2.4. Mutations in mtDNA as causes of diseases
The biochemical and genetic complexity of the respiratory chain accounts for the extraordinarily wide range of clinical presentations of mitochondrial disorders (Table 1). In general, the organs with the highest aerobic demand and the lowest regenerative capacity, such as the brain, heart and skeletal muscle, are the ones that are most severely involved, but virtually any organ or tissue in the body can be affected, including the gastrointestinal tract (Bardosi et al. 1987), liver (Mazziotta et al. 1992), kidney (Manouvrier et al. 1995) and the endocrine systems (Shoffner et al. 1995a, Manouvrier et al. 1995). Each tissue can be affected alone, e.g. pure mitochondrial myopathy, encephalopathy or cardiomyopathy, or more often in combination, e.g. mitochondrial encephalomyopathy.
The two main biochemical features in most mitochondrial diseases are respiratory chain deficiency and lactic acidosis. Morphologically, patients often display ragged-red fibers (RRF) in the muscle due to the accumulation of structurally abnormal subsarcolemmal mitochondria (Rowland et al. 1991).
Some mitochondrial syndromes are well established, having a certain molecular genetic background and are nosologically defined entities. There are many disorders, however, that are defined on the basis of morphological or biochemical findings and still lack a molecular genetic definition. In addition, overlap syndromes and non-specific phenotypes mean that clinical data are not sufficient to provide a systematic classification of mitochondrial diseases (Zeviani et al. 1996).
Genetically, mitochondrial diseases can be divided into three groups: those characterized by the presence of sporadic or maternally inherited mtDNA mutations, those characterized by the association of mtDNA abnormalities with mendelian transmission of the trait, i.e. disorders believed to be due to mutations in nuclear genes that control mitochondrial biogenesis, and those that lack a mtDNA defect but are thought on the basis of biochemical findings to be caused by mutations in nuclear genes.
Table 1. Common clinical manifestations of mitochondrial disorders.
| Neurological manifestations | Systemic manifestations |
|---|---|
| Ptosis, ophthalmoplegia | Metabolic acidosis |
| Optic neuropathy | Nausea and vomiting |
| Sensorineural hearing loss | Cardiomyopathy |
| Headache | Cardiac conduction defects |
| Seizures | Short stature |
| Stroke | Pigmentary retinopathy |
| Dementia | Hepatopathy |
| Myelopathy | Nephropathy |
| Peripheral neuropathy | Diabetes mellitus |
| Myopathy | Exocrine pancreatic dysfunction |
| Exercise intolerance, fatigability | Hypoparathyroidism |
| Ataxia | Intestinal pseudo-obstruction |
| Myoclonus | Cataract |
| Dystonia | Pancytopenia |
| Sideroblastic anaemia |
2.4.1. Point mutations
Many but not all pathogenic mtDNA point mutations are heteroplasmic. When the proportion of the mutant genome exceeds a certain threshold, the deleterious effects of the mutation will no longer be complemented by the coexisting wild-type mtDNA and will be expressed phenotypically as a cellular dysfunction leading to disease (Wallace et al. 1997a). Phenotypic expression will depend on the nature of the mutation, its tissue distribution and the relative reliance of each organ system on the mitochondrial energy supply (Schon et al. 1997). The influence of nuclear genes, coexisting mitochondrial polymorphisms, the age and sex of the individual and environmental factors may also play an important, although poorly understood, role in the phenotypic expression of mtDNA point mutations (Wallace et al. 1997a).
Mitochondrial DNA point mutations are maternally inherited and can occur in rRNA or tRNA genes, or in genes coding for proteins of respiratory chain complexes. Although more than 50 deleterious point mutations have been identified to date, four mutations are by far the most frequent (Wallace et al. 1997a). These are the 3243A>G ‘MELAS’, the 8344A>G ‘MERRF’, the 8993T>G ‘NARP’ and the 11778G>A ‘LHON’ mutations. Others are found less often, while still others have been described only in single individuals or families.
The investigation of pathogenic mitochondrial DNA mutations has revealed a complex relation between patient genotype and phenotype (Schon et al. 1997). For unknown reasons, some mtDNA mutations lead to specific clinical manifestations, an example being 3243A>G, causing the MELAS syndrome, one of the classic mitochondrial syndromes. Moreover, the MELAS syndrome has a high prevalence in the adult population, suggesting that mitochondrial disorders constitute one of the largest diagnostic categories of neurogenetic diseases (Majamaa et al. 1998).
2.4.1.1. Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)
Pavlakis et al. (1984) described two patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes. These two patients and nine others reported earlier (Gardner-Medwin et al. 1975, Shapira et al. 1975, Koenigsberger et al. 1976, Hart et al. 1977, Askanas et al. 1978, Skoglund 1979) presented with normal early development, short stature, seizures and alternating hemiparesis, hemianopia or cortical blindness. All these eleven patients had ragged-red fibers in their skeletal muscle, and most had elevated blood lactate concentrations. The cause of this syndrome was uncertain, but Pavlakis et al. (1984) presumed that there was an underlying biochemical defect in the mitochondria.
Genetics of the MELAS syndrome. MELAS is most commonly associated with a heteroplasmic point mutation in the tRNALeu(UUR) gene, an A to G transition at position 3243 (Goto et al. 1990), and approximately 80% of the cases of MELAS harbour this mutation (Ino et al. 1991, Goto et al. 1990, 1991, Goto 1995). Another 7–15% of MELAS patients have been found to have a T to C transition mutation at position 3271 in the same tRNALeu(UUR) gene (Goto et al. 1991, Togunaga et al. 1993, Marie et al. 1994, Goto 1995, Tarnopolsky et al. 1998). Other MELAS-associated point mutations have also been reported in the tRNALeu(UUR) gene, at np 3252 (Morten et al. 1992), np 3256 (Moraes et al. 1993b, Sato et al. 1994) and np 3291 (Goto et al. 1994) (Figure 3). In addition, mutations elsewhere in mtDNA can give rise to a MELAS-like phenotype, including the 8344A>G MERRF mutation. Some tRNA mutations, such as the 3256C>T in tRNALeu(UUR) and 7512T>C in tRNASer(UCN), can give rise to a MERRF/MELAS overlap syndrome (Moraes et al. 1993a, 1993b, Nakamura et al. 1995, Sato et al. 1994). One mis-sense mutation in a polypeptide-coding gene, at nt 11084 in ND4, has also been associated with MELAS (Lertrit et al. 1992), but there is evidence that it is a rare polymorphism (Ozawa et al. 1991, Sakuta et al. 1993) rather than a disease-causing mutation.
The mitochondrial tRNALeu(UUR) gene appears to be an aetiological hot spot for mtDNA mutations (Moraes et al. 1993b). In addition to mutations causing the MELAS syndrome, several others are located in this gene. Mutations at nt 3250 (Goto et al. 1992), nt 3251 (Sweeney et al. 1993) and nt 3302 (Bindoff et al. 1992) are associated with myopathy, while mutations at nt 3260 (Zeviani et al. 1991) and nt 3303 (Silvestri et al. 1993) are thought to cause both myopathy and cardiomyopathy. A 2 bp nucleotide pair deletion in the tRNALeu(UUR) gene involving positions 3271 to 3273 is expressed in severe intracerebellar calcifications with complex neurological manifestations (Shoffner et al. 1995a).
All mutations associated with the MELAS syndrome are heteroplasmic, with a high proportion (>80%) of mutant mtDNAs in muscle (Goto et al. 1992, Ciafaloni et al. 1992). It has been suggested that the onset of the MELAS syndrome is precipitated by proportions of mutant genome in muscle exceeding a threshold of about 60% (Miyabayashi et al. 1992). The percentage is always lower in blood than in muscle (Ciafaloni et al. 1992, Inui et al. 1992), but there is no correlation between the percentage of blood heteroplasmy and the phenotypic expression of the disorder (Tarnopolsky et al. 1998).
Figure 3. tRNALeu(UUR). The nucleotide transitions 3243A>G, 3252A>G, 3256C>T, 3271T>C and 3291T>C are associated with the MELAS syndrome.
Phenotype of the MELAS syndrome. The genotype to phenotype correlation of the 3243 A>G mutation is fairly loose, since not all patients with this mutation have the full-blown MELAS syndrome. For instance, the 3243 A>G mutation has been detected in several patients and families with maternally inherited Kearns-Sayre or chronic progressive external ophthalmoplegia (CPEO) syndromes, mitochondrial encephalomyopathies with or without ragged-red fibers, isolated myopathy, cardiomyopathy, or pedigrees with maternally inherited diabetes mellitus and deafness (Goto et al. 1990, Hammans et al. 1991, Ciafaloni et al. 1992, Reardon et al. 1992, van den Ouweland et al. 1992, Hirano et al. 1992, Mariotti et al. 1995).
The MELAS syndrome is first suspected when the patient has focal or generalized seizures, recurrent migraine headaches, vomiting, and stroke (Pavlakis et al. 1984). Recurrent strokes, perhaps the sole exclusive clinical manifestation, may be a late feature of the disease in the majority of cases (Morgan-Hughes et al. 1995). The posterior cerebral hemispheres are particularly vulnerable and the stroke commonly causes hemianopia and cortical blindness. The aetiology of the stroke is unknown.
Other manifestations in MELAS patients may include a ragged-red fiber myopathy, lactic acidaemia, sensorineural hearing loss, short stature, dementia, pigmentary retinal degeneration, hypertrophic cardiomyopathy, cardiac conduction abnormalities, renal failure, diabetes mellitus and basal ganglia calcifications (Kobayashi et al. 1990, Goto et al. 1990, 1992, Hammans et al. 1991, Ciafaloni et al. 1992, Moraes et al. 1992, Inui et al. 1992, Hirano et al. 1992, Togunaga et al. 1993, Sakuta et al. 1993, de Vries et al. 1994, Manouvrier et al. 1995).
The phenotype associated with the 3271T>C mutation is very similar, but expression may occur at a later age (Sakuta et al. 1993, Tarnopolsky et al. 1998).
The highest levels of mutant mtDNA occur in patients with an early onset of the disease (Morgan-Hughes et al. 1995). Furthermore, the proportion of mutant mtDNA is generally higher in patients with recurrent strokes than in those without strokes (Ciafaloni et al. 1992). Differences in the proportions of mutant mtDNA may not be the sole determinants of disease expression, however, and additional genetic mechanisms may be involved in defining the range of clinical and biochemical phenotypes associated with this aberrant mitochondrial genome (Morgan-Hughes et al. 1995).
Biochemical pathophysiology of the 3243A>G mutation. The mechanism by which the 3243 A>G transition leads to disease remains unclear. It alters a highly conserved dihydrouridine stem region in tRNALeu(UUR) (Goto et al. 1990), and thus may possibly alter the stability and functioning of the tRNA molecule. Furthermore, the mutation is located within a 13-nucleotide segment which binds the factor (mTERF) that promotes termination of transcription at the 16S rRNA/tRNALeu(UUR) gene boundary (Hess et al. 1991, Daga et al. 1993). Cybrids harbouring 3243A>G accumulate a precursor RNA 19, which corresponds to a transcript containing the contiguous 16S rRNA + tRNALeu(UUR) + ND1 genes (King et al. 1992, Koga et al. 1995). Furthermore, decreased 5’ and 3’ processing of tRNALeu(UUR) has been observed in these cybrids (King et al. 1992).
The complex I subunits ND6 and ND3 are mtDNA encoded polypeptides with the highest proportion of leucine residues translated by means of the UUR codon. Complex I deficiency is the most common respiratory chain defect observed in patients with the 3243A>G mutation (Goto et al. 1992). A deficiency in complex I or IV (Kobayashi et al. 1991, Chomyn et al. 1992, King et al. 1992, Dunbar et al. 1996) will lower proton pumping, reduce the electrochemical potential gradient across the mitochondrial inner membrane (Moudy et al. 1995, James et al. 1996) and lead to decreased respiration and a lower rate of ATP synthesis.
Inhibition of the respiratory chain increases the production of reactive oxygen species. The severity of complex I deficiency has been correlated with the production of superoxide anions and the induction of mitochondrial Mn superoxide dismutase (Pitkänen & Robinson 1996). Chronic generation of reactive oxygen species can result in oxidative damage to mitochondrial and cellular proteins, lipids and nucleic acids (Wallace & Melov 1998, Wallace 1999). Moreover, increased oxidative stress and decreased energy levels could activate the mitochondrial permeability transition pore, leading to apoptosis (Petit et al. 1996, Green & Reed 1998, Zoratti & Szabo 1995).
2.4.1.2. Other mtDNA point mutations
Together with the MELAS syndrome, Leber’s hereditary optic neuropathy (LHON), myoclonic epilepsy with ragged-red fibers (MERRF) and neurogenic weakness, ataxia, and retinitis pigmentosa (NARP) are by far the most frequent mitochondrial diseases caused by mtDNA point mutations.
Several other point mutations have been detected in single patients or in pedigrees affected with various phenotypes (Servidei 1997). For instance, myopathy and cardiomyopathy is associated with a number of tRNA gene mutations (Zeviani et al. 1991, Servidei 1997). One interesting phenomenon is aminoglycoside-induced or spontaneous, non-syndromic progressive deafness, which has been associated with a homoplasmic 1555A>G transition in the 12S RNA gene. It has been postulated that the 1555 mutation elongates the region where the tRNA binds to the ribosome, thus facilitating the binding of aminoglycosides and thereby potentiating their effects on the fidelity of the translation of the mRNA (Prezant et al. 1993). The cochlear cell death observed in these patients is thought to be due to misreading of mRNA during mitochondrial protein synthesis in this highly energy-dependent organ.
Leber's hereditary optic neuropathy (LHON). Leber’s hereditary optic neuropathy is a maternally inherited form of blindness predominantly affecting men and with onset in the second or third decade of life. Painless loss of vision begins in the central visual field, usually in one eye, and subsequently affects the other eye weeks or months later. Recovery of vision has been reported in some patients (Mackey & Howell 1992) and seems to depend on the particular pathogenic mtDNA mutation present.
LHON was the first mitochondrial disease to be defined at the molecular level. Wallace et al. (1988a) found a G>A transition at position 11778 in the ND4 gene of complex I in pedigrees with maternally inherited LHON, and since then 18 novel mis-sense mutations have been associated with the disease. Only a few of them are ‘primary’ mutations: 14459G>A in the ND6 gene (Jun et al. 1994, Shoffner et al. 1995b), 11778G>A in the ND4 gene (Wallace et al. 1988a), 3460G>A in the ND1 gene (Howell et al. 1991, Huoponen et al. 1991, 1993) and 14484T>C in the ND6 gene (Johns et al. 1992, Mackey & Howell 1992). The remaining mutations may either increase the probability of expressing the phenotype or are polymorphisms frequently linked to one of the clinically important mutations rather than contributing substantially to the potential for developing blindness. The 3460G>A mutation is associated with a variety of mtDNA haplotypes, demonstrating that this mutation has arisen independently on multiple occasions, each resulting in LHON (Brown et al. 1995, Howell et al. 1995). Similarly, all 14459G>A pedigrees have different mtDNA haplotypes (Jun et al. 1994, Shoffer et al. 1995b). By contrast, most patients harbouring the 11778G>A or 14484T>C mutation belong to a European haplogroup J (Torroni et al. 1994, 1997, Brown et al. 1997, Lamminen et al. 1997). This implies that the mtDNA haplogroup J may contribute to expression of the LHON phenotype in the presence of the 11778G>A or 14484T>C mutation. Furthermore, several additional mutations of unknown pathogenic significance have been found, and LHON may be a result of the cumulative effects of such ‘secondary’ mutations (Mackey & Howell 1992).
Analyse of the LHON mutations have provided a new insight into the phenotypic expression of mtDNA mutations. It appears that specific mutations may act together, perhaps synergistically, to produce the LHON phenotype (Howell et al. 1991, Mackey & Howell 1992, Brown et al. 1992), in addition to which the mtDNA haplogroup J may contribute to its expression (Torroni et al. 1996b).
Myoclonus epilepsy with ragged-red fibers (MERRF). MERRF is a maternally inherited neuromuscular disorder characterized by progressive myoclonus, epilepsy, muscle weakness and wasting, cerebellar ataxia, deafness and dementia (Wallace et al. 1988b). The most commonly observed mutation is an A>G transition at nt 8344 in the tRNALys gene in mtDNA (Shoffner et al. 1990), and a second mutation has been reported in the same gene, at position 8356 (Silvestri et al. 1992, Zeviani et al. 1993). The genotype to phenotype correlation with the 8344A>G mutation is stronger than in the case of the other mtDNA mutations (Silvestri et al. 1992). There is a positive correlation between the severity of the disease, age at onset, mtDNA heteroplasmy (Chinnery et al. 1997) and reduced activity of the respiratory chain complexes I and IV in skeletal muscle (Boulet et al. 1992).
Neurogenic weakness, ataxia and retinitis pigmentosa (NARP). NARP is a maternally inherited, adult-onset syndrome associated with a heteroplasmic T>G transversion at position 8993 in the ATPase 6 subunit gene (Holt et al. 1990). A transition 8993T>C was described later in other patients with NARP (de Vries et al. 1993). RRFs are absent in a muscle biopsy. The degree of heteroplasmy is correlated with the severity of the disease, so that when the proportion of mutant mtDNA is more than 95%, patients show clinical, neuroradiological and neuropathological findings of Leigh syndrome (see chapter 2.5). Hence it is called maternally inherited Leigh syndrome (MILS) (Tatuch et al. 1992). The two phenotypes, NARP and MILS, may coexist in the same family. Impairment of ATP synthesis has been reported in cell cultures harbouring the 8993T>G mutation, possibly because of defective assembly of the enzyme complex (Houstek et al. 1995).
2.4.2. Large-scale rearrangements of mtDNA
Large-scale rearrangements of mtDNA can be either mtDNA deletions or, more rarely, duplications. Both types of mutation are heteroplasmic, and they can occasionally exist simultaneously in patient tissues (Ballinger et al. 1992). More than 120 mtDNA deletions have been identified, most within direct repeats of 3–13 nucleotides in length (Kogelnik et al. 1998). The most common large-scale deletions are between a 13-base pair direct repeat from nt 8470 to nt 8482 in the ATPase8 gene and from nt 13447 to nt 13459 in the ND5 gene (Moraes et al. 1989). The resulting 4997 bp deletion (the common deletion) has occurred independently over 200 times and accounts for perhaps 50% of ocular myopathy patients (Kogelnik et al. 1998). Because virtually all deletions eliminate at least one tRNA, it is likely that they result in a generalized translational defect. Deletions are usually sporadic, non-transmittable mutagenic events (Brown & Wallace 1994).
The three main clinical phenotypes associated with large-scale mtDNA deletions are the Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia (CPEO) and the Pearson syndrome (Zeviani et al. 1988, Moraes et al. 1989, Poulton et al. 1989, Rötig et al. 1990, McShane et al. 1991). The Kearns-Sayre syndrome is characterized by an invariant triad of PEO, pigmentary retinopathy and onset before 20 years of age. Frequent additional symptoms are a progressive cerebellar syndrome, heart block and increased protein content in the cerebrospinal fluid. CPEO is characterized by bilateral ptosis and ophtalmoplegia, frequently associated with variable degrees of proximal muscle weakness and wasting, and exercise intolerance. The Pearson bone marrow-pancreas syndrome is a rare disorder of early infancy characterized by sideroblastic anaemia with pancytopaenia and exocrine pancreatic insufficiency. Infants surviving into childhood may develop the clinical features of Kearns-Sayre syndrome (McShane et al. 1991, Rötig et al. 1990).
Partial duplications of mtDNA has been detected in ocular myopathy and Pearson’s syndrome patients, although duplications are much rarer in these patients than deletions. Duplications can be sporadic (Poulton et al. 1991) or maternally transmitted (Rötig et al. 1992).
2.4.3. MtDNA genotypes conferring increased risk of disease
The high mutation rate has resulted in the accumulation of a wide range of population-specific base substitutions in mtDNA. While most of these variants are neutral, some are mildly deleterious. The latter, although they do not significantly reduce fitness, may interact with nuclear and environmental factors, predisposing individuals to an increased risk of developing neurodegenerative diseases late in life (Shoffner et al. 1993, Wallace 1994). Moreover, mildly deleterious polymorphisms may synergistically compromise mitochondrial function and contribute to the pathogenesis of a mitochondrial disorder (Lertrit et al. 1994).
It has been shown that the expression of some pathogenic mtDNA mutations depends on the mtDNA background against which they occur. The risk of expression of LHON in the presence of the primary mutation 14484T>C is eight-fold higher when this mutation occurs in the specifically European haplogroup J (Torroni et al. 1996b). The 11778G>A mutation was observed in a wide range of mtDNA haplogroups but has shown an almost six-fold preferential association for haplogroup J (Torroni et al. 1997). The mutation 4336T>C is associated with late-onset Alzheimer disease (Shoffner et al. 1993, Hutchin & Cortopassi 1995), and has arisen as a single mutational event in the European haplogroup H (Torroni et al. 1994).
The association of a particular mtDNA sequence variant with a particular disease is not an unambiguous indicator of aetiological significance (Chinnery et al. 1999), however, as the mtDNA sequence may act as a surrogate marker for a nuclear gene defect, particularly in isolated populations that have experienced a marked founder effect (Heyer 1995, Jorde et al. 1995). Similarly, a particular mtDNA haplotype may signal, through a founder effect, a population subgroup that has inherited a group of detrimental or protective nuclear genes.