA1 (dynamin-like protein1). 3. Defects of mitochondrial protein synthesis: e.g., mutations in genes of tRNA-modifying enzymes (PUS1, TRMU), elongation factors (TUFM, TSFM), or mitochondrial aminoacyl tRNA synthetases (RARS2, DARS2, YARS2). 4. Defects in lipid milieu/transporter carriers of the inner mitochondrial membrane, like TAZ gene (taffazin transacylase) catalyzing cardiolipin maturation, or DDP1 (TIMM8A), causing Mohr–Tranebjaerg syndrome. 5. Defects of mtDNA maintenance (see the next section).
DEFECTS OF MTDNA MAINTENANCE Cross-talk between nuclear and mitochondrial genomes is crucial for mitochondrial biogenesis and function, and the two genomes are probably subjected to coevolutionary processes. The defect of intergenomic signaling can affect mtDNA quantitatively (mtDNA depletion) and qualitatively (multiple mtDNA deletion). Nuclear genes responsible for the integrity of the mtDNA can be classified as follows: genes responsible (1) for mitochondrial DNA replication and repair (POLG, Twinkle); (2) for mitochondrion biogenesis and for the maintenance of nucleotide pool (SLC25A3, SLC25A4, TYMP, DGUOK, TK2, RRM2B, MVP17, SUCLAA, SUCLG1); and (3) for mitochondrial translation (PUS1, TRMU, LRPPRC, TUFM, TSFM and GFM1, MRPS16 and MRPS22, RARS2, DARS2, and YARS2). The most frequent gene, affected in intergenomic communication disturbances is POLG (polymerase gamma). Mutations in
Table 10.1 Phenotype–genotype correlation in some mitochondrial disorders due to nDNA mutations Phenotype Respiratory complex disorders Childhood encephalopathy Cardiomyopathy and encephalopathy with complex I deficiency Encephalopathy, hypertrophic cardiomyopathy Multisystemic complex I deficiency Leigh syndrome with complex 1 deficiency
Gene
NDUFS1 NDUFS2 NDUFS2, NDUFV2 NDUFS4 NDUFS1, NDUFS4, NDUFS4, NDUFS7, NDUFS8, NDUFV1, SDHAF1 NDUFV1 SDHB, SDHC, SDHD, and SDHAF2 L SDHA BCSL1 COX10 COX15, SCO2 SCO1 ETHE-1
Leigh syndrome with complex 2 deficiency Leukodsytrophy, myoclonic epilepsy Hereditary paraganglioma, pheochromocytoma Optic atrophy and ataxia (complex II deficiency GRACILE syndrome Encephalomyopathy, renal tubulopathy Infantile cardioencephalomyopathy Hepatoketoacidotic coma Ethylmalonic encephalopathy Disorders of mitochondrial protein synthesis Leukoencephalopathy DARS2 Myopathy, lactic acidosis, sideroblastic anaemia YARS2 Disorders of mtDNA maintenance (disorders associated with mtDNA deletion or depletion) CPEO, myopathy POLG1, POLG2, Twinkle, RRM2Bm SLC25A4 IOSCA POLG1 Alpers–Huttenlocher syndrome POLG1 Ataxia, neuropathy syndromes POLG1, Twinkle,OPA1 MIRAS POLG1 MNGIE TP SMA-like myopathy TK Hypotonia, movement disorder, and/or Leigh SUCLA2 syndrome with methylmalonic aciduria Hepatoencephalopathy DGUOK Defects of mitochondrial dynamics Charcot–Marie–Tooth II MNF2 Hereditary spastic paraplegia KIF5A Optic atrophy OPA1, OPA2 Defects of the lipid milieu Barth syndrome Tafazzin Other nDNA encoded mitochondrial disorders Dilatated cardiomyopathy with ataxia (DCMA) DNAJC19 Epilepsy, episodic ataxia, encephalopathy Pyruvate dehydrogenase Encephalopathy, hepatomegaly HMC-CoA-lyase Epilepsy, encephalopathy HMGCS2 Friedreich ataxia FXN Hepatopathy, hypotonia, failure to thrive DGUOK Hereditary spastic paraplegia SPG7 Hypocarnitinemia, hypolysinemia DCAR Menkes disease, occipital horn syndrome ATP7A Microcephaly SCL25A19 Mohr–Tranebjaerg syndrome DDP1/TIMM8A Myopathy, retinopathy, hepatomegaly HADHA Wolfram syndrome WFS1
GRACILE, growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis, and early death; IOSCA, infantile-onset spinocerebellar ataxia; MIRAS, mitochondrial recessive ataxia syndrome; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; SMA, spinal muscular atrophy.
MITOCHONDRIAL DISEASES the POLG gene, which encodes for the catalytic subunit of the mitochondrial DNA polymerase (pol-g), have been related to a wide variety of autosomal disorders, inherited both in a dominant and recessive fashion (Milone and Massie, 2010). POLG mutation may result in secondary mtDNA multiple deletions or depletions. The most common phenotype is CPEO. The bestknown autosomal-recessive inherited disorders are the sensory-ataxic neuropathy and dysarthria with external ophthalmoplegia (SANDO), Alpers-Huttenlocher syndrome, a severe pediatric hepatoencephalopathy, mitochondrial recessive ataxia syndrome, and myoclonic epilepsy myopathy sensory ataxia. Mutations in Twinkle and ANT1 genes provoke multiplex deletion with autosomal-dominant CPEO phenotype. Proper balance of the mitochondrial deoxynucleotide pools is essential for the maintenance of mtDNA copy number. Defects in these genes lead to depletion of mtDNA. The mitochondrial depletion syndrome may manifest as myopathic, hepatocerebral, and encephalomyopathic forms. The myopathic form usually occurs in the first year of life with feeding difficulty, failure to thrive, hypotonia, muscle weakness, and occasionally CPEO. It is due to mutations mostly in ANT1 (responsible for ADP/ATP balance), TK2 (in charge of pyrimidine biosynthesis), or RRM2B, and POLG. The hepatocerebral form is frequently due to mutated DGUOK (in charge of purin biosynthesis), POLG, or Twinkle. A peculiar form of hepatocerebral mitochondrial depletion syndrome is the Alpers–Huttenlocher syndrome, an early-onset, fatal disease, characterized by hepatic failure and intractable seizures. In these cases, due to POLG single-nucleotide polymorphism severe valproate hepatotoxicity is observed (Delarue et al., 2000). The encephalomyopathy usually is due to mutated SUCLA and RRM2B genes and characterized by infantile onset of hypotonia with severe psychomotor retardation, high lactate in blood, progressive neurologic deterioration, a hyperkinetic-dystonic movement disorder, external ophthalmoplegia, deafness, generalized seizures, and variable renal tubular dysfunction. Brain magnetic resonance imaging could be suggestive of Leigh syndrome (Spinazzola and Zeviani, 2007). The second large group of the mitochondrial integrity disorders are the mtDNA deletion syndromes. The main clinical manifestations associated with multiple deletions are: 1.
CPEO (autosomal-dominant or recessive) (Filosto et al., 2003; Luoma et al., 2004). Additional symptoms may include cataracts, hearing loss, sensory axonal neuropathy, ataxia, depression, and parkinsonism.
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2. SANDO is an autosomal-recessive systemic disorder characterized mainly by adult-onset sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (Spinazzola and Zeviani, 2007). 3. Mitochondrial neurogastrointestinal encephalomyopathy, an autosomal-recessive disorder clinically characterized by young age at onset, gastrointestinal dysmotility (often pseudo-obstruction), cachexia, diffuse leukoencephalopathy, peripheral neuropathy, and early death. mDNA abnormalities can include depletion, multiple deletions, and point mutations (Hirano et al., 1994). 4. Spinocerebellar ataxia – epilepsy syndrome disorder is similar to SANDO but with a higher frequency of migraine headaches and seizures (Winterthun et al., 2005).
NEUROPATHOLOGIC CHANGES IN MITOCHONDRIAL DISORDERS Muscle Muscle biopsy is a very common diagnostic procedure in the diagnosis of the mitochondrial disorders. The pathologic changes of the muscle are variable and can range from striking abnormalities, highly evocative of a mitochondrial disease, to nonspecific or minimal. The absence of pathologic changes does not exclude a mitochondrial problem. The two most valuable tools in pathologic assessment of muscle biopsy for suspected mitochondrial myopathy are the succinate dehydrogenase (SDH) enzyme histochemical reaction and the cytochrome oxidase (COX) reaction, which can be performed together (Fig. 10.1). The modified G€om€ori trichrome reaction is also useful, but less specific and less sensitive because only well-developed ragged red fibers with prominent red stain throughout the fiber, and particularly at the periphery, reflecting the presence of structurally abnormal mitochondria (Fig. 10.1). Using hematoxylin and eosin staining these fibers have a granular, basophilic appearance and react intensely for SDH and NADH-TR (Fig. 10.1). Modification of the SDH reaction by the addition of phenazine methosulfate is useful because it suppresses the reactivity of normal mitochondria. It shows both ragged red fibers and ragged red equivalents. The fibers that are positive with the modified SDH have been called ragged blue fibers. Further pathologic features in mitochondrial myopathies include variation in fiber size, with atrophy but little or no hypertrophy, an increase in internal nuclei, necrosis and fiber regeneration in cases with myoglobinuria. In conditions in which there is associated peripheral nerve involvement there may be associated fiber-type grouping and the presence of angular atrophic fibers.
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Fig. 10.1. Myopathologic alterations in mitochondrial disorders. (A) (Cytochrome oxidase (COX) staining. (B) Succinate dehydrogenase (SDH) staining. (C) Combined COX/SDH staining: the strongly blue fibers indicate loss of COX staining. (D) Hematoxylin and eosin staining showing fibers with basophilic rim representing ragged-red fibers. (E) Combined COX/SDH staining reveals a ragged-blue fiber. (F) Typical ragged-red fiber as seen in Gomori-trichrome staining (the same fiber as shown in E).
The ultrastructural abnormalities of mitochondria include an alteration in shape, increase in size, disruption and/or distortion of the cristae, and paracrystalline or osmiophilic inclusions (Fig. 10.2). Electron microscopy may reveal varying degrees of myofibrillar loss and disruption and an increase in intracellular lipid, which can often be detected with oil red O or Sudan black staining as well. It must be emphasized that ragged red and COXnegative fibers are not specific for mitochondrial myopathies and may be a secondary feature in some muscular dystrophies and inflammatory myopathies, in particular inclusion body myositis. As the number of COXnegative fibers increases with age the observation of 2-3 COX-negative fibers in a sample of an elderly patient should not be interpreted as significant.
Brain Neuropathologic alterations in the brain show common features in disorders with different genetic backgrounds. The distribution of neuropathologic alterations is usually characteristic for a syndrome (Table 10.2). Various
degrees of vacuolation in the white and gray matter, regional neurodegeneration with reactive astrogliosis, loss of oligodendrocytes, presence of macrophages and microgliosis, capillary proliferation, and mineralization of vessel walls can be seen in most of those, which are associated with central nervous system symptoms (Fig. 10.3). Neurons may be preserved in the vacuolated gray-matter areas, giving the appearance of pseudonecrosis. A recent study suggested an intramyelinic nature of white and gray-matter vacuolation and presence of intraoligodendroglial vacuoles (Szalardy et al., 2016). It has been suggested that oligodendrocytes are important players of the pathogenesis of central nervous system lesioning (Szalardy et al., 2016). The affection of myelin is supported also by the observation of syndromes with hypomyelination in the brainstem and spinal cord (Taft et al., 2013). There are rare reports of multiple sclerosis-like lesioning with selective demyelination and prominent inflammatory infiltrates in the brain in LHON (Kovacs et al., 2005), which, on the other hand highlights the role of mitochondria in the pathogenesis of multiple sclerosis.
MITOCHONDRIAL DISEASES
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Fig. 10.2. (A–D) Representative electron microscopy images of paracrystalline mitochondrial inclusions. Table 10.2 Characteristic neuropathologic findings in the brain in mitochondrial disorders with brain involvement Neuropathologic feature
Syndrome
Remark
Vacuolation of the WM
KSS MDS; LS, MERRF, LHON, MELAS AHS KSS
Prominent and characteristic in cerebral and cerebellar WM Variably present in cerebral WM
LS
Characteristic feature in subcortical areas (i.e., thalamus) and brainstem but not in the cortex Only mild degree, mostly in cortex Focal or missing Not seen Characteristic feature in the cortex (i.e., striate) and mild in subcortical areas Not seen Associates with spongy degeneration of the GM Characteristically seen with GM lesions Seen in the cerebral and cerebellar cortex In the hepatocerebral form Purkinje cell loss Retinal ganglion cells Focal/mild or not seen
Spongy degeneration of the GM
MELAS MERRF LHON AHS
Neuron loss/astrogliosis
Capillary proliferation/ prominence Other
MDS KSS MELAS, MERRF, LS AHS MDS LHON KSS, LHON, MERRF, AHS MELAS, LS MELAS LS
Usually not involved Present mostly in the brainstem
Characteristically seen Cortical laminar necrosis: infarct-like lesions “Vasculonecrotic” lesions
AHS, Alpers–Huttenlocher syndrome; GM, gray matter; KSS, Kearns–Sayre syndrome; LS, Leigh syndrome; LHON, Leber hereditary optic neuropathy; MDS, mitochondrial depletion syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndromes; MERRF, myoclonus epilepsy with ragged-red fibers; MDS, mitochondrial depletion syndrome; WM, white matter.
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Fig. 10.3. Neuropathologic alterations in the brain in mitochondrial disorders. (A and B) Spongy vacuolation in the white matter in Kearns–Sayre syndrome. (C) Capillary proliferation in the basal ganglia (left side of image) in Leigh syndrome associated with spongy degeneration of the thalamus (D), the pontine tegmentum (E), and vacuolation of the white and gray matter of the spinal cord (F).
DIAGNOSTIC PRINCIPLES During the diagnostic evaluation of suspected mitochondrial disease, the following strategy is recommended (Shoubridge and Molnar, 2003). The most important laboratory parameters are resting serum lactate and pyruvate, which are frequently increased. The ratio of lactate/pyruvate is increased in many cases. Serum creatine kinase levels are either normal or slightly elevated. Serum lactate increases during slight exercise in mitochondrial patients and 30 minutes after the exercise will not decline to the baseline. The use of a biomarker such as serum of fibroblast growth factor 21 levels is a promising new approach in the diagnostic pathway for patients with mitochondrial disease (Suomalainen et al., 2011). Electromyogram is normal, neurogenic or myogenic, or not specific. The muscle biopsy mostly displays the characteristic ragged red or ragged blue fiber pathology. The ragged red fibers usually do not have COX activity. Ultrastructural analysis of the muscle reveals aberrant and enlarged mitochondria, usually with paracrystalline inclusions or abnormally organized cristae. Biochemical investigations detect the reduced activity of the affected
enzyme. Searching for mtDNA mutation genetic testing is recommended on postmitotic tissue (e.g., muscle specimen). In the routine diagnostic for the mtDNA mutation hotspots, mtDNA deletion and depletion are screened. In many cases the whole mtDNA is sequenced. Mutations in the nuclear genes were searched based on the clinical and imaging phenotype for many years. In the near future the targeted panel sequencing of these nuclear genes and whole-exome analysis by next-generation sequencing will improve the diagnostics of mitochondrial disorders due to nuclear gene mutations.
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