Mitochondrial genome and human mitochondrial diseases

ISSN 0026�8933, Molecular Biology, 2010, Vol. 44, No. 5, pp. 665–681. © Pleiades Publishing, Inc., 2010.Original Russian Text © I.O. Mazunin, N.V. Volodko, E.B. Starikovskaya, R.I. Sukernik, 2010, published in Molekulyarnaya Biologiya, 2010, Vol. 44, No. 5, pp. 755–772.

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INTRODUCTION

Mitochondria play many roles in the cells, but theirmost important function is to generate energy via oxi�dative phosphorylation (OP). In contrast to otherorganelles, mitochondria have their own DNA(mtDNA), which codes for several subunits of thecomplexes involved in OP. Mutations arising inmtDNA may distort energy production and, eventu�ally, cause cell death. Such defects of highly differenti�ated cells of various human tissues and organs lead tovarious medical conditions [1–3]. It was known for along time that defects in energy (ATP) productioncause certain neuromuscular syndromes [4, 5], but thecause� and� effect relations between known disor�ders/syndromes and mutations of the mtDNA codingregion was established far more recently [6–9]. Mito�chondrial disease affects approximately 1 in 10000adults in the global population.

The review considers the modern views of thestructure and organization of the mitochondrialgenome and the molecular mechanisms of mitochon�drial disorders due to mtDNA mutations. In addition,we compare the molecular methods used to detectmtDNA mutations and experimental strategies aimedat correcting OXPHOS defects. Finally, the means toprevent inheritance of mtDNA mutations are dis�cussed, since this is a pressing problem of mitochon�drial medicine and, in particular, medical geneticcounseling.

THE STRUCTURE OF THE MITOCHONDRIAL GENOME

Human mtDNA is a 16568�bp circular double�stranded molecule and harbors 37 genes, which areinvolved in energy production taking place in mito�chondrial respiratory chain. The gene set includes13 structural genes, which code for subunits of theOXPHOS complexes, and genes for 22 tRNAs and2 rRNAs, which are involved in protein synthesisdirectly in mitochondria. The majority of regulatoryregions occur in a noncoding region, which is known asthe control region (CR) and is 1122 bp in size. DuringmtDNA replication, the CR forms a 710�bp triple�stranded region the so�called displacement loop (D�loop).The coding sequence accounts for a major part of themitochondrial genome, and only 87 bp correspond tointercistron regions within this sequence. The CR har�bors heavy�strand (HSP1 and HSP2) and light�strand(LSP) promoters and a heavy�strand replication origin(OH). A light�strand replication (OL) origin is outsidethe CR. The mtDNA strands are characterized by anasymmetric distribution of G/C pairs. The G�richheavy strand contains both of the rRNA genes, 12structural genes, and 14 tRNA genes. The remainingeight tRNA genes and one structural gene (ND6)occur in the light strand (Fig. 1a) [12].

Although human mtDNA and prokaryotic DNAshare certain structural features (for instance, lack ofintrons and overlapping genes), the structural organi�zation of the mitochondrial genome is far more intri�cate [13]. It was found that five to seven mtDNA mol�

Mitochondrial Genome and Human Mitochondrial DiseasesI. O. Mazunin, N. V. Volodko, E. B. Starikovskaya, and R. I. Sukernik

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia;

e�mail: [email protected] March 25, 2010

Accepted for publication April 8, 2010

Abstract—Today there are described more than 400 point mutations and more than hundred of structuralrearrangements of mitochondrial DNA associated with characteristic neuromuscular and other mitochon�drial syndromes, from lethal in the neonatal period of life to the disease with late onset. The defects of oxida�tive phosphorylation are the main reasons of mitochondrial disease development. Phenotypic diversity andphenomenon of heteroplasmy are the hallmark of mitochondrial human diseases. It is necessary to assess theamount of mutant mtDNA accurately, since the level of heteroplasmy largely determines the phenotypicmanifestation. In spite of tremendous progress in mitochondrial biology since the cause�and�effect relationsbetween mtDNA mutation and the human diseases was established over 20 years ago, there is still no cure formitochondrial diseases.

DOI: 10.1134/S0026893310050018

Key words: mitochondrial genome, oxidative phosphorylation, mtDNA mutations, heteroplasmy, mitochon�drial diseases, mitochondrial respiratory chain defect therapy

REVIEWS

UDC 576.311.347

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HSP OH

D�loop

T

Cytb

PLSP

E

ND6

ND5

LS

H

ND4

RND3G

COIIIATP6ATP8K

COII

D

COI

W

ND2

MI

ND1

L

Q

OL

ANCY

16S

V12S F

S

ND4L

Matrix

Inner membrane

Intermembrane space

succinate fumarate

Subunits

mtDNA genes:

nDNA genes:

Complex I Complex II Complex III Complex IV Complex V

ND2ND3

ND4LND4

ND5

ND6

H+

e–CoQ

Cytb

Cytc

COICOII

COIII

H2O ADP ATP

7

~39

0

4

1

10

3

10

2

~14

ND1

H+ H+H+

e–e– e–

O2

(a)

(b)

ATP8

ATP6

Fig. 1. (a) Map of the human mitochondrial genome and (b) scheme of oxidative phosphorylation. The genome includes37 genes, of which 13 (ND1�ND6, ND4L, Cytb, COI�COIII, ATP6, and ATP8) code for subunits of the OXPHOS complexes, 2code for rRNAs (12S and 16S), and 22 (designated with Latin capitals) code for tRNAs. The D�loop is a triple�stranded segmentof the mtDNA control region that forms during replication; the control region additionally harbors transcription initiation sitesfor the heavy (HSP) and light (LSP) strands and a heavy�strand translation initiation site (OH). A light�strand translation initia�tion site (OL) is outside the control region. The OXPHOS system includes five complexes. Complex I consists of 46 subunits, ofwhich 7 are encoded by mtDNA and 39 are encoded by nDNA. Complex II consists of four subunits (nDNA). Complex III con�sists of 11 subunits (one encoded by mtDNA and ten encoded by nDNA). Complex IV consists of 13 subunits (three encoded bymtDNA and ten encoded by nDNA). Complex V consists of 16 subunits (2 encoded by mtDNA and 14 encoded by nDNA). Inaddition, there are two electron transporters, CoQ and Cytc. As electrons move along the respiratory chain, protons are pumpedfrom the matrix into the intermembrane space by complexes I, III, and IV and then return to the matrix through complex V. Com�plex V synthesizes ATP from ADP and inorganic phosphate owing to Ψp. Adapted from [2].

ecules of somatic cells form nucleoids, which includehistone�like proteins and proteins involved in tran�scriptional regulation and replication of mtDNA; themajor proteins are mtSSB, POLG, TFAM, and Twin�kle [13–16]. Nucleoids interact with the inner mito�chondrial membrane via the proteins that specificallybind to the mtDNA CR (presumably, with the D�loop) and the inner mitochondrial membrane, thusuniting and stabilizing several mtDNA molecules [17,18]. It is thought that nucleoids have a multilayerstructure with replication and transcription taking

place in the central region and RNA processing andtranslation occurring at the periphery [16]. The nucle�oid organization probably protects mtDNA fromdamage, and the relative arrangement of mtDNAmolecules within a nucleoid facilitates repair via geneconversion. In addition, a nucleoid is considered to bea main unit of mtDNA segregation [15].

There is evidence that mtDNA exchange betweenindividual nucleoids is extremely rare [19], whichindirectly supports a faithful nucleoid hypothesis [20].According to an alternative dynamic nucleoid hypoth�

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esis, mtDNA moves between nucleoids freely withsubsequent recombination [21].

SPECIFICS OF mtDNA REPLICATION, TRANSCRIPTION, AND TRANSLATION

Two possible models of mtDNA replication are dis�cussed now. According to one model, replication fol�lows a conventional asynchronous mechanism, startsin OH, and proceeds along the heavy strand up to OL;then, the light strand is replicated in the oppositedirection [22, 23]. The alternative model suggests thatreplication similarly starts at OH, but the two strandsare synthesized simultaneously [24, 25]. One oranother replication mechanism is utilized dependingon the cell state. The synchronous mechanism ofmtDNA replication is probably used at the steady�state conditions and is switched to the asynchronousone when the number of mitochondria should beincreased rapidly [24, 25]. Replication is known toinvolve nuclear DNA (nDNA)�encoded proteins:mitochondrial DNA polymerase (POLG), helicase(Twinkle), and single�stranded DNA�binding protein(mtSSB) [26, 27].

Transcription of mtDNA starts from two promoterson the heavy strand (HSP1 and HSP2) and from onepromoter on the light strand (LSP). Transcriptionfrom LSP yields a polycistronic RNA, which consistsof eight tRNAs and one mRNA for an ND6 subunit.The transcripts synthesized from HSP1 and HSP2include the 14 other tRNAs, 2 rRNAs, and 12mRNAs. The amount of the transcript including tworRNAs and two tRNAs (short transcript synthesizedfrom HSP1) is one order of magnitude higher thanthat of the other transcript. Maturation of individualmRNAs has its specifics: the mRNAs are excised fromthe polycistronic transcript via recognition of second�ary structures in the tRNAs whose genes are betweenthe structural genes [28]. Polyadenylation is a key stepin the expression of mitochondrial mRNAs, sincepolyadenylation provides certain mRNAs with a stopcodon (UAA), which is absent from pre�mRNA [29].The main proteins of the transcription machineryinclude mitochondrial RNA polymerase (POLRMT);mitochondrial transcriptional activators A (TFAM)B1 (TFB1M), and B2 (TFB2M); and a transcriptiontermination factor (mTERF) [30–32].

The mtDNA�encoded proteins are synthesized inthe matrix on mitochondrial ribosomes (mitoribo�somes), which have fewer rRNAs and more ribosomalproteins as compared with bacterial or eukaryoticribosomes. The translation machinery of human mito�chondria includes two initiation factors (IF2 and IF3),three elongation factors (EFG, EFTs, and EFTu), andat least one termination factor (mtRF1). Some of thespecifics of mitochondrial translation are that a uniquegenetic code is used, there are 22 tRNAs, and mRNAslack caps, which are necessary for mRNA to recognizethe binding site on the ribosome [33–35].

OXIDATIVE PHOSPHORYLATION

OXPHOS is one of the fundamental metabolicreactions, and it takes place on the inner mitochon�drial membrane. The reaction consists in a coupling ofelectron transport with ATP production. TheOXPHOS system includes five protein complexes,each consisting of several subunits (Fig. 1b). Ineukaryotes, electrons are transported via the respira�tory chain from NADH through complex I (NADH�coenzyme Q reductase) or from succinate throughcomplex II (succinate�CoQ reductase) and then, con�secutively, through the integral membrane electrontransporter CoQ, complex III (ubiquinol�cytochromec reductase), the electron transporter cytochrome c(Cytc), and complex IV (cytochrome c oxidase) tomolecular oxygen. [39–42]. This creates an electro�chemical potential difference (Ψp, a proton gradient)across the inner membrane. The energy stored in theform of Ψp is utilized by complex V (ATP synthase).As protons transported back into the matrix throughthe proton channel (the Fо–subunit of ATP synthase),ADP is phosphorylated with inorganic phosphate toyield ATP [42]. Substrate oxidation and oxygen reduc�tion are, thus, coupled with ATP production.

The OXPHOS complexes were found to driftwithin the inner membrane as a single molecularsupercomplex termed the respirasome, rather than asindividual structures [43–45]. The proportion of thecomplexes in the respirasome is probably species spe�cific [46–49]. It is clear that a true respirasome, whichis capable of transferring electrons from NADH tomolecular oxygen, is a supercomplex that includescomplexes I, II, III, and IV and the specific transport�ers CoQ and Cytc [50]. An ATP synthasome washypothesized to include complex V, the inorganicphosphate transporter, and adenine nucleotide trans�locase (ANT) at a 1 : 1 : 1 ratio [51, 52]. However, thereis evidence that these components function indepen�dently [53].

Although the Mitchell theory is commonlyaccepted [54], the mechanism of proton transfer fromthe matrix into the intermembrane space is still poorlyunderstood, and the structures of the complexes thatare involved in this process were not identified. How�ever, a comparison of the OXPHOS complexes for dif�ferent species showed that proton and electron trans�ports involve the mtDNA�encoded subunits.

In addition to producing ATP, OXPHOS acts as anendogenous source of reactive oxygen species (ROS),including (superoxide), Н2О2 (hydrogen perox�

ide), and ОН– (hydroxyl radical) [55–57]. Superoxideforms mostly in complexes I and III [39, 58, 59].Mitochondrial Mn�dependent superoxide dismutaseor Cu–Zn�dependent superoxide dismutase convert

to H2O2, which is converted to H2O by glutathioneperoxidase. In addition, H2O2 may be converted toOH– in the presence of Fe2+ and Сu2+. is capable

O2–

O2–

O2–

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of reacting with NO (nitric oxide), which is producedin mitochondria endogenously by mitochondrial NOsynthase [60, 61]. The reaction yields ONOO– (perox�ynitrite). Complex IV was shown to play a role in theproduction of reactive nitrogen species [56]. ChronicROS exposure of the cell leads to oxidative damage toproteins, lipids, and nucleic acids, while acute ROSexposure inactivates the Fe–S centers of the enzy�matic OXPHOS complexes and aconitase, which isinvolved in the tricarboxylic acid (TCA) cycle [62],thus, decreasing the ATP production [63, 64]. Highlyreactive ONOO– nitrifies tyrosine residues in sur�rounding proteins, damaging complex I and mito�chondrial superoxide dismutase [65, 66]. In addition,complex I may be nitrosylated at the sulfhydryl groups,which inhibits its activity [67]. The effect of ROS onmtDNA leads to accumulation of multiple mutations,decreases the OXPHOS rate, and further stimulatesROS accumulation. Taken together, these alterationseventually distort the cell function and cause pro�grammed cell death, or apoptosis [68–70].

PATHOGENIC mtDNA MUTATIONS AND MITOCHONDRIAL DISEASES

The mutation rate of mtDNA is approximately 17�fold higher than in nDNA [71]. This is determined bya set of factors, including the specific structural orga�nization of the mitochondrial genome, the functionalstate of ribonucleotide reductase, replication errors,and mutations of the nuclear genes, whose proteinproducts function in mitochondria. The greatest con�tribution is made by ROS [72]. The pathway from amutation arising in mtDNA to the clinical manifesta�tion of disease is poorly understood. It is thought thatmtDNA mutations lead to ROS accumulation,change calcium metabolism, activate mitochondrialpermeability transition pores (mtPTPs), and, eventu�ally, trigger apoptosis. This scenario is probably char�acteristic of neurodegenerative processes due tomtDNA mutations [73, 74].

To date, mitochondrial diseases have been wellcharacterized both clinically and biochemically [75–80]. However, a molecular analysis of mtDNA is nec�essary for diagnosing the disease and evaluating theprognosis for affected patients and the risk for healthycarriers. Diseases are commonly described using aclassification based on the mtDNA region affected bya mutation. Accordingly, pathogenic mtDNA muta�tions are divided into (1) mutations of the structuralgenes, (2) mutations of the rRNA and tRNA genes,and (3) structural rearrangements involving largemtDNA segments.

Pathogenic Mutations of the Structural Genes

Pathogenic mutations that change the nucleotidesequences of the mtDNA structural genes are divided

into four groups depending on the affected OXPHOScomplex.

Mutations of the mitochondrial genes for complex I.The greatest number of pathogenic mutations wereidentified in the structural genes of complex I [81, 82].As of February 9, 2010, the MITOMAP database [10]contained data on a total of 110 pathogenic mutations,including 33 mutations of ND1, 12 of ND2, 6 of ND3,5 of ND4L, 14 of ND4, 22 of ND5, and 18 of ND6.

Leber’s hereditary optic neuropathy (LHON)(atrophy) is the most common mitochondrial diseaseand is due to mutations of mtDNA structural genes;the ND genes are affected in most cases (Table 1).Clinically, LHON is characterized by degeneration ofthe retinal ganglion cell layer and atrophy of the opticnerve. Approximately 95% of LHON cases in the Cau�casian population are due to three primary risk muta�tions: A3460G (ND1), G11778A (ND4), andT14484C (ND6). Many rare LHON�associated muta�tions were identified in the mtDNA genes, and theirnumber is continuously increasing [83, 84]. LHON isone of a few mitochondrial diseases where the expres�sion of a pathogenic mutation was correlated with acertain phyletic lineage (mtDNA haplogroup). Thus,the G11778A and T14484C mutations are often asso�ciated with haplogroup J, while the G3460A mutationis associated with haplogroup K [85, 86]. In particular,we found that the G3460A mutation identified inSiberia is associated with haplogroups deriving frommacrohaplogroup M, which occurs at the highest fre�quency in indigenous Siberian populations (Altaians,Tuvinians, and Buryats), while the G11778A mutationis expressed when occurring in haplogroups of clusterTJ according to published data [85, 87].

Leigh syndrome (LS) is another common diseaseassociated with a mutation of the ND genes. LS is aprogressive neurodegenerative condition that affectsthe brainstem and basal ganglia and is accompanied bythe formation of characteristic symmetrical necroticchanges. Similar symptoms are caused by mutations ofCOIII, ATP6, and certain tRNA genes [88].

Mutations of the mitochondrial genes for complexIII. In total, 29 pathogenic mutations were identifiedin the Cytb gene; these mutations usually cause myop�athy [10, 81]. In addition, Cytb mutations are associ�ated with encephalomyopathy, cardiomyopathy, tubu�lopathy, and LHON [89–91].

Mutations of mitochondrial genes for complex IV.At present, there are 33 known pathogenic mutationsof COI, 14 mutations of COII, and 13 mutations ofCOIII [10]. Most patients with mutations of thesegenes develop neuromuscular syndromes, while cer�tain mutations are associated with LHON and sen�sorineural hearing loss (SNHL) [91, 92], and cer�tain COI mutations are associated with prostatecancer [93].

Mutations of mitochondrial genes for complex V. Atotal of 19 pathogenic mutations were identified in

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ATP6, which codes for an ATP synthase subunit [10].Only one mutation, A8381G, was identified in theATP8 gene, and this mutation leads to maternallyinherited diabetes mellitus and sensorineural deafness(MIDD) [94].

The most common disorder associated with theT8993G mutation of ATP6 is a complex of symptomsincluding neuropathy, ataxia, and retinitis pigmentosa(NARP). It is of interest that the T8993G mutation isexpressed as NARP when mutant mtDNA accountsfor 70–90% of total cell mtDNA and as maternallyinherited Leigh syndrome (MILS) when the propor�tion is 90–95%. Similar syndromes are associated withthe mutations T8993C, T9176G, and T9176C [95].The T8993G and T8993C pathogenic mutations,which change highly conserved Leu156 to Pro or Arg,respectively, decrease the proton flow through ATPsynthase by 30% [96]. The pathogeny of the diseasemay be affected by a certain mitochondrial haplo�group [97].

Pathogenic Mutations of the rRNA and tRNA Genes

Mutations of the genes for rRNAs and tRNAsinvolved in protein biosynthesis in mitochondria maycause several mitochondrial diseases [10].

Pathogenic mutations of the rRNA genes. In total,16 pathogenic mutations were found to alter the struc�ture of the 12S rRNA; no mutation leading to diseasewas identified in the 16S rRNA gene.

The G1555A transition is the most common inrRNA and is phenotypically expressed as SNHL. Themutation affects the highly conserved region of the12S rRNA, which is a component of the small riboso�mal subunit. The mutation changes the aminoglyco�side�binding site of the 12S rRNA, and the patientsare, consequently, sensitive to ototoxic aminoglyco�sides [98]. All other pathogenic mutations of the 12SrRNA gene also lead to SNHL [99].

Pathogenic mutations of the tRNA genes. Approxi�mately two�thirds (166) of the pathogenic point muta�tions of mtDNA occur in the tRNA genes. Mutationsaffecting various tRNAs are expressed as various clin�ical syndromes. The most common mutations affectthe tRNALeu and tRNALys genes.

The A3243G mutation is diagnosed in approxi�mately 80% of cases of mitochondrial encephalopathywith stroke�like episodes and lactic acidosis(MELAS). This transition affects the tertiary structuretRNALys, as well as methylation, acetylation, and tau�rine modification of the anticodon, thus distortingtranslation [100]. It is of interest that the A3243Gmutation is usually heteroplasmic. The proportion ofmutant and wild�type mtDNAs greatly varies amongtissues; the portion of mutant mtDNA is the highest inmuscle tissue and brain cells and the lowest in bloodleukocytes [100, 101]. The mutant mtDNA portionmay increase with age in all tissues except blood cells,which is apparently due to specific selection [102].Apart from MELAS, the disorders associated with the

Table 1. Pathogenic point mutations of mtDNA

Disease

Pathogenic mutations of structural genes Pathogenic mutations of rRNA and tRNA genes

complex I (ND genes)

complex III (Cytb)

complex IV (CO genes)

complex V (ATP6 and ATP8) rRNA tRNA

LHON 43 6 8 4 – 3

LS 13 – 1 4 – 5

NARP/MILS – – – 5 – –

SNHL/DEAF 1 1 6 – 15 10

MELAS 14 1 2 1 15

MERRF – – – – – 5

KSS – – – – – 3

CPEO 1 – – – – 17

Other 39 21 43 7 4 112

Total mutations 111 29 60 20 20 170

Note: ND—NADH dehydrogenase; Cytb—ubiquinol–cytochrome c oxidoreductase; CO—cytochrome c oxidase; ATP—ATP synthase;LHON—Leber’s hereditary optic neuropathy (atrophy); LS—Leigh syndrome; NARP/MILS—neuropathy, ataxia, retinitis pigmen�tosa/maternally inherited Leigh syndrome; SNHL/DEAF—sensorineural hearing loss/aminoglycoside�induced deafness; MELAS—mitochondrial encephalopathy with stroke�like episodes and lactic acidosis; MERRF—myoclonic epilepsy with ragged�red fibers;KSS—Kearns–Sayre syndrome; CPEO—chronic progressive external ophthalmoplegia.

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A3243G mutation include myoclonic epilepsy withragged�red fibers (MERRF), chronic progressiveexternal ophthalmoplegia (CPEO), Kearns–Sayresyndrome (KSS), SNHL, and LS [100].

Another common mutation is the A8344G transi�tion in the tRNALys gene; the mutation is associatedwith MERRF in 80% of cases [103]. The mutationaffects a highly conserved nucleotide in the pseudouri�dine loop of tRNA, thus blocking mitochondrial proteinsynthesis. The disease is not expressed phenotypicallyuntil the heteroplasmy level reaches 85–90% [104].

Structural Rearrangements Involving Large mtDNA Segments

Deletions of mtDNA underlie certain mitochon�drial diseases and, probably, play a key role in the agingof postmitotic tissues. Two models of the origin ofmtDNA deletions are considered now [105]. Onemodel suggests that deletions arise during mtDNAreplication via the asynchronous mechanism. Theother model postulates that deletions form duringdouble�strand DNA repair in mtDNA. Deletions usu�ally arise sporadically and are not transmitted to thenext generation [106].

An extended 4977�bp mtDNA deletion (region8488–13 460) is considered to be the most commoncause of KSS, which is characterized by progressiveexternal ophthalmoplegia, retinitis pigmentosa, andearly onset (until 20 years of age) [107].

The main cause of CPEO is either one extendeddeletion or multiple short deletions. CPEO is charac�terized by progressive paralysis of the oculomotormuscles, leading to impaired eye mobility and ptosis[108].

Pearson’s syndrome (PS) is a rare disease thatoccurs in early childhood and is characterized by sid�eroblastic anemia, pancytopenia, and exocrine insuf�ficiency of the pancreas. The disease is extremelysevere and early leads to a fatal outcome; survivingpatients develop the clinical symptoms of KSS. All tis�sues and organs usually contain a large portion ofmtDNA with deletions in these syndromes [109, 110].

Each of the three above syndromes is associatedwith mtDNA deletions of certain sizes and localiza�tions, which should be considered in diagnosis [111].

PATHOGENIC MUTATIONS OF nDNA AND MITOCHONDRIAL DISEASES

Since approximately 2000 genes of the nucleargenome are involved in the biogenesis of mitochondria[112, 113], it is clear that nDNA lesions may alsocause mitochondrial diseases. Defects in nDNA arefar more diverse than mtDNA defects and includemutations of the genes involved in OP, the mitochon�drial protein synthesis machinery, mitochondrialimport and export, movements of mitochondria, theirfusion and division, mtDNA transcription and repli�

cation, various enzymatic cycles (TCA cycle and fattyacid β�oxidation), and other metabolic pathwaysrelated to the function of mitochondria [82, 114, 115].These nDNA defects and the associated mitochon�drial diseases, which clinically differ from the classicalmitochondrial diseases, are beyond the scope of thisreview.

FACTORS AFFECTING THE MANIFESTATION OF MITOCHONDRIAL DISEASES

The observed diversity of clinical symptoms inmitochondrial diseases is due to several factors, suchas heteroplasmy, a threshold effect, and a bottleneckeffect.

The multiplicity of mtDNA copies in the cell oftenleads to heteroplasmy, where several mtDNA variantsoccur in one mitochondrion, one cell, or one organ incontrast to homoplasmy, where all mtDNA copies areidentical [116]. When the cell is divided, mitochondriaare distributed between the daughter cells at randomvia mitotic segregation, and the daughter cells mayconsequently differ in the level of heteroplasmy [117].The rate of a shift towards mutant or wild�typemtDNA in daughter (somatic) cells presumablydepends on the nucleoid composition of the parentalcell. Both mutant and wild�type mtDNAs may appearin one nucleoid (a heteroplasmic nucleoid) or in dif�ferent nucleodis (homoplasmic nucleoids). When amaternal cell contains heteroplasmic nucleoids theheteroplasmy level changes insignificantly in thedaughter cells. When maternal nucleoids arehomoplasmic, the heteroplasmy level may dramati�cally differ between the daughter cells and depends onselection and genetic drift [118, 119].

The heteroplasmy level of a pathogenic mtDNAmutation usually determines the severity of the mito�chondrial disease [120]. The disease does not manifestitself until the mutant mtDNA portion increases overa certain level; the phenomenon is known as thethreshold effect. For instance, the threshold portion ofmtDNA with the A8344G mutation is 85–90% in thecase of MERRF. The T8993G mutation leads to onedisease (NARP) when its content (heteroplasmy level)reaches 70–90% and another disease (MILS) at ahigher (90–95%) heteroplasmy level [121].

With a few exceptions, mammalian mtDNA isinherited maternally. Mature eggs contain at least100000 mtDNA copies, approximately one or twocopies per mitochondrion [122–124]. In spite of themultiplicity of mtDNA copies in the egg, new mtDNAvariants may appear as early as the next generation.Rapid segregation of new mtDNA variants (D�loopmutations) in cattle occurred within a few generations[125]. Based on these findings, a bottleneck wasassumed for one of the egg development stages(Fig. 2). Further ultrastructural studies showed thatfertilization is followed by a number of zygote divi�sions without a division of mitochondria (and, accord�

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ingly, without mtDNA replication); i.e., the mito�chondrial pool is halved at each cell division. Forinstance, a mouse primary germline cell containsapproximately 10 mitochondria [126, 127]. Thus, whenprecursor germline cells form, their mitochondriaaccount for only a minor portion (0.01%) of the initialmitochondrial pool of the zygote [128–130]. It isthought that the mitochondrial pool characteristic of amature egg is restored owing to only some of the mito�chondrial subpopulations of primordial follicles [131].

Since the number of mitochondria characteristic ofa mature egg originates from a highly limited mito�chondrial pool of primary germline cells; newlyformed mitochondria are homogeneous (or almosthomogeneous) in composition. In other words, a fun�damental evolutionary effect of the genetic bottleneckis to maintain mtDNA homoplasmy and to minimizeheteroplasmy [132].

DETECTION OF mtDNA MUTATIONS AND ESTIMATION OF THE HETEROPLASMY

LEVEL

Since the level of heteroplasmy is known to deter�mine the phenotypic expression of a mutation to asubstantial extent, it is necessary to estimate the pro�portion of mutant mtDNA when performing a molec�ular analysis. It should be noted that estimation of theheteroplasmy level already includes the identificationof a mutation, while the methods used to detect amutation fail to estimate its heteroplasmy in somecases.

The main methods used to estimate the hetero�plasmy level for mtDNA mutations are summarized inTable 2. Cloning, which produces reliable quantitativeresults, is the most laborious and time consuming.Fluorescent PCR yields more accurate results and isless laborious, but small deletions or insertions remainundetectable in this case. Denaturing high�resolutionliquid chromatography yields reproducible results forall types of heteroplasmic mutations (deletions, inser�tions, and point mutations). This method estimatesthe heteroplasmy level more accurately than cloningand fluorescent PCR [133]. Real�time PCR was alsoemployed in detecting and quantifying mtDNA muta�tions, and excellent results were obtained with bothhydrolysable probes (TaqMan) and the intercalatingdye SYBR [134]. Molecular beacons were proposedfor detecting mtDNA mutations and estimating thelevel of their heteroplasmy [135]. Modification of theTaqMan system to utilize specific primers was used toestimate the heteroplasmy level for the A3243G muta�tion [136]. A comparison of three methods estimatingthe heteroplasmy level—DNA sequencing, Southernblot hybridization, and a combination of the peptidenucleic acid (PNA) technique with real�time PCR—showed that PNA/real�time PCR more accurately(quantitatively) distinguishes mutant and wild�typemtDNAs as compared with sequencing, while South�

ern blotting fails to reveal the actual level of hetero�plasmy [137]. The most accurate estimates are pro�duced by three methods: SNaPshot [138], pyrose�quencing [139], and Biplex Invader [140]. While theyare comparable in accuracy, Biplex Invader is the sim�plest to use and SNaPshot is the most expensive [141].As detection of mtDNA mutations becomes a routinetest, preference is given to microarray techniques,which make it possible to simultaneously test manymtDNA samples for the most common pathogenicmutations and to establish the level of heteroplasmyfor each individual mutation [142, 143].

THERAPY OF RESPIRATORY CHAIN DEFECTS

Mitochondrial diseases are still incurable. Symp�tomatic therapies used in medicine include the use ofmedications, special diets [144–149], and physicalexercise [150–153]. Certain pathologies due tomtDNA mutations are corrected surgically. Forinstance, cochlear implants are used for sensorineuralhearing loss in MELAS, SNHL, and KSS, and a pace�

Number of mitochondria per cell

100000

1000

10

200

5000

Fertilizedovicell

Blastocyst

Primarygermcells

Oogonia

Primordialfollicle

Matureovicell

Bottleneck

100000

50

20000

4000

400

Fig. 2. Changes in the number of mitochondria duringfemale germline cell development in mice. The number ofmitochondria is indicated for each developmental stage. Abottleneck is observed at the stage of primary germline celldevelopment. An approximate number of germline cells inmice is indicated on the right. Adapted from [127].

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maker is implanted to correct alterations in cardiacconduction in KSS [146, 153, 154].

Experimental methods (Table 3) aimed at correct�ing the respiratory chain defects by affecting thegenetic apparatus of mitochondria are developed, buttheir clinical use is hardly possible in the nearest future[155]. The main strategies used to correct the defectsin the mitochondrial respiratory chain are consideredbelow.

An allotopic expression strategy is based on gener�ation of a genetic construct carrying the wild�typecopy of a mitochondrial gene. Cells are transformedwith the construct, and the wild�type mitochondrialgene is expressed after construct integration into thenuclear genome. However, certain mitochondrial

genes cannot be expressed in this manner [156]. Todate, similar procedures have been performed with celllines to correct the defects in ND1, ND4, and ATP6 [9,157–162].

A xenotopic expression strategy implies the use ofthe genes for subunits of the OXPHOS complexesfrom other organisms. For instance, yeast NADH oxi�dase (Ndi1) was used to compensate for a defect incomplex I in mammalian cells [163–165]. The ascid�ian cyanide�insensitive alternative oxidase (AOX) genewas delivered into the nucleus and expressed to correctcomplex I defects [166]. Theoretically, alternativeOXPHOS complexes may functionally compensate fora defective complex regardless of the mutation thatdistorts the function of the complex. However, themajority of alternative complexes are incapable ofpumping protons from the matrix into the intermem�brane space [38].

Although the possibility of tRNA import into mito�chondria is questionable (tRNAs are normally synthe�sized in mitochondria rather than imported), experi�ments in the field are in progress [167–169]. Forinstance, the Leishmania tropica tRNA import complex(RIC) was successfully used to deliver tRNALys intomitochondria, thus compensating for a translationaldefect, and to restore cell respiration [170, 171].Pathogenic tRNA mutations were compensated for viamodification or overexpression of the correspondingaminoacyl�tRNA synthetases [172–174].

A strategy aimed at changing the heteroplasmylevel utilizes molecular constructs that specificallybind to a certain mtDNA nucleotide sequence toblock its transcription and/or replication. A hetero�plasmy level may be shifted towards wild�type mtDNAwith restriction endonucleases that recognize certainsites generated by the mutation and specifically cleavemutant mtDNA [175]. It is of interest that the recog�nition site for a particular restriction endonuclease is

Table 2. Methods for detecting heteroplasmy of mtDNA mu�tations

Method Reference

Cloning [133]

Fluorescent PCR [133]

Denaturing high�resolution liquid chroma�tography

[133, 214]

High�resolution melting analysis [133, 215]

Southern blot hybridization [137]

Biplex Invader® assay [140]

Combined PNA/real�time PCR method [137]

Quantitative real�time PCR [134, 135]

Minisequencing (SNaPshot) [138]

Pyrosequencing [139]

Microarray techniques [142, 143]

Table 3. Methods of gene therapy for respiratory chain defects

Disease mtDNA mutation

Method for gene therapy for respiratory chain defects

allotopic expression

xenotopic expression

correction of the transla�tion machin�ery (tRNA)

restriction endonucleases

peptide�nucleic acids

zinc�finger methylases

LHON A11778G [158, 161, 162] [164]

G3460G [160]

NARP/MILS T8993G [9, 157, 159, 161] [216] [175, 176] [183]

MERRF A8344G [169, 170] [177, 178]

G611A [172]

MELAS A3234G [174]

Note: See Table 1 for abbreviations.

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MITOCHONDRIAL GENOME AND HUMAN DISEASES 673

not necessarily unique, since mutant and wild�typemtDNAs may differ in the number of restriction sites[176]. A promising approach to changing the propor�tion of mutant and wild�type mtDNAs is to use PNAs,which are linear polymers of N�(2�aminoethyl)�gly�cine substituted with nucleic base derivatives at nitro�gen of the aminoethyl group and are capable of nonco�valent interactions with DNA and RNA bases. PNAsspecifically bind to mutant mtDNA, blocking its rep�lication [177–181]. Modified PNAs known as cellmembrane crossing oligomers (CMCO) have a higherpolarity than PNAs and better penetrate into mito�chondria [182]. In addition, zinc�finger proteinsproved capable of binding to a certain nucleotidesequence in mtDNA [183].

A transfer of normal mitochondria from stem andsomatic cells into cells with defective mitochondria torestore cell respiration is a promising strategy for treat�ing mitochondrial diseases [184–186].

An interesting approach to treatment of mitochon�drial diseases is based on delivering DNA or a proteindirectly into defective mitochondria. For this purpose,DNA or proteins are packaged into liposomes, whichserve as lipid�soluble capsules. Such capsules haveaffinity for the mitochondrial membrane, specificallybind to and fuse with mitochondria, and release theircontents into the mitochondrial matrix [187, 188].

As with other hereditary disorders, the main diffi�culty in therapy of mitochondrial diseases is that tar�geted delivery of a necessary compound to all mito�chondria of all (or certain) human cells is impossibleas of yet. Thus, the only alternative available now is toprevent transmission of pathogenic mtDNA muta�tions from a mother to her children.

STRATEGIES AIMED AT PREVENTING TRANSMISSION OF PATHOGENIC mtDNA

MUTATIONS

To prevent mutant mtDNA transmission to theprogeny is the most pressing problem of mitochondrialmedicine at the current stage of its development [189].Oocyte donation may help to prevent the transmissionof mutant mtDNA to the next generation. An embryois obtained via in vitro fertilization (IVF) andimplanted into the uterus, and the child does not havethe mother’s mitochondrial disease as a result [189,190]. It should be noted that the mother’s female rela�tives are not recommended as ovicell donors, sincethey may carry the pathogenic mtDNA mutation.

Prenatal diagnosis (PND) via sampling fetal mate�rial for laboratory testing [191] has substantial limita�tions because mtDNA mutations are nonuniformlyspread through different tissues and organs. Criteriafor PND of mitochondrial diseases were established[192]. According to the criteria, reliable interpretationof the PND results is possible only for the mutationsthat display a significant correlation between the het�eroplasmy level and disease severity and are uniformly

spread through all tissues, and whose heteroplasmylevel remains unchanged throughout life. As wasfound, only the T8993G/C mutations meet theserequirements.

Preimplantation genetic diagnosis (PGD) is thediagnosis of genetic abnormalities in embryos prior totheir implantation into the uterus. PGD is performedwith individual cells of embryos obtained via IVF[193]. Both the polar body and one or two blastomersof an early embryo (up to the eight�cell stage) are suit�able for PGD, since all of these cells have the samelevel of heteroplasmy. The heteroplasmy level is farmore efficient to estimate with blastomers than withthe polar body [194]. The embryo is implanted into theuterus only when pathogenic mutations are com�pletely absent or the heteroplasmy level is low, sincethe PND criteria [192] are applicable for PGD as well.It should be noted that the procedure was performedonly in two cases [195, 196].

An apparent advantage of PGD over PND is thatthe pregnancy may be preserved [197–199].

Cytoplasmic transport is a transfer of normallyfunctioning mitochondria from another ovicell orzygote to an ovicell with defective mitochondria inorder to reduce the portion of defective mitochondriaand to compensate for the defect in energy production[200]. However, disappointing results were obtainedwhen a donor cytoplasm was experimentally trans�ferred into a defective ovicell: the incidence of chro�mosome aberrations in newborns was significantlyhigher than average [201, 202]. It was found thatmRNAs, proteins, and other factors are transferredwith the cytoplasm in addition to mitochondria, con�tributing to a new surrounding of the nuclear genome[203, 204].

Nuclear transport is theoretically feasible at variousstages of ovicell or zygote development when thematerial to be transferred is (i) a germinal vesicle,(ii) the chromosomes of a mature ovicell, (iii) pronu�clei, or (iv) the nucleus of one of the blastomers [205–209]. It should be noted in connection with the ethicalproblems of cloning that the first three variants are notassociated with cloning, since nDNA is not doubled atthe corresponding stages. However, the use of a blas�tomer nucleus is cloning by definition, which is pro�hibited in humans (United Nations Declaration onHuman Cloning 59/280 dated March 8, 2005, and theBill on Continuing the Ban on Human Cloning, Rus�sia, January 22, 2010).

In a recent study, nuclear material (sprindle�chromo�some complex of a mature primate (Macaca mulatta))ovicell in metaphase II was successfully transferredinto an enucleated ovicell [210]. As a mtDNA analysisshowed, mitochondria were not transferred togetherwith the chromosomes. A unique feature of the proce�dure is selection of a proper stage (metaphase II) whenthe ovicell karyoplast is free from mitochondria.Another method to prevent the transfer of mitochon�dria along with nDNA is to destroy them [209].

674

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CONCLUSIONS

In spite of the substantial advances made since thecause�and�effect relations had been establishedbetween mtDNA mutations and human diseases [6–9], mitochondrial diseases are still incurable now.First, this is determined by gaps in the understandingof the biogenesis of mitochondria. The available dataon the structure and function of mitochondria arecontinuously corrected and augmented with thedevelopment of new physico�chemical, moleculargenetic, and bioinformatics methods. In addition,there is an abyss between molecular and pathophysio�logical studies because humans remain the only sub�ject apart from mouse models (mitomice) and celllines [211–213], and this imposes many limitationsbecause of the possible risk of health injury or life�threatening consequences. However, there are meansto avoid the inheritance of a pathogenic mitochondrialmutation or to defer the onset of a disease due mito�chondrial dysfunction.

ACKNOWLEDGMENTS

We are grateful to Professor G.M. Dymshits (Insti�tute of Cytology and Genetics, Siberian Branch, Rus�sian Academy of Sciences) and Dr. K.Yu. Popadin(Institute of Information Transmission Problems,Russian Academy of Sciences) for reading the manu�script and helpful comments.

This work was supported by the Russian Founda�tion for Basic Research (project no. 09�04�00183a).

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