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 that potentially causes disease but that heteroplasmic germ-line pathogenic mtDNA mutations affect up to approximately 1 in 5000 individuals. The true overall impact of mtDNA mutation in human health and disease may be much greater if the potential contribution of homoplasmic mtDNA sequence variation to common complex diseases that appear in the postreproductive age also is considered.
The true disease burden will be known only with the ability to distinguish a completely neutral sequence variant from a true phenotype-modifying or pathogenic mutation, when an accurate assessment of heteroplasmy can be determined with fidelity, and when the epistatic interactions of mtDNA sequence variations with mutations in the nuclear genome can be expressed using a systems biology approach (Chap. e19).
Overview of Clinical and Pathologic Features of Human MTDNA Disease
In light of 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 that affect ETC function. Figure e18-4 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), and (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 hereditary optic neuropathy; LS, Leigh syndrome; MELAS, mitochondrial encephalomyopathy, 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; PPK, palmoplantar keratoderma; SIDS, sudden infant death syndrome. (Reproduced with permission from S DiMauro, E Schon: N Engl J Med 348: 2656, 2003.)
Heteroplasmy sometimes can be demonstrated elegantly at the tissue level by 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 encoded exclusively 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; therefore, this enzyme has the lowest threshold for dysfunction when a threshold 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. e18-5). Heteroplasmy also can be detected at the genetic level through direct mtDNA genotyping in special conditions, though clinically significant low levels of heteroplasmy can escape detection in genomic samples extracted from whole blood using conventional genotyping and sequencing techniques.
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 encountered, rendering genotype-phenotype correlation challenging. In both classic and nonclassic mtDNA disorders, there is often a clustering of some combination of abnormalities affecting the neurologic system (including optic nerve atrophy, pigment retinopathy, 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, though these systems are involved more frequently 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, though 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 reactive oxygen species 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 e18-2) 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 e18-2 Common Features of mtDNA-Associated Diseases in Adults |Favorite Table|Download (.pdf)
Table e18-2 Common 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 e18-3 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 germ line.
Table e18-3 Mitochondrial Diseases Due to mtDNA Point Mutations and Large-Scale Rearrangements |Favorite Table|Download (.pdf)
Table e18-3 Mitochondrial Diseases Due to mtDNA Point Mutations and Large-Scale Rearrangements
|Disease||Phenotype||Most Common mtDNA Mutations||Homoplasmic (usually)||Maternal|
|Leber Hereditary Optic Neuropathy (LHON)||Bilateral subacute or acute painless optic atrophy||G11778A, T14484C, G3460A||Homoplasmic||Maternal|
|NARP, Leigh disease||Loss of central vision leading to blindness in young adult life||G1778A, T14484C, G3460A||Heteroplasmic||Maternal|
|MELAS||Mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes; may manifest only as diabetes||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.||A1555G mutation in 12S rRNA||Homoplasmic||Maternal|
|Nonsyndromic sensorineural deafness||A7445G 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|
|Kearn-Sayre syndrome (KSS)||External ophthalmoplegia, heart block, retinal pigmentation, ataxia||The 5-kb “common deletion”||Heteroplasmic||Sporadic, somatic mutations|
Leber 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 that encode different subunits of complex I of the mitochondrial ETC; however, not all individuals who inherit a primary LHON mtDNA mutation develop optic neuropathy, indicating that additional environmental (e.g., tobacco exposure) or genetic factors are important in the etiology of the disorder. Both the nuclear and the mitochondrial genomic background modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk haplotype for LHON has been 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 the modifying effect of protective background nuclear genomic haplotypes may have enabled homoplasmic pathogenic mutations to escape evolutionary censoring.
Mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) is probably the most common mtDNA disease, consisting of a progressive encephalomyopathy characterized by repeated strokelike events involving mainly posterior cerebral areas. Of note, brain lesions do not respect the distribution of vascular territories. Recurrent migraine-like headache and vomiting, exercise intolerance, seizures, short stature, and lactic acidosis are other common clinical features. The most commonly described pathogenic point mutations are A3243G and T3271C in the gene encoding the leucine tRNA.
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 A8344G mutation in the mtDNA gene encoding the lysine amino acid tRNA is responsible for 80–90% of MERRF cases.
Neurogenic weakness, ataxia, and retinitis pigmentosa (NARP) is characterized by moderate diffuse cerebral and cerebellar atrophy and symmetric lesions of the basal ganglia on MRI. A heteroplasmic T8993G mutation in the gene 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. Since germ-line 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, and short stature are also part of the syndrome. Single deletions/duplication also can 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 common accompaniments. Pearson syndrome also is 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 appropriately has been attributed mostly to the high energy utilization of the involved tissues and organ systems and, hence, greater dependency on mitochondrial ETC integrity and health. However, since 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 A3243G 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 in reducing 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 affected the most.
Another important question of interest arises from the observation that only a subset of mtDNA mutations account 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 utilizing the introduction of one severe and one mild point mutation into the female germ line of experimental animals demonstrated selective elimination during oogenesis of the severe and selective retention of the milder mutations, 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. e18-6. 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 that present 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 also may be abnormal, reflecting metabolic and kidney proximal tubule dysfunction. Every patient with seizures or cognitive decline should have an electroencephalogram. A brain CT scan may show calcified basal ganglia or bilateral hypodense regions with cortical atrophy. MRI is indicated in patients with brainstem signs or strokelike episodes.
Clinical and laboratory investigation of suspected mtDNA disorder. CSF, cerebrospinal fluid; ECG, electrocardiogram; EEG, electroencephalogram; EMG, electromyogram; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes; MERFF, myoclonic epilepsy with ragged red fibers; LHON, Leber hereditary optic neuropathy; 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 (A11778G, A3460G, and T14484C). These patients have very high levels of mutated mtDNA in peripheral blood cells, and it is therefore 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 A3243G MELAS mutation often have low levels of mutated mtDNA in blood. If clinical suspicion is strong enough to warrant peripheral blood testing, patients with a negative result should be investigated further with 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 may show abnormal mitochondria with paracrystalline inclusions. Muscle histochemistry may show cytochrome c oxidase (COX)–deficient fibers, which indicate mitochondrial dysfunction (Fig. e18-5). Respiratory chain complex assays also may 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.
Relevant 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 these are not strictly considered as mtDNA genetic disorders, their manifestations overlap with those highlighted above.