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The mtDNA is prone to high levels of mutagenesis, with a mutation rate

approximately 10 times higher than that of the nuclear genome (Brown et al., 1979).

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The high mutation rate may be due to a number of factors and more than likely to a combination of these factors. The first to consider is the close proximity of the mtDNA to the respiratory chain complex and thus the exposure to damaging ROS produced by the respiratory chain. A second issue is that the mtDNA polymerase POLG has a much lower fidelity than that of the nuclear polymerase (Kunkel and Loeb, 1981).

Further to this, the mitochondrial DNA is replicated much more frequently than the nuclear genome (Bogenhagen and Clayton, 1977) and therefore the probability of an error during replication that is incorrectly repaired is much more likely. In addition to the high mutation rate, since the mitochondrial genome consists almost entirely of coding regions mutations are much more likely to occur in a coding region and as such be pathogenic.

1.6.1 Point mutations

Point mutations are a single base substitution and occurrence in the mitochondrial genome is highest in the tRNA genes, with 50% of all point mutations occurring here (Schaefer et al., 2008). Pathogenic point mutations are known to cause a number of mitochondrial diseases and can be maternally inherited or sporadic. The most common and thus most well characterised point mutations are the m.3243A>G tRNA^Leu(UUR) and the m.8344A>G tRNA^Lys (Gorman et al., 2015).

The m.3243A>G mutation is the most common cause of MELAS causing approximately 80% of all cases (Urata et al., 2004). However, the phenotypic spectrum of m.3243A>G mutations is very variable. MIDD is most common but patients also present with Leigh’s syndrome and CPEO, and isolated symptoms are also reported (Nesbitt and McFarland, 2011; Mancuso et al., 2014). The m.8344A>G point mutations has been reported to cause 80-90% of all myoclonic epilepsy with ragged red fibres (MERRF) cases (Wu et al., 2010). However, m.8344A>G, similar to m.3243A>G, also causes a wider variety of symptoms including migraine, psychiatric disorders, hearing impairment, respiratory and gastrointestinal symptoms (Mancuso et al., 2013; Altmann et al., 2016).

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1.6.2 Single large-scale mtDNA deletions

Large-scale single mtDNA deletions arise sporadically during embryogenesis due to errors in either mtDNA replication or repair (Shoffner et al., 1989; Krishnan et al., 2008; Dong et al., 2014) and the same deletion species is found at variable heteroplasmy in each cell. Clinically these account for 12% of adult mitochondrial disease cases (Gorman et al., 2015). Patients can present with chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre Syndrome and Pearson Syndrome as well as non-syndromic disease symptoms, with the most common symptoms being ptosis, ophthalmoparesis and muscle weakness (Mancuso et al., 2015). The most common single, large-scale mtDNA deletion is the 4977bp common deletion between nucleotides 8482 and 13460 of the mtDNA, and typically has a 13bp repeat either side of the breakpoint as reported in MitoBreak (Damas et al., 2014a). The common deletion has been reported to account for approximately one third of all single mtDNA deletions (Pitceathly et al., 2012a), but also commonly accumulates with age (Williams et al., 2013).

Early investigations into single, large-scale mtDNA deletions found there to be a lack of relationship between mtDNA genetics, biochemical function and clinical phenotype (Zeviani et al., 1988; Holt et al., 1989; Moraes et al., 1989). However, more recently reports have found relationships between mtDNA deletion heteroplasmy, age of onset and disease phenotype i.e. larger deletions lead to a more severe phenotype (Yamashita et al., 2008; Lopez-Gallardo et al., 2009). However, mtDNA deletion size is found to both have a correlation with disease phenotype (Yamashita et al., 2008) and not to have a relationship (Lopez-Gallardo et al., 2009). Furthermore, both of the above studies found that deletion location can impact disease severity. (Yamashita et al., 2008), find that a deletion affecting MT-COI, MT-COII, MT-COIII, MT-ATP6 or MT-ATP8 have significantly earlier onset, while (Lopez-Gallardo et al., 2009) found that deletions affecting MT-CYB are associated with a severe KSS phenotype.

1.6.3 Disorders of mtDNA maintenance

Mitochondrial DNA deletions have been associated secondary mitochondrial

diseases due to nuclear mutations and also with a number of neuromuscular (Oldfors et al., 2006; Rygiel et al., 2016) and neurodegenerative disorders (Bender et al.,

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2006), as well as accumulating with age in healthy post-mitotic tissues (Muller-Hocker et al., 1993; Brierley et al., 1998; Kraytsberg et al., 2006; Meissner et al., 2006).

They most commonly occur in the major arc of the mtDNA, however the frequency at which deletions are detected in the minor arc has increased in recent years. They are typically found to accumulate throughout life, with the highest levels identified in the post-mitotic tissues e.g. neurons and myofibres (Bender et al., 2006). Mitochondrial DNA deletions which typically arise secondary to a nuclear mutations in a gene involved in the maintenance or replication of the mtDNA, but are also associated with neuromuscular and neurodegenerative diseases and healthy ageing. Genes

associated with mtDNA deletions encode proteins involved in replication (e.g. POLG (Van Goethem et al., 2001), TWNK (Spelbrink et al., 2001)), synthesis of dNTPs (e.g.

RRM2B (Tyynismaa et al., 2009)) and mitochondrial fission and fusion (e.g. OPA1 and MFN2 (Amati-Bonneau et al., 2008; Rouzier et al., 2012)). Additional genes that cause multiple mtDNA deletions are continually being identified. Phenotypically mtDNA deletions range from isolated progressive external opthalmoplegia (PEO) to fatal multi-systemic PEO-plus syndromes. The other symptoms encompassed in the PEO-plus category can be very diverse, and disease onset ranges from infantile to late adulthood. Accumulation of deletions with age is termed clonal expansion, theories of how this occurs and the process by which deletions are expected to form is explored in more detail in Chapter 4.

1.10 Mitochondrial disease 1.7.1 Disease prevalence

Mitochondrial diseases are multi-systemic disorders that typically present with symptoms affecting some of the most energetically demanding organs such as the central and peripheral nervous systems and the skeletal muscle. The prevalence of mitochondrial disease is difficult to accurately assess, due to the clinical and genetic heterogeneity. The most recent assessment in the North East of England finds the prevalence of mitochondrial disease caused by mutations of both the mitochondrial and nuclear genomes to be approximately 1 in 4300 (Gorman et al., 2015). In comparison, the minimum prevalence of mtDNA mutations is reported to be 1 in

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5000. The study finds the m.3243A>G mutation to be the most prevalent pathogenic point mutation with a prevalence of 7.8 per 1000000. Although, single deletions are still the most common cause of mitochondrial disease (minimum prevalence = 1.5 × 10⁻⁵).