Introduction
Mitochondria are essential organelles found in nearly all eukaryotic cells (Vafai and Mootha
2012), where they are mainly related to energy metabolism. The most prominent role for mitochondria is to generate ATP through the respiratory chain (RC) by means of oxidative phosphorylation (OXPHOS). However, mitochondria also take part in a vast array of important biochemical pathways including, among others, heat production, apoptosis, generation and detoxification of reactive oxygen species (ROS), intracellular Ca
2+ regulation, steroid hormone and heme synthesis and lipid metabolism (Wallace
2005).
The RC is composed of four multiheteromeric complexes (CI-IV), which transfer the electrons extracted from nutrients by means of sequential redox reactions to molecular oxygen to form water. This process is coupled to the translocation of proton across the IM by three of the four canonical respiratory chain complexes (CI, CIII, and CIV), generating a proton gradient, which is then exploited by ATP synthase (or complex V, CV) to convert ADP into ATP.
Mitochondria are semi-autonomous organelles because they have their own DNA (mitochondrial DNA, mtDNA), which encodes for 13 essential subunits of CI, CIII, CIV, and CV (Schon et al
2012), whereas all the other components of the RC, and indeed all of the other ≈1500 polypeptides constituting the mitochondrial proteome, are encoded by the nuclear genome. Therefore, mitochondrial bioenergetics and related homeostatic and execution pathways are under the double genetic control of both nuclear and mtDNA. The genetic duality of mitochondrial metabolism has relevant consequences for human pathology, as mutations in either nuclear or mtDNA can lead to mitochondrial dysfunction and disease. These genetic features explain why mitochondrial disorders can be transmitted as dominant, recessive, X-linked or maternally-inherited traits (Zeviani and Di Donato
2004). Hundreds of pathogenic mtDNA mutations have been reported (MITO-MAP 2012). Whilst mtDNA in normal conditions is essentially uniform, a condition called homoplasmy, most of the pathogenic mutations of mtDNA co-exist with a variable percentage of wild-type mtDNA, in a condition termed heteroplasmy. Mutations of mtDNA or of OXPHOS-related nuclear genes can affect virtually every tissue in the body, and lead to different phenotypes depending on the organ involved, intrinsic severity of the mutation, targeted gene, and, in case of mtDNA mutations, heteroplasmy levels. Tissue and organ functions critically depend on adequate ATP production, especially when energy demand is high, like in neurons and muscle fibers (Koopman et al
2013). This explains why primary disorders of mitochondrial bioenergetics usually cause neurodegeneration and/or myopathy, often in combination, to determine encephalomyopathies affecting children or adults. However, many mitochondrial disorders also involve additional organs, e.g., causing heart impairment (for instance cardiac failure due to cardiomyopathy, or heart conduction defects), liver dysfunction, diabetes mellitus, or alterations in special senses or specific districts, e.g. sensory-neural deafness, ophthalmoparesis (due to weakness of the extrinsic eye muscles), ptosis (drooping eyelids), retinitis pigmentosa, and optic neuropathy causing blindness due to degeneration of the optic nerve. Mutations in mtDNA can affect specific proteins of the respiratory chain or the in situ, autochthonous synthesis of mitochondrial proteins as a whole (when mutations or deletions involve tRNA or rRNA genes) and can be in turn classified into large-scale rearrangements (i.e., partial deletions or duplications) and point mutations. Both groups have been associated with well-defined clinical syndromes. Whilst single large-scale rearrangements are usually sporadic, point mutations are tipically maternally inherited. Large-scale rearrangements include several genes and are invariably heteroplasmic. In contrast, point mutations may be heteroplasmic or homoplasmic, the latter being characterized by incomplete penetrance. (e.g., Leber’s hereditary optic neuropathy).
In addition to sporadic or maternally inherited disorders due to mutations of the mitochondrial genome, mitochondrial diseases can also be transmitted as Mendelian traits. Here, we shall focus on those Mendelian disorders that alter the stability and the integrity of mtDNA. In 1989, Zeviani and colleagues described an Italian family with adult-onset mitochondrial myopathy characterized by chronic progressive external ophthalmoplegia, CPEO, and inherited in an autosomal dominant fashion (Zeviani et al
1989). Maternal inheritance was excluded because the male patients also transmitted the disease to their offspring. Since then, many additional autosomal dominant CPEO families have been described. A second group of syndromes, characterized by infantile myopathy or hepatopathy, was then associated with depletion of mitochondrial DNA in affected tissues (Moraes et al
1991). Multiple deletions and depletion of mitochondrial DNA were also found in skeletal muscle in a complex, multisystem syndrome combining muscle, brain, and gastrointestinal symptoms (mitochondrial neurogastrointestinal encephalomyopathy or MNGIE) (Hirano et al
1998). Finally, mutations in the gene encoding the DNA polymerase gamma (
POLG), the master enzyme of mitochondrial DNA replication, were found in severe, early-onset neurologic disorders, namely Alpers-Huttenlocher hepatopathic poliodystrophy, sensory-ataxia neuropathy with dysarthria and ophthalmoplegia, and spinocerebellar ataxia-epilepsy syndrome (Naviaux and Nguyen
2005).
Over the last decade, an increasing number of genes have been identified in association with mtDNA multiple deletions or depletion with variable phenotypes hallmarked by syndromic CPEO, encephalomyopathy, and cardiomyopathy. The vast majority of them involve proteins with a role in the mtDNA replisome (POLG and 2, TWNK, DNA2, MGME1) or dNTP supply for mtDNA synthesis (TP, TK2, DGUOK, RRM2B, SUCLA2, SUCLG1). A novel category of proteins involved in accumulation of mtDNA multiple deletions is represented by OPA1 and MFN2, which are part of the complex machinery regulating mitochondrial dynamics, specifically mitochondrial fusion; and paraplegin and AFG3L2, which play an important role in the protein quality control of mitochondria.
Clinical features
Autosomal disorders classified as defects of mtDNA maintenance due to disturbed nuclear-mitochondrial intergenomic communication can be associated with the accumulation of mtDNA large-scale rearrangements (mtDNA multiple deletions) or by severe reduction of the mtDNA copy number (mtDNA depletion syndromes) (Spinazzola and Zeviani
2005). The most frequent clinical presentations (Table
1) are:
(1)
adult-onset encephalomyopathy, defined clinically by CPEO, genetically by autosomal dominant or recessive transmission, and molecularly by the presence of multiple deletions of mtDNA.
(2)
an autosomal recessive multisystem disorder known as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), characterized by combined accumulation of multiple deletions and partial depletion of mtDNA.
(3)
a spectrum of recessive neurologic syndromes ranging from typical infantile hepatopathic poliodystrophy (Alpers-Huttenlocher syndrome) to juvenile-onset sensory-ataxia neuropathy, dysarthria, and ophthalmoplegia (SANDO) to a combination of spinocerebellar ataxia and epilepsy (SCAE) with or without external ophthalmoplegia.
(4)
early-onset, organ-specific autosomal recessive syndromes associated with profound mtDNA depletion.
Table 1
Genes and phenotypes affecting mtDNA maintenance
SLC25A4
| Multiple deletions | AD | ad/arCPEO | 157640/258450 |
| Multiple deletions | AR | myopathy and cardiomyopathy | 615418 |
| Depletion | AD | myopathy and cardiomyopathy | |
TWNK
| Multiple deletions | AD | adCPEO | 609286 |
| Multiple deletions | AR | IOSCA | 271245 |
| Depletion | AR | Alpers-like | |
POLG
| Multiple deletions | AD | adCPEO | 157640 |
| Multiple deletions | AR | arCPEO | 258450 |
| Depletion | AR | Alpers-Huntenlocher | 203700 |
| Multiple deletions | AR | SANDO/SCAE | 607459 |
POLG2
| Multiple deletions | AD | adCPEO | 610131 |
TFAM
| Depletion | AR | Hepatocerebral syndrome | 617156 |
MGME1
| Multiple deletions | AR | arCPEO | 615076 |
DNA2
| Multiple deletions | AD | adCPEO | 615156 |
RNASEH1
| Multiple deletions | AR | arCPEO | 616479 |
RRM2B
| Multiple deletions | AD | adCPEO | 613077 |
| Depletion | AR | myopathy and tubulopathy | 612075 |
TK2
| Depletion | AR | myopathy | 609560 |
| Multiple deletions | AR | arCPEO | 617069 |
DGUOK
| Depletion | AR | Hepatocerebral syndrome | 251880 |
| Multiple deletions | AR | Myopathy with or w/o CPEO | 617070 |
| Multiple deletions | AR | lower motor neuron syndrome | |
MPV17
| Depletion | AR | Hepatocerebral syndrome | 256810 |
| Multiple deletions | AR | arCPEO, leukoencephalopathy and parkinsonism | |
OPA1
| Multiple deletions | AD | DOA | 165500 |
| Multiple deletions | AD | DOA plus | 125250 |
MFN2
| Multiple deletions | AD | DOA plus | 608507 |
SPG7
| Multiple deletions | AR | arCPEO and ataxia | 602783 |
AFG3L2
| Multiple deletions | AD | arCPEO and ataxia | 604581 |
TYMP
| Multiple deletions and depletion | AR | MNGIE | 603041 |
SUCLA2
| Depletion | AR | Hepatocerebral syndrome | 612073 |
SUCLG1
| Depletion | AR | Hepatocerebral syndrome | 245400 |
ABAT
| Multiple deletions | AR | Encephalomyopathy | 613163 |
FBXL4
| Depletion | AR | Encephalomyopathy | 615471 |
GFER
| Multiple deletions | AR | myopathy | 613076 |
Pathogenetic considerations
The CPEO phenotype is commonly associated with single mtDNA deletions in sporadic patients (Spinazzola and Zeviani
2005). A pathogenetic role of multiple mtDNA deletions in autosomal dominant or recessive CPEO is supported by the evidence of tight segregation of the molecular lesions with the onset and severity of the disease. For instance, Lamantea and colleagues have reported that, in a series of
POLG-positive CPEO families, homozygous individuals appeared more severely affected and showed the presence of much higher amounts of multiple mtDNA deletions in muscle than their heterozygous relatives (Lamantea et al
2002). Moslemi and colleagues have demonstrated close correlation between the accumulation of deletions and the segmental ragged-red cytochrome
c oxidase-negative transformation of muscle fibers (Moslemi et al
1996). These authors showed that within a single cytochrome
c oxidase-deficient muscle fiber segment, only one single deletion could be detected. However, different deletions were identified in different segments. These results indicate clonal expansion of a single deleted mtDNA in each cytochrome
c oxidase-deficient muscle fiber segment. A two-hit mechanism can, therefore, be hypothesized, consisting of the combination of a nuclear factor that somehow predisposes to mtDNA deletions, followed by clonal expansion of each deleted mtDNA molecule in muscle and other stable tissues (Moslemi et al
1996). Deletions are absent in cultured fibroblasts, peripheral blood cells, and cultured myoblasts but can be detected in stable tissues, including (besides skeletal muscle) brain, heart, and in lesser amount, kidney, and liver. Disturbances of the nucleotide pool available for mtDNA replication, as well as abnormalities in either the mitochondrial helicase or mtDNA polymerase, are likely to affect the rate or process of DNA replication, which could ultimately lead to the exaggerated production of rearranged mtDNA molecules (Graziewicz et al
2004). In addition to large-scale rearrangements, increased frequency of mtDNA point mutations have been reported in MNGIE (Nishigaki et al
2003). Several somatic mutations, mostly T > C transitions preceded by 5′-An sequences, were scattered throughout the mtDNA molecule of tissues and cultured cells from MNGIE patients. Some mutations were clearly pathogenic, as they predict loss or abnormal function of mtDNA-encoded proteins or tRNA. The accumulation of these mutations is likely to be due to next-nucleotide effects and dislocation mutagenesis, as a consequence of increased levels of mitochondrial deoxy-thymidine and deoxy-uridine pools (Nishigaki et al
2003).
Finally, the mtDNA damage caused by
POLG mutations in Alpers-Huttenlocher syndrome, SANDO, and SCAE syndrome is unclear. Depletion of mtDNA in critical organs (liver and brain) has been demonstrated in a few cases (Ferrari et al
2005; Tzoulis et al
2014), which may explain the early onset and severity of these conditions.
Balance and control of the mitochondrial deoxynucleotide pools are essential for the maintenance of mtDNA integrity and copy number. Perturbation of this homeostatic control, as determined by defects of dGK and TK2, and possibly of thymidine phosphorylase, RRM2B, and ANT1 as well, can lead to mtDNA depletion or multiple deletions. In particular, dGK and TK2 are involved in the salvage pathways of mitochondrial deoxynucleotides, which constitute the major source of mtDNA precursors in stable tissues such as liver, brain, and muscle. Although a role for MPV17 has been proposed in the cellular response to metabolic stress and maintenance of nucleotide pools (Dalla Rosa et al
2016; Spinazzola et al
2006), its function remains largely unknown.
The identification of
OPA1 mutations as a cause of mtDNA multiple deletions in skeletal muscle, which is now extended to the cognate mitochondrial fusion protein MFN2 (Rouzier et al
2012), points to the role played by mitochondrial network dynamics in mtDNA maintenance (Yu-Wai-Man and Chinnery
2012; Zeviani
2008). Impaired mitochondrial fusion in cells and in recombinant mouse models is indeed associated with mtDNA instability and worsening of the phenotype effects of mtDNA mutations (Chen et al
2010). Likewise, multiple deletions associated with mutations in
SPG7 (paraplegin) and
AFG3L2 indicate a causative association between mtDNA instability and impaired protein quality control in mitochondria.
Additional genes
The mechanisms leading to mtDNA instability are rather controversial for several of the genes associated with the disease.
For instance, SUCLA1 and SUCLG1 encode for components of the Krebs cycle and their role in the last step of the mitochondria dNTPs salvage pathway, because their association with NDPK, has been postulated but not conclusively proven. Similarly, ABAT is involved in GABA biosynthesis but also interacts with SCS and has been proposed to have a role in dNTP salvage pathway.
Another gene with an unclear role in mtDNA maintenance is
FBXL4. This gene has been recently associated with early onset encephalopathy with mtDNA depletion (Bonnen et al
2013; Gai et al
2013).
FBXL4 encodes for F-box and leucine-rich repeat 4 protein targeted to the intermembrane space of mitochondria. Although pathogenic mutations are associated with substantially decreased mtDNA content and mitochondrial respiratory chain deficiency in muscle and fibroblasts, its role in mtDNA maintenance is still unclear.
Finally, a homozygous mutation in the human
GFER gene, coding for a sulfhydryl oxidase (DRS) of the mitochondrial intermembrane space, has been reported in an inbred Moroccan family (Di Fonzo et al
2009). Three siblings were affected by congenital cataract, progressive muscular hypotonia, sensorineural hearing loss, and developmental delay. Muscle biopsy showed scattered COX deficiency and mtDNA multiple deletions. DRS is a protein involved in one of the mitochondrial protein import pathways. The pathogenic link between DRS mutation and mtDNA multiple deletions is presently unknown.
The development of next-generation sequencing (NGS) technologies (whole exome sequencing and whole genome sequencing) and their use in the diagnostic workout for mitochondrial disease has had a tremendous impact on the pace of gene discovery in recent years, and a huge set of new genes have indeed been associated with either mtDNA multiple deletions or mtDNA depletion. Nevertheless, a substantial fraction of the cases associated with mtDNA instability remains unsolved. This may be due to a number of reasons. First, we now know that genes encoding proteins whose function is not directly related to mtDNA replication or repair, such as OPA1, MFN2, FBXL4, can affect mtDNA metabolism. This consideration requires a substantial expansion of the territory of gene hunting, well beyond that of the “usual suspects”. Second, mtDNA defects can be due to sporadic, heterozygous mutations, affecting singleton cases, as in the case of the recent identification of lethal de novo mutations in ANT1 (Thompson et al
2016). These cases can escape detection, particularly if the screening does not include parents and direct relatives of the proband. Third, the mutation can affect deep intronic regions, and may escape detection if ad hoc gene panels, or whole exome sequencing, WES, are carried out or are considered a benign variant. Fourth, in case of sporadic patients, oligo- or multi-genic inheritance cannot be excluded, which complicates the diagnostic work-up. Fifth, the expression of a pathological phenotype may depend on dual or multiple mechanisms, for instance the mtDNA haplogroup, the gender, or behavioral habits. Finally, in several cases the function of the culprit gene is unknown, and the attribution of a pathogenic role to these “unknown factors” may not be obvious. The accumulation of more WES and whole genome sequencing, WGS, data in the human population and the systematic use of trio sequencing will possibly reduce the gap between the biochemical and genetic diagnosis, not only in the syndromes associated with mtDNA instability but in mitochondrial medicine as a whole.
A tissue biopsy, typically muscle, remains the gold standard for the diagnosis of mtDNA instability syndromes. However, in an increasing number of cases the syndromic characterization is specific enough to consent the screening of suspected genes, by ad hoc gene panels or WES, before or alternative to the investigation of muscle biopsy. Muscle and, in specific cases, liver biopsies should be performed in the routine analysis for mitochondrial disease when the diagnosis cannot be confirmed by direct targeted DNA testing.
Functional in vitro assays in tissue (typically muscle) have been the mainstay of diagnosis of mitochondrial disorders, especially prior to the recent advances in genomics. Functional assays remain important measures of mitochondrial function. All of the mitochondrial disease guidelines and diagnostic criteria developed prior to the recent advances in genetic techniques and understanding include results of such biochemical studies to help establish a mitochondrial disease diagnostic workup (Nishigaki et al
2003; Nishino et al
1999; Nolden et al
2005; Oskoui et al
2006; Ostergaard et al
2007a).
These tests evaluate the various functions of the mitochondrial ETC or respiratory chain. Functional assays include enzyme activities of the individual components of the ETC, measurements of the activity of respiratory chain complexes and ATP synthase, blue-native gel electrophoresis, measurement of the presence of various protein components within complexes and supercomplexes (achieved via western blots and gel electrophoresis), and consumption of oxygen using various substrates and inhibitors. In any case we recommend that these assays should be carried out in specialized centers, as they require ad hoc expertise and standardized, verified and complex technologies and diagnostic protocols.
As for the majority of mitochondrial disorders, no treatment is currently available for disease associated with mtDNA instability. However, it has been recently demonstrated in mouse models knockout for Tymp and knockin for the Tk2 H126N mutation that the supplementation of deoxyribonucleotides can effectively correct mtDNA depletion (Camara et al
2014; Garone et al
2014). Albeit preliminary, these new experimental approaches open the possibility for ad hoc therapies for diseases due to mtDNA instability.