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Mitochondrial myopathies are regulated by two genomes: the nuclear DNA, and the mitochondrial DNA. While, so far, most studies have dealt with mitochondrial myopathies due to deletions or point mutations in the mitochondrial DNA, a new field of investigation is that of syndromes due to mutations in the nuclear DNA. These latter disorders have mendelian inheritance.
Three representative cases have been selected: one with COX deficiency and a Leigh syndrome due to a SURF1 gene mutation, one due to a defect of Coenzyme Q synthesis and one with dominant optic atrophy due to a mutation in the OPA1 gene.
Future developments will show that many neurodegenerative disorders are due to mutations of nuclear genes controlling mitochondrial function, fusion and fission.
Production of energy in mitochondria, by means of oxidative phosphorylation, strictly depends upon factors which are encoded both by the mitochondrial DNA (mtDNA) and the nuclear genome (nDNA). Respiratory chain complexes are formed, for the most part by subunits of nuclear origin, as are several indispensable complex-assembling proteins. Accurate replication and efficient maintenance of mtDNA are also essential for the respiratory chain to function properly.
Many metabolic processes, distinct from ATP production, are fulfilled in mitochondria: for instance, important steps of metal cation metabolism take place in the mitochondrial matrix. Furthermore, mitochondria actively fuse and divide, and move interacting with the cytoskeleton. All these functions require the expression of nuclear genes. Mitochondrial disorders caused by nDNA defects have been the object of increasing attention in the past few years, establishing themselves as an important and relatively prevalent group of pathologies, and challenging the relevance of disease caused by inherited mutations of mtDNA itself.
Disorders involving mtDNA replication are referred to as “intergenomic signalling defects” (1), and may be divided into two main categories: qualitative defects, i.e., multiple mtDNA deletions, and quantitative defects, i.e., mtDNA depletion syndromes. Other relevant genes include those for respiratory chain subunits and their ancillary proteins, as well as those for enzymes performing critical steps in metabolic pathways, or proteins involved in mediating mitochondrial fusion and fission.
Major nuclear genes giving rise to mitochondrial disease have been reviewed, with reference to pathophysiologic mechanisms and clinical phenotypes (Table (Table1).1). Three cases are reported as an example of the clinical spectrum of these disorders.
The first nDNA mutations to be associated with secondary multiple deletions of mtDNA were described due to a thymidine phosphorylase gene (TP) defect by Nishino et al. in 1999 (2). TP does not localize in mitochondria, but it is functionally related to mtDNA replication, as its impairment alters the composition of the nucleotide pool within the mitochondrial matrix. The resulting phenotype is Mitochondrial Neuro-Gastro-Intestinal leukoEncephalopathy (MNGIE), an autosomal recessive (AR) disease of young adulthood which severely reduces life expectancy, mostly due to intestinal malabsorption and cachexia. External ophthalmoplegia and neuropathy may also be present (3).
In the following years, other nuclear genes were found to be mutated in patients affected by progressive external ophthalmoplegia with a mendelian pattern of inheritance. Evidence was produced in animal models that defective nucleotide exchange across the mitochondrial membrane eventually leads to damage of mtDNA, possibly through a mechanism which involves production of reactive oxygen species and oxidative stress. These experiments employed adenine nucleotide translocator 1 (ANT1) null mice (4). The corresponding human gene, when altered, causes adult-onset autosomal dominant progressive external ophthalmoplegia (adPEO) (5).
The same clinical syndrome may similarly originate from mutations in TWINKLE. The protein product of this gene was found to be a helicase, to co-localize with mtDNA, and to lead to accumulation of multiple mtDNA deletions when altered (6, 7). More recently, an additional phenotype, Infantile Onset SpinoCerebellar Ataxia (IOSCA), has been repoted to be caused by alterations of the Twinkle helicase or of its splicing variant Twinky. This severe disease is the only AR phenotype to be associated with TWINKLE mutations (8).
Mutations in the POLG gene, which encodes for the catalytic subunit of the mitochondrial DNA polymerase (pol-γ), have been related to a wide variety of autosomal disorders, inherited both in a dominant and recessive fashion. Structural defects in pol-γ lead to age-related accumulation of multiple mtDNA deletions. Less frequently, similar or identical mutations may alternatively cause mtDNA depletion, and thus more severe, early-onset phenotypes. Molecular mechanisms underlying such variability have not yet been completely elucidated (9).
The first phenotype to be described in association with POLG mutations was again autosomal dominant AD-PEO (10), followed by other AD syndromes such as parkinsonism and premature ovarian failure (11), and by AR disorders characterized by sensory-ataxic neuropathy and dysarthria together with external ophthalmoplegia (SANDO) (12). AR-inherited POLG mutations also cause Alpers syndrome, a severe paediatric hepato-encephalopathy. Such phenotypes present critical mtDNA depletion from birth, rather than a slow accumulation of deletions over the years, suggesting a greater functional impairment of the polymerase (13).
Myopathic and hepato-cerebral syndromes are the main phenotypes to be observed in patients with consistent quantitative reduction of mtDNA. These groups of AR disease resemble multiple deletion syndromes in that they are caused by alterations in deoxyribonucleotide triphosphate metabolic pathways, but are usually severe and present earlier in life.
Myopathic forms are related to mutations in TK2, which encodes for thymidine kinase 2, and show an AR pattern of inheritance (14,15). In most cases, a slowly progressive mitochondrial myopathy develops, but the clinical spectrum for this gene has recently spanned to include spinal muscular atrophy and severe congenital myopathy (16, 17).
Encephalomyopathic variants, reminiscent of Leigh syndrome, were found in patients with SUCLA2 homozygous mutations, who carried a subsequent defect of the beta-subunit of the ADP-forming succinyl-CoA synthase (SCS-A) (18).
Deoxyguanosine kinase deficiency, arising from mutations in DGUOK (19), and, as very recently discovered, MPV17 mutations (20), are the basis for hepato-cerebral degenerative syndromes with mtDNA depletion. From a clinical standpoint, these disorders can be grouped under the eponym of Alpers syndrome, together with the afore-mentioned severe varieties of pol-γ deficiencies.
More than 70 out of >90 polypeptides which constitute the RC are encoded by the nucleus. Many of the respective genes have been shown to be mutated in human diseases.
Complex I is that most frequently involved in these pathologies. Several culprit genes have been identified: NDUSF2, NDUSF4, NDUSF6, NDUSF7, NDUSF8, NDUFV1 and NDUFV2 (21, 22), but they are not sufficient to explain all cases of complex I deficiency of mendelian inheritance, therefore leaving space for speculation about possible, as yet unidentified, ancillary protein defects.
Complex II deficiency is a rare cause of mitochondrial encephalomyopathy, caused by homozygous mutations in SDHA (23). This gene encodes for one of the four subunits of complex II, which is the smallest complex of the RC, and the only one to be entirely nuclear in origin. Mutations of the other three subunits have been associated with familiar endocrine tumours (24).
Many cases of specific RC complex deficiency, asrevealed by biochemical assays, show a quantitative reduction or structural disruption of the affected complex, which cannot be explained by mutations in mtDNA or nDNA encoded subunits. This scenario is typical of assembly protein defects, many of which have already been identified.
Complex III deficiencies are commonly caused by a defect of cytochrome b, which is encoded by mtDNA. However, some patients with complex III deficiency and mitochondrial encephalopathy have a normal cytochrome b. These particular patients were found to carry a point mutation in the ancillary protein gene BCS1L (25). Phenotype is severe with features of multisystemic failure, as summarized by the acronym GRACILE (Growth Retardation, Aminoaciduria, Cholestasis, Iron overload, Lactacidosis, Early death) (26).
Deficiency of cytochrome C oxidase (COX, complex IV) is one of the most frequent causes of Leigh syndrome. There are no known mutations of nuclear genes encoding for COX subunits, as autosomal COX deficiency seems to be entirely due to lack of assembly proteins. SURF1 was the first candidate nuclear gene to be connected to COX-deficient encephalomyopathy, as illustrated in Case 1 (27, 28). Other genes were subsequently linked to COX deficiency, notably SCO2 and COX15. Clinical features were comprehensive of encephalomyopathy, early-onset fatal hypertrophic cardiomyopathy, neuropathy (29–31). Both SCO2 and COX15 play relevant roles in the regulation of cytochrome C prosthetic groups, respectively, binding copper and heme iron (32, 33).
Case 1: Childhood encephalomyopathy with COX deficiency, ataxia, muscle wasting, and mental impairment (27). Case 1 refers to a boy with consanguineous parents (third cousins) born after an uneventful pregnancy and delivery. Psychomotor development delay, muscle hypotonia and gait disturbances were evident at age 2 years. At the time of muscle biopsy (age 8 years), the boy presented hypotonia, muscle hypotrophy, axial and limb ataxia, and was unable to walk unassisted. Mental impairment was mild. Ocular motility was impaired with a bilateral abduction defect resulting in a converging squint. His face and lower extremities were hairy. Electromyography (EMG) showed myopathic features, and a right bundle branch block was found on electrocardiography. He had a rapid right frontoparietal rhythm on electroencephalography and delayed brainstem evoked potentials.
Muscle biopsy demonstrated many atrophic vacuolated fibres with numerous oil red O positive lipid droplets, and a faint histo-enzymatic reaction for COX. COX activity was decreased to 7% of the normal mean in muscle homogenate, while other mitochondrial enzymes were normal. A homozygous mutation in SURF1 was subsequently demonstrated.
Complex V, i.e., the F0F1 ATPase, has also been found to be a cause of encephalomyopathy due to a mutation in an assembly protein gene, ATP12 (34). As seen with complex IV, no nuclear-encoded subunit defects are known.
To be included in this group are the defects of coenzyme Q (CoQ) synthesizing proteins (Case 2), which have only very recently started to be genetically defined. Inheritance is AR and clinical characteristics are heterogeneous, ranging from encephalomyopathy, to renal tubulopathy and ataxic neuropathy. Mutations have been described in the para-hydroxybenzoate-polyprenyl transferase gene COQ2 -as exemplified in Case 2 (35, 36) and in PDSS2, which encodes for subunit 2 of decaprenyl-diphosphate-synthase (37).
Case 2: Infantile encephalomyopathy, nephrotic syndrome CoQ deficiency. Patient 2 refers to a boy born with consanguineous parents (first cousins) of North African origin, after an uneventful pregnancy and delivery. At age 2 months, nystagmus was noted. At age 12 months, the boy developed a severe, corticosteroid-resistant nephrotic syndrome. Renal biopsy revealed focal and segmental glomerulosclerosis. Acquisition of developmental milestones was slightly delayed, and a mild hypotonia was evident on neurologic examination. Fundoscopy revealed optic atrophy. Plasma creatine kinase and lactate were normal. Brain magnetic resonance imaging (MRI), echocardiography and brainstem auditory evoked potentials were normal. Visual evoked potentials showed an altered retinocortical transmission, and an electroretinogram indicated rod cone retinopathy. In the following months, a deterioration was observed both in renal function, requiring dialysis, and in brain function: at age 18 months, the boy lost the ability to walk and stand unassisted. Cerebral spinal fluid lactate, serum amino acids and urinary organic acids were within normal limits. He developed frequent vomiting, psychomotor regression and focal status epilepticus. A new brain MRI showed diffuse cerebral atrophy, mild cerebellar atrophy and lesions in the cingulate cortex and subcortical area. At the age when muscle biopsy was performed (22 months) the patient had developed right hemiplegia and swallowing difficulties. The muscle biopsy had an essentially normal morphology. Subsarcolemmal accumulation of succinate dehydrogenase stain was evident in some fibers. Activity of complex II + III, which depends on coenzyme Q (CoQ), was decreased. Subministration of CoQ resulted in prompt and dramatic improvement of muscle tone and strength, remission of vomiting and myoclonus, and regain of the ability of standing and walking unassisted, as well as of other developmental milestones that had been previously lost. Unfortunately, CoQ therapy was not sufficient to correct renal dysfunction, and the boy remained dependent on dialysis. A homozygous missense mutation was identified in COQ2 by analysis of the eight known human genes (COQ1-8) which encode for CoQ biosynthetic proteins. An A→G transition at a highly conserved position (nucleotide 890) caused amino acid 297 to change from tyrosine to cysteine.
The mitochondrial matrix is a major site for heme and [Fe-S] cluster synthesis. When these metabolic pathways are disrupted, damage is caused to the cell not only by the lack of prosthetic groups for several mitochondrial and non-mitochondrial proteins, but also by the ungovernable production of reactive oxygen species which the free forms of metal cations catalyze (38).
The first step in heme synthesis, i.e., condensation between glycine and succinyl-CoA resulting in δ-aminolevulinic acid (ALA), is mediated by δ-aminolevulinic acid synthetase (ALAS) (39). The erythroid isoform of ALAS is encoded by ALAS2, and its deficiency causes X-linked sideroblastic anaemia (40). It is interesting to note that no neuropathy is observed in this disease, as the neural isoform of ALAS is encoded by a distinct gene, ALAS1 (41). X-linked sideroblastic aneamia with ataxia, on the other hand, is caused by an alteration of a transporter protein of the ATP-binding cassette superfamily (ABCB7 mutations): [Fe-S] cluster transportation from the mitochondrial matrix to the cytosol. If this is impaired, leads to iron accumulation in mitochondria and multisystemic dysfunction of iron metabolism (42).
Metal cation accumulation and consequent oxidative damage, mainly within tissues of high mitochondrial content, such as CNS and nerve, is widely thought to be a pathogenetic feature common to many neurodegenerative diseases. Friedreich’s ataxia is the most frequent AR hereditary ataxia, and is caused by intronic GAA repeats in the gene for frataxin, FRDA (43). Mitochondrial localization of frataxin (44) and iron overload in afflected tissues, such as nerve and myocardium (45), point to a deficit in mitochondrial trafficking of iron atoms. Furthermore, the partial benefit obtained with free-radical scavengers such as idebenone (46) would suggest an increased oxidative stress associated with iron overload in Friedreich’s ataxia pathogenesis, although the specific role of frataxin in the process has not yet been univocally defined.
Friedreich’s ataxia is a model for the crucial role of mitochondria in neurodegeneration. Similar mechanisms may have a role in the genesis of other, far more prevalent degenerative diseases (such as Alzheimer’s and Parkinson’s) which affect a large part of the senile population (47, 48). Mitochondrial dysfunction enhances production of free radicals, the immediate consequences of which are mtDNA replication errors and/or direct mtDNA damage, leading to further mitochondrial dysfunction. This vicious cycle may be relevant to the physiologic process of ageing itself, even if some experimental findings do not indicate this mechanism (49).
Mitochondria, in no different way than other intracellular organelles, are known to exert a dynamic relationship with the cytoskeleton, which allows them to move within the cell. Balance of fusion and fission forces determines morphologic and functional aspects of mitochondria (50). A kinesin, KIF5A, is altered in a pure form of hereditary spastic paraplegia (51), while mutations of dynamin related GTPase OPA1 provoke AD optic nerve atrophy, a mitochondrial cause of vision loss distinct from maternally inherited Leber’s optic neuropathy (52). A peculiar association of AD optic atrophy with neuropathy has been proposed in Case 3 (53). MFN2 (mitofusin 2) mutations, on the other hand, produce the hereditary sensorimotor neuropathy classified as CMT-2A, caused by a disturbance of mitochondrial fusion pathways (54).
Case 3: Autosomal dominant optic atrophy and neuropathy (53). A 46-year-old female complained of slowly progressive visual loss and dyschromatopsia since 10 years of age. She also complained of nocturnal distal paresthesias in the four limbs. She had optic atrophy (Fig. (Fig.1)1) both upon fundoscopic examination and brain MRI (Fig. (Fig.2).2). EMG showed a severe sensory-motor axonal neuropathy. The patient’s sister, aged 45 years, also complained of visual loss, also present in several other members of her family with a pattern suggesting an autosomal dominant inheritance.
Muscle biopsy was performed, showing neurogenic atrophy and aggregates of abnormally-shaped mitochondria on electron microscopy (Fig. (Fig.3).3). Molecular analysis of the OPA1 gene showed a new 38 base pair deletion in the junction between exon and intron 14, causing a splice site defect.
It was not until very recently that a disease, due to mitochondrial fission disturbances, was demonstrated. A dominant-negative mutation in dynamin-like protein 1 gene (DLP1) was found in a girl with microcephaly, optic atrophy and lactic acidosis, and a defect in fission capabilities both of mitochondria and peroxisomes was noted (55).
The cases reported here illustrate the wide clinical spectrum of mitochondrial disorders caused by nuclear genes. Diagnostic suspicion is arisen by a multisystemic pattern of involvement, typical of mitochondriopathies, in the presence of apparent mendelian inheritance. Reaching a correct diagnosis may have remarkable therapeutic implications, as emphasized in Case 2.
New syndromes and mutations are being described with growing frequency, but we are not yet able to quantify the overall impact, in terms of incidence, of mitochondrial disorders depending on the nuclear genome. While epidemiologic studies of primary mtDNA alterations have produced figures of a minimum 1/5000 prevalence (56, 57), the spectrum of these disorders has not yet been systematically defined from an epidemiological standpoint. Yet, as knowledge increases (and consequentially as more cases are correctly diagnosed), these disorders might be recognized as an important group of mitochondrial diseases, imposing a heavy burden on the community, both from a sanitary and social point of view. Further insight into the molecular mechanisms should provide hope for new therapeutic strategies to be experimented and applied.
Study conducted with support of grants from Telethon-Italy (GTB07001) and EuroBioBank network partner of Treat-NMD Neuromuscular Network (6th framework programme).