The true prevalence of mtDNA disease is difficult to estimate because of the phenotypic heterogeneity that occurs as a function of heteroplasmy, the challenge of detecting and assessing heteroplasmy in different affected tissues, and the other unique features of mtDNA function and inheritance described above. It is estimated that at least 1 in 200 healthy humans harbors a pathogenic mtDNA mutation with the potential to causes disease, but that heteroplasmic germline pathogenic mtDNA mutations actually affect up to approximately 1 in 8500 individuals.
The true disease burden relating to mtDNA sequence variation will only be known when the following capabilities become available: (1) ability to distinguish a completely neutral sequence variant from a true phenotype-modifying or pathogenic mutation, (2) accurate assessment of heteroplasmy that can be determined with fidelity, and (3) a systems biology approach (Chap. 87e) to determine the network of epistatic interactions of mtDNA sequence variations with mutations in the nuclear genome.
OVERVIEW OF CLINICAL AND PATHOLOGIC FEATURES OF HUMAN MTDNA DISEASE
Given the vital roles of mitochondria in all nucleated cells, it is not surprising that mtDNA mutations can affect numerous tissues with pleiotropic effects. More than 200 different disease-causing, mostly heteroplasmic mtDNA mutations have been described affecting ETC function. Figure 85e-4 provides a partial mtDNA map of some of the better characterized of these disorders. A number of clinical clues can increase the index of suspicion for a heteroplasmic mtDNA mutation as an etiology of a heritable trait or disease, including (1) familial clustering with absence of paternal transmission; (2) adherence to one of the classic syndromes (see below) or paradigmatic combinations of disease phenotypes involving several organ systems that normally do not fit together within a single nuclear genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that reflect disruption in cellular energetics (e.g., lactic acidosis and neurodegenerative and myodegenerative symptoms with the finding of ragged red fibers, reflecting the accumulation of abnormal mitochondria under the muscle sarcolemmal membrane); or (4) a mosaic pattern reflecting a heteroplasmic state.
Mutations in the human mitochondrial genome known to cause disease. Disorders that are frequently or prominently associated with mutations in a particular gene are shown in boldface. Diseases due to mutations that impair mitochondrial protein synthesis are shown in blue. Diseases due to mutations in protein-coding genes are shown in red. ECM, encephalomyopathy; FBSN, familial bilateral striatal necrosis; LHON, Leber’s hereditary optic neuropathy; LS, Leigh syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fibers; MILS, maternally inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; PEO, progressive external ophthalmoplegia; PPK, palmoplantar keratoderma; SIDS, sudden infant death syndrome. (Reproduced with permission from S DiMauro, E Schon: Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656, 2003.)
Heteroplasmy can sometimes be elegantly demonstrated at the tissue level using histochemical staining for enzymes in the oxidative phosphorylation pathway, with a mosaic pattern indicating heterogeneity of the genotype for the coding region for the mtDNA-encoded enzyme. Complex II, CoQ, and cytochrome c are exclusively encoded by nuclear DNA. In contrast, complexes I, III, IV, and V contain at least some subunits encoded by mtDNA. Just 3 of the 13 subunits of the ETC complex IV enzyme, cytochrome c oxidase, are encoded by mtDNA, and, therefore, this enzyme has the lowest threshold for dysfunction when a threshold level of mutated mtDNA is reached. Histochemical staining for cytochrome c oxidase activity in tissues of patients affected with heteroplasmic inherited mtDNA mutations (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern of reduced histochemical staining in comparison with histochemical staining for the complex II enzyme, succinate dehydrogenase (Fig. 85e-5). Heteroplasmy can also be detected at the genetic level through direct Sanger-type mtDNA genotyping under special conditions, although clinically significant low levels of heteroplasmy can escape detection in genomic samples extracted from whole blood using conventional genotyping and sequencing techniques.
Cytochrome c oxidase (COX) deficiency in mitochondrial DNA (mtDNA)–associated disease. Transverse tissue sections that have been stained for COX and succinate dehydrogenase (SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient cells shown in blue. A. Skeletal muscle from a patient with a heteroplasmic mitochondrial tRNA point mutation. The section shows a typical “mosaic” pattern of COX activity, with many muscle fibers harboring levels of mutated mtDNA that are above the crucial threshold to produce a functional enzyme complex. B. Cardiac tissue (left ventricle) from a patient with a homoplasmic tRNA mutation that causes hypertrophic cardiomyopathy, which demonstrates an absence of COX in most cells. C. A section of cerebellum from a patient with mtDNA rearrangement that highlights the presence of COX-deficient neurons. D, E. Tissues that show COX deficiency due to clonal expansion of somatic mtDNA mutations within single cells—a phenomenon that is seen in both postmitotic cells (D; extraocular muscles) and rapidly dividing cells (E; colonic crypt) in aging humans. (Reproduced with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. Nat Rev Genetics 6:389, 2005.)
The emerging next-generation sequencing (NGS) techniques and their rapid penetration and recognition as useful clinical diagnostic tools are expected to also dramatically improve the clinical genetic diagnostic evaluation of mitochondrial diseases at the level of both the nuclear genome and mtDNA. In the context of the larger nuclear genome, the ability of NGS techniques to dramatically increase the speed at which DNA can be sequenced at a fraction of the cost of conventional Sanger-type sequencing technology is particularly beneficial. Low sequencing costs and short turnaround time expedite “first-tier” screening of panels of hundreds of previously known or suspected mitochondrial disease genes or screening for the entire exome or genome in an attempt to identify novel genes and mutations affecting different patients or families. In the context of the mtDNA, NGS approaches hold the particular promise for rapid and reliable detection of heteroplasmy in different affected tissues. Although Sanger sequencing allows for complete coverage of the mtDNA, it is limited by the lack of deep coverage and low sensitivity for heteroplasmy detection when it is much less than 50%. In contrast, NGS technology is an excellent tool for rapidly and accurately obtaining a patient’s predominant mtDNA sequence and also lower frequency heteroplasmic variants. This is enabled by deep coverage of the genome through multiple independent sequence reads. Accordingly, recent studies making use of NGS techniques have demonstrated sequence accuracy equivalent to Sanger-type sequencing, but also have uncovered heretofore unappreciated heteroplasmy rates ranging between 10 and 50% and detection of single-nucleotide heteroplasmy down to levels of <10%.
Clinically, the most striking overall characteristic of mitochondrial genetic disease is the phenotypic heterogeneity associated with mtDNA mutations. This extends to intrafamilial phenotypic heterogeneity for the same mtDNA pathogenic mutation and, conversely, to the overlap of phenotypic disease manifestations with distinct mutations. Thus, although fairly consistent and well-defined “classic” syndromes have been attributed to specific mutations, frequently “nonclassic” combinations of disease phenotypes ranging from isolated myopathy to extensive multisystem disease are often encountered, rendering genotype-phenotype correlation challenging. In both classical and nonclassical mtDNA disorders, there is often a clustering of some combination of abnormalities affecting the neurologic system (including optic nerve atrophy, pigment retinopathy, and sensorineural hearing loss), cardiac and skeletal muscle (including extraocular muscles), and endocrine and metabolic systems (including diabetes mellitus). Additional organ systems that may be affected include the hematopoietic, renal, hepatic, and gastrointestinal systems, although these are more frequently involved in infants and children. Disease-causing mtDNA coding region mutations can affect either one of the 13 protein encoding genes or one of the 24 protein synthetic genes. Clinical manifestations do not readily distinguish these two categories, although lactic acidosis and muscle pathologic findings tend to be more prominent in the latter. In all cases, either defective ATP production due to disturbances in the ETC or enhanced generation of ROS has been invoked as the mediating biochemical mechanism between mtDNA mutation and disease manifestation.
mtDNA DISEASE PRESENTATIONS
The clinical presentation of adult patients with mtDNA disease can be divided into three categories: (1) clinical features suggestive of mitochondrial disease (Table 85e-2), but not a well-defined classic syndrome; (2) classic mtDNA syndromes; and (3) clinical presentation confined to one organ system (e.g., isolated sensorineural deafness, cardiomyopathy, or diabetes mellitus).
TABLE 85e-2Common Features of mtDNA-Associated Diseases in Adults ||Download (.pdf) TABLE 85e-2Common Features of mtDNA-Associated Diseases in Adults
|Neurologic: stroke, epilepsy, migraine headache, peripheral neuropathy, cranial neuropathy (optic atrophy, sensorineural deafness, dysphagia, dysphasia) |
|Skeletal myopathy: ophthalmoplegia, exercise intolerance, myalgia |
|Cardiac: conduction block, cardiomyopathy |
|Respiratory: hypoventilation, aspiration pneumonitis |
|Endocrine: diabetes mellitus, premature ovarian failure, hypothyroidism, hypoparathyroidism |
|Ophthalmologic: cataracts, pigment retinopathy, neurologic and myopathic (optic atrophy, ophthalmoplegia) |
Table 85e-3 provides a summary of eight illustrative classic mtDNA syndromes or disorders that affect adult patients and highlights some of the most interesting features of mtDNA disease in terms of molecular pathogenesis, inheritance, and clinical presentation. The first five of these syndromes result from heritable point mutations in either protein-encoding or protein synthetic mtDNA genes; the other three result from rearrangements or deletions that usually do not involve the germline.
TABLE 85e-3Mitochondrial Diseases Due to mtDNA Point Mutations and Large-Scale Rearrangements ||Download (.pdf) TABLE 85e-3Mitochondrial Diseases Due to mtDNA Point Mutations and Large-Scale Rearrangements
|Disease ||Phenotype ||Most Frequent mtDNA Mutations ||Homoplasmic (usually) ||Maternal |
|NARP, Leigh disease ||Loss of central vision leading to blindness in young adult life ||m.1778G>A, m.14484T>C, m.3460G>A ||Heteroplasmic ||Maternal |
|MELAS ||Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; may manifest only as diabetes mellitus ||Point mutation in tRNAleu ||Heteroplasmic ||Maternal |
|MERRF ||Myoclonic epilepsy, ragged red fibers in muscle, ataxia, increased CSF protein, sensorineural deafness, dementia ||Point mutation in tRNAlys ||Heteroplasmic ||Maternal |
|Deafness ||Progressive sensorineural deafness, often induced by aminoglycoside antibiotics ||m.1555A>G mutation in 12S rRNA ||Homoplasmic ||Maternal |
| ||Nonsyndromic sensorineural deafness ||m.7445A>G mutation in 12S rRNA ||Homoplasmic ||Maternal |
|Chronic progressive external ophthalmoplegia (PEO) ||Late-onset bilateral ptosis and ophthalmoplegia, proximal muscle weakness, and exercise intolerance ||Single deletions or duplications ||Heteroplasmic ||Mostly sporadic, somatic mutations |
|Pearson syndrome ||Pancreatic insufficiency, pancytopenia, lactic acidosis ||Large deletion ||Heteroplasmic ||Sporadic, somatic mutations |
|Kearns-Sayre syndrome (KSS) ||External ophthalmoplegia, heart block, retinal pigmentation, ataxia ||The 5-kb “common deletion” ||Heteroplasmic ||Sporadic, somatic mutations |
Leber’s hereditary optic neuropathy (LHON) is a common cause of maternally inherited visual failure. LHON typically presents during young adulthood with subacute painless loss of vision in one eye, with symptoms developing in the other eye 6–12 weeks after the initial onset. In some instances, cerebellar ataxia, peripheral neuropathy, and cardiac conduction defects are observed. In >95% of cases, LHON is due to one of three homoplasmic point mutations of mtDNA that affect genes encoding different subunits of complex I of the mitochondrial ETC; however, not all individuals who inherit a primary LHON mtDNA mutation develop optic neuropathy, and males are four to five times more likely than females to be affected, indicating that additional environmental (e.g., tobacco exposure) or genetic factors are important in the etiology of the disorder. Both the nuclear and mitochondrial genomic backgrounds modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk haplotype for LHON was recently identified, supporting the formulation that nuclear genes act as modifiers and affording an explanation for the male prevalence of LHON. This haplotype can be used in predictive genomic testing and prenatal screening for this disease. In contrast to the other classic mtDNA disorders, it is of interest that patients with this syndrome are often homoplasmic for the disease-causing mutation. The somewhat later onset in young adulthood and modifying effect of protective background nuclear genomic haplotypes may have enabled homoplasmic pathogenic mutations to have escaped evolutionary censoring.
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a multisystem disorder with a typical onset between 2 to 10 years of age. Following normal early psychomotor development, the most common initial symptoms are seizures, recurrent headaches, anorexia, and recurrent vomiting. Exercise intolerance or proximal limb weakness can be the initial manifestation, followed by generalized tonic-clonic seizures. Short stature is common. Seizures are often associated with stroke-like episodes of transient hemiparesis or cortical blindness that may produce altered consciousness and may recur. The cumulative residual effects of the stroke-like episodes gradually impair motor abilities, vision, and cognition, often by adolescence or young adulthood. Sensorineural hearing loss adds to the progressive decline of these individuals. A plethora of less common symptoms have been described including myoclonus, ataxia, episodic coma, optic atrophy, cardiomyopathy, pigmentary retinopathy, ophthalmoplegia, diabetes mellitus, hirsutism, gastrointestinal dysmotility, and nephropathy. The typical age of death ranges from 10 to 35 years, but some individuals live into their sixth decade. Intercurrent infections or intestinal obstructions are often the terminal events. Laboratory investigation commonly demonstrates elevated lactate concentrations at rest with excessive increase after moderate exercise. Brain imaging during stroke-like episodes shows areas of increased T2 signal, typically involving the posterior cerebrum and not conforming to the distribution of major arteries. Electrocardiogram (ECG) may show evidence of cardiomyopathy, preexcitation, or incomplete heart block. Electromyography and nerve conduction studies are consistent with a myopathic process, but axonal and sensory neuropathy may coexist. Muscle biopsy typically shows ragged red fibers with the modified Gomori trichrome stain or “ragged blue fibers” resulting from the hyperintense reaction with the histochemical staining for succinate dehydrogenase. The diagnosis of MELAS is based on a combination of clinical findings and molecular genetic testing. Mutations in the mtDNA gene MT-TL1 encoding tRNAleu are causative. The most common mutation, present in approximately 80% of individuals with typical clinical findings, is an A-to-G transition at nucleotide 3243 (m.3243A>G). Mutations can usually be detected in mtDNA from leukocytes in individuals with typical MELAS; however, the occurrence of heteroplasmy can result in varying tissue distribution of mutated mtDNA. In the absence of specific treatment, various manifestations of MELAS are treated according to standard modalities for prevention, surveillance, and treatment.
Myoclonic epilepsy with ragged red fibers (MERRF) is a multisystem disorder characterized by myoclonus, seizures, ataxia, and myopathy with ragged red fibers. Hearing loss, exercise intolerance, neuropathy, and short stature are often present. Almost all MERRF patients have mutation in the mtDNA tRNAlys gene, and the m.8344A>G mutation in the mtDNA gene encoding the lysine amino acid tRNA is responsible for 80–90% of MERRF cases.
Neuropathy, ataxia, and retinitis pigmentosa (NARP) is characterized by moderate diffuse cerebral and cerebellar atrophy and symmetric lesions of the basal ganglia on magnetic resonance imaging (MRI). A heteroplasmic m.8993T>G mutation in the ATPase 6 subunit gene has been identified as causative. Ragged red fibers are not observed in muscle biopsy. When >95% of mtDNA molecules are mutant, a more severe clinical, neuroradiologic. and neuropathologic picture (Leigh syndrome) emerges. Point mutations in the mtDNA gene encoding the 12S rRNA result in heritable nonsyndromic hearing loss. One such mutation causes heritable ototoxic susceptibility to aminoglycoside antibiotics, which opens a pathway for a simple pharmacogenetic test in the appropriate clinical settings.
Kearns-Sayre syndrome (KSS), sporadic progressive external ophthalmoplegia (PEO), and Pearson syndrome are three disease phenotypes caused by large-scale mtDNA rearrangements including partial deletions or partial duplication. The majority of single large-scale rearrangements of mtDNA are thought to result from clonal amplification of a single sporadic mutational event, occurring in the maternal oocyte or during early embryonic development. Because germline involvement is rare, most cases are sporadic rather than inherited. KSS is characterized by the triad of onset before age 20, chronic progressive external ophthalmoplegia, and pigmentary retinopathy. Cerebellar syndrome, heart block, increased cerebrospinal fluid protein content, diabetes mellitus, and short stature are also part of the syndrome. Single deletions/duplication can also result in milder phenotypes such as PEO, characterized by late-onset progressive external ophthalmoplegia, proximal myopathy, and exercise intolerance. In both KSS and PEO, diabetes mellitus and hearing loss are frequent accompaniments. Pearson syndrome is also characterized by diabetes mellitus from pancreatic insufficiency, together with pancytopenia and lactic acidosis, caused by the large-scale sporadic deletion of several mtDNA genes.
Two important dilemmas in classic mtDNA disease have benefited from recent important research insights. The first relates to the greater involvement of neuronal, muscular, renal, hepatic, and pancreatic manifestations in mtDNA disease in these syndromes. This observation has appropriately been mostly attributed to the high energy utilization of the involved tissues and organ systems and, hence, greater dependency on mitochondrial ETC integrity and health. However, because mutations are stochastic events, mitochondrial mutations should occur in any organ during embryogenesis and development. Recently, additional explanations have been suggested based on studies of the common m.3243A>G transition. The proportion of this mutation in peripheral blood cells was shown to decrease exponentially with age. A selective process acting at the stem cell level with a strong bias against the mutated form would have its greatest effect to reduce the mutant mtDNA only in highly proliferating cells, such as those derived from the hematopoietic system. Tissues and organs with lower cell turnover, such as those involved with mtDNA mutations, would not benefit from this effect and, thus, would be the most affected.
The other dilemma arises from the observation that only a subset of mtDNA mutations accounts for the majority of the familial mtDNA diseases. The random occurrence of mutations in the mtDNA sequence should yield a more uniform distribution of disease-causing mutations. However, recent studies using the introduction of one severe and one mild point mutation into the female germline of experimental animals demonstrated selective elimination during oogenesis of the severe mutation and selective retention of the milder mutation, with the emergence of mitochondrial disease in offspring after multiple generations. Thus, oogenesis itself can act as an “evolutionary” filter for mtDNA disease.
THE INVESTIGATION OF SUSPECTED mtDNA DISEASE
The clinical presentations of classic syndromes, groupings of disease manifestations in multiple organ systems, or unexplained isolated presentations of one of the disease features of a classic mtDNA syndrome should prompt a systematic clinical investigation as outlined in Fig. 85e-6. Indeed, mitochondrial disease should be considered in the differential diagnosis of any progressive multisystem disorder. Despite the centrality of disruptive oxidative phosphorylation, an elevated blood lactate level is neither specific nor sensitive because there are many causes of blood lactic acidosis, and many patients with mtDNA defects presenting in adulthood have normal blood lactate. An elevated cerebrospinal fluid lactate is a more specific test for mitochondrial disease if there is central nervous system involvement. The serum creatine kinase may be elevated but is often normal, even in the presence of a proximal myopathy. Urinary organic and amino acids may also be abnormal, reflecting metabolic and kidney proximal tubule dysfunction. Every patient with seizures or cognitive decline should have an electroencephalogram. A brain computed tomography (CT) scan may show calcified basal ganglia or bilateral hypodense regions with cortical atrophy. MRI is indicated in patients with brainstem signs or stroke-like episodes.
Clinical and laboratory investigation of a suspected mitochondrial DNA (mtDNA) disorder. CSF, cerebrospinal fluid; CT, computed tomography; ECG, electrocardiogram; ECHO, echocardiography; EEG, electroencephalogram; EMG, electromyogram; LHON, Leber’s hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stoke-like episodes; MERFF, myoclonic epilepsy with ragged red fibers; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.
For some mitochondrial diseases, it is possible to obtain an accurate diagnosis with a simple molecular genetic screen. For examples, 95% of patients with LHON harbor one of three mtDNA point mutations (m.11778A>G, m.A3460A>G, or m.14484T>C). These patients have very high levels of mutated mtDNA in peripheral blood cells, and therefore, it is appropriate to send a blood sample for molecular genetic analysis by polymerase chain reaction (PCR) or restriction fragment length polymorphism. The same is true for most MERRF patients who harbor a point mutation in the lysine tRNA gene at position 8344. In contrast, patients with the m.3243A>G MELAS mutation often have low levels of mutated mtDNA in blood. If clinical suspicion is strong enough to warrant peripheral blood testing, then patients with a negative result should be investigated further by performing a skeletal muscle biopsy.
Muscle biopsy histochemical analysis is the cornerstone for investigation of patients with suspected mitochondrial disease. Histochemical analysis may show subsarcolemmal accumulation of mitochondria with the appearance of ragged red fibers. Electron microscopy might show abnormal mitochondria with paracrystalline inclusions. Muscle histochemistry may show cytochrome c oxidase (COX)–deficient fibers, which indicate mitochondrial dysfunction (Fig. 85e-5). Respiratory chain complex assays may also show reduced enzyme function. Either of these two abnormalities confirms the presence of a mitochondrial disease, to be followed by an in-depth molecular genetic analysis.
Recent evidence has provided important insights into the importance of nuclear-mtDNA genomic cross-talk and has provided a descriptive framework for classifying and understanding disorders that emanate from perturbations in this cross-talk. Although not strictly considered as mtDNA genetic disorders, manifestations do overlap those highlighted above (Fig. 85e-7).
Disorders associated with perturbations in nuclear-mitochondrial genomic cross-talk. Clinical features and genes associated with multiple mitochondrial DNA (mtDNA) deletions, mtDNA depletion, and mitochondrial neurogastrointestinal encephalomyopathy syndromes. ANT, adenine nucleotide translocators; adPEO, autosomal dominant progressive external ophthalmoplegia; arPEO, autosomal recessive progressive external ophthalmoplegia; IOSCA, infantile-onset spinocerebellar ataxia; SCAE, spinocerebellar ataxia and epilepsy. (Reproduced with permission from A Spinazzola, M Zeviani: Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk. J Intern Med 265:174, 2009.)