Mitochondrial DNA ageing and disease


Histochemical studies have shown that a small proportion of non-dividing cells from healthy humans acquire a respiratory chain defect throughout life. Single-cell studies have identified pathogenic mutations of mtDNA within the majority of these cells, explaining the accumulation of potentially pathogenic mtDNA mutations with age in many human tissues (Michikawa et al., 1999b; Attardi, 2002; Fayet et al., 2002). Both point mutations and deletions have been detected, but deletions appear to be the most common cause of age-related COX deficiency. The majority of mtDNA deletions appear to occur between sites of direct DNA sequence repeats of 4 to 15 bp. This suggests that the mechanism by which deletions occur is by slippage during replication or by homologous recombination. The majority of deletions are found on the major arc of the mitochondrial genome, a region defined as being between the origin of light strand replication (OL) and the origin of heavy strand replication (OH). Approximately 95% of reported deletions occur in this region and remove genes encoding subunits of COX, ATP synthase and NADH dehydrogenase. The first mtDNA deletion reported to accumulate with age was a deletion of 4,977 bp. The so-called 'common deletion' occurs between two 13 bp repeat sites, at nucleotide positions 8,470 to 8,483 and 13,447 to 13,459, removing approximately 5 kb of mtDNA between the ATPase8 and ND5 genes. In accordance with the MITOMAP website, all base positions are numbered from the light strand (L strand). Table 11.1 lists some of the mtDNA mutations associated with ageing.

Primary mitochondrial DNA diseases

Primary mitochondrial diseases are those caused by a mutation, either a point mutation or deletion, in the mitochondrial genome. Mitochondrial disease is a term which covers a broad spectrum of multi-systemic disorders. The pathogenic mutations compromise respiratory chain function leading to cellular dysfunction and ultimately cell death. Table 11.2 summarizes some of the more common mtDNA disorders.

Table 11.1 Examples of mtDNA deletions and base substitutions which have been reported at higher prevalences in healthy aged individuals with the tissues each has been seen in. Deletion start and end points are shown. CSB2 - Conserved Sequence Block 2; OH and OL -origins of replication for the heavy and light strand respectively; PH and PL - promoters for heavy and light strand respectively.

Mutation mtDNA deletions 4977 bp 6063 bp 7400 bp 3895 bp

Multiple deletions mtDNA point mutations





Cins 310






8468 to 13446 7842 to 13905 8648 to 16085 548 to 4442 Various tRNALys tRNALeu(UUR) tRNAMet tRNAMet D-Loop (CSB2) D-Loop (PL) D-Loop (oh) D-Loop (PL) D-Loop (oh)

Affected tissue

Heart, liver, brain, skeletal muscle and other tissues Liver

Myocardium, brain cortex and putamen

Skeletal muscle


Extraocular muscle Extraocular muscle Skeletal muscle Skeletal muscle Skin fibroblasts Skin fibroblasts Skeletal muscle Skeletal muscle Leukocytes

Table 11.2 Common mitochondrial disorders and associated mtDNA mutations.


Clinical features

mtDNA mutation

Chronic Progressive External

External ophthalmoplegia and

Large Scale

Ophthalmoplegia (CPEO)

bilateral ptosis

deletions or

Kearns Sayre Syndrome (KSS)

Early PEO onset (<20 yrs) with pigmentary retinopathy with one of; elevated CSF, cerebellar ataxia and heart block


Pearson Syndrome (PS)

Sideroblastic anemia in childhood, pan-cytopenia and exocrine pancreatic failure

Lebers Hereditary Optic

Subacute painless bilateral loss of vision,


Neuropathy (LHON)

onset ~20 yrs. Male affected bias

11778G>A 14484T>C

Neurogenic weakness with

Early adult/late childhood onset peripheral


ataxia and retinitis pigmentosa

neuropathy with ataxia and pigmentary



Mitochondrial encephalomyopathy

Stroke like episodes, onset <40 yrs.


with lactic acidosis and stroke-like

Seizures, dementia, ragged red fibers and


episodes (MELAS)

lactic acidosis


Myoclonic epilepsy with ragged

Myoclonus, seizures, cerebellar ataxia and


red fibers (MERRF)



Leigh Syndrome (LS)

Subacute relapsing encephalopathy with cerebellar and brain stem signs, onset in infancy


Non-syndromic sensorineural deafness


Aminoglycoside induced


non-syndromic deafness

Designing as assay for the detection of mtDNA point mutations is, in essence, no different to detecting nDNA mutations by allelic discrimination. Examples of allelic discrimination assays are presented in Chapters 8, 9, and 17 and will not be repeated here. The major difference is that when measuring nuclear alleles the percentage values are either 50% heterozygous or 100% homozygous. When measuring the mutant load in the mtDNA, values of heteroplasmy from 1 to 99% can be present. These need to be accurately measured when investigating a pathogenic point mutation, e.g. 8344A>G (Szuhai et al., 2001), as the level of heteroplasmy has important implications on the clinical phenotype and disease progression. Validation across the full range of heteroplasmy values is required to enable meaningful data to be obtained from the assay and for valid conclusions to be drawn.

Nuclear mitochondrial disorders

Nuclear mitochondrial disorders are those conditions where the primary genetic defect is encoded on the nuclear genome but the effect of this defect is realized in mitochondria (Spinazzola and Zeviani, 2005). This effect is either directly on mtDNA maintenance or on mitochondrial processes, e.g. OXPHOS and protein import. Some examples of these disorders where the effect is on the mtDNA are shown in Table 11.3. When considering realtime PCR for these nuclear mutations the primary genetic defect is either homozygous or heterozygous, so quantification of the mutant load is unnecessary. Detection of the presence of the nuclear mutation can be easily achieved by another means, e.g. sequencing, RFLP, primer extension assays. It is the secondary defect of the mtDNA which is of relevance here, be it the level of heteroplasmy (point mutations or deletions, single or multiple) or the absolute quantity of mtDNA (depletion).

mtDNA mutations and cancer mtDNA substitutions have been detected in a number of different human cancers (Penta et al., 2001). It is currently unclear whether these mutations are a secondary phenomenon, and are of no direct relevance to carcinogenesis, or whether they are primarily involved in the disease (Coller et al., 2001). In

Table 11.3 Nuclear-mitochondrial disorders where the primary genetic defect is on the nuclear genome and results in a secondary mtDNA genetic defect.


Autosomal dominant PEO

Mitochondrial neuro-gastrointestinal encephalomyopathy

Myopathy with mtDNA depletion

Myopathy with hepatic failure

Nuclear defect

Adenine Nucleotide Translocator (ANT1) mtDNA polymerase y (POLG) Twinkle helicase (C10orf2)

Thymidine Phosphorylase (TP)

Thymidine Kinase (TK2) Deoxyguanosine Kinase (DGOUK)

mtDNA mutation

Multiple mtDNA deletions mtDNA depletion most cases the mutations are homoplasmic (i.e. all of the mtDNA is mutated within individual cells). Each tumor contains a specific point mutation (or mutations), and allele detection is usually achieved by sequencing.

Toxin-induced mtDNA depletion

Shortly after the introduction of nucleoside analogue treatment for acquired immunodeficiency due to HIV infection, it became clear that some patients receiving AZT (azydothymidine) developed a mitochondrial myopathy due to mtDNA depletion. mtDNA is replicated by pol y, which is inhibited by a variety of nucleoside analogues (part of the HAART, or highly active anti-retroviral therapy regime) (Lewis et al., 2003). Acute and subacute nucleoside toxicity can be life-threatening, due to the lactic acido-sis and liver failure. A number of studies have described mtDNA depletion in peripheral blood immediately preceding the clinical deterioration, raising the possibility that measuring mtDNA levels (relative to a single copy nuclear gene), might be used to monitor therapy and prevent fatal complications (de Mendoza et al., 2004) or the more chronic complications such as lipodystrophy (McComsey et al., 2005b). However, not all studies are in agreement, and it may be necessary to study a specific group of blood monocytes to gain clinically useful data (Hoy et al., 2004). Moreover, with the advent of new generation therapies, nucleoside toxicity is less of a problem (Lewis et al., 2003; McComsey et al., 2005a). This chapter will not discuss mtDNA assays in HAART in greater detail, but the basic principle of measuring mtDNA copy number will be considered in greater depth and has broad applications across this and other disciplines.

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