In 1988, a major breakthrough in our understanding of mitochondrial disorders occurred with the report of the association of sporadic human encephalomyopathies with large deletions of the mtDNA (Holt et al., 1988) and of Leber hereditary optic neuropathy (LHON) with a point mutation at nucleotide pair (np) 11778 in the mtDNA. More than 170 pathogenic point mutations of the mtDNA, large-scale mtDNA deletions, and rearrangements have been subsequently reported (Schon and DiMauro, 2007). More than 85% of mitochondrial proteins are encoded by the nuclear DNA (nDNA), and therefore many unknown mitochondrial disorders due to nDNA defects may exist. The list of nDNA defects affecting mitochondrial function, including mtDNA replication and integrity, has been growing in recent years (DiMauro and Hirano, 2005). The delineation of human mtDNA variation and genetics has also provided startling new insights into the evolution and migration of human populations (Wallace, 1995). Mitochondria may also play an important role in neurodegenerative diseases and the aging process (Schapira, 2006, 2008; Turner and Schapira, 2010).

The diseases included under the term mitochondrial disorders are so diverse and involve so many parts of the nervous system and other organs that the whole spectrum cannot be easily addressed in any one chapter. Therefore the disorders related to intermediate metabolism and mitochondrial Krebs cycle are discussed with inborn errors of metabolism in Chapter 62. The syndrome that combines epilepsy and ragged-red muscle fibers (myoclonic epilepsy and ragged-red fibers, MERRF) is discussed in Chapters 40 and 67. The syndrome of progressive external ophthalmoplegia (PEO) is discussed with other abnormalities of eye movement (Chapter 16), and LHON with other causes of vision loss (Chapter 14). This chapter overviews the principles of mitochondrial genetics and mitochondrial pathophysiology and provides a summary of the clinical features and management of patients with mitochondrial disorders.

Genetics of Mitochondrial Disorders

MtDNA is a 16,569-np double-stranded, closed, circular molecule located within the matrix of the double-membrane mitochondrion. Each human cell contains a dynamic network of mitochondria and hundreds of mtDNA molecules. Human mtDNA encodes 13 of the 89 subunits of the mitochondrial respiratory chain, as well as the small (12S) and large (16S) ribosomal RNAs (rRNAs) and 22 tRNAs necessary for intramitochondrial protein synthesis (Fig. 63.1, A).

Fig. 63.1 A, Human mtDNA map. The mtDNA encompasses 16,569 np, with numbering starting at OH and proceeding counterclockwise around the circular map. Each gene is identified by shading, and the tRNA gene is identified by the letter of its cognate amino acid. Mutation sites of common mitochondrial diseases are indicated. B, Schematic representation of the mitochondrial respiratory chain located in the inner mitochondrial membrane. Electrons are transferred from complexes I and II to complex III via the mobile lipid carrier, coenzyme Q10 (CoQ10 [ubiquinone]). Electrons are then transferred to complex IV through the protein mobile carrier, cytochrome c (CYT c). Electrons are finally transferred to molecular oxygen by complex IV. Three of the respiratory complexes (I, III, and IV) have a proton pumping mechanism coupled to the electron transfer. Pumping of protons to the intermembrane space generates a membrane potential and a proton gradient that flows back through complex V. The flow of protons through complex V enables ATP synthesis. LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes; MERRF, myoclonic epilepsy with ragged-red fibers; MILS, maternally inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; PEO, progressive external ophthalmoplegia.

The mtDNA is replicated and transcribed by using an origin and promoter for each of the two DNA strands, the G-rich heavy (H) strand and the C-rich light (L) strand. The H- and L-strand replication origins (OH and OL; see Fig. 63.1, A) are relatively distant within the molecule, but the H- and L-strand promoters (PH and PL) are closely spaced and located adjacent to OH in the approximately 1000-np noncoding control region. This region also encompasses the D-loop, which is formed by replication initiation events from the OH. Research involving evolution of mammalian species and origin and migration of humans on earth has focused on the variation in a small hypervariable noncoding region within the D-loop. The replication mechanism of mtDNA may be more complex than originally suggested, in that the mtDNA can also replicate by the extension of both leading and lagging strands, a process resembling nDNA replication (Holt et al., 2000).

The respiratory chain (see Fig. 63.1, B) is located within the mitochondrial inner membrane and is composed of five multimeric enzyme complexes whose genes are dispersed between the mtDNA and nDNA. Complex I (NADH–ubiquinone oxidoreductase) accepts electrons from the reduced form of nicotinamide adenine dinucleotide (NADH), whereas complex II (succinate–ubiquinone oxidoreductase) collects electrons from succinate. Both NADH and succinate are the products of the Krebs cycle. Enzyme complexes I and II transfer electrons to coenzyme Q10 (CoQ10). From CoQ10, the electrons flow through complex III (ubiquinone–cytochrome c oxidoreductase) to cytochrome c, then to complex IV (cytochrome c oxidase), and finally to oxygen to yield water. The electron transfer is coupled to proton (H⁺) pumping by complexes I, III, and IV from the matrix to the intermembrane space, creating an electrochemical gradient across the inner membrane. This electrochemical gradient is utilized by complex V (adenosine triphosphate [ATP] synthase) as a source of energy to condense adenosine diphosphate (ADP) and inorganic phosphate (Pi) to synthesize ATP. ATP and ADP are then exchanged across the mitochondrial membrane by the adenine nucleotide translocator (ANT).

Complex I comprises approximately 46 subunits, 7 of which (ND-1, -2, -3, -4, -4L, -5, and -6) are encoded by the mtDNA; complex II has 4 subunits, none from the mtDNA; complex III includes 11 subunits, 1 (cytochrome b) from the mtDNA; complex IV contains 13 subunits, 3 (COX-1, -2, and -3) from the mtDNA; and complex V contains 13 subunits, 2 (ATPase-6 and -8) from the mtDNA. The remaining subunits of complexes I, III, IV, and V; the entire complex II; the 2 small electron carriers, CoQ10 and cytochrome c, and ANT are encoded by the nDNA. The mitochondrial rRNA and tRNA genes provide the structural RNAs for mitochondrial protein synthesis (i.e., for the expression of 13 mtDNA-encoded polypeptides). The majority of mitochondrial respiratory chain proteins are encoded by the nDNA in the cytosol. A complex mitochondrial importation process therefore enables the cytosolically synthesized nuclear-encoded mitochondrial respiratory chain subunits to be co-assembled with mtDNA encoded counterparts in the inner mitochondrial membrane. There are over 1000 other nuclear genes which express mitochondrial proteins that are important for mitochondrial function.

Mitochondrial diseases therefore can arise from defects in the mtDNA (sporadic or maternal inheritance, see following section) or nDNA (sporadic or Mendelian inheritance). Nuclear DNA-related mitochondrial disorders result from defects involving nDNA-encoded mitochondrial polypeptides including respiratory chain complexes, respiratory chain assembly, mtDNA maintenance, protein import, lipid dynamics, and biosynthesis of CoQ10. Table 63.1 summarizes a simplified clinical and genetic classification of mitochondrial diseases.

Table 63.1 Major Gene Mutations in Mitochondrial Disorders

Maternal Inheritance of Mitochondrial DNA

Patients inherit mtDNA from their mothers, and therefore the mode of transmission of mtDNA, including pathogenic mtDNA mutations, follows maternal line inheritance. A single case report has demonstrated paternal transmission of a mtDNA mutation (Schwartz and Vissing, 2002). Maternal inheritance implies maternal transmission of mtDNA to all offspring but subsequent transmission only by females. Thus a disease expressed in all children of both sexes of an affected individual, without evidence of paternal inheritance, strongly suggests an mtDNA point mutation. However, exceptions to this general rule are encountered in clinical practice. First, a de novo point mutation in the mtDNA of the maternal germ cell line will not necessarily be transmitted to all children. Second, for unknown reasons, mtDNA mutations involving large-scale deletions, rearrangements, and point mutations in some protein-coding genes may occur sporadically and be due to mutations arising in the oocyte. Bottleneck expression of an mtDNA mutation may be enhanced in the fetus. Multiple mtDNA deletions and depletions are autosomally transmitted, as they are the consequence of mutations in nuclear-encoded factors involved in mtDNA metabolism or replication. Third, maternal inheritance may not always be clinically evident because of extreme variability of clinical expression among family members due to heteroplasmy and the threshold effect (described in the following section).

Heteroplasmy and Mitotic Segregation of Mitochondrial DNA

Each cell contains thousands of mtDNA copies in a network of cytoplasmic mitochondria. MtDNA is constantly undergoing replication, fission, and fusion even in terminally differentiated cells. When all mtDNA molecules are identical, the mtDNA is homoplasmic. If pathogenic mtDNA mutations exist with normal (wild-type) mtDNA, then there are two populations of mtDNA in the system, and this is heteroplasmy. The severity of the phenotypic effects of the mtDNA mutations in heteroplasmic cells is determined by the proportion of the wild-type and mutant mtDNA. Neutral polymorphic sites (nonpathogenic mutations) of mtDNA are generally homoplasmic, whereas pathogenic mutations are mostly, but not invariably, heteroplasmic.

Heteroplasmy may occur at the level of the cell or at the level of the individual mitochondrion (intramitochondrial heteroplasmy). At cell division, mitochondria and mtDNA are randomly partitioned to be carried into the two daughter cells (mitotic segregation). Therefore, mtDNA mutation is typically represented in a variable proportion of mitochondrial genomes, with the consequence that cells, tissues, and the whole individual would harbor variable proportions of mutant and wild-type mtDNA. Mitotic segregation explains how certain patients with mtDNA-related disorders may present with one manifestation at an early age and shift to another as they grow older as the proportions of heteroplasmy change with time in different tissues. For a disease to manifest in a tissue, the proportion of mutant mtDNA must be greater than the threshold level.

Threshold Effect of Mitochondrial DNA Mutations

Threshold effect denotes the minimal critical number of mutated mtDNA molecules that would cause mitochondrial dysfunction in one or more tissues or organ systems. Variable somatic load of mtDNA mutation in different tissues and organ systems, or skewed heteroplasmy is a universal finding in heteroplasmic states. It often changes over time, particularly in postmitotic cells such as neurons, and it increases with age. This in part explains the age-dependent penetration of many mitochondrial clinical phenotypes and age-related variability in their clinical features. The mtDNA mutation load needed for clinical expression is typically high and in the range of 70% to 90% for both point mutations and deletions. The mutation threshold effect is believed to be affected by the oxidative metabolic requirement of a particular tissue. For example, the threshold effect may manifest itself at lower concentrations of mutated mtDNA in tissues that are inherently dependent on high oxidative metabolism, such as brain, eye, myocardium, and skeletal muscle.

Examples of threshold effect and mitotic segregation include a patient with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) who may present with only episodic headache in childhood but with stroke-like episodes and neurological deficits as they age and the pathogenic mutations accumulate in brain and cerebral vasculature. Infants with Pearson syndrome (a hematopoietic disease) may survive their disease because the proportion of mtDNA with deletions may decrease as bone marrow cells with heavy mutation loads are selected against. Following this process of outgrowing the hematopoietic disease in childhood, individuals with Pearson syndrome may later develop a different clinical phenotype such as Kearns-Sayre syndrome (KSS) as the mutation accumulates in their postmitotic neurons and muscle fibers.

Pathophysiology of Mitochondrial Disorders

Mitochondria have several functions in maintaining cellular homeostasis, such as transient storage of intracellular calcium, fatty acid oxidation, the Krebs cycle, and iron metabolism (McBride et al., 2006; Schapira, 2006). Mitochondria also have a key role in the regulation of apoptosis (Wang, 2001). One of the most important roles of mitochondria is to catalyze the phosphorylation of the majority of cellular ADP to ATP. ATP is generated by oxidation of intermediates such as NADH and FADH2 via the process of oxidative phosphorylation within mitochondria by the respiratory chain enzymes. Several intermediary metabolic pathways from carbohydrates, fatty acids, and amino acids converge in mitochondria at the level of acetyl-CoA for the final conversion of the fuel into ATP. Pyruvate is the terminal product of anaerobic glycolysis and is transported across the inner mitochondrial membrane to the mitochondrial matrix where it is converted to acetyl CoA. The transportation of pyruvate is coupled with the influx of hydrogen ions down their electrochemical gradient across the inner mitochondrial membrane. Pyruvate can also be generated during the catabolism of the amino acids, alanine, serine, glycine, and cysteine. Transport of free fatty acids across the mitochondrial membrane requires two enzymes (carnitine palmitoyltransferases [CPT] I and II), a carrier molecule (l-carnitine), and a translocase (carnitine-acylcarnitine translocase). Fatty acids are also metabolized into acetyl-CoA. Acetyl-CoA enters the Krebs cycle, where three molecules of NADH and one molecule of the reduced form of flavin adenine dinucleotide (FADH₂) are produced from each acetyl-CoA. Molecules of NADH and FADH₂ donate electrons to the electron transport chain (NADH to complex I and succinate to FAD in complex II). The functional unit comprising the electron transport chain (complex I to IV) and complex V (ATP synthase) constitutes the oxidative phosphorylation (OXPHOS) system (see Fig. 63.1, B).

Lactic acid is the end product of glycolytic anaerobic metabolism and acts as a reservoir for excess pyruvate. Physiological states such as exercise may cause transient lactic acidosis. Pathological lactic acidosis occurs during anoxia/ischemia, in metabolic failure from liver disease and diabetes mellitus (secondary lactic acidosis), and in defects of OXPHOS (primary lactic acidosis) such as mitochondrial disorders. The blood lactate-to-pyruvate ratio reflects the NADH to NAD⁺ ratio, or redox state, and is useful in the diagnosis of primary lactic acidosis. Defects in pyruvate dehydrogenase, which catalyses the conversion of pyruvate to acetyl CoA with the conversion of NAD⁺ to NADH, are associated with a low redox state (due to increased NAD⁺) and usually produce elevated levels of lactate and pyruvate with normal or slightly low lactate/pyruvate ratios (<20). OXPHOS defects often cause a high redox state (due to increased NADH accumulation) and generally produce a high lactate/pyruvate ratio (>20) as NADH and pyruvate are converted to lactate by lactate dehydrogenase.

Neurons, myocardial and skeletal muscle, liver, and renal tissues are highly dependent on oxidative metabolism and are most commonly involved in mitochondrial diseases. Neurons especially require high levels of ATP production to maintain ion homeostasis following controlled flux of ions across the cell membrane during electrical signaling. Within the brain, the high metabolic activity of the basal ganglia makes them vulnerable to oxidative metabolic defects. Necrosis of the basal ganglia and brainstem is an early feature of Leigh syndrome and is common in other mitochondrial cytopathies.

In skeletal muscle, some fibers are severely involved and others may appear normal on histological analysis. With more severe involvement, the combination of patchy myofibrillar degeneration along with mitochondrial proliferation gives rise to the so-called ragged-red appearance of fibers on modified Gomori trichrome staining (Fig. 63.2, A). Defective OXPHOS may result in compensatory mitochondrial proliferation, particularly in type I and IIA muscle fibers. Ultrastructural analysis can demonstrate abnormal mitochondrial morphology such as intramitochondrial paracrystalline inclusions (see Fig. 63.2, B). Patients with mitochondrial disorders can show a wide range of symptoms, including any combination of developmental delay, short stature, small muscle bulk, seizures, vision loss, hearing impairment, peripheral neuropathy, autonomic nervous system difficulties, gastrointestinal dysfunction, endocrine problems, hematopoietic disease, and failure to thrive (Box 63.1). The presentation of a particular mitochondrial disorder can be variable, even among affected individuals in the same family. Conversely, more than one pathogenic mutation can give rise to similar clinical phenotypes.