Scheffler Immo E. Mitochondria

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A potentially very interesting mutant is a mutant with a severe defect in

mitochondrial protein synthesis (31 – 33) . DNA replication and maintenance

are unaffected, in contrast to similar mutants of yeast. RNA transcription and

processing are also normal, but the failure to translate the mitochondrial

mRNAs makes some of these quite unstable, and their steady - state levels are

far below normal. cDNAs for mitochondrial elongation factors (mtEF - Tu

and EF - Ts) cannot complement the mutation, and a search is in progress to

clone the complementing cDNA from a mammalian library and to identify a

perhaps novel factor required for translational initiation in mammalian

mitochondria.

The biochemical characterization of the speciﬁ c defect in many of these

mutants has been slow. Cloning mammalian genes by complementation with

genomic or cDNA libraries is unpredictable and time - consuming. The mutant

cells lacking succinate dehydrogenase activity were shown to have a point

mutation in the nuclear gene for the anchor protein C

II - 3

, yielding a truncated

peptide and resulting in the failure to assemble a functional complex II (34,

35) . Five complementation groups of mutants defective in complex I were ﬁ rst

characterized biochemically, and the genes for three of these groups were

shown to be X - linked in 1982 (25) . More recently, two of these mutated X -

linked genes were identiﬁ ed: (a) the NDUFA1 gene encoding the MWFE

subunit and (b) the NDUFB11 gene encoding the ESSS subunit (36 – 38) . The

ﬁ nding of the third group suggests the existence of a gene encoding either an

as yet unidentiﬁ ed subunit of complex I, or an essential assembly factor (see

Chapter 5 ).

The demonstration that mammalian ﬁ broblasts can proliferate in tissue

culture in the absence of respiration created a basis for the efforts to obtain

mutant cells in tissue culture equivalent to the ρ

mutants of yeast. Several

groups have been successful in such efforts. The ﬁ rst report described ρ

chicken cells (39) , and subsequently ρ

human tumor cells (40, 41) , ρ

mouse

cells, and ρ

primary human ﬁ broblasts have been successfully isolated. The

methodology is based on ﬁ rst deﬁ ning a medium that makes respiration obso-

lete and then using speciﬁ c inhibitors of mitochondrial DNA replication to

deplete cells of mtDNA over a prolonged period of time. Ethidium bromide

at sublethal concentrations was used effectively with human tumor cells, but

it was less effective with mouse cell lines. Other inhibitors investigated are

dideoxy cytidine (ddCTP inhibits γ - DNA polymerase in mitochondria) or bis -

intercalating antitumor drugs such as ditercalinium (42) .

The ρ

human cell lines have found very interesting applications in the study

of mitochondrial mutations isolated from human patients, since they can be

repopulated with mitochondria from the same species by fusion with enucle-

ated cytoplasts. Thus, mutant mitochondria can be introduced into ρ

cells with

a different nuclear genome, and effects of mitochondrial mutations can be

clearly separated from potential effects of a speciﬁ c nuclear background. It

should be noted that experiments of this type have also given the clearest

indication that mitochondria from one species are not compatible with the

IN CELL CULTURE 351

352 MITOCHONDRIAL MUTATIONS AND DISEASE

nuclear genome from another species (43) . A more detailed description of

such experiments and their usefulness will be presented in a later section.

While yeast rho

−

mutants are generated spontaneously at a high frequency,

mammalian cells in culture appear to have a stable mitochondrial genome over

many generations, and even a mutant cell line with no mitochondrial protein

synthesis (31 – 33) has maintained an intact mtDNA. If there are mutations or

deletions, they are present at a very low frequency. In the early 1970s, when

mammalian cells were beginning to be regarded as microorganisms from which

useful and informative mutants could be isolated, a challenging question was

whether mitochondrial mutations could also be isolated. Few people thought

about mtDNA, but those who did were aware that there were thousands of

copies per cell. A priori , one would therefore predict that a single mutation on

one mtDNA molecule in the entire population would not lead to a phenotype,

and a phenotype could arise only if there was a mechanism for segregation. It

was quite a surprise when the Eisenstadt laboratory announced in 1974 that

chloramphenicol resistance in a mouse cell line A9 was cytoplasmically inher-

ited (44) . The targets for chloramphenicol were mitochondrial ribosomes, and

there was an expectation from experience with bacteria and yeast that one or

more mutations in the large rRNA could lead to antibiotic resistance. A year

earlier the same laboratory had described chloramphenicol - resistant HeLA

cells, but the proof of cytoplasmic inheritance required enucleated cells (by a

technique that had just been introduced) and the fusion of such cytoplasts

containing the mutated mitochondria with nucleated cells containing wild - type

mitochondria. The resulting cells have been referred to as “ cybrids ” . Curiously,

the ﬁ rst paper reporting cytoplasmic inheritance concluded that “ CAP resis-

tance . . . may be encoded in mtDNA, or possibly in one of the other types of

cytoplasmic DNAs reported in mammalian cells . . . (spcDNA, microsome -

associated DNA, informational or I - DNA and membrane associated cmDNA). ”

Of course the latter types of DNA have now been lost in history, since they

most likely were simply contaminants derived from nuclear DNA.

The method of obtaining such mutants is worth recounting. Cells were

treated for about a day with ethidium bromide, followed by incubation with

50 μ g/ml of chloramphenicol. Two and a half months later, colonies appeared

which could be cloned and propagated in the presence of the drug. Clearly, at

the later stages the cells had a mixed population of mtDNAs with the mole-

cules containing the mutation in the majority. The cells were heteroplasmic. It

is necessary to postulate that during the prolonged exposure to the antibiotic

the cells initially did not proliferate, and a majority of the cells in fact died. By

a mechanism that is still quite obscure, in some cells a buildup of mutated

mtDNA relative to wild - type mtDNA must have occurred. As the proportion

increased, mitochondrial protein synthesis could proceed, and eventually the

cells gained the level of energy metabolism necessary to sustain cell divisions.

With hindsight, one might also wonder why such cells did not become rho

cells, but one must presume that the media chosen would not have supported

such cells (glucose level, no uridine or pyruvate present).

The original discoveries were followed by a series of interesting and infor-

mative studies on intra - and interspeciﬁ c hybrids and cybrids (45) . It was

shown that the mitochondrial and nuclear genome must be from the same

organism to be compatible and to yield viable cells. For example, when CAP -

resistant moue mitochondria were transferred into Chinese hamster cells, the

cybrids had a very limited life span. When interspecies hybrids were made and

selected directly in chloramphenicol, the chromosomes of the parent with

the resistant mitochondria were retained. In some examples this represents a

reversal of the usual loss of human chromosomes from a rodent – human hybrid

cell line. By making intraspeciﬁ c hybrids, it was possible to make cells that

were deliberately heteroplasmic and had a roughly known proportion of wild -

type and mutant mtDNAs. Continued culture under selective or nonselective

conditions allowed an assessment of the rates of segregation of these genomes

from each other under selective pressure or in its absence. So - called xeno-

mitochondrial cybrids have been studied in which the nuclear genes are human

and the mitochondrial genome is derived from other primates (46) . A func-

tional electron transport chain can be produced when the mtDNA is from the

chimpanzee or gorilla, but not when the mtDNA is from the orangutan.

The ﬁ rst success in producing xenomitochondrial mice harboring trans -

species mitochondria on a Mus musculus domesticus (MD) nuclear back-

ground has been reported. First, xenomitochondrial ES cell cybrids were

produced by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or

Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6 - G treated

CC9.3.1 or PC4 ES cells. Homoplasmic (MS, MC, Mdu, and MP) and hetero-

plasmic (MC) cell lines were injected into MD embryos, and liveborn chimeric

mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140,

and MP/MD l of 9 founder chimeras, respectively). Chimeric founder females

were mated with wild - type MD males producing homoplasmic offspring with

low efﬁ ciency (5%) (47) . Heteroplasmic mice carrying wild - type and mutated

mtDNA have also been reported (48, 49) .

Other antibiotics were used successfully in other laboratories to obtain

resistant cell lines in which drug resistance was cytoplasmically inherited (50 –

53) . Oligomycin (or rutamycin) resistance could arise from a mitochondrial

mutation in the “ oligomycin - sensitivity - conferring protein ” of complex V

(ATPsynthase), and antimycin resistance resulted from a mutation in the

cytochrome b gene encoded by mtDNA. In all cases the cytoplasmic inheri-

tance had to be carefully documented, since other, nuclear alterations (affect-

ing uptake of the drug, for example) could also give rise to such phenotypes.

Later, the mutation responsible for oligomycin resistance in Chinese hamster

ovary cell mutants was shown to be in the ATPase 6 gene on the mitochondrial

DNA (54) , and the formation of an altered form of this subunit was also dem-

onstrated by immunological methods (55) .

In conclusion, mutations on mammalian mtDNA were ﬁ rst deﬁ nitively

demonstrated in tissue culture cells. The astonishment and continued puzzle-

ment arises not from the existence of such mutations, but about the

IN CELL CULTURE 353

354 MITOCHONDRIAL MUTATIONS AND DISEASE

mechanisms that operate on a population of approximately 1000 mitochon-

drial genomes/cell to segregate and accumulate a sufﬁ ciently large fraction of

mutant mtDNAs for a phenotype to be expressed. The extreme selective pres-

sure exerted during the culture with the drugs is responsible for the ultimate

selection, but the shift in the mtDNA populations takes weeks or months,

while cell division is arrested or extremely slow. This issue will become relevant

again in the discussion of human mitochondrial diseases and similar or related

mechanisms occurring in vivo .

7.3 MOLECULAR GENETICS OF HUMAN

MITOCHONDRIAL DISEASES

7.3.1 Introduction

Until about 1988, human patients with myopathies, neuropathies, or encepha-

lomyopathies had attracted attention mainly of neurologists, pathologists, and

histologists. Abnormal staining of muscle biopsies viewed in the light micro-

scope had been detected ( “ ragged red ﬁ bers ” ), and later electron microscopic

investigations had indicated that such abnormalities were associated with

morphologically abnormal mitochondria. The term mitochondrial encephalo-

myopathies (MEM) described a combination of symptoms and morphological

characteristics with signiﬁ cant variability and severity, which made a genetic

analysis complicated and difﬁ cult to interpret. Was it a complex genetic trait?

Nevertheless, as examples accumulated and reliable pedigree studies became

possible with larger samples, there were indications that MEM may follow a

pattern of maternal inheritance. The maternal inheritance of human mtDNA

had been ﬁ rmly established in 1980 (56) , after related studies had reached the

same conclusion in horse – donkey hybrids (56a) , in the rat (56b, 56c) , in the

white - footed mouse Peromyscus polionotus (56d) , and in frogs, Xenopus laevis

(56e) , and Drosophila melanogaster (56f) . The ideas converged dramatically

in 1988 with two publications describing mutations in mtDNA being linked to

“ mitochondrial diseases. ” In one report, Wallace et al. (57) described a point

mutation in the ND4 gene (complex I) in several patients with Leber ’ s heredi-

tary optical neuropathy (LHON). The other, by Holt et al. (58) , described

deletions in mtDNA in patients with mitochondrial myopathies. It has been

difﬁ cult to keep up with the ﬁ eld ever since. A ﬂ ood of publications from these

and other laboratories rapidly established mtDNA mutations as the genetic

basis for a variety of clinical syndromes, in the process deﬁ ning concepts and

generalizations, but also raising new questions of fundamental importance

for understanding mitochondrial inheritance and the phenotypic expression

of mitochondrial mutations (59 – 62) . To date (October 2006), there have been

reports on 126 pathogenic mtDNA point mutations affecting the function of

rRNAs and tRNAs (63, 64) , and 103 point mutations in the thirteen mt genes

encoding polypeptide (D. Wallace, personal communication). It is not the

MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 355

intent in this chapter to discuss these mutations exhaustively. Select examples

will be used to illustrate some fundamental and general principles.

The idea of uniparental transmission of mtDNA has been challenged in

several publications, for species in which intact sperm is found in the egg

cytoplasm after fertilization. Back crosses between Mus musculus and Mus

spretus (an interspeciﬁ c cross) had yielded offspring in which a very small

proportion of paternal mtDNA (0.01 – 0.1%) could be detected by sensitive

PCR techniques (65) . This prompted a reexamination for the much more

common intraspeciﬁ c crosses between mammals, from which the authors con-

cluded that in intraspeciﬁ c crosses ( Mus musculus ) the paternal mtDNA is

eliminated at the late pronucleus stage (66) . Such a mechanism requires a

species - speciﬁ c recognition system by which egg cytoplasm identiﬁ es and

destroys sperm mitochondria and mtDNA (67 – 71) . Paternal transmission in

rare circumstances may be irrelevant for the present discussion, but it will

become a potential issue again in the discussion of mitochondria and human

evolution (72) .

7.3.2 Maternal Versus Sporadic Inheritance

As one of the pioneers in these investigations pointed out (73) , it is perhaps

“ ironic that the ﬁ rst mtDNA defects identiﬁ ed, large deletions, are not usually

inherited ” ; that is, the observation of maternal inheritance of MEM, though a

clue for other disorders, was not relevant in the ﬁ rst studies reported by Holt

et al. (58) . Patients with MEM had muscle biopsies, and a substantial fraction

of such patients (9/25) exhibited heteroplasmy in muscle: Up to 7 kb of mtDNA

were deleted in 20 – 70% of the population of mtDNAs. A detailed description

of the size of the deletions and the region of the mitochondrial genome

affected will be given in a later section. At this point it is to be stressed that

there was heteroplasmy, but this heteroplasmy was found originally only in

muscle, not in cells obtained from blood. The defects were not maternally

inherited. The generation of deletions and the accumulation of deleted

mtDNAs in speciﬁ c tissues may have been a postzygotic event.

As more patients with mtDNA deletions and rearrangements were identi-

ﬁ ed, a fairly complex genetic picture emerged (73, 74) . Only one family was

found in which the same deletion was transmitted over three generations. In

another family a mother and daughter with similar symptoms had different

deletions. The deletions found in several patients could not be detected in their

mothers. The child of one patient did not have any deletions detectable by

Southern blotting. It is important to recognize that Southern blots may be

quite insensitive in the detection of a small fraction of deleted mtDNA mole-

cules. PCR - based methods are very suitable, if primers are chosen which ﬂ ank

a relatively large deletion. Efﬁ cient ampliﬁ cation occurs only when the dele-

tion is present, since in the normal mtDNA the primers are too far apart for

standard PCR conditions. In general, this group of mtDNA deletions and the

associated symptoms are classiﬁ ed as spontaneous, because there is no appar-

356 MITOCHONDRIAL MUTATIONS AND DISEASE

ent Mendelian pattern of inheritance and there is also no predictable maternal

transmission over several generations.

The most likely explanation for the observed distributions of mtDNAs in

a pedigree is that the deletions occurred either during oogenesis, leading

to the formation of the occasional oocyte with a subpopulation of mutated

mtDNAs, or very early during embryogenesis. If the deletion occurred before

fertilization, one would expect a widespread distribution of deleted mtDNAs

in different tissues. On the other hand, such a distribution could be strongly

inﬂ uenced by unequal segregation of wild - type and mutant mtDNAs during

embryogenesis, or by a selection against cells with a large proportion of

mutated mtDNAs and hence partially defective mitochondria. It is conceiv-

able that mtDNAs with substantial deletions are replicated more rapidly and

thus have a selective advantage over normal mtDNAs. Such an explanation

may account for several observations: (1) Depending on the syndrome and its

severity, high proportions of deleted mtDNA were found frequently in the

muscle and brain (after autopsy, where possible), while other tissues (e.g., liver

or blood) had a lower proportion, sometimes only detectable by PCR. (2) The

fraction of deleted mtDNAs increases with age in muscle and possibly the

brain. In a situation where the total number of muscle ﬁ bers does not change

appreciably after birth, mitochondrial DNA replication must be dissociated

from cell division, selection against defective cells ceases, and shorter mtDNA

molecules gain a replicative advantage. Direct tests of such a hypothesis are

becoming possible through the use of ρ

human cell lines that can be repopu-

lated with mitochondria from enucleated cells with a known proportion of

deleted mtDNAs. In one such experiment the fraction of deleted mtDNAs

increased from 40 – 50% to about 80% in a period of 10 weeks. (3) There is a

wide range of age of onset of neurological symptoms from between birth to

the age of 60. The factor determining the age of onset could be the percentage

of mutated mtDNAs present in certain tissues at birth, or the rate at which

mutated mtDNAs accumulate relative to normal mtDNAs during the period

after birth.

A most impressive example of a mtDNA mutation accumulating with time

in muscle has been reported by Weber et al. (75) . A patient, healthy and ath-

letically active as a teenager, experienced the ﬁ rst symptoms at the age of 34

(joint pains and muscle weakness), and she was subsequently observed and

examined over a period of 12 years, during which the symptoms progressively

worsened to the point where she became dependent on a wheelchair, and

suffered from dysphagia to solids and liquids. Ragged red ﬁ bers and COX -

negative ﬁ bers were observed to increase from 2% and 4.5%, respectively, to

18% and 68%, respectively, over the 12 - year period. Finally, mtDNA sequenc-

ing established a single - point mutation in the tRNA

Leu(CUN)

gene at position

12320, affecting the TYC loop at a conserved site. It is difﬁ cult to argue that

mtDNA with a point mutation has a replicative advantage. The mutation is

found in heteroplasmic cells and is restricted to skeletal muscle, the tissue that

MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 357

is clinically and biochemically affected. Within muscle, it was highest in the

COX - deﬁ cient ﬁ bers. In the muscle biopsy specimens that had been collected

between 1983 and 1995, the fraction of mutant mitochondrial genomes

increased from 0.7 to 0.9. It was absent in ﬁ broblasts and white blood cells.

Speculation is that the mutation occurred postzygotically and may already

have been restricted to speciﬁ c cell lineages, including myoblasts. However,

the signiﬁ cant expansion of the mutation in skeletal muscle is dramatically

demonstrated here for a postmitotic tissue.

The patient has daughters over the age of 20, and examining them

might be reassuring to conﬁ rm that it is indeed a somatic mutation in the

mother and hence absent in the offspring, but they have opted not to be

tested at this time. This is not an atypical situation encountered in genetic

counseling.

The same female patient provided biopsy material to support another very

intriguing observation made in a number of laboratories (76) . In patients with

myopathies caused by mt mutations, even when the mutated mtDNA was

present in large excess, progenitor satellite cells contain no, or only very low

levels of, mutated mt genomes. Such satellite cells can form regenerated muscle

following induction of necrosis. The authors were successful in using a local

anesthetic, bupivicaine hydrochloride, to induce a localized necrosis, and three

weeks later the same region of muscle showed clear muscle regeneration, but

now a very signiﬁ cant portion of ﬁ bers was COX - positive, and in such ﬁ bers

the levels of mutated mtDNA was very low or undetectable (76) . The stimula-

tion of muscle regeneration therefore offers the prospect of reversing severe

biochemical and physiological defects in such patients, as an alternative to

gene therapy. For obvious reasons, the obstacles to gene therapy with mito-

chondrial DNA are even greater than those with nuclear genes.

In a pioneering experiment, mitochondrial DNA in normal human oocytes

has been investigated by Chen et al. (77) . Using sensitive PCR - based tech-

niques, these investigators were able to detect rearranged mtDNAs in single

oocytes. Among ∼ 100,000 normal mtDNAs less than 0.1% had deletions/dupli-

cations or other structural changes responsible for abnormal PCR products.

For most practical reasons, one would consider this homoplasmy. But can such

a minority population of mtDNAs become the templates from which the

embryo is equipped with mutated mitochondrial genomes, possibly differen-

tially in different tissues? A further discussion of this subject will be resumed

below. The consideration of mtDNA in oocytes has led to an interesting addi-

tional speculation. MtDNA deletions have been found to accumulate in an

age - related fashion in tissues with slowly or nondividing cells. Oocytes in

humans are also arrested (at meiosis I) for a prolonged period of time (years),

and it is conceivable that mtDNA deletions might arise during this time. The

sample size may be too small at this stage, but one could ask whether the

incidence or risk of mitochondrial diseases due to mtDNA deletions could

increase with advancing maternal age (78) .

358 MITOCHONDRIAL MUTATIONS AND DISEASE

7.3.3 Mapping mtDNA Deletions/Rearrangements

A signiﬁ cant effort has been expended to date to map the mtDNA deletions

precisely and to deﬁ ne the deletion junctions at high resolution by sequencing.

The mapping of the deletions has been pursued with several goals in mind.

First, it is desirable to establish precisely which genes or gene functions are

affected and to relate such deletions to measurements of residual activities of

the mitochondrial electron transport chain. Second, one can ask whether the

deletions are entirely random, or whether there are common regions that are

most frequently deleted. And ﬁ nally, if there is a “ common deletion, ” do the

deletion junctions provide any evidence for a likely mechanism by which such

deletions could be generated?

The results are provocative, but not uncomplicated to interpret. Not surpris-

ingly, few deletions have been found so far which delete either one of the

origins of replication on either strand, and transcriptional promoters have not

been found to be missing. These are the important cis - acting elements required

for replication, and their loss would also lead quickly to the loss of the mutated

mtDNA. Exceptions can be found in the case of a maternally inherited mtDNA

deletion (10.4 kb) (79) , as well as in four patients in which the heavy - strand

promoter and the adjacent binding site for the transcription factor mtTFA

have been deleted (73, 80) . Initiation of DNA replication requires transcrip-

tion from the light - strand promoter, and its continued presence makes the

propagation of the deleted mtDNA possible. The two promoters are close

together, and deletions starting within the interval are rare. As predicted, and

in conﬁ rmation of earlier conclusions (see Chapter 4 ), transcripts dependent

on the light - strand promoter were abundant in these muscle ﬁ bers, but tran-

scripts from the other strand were missing, regardless of whether the genes

were deleted or not (80) .

The junctions and ﬂ anking regions of mtDNA deletions have been investi-

gated in more than 150 patients (as of 1993 (73) ) after ampliﬁ cation by PCR

and sequencing. Signiﬁ cantly, in about half of the patients there is a “ common

deletion ” of 4.9 - kb mapping in the interval between bp 8000 and bp 13,600.

More precisely, the sequence up to 8482 is generally retained, and the sequence

following 13,460 is also present. Further inspection of the human mtDNA

sequence reveals a 13 - bp sequence (8470 – 8482) at one breakpoint region that

is directly repeated at the other breakpoint region (13447 – 13459). Loop for-

mation and recombination between the direct repeats is one mechanism sug-

gested by these observations. One plausible mechanism for the formation of

such molecules starts with mtDNA dimers that are formed during replication,

followed by a recombination event generating the deletion. This model is

strengthened by additional data which show that direct repeats of 3 to 13 bp

are found ﬂ anking the deleted sequences in 80 – 90% of all the patients exam-

ined (73) . Although the model is attractive, it should be considered in light of

the knowledge that recombination is generally thought to be absent or very

infrequent in mammalian mitochondria, as evidenced also by the lack of active

MOLECULAR GENETICS OF HUMAN MITOCHONDRIAL DISEASES 359

repair systems of the type familiar to us from the nucleus (81) . Therefore,

recombination events giving rise to deleted mtDNAs are rare, and, fortunately,

patients suffering the consequences are also relatively rare. Another interest-

ing question is whether the postulated recombination activity is restricted to

or particularly active at speciﬁ c stages in the life cycle of an individual — for

example, during rapid mitotic divisions preceding oogenesis or during the

rapid early zygotic divisions in the embryo. Such a restricted period of activity

might help explain the origin and distribution of these deletions in families

and in different tissues of an affected individual. An alternative to the recom-

bination mechanism for generating deletions is slippage and mispairing during

replication of the mtDNA. The issue remains unsettled at this time.

With the mitochondrial genome so packed with genes, every major deletion

clearly includes one or more genes, and it is nowadays straightforward to

predict the consequences at the biochemical level. It should be emphasized

that patients with the diagnostic symptoms have been found in which mtDNA

deletions were not detectable. Obviously, very small deletions or even single

nucleotide changes can cause serious problems. Such single nucleotide changes

will be discussed below for the cases where maternal inheritance is observed,

but clearly, small changes can also occur spontaneously by replication errors,

and so far in the discussion the focus has been on spontaneous errors observed

in individual patients and, rarely, in multiple offspring from the same

mother.

A description of the pathologies and symptoms and their relationship to

mitochondrial functions will be presented later. It need not be stressed that

individuals would not be alive if at least some of their mitochondrial genomes

were not intact and capable of sustaining the biogenesis of the complexes for

oxidative phosphorylation. A generalization that can be made, therefore, is

that heteroplasmy is the rule in patients with mtDNA deletions .

7.3.4 mtDNA Point Mutations and Maternal Inheritance

It has been mentioned before that numerous families with incidents of mito-

chondrial encephalomyopathies show maternal inheritance, a strong hint that

an inheritable mitochondrial mutation is involved. The ﬁ rst report with strong

experimental support for this genetic basis of the transmission of a disease

appeared in 1988 by Wallace et al. (57) . The group at Emory University

sequenced most of the mtDNA of a patient with Leber ’ s hereditary optic

neuropathy (LHON) and compared it with the published nucleotide sequence

for human mtDNA. It is important to point out here that a total of 25 “ muta-

tions ” were found — that is, nucleotide differences between the Cambridge

sequence and the sequence from an African American LHON patient. Which

is the deleterious mutation and which represents a harmless single nucleotide

polymorphism (SNP or “ snip ” )? An answer to this question continues to be

crucial for all correlations of mitochondrial point mutations with a particular

disease (symptom), since it is not yet possible to recreate the same mutation

360 MITOCHONDRIAL MUTATIONS AND DISEASE

by recombinant DNA technology to verify that it alone is responsible for the

defect. In 1988 the rationale for identifying the true mutations causing the

disease was as follows: Two of the nucleotide changes were in the rRNA genes

and were thought to be of little consequence. Fifteen of the changes were base

changes that would not have caused any change in the amino acid sequence

of the encoded peptides. No changes were found in any of the tRNA genes in

this patient (however, see below). This narrowed the choice to eight potential

sites. The extreme variability in metazoan mtDNA sequences, between closely

related species, and even within a species has been noted (see Chapters 4 and

8 ). Phylogenies can be constructed providing very valuable information on

evolution, and because of the speed of the mitochondrial clock, a time scale

of tens of thousands of years can be resolved (see Section 8.2 , “ Human Evolu-

tion ” ). Investigators had only begun to become aware of the variability among

human populations, but sufﬁ cient sequence data were available in 1988 to

suggest that ﬁ ve of the changes were likely to be of no consequence since they

were also found in other humans not suffering the neuropathy (LHON). Two

additional nucleotide changes were found in various ethnic groups and/or in

other members of the Georgia pedigree unaffected by LHON. This left the

change at nucleotide 11,778 as the likely candidate mutation. A codon change

replaces an arginine by a histidine in subunit 4 (ND4 gene) of complex 1. The

homologous protein has an invariant arginine at that site in species ranging

from fungi to fruit ﬂ ies to humans. Luckily, the nucleotide change also elimi-

nated an SFaNI restriction site found in normal mtDNA, and it became pos-

sible to check this site quickly in several other LHON patients of diverse

ethnic origin. All LHON patients in the limited sample investigated in 1988

had this mutation.

A later analysis employed a parsimony computer program PAUP to con-

struct a number of plausible phylogenetic trees for European, African, African -

American, and Asian populations, based on nucleotide changes in mtDNA

(82) . Details will be discussed in a more general context of human evolution

in one of the following chapters, but here it is relevant that the authors dem-

onstrated that the SfaNI mutation at position 11778 appears to be quite recent,

has occurred twice, and in each case was associated with LHON. It was unlikely,

therefore, that the mutation at 11778 was a silent or neutral mutation.

In the intervening years a number of other syndromes with maternal

inheritance have been investigated at the molecular level and found to result

from single nucleotide changes in mtDNA. Point mutations were found in

structural mitochondrial genes as well as in rRNA and tRNA genes (Table

7.1 ).

Before describing LHON and other deﬁ ned clinical syndromes with mater-

nal inheritance in more detail from a biochemical and physiological stand-

point, some important distinctions between single - point mutations and larger

deletions must be emphasized. Individuals with point mutations in their

mtDNA are generally heteroplasmic, but frequently they are homoplasmic,

that is, all mtDNAs examined contain the mutation. This astonishing observa-