Mitochondria and Mitochondrial Disorders

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E. Morava,J.A.M. Smeitink

23.1 Mitochondrial Structure and Function

Mitochondria are membranous organelles that provide the energy necessary for the different cell functions. 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 compartments: 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 contain many mitochondria, the actual number differing in relation to the energy requirements of the type of cell. Mitochondria are dynamic organelles. They undergo 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 energy by mitochondria is called oxidative phosphorylation. 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. Subsequently it is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHc),which produces CO2 and acetyl-CoA, while reducing one nicotinamide adenine dinucleotide (NAD+) to NADH + H+.

The pyruvate dehydrogenase complex is located at the inner border of the inner mitochondrial membrane.

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 activated 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 (CPT1), 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 reaction 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 mitochondria 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 ß-oxidation. This ß-oxidation spiral involves four successive reactions, 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 adenine dinucleotide (FAD) to FADH2 and NAD+ to NADH + H+. An acyl-CoA can recycle through ß-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 dehydrogenase from 4 to 12 carbons; and short-chain acyl-CoA dehydrogenase from 4 to 6 carbons. Fatty acids with double bonds require additional enzymes, 63, 62-enoyl-CoA isomerase and 2,4-dienoyl-CoA re-ductase. In the liver, the metabolic end product of each cycle of ß-oxidation, acetyl-CoA, is primarily converted to ketone bodies, while in other tissues acetyl-CoA is completely oxidized via the citric acid

Fatty Acid Oxidation Disorder

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


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

Oxidation Fatty Acid


Fig. 23.2. Citric acid cycle in the mitochondrial matrix


Fig. 23.2. Citric acid cycle in the mitochondrial matrix

23.1 Mitochondrial Structure and Function 197

23.1 Mitochondrial Structure and Function 197

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

cycle. Electrons from the acyl-CoA dehydrogenase-mediated reactions are transferred to electron transfer 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-oxidation of fatty acids. It can also be derived from breakdown of certain amino acids (for instance, leucine and isoleucine). Acetyl-CoA and oxaloacetate condense 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 CO2 and reducing equivalents in the form of NADH + H+ and FADH2. Certain amino acids can be converted to citric acid cycle intermediates, such as glutamate, aspartate, alanine, proline, and glutamine.

The reducing equivalents NADH and FADH2, produced by b-oxidation of fatty acids and the citric acid cycle, are reoxidized to NAD+ and FAD by the respiratory chain. This oxidation sequence is tightly coupled to the phosphorylation of ADP to yield ATP. This process of so-called oxidative phosphorylation is responsible 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 (succinate ubiquinone reductase) accepts reducing equivalents from succinate and also passes electrons down the chain to ubiquinone to yield ubiquinol. The electrons from ubiquinol are transferred to complex III (ubiquinol: cytochrome-c reductase),subsequently to cytochrome c, then to complex IV (cytochrome-c oxidase), 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 complexes I, III, and IV. The resulting electrochemical gradient 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 adenine 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 substrate 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 respiration.

Each complex of the respiratory chain is made up of a number of protein components. Complex I contains 46 polypeptides and has as prosthetic groups flavin mononucleotide and several nonheme iron-sulfur clusters. Complex II consists of 4 sub-units. Complex III is composed of 11 subunits including cytochrome ^cytochrome c1,and a nonheme iron protein. Complex IV is composed of 13 different protein units, 2 cytochromes (a and a3) and two copper atoms. Complex V is composed of 16 different polypeptides. Two small electron carriers, ubiquinone (coenzyme Q10) and cytochrome c (a low molecular weight hemoprotein) act as shuttles between the complexes.

23.2 Mitochondrial Genetics

Human cells possess two different genomes: nuclear DNA (nDNA), a 3x109-base-pair-long genome, present in two copies in each cell, and mitochondrial DNA (mtDNA), a 16,569-base pair-long genome, present 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 mitochondrial 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 mitochondria (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) necessary for expression of these genes (13 messenger RNAs [mRNAs], 22 transfer RNAs [tRNAs], and 2 ribosomal RNAs [rRNAs]). The 13 mRNAs specify as many polypeptides of the respiratory chain: 7 sub-units of complex I, the cytochrome b subunit of complex III, 3 subunits of complex IV and 2 subunits of complex V.

In mtDNA, the gene organization is highly compact; 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 important 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 spontaneous 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 individual, either male or female, is inherited from the mother, 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 almost no cytoplasm to the zygote.

The replication and expression of mtDNA are controlled by nuclear genes. The nuclear genome furthermore 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 synthesized in the cellular cytoplasm on free polyribo-somes and have to be imported from the cytoplasm into the mitochondria through a complicated translocation machine, which is under the control of the nuclear genome. In addition, nDNA encodes several factors controlling mtDNA replication, transcription and translation.

Most nuclear-encoded mitochondrial proteins destined for the inner three mitochondrial compartments (the intermembrane space, the inner membrane, 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 apparent 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 structure. Most mitochondrial proteins, in particular matrix 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-

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 occurs 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 translocation 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 membrane. Completely folded polypeptides are unable to traverse the membrane. Premature folding in the cellular cytoplasm of the precursor proteins is prevented 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 protein structure but prevent nondesired folding and interactions 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 currently identified molecular chaperones belong to the class of so-called "heat shock proteins." A different kind of molecular chaperones involved in mitochondrial precursor proteins consists of the so-called "pre-sequence binding factors." These proteins, through their interaction with the presequences of mitochondrial precursors, are also essential in the import pathway of proteins into mitochondria.

After import across the mitochondrial membrane, presequences are proteolytically removed in the mi-tochondrial matrix. This cleavage is necessary for further assembly of the newly imported polypeptides into functional proteins and complexes. Two different proteins are required for full protease activity: mito-chondrial processing peptidase (MPP) and processing enhancing protein (PEP). The MPP component contains the catalytic activity, which is stimulated by PEP. After that, the proteins are sorted to their respective 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 important 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 voltage-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 outer membrane. Probably the outer membrane has several permeability pathways differing in selectivity, regulation, and function.

23.3 Mitochondrial Disorders

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

Many mitochondrial disorders follow maternal inheritance. 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 segregation, and threshold effect, different individuals in the matrilinear lineage may differ in symptomatology and in organ involvement; some may even be asymptomatic. In most cases of mitochondrial genomic defects, heteroplasmy is present, with occurrence of both wild type and mutant mtDNA. At cell division mitochondria and mtDNA are haphazardly distributed 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 different 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 actually 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 severely enough to cause dysfunction of that particular organ or tissue. This phenomenon is known as the threshold effect. The number of affected mitochondria and cells needed to cause organ dysfunction varies from tissue to tissue depending on the vulnerability of that particular tissue to impairments of ox-idative phosphorylation. The relative reliance of tissues on oxidative phosphorylation energy decreases in the following order: CNS, skeletal muscle, heart, kidney, and liver. The presence of a mutation in a particular percentage of mitochondrial genomes may lead to signs of encephalopathy and/or myopathy, without any sign of dysfunction of other organs. The metabolic vulnerability may also vary in the same tissue 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 insufficient ability to repair DNA alterations. This phenomenon may explain the late age of onset of clinical signs and symptoms in some patients, and the increase in severity of the disease with age.

The mitochondrial genome depends heavily on the nuclear genome, which encodes several factors involved in mtDNA replication, transcription, and translation. Faulty communications between nuclear and mitochondrial genomes may lead to either multiple mtDNA deletions or mtDNA depletion. These disorders are transmitted by mendelian inheritance, because the primary genetic defect resides in nDNA. The nuclear genome also contains the genes encoding structural mitochondrial proteins, most of the respiratory 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. Classification 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 respiratory chain

(b) Mutations in ancillary proteins of the respiratory 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 respiratory chain

(a) Defects of mitochondrial protein importation

(b) Defects in mitochondrial motility

(c) Neurodegenerative disorders

For the clinician, the genetic origin of the mitochondrial defect is the most important item in the classifi-cation,recognizing two major groups: mtDNA defects (point mutations in structural proteins of the respiratory chain, point mutations in tRNAs and rRNAs, and major mtDNA rearrangements) and nDNA defects (defects in structural oxidative phosphorylation proteins, defects in genes encoding assembly factors, defects in genes involved in the biogenesis of the oxida-tive phosphorylation system, and defects in the maintenance of mtDNA).

In a simplified view, a positive family history suggestive of maternal inheritance may help in the early diagnosis in mtDNA defects,but the clinical presentation 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 symptoms frequently arise soon after birth, and when progressive, 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 coding 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, biochemical analysis reveals a defect restricted to one respiratory enzyme complex (complex I, II, II, IV, or V). In such patients the primary biochemical analysis, demonstrating a usually partial isolated complex deficiency, leads to targeted mutation analysis of particular mitochondrial structural genes. Different point mutations in genes coding for components of complex I have been observed in exercise intolerance, Leber hereditary optic neuropathy (LHON), Leigh syndrome, Leigh-like syndrome, progressive epilepsy, adult-onset dystonia associated with neurological and ophthalmological symptoms, and in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome. mtDNA mutations involving components of complex III have been reported in patients with progressive exercise intolerance, 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, Leighlike syndrome, MELAS, proximal myopathy with lactic 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 (complex 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, although some phenotypic overlap between the different 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. Furthermore, 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 syndrome, and Kearns-Sayre syndrome (KSS). These syndromes are clinically overlapping. Muscle weakness and chronic progressive external ophthalmople-gia are also present in KSS. Many patients with Pearson syndrome die in early childhood from a combination 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 childhood. The deletions encompass many of the genes encoding for proteins of the respiratory chain, and also several tRNAs. This explains the decrease in presence of all mitochondrial translation products demonstrated by immunological analyses. Biochemical analysis reveals decreased activity of complexes I, III, and IV, individually or in combination. The percentage 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 marrow precursor cells. The proportion probably falls with age if the patient survives; however, repeated investigation has shown an increased proportion of deleted mtDNA in muscle tissue. Most (or all) of the cases associated with single DNA deletions are sporadic. The deletion probably occurs after egg fertilization and owing to unequal mitotic segregation is present in only some of the cells. The replication of deleted 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. Sporadic cases of KSS with heteroplasmic mtDNA duplications have been described.

The relationship between mtDNA changes, biochemical 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 maternally 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 mutations (see Chap. 25). Combined features of different syndromes have been observed in one patient (KSS combined with MELAS; MERRF with MELAS; Pearson syndrome progressing to KSS). Part of the relationship 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 functionally into genes encoding structural components of the oxidative phosphorylation system, genes encoding assembly factors of the oxidative phosphory-lation complexes, genes involved in the biogenesis of the oxidative phosphorylation system, and genes involved in the maintenance of the mtDNA.

Among the defects in structural components of the oxidative phosphorylation system, complex I deficiency is the most common. In general, isolated complex I

deficiency is one of the most frequent disturbances of the oxidative phosphorylation system and often follows 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 cardiomyopathy. Mutations have been identified in seven of the 39 nuclear genes encoding structural subunits of complex I. Complex II comprises four nuclear encoded 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 consists of a flavoprotein (active site with covalently bound FAD) and an iron-sulfur protein, and is anchored to the membrane by two transmembrane proteins. 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 encoding 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 often associated with encephalomyopathy or cardiomyopathy. However, in the patient with the first mutation described in a nuclear-encoded subunit of complex III (an enzyme-binding protein), no psychomotor retardation or neurological impairment was detected, but hypoglycemia and lactic acidemia were. To date no mutations have been reported in the structural nuclear components of complex IV and complex V. Most syndromes with isolated complex IV deficiency are caused by mutations in genes encoding proteins involved 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 complex 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 necessary for the assembly and maintenance of the active complex, and mutations in several of these factors have been described. The first gene encoding an assembly factor known to be responsible for COX deficiency is the SURF1 gene. Leigh syndrome is the most common clinical manifestation observed in patients with SURF1 mutation, but milder neurological involvement 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 involved 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 microcephaly, hepatomegaly, and early death.

As to defects in the biogenesis of the oxidative phos-phorylation system, oxidative phosphorylation deficiencies can caused by defects in the import of nuclear-encoded mitochondrial proteins into the mitochondrion. A mutation in the gene for subunit 8a of the translocase of the inner mitochondrial mem-brane,the TIMM8A or DDP1 gene,causes human dystonia 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 genetically 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 encodes a nuclear-encoded mitochondrial metallopro-tease protein, which has both proteolytic and chaper-one-like activities at the inner mitochondrial membrane. The oxidative phosphorylation system may be disturbed not only by defects in factors which are directly part of the system or involved in its biogenesis, but also by defects in factors that indirectly contribute to its function, such as alterations in the lipid composition of the inner membrane (Barth syndrome). 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 understandable since the nuclear gene defect causes secondary mtDNA loss (mtDNA depletion) or formation of multiple mtDNA deletions. However, these disor 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 external ophthalmoplegia (AD PEO), for which three different disease genes have been identified: ANT1, encoding the adenine nucleotide translocator or ADP/ATP translocator; POLG1, encoding the catalytic 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 defects. Clinically three syndromes are distinguished: fatal infantile hepatopathy, congenital myopathy with or without nephropathy, and later-onset infantile or childhood progressive encephalomyopathy. Two genes involved in mitochondrial deoxyribonucleo-side metabolism, TK2, encoding thymidine kinase 2, and DGUOK, encoding deoxyguanosine kinase, have been associated with a myopathic form and a hepa-toencephalopathic form of mtDNA depletion syndrome, respectively. Thymidine kinase 2 and de-oxyguanosine kinase are both enzymes of the "salvage pathway," which is the main supply of deoxyri-bonucleoside triphosphates (dNTPs) for mtDNA synthesis. The link between these salvage pathway enzymes and mtDNA depletion suggests their involvement in the maintenance of balanced mitochondrial dNTP pools. Deficiency of thymidine phosphorylase causes multiple mtDNA deletions and/or mtDNA depletion leading to mitochondrial neurogastrointesti-nal encephalomyopathy (MNGIE). Recently, mutations in a mitochondrial deoxynucleotide carrier (DNC) have been shown to cause failure of deoxynu-cleotide transport across the inner mitochondrial membrane in Amish congenital microcephaly.

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