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. 93.1, A ).

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. 93.1, 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 ( ).

The respiratory chain (see Fig. 93.1, 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 (nicotinamide adenine dinucleotide [NADH]–ubiquinone oxidoreductase) accepts electrons from the reduced form of 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 Q ₁₀ (CoQ ₁₀ ). From CoQ ₁₀ , 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 used 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 two small electron carriers, CoQ ₁₀ 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 coassembled with mtDNA-encoded counterparts in the inner mitochondrial membrane. There are more than 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). nDNA-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 CoQ ₁₀ . Table 93.1 summarizes a simplified clinical and genetic classification of mitochondrial diseases.

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 ( ). Mitochondria also have a key role in the regulation of apoptosis ( ). 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 reduced flavin adenine dinucleotide (FADH2) via the process of OXPHOS 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 coenzyme A (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 (CPTs 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 FADH ₂ are produced from each acetyl CoA. Molecules of NADH and FADH ₂ 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–IV) and complex V (ATP synthase) constitutes the OXPHOS system (see Fig. 93.1 , 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 (PDH), which catalyzes 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. 93.2 , 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. 93.2 , 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 93.1 ). 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.

Approach to the Diagnosis of Mitochondrial Disorders

The complex inheritance patterns and clinical heterogeneity of mitochondrial diseases often result in incorrect or delayed diagnosis of the affected individuals. This delay can result in increased morbidity or mortality. For example, patients with specific metabolic defects such as β-oxidation of fatty acids can benefit from appropriate manipulations of diet and physical activity. CoQ ₁₀ (ubiquinone) and l -carnitine replacements are often effective in rare metabolic disorders of CoQ ₁₀ and primary systemic carnitine deficiencies. Failure to make a precise genetic diagnosis and, in turn, the lack of appropriate genetic counseling, can lead to the subsequent birth of affected children in unsuspecting families. Leigh syndrome is a good example in that the phenotype can vary among members of the same family and can be inherited in the maternal line (mtDNA-related mutations) or in Mendelian (autosomal or sex-linked) inheritance patterns.

A detailed clinical history and examination in conjunction with experienced interpretation of a battery of complex laboratory results are often required to make an accurate diagnosis. This process is often best carried out in a specialist mitochondrial clinic and subsequently discussed at a multidisciplinary meeting involving neurologists, pediatricians, geneticists, pathologists, and biochemists. Diagnostic criteria for mitochondrial disorders have previously been proposed ( ). However, the usefulness of categorizing patients as having “possible” mitochondrial disease has recently been questioned ( ).

Although the overall clinical spectrum of mitochondrial disorders is broad, recognized patterns of clinical presentations, clinical signs, and investigations have emerged. A detailed extended family history is essential in deciphering subtle clues suggesting a maternal line of inheritance. Any patient with unexplained multisystem problems, particularly affecting the nervous system, skeletal muscle, liver, kidney, and heart, may have mitochondrial disease. Rare presentations can be of mitochondrial origin, such as stroke-like episodes with MELAS, chronic ophthalmoplegia in KSS, and a movement disorder in children or young adults with Leigh syndrome. Patients and families often report a history of periods of severe fatigue with intercurrent illnesses, trauma, or surgery. Affected individuals may develop exacerbations, such as an increase in seizures, or new symptoms, such as an episode of lactic acidosis, during a seemingly minor illness. The patient may develop a permanent neurological deficit following these physiological stressors.

Neuroradiology

The use of neuroimaging, especially brain magnetic resonance imaging (MRI), has greatly facilitated the detection of CNS involvement in mitochondrial disorders. Brain atrophy is common in children with mitochondrial disease. Developmental delay and basal ganglia calcification are common in KSS and MELAS, and diffuse signal abnormalities of the white matter are characteristic of KSS and myoneurogastrointestinal encephalopathy (MNGIE) ( Fig. 93.3 ). The diagnosis of MELAS can be aided by the clinical association of stroke-like episodes with radiological lesions that do not conform to the anatomical territories of blood vessels and predominantly involve cortical gray matter ( Fig. 93.4 ). The initial or predominant lesions in MELAS are characteristically in the parietal-occipital region, and new lesions generally appear with acute illness and elevated CSF lactate levels. Leigh syndrome characteristically shows bilateral hyperintense signals on T2-weighted and fluid-attenuated inversion recovery (FLAIR) MRIs in the putamen, globus pallidus ( Fig. 93.5 ), and thalamus.

Extraocular muscle T2 signal is elevated in chronic progressive external ophthalmoplegia (CPEO) and correlates negatively with ocular movements, thus providing a potential quantitative measure of disease severity ( ). ¹ H-MRS often detects lactate accumulation in the CSF and in specific areas of the brain, whereas positron emission tomography (PET), which measures metabolic flux, has identified several metabolic abnormalities in mitochondrial disease patients using radioisotopically labeled metabolites relevant to the study of bioenergetics.