mitochondrial dna mutations in human disease

Critical Review

Mitochondrial DNA Mutations in Human Disease

Laura C. Greaves and Robert W. TaylorMitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry,University of Newcastle upon Tyne, UK

Summary

Since their rst association with human disease in 1988, more than250 pathogenic point mutations and rearrangements of the 16.6 kbmitochondrial genome (mtDNA) have been reported in a spectrum ofclinical disorders which exhibit prominent muscle and central nervoussystem involvement. With novel mutations and disease phenotypesstill being described, mtDNA disorders are recognized collectively ascommon, inherited genetic diseases although relatively little is stillknown concerning the precise pathophysiological mechanismsthat lead to cell dysfunction and pathology. This review considersthe basic principles of mitochondrial genetics which govern boththe behaviour and investigation of pathogenic mtDNA mutationssummarizing recent advances in this area, and an assessment ofthe ongoing debate into the role of somatic mtDNA mutations inneurodegenerative disease, ageing and cancer.

IUBMB Life, 58: 143 151, 2006

Keywords mtDNA disorders; mitochondrial genetics; neurodegen-erative disease; ageing; cancer.

INTRODUCTION

Mitochondria are ubiquitous organelles found in all

nucleated cells and are the major generators of cellular ATP

by oxidative phosphorylation (OXPHOS). Electrons gener-

ated by oxidation of fat and carbohydrates are transferred to

oxygen via the redox components of the respiratory chain

(complexes I IV), forming water. Protons are pumped across

the inner membrane from the matrix to the intermembrane

space forming an electrochemical gradient which is used by the

ATP synthase (complex V) to synthesize ATP. Thirteen of the

90 or so protein subunits that make up the OXPHOS

complexes are encoded by the mitochondrial genome

(mtDNA), making this organelle the only location of

extrachromosomal DNA within mammalian cells. Human

mtDNA is a multicopy, circular double-stranded DNA

molecule of 16.6 kb which encodes 37 contiguous genes 13

polypeptides of the OXPHOS system and the necessary RNA

machinery (2 rRNAs and 22 tRNAs) for their translation

within the organelle (Fig. 1). MtDNA has no introns, the only

non-coding region being the 1.1 kb displacement (D-) loop

which contains transcriptional promoters and one of the

proposed replication origins, OH. All the remaining respira-

tory chain subunits, together with several hundred other

proteins including the entire complement of enzymes and

factors that comprise the mtDNA replisome and transcription

machinery are encoded by nuclear genes. Mitochondrial

function is therefore wholly dependent upon the coordination

and cooperation of both genomes.

Whilst this review will focus exclusively on disease-causing

mtDNA mutations, the contribution of mtDNA replication,

transcription and translation to mitochondrial disease pathol-

ogy cannot be neglected, not only in terms of their role in the

expression of specic mtDNA mutations and ensuing bio-

chemical consequences, but also as mutations of proteins

critical for the maintenance of mtDNA are increasingly

recognized as causing specic clinical disorders. Perhaps the

best example is that of the POLG1 gene, which encodes the

140-kDa catalytic (DNA polymerase and 30-50 exonucleaseactivities) subunit of mtDNA polymerase g, forming aheterodimeric enzyme together with the 55-kDa accessory

subunit, POLG2. The rst pathogenic mutations of POLG1

were identied in families with autosomal dominant chronic

progressive external ophthalmoplegia (adPEO) and an accu-

mulation of multiple mtDNA deletions was demonstrated in

aected tissues. Since then, both compound heterozygous and

homozygous POLG1 mutations have been described in

recessive PEO, adult-onset spinocerebellar ataxia with multi-

ple mtDNA deletions and Alpers syndrome due to

quantitative mtDNA depletion. POLG1 mutations have also

been linked to Parkinsonism and premature ovarian failure

(1), whilst mice expressing a mutator (proofreading-decient)

POLG1 enzyme accumulate mtDNA mutations at a higher

Received 14 February 2006; accepted 2 March 2006Address correspondence to: Dr R. W. Taylor, Mitochondrial

Research Group, School of Neurology, Neurobiology and Psychiatry,University of Newcastle upon Tyne, Newcastle upon Tyne, NE24HH, UK. Tel: 44 (0)191 2223685. Fax: 44 (0)191 2228553.E-mail: [email protected]

IUBMBLife, 58(3): 143 151, March 2006

ISSN 1521-6543 print/ISSN 1521-6551 online 2006 IUBMBDOI: 10.1080/15216540600686888

rate than normal and display features of accelerated aging

(2, 3).

BASIC MITOCHONDRIAL GENETICS

Mitochondrial DNA has dierent genetic rules that set it

apart from Mendelian genetics and which strongly dictate the

functional consequences of pathogenic mtDNA mutations (4).

These include heteroplasmy and the threshold eect, a strict

maternal pattern of inheritance and mitotic segregation.

The mitochondrial genome is polyploid, with several

hundreds or thousands of copies present in a single cell which

distribute randomly to daughter cells during cell division.

Homoplasmy describes the existence of identical mtDNA

copies within a cell and heteroplasmy is when there is a

mixture of two or more mitochondrial genotypes. The

majority of deleterious mtDNA mutations are heteroplasmic

and exhibit a threshold level of mutation load which is

required to cause a biochemical defect, and hence clinical

expression of the disease. This critical proportion of wild type:

mutated mtDNA molecules is dierent for specic mutations,

varies amongst tissues even in an individual and is demon-

strated for a number of mtDNA mutations by the

histocytochemical assessment of cytochrome c oxidase

(complex IV) activity in individual cells. Although there are

pathogenic mutations that are truly homoplasmic, the concept

Figure 1.Map of the human mitochondrial genome depicting common genotype:phenotype correlations. Human mitochondrial

DNA is a 16,569 bp circle of double stranded DNA that encodes 13 essential respiratory chain subunits: ND1-ND6, ND4L are

seven subunits of complex I (NADH:ubiquione oxidoreductase), CYT b of complex III (ubiqionol:cytochrome c

oxidoreductase), COX I COX III are the three catalytic subunits of complex IV (cytochrome c oxidase), and the ATPase6

and ATPase8 subunits of complex V (ATP synthase). Also shown are the two ribosomal RNA (12S rRNA and 16S rRNA) genes

and the twenty-two transfer RNA genes (depicted by their single letter abbreviation) required for intramitochondrial protein

synthesis. The only major non-coding regions are the 1.1 kb displacement (D-) loop that contains the origins of heavy- and light-

strand transcription and heavy-strand replication (shown as OH), and the origin of light-strand replication (shown as OL). In

addition, the sites of the common mtDNA mutations are indicated, together with their associated clinical presentations. CPEO,

chronic progressive external ophthalmoplegia; LHON, Leber hereditary optic neuropathy; MELAS, Mitochondrial myopathy,

encephalopathy, lactic acidosis and stroke-like episodes; MERRF, Myoclonic epilepsy and ragged-red bres; MIDD,

Maternally-inherited diabetes and deafness; MILS, Maternally-inherited Leigh Syndrome; NARP, Neurogenic weakness, ataxia

and retinitis pigmentosa.

144 GREAVES AND TAYLOR

of homoplasmy is rather more apparent than real as mtDNA

is constantly undergoing mutation with the clonal expansion

or loss of either point mutations or deletions; in some tissues,

such as the human colon, this occurs at a high frequency (5).

Since these mutational events occur stochastically, all acquired

mutations are present at very low levels and will only be

detectable in single cell experiments and not by the analysis of

whole tissue homogenates or blood sample.

The inheritance of the mitochondrial genome, organized

in vivo as discrete DNA-protein structures termed nucleoids,

is strictly maternal. Every mtDNA copy present in the

fertilized egg is derived from the oocyte, with no apparent

contribution from paternal mtDNA these organelles are

ubiquitinated and are selectively destroyed in the fertilized

embryo prior to the breakdown of mitochondrial structures

(6). There are challenges to this paradigm, with evidence of

low levels of paternal transmission of mtDNA in crosses

between, but not within, species, though data regarding

mtDNA recombination within the human population re-

mains contentious. A recent case of paternal mtDNA

inheritance in a patient with a sporadic mtDNA mutation

(2-bp microdeletion in the MTND2 gene) has documented

mtDNA recombination at the cellular level (7) although

studies of larger cohorts of patients with sporadic mtDNA

mutations have shown this to be a rare event (8). The genetic

advice to patients harbouring pathogenic, germline point

mutations remains rmly based on the strict maternal

transmission of mtDNA.

Mitotic segregation describes the random distribution of

mtDNA genotypes to daughter cells during cell division, and

accounts for the remarkable change in mutation load

between generations that has been documented in some

families harbouring pathogenic, heteroplasmic mtDNA

mutations. Such a phenomenon explains how patients may

develop specic clinical phenotypes over time, and together

with a number of other factors (the specic gene that is

mutated, the mutation distribution within a tissue and the

reliance of that tissue on mitochondrial-generated ATP as

energy source) gives rise to the astonishing range of diverse

clinical presentations of mtDNA disorders presenting in both

childhood and adulthood (9). Relatively little is known

about this process which is controlled by nuclear genetic

factors (10).

CLINICAL FEATURES OF mtDNA DISEASE

Disease-related mutations of mtDNA can be divided into

two groups maternally-inherited point mutations which may

be heteroplasmic or homoplasmic and aect protein, tRNA or

rRNA genes, and large-scale rearrangements (mainly single

deletions) which span several genes (Fig. 1). More than 250

pathogenic mutations have now been described (see also

MITOMAP Database, http://www.mitomap.org) associated

with an impressive array of clinical features ensuring patients

present to every clinical speciality (summarized in Table 1);

whilst muscle and brain involvement remain prominent,

patients with mtDNA disease frequently present outside of

neurology. Some classical clinical syndromes are widely

recognized and associated with specic genetic defects making

diagnosis straightforward; the most common of these are

highlighted in Fig. 1. For many patients with suspected

mtDNA disease, the clinical symptoms might be relatively

non-specic, whilst in others there is a mtDNA mutation but

aecting another nucleotide or gene, giving rise to the

widespread genetic heterogeneity that characterizes these

disorders; perhaps the best example of this is the syndrome

of MELAS (mitochondrial myopathy, encephalopathy, lactic

acidosis and stroke-like episodes) which in 480% of cases iscaused by a specic tRNA gene mutation (3243A4G intRNALeu(UUR)) yielding a range of biochemical defects. Many

other point mutations, however, in this gene, other tRNA

genes or even protein-encoding genes such as MTND5 and

MTND1 can also cause MELAS. In cases where mtDNA

disease is suspected, a diagnosis is often only made following

an approach that integrates several lines of investigation

including clinical, histochemical, biochemical and ultimately

molecular genetic tests. Such investigations continue to rely

heavily on the study of a clinically-aected tissue, often

skeletal muscle, in which characteristic, cytochemical features

such as ragged-red bres and cytochrome c oxidase decient

Table 1Common clinical features of mtDNA disease

Adult presentations

Neurological migraine, strokes, epilepsy, myopathy,

peripheral neuropathy, ataxia, sensorineural deafness

Gastrointestinal irritable bowel, dysphagia

Cardiac heart failure, heart block, cardiomyopathy

Respiratory respiratory failure, nocturnal hypoventilation

Endocrine diabetes, thyroid and parathyroid disease,

ovarian failure

Ophthalmology ophthalmoplegia, ptosis, optic atrophy,

cataracts

Paediatric presentations

Neurological epilepsy, myopathy, psychomotor retardation,

ataxia, spasticity, dystonia, sensorineural deafness

Gastrointestinal vomiting, failure to thrive, dysphagia

Cardiac hypertrophic cardiomyopathy, rhythm

abnormalities

Respiratory central hypoventilation/apnoea

Endocrine diabetes, adrenal failure

Ophthalmology optic atrophy

Haematological anaemia, pancytopenia

Renal renal tubular defects

Liver hepatic failure

MITOCHONDRIAL DNA MUTATIONS IN HUMAN DISEASE 145

bres are apparent (Fig. 2). Whole genome sequencing is now

commonplace in many diagnostic centres, with novel mtDNA

mutations still regularly being discovered, ever-widening the

clinical spectrum of disease phenotypes (11). Such an

increasing recognition of clinical presentations, together with

the development of methods (e.g., screening urinary sedi-

ments) for the non-invasive testing of patients and their

asymptomatic relatives has helped to conrm mtDNA

disorders as common genetic diseases with a minimum

prevalence of at least 1 in 5000 aected individuals in some

populations (12).

The following sections of this review consider recent

progress in understanding the role of mtDNA mutations in

all aspects of human disease including ageing and cancer.

CLASSICAL AND NON-CLASSICAL PRESENTATIONSASSOCIATED WITH mtDNA MUTATIONS

The historical classication of mitochondrial disease was a

cause of much debate between splitters and lumpers those

who predicted a tight correlation between genotype and

phenotype versus a belief that considerable overlap exists

leading to genetic and phenotypic heterogeneity. Although

there is evidence for and against both arguments, the diversity

of presentations has made it dicult to dene the impact of

mtDNA mutations on human health and as such it is helpful

to stratify mtDNA mutations into one of the following

groups: classic mtDNA syndromes (e.g., MELAS, MERRF,

CPEO, NARP and LHON as indicated in Fig. 1), clinical

syndromes with a high risk of mtDNAmutations, involvement

in common disease phenotypes and mtDNA as a predisposi-

tion for common disease (4). Well-recognized clinical

syndromes in which mtDNA mutations have recently been

demonstrated by whole mitochondrial genome sequencing

include Leigh or Leigh-like encephalopathy (recurrent MTND

and MTATP gene mutations) and exercise intolerance and

rhabdomyolysis (several dierent MTCYB or tRNA muta-

tions). Dening pathogenicity of specic base substitutions

identied by sequencing may be dicult however due to the

polymorphic nature of mtDNA and should be based on a

number of functional and structural canonical criteria (13, 14).

Misattribution of pathogenicity to a neutral mtDNA sequence

variant clearly has important ramications for families, but

this is not straightforward, especially as several homoplasmic

mutations in mitochondrial tRNA genes are now recognized

as causative (15).

The role of mtDNA mutations in common disease

phenotypes has been recognized for some time. The classic

Figure 2. Cytochrome c oxidase decient cells a marker of mitochondrial disease and ageing. The rst three panels show

transversely-orientated sections of skeletal muscle from a patient with an mtDNA-tRNA point mutation reacted for: (a)

cytochrome c oxidase cytochemical activity alone, (b) succinate dehydrogenase (entirely encoded by nuclear DNA) activity, and

(c) cytochrome c oxidase and succinate dehydrogenase activities sequentially. The latter activity stain exentuates the cytochrome

c oxidase decient (blue) bres; the asterisked bre shows clear evidence of subsarcolemmal mitochondrial accumulation, a

typical ragged-red bre. (d) transverse section of human colonic epithelium showing marked cytochrome c oxidase deciency

due to the accumulation of somatic mtDNA mutations in the stem cells. The presence of a single mtDNA mutation (7275T4CinMTCO1) in the cytochrome c oxidase decient crypts (panel e) that is absent in surrounding positive staining crypts (panel f)

is strong evidence that crypts expand throughout the colon by the process of crypt ssion (see ref. 42 for details).


example is diabetes mellitus which often presents in association

with other phenotypes including deafness and myopathy (Fig.

1). Well-characterized, causative mtDNA mutations have only

been described however in a small (0.1 3%) proportion of

diabetic populations, although this does seem to be more

prevalent in Japanese diabetics, suggesting a role for nuclear

or other mtDNA genetic inuences. Polymorphic length

variation in a poly-C tract of the mtDNA non-coding region

has been demonstrated in several, late-onset multifactorial

diseases including diabetes and was postulated to inuence

pathophysiology through subtle eects on mtDNA replication

(16) but a recent meta-analysis failed to conrm this

association (17). Further interest in this area has been sparked

by the report of a large pedigree with metabolic syndrome,

characterized by hypertension, hypomagnesaemia, hypercho-

lesterolaemia and often insulin resistance, in which the authors

documented a marked association between aected family

members and a homoplasmic mitochondrial tRNAIle muta-

tion (18).

Finally, considerable interest remains in the possibility that

mtDNA mutation as a result of increased oxidative stress

and free radical damage may either contribute to the

neuronal cell damage or genetically predispose to common

neurodegenerative diseases such as Alzheimers disease (AD),

Parkinsons disease (PD) and amyotrophic lateral sclerosis

(ALS). Whilst case-control studies have identied genuine,

heteroplasmic pathogenic mtDNA mutations causing neuro-

degenerative disease phenotypes (19), much interest has

focussed on the phylogenetic analysis of mtDNA haplotypes.

Ethnic populations can be divided into several mtDNA

haplogroups dened by specic, single nucleotide polymorph-

isms in both coding and non-coding mtDNA regions

which have become xed by discrete maternal lineages,

and it has been suggested that accumulations of these

natural genetic variants might have subtle eects on respira-

tory chain function, thus contributing to disease expression.

Recent studies of two European populations have suggested

that haplogroups J and K (20) and the haplogroup cluster

UKJT (21) were associated with a decreased risk of PD, yet a

similar association was not apparent in AD. Further

considerations for a role of mtDNA mutations in both AD

and PD are discussed in a recent review by Howell and

colleagues (22).

PATHOGENICITY: MODIFICATIONS ANDMECHANISMS

Understanding the phenotypic and genotypic diversity and

dissecting the molecular mechanisms by which mtDNA

mutations result in disease remains a signicant challenge.

For some mutations, particularly those that occur in

mitochondrial tRNA genes and impair mitochondrial protein

synthesis, the cybrid transfer of mutant mitochondria to the

control nuclear background of either osteosarcoma or lung

carcinoma r0 cells has yielded important information aboutthe precise molecular mechanisms (impairment of tRNA

stability, aminoacylation,maturationor evenpost-transcriptional

modication (23)) by which they impair cellular function.

Transmitochondrial cybrid cells have proved to be a particu-

larly useful system for the study of mtDNA disorders, not

least because the generation of mice harbouring pathogenic,

heteroplasmic mtDNA mutations has proved frustratingly

problematic.

Many dierent molecular defects result in the same

endpoint a compromised respiratory chain activity leading

to a reduction in ATP synthesis yet how can a biochemical

defect involving a single enzyme complex, say complex I, lead

to very dierent clinical phenotypes including Leigh syn-

drome, LHON and MELAS and exercise intolerance? The

tissue segregation of mutant mtDNA is an important factor,

but not relevant to diseases caused by homoplasmic mtDNA

mutations where matrilineal relatives with an identical

mtDNA mutation can exhibit marked inconsistency in disease

severity and progression. Two such examples are the

1555A4Gmutation in the small (12S) rRNA subunit causingnonsyndromic deafness and the primary MTND1, MTND4

and MTND6 LHON mutations which are usually homo-

plasmic. Both disorders show incomplete penetrance and

variable expressivity of either hearing loss (in the case of the

1555A4G mutation) or visual loss in patients with LHONassociated with the specic mtDNA mutation, which is

thought to be due to the interaction between other genetic

factors and environmental factors. For example, the pheno-

typic expression of the 11778G4A LHON mutation can beinuenced by both mtDNA haplogroup (24) and a homo-

plasmic mitochondrial tRNAMet mutation (25) whilst a

collaborative study of a large number of LHON pedigrees

to search for nuclear genetic modiers has identied an

X-chromosomal haplotype which may explain variable pene-

trance and male sex bias (26). The inuence of environmental

factors is best illustrated by patients with the 1555A4Gmutation who exhibit sensitivity to aminoglycoside antibiotics,

an interaction enhanced by a conformational change in the

secondary structure of the 12S rRNA molecule of the

ribosome.

ATP production aside, the role of mitochondria in the

generation of reactive oxygen species (ROS) and regulation

of the apoptotic cascade has prompted a closer inspection of

these pathways in patient cell lines and tissues harbouring

mtDNA mutations. Neuronal NT2 cybrid cells harbouring

pathogenic LHON mutations show increased ROS production

(27), whilst the ROS-mediated oxidative phosphorylation

defect in cybrids carrying the 8993T4G NARP mutationcan be rescued by administering various antioxidants (28). The

data concerning the eect of mtDNA mutations on apoptosis

is a little more ambiguous; there is evidence from patient

muscle biopsies and cell culture studies that certain mutations

lead to an increase in programmed cell death, but data


from the POLG1 mutator mice which accumulate mtDNA

mutations sucient to cause severe respiratory chain dysfunc-

tion showed normal levels of ROS and no increased sensitivity

to oxidative stress-induced apoptotic cell death (29).

mtDNA MUTATIONS IN AGEING

Ageing is dened as a multi-factorial decline in an

organisms physiological function, accompanied by a decline

in fertility and an increasing risk of death. DNA damage

including damge to mtDNA has been proposed to be a major

contributing factor in the ageing process as protein, RNA and

other macromolecules are rapidly turned over and therefore

are unlikely candidates for a lifelong accumulation of damage.

Given its essential role for the normal physiological function

of the cell, the mitochondrial theory of ageing predicts that the

progressive accumulation of ROS-induced, somatic mtDNA

mutations during a lifetime, leads to an inevitable decline in

mitochondrial function. ROS are produced at very low levels

during the normal process of oxidative phosphorylation, and

due to the close proximity of the respiratory chain and its lack

of protective histone protein, mtDNA is a prime target for

ROS-mediated damage. Mutations induced by ROS are

thought to impair cellular OXPHOS leading to enhanced

ROS production and further mtDNA mutation, and an

exponentially increasing load of oxidative damage and

mitochondrial dysfunction is produced (30). This results in

eventual loss of cellular and tissue function through a

combination of mechanisms including diminished energy

supplies, signalling defects and apoptosis.

An accumulation of mtDNA mutations with age was rst

shown in the early 1990s with the 4,977-bp common mtDNA

deletion being shown to accumulate to high levels in cells from

a number of post-mitotic tissues (31). This followed the

observation of cells in post mitotic tissues reecting an age-

related deciency in cytochrome c oxidase (32). More recently

studies have been carried out to see if this phenomenon occurs

in dividing cells. Colonic epithelium (5), buccal epithelium (33)

and CD34 cells from bone marrow (34) have all been shownto harbour an age-related accumulation of mtDNA mutations

and the colonic epithelium has been shown to have an

exponential age-related increase in the incidence of cyto-

chrome c oxidase decient crypts (5). These data indicate that

the mutational events are occurring in the stem cells which give

rise to these tissues. It is interesting that large-scale mtDNA

deletions have not been observed in any of the dividing cell

populations investigated so far, only mtDNA point mutations

have been reported. This suggests that perhaps there is either a

dierent mechanism of mutation accumulation in mitotic and

post-mitotic cells, or that there are dierent mechanisms of

DNA damage detection and/or repair. One thing both cell

types have in common is that there are not multiple dierent

mutations within a cell, but clonal expansion of a single

mutated genotype to high levels with in the cell. The

mechanism by which clonal expansion occurs is unknown

though mathematical modelling studies favour the theory of

random genetic drift (35).

The above data provide a correlative link between mtDNA

mutations and ageing, but in order to investigate whether there

is a causative link between mtDNA mutations and ageing mice

have been developed which have a defect in the proof-reading

portion of POLG1, the nuclear encoded catalytic subunit of

mtDNA polymerase. This knock-in mutation causes a pro-

found reduction in the exonuclease activity of the polymerase

which is necessary for proof-reading of newly synthesized

mtDNA. The mice have a greatly increased incidence of

mtDNA point mutations and deletions, and show many of the

classical features of premature ageing including osteoporosis,

reduced subcutaneous fat, reduced muscle mass and alopecia

(2, 3). Cytochrome c oxidase-decient cells were also observed

in a number of tissues (2). ROS play a central role in the

mitochondrial theory of ageing, however when ROS levels and

oxidative stress levels were measured in the mutator mice, ROS

production was found to be normal, and levels of oxidative

stress to be only slightly elevated (3, 29). This has added to the

intense debate about the exact role of ROS in ageing.

Transgenic mice have been developed which over-express

human catalase targeted to mitochondria, peroxisomes and

the nucleus (36). Mice expressing mitochondrially-targeted

catalase were found to have extended lifespan, delayed cardiac

pathology and cataract development, associated with a decrease

in the frequency of mtDNA deletions. Targeting catalase to the

nucleus or peroxisomes had no eect on extending lifespan.

Further studies on levels of ROS production and mtDNA

mutations are clearly required to attempt to elucidate the role of

these processes in ageing.

mtDNA MUTATIONS IN TUMOURS

Following the publication in 1998 of a paper by Polyak

et al. which reported that 70% of colorectal cancers have

somatic mtDNA mutations (37), there has been immense

interest in the association of mtDNA mutations with cancer.

This debate remains highly contentious with recent data

supporting both sides of the argument. A recent paper

investigating the incidence of mtDNA mutations in prostate

cancer described an increase in the number of mutations in the

MTCO1 gene in prostate cancer patients compared to controls

(12% of patients to 2% of controls harbouring mutations)

(38). These authors also introduced a pathogenic MTATP6

mtDNA mutation (8993T4G) into a prostate cancer cell lineand found that when this was injected into nude mice, tumours

were seven times larger than those without the pathogenic

mutation. A similar study constructed cybrids using a HeLa

nucleus and mtDNA containing either the 8993T4G NARPmutation or a second pathogenic MTATP6 mutation,

9176T4C (39). Similarly, it was reported that the mutantsconferred a growth advantage in the early stages of the


tumour, the authors documenting that apoptosis occurred less

frequently in mutant cybrid cultures compared to wild-type

cybrids. The decreased levels of apoptosis may be the

mechanism by which tumour growth is promoted in the

mutant cybrids.

Although there are many reports describing the association

with mtDNA mutations and tumours, there have been

criticisms of the way in which the analyses have been carried

out. A recent paper published by Bandelt and colleagues in

PLoS Medicine reassessed many of these studies (40). They

concluded that many of the authors appear to have scant

knowledge of human phylogenies and that when numerous

mtDNA sequence changes between the tumour and control

sequence are documented, often these variants are present on a

haplogroup other than that of the patient, and are therefore

indicative of sample contamination. A second point is that

although somatic mtDNAmutations do exist in many dierent

tumours, there is little evidence to suggest that they are a cause

of tumour formation; perhaps they are present in the normal

tissue prior to tumourigenesis. In epithelial tissues such as the

colonic (5) and buccal (33) epithelium, there are high levels of

somatic mtDNA mutations in normal aged human tissue,

suggesting that in those cancers the mutations are likely to be

passive bystanders carried along during the formation of the

tumour. However this might prove to be useful in deciphering

the mechanism by which tumours form. In the development of

colorectal cancer, which is considered to be a disease of colonic

stem cells, there is controversy concerning the mechanism by

which the tumour spreads following the initial transformation

event. Crypt ssion is a process by which one crypt divides to

form 2 daughter crypts and as such, the colon is thought to be

composed of patches of genotypically-related crypts. This

process has been proposed to be an important part of the

mechanism by which tumours spread (41). Greaves et al. used

cytochrome c oxidase deciency to identify crypts which might

be related based on their mitochondrial phenotype. They

sequenced the mitochondrial genome of two small patches (2

and 3 crypts) of cytochrome c oxidase decient crypts and

adjacent cytochrome c oxidase positive crypts, showing that

each patch of decient crypts contained a somatic mtDNA

mutation which was not present in adjacent positive crypts or

in bulk homogenate DNA from that patient (see Fig. 2).

Subsequent sequencing of a pair of cytochrome c oxidase

decient crypts in the process of ssion revealed these to have a

somatic mtDNA mutation not present in adjacent cytochrome

c oxidase positive crypts. These data predict that an mtDNA

mutation originating in the stem cell of a colonic crypt can go

on to occupy the whole crypt, and that mutated clones expand

by crypt ssion (42).

CONCLUSION

Although pathogenic mtDNA mutations were rst

described nearly 20 years ago, their rightful impact on human

health is only now becoming apparent. Despite the availability

of cell culture and some animal models of disease, surprisingly

little is known of the pathophysiological mechanisms by which

mtDNA mutations, through respiratory chain deciency,

cause cell dysfunction and death. Sadly, eective treatment

strategies for these disorders remain lacking despite many

novel experimental approaches to the problems associated

with providing cures (43). Current clinical practice for patients

and their families is based upon supportive measures and

genetic counselling, yet given the inherent diculties in

treating these disorders, a dierent line of attack, the

prevention of mtDNA disease transmission, is being actively

pursued. Preimplantation genetic diagnosis (44) and pro-

nuclear transfer between single cell embryos (45) are two such

avenues, and whilst there are considerable ethical and moral

issues to be considered, clearly developing techniques to

improve the chances of a mother with mtDNA disease having

a healthy child is a major priority for families, scientists and

clinicians alike (46).

ACKNOWLEDGEMENTS

We thank the Wellcome Trust, Muscular Dystrophy Cam-

paign and Newcastle upon Tyne NHS Hospitals Trust for

their continuing nancial support of this work.

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