Mitochondria and Mitochondrial Disorders E. Morava, J.A.M. Smeitink

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23.1 Mitochondrial Structure and Function

Mitochondria are membranous organelles that pro- vide the energy necessary for the different cell func- tions. They are called mitochondria because of their threadlike appearance (Greek mitos = thread) on light microscopy. On electron microscopy they appear as vesicles bounded by two membranes. The inner membrane is thrown into folds that project like shelves into the mitochondria. These projections are called cristae. Mitochondria consist of four compart- ments: the outer membrane, the intermembrane space, the inner membrane, and the mitochondrial matrix. Mitochondria vary considerably in size in any one cell type, but most have a diameter of between 0.1 and 1.0 mm. In different cell types the size, shape, and number of cristae vary considerably. Most cells con- tain many mitochondria, the actual number differing in relation to the energy requirements of the type of cell. Mitochondria are dynamic organelles. They un- dergo frequent fission and fusion or branching, and they may alter their size and location in the cell under different conditions.

The main role of mitochondria is to synthesize adenosine triphosphate (ATP), the universal source of energy for the cell. Mitochondria convert the energy derived from oxidation of substrates into the high- energy bond of ATP, which is then transported into the cytosol in exchange for adenosine diphosphate (ADP). The process of production and storage of en- ergy by mitochondria is called oxidative phosphoryla- tion. In addition to this process, mitochondria also perform many other functions, including the first two reactions of the urea cycle, the synthesis of ketone bodies, and propionate metabolism.

Pyruvate and fatty acids are the most important substrates for energy production, although amino acids may also contribute in certain conditions, for instance during fasting.

Pyruvate represents the metabolic end point of glycolysis, which occurs in the cytoplasm and yields a small amount of ATP. Pyruvate is carried across the mitochondrial membrane into the mitochondrial matrix space by monocarboxylate translocase. Subse- quently it is oxidatively decarboxylated by the pyru- vate dehydrogenase complex (PDHc), which produces CO

2

and acetyl-CoA, while reducing one nicoti- namide adenine dinucleotide (NAD

⁺

) to NADH + H

⁺

.

The pyruvate dehydrogenase complex is located at the inner border of the inner mitochondrial mem- brane.

Fatty acids with more than eight or ten carbon atoms require a specific carrier system to enter the mitochondrial matrix space (Fig. 23.1). They are acti- vated to acyl-CoA by acyl-CoA ligase (= acyl-CoA synthetase) on the mitochondrial outer membrane.

Fatty acyl-CoA is subsequently converted to fatty acyl carnitine in the intermembrane space by carnitine palmitoyl transferase 1 (CPT 1), which is located in the outer mitochondrial membrane. Fatty acyl carni- tine is then translocated across the inner mitochon- drial membrane in exchange for free carnitine, a reac- tion catalyzed by carnitine:acylcarnitine translocase, located in the inner mitochondrial membrane. On the inner surface of the inner membrane a second carni- tine palmitoyl transferase, CPT 2, converts fatty acyl carnitine to acyl-CoA and free carnitine. Shorter- chain fatty acids (ten or fewer carbons) enter the mi- tochondria independently of the carnitine-requiring transport system, and are activated by short- and medium-chain acyl-CoA ligase to the respective acyl- CoA esters in the mitochondrial matrix. Acyl-CoA is the primary substrate for mitochondrial b-oxidation.

This b-oxidation spiral involves four successive reac- tions, mediated by acyl-CoA dehydrogenase, 2-enoyl- CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. The products of each turn of the spiral are acetyl-CoA and acyl-CoA, now two carbons shorter, with conversion of flavin ade- nine dinucleotide (FAD) to FADH

2

and NAD

⁺

to NADH + H

⁺

. An acyl-CoA can recycle through b-oxi- dation spirals as many times as it can yield acetyl- CoA fragments. With each turn of the spiral, as the acyl-CoA becomes shorter, it encounters enzymes with different substrate specificities. An example of this is the series of chain-length-specific acyl-CoA dehydrogenases: long-chain acyl-CoA dehydrogenase mediates the reaction for acyl-CoA compounds from 8 carbons to 18 carbons; medium-chain acyl-CoA de- hydrogenase from 4 to 12 carbons; and short-chain acyl-CoA dehydrogenase from 4 to 6 carbons. Fatty acids with double bonds require additional enzymes, d

³

, d

²

-enoyl-CoA isomerase and 2,4-dienoyl-CoA re- ductase. In the liver, the metabolic end product of each cycle of b-oxidation, acetyl-CoA, is primarily converted to ketone bodies, while in other tissues acetyl-CoA is completely oxidized via the citric acid

Mitochondria and Mitochondrial Disorders

E. Morava, J.A.M. Smeitink

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Chapter 23 Mitochondria and Mitochondrial Disorders 196

Fig. 23.1. Import and b-oxidation of fatty acids in mitochondria

Fig. 23.2. Citric acid cycle in the mitochondrial matrix

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cycle. Electrons from the acyl-CoA dehydrogenase- mediated reactions are transferred to electron trans- fer flavoprotein to the electron transport chain.

Therefore, acetyl-CoA is produced from different sources. Carboxylation of pyruvate yields oxalo- acetate and decarboxylation of pyruvate yields acetyl-CoA. Acetyl-CoA is produced in the b-oxida- tion of fatty acids. It can also be derived from break- down of certain amino acids (for instance, leucine and isoleucine). Acetyl-CoA and oxaloacetate con- dense to form citrate. Citrate is decarboxylated in the citric acid cycle (also called Krebs cycle, tricarboxylic acid cycle, or TCA cycle, see Fig. 23.2), yielding CO

2

and reducing equivalents in the form of NADH + H

⁺

and FADH

2

. Certain amino acids can be converted to citric acid cycle intermediates, such as glutamate, aspartate, alanine, proline, and glutamine.

The reducing equivalents NADH and FADH

2

, pro- duced by b-oxidation of fatty acids and the citric acid cycle, are reoxidized to NAD

⁺

and FAD by the respira- tory chain. This oxidation sequence is tightly coupled to the phosphorylation of ADP to yield ATP. This process of so-called oxidative phosphorylation is re- sponsible for the main production of ATP in most cells.

Oxidative phosphorylation (Fig. 23.3) is achieved by five multisubunit enzyme complexes (complexes I–V), all located within the mitochondrial inner membrane. Complexes I–IV constitute the electron transport chain (or respiratory chain). Complex I (NADH ubiquinone reductase = NADH-coenzyme Q

reductase) oxidizes NADH to NAD

⁺

and the electrons of the hydrogen are transferred to ubiquinone (CoQ) to yield ubiquinol (reduced CoQ). Complex II (succi- nate ubiquinone reductase) accepts reducing equiva- lents from succinate and also passes electrons down the chain to ubiquinone to yield ubiquinol. The elec- trons from ubiquinol are transferred to complex III (ubiquinol: cytochrome-c reductase), subsequently to cytochrome c, then to complex IV (cytochrome-c ox- idase), and finally to oxygen, which combines with protons to form water. The energy released in the electron transport is used to pump hydrogen ions out of the mitochondrial inner membrane through com- plexes I, III, and IV. The resulting electrochemical gra- dient is exploited by complex V (ATP synthetase = ATPase). Complex V allows some of the protons to flow back into the mitochondria and uses the energy so generated for the synthesis of ATP from ADP and inorganic phosphate. ATP formed in the matrix space is then transported out of the mitochondria by ade- nine nucleotide translocase in exchange for ADP. The production and storage of energy by mitochondrial oxidative phosphorylation is a very efficient process with a high ATP yield. The rate of mitochondrial sub- strate oxidation is finely geared to the needs of the cell. The main control mechanism is the ratio of ATP to ADP. ADP is an activator of mitochondrial respira- tion.

Each complex of the respiratory chain is made up of a number of protein components. Complex I con- tains 46 polypeptides and has as prosthetic groups

Fig. 23.3. Electron (e) transport chain in mitochondrial inner membrane

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flavin mononucleotide and several nonheme iron–sulfur clusters. Complex II consists of 4 sub- units. Complex III is composed of 11 subunits includ- ing cytochrome b, cytochrome c

1

, and a nonheme iron protein. Complex IV is composed of 13 different protein units, 2 cytochromes (a and a

3

) and two copper atoms. Complex V is composed of 16 different polypeptides. Two small electron carriers, ubi- quinone (coenzyme Q

10

) and cytochrome c (a low molecular weight hemoprotein) act as shuttles be- tween the complexes.

23.2 Mitochondrial Genetics

Human cells possess two different genomes: nuclear DNA (nDNA), a 3¥10

⁹

-base-pair-long genome, pre- sent in two copies in each cell, and mitochondrial DNA (mtDNA), a 16,569-base pair-long genome, pre- sent in 2–10 copies per mitochondrion. Mitochondria are dependent upon the coordinated expression of these two parallel genetic systems.

Each mitochondrion contains on average five mi- tochondrial genomes and each cell contains hundreds to thousands of mitochondria. Consequently, there are hundreds or thousands of copies of mtDNA in each cell. In normal individuals all of these copies of mtDNA are identical (homoplasmy), but in disease there may be more than one distinct population of mtDNA (heteroplasmy), one being normal and the other being mutant. During cell division mitochon- dria (and mtDNA) are randomly distributed between daughter cells. As a consequence, the proportion of mutant genomes may shift in daughter cells.

mtDNA consists of two complementary strands:

one filament is rich in guanine nucleotide residues, while the other is rich in cytosine residues. They are conventionally called heavy (H) and light (L) strands, respectively. mtDNA contains only 37 genes and codes for different proteins of the respiratory chain and the RNAs (transfer and ribosomal RNAs) neces- sary for expression of these genes (13 messenger RNAs [mRNAs], 22 transfer RNAs [tRNAs], and 2 ri- bosomal RNAs [rRNAs]). The 13 mRNAs specify as many polypeptides of the respiratory chain: 7 sub- units of complex I, the cytochrome b subunit of com- plex III, 3 subunits of complex IV and 2 subunits of complex V.

In mtDNA, the gene organization is highly com- pact; all of the coding sequences are contiguous with each other and there are no introns. The only noncod- ing stretch of DNA is the displacement loop (D loop), a region of 1123 base pairs that contains the origin of replication of the H strand and the promoters for L- and H-strand transcription. The D loop is an im- portant area of interaction of mtDNA with nuclear- encoded proteins regulating mtDNA housekeeping

functions. The genetic code used in the mitochondria differs from the universal code, making nDNA and mtDNA reciprocally untranslatable. The rate of spon- taneous mutations of mtDNA genes is much higher than that of nDNA genes and repair mechanisms are limited and less efficient.

The entire mitochondrial genome of each individ- ual, either male or female, is inherited from the moth- er, although the possibility is not excluded that a small contribution of the mitochondrial genotype may be of paternal origin. This is due to the fact that during egg fertilization the sperm cell contributes al- most no cytoplasm to the zygote.

The replication and expression of mtDNA are con- trolled by nuclear genes. The nuclear genome further- more encodes for most of the mitochondrial proteins and cooperates with the mitochondrial genome in the assembly of the multisubunit enzyme complexes of the oxidative phosphorylation apparatus. The D-loop region is an important area of interaction between nDNA and mtDNA.

The structural proteins of the oxidative phospho- rylation (complexes I–V) are all dual-coded, except for complex II. Most mitochondrial membrane and matrix proteins are coded by nuclear genes and syn- thesized in the cellular cytoplasm on free polyribo- somes and have to be imported from the cytoplasm into the mitochondria through a complicated translo- cation machine, which is under the control of the nu- clear genome. In addition, nDNA encodes several fac- tors controlling mtDNA replication, transcription and translation.

Most nuclear-encoded mitochondrial proteins destined for the inner three mitochondrial compart- ments (the intermembrane space, the inner mem- brane, and the mitochondrial matrix) are synthesized as larger precursors containing an amino-terminal extension (presequence). The presequences are the targeting signals of the precursor proteins and direct these proteins to mitochondria. The presequences consist of 20–80 amino acid residues. There is no ap- parent sequence identity among the different mito- chondrial protein presequences, but a characteristic common to all of them is a relatively high content of positively charged and nonpolar residues and an almost complete absence of negatively charged residues. This finding suggests that the specificity of the presequences resides in a structural configuration rather than a particular biochemical motif. Most of the presequences probably adopt an a-helical struc- ture. Most mitochondrial proteins, in particular ma- trix proteins, contain their targeting signal in a prese- quence, but there are a few exceptions. A few proteins do not have presequences and apparently contain a targeting signal in their mature form.

Presequences are recognized by specific receptors on the mitochondrial surface (a special class of mito-

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chondrial outer membrane or MOM proteins). The receptors directly interact with another protein, the general insertion protein (GIP), and thereby donate the precursor proteins to this membrane insertion site. The further transport of precursor proteins oc- curs through contact sites, where the mitochondrial outer and inner membranes are closely apposed. The inner membrane has a translocation channel distinct from, but in dynamic interaction with, the transloca- tion channel of the outer membrane. Import across the inner mitochondrial membrane requires ATP and the presence of a membrane potential across this membrane.

The configuration of proteins is important in the process of import across the mitochondrial mem- brane. Completely folded polypeptides are unable to traverse the membrane. Premature folding in the cel- lular cytoplasm of the precursor proteins is prevent- ed by stabilization of the proteins in a translocation- competent, unfolded conformation with help of so- called molecular chaperones. Molecular chaperones do not form part of the final folded or assembled pro- tein structure but prevent nondesired folding and in- teractions of the substrate protein along its transport, folding, and assembly pathways. Appropriate peptide folding is attained after the chaperone has released the polypeptide substrate. The majority of the cur- rently identified molecular chaperones belong to the class of so-called “heat shock proteins.” A different kind of molecular chaperones involved in mitochon- drial precursor proteins consists of the so-called “pre- sequence binding factors.” These proteins, through their interaction with the presequences of mitochon- drial precursors, are also essential in the import path- way of proteins into mitochondria.

After import across the mitochondrial membrane, presequences are proteolytically removed in the mi- tochondrial matrix. This cleavage is necessary for fur- ther assembly of the newly imported polypeptides in- to functional proteins and complexes. Two different proteins are required for full protease activity: mito- chondrial processing peptidase (MPP) and process- ing enhancing protein (PEP). The MPP component contains the catalytic activity, which is stimulated by PEP. After that, the proteins are sorted to their respec- tive mitochondrial subcompartments.

The outer and inner mitochondrial membranes differ in permeability. The transport of metabolites and inorganic ions across the inner membrane is highly regulated. This relative impermeability is im- portant in maintaining a membrane potential and pH gradient across the membrane necessary for oxida- tive phosphorylation. The permeability of the outer membrane is less restricted. The outer membrane contains pore-forming proteins called porins or volt- age-dependent, anion-selective channels (VDAC).

Whether the pore is open or partially closed is depen-

dent on the membrane potential. In addition to VDAC, other types of channels are present in the out- er membrane. Probably the outer membrane has sev- eral permeability pathways differing in selectivity, regulation, and function.

23.3 Mitochondrial Disorders

Mitochondrial disorders are the most common in- born errors of metabolism, with an estimated inci- dence of 1 per 10,000 live births. They present as an extremely heterogeneous group of disorders with variable age of onset, progression, and severity. Con- sidering the central role of mitochondria in cellular metabolism, it is not surprising that mitochondrial diseases are often multiorgan disorders with predom- inant involvement of brain and muscles. Mitochondr- ial disorders can follow either a maternal, autosomal, or X-linked mode of inheritance.

Many mitochondrial disorders follow maternal in-

heritance. A mother carrying a mtDNA mutation will

transmit it to all her children, male and female, but

only her daughters will pass it on to their children. At

a clinical level maternal transmission may be difficult

to detect. Due to heteroplasmy, unequal mitotic segre-

gation, and threshold effect, different individuals in

the matrilinear lineage may differ in symptomatology

and in organ involvement; some may even be asymp-

tomatic. In most cases of mitochondrial genomic de-

fects, heteroplasmy is present, with occurrence of

both wild type and mutant mtDNA. At cell division

mitochondria and mtDNA are haphazardly distrib-

uted between the daughter cells and consequently the

proportion of mutant genomes may differ between

daughter cells. The percentage of mutant DNA versus

normal DNA may, therefore, be very different in dif-

ferent children of the same mother, and in different

tissues in the same person, and may alter in the course

of time. Whether or not the mtDNA mutation is actu-

ally expressed is largely determined by the relative

proportion of normal versus mutant genomes in a

given tissue. A minimum critical number of mutant

DNA is necessary to impair energy metabolism se-

verely enough to cause dysfunction of that particular

organ or tissue. This phenomenon is known as the

threshold effect. The number of affected mitochon-

dria and cells needed to cause organ dysfunction

varies from tissue to tissue depending on the vulner-

ability of that particular tissue to impairments of ox-

idative phosphorylation. The relative reliance of tis-

sues on oxidative phosphorylation energy decreases

in the following order: CNS, skeletal muscle, heart,

kidney, and liver. The presence of a mutation in a par-

ticular percentage of mitochondrial genomes may

lead to signs of encephalopathy and/or myopathy,

without any sign of dysfunction of other organs. The

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metabolic vulnerability may also vary in the same tis- sue with time and according to functional demands.

As the proportion of mutant mitochondrial genomes may shift in daughter cells, this may also be the cause of a change in phenotype. In general, there is a decline in oxidative phosphorylation capacity with age. The most likely mechanism for this phenomenon is the accumulation of damage to mtDNA in the face of in- sufficient ability to repair DNA alterations. This phe- nomenon may explain the late age of onset of clinical signs and symptoms in some patients, and the in- crease in severity of the disease with age.

The mitochondrial genome depends heavily on the nuclear genome, which encodes several factors in- volved in mtDNA replication, transcription, and translation. Faulty communications between nuclear and mitochondrial genomes may lead to either multi- ple mtDNA deletions or mtDNA depletion. These dis- orders are transmitted by mendelian inheritance, be- cause the primary genetic defect resides in nDNA.

The nuclear genome also contains the genes encoding structural mitochondrial proteins, most of the respi- ratory chain proteins, and the proteins necessary for the proper assembly of the oxidative phosphorylation complexes.

The classification of these pheno- and genotypi- cally heterogeneous diseases is very difficult. Classifi- cation may be based on the clinical presentation, on the age of presentation and possible progression, or on the underlying biochemical or genetic defects.

A previous classification by DeVivo (1993) is based on a genetic framework. The major subdivision is into nDNA defects, mtDNA defects, and intergenomic signaling defects.

This original classification developed further with time, e.g., with the description of defects in assembly factors, defects in mtDNA maintenance (replication control), and defects in oxidative phosphorylation system biogenesis. The classification of DiMauro and Schon (2003) has incorporated these recent insights:

1. Respiratory chain disorders due to defects in mtDNA

(a) Mutations in protein synthesis genes (b) Mutations in protein coding genes

2. Respiratory chain disorders due to defects in nDNA

(a) Mutations in structural components of the res- piratory chain

(b) Mutations in ancillary proteins of the respira- tory chain

(c) Defects in intergenomic signaling affecting respiratory function

(d) Defects of the membrane lipid milieu

(e) Defects in mitochondrial motility, fusion, and fission

3. Disorders with indirect involvement of the respi- ratory chain

(a) Defects of mitochondrial protein importation (b) Defects in mitochondrial motility

(c) Neurodegenerative disorders

For the clinician, the genetic origin of the mitochon- drial defect is the most important item in the classifi- cation, recognizing two major groups: mtDNA defects (point mutations in structural proteins of the respira- tory chain, point mutations in tRNAs and rRNAs, and major mtDNA rearrangements) and nDNA defects (defects in structural oxidative phosphorylation pro- teins, defects in genes encoding assembly factors, de- fects in genes involved in the biogenesis of the oxida- tive phosphorylation system, and defects in the main- tenance of mtDNA).

In a simplified view, a positive family history sug- gestive of maternal inheritance may help in the early diagnosis in mtDNA defects, but the clinical presenta- tion may be extremely variable in different family members. In many affected individuals the disease becomes evident only in early adulthood and may be organ-specific. Mitochondrial deletions, however, are most often sporadic, the symptoms may appear early, and the family history is negative.

In nDNA defects, the family history is noncontrib- utory in most cases, the parents are healthy, the symp- toms frequently arise soon after birth, and when pro- gressive, the disease may be fatal in early childhood.

Sibs may be affected to a variable degree (genetic and environmental factors), but the clinical variability is usually much less marked than in mtDNA defects.

23.3.1 Mitochondrial DNA Defects

A variety of defects in mtDNA can be distinguished:

point mutations, deletions, and duplications. The DNA defects may involve genes coding for proteins of the respiratory chain (structural proteins) or genes cod- ing for tRNA or rRNA. mtDNA depletion is related to a nDNA defect and is included under nuclear defects of mtDNA maintenance.

In cases of mutations involving genes encoding structural proteins of the respiratory chain, biochemi- cal analysis reveals a defect restricted to one respira- tory enzyme complex (complex I, II, II, IV, or V). In such patients the primary biochemical analysis, demonstrating a usually partial isolated complex de- ficiency, leads to targeted mutation analysis of partic- ular mitochondrial structural genes. Different point mutations in genes coding for components of com- plex I have been observed in exercise intolerance, Leber hereditary optic neuropathy (LHON), Leigh

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syndrome, Leigh-like syndrome, progressive epilepsy, adult-onset dystonia associated with neurological and ophthalmological symptoms, and in mitochon- drial encephalomyopathy, lactic acidosis, and stroke- like episodes (MELAS) syndrome. mtDNA mutations involving components of complex III have been re- ported in patients with progressive exercise intoler- ance, proximal limb weakness with myoglobinuria, Parkinsonism, and LHON. In complex IV defects, structural mutations have been detected in patients presenting with isolated motor neuron disease, Leigh- like syndrome, MELAS, proximal myopathy with lac- tic acidosis, and LHON. In patients with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and Leigh syndrome, common mutations are known in a gene coding for an ATP synthetase (com- plex V) subunit.

A number of point mutations in tRNA and rRNA genes have been identified. Each tRNA mutation is generally associated with a distinctive phenotype, al- though some phenotypic overlap between the differ- ent tRNA defects is common. Well-known clinical phenotypes include myoclonic epilepsy with ragged red fibers (MERRF), MELAS, Leigh syndrome and variable combinations of myopathy, cardiomyopathy, chronic progressive external ophthalmoplegia (CPEO), diabetes mellitus, and hearing loss. Further- more, a few rRNA mutations have been also described with a very similar phenotype of CNS involvement, deafness, neuropathy, and diabetes mellitus. Despite the overlap in the clinical symptoms mentioned, the association of different tRNA mutations with clinical syndromes is surprisingly specific considering that these mutations all act on the same mitochondrial function, i.e., the ability of mitochondria to translate their own genes. Since mutant tRNA is unavailable for translation, all mitochondrially encoded proteins are present in decreased amounts. Among the different clinical phenotypes the biochemical abnormalities are virtually indistinguishable, revealing multiple partial complex defects.

Large, single deletions of a substantial proportion of mtDNA have been described in CPEO, Pearson syn- drome, and Kearns–Sayre syndrome (KSS). These syndromes are clinically overlapping. Muscle weak- ness and chronic progressive external ophthalmople- gia are also present in KSS. Many patients with Pear- son syndrome die in early childhood from a combina- tion of pancreatic, hepatic, renal, and bone marrow insufficiency with pancytopenia, but the few patients who improve and survive develop KSS and histologi- cal signs of a mitochondrial myopathy in later child- hood. The deletions encompass many of the genes en- coding for proteins of the respiratory chain, and also several tRNAs. This explains the decrease in presence of all mitochondrial translation products demon- strated by immunological analyses. Biochemical

analysis reveals decreased activity of complexes I, III, and IV, individually or in combination. The percent- age of deleted mtDNA is similar in muscles of both CPEO and KSS patients, but deletions are restricted to skeletal muscle in CPEO and widely distributed in ex- tramuscular tissue in KSS. In Pearson syndrome high amounts of mtDNA defects are present in bone mar- row precursor cells. The proportion probably falls with age if the patient survives; however, repeated in- vestigation has shown an increased proportion of deleted mtDNA in muscle tissue. Most (or all) of the cases associated with single DNA deletions are spo- radic. The deletion probably occurs after egg fertiliza- tion and owing to unequal mitotic segregation is pre- sent in only some of the cells. The replication of delet- ed mtDNA occurs more rapidly and, in consequence, positive selection of respiratory-deficient cells may occur in tissues with rapid cell turnover.

Duplications of mtDNA are very rare in man. Spo- radic cases of KSS with heteroplasmic mtDNA dupli- cations have been described.

The relationship between mtDNA changes, bio- chemical defects of the respiratory chain, and clinical phenotype remains difficult to understand (genetic and phenotypic heterogeneity). One point mutation can be associated with different clinical phenotypes (for instance, the same mutation is present in both NARP and some cases of Leigh syndrome; the same mutation in a tRNA gene is found in MELAS, in ma- ternally inherited myopathy and cardiomyopathy, and in maternally inherited diabetes mellitus). The phenotype related to the same abnormality in mtDNA may also vary within a single kindred. The same disease can be caused by different point muta- tions (see Chap. 25). Combined features of different syndromes have been observed in one patient (KSS combined with MELAS; MERRF with MELAS; Pear- son syndrome progressing to KSS). Part of the rela- tionship between genotype and phenotype can be explained by heteroplasmy with different percentages of mutant mtDNA in different tissues and changes of percentages over the course of time.

23.3.2 Nuclear DNA Defects

Nuclear genes responsible for oxidative phosphoryla- tion defects can be categorized structurally and func- tionally into genes encoding structural components of the oxidative phosphorylation system, genes en- coding assembly factors of the oxidative phosphory- lation complexes, genes involved in the biogenesis of the oxidative phosphorylation system, and genes in- volved in the maintenance of the mtDNA.

Among the defects in structural components of the

oxidative phosphorylation system, complex I deficien-

cy is the most common. In general, isolated complex I

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deficiency is one of the most frequent disturbances of the oxidative phosphorylation system and often fol- lows an autosomal recessive inheritance. The clinical presentation in the majority of cases is of a Leigh or a Leigh-like syndrome, a leukodystrophy, or a car- diomyopathy. Mutations have been identified in seven of the 39 nuclear genes encoding structural subunits of complex I. Complex II comprises four nuclear en- coded subunits. It has a dual function as an enzyme complex of the oxidative phosphorylation system and as an essential enzyme of the citric acid cycle. It con- sists of a flavoprotein (active site with covalently bound FAD) and an iron–sulfur protein, and is an- chored to the membrane by two transmembrane pro- teins. A mutation in the flavoprotein coding gene of a patient with Leigh syndrome was the first mutation identified in a nuclear gene causing oxidative phos- phorylation deficiency. Mutations in the genes encod- ing the iron–sulfur protein and the anchor proteins cause a different phenotype of hereditary paragan- gliomas and/or pheochromocytomas. Complex III transfers electrons from the ubiquinone pool to cy- tochrome c, and is composed of 11 subunits, of which ten are nuclear-encoded. Complex III deficiency is of- ten associated with encephalomyopathy or cardiomy- opathy. However, in the patient with the first mutation described in a nuclear-encoded subunit of complex III (an enzyme-binding protein), no psychomotor re- tardation or neurological impairment was detected, but hypoglycemia and lactic acidemia were. To date no mutations have been reported in the structural nu- clear components of complex IV and complex V. Most syndromes with isolated complex IV deficiency are caused by mutations in genes encoding proteins in- volved in the proper assembly of the complex.

Assembly factors are proteins which mediate the process of assemblage of subunits and intermediate complexes into fully assembled oxidative phosphory- lation complexes, and have chaperone-like functions.

A few assembly factors have been identified for com- plex I and III, and many for complex IV. Mutations of the only known assembly factor gene responsible for isolated complex III deficiency (Rieske iron–sulfur subunit) are associated with neonatal proximal tubu- lopathy, hepatic involvement and encephalopathy, and with GRACILE syndrome (growth retardation, amino aciduria, cholestasis, iron overload, lactic aci- dosis, and early death), a fatal metabolic disorder with iron overload. Complex IV (COX) is composed of 13 structural subunits, ten of which are encoded by the nucleus. A large number of accessory factors are nec- essary for the assembly and maintenance of the active complex, and mutations in several of these factors have been described. The first gene encoding an as- sembly factor known to be responsible for COX defi- ciency is the SURF1 gene. Leigh syndrome is the most common clinical manifestation observed in patients

with SURF1 mutation, but milder neurological in- volvement with a malabsorption syndrome and cases with a leukoencephalopathy have been described in association with SURF1 mutations as well. Mutations in two other COX assembly genes, SCO1 and SCO2, frequently present with hypertrophic cardiomyopa- thy and encephalopathy, sometimes combined with hepatic failure. The COX10 and COX15 genes are in- volved in the synthesis of heme A, a prosthetic group of COX. Mutations in these genes result in a clinical manifestation comparable to that of SCO2 mutations.

A novel mutation has been recently described in the ATP12 assembly gene of complex V with severe mi- crocephaly, hepatomegaly, and early death.

As to defects in the biogenesis of the oxidative phos- phorylation system, oxidative phosphorylation defi- ciencies can caused by defects in the import of nu- clear-encoded mitochondrial proteins into the mito- chondrion. A mutation in the gene for subunit 8a of the translocase of the inner mitochondrial mem- brane, the TIMM8A or DDP1 gene, causes human dys- tonia deafness syndrome, also known as Mohr–Tra- jenberg syndrome, a progressive neurodegenerative disorder. Friedreich ataxia, an autosomal recessive disorder, is caused by disturbances of the iron–sulfur cluster formation, related to an abnormal expansion of a GAA repeat in the first intron of the frataxin gene, which encodes a mitochondrial protein of unknown function. The defect in frataxin results in oxidative damage of the highly sensitive iron–sulfur protein complexes I, II, III, and the Krebs cycle enzyme aconi- tase. Hereditary spastic paraplegia (HSP) is a geneti- cally heterogeneous neurodegenerative disorder characterized by progressive spasticity and weakness of the legs.A subtype of HSP is related to mutations in SPG7. Patients with this subtype show typical signs of mitochondrial disease (ragged red fibers and COX- negative fibers). The spastic paraplegia gene SPG7 en- codes a nuclear-encoded mitochondrial metallopro- tease protein, which has both proteolytic and chaper- one-like activities at the inner mitochondrial mem- brane. The oxidative phosphorylation system may be disturbed not only by defects in factors which are di- rectly part of the system or involved in its biogenesis, but also by defects in factors that indirectly con- tribute to its function, such as alterations in the lipid composition of the inner membrane (Barth syn- drome). Dominant optic atrophy (DOA) is caused by defects in the OPA1 gene, which encodes a mitochon- drial GTPase that is important for the formation and maintenance of the mitochondrial network.

Mutations in nuclear genes involved in mtDNA maintenance cause disorders that clinically resemble disorders caused by mtDNA mutations. This is under- standable since the nuclear gene defect causes sec- ondary mtDNA loss (mtDNA depletion) or formation of multiple mtDNA deletions. However, these disor-

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ders follow a mendelian inheritance pattern. Multiple deletions of the mtDNA have been described in a number of clinical syndromes. The most frequently described is autosomal dominant progressive exter- nal ophthalmoplegia (AD PEO), for which three dif- ferent disease genes have been identified: ANT1, en- coding the adenine nucleotide translocator or ADP/ATP translocator; POLG1, encoding the catalyt- ic subunit of the mtDNA-specific polymerase g; and C10ORF2 encoding the Twinkle protein, a putative mtDNA helicase. For defects in POLG1, mutations with a recessive mode of inheritance have also been reported. The mtDNA depletion syndrome causes a rapidly fatal mitochondrial disorder in infancy.

Quantitative analysis of the mtDNA reveals a severe depletion, varying from 50% up to 98% as compared to normal controls. Depletion of mtDNA correlates with the presence of multiple respiratory chain de- fects. Clinically three syndromes are distinguished:

fatal infantile hepatopathy, congenital myopathy with or without nephropathy, and later-onset infantile