Description

General overview. Mitochondrial disorders describe a broad range of disorders that have multiple genetic causes and variable symptoms and signs. Incidence is estimated to be as high as 1:4300 of all types of mitochondrial disorders (46). They are, by definition, dysfunction of mitochondria, which normally provide cell energy. Thus, the dysfunction of high-energy organs, including the brain, muscle, nervous system, eyes, heart, kidneys, pancreas, liver, and many more organs, is common yet frequently nonspecific (89; 04).

Categorizing and describing a mitochondrial disorder is complicated in part because there are three ways to do so. This reflects the history of understanding these disorders (92). Initially, these disorders were named according to symptoms (later the cause was found to be mitochondrial dysfunction and, more specifically, maternally inherited mitochondrial DNA variations). Then biochemical assays such as oxidative phosphorylation activity by respiratory chain enzymes were used to characterize enzyme disorders (eg, complex 1 or complex 3 deficiencies). Finally, we have entered the era of genetic diagnosis. Now, a variety of genes that cause mitochondrial disease have been discovered. There are over 350, with an additional 16 recognized in the last 1 year (78). The named and biochemical disorders may or may not have been caused by their initially described genetic variation but potentially by other gene variations. Complicating this picture is that there are two sources of genetic material—maternal inherited mtDNA and nuclear DNA.

In general, mitochondrial disorders are disorders of energy production. Mitochondria, according to the widely accepted “endosymbiotic hypothesis,” are the relics of protobacteria that populated anaerobic nucleated cells and endowed them with oxidative metabolism. Thus, mitochondria are the main source of energy for all human tissues. Mitochondria do not only contain oxidative phosphorylation (and the electron chain transport) but also contain many metabolic pathways, including the pyruvate dehydrogenase complex, protein amino acid catabolic pathways, the carnitine cycle, beta-oxidation, and the tricarboxylic acid cycle (also known as Krebs cycle or citric acid cycle) (99).

Although defects in all of these pathways are, by definition, mitochondrial diseases, the term “mitochondrial encephalomyopathy” has come to indicate disorders due to defects in the respiratory chain (30; 27). This is the “business end” of oxidative metabolism, where ATP is generated. Reducing equivalents produced in the Krebs cycle and in the beta-oxidation spirals are passed along a series of protein complexes embedded in the inner mitochondrial membrane (the electron transport chain). The electron transport chain consists of four multimeric complexes (I to IV) plus two small electron carriers, coenzyme Q10 (or ubiquinone) and cytochrome c. The energy generated by these reactions is used to pump protons from the mitochondrial matrix into the space between the inner and outer mitochondrial membranes. This creates an electrochemical proton gradient, which is utilized by complex V (or ATP synthase) to produce ATP in a process known as oxidation/phosphorylation coupling.

A unique feature of the respiratory chain is its dual genetic control: mitochondrial DNA (mtDNA) encodes 13 of the approximately 89 proteins that compose the respiratory chain, and nuclear DNA (nDNA) encodes all the others. Notably, complex II (also known as succinate dehydrogenase—a TCA cycle enzyme), coenzyme Q10, and cytochrome c are exclusively encoded by nDNA. In contrast, complexes I, III, IV, and V contain some subunits encoded by mtDNA: seven for complex I (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6), one for complex III (cytochrome b), three for complex IV (COX I, COX II, and COX III), and two for complex V (ATPase 6 and ATPase 8) (30; 27).

History. Initially, mitochondrial disorders were described by symptoms, and the first were the mitochondrial myopathies (affecting muscles). Mitochondrial myopathies were described in the early 1960s when systematic ultrastructural and histochemical studies revealed excessive proliferation of normal- or abnormal-looking mitochondria in muscle of patients with weakness or exercise intolerance (111; 112). Because, with the modified Gomori trichrome stain, the areas of mitochondrial accumulation appeared crimson, the abnormal fibers were dubbed “ragged-red fibers” (38) and came to be considered the pathological hallmark of mitochondrial disease.

However, it soon became apparent that in many patients with ragged-red fibers, the myopathy is associated with symptoms and signs of brain involvement, and the term “mitochondrial encephalomyopathies” was introduced. It also became clear that the lack of ragged-red fibers in the biopsy does not exclude a mitochondrial etiology, as exemplified by Leigh syndrome, an encephalopathy of infancy or childhood invariably due to mitochondrial dysfunction but rarely accompanied by ragged-red fibers.

Through the 1970s and 1980s, abnormalities of oxidative phosphorylation were identified (29), so this was the assay often done. Multisystem disorders such as Kearns-Sayre (limb weakness, progressive ataxia, and ophthalmologic abnormalities) and Leigh syndrome (brain MRI findings consistent with encephalopathy) were associated with mitochondrial disease. In the late 1970s, Koeningsberger described mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), as well as myoclonic epilepsy with ragged red fibers (MERRF) (64). Once mitochondrial DNA was characterized in 1988 (54), variations were quickly associated with diseases that had been described. However, not all patients with clear mitochondrial disease (by symptoms or respiratory chain enzyme assays) had abnormal mtDNA, and with the advent of nuclear DNA sequencing, more and more nuclear genes were associated with mitochondrial disease (12). The list of nuclear genes that cause mitochondrial disease continues to expand.

mtDNA. Human mtDNA is a 16,569-kb circular, double-stranded molecule containing 37 genes: two rRNA genes, 22 tRNA genes, and 13 structural genes encoding the respiratory chain subunits listed above.

In the course of evolution, mtDNA has lost much of its original autonomy and now depends heavily on the nuclear genome for the production of factors needed for mtDNA integrity; transcription, translation, and replication (“maintenance”); inner membrane integrity; and mitochondrial dynamics (27). Mitochondrial disease can be inherited through the human mitochondrial DNA (mtDNA), which is inherited through the maternal line because it is a circular double-stranded molecule. It can also be inherited through nuclear inheritance patterns, including X-linked, autosomal recessive, and autosomal dominant manners.

Since 1988, the circle of mtDNA has become crowded with pathogenic mutations, and the principles of mitochondrial genetics should, therefore, be familiar to the practicing physician.

Heteroplasmy and threshold effect. Each cell contains hundreds or thousands of mtDNA copies, which, at cell division, distribute randomly among daughter cells. In normal tissues, all mtDNA molecules are identical (homoplasmy). Deleterious mtDNA mutations usually (but not always) affect only some (but not all) mtDNAs. The clinical expression of a pathogenic mtDNA mutation is largely determined by the relative proportion of normal and mutant genomes in different tissues. A minimum critical number of mutant mtDNAs is required to cause mitochondrial dysfunction in a particular organ or tissue and mitochondrial disease in an individual (threshold effect). Studies examining a known mtDNA mutation, m.3243 A>G, demonstrated that the amount of heteroplasmy in blood and urine > muscle and blood > muscle and urine seemed to correlate with disease severity (47).

Mitotic segregation. At cell division, the proportion of mutant mtDNAs in daughter cells may shift, and the phenotype may change accordingly. This phenomenon, called “mitotic segregation,” explains how the clinical phenotype may change in certain patients with mtDNA-related disorders as they grow older.

Maternal inheritance. At fertilization, all mtDNA derives from the oocyte. Therefore, the mode of transmission of mtDNA and mtDNA point mutations (single deletions of mtDNA are usually sporadic events) differs from Mendelian inheritance. A mother carrying an mtDNA point mutation will pass it on to all her children (boys and girls), but only her daughters will transmit it to their progeny.

Nuclear DNA (nDNA). In the last several years, there has been an increasing recognition that mitochondrial disease is not just inherited as mtDNA but also is from both parents through genes passed on by the nucleus.

For the most part, most nuclear-inherited mitochondrial disorders are inherited in an autosomal recessive manner such that both parents are carriers and the affected offspring has two defective copies of the gene causing the mitochondrial disease. However, X-linked and autosomal dominant inheritance also occur. A common example of X-linked is pyruvate dehydrogenase deficiency from mutations in the PDHA1 subunit located on the X chromosome. Disorders from the autosomally located POLG can be inherited in an autosomal dominant and an autosomal recessive manner, depending on the type of variation.

Abnormal oxidative phosphorylation from abnormal respiratory chain enzymes is not the only cause of mitochondrial disease. Mitochondrial oxidative phosphorylation is dependent on numerous processes. The respiratory chain subunits must be encoded. The actual electron channel is usually encoded by mtDNA and the support system by nuclear DNA. These subunits must be assembled (usually inherited through the nuclear DNA). Protein synthesis within the mitochondria is dependent on translation by mitochondrial-specific ribosomal RNA (encoded by mtDNA) as well as transfer RNA (encoded by mtDNA), which are loaded by tRNA synthases (which are encoded by nuclear DNA). Nuclear genes encode mtDNA maintenance and replication. The mitochondria themselves move, join, and separate (fusion, fission, and motility) and are encoded by nuclear genes. Mitochondria are also dependent on having appropriate lipid bilayers that are built from proteins encoded by nuclear genes. As you can see, normal mitochondrial function is a complex orchestration between mtDNA and nuclear DNA and is still not entirely understood.

To best understand testing, a classification using the two major categories, disorders due to defects of mtDNA and disorders due to defects of nDNA (see Table 1) can be used.

Table 1. Mitochondrial Disorders Category Types by Inheritance Type

Disorders due to defects of mtDNA

Clinically named disorders. Many of the initial mitochondrial disorders were diagnosed based on clinical findings. Several of these were then found to have mtDNA causes. Of important note, as nuclear genes have also been identified, this association is not always true, so the clinical phenotype is important to naming, but the initial description of cause (mtDNA point mutation or deletion) is not always found, and a nuclear mutation is found instead.

Causes of these named disorders include mitochondrial DNA rearrangements (single deletions or duplications) and point mutations.

Disorders initially attributed to mtDNA rearrangements and single deletions of mtDNA. These include three sporadic conditions: Pearson syndrome, Kearns-Sayre syndrome, and progressive external ophthalmoplegia with or without proximal limb weakness.

Pearson syndrome is usually a fatal disorder in infancy characterized by sideroblastic anemia and exocrine pancreas dysfunction.

Kearns-Sayre syndrome is a multisystem disorder of impaired eye movements (progressive external ophthalmoplegia), pigmentary retinopathy, and heart block, with onset before the age of 20. Frequent additional signs include ataxia, dementia, and endocrinopathies (diabetes mellitus, short stature, hypoparathyroidism). Lactic acidosis, elevated CSF protein (over 100mg/dl), and scattered COX-negative ragged-red fibers in the muscle biopsy are typical laboratory abnormalities. This is one of the earlier described disorders.

The third condition is progressive external ophthalmoplegia with or without proximal limb weakness. It is often compatible with a normal lifespan. Deletions within the mtDNA vary in size and location, but a “common” deletion of 5 kb is frequently seen in patients and in aged individuals (108; 106).

Point mutations within the mtDNA. These are common, with over 250 pathogenic point mutations having been identified in mtDNA from patients with various disorders. Most are maternally inherited and multisystemic (81), but some are sporadic and tissue-specific. Among the maternally inherited encephalomyopathies, four syndromes are more common.

The first is MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), which typically but not always presents in children or young adults after normal early development. Symptoms include recurrent vomiting, migraine-like headache, and stroke-like episodes causing cortical blindness, hemiparesis, or hemianopia. These symptoms often are progressive, leading to greater morbidity and, potentially, mortality. MRI of the brain shows “infarcts” that do not correspond to the distribution of major vessels, raising the question of whether the strokes are vascular or metabolic in nature (73). The most common mtDNA mutation is m.3243A>G in the tRNA(Leu)(UUR) gene, but about a dozen other mutations have been associated with MELAS, most notably a mutation (m.13513G> A) in the ND5 gene, which encodes subunit 5 of complex I (110). Notably, most maternal relatives of a typical MELAS patient carry the mutation in low abundance and are either mildly affected or unaffected due to heteroplasmy. In agreement with this observation, two epidemiological studies have reported comparably high prevalence (about 1 in 750) of the m.3243A>G mutation in the normal population (74; 36), and 1 of them has found that the prevalence of pathogenic mtDNA mutations is 1 in 200 individuals in northern England.

The second syndrome is MERRF (myoclonus epilepsy with ragged-red fibers), characterized by myoclonus, weakness, ataxia, seizures, hearing loss, dementia, multiple lipomas, and neuropathy (72). Three mtDNA mutations (m.8344A> G, m.8356T> C, m.8363G> A) have been associated with MERRF, and all are in the tRNA(Lys) gene.

The third syndrome comes in two phenotypes: (1) neuropathy, ataxia, and retinitis pigmentosa (NARP) usually affecting young adults and causing retinitis pigmentosa, dementia, seizures, ataxia, proximal weakness, and sensory neuropathy (53) or (2) maternally inherited Leigh syndrome (MILS), a severe infantile encephalopathy with characteristic symmetrical lesions in the basal ganglia and the brainstem (103).

The fourth syndrome, Leber hereditary optic neuropathy (LHON), is characterized by acute or subacute loss of vision in young adults, more frequently males, due to bilateral optic atrophy (16). Three mtDNA point mutations in ND genes are often homoplasmic and account for more than 90% of Leber hereditary optic neuropathy cases. These are m.11778G>A in ND4, m.3460G>A in ND1, and m.14484T>C in ND6.

Because mitochondria are present in all tissues, syndromes associated with mtDNA mutations can affect every system in the body, including the eye (optic atrophy, retinitis pigmentosa, cataracts), hearing (sensorineural deafness), endocrine system (short stature, diabetes mellitus, hypoparathyroidism), heart (familial cardiomyopathies, conduction blocks), gastrointestinal tract (exocrine pancreas dysfunction, intestinal pseudo-obstruction, gastroesophageal reflux), and kidney (renal tubular acidosis, nephrotic syndrome). Any combination of the symptoms and signs listed above should raise the suspicion of a mitochondrial disorder, especially if there is evidence of maternal transmission.

On the other hand, point mutations in mtDNA protein-coding genes often escape the rules of mitochondrial genetics in that they affect single individuals and single tissues, most commonly skeletal muscle (02). Thus, patients with exercise intolerance, myalgia, and sometimes recurrent myoglobinuria may have isolated defects of complex I, complex III, or complex IV due to pathogenic mutations in genes encoding ND subunits, cytochrome b, or COX subunits. The lack of maternal inheritance and the involvement of muscle alone suggest that mutations arose de novo in myogenic stem cells after germ-layer differentiation (“somatic mutations”) (28).

Respiratory chain oxidative phosphorylation enzymes as the description. Oxidative phosphorylation by the respiratory chain utilizes reducing substances to produce an electron gradient that drives the ATP synthase. Abnormalities of specific complexes of the respiratory chain (eg, complex 1/COX1) can be seen in biopsy specimens of affected tissues. Historically, muscle biopsies were used for diagnosis. Biochemical testing of a tissue that shows abnormalities of oxidative phosphorylation may not be abnormal in the genes that encode the complexes because abnormal enzyme activity can also be seen in secondary mitochondrial disease (eg, abnormal TCA cycle, pathways of abnormal amino acid catabolism), physiological or psychiatric stress, or abnormal biopsy collection (90). Biopsy enzyme assays and staining are more likely to be useful in confirming genetic diagnosis or if a genetic diagnosis cannot be identified (90).

Disorders due to defects of nDNA

These are all transmitted by Mendelian inheritance and include six major subgroups.

Mutations in genes encoding subunits of the respiratory chain. As noted above, mtDNA encodes only 13 subunits of the respiratory chain, whereas nDNA encodes all subunits of complex II, most subunits of the other four complexes, as well as CoQ10 and cytochrome c. Nuclear DNA mutations can affect respiratory chain complexes directly or indirectly.

Mutations in genes encoding respiratory chain subunits are known mostly for two complexes (complex 1 and complex 2) because these contain the greatest number of subunits (complex 1) and the greatest percentage of nuclear-encoded genes (complex 2). In general – and in keeping with the all-or-none effects of mendelian mutations (most of which are recessive), as opposed to the variegated effects of heteroplasmic mtDNA mutations – disorders due to respiratory chain enzyme abnormalities manifest at or soon after birth and are very severe, often lethal in infancy. Most of these mutations have been associated with autosomal recessive forms of Leigh syndrome, a disorder that reflects the ravages of energy shortage on the developing nervous system. The neuropathological (or neuroradiological) hallmarks of Leigh syndrome are bilateral symmetric lesions all along the nervous system, but especially in the basal ganglia, thalamus, brainstem, and cerebellar roof nuclei. Microscopically, there is neuronal loss, proportional loss of myelin, reactive astrocytosis, and proliferation of cerebral microvessels (27).

Mutations in assembly proteins. Even when all nDNA-encoded subunits of the various complexes are expressed correctly, they have to be translated, imported into mitochondria, and directed to the mitochondrial inner membrane, where they assemble with their mtDNA-encoded and nDNA-encoded counterparts.

The search for the molecular basis of COX-deficient Leigh syndrome led to the simultaneous discovery by two laboratories of the first mutant assembly gene, SURF1 (123; 133). Mutant assembly factors have been found through whole exome sequencing (WES) of patients with specific respiratory chain complex deficiencies, such as children with complex I deficiency (14).

The clinical manifestations of indirect hits in complex I tend to be more heterogeneous than those associated with direct hits (40; 125). All described patients have encephalopathy clinically resembling Leigh syndrome, but often with leukodystrophy rather than grey matter involvement. Cardiomyopathy is more common and often the dominating feature.

The first assembly defect in complex III was identified in 2002 in Finnish infants with an extremely severe syndrome named GRACILE that summarizes acronymically the main symptoms and signs: growth retardation, aminoaciduria, cholestasis, iron overload, and early death (before 5 months of age) (127). The mutated protein, BCS1L, is needed to insert the Rieske FeS subunit into the complex.

Mutations in the assembly protein SURF1 are among the most common causes of Leigh syndrome, and SURF1 ought to be sequenced in all children with COX-deficient Leigh syndrome.

Mutations in at least seven more COX assembly factors have been associated with human diseases; although all cause encephalopathy, each also involves one other tissue preferentially. Thus, mutations in SCO2 cause severe cardiomyopathy, as do mutations in COX10, COX15, and COA5, whereas mutations in SCO1 cause hepatopathy (32).

The function of complex V depends on the integrity of two partners, the adenine nucleotide transporter (ANT1) and the inorganic phosphate carrier (PIC). Whereas mutations in ANT1 affect mostly mtDNA maintenance (see below), mutations in SCL25A3, the gene encoding the heart and muscle isoform of PIC, affect ATP synthesis and caused infantile and rapidly fatal cardiopathy and myopathy in two sisters (77).

We include among the indirect hits a relatively new group of disorders due to coenzyme Q10 (CoQ10) deficiency. In this case, mutations in a cascade of biosynthetic enzymes result in the deficiency of one relatively simple component of the respiratory chain. CoQ10 (ubiquinone) is a small lipophilic molecule comprising a redox-active benzoquinone ring and a polyisoprene tail consisting of 10 units in humans.

PrimaryCoQ10 deficiencies can cause five major syndromes: (1) a predominantly myopathic disorder with recurrent myoglobinuria but also CNS involvement (seizures, ataxia, mental retardation) (87); (2) a predominantly encephalopathic disorder with ataxia and cerebellar atrophy; (3) an isolated myopathy, with ragged-red fibers and lipid storage; (4) a generalized mitochondrial encephalomyopathy, usually with onset in infancy; (5) nephropathy alone or associated with encephalopathy (98; 37); and (6) a wide spectrum of mitochondrial disorders due to mutations in COQ4 (13). Examples of secondary CoQ10 deficiency include ataxia oculomotor apraxia (AOA1) associated with mutations in the aprataxin (APTX) gene, the myopathic presentation of glutaric aciduria type II due to mutations in the electron transfer flavoprotein dehydrogenase (ETFDH) gene (44), and autosomal recessive spinocerebellar ataxia (SCAR) due to mutations in ANO10 (03). Examples of primary CoQ10 deficiency include mutations in the biosynthetic genes, COQ1 (PDSS1 and PDSS2), COQ2, COQ9, COQ6, COQ4, and CABC1/ADCK3. Irrespective of etiology, diagnosis is important because most patients with CoQ10 deficiency respond to high-dose CoQ10 supplementation (98; 37).

In a novel type of assembly protein deficiency, toxins impacting the assembly are illustrated by ethylmalonic encephalomyopathy (EE), a devastating early-onset disorder with encephalopathy, microangiopathy, chronic diarrhea, and massively increased levels of ethylmalonic acid and short-chain acylcarnitines in body fluids. This disorder is due to mutations in the ETHE1 gene, whose product, ETHE1, is a mitochondrial matrix thioesterase. Studies of an Ethe1-null mouse led to the discovery that thiosulfate and sulfide accumulate excessively both in the animal model and in affected children due to the lack of sulfur dioxygenase activity (124). As sulfide is a powerful COX inhibitor, this turned out to be an indirect toxic cause and likely the prototype of other similar pathogenic mechanisms.

Because a prerequisite for the assembly of any respiratory chain complex is the import of nDNA-encoded subunits from the cytoplasm into mitochondria, it seems plausible to add one more category to the assembly and transport category, defects of mitochondrial protein importation. An example is the X-linked deafness-dystonia syndrome (Mohr-Tranebjaerg syndrome), characterized by progressive sensorineural deafness, dystonia, cortical blindness, and psychiatric symptoms. This disorder is due to mutations in TIMM8A, which encodes the deafness-dystonia protein (DDP1), a component of the mitochondrial protein import machinery located in the intermembrane space (101).

This field of research has important theoretical and practical implications. From an investigative point of view, these disorders are teaching us a lot about the structural and functional complexity of the respiratory chain. At a more practical level, identification of mutations in these genes allows prenatal diagnosis and suggests approaches to therapy.

Defects of mtDNA maintenance. As noted above, the mtDNA has become the slave of nDNA and depends on numerous factors encoded by nuclear genes. Mutations in these genes cause Mendelian disorders characterized by qualitative or quantitative alterations of mtDNA. Two important features of this group of diseases ought to be considered. First, although inheritance is unequivocally Mendelian, these disorders share much of the clinical heterogeneity of primary mtDNA-related diseases, presumably because the polyploid mtDNA is involved in both conditions. Second, we had been lured into the simplistic concept that mtDNA depletion and mtDNA multiple deletions were distinct conditions with characteristic and distinguishable phenotypes. Largely due to the application of whole genome sequencing, we have been disabused of this idea and now realize that the two conditions often coexist and that mutations in the same genes can cause either predominantly mtDNA depletion in infants or children or predominantly mtDNA multiple deletions in adults (27).

Examples of qualitative alterations include autosomal dominant or recessive multiple mtDNA deletions of mtDNA, usually accompanied clinically by progressive external ophthalmoplegia (PEO), plus various other symptoms and signs. Several of these conditions have been characterized at the molecular level. Mutations in the gene TYMP for thymidine phosphorylase are responsible for an autosomal recessive multisystemic syndrome called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) (52). Mutations in the gene for one isoform of the adenine nucleotide translocator (ANT1) have been identified in patients with autosomal dominant progressive external ophthalmoplegia (59). Mutations in the PEO1 gene, encoding Twinkle, a helicase, are also associated with autosomal dominant progressive external ophthalmoplegia (114).

The ultimate example of a single gene causing either mtDNA depletion or multiple mtDNA deletions and resulting in extremely diverse syndromes is POLG, the gene encoding Polg-A. Depending largely on which of the three domains of POLG (polymerase, exonuclease, linker region) harbors the mutation(s), the clinical phenotype ranges from a severe hepatocerebral disorder of infancy or childhood (Alpers syndrome) to adult-onset autosomal dominant or recessive progressive external ophthalmoplegia (AD- or AR-PEO) to parkinsonism and other clinical phenotypes, including sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO), and mitochondrial recessive ataxia syndrome (MIRAS) (25; 131). Similarly, mutations in the PEO1 gene encoding the Twinkle helicase usually cause adult-onset AD-PEO with multiple mtDNA deletions but can also cause Alpers-like autosomal recessive hepatocerebral syndrome with mtDNA liver depletion or infantile-onset spinocerebellar ataxia (IOSCA), a disease prevalent in the Finnish population and characterized by mtDNA depletion in the brain (131).

Mutations in the gene POLG2, encoding the accessory subunit of POLG, can also cause autosomal dominant progressive external ophthalmoplegia and multiple deletions (126; 68). Finally, mutations in OPA1, which encodes a protein involved in mitochondrial dynamics (see below), besides causing dominant optic atrophy can also result in a syndrome that includes optic neuropathy, progressive external ophthalmoplegia, deafness, ataxia, and axonal neuropathy associated with multiple mtDNA deletions in the muscle biopsy (01; 55).

An iatrogenic form of mtDNA depletion may follow treatment with nucleoside analogs such as zidovudine (AZT) and will cause multiple mtDNA deletions (93).

Examples of quantitative alterations of mtDNA include severe or partial mtDNA depletion, usually characterized clinically by congenital or childhood forms of autosomal recessively inherited myopathy or hepatopathy. Mutations in nine genes, seven of them involved in mitochondrial nucleotide homeostasis, have been associated with mtDNA depletion syndromes, although they still do not explain all cases (117). Mutations in the gene encoding thymidine kinase 2 (TK2) are typically seen in patients with myopathic mtDNA depletion syndromes, whereas mutations in the genes encoding the beta subunit (SUCLA2) or the alpha subunit (SUCLG1) of the mitochondrial matrix enzyme succinyl-CoA synthetase (SCS-A, which is the reverse reaction to the TCA cycle enzyme succinate synthase) cause both myopathy and encephalopathy. Mutations in DGUOK, encoding deoxyguanosine kinase, predominate in patients with hepatic or hepatocerebral mtDNA depletion syndromes, but mutations in POLG are the major causes of Alpers-Huttenlocher syndrome, a severe hepatocerebral syndrome with vulnerability to valproic acid (85). Mutations in the gene MPV17, not involved in nucleotide pool homeostasis, have been associated with hepatocerebral syndrome (116) and with the Navajo neurohepatopathy syndrome, prevalent in the Navajo population of the southwestern United States (56). Mutations in FBXL4 cause mtDNA depletion accompanied by early-onset mitochondrial encephalomyopathy (11; 42). FBXL4 is localized in the intermembrane space, where it controls bioenergetic homeostasis and mtDNA maintenance.

Defects of mtDNA translation. The mitochondrial genome is translated into nine monocistronic and two dicistronic mRNAs. Translation of these mRNAs into the 13 mtDNA-encoded respiratory chain subunits is effected by mitoribosomes that consist of one large subunit (48 proteins) and one small subunit (29 proteins). The translation process can be broken down into four phases, each requiring multiple ancillary factors (22; 94).

A group of defects of intergenomic communication is due to mutations in genes encoding those factors necessary for the faithful translation of mtDNA-encoded proteins (22; 94), including EFG1 (encoding elongation factor 1), MRPS16 (encoding small subunit protein), EFTu (encoding elongation factor Tu), TSFM (controlling the expression of both EFTs and EFTu), and PUS1 (encoding pseudouridine synthase 1). The resulting disorders usually affect infants and cause severe encephalomyopathy, cardiomyopathy, or sideroblastic anemia. Typically, both quality and quantity of mtDNA are normal in these patients, but there are – not surprisingly – multiple respiratory chain defects involving all complexes containing mtDNA-encoded subunits. Mutations in genes encoding mitochondrial amino acyl-tRNA synthetases cause a variety of syndromes. For example, mutations in DARS2 (encoding mitochondrial aspartyl-tRNA synthetase) cause leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL): surprisingly, no defects of respiratory chain enzymes were found in patients with LBSL, at least in fibroblasts and lymphoblasts (104). A novel mutation in the gene encoding alanyl-acyl-tRNA synthetase (AARS) caused Charcot-Marie-Tooth neuropathy in one family (84). RARS2 encodes the argino-tRNA synthetase and can present with pontocerebral hypoplasia or with lactic acidosis and encephalomyopathy (71). A growing number of these mitochondrial aminoacyl-tRNA synthetases have been described to cause disease (113).

Defects of mtDNA transcription are much less frequent, but mutations in the gene (ELAC2) encoding the human ortholog of bacterial RNase Z have been associated with infantile hypertrophic cardiomyopathy and complex I deficiency (50).

Defects of the inner membrane lipid milieu. The function of the respiratory chain can be impaired by alterations in the lipid composition of the inner mitochondrial membrane, which does not act simply as a scaffold but participates in the function of the respiratory chain. The first and prototypical disorder in this group is Barth syndrome, an X-linked recessive disorder characterized by mitochondrial myopathy, cardiopathy, and leukopenia (06). The gene responsible for this disorder (TAZ) (08) encodes a family of proteins (“tafazzins”) involved in the synthesis of phospholipids, and biochemical analysis has shown altered amounts and composition of cardiolipin, the main phospholipid component of the inner mitochondrial membrane (105).

Like Barth syndrome, Sengers syndrome also affects primarily heart and muscle, with the distinctive additional clinical feature of congenital cataracts. Muscle biopsy shows mitochondrial myopathy and respiratory chain dysfunction, with lactic acidosis and decreased activities of multiple respiratory chain enzymes. Although there was convincing evidence that ANT1 was missing in muscle of patients, no mutation was found in that gene, leading to postulations of defective transcription or translation. Whole exome sequencing solved the riddle when it revealed mutations in AGK, the gene encoding acylglycerol kinase (76). AGK catalyzes the phosphorylation of diacyl- and monoacylglycerol to form phosphatidic acid or lysophosphatidic acid, important intermediates in the synthesis of phospholipids. As phosphatidic acid is also a precursor of cardiolipin, there is a point of convergence with the pathogenesis of Barth syndrome, probably explaining some of the clinical similarities between the two disorders.

A third defect of the inner mitochondrial membrane lipid milieu is due to mutations in the gene encoding choline kinase beta (CHKB) that catalyzes the first step in the biosynthesis of phosphatidylcholine. The resulting clinical picture is a congenital mitochondrial myopathy morphologically characterized by giant mitochondria (“megaconial myopathy”) displaced to the periphery of the fibers (82; 83; 49; 96).

Altered inner membrane phospholipid composition was documented in another disorder dubbed MEGDEL, an acronym referring to a syndrome characterized by 3-methylglutaconic aciduria type IV, deafness, and Leigh-like encephalopathy (129). In this case, whole exome sequencing revealed mutations in the SERAC1 gene: SERAC controls the exchange of phospholipids between the endoplasmic reticulum and mitochondria (130). Analysis of patient fibroblasts showed both altered distribution of phosphatidylglycerol species and altered composition of cardiolipin subspecies. Thus, quantitative or qualitative alterations in cardiolipin may be a common denominator in the pathogenesis of disorders other than Barth syndrome.

Finally, at least for now, recurrent muscle breakdown and myoglobinuria in children, a syndrome often occurring during febrile illnesses and rarely associated with known inborn errors of energy metabolism, has been attributed to mutations in LPIN1, a gene encoding the muscle-specific isoform of phosphatidic acid phosphatase (132). Here, there is no direct evidence of mitochondrial dysfunction: rather, damage to the sarcolemma is postulated to occur through the detergent action of accumulated lysophospholipids.

Defects of mitochondrial motility, fusion, and fission. Defects of mitochondrial dynamics are taking center stage as causes of neurodegenerative disorders and do belong to the mitochondrial diseases sensu stricto because impairment of oxidative phosphorylation has been documented. Genes causing loss of motility, fusion, or fission are becoming more apparent in mitochondrial disorders. Mitochondria travel on microtubular rails, propelled by motor proteins, usually GTPases, called kinesins or dyneins (20). The first defect of mitochondrial motility was identified in a family with autosomal dominant hereditary spastic paraplegia and mutations in a gene (KIF5A) encoding one of the kinesins. Interestingly, the mutation affects a region of the protein involved in microtubule binding (41).

Mutations in OPA1 cause autosomal dominant optic atrophy, the Mendelian counterpart of Leber hereditary optic neuropathy. Mutations in MFN2, encoding mitofusin 2, cause an autosomal dominant axonal variant of Charcot-Marie-Tooth disease. Also, mutations in GDAP1, the gene encoding ganglioside-induced differentiation protein 1, which is located in the mitochondrial outer membrane and regulates the mitochondrial network, cause Charcot-Marie-Tooth disease type 4A, an autosomal recessive, severe, early-onset form of either demyelinating or axonal neuropathy (27).

Pathogenesis

From the standard textbook gloss that mitochondria are the “powerhouses” of the cell, most people would reasonably deduce that mitochondrial diseases are energy failures, blackouts, or brownouts affecting various tissues. Although defective ATP production undoubtedly has an important pathogenic role, mitochondria perform multiple additional functions that are important for cell life and death, including the generation of reactive oxygen species (ROS), the control of calcium homeostasis, and the regulation of programmed cell death (apoptosis). It is likely that the pathogenesis of any mitochondrial disease will involve, at least to some extent, all of these functions.

If we focus on the “primary” mitochondrial diseases outlined above, ie, defects of the respiratory chain, two pathogenic mechanisms are most commonly considered – impaired ATP synthesis and excessive reactive oxygen species production. Due to the lack of animal models for mtDNA-related disorders, the biochemical and functional consequences of mtDNA mutations have been gleaned from studies of cybrid cell lines, that is, immortalized human cells that have been “emptied” of their mtDNA and repopulated with mutant mtDNA from patients (60). In particular, cybrid cell lines harboring varying mutant loads have been used to identify the threshold of pathogenicity for various mtDNA mutations. For all major mutations, the thresholds assessed in vitro appeared to be both high and steep: for the m.3243A>G MELAS mutation, as one example, the threshold was around 90%. However, these data cannot be extrapolated to the in vivo situation, as shown by oligosymptomatic carriers of the m.3243A>G mutation, who had abnormal 31P-MRS studies of muscle and abnormal lactate peaks in both cerebrospinal fluid and brain parenchyma by 1H-MRS (21; 58).

In mtDNA-related disorders, apparent tissue-specificity can occur in two situations, best exemplified by mitochondrial myopathies. There are numerous examples of mutations in tRNA or protein-coding genes arising de novo and affecting selectively the progenitor cells of skeletal muscle: these often create diagnostic conundrums because they contradict at least two “rules” of mitochondrial genetics, maternal inheritance, and multisystem distribution. An example of what could be called “pseudo tissue specificity” is extreme skewed heteroplasmy of a generalized mutation: as the pathogenic threshold is surpassed only in skeletal muscle, the resulting phenotype will be a pure myopathy.

A more puzzling example of tissue specificity is offered by homoplasmic mtDNA mutations, such as most Leber hereditary optic neuropathy (LHON)-associated mutations in ND genes: obviously, there is a special vulnerability of the retinal ganglion cells to the consequences of these mutations (be it ATP deprivation, reactive oxygen species excess, or both), probably exacerbated by the extraordinary dependence of these cells on oxidative metabolism. Similarly, a homoplasmic mutation in tRNA(Ile) causes a selective cardiomyopathy (122), whereas a homoplasmic mutation in tRNA(Glu) causes a selective and largely reversible COX-deficient myopathy, which can only be explained by the coexistence of a modifier nuclear gene, TRMU (10). Clearly, the cross-talk between the two genomes goes beyond the classical defects of intergenomic signaling and remains to be fully elucidated. Epigenetics also goes beyond tissue specificity to affect, for example, gender vulnerability: the predominance of affected males in Leber hereditary optic neuropathy has been attributed to two loci in the X-chromosome, but more recently (and more likely), to a protective effect of estrogens in women, an example of epigenetic effect (45).

The relative contribution of ATP shortage and reactive oxygen species excess to pathogenesis and clinical expression is evident in both mtDNA-related and Mendelian mitochondrial diseases. A correlation between biochemical data and severity of clinical presentation was observed in studies of fibroblasts from two patients with different primary CoQ10 deficiency, one due to mutations in COQ2 and causing a severe but treatable infantile encephalomyopathy and the other due to mutations in PDSS2 and causing fatal infantile Leigh syndrome. PDSS2 mutant cells showed severely reduced ATP synthesis but no reactive oxygen species overproduction or compensatory increase of antioxidant defense markers, whereas COQ2 mutant cells showed only a partial defect of ATP synthesis but marked increase of reactive oxygen species with attending oxidation of lipids and proteins (97).

Therapy

Three strategies toshould be considered in treating mitochondrial diseases (107; 27; 89; 99). The first strategy aims at modifying the consequences, ie, the symptoms, of these disorders: this is symptomatic therapy. The second strategy is both more ambitious and more difficult because it aims at attacking the causes of these disorders and is, therefore, based either on gene therapy or on enzyme replacement therapy. The third therapeutic approach falls between the other two and it aims to interrupt or modifyi the pathogenic mechanisms, thus interrupting or, more likely, slowing the course of diseases.

There is a significant move to improve clinical therapies as more therapeutic studies become available for this cohort (99); a consensus statement has been published concerning the measured clinical outcomes for mitochondrial myopathies and encephalo(myo)opathy (63) and should be considered when evaluating newer therapies.

Symptomatic therapy. The fact that mitochondrial disorders can affect every tissue in the body requires the application of symptomatic therapy already used in different subspecialties of medicine. These symptomatic therapies can be divided into general and situational-specific therapies.

General therapies are used to approach the symptoms that include fatigue and exercise intolerance. They include roles for small molecule, vitamin, and nutritional therapy.

Exercise intolerance is a common complaint, which may lead to inactivity, deconditioning, and further deterioration of muscle function. Aerobic exercise under proper supervision has proven helpful in staving off this downhill course (120).

More situation-specific therapies attempt to treat the symptoms of specific organ systems or specific situations (eg, surgery, anesthesia, pregnancy).

Treatment of the underlying organ system abnormalities.

Audiology: hearing loss. Hearing should be screened every 1 to 2 years because hearing loss can be progressive (89). Avoidance of excessive noise exposure should be encouraged. Neurosensory hearing loss is a common consequence of mitochondrial-related disorders and, when severe, can be effectively treated in most patients with cochlear implants.

Neurology. Seizures, stroke-like episodes, psychiatric disease, migraines/headaches, developmental delay/intellectual disabilities, myopathy, neuropathies, ataxia.

Developmental delay and intellectual disabilities can be seen (99). Awareness and screening with appropriate referrals are very important for these patients. Occasionally, individuals will have a regression, and ruling out treatable etiologies is important. Autism and attention deficit should be treated in the conventional methods.

Seizures are treated with conventional anticonvulsants, except that valproic acid should be avoided in children with Alpers-Huttenlocher syndrome and POLG mutations because it often triggers acute hepatic failure. Vigabatrin should be avoided in mtDNA depletion patients because it inhibits deoxyribonucleoside diphosphate conversion to deoxyribonucleoside triphosphate (07). Topiramate can worsen acidosis and, thus, should be avoided in those who are at risk (80).

Mitochondrial encephalopathy (historically called mitochondrial stroke-like episodes) can occur, and the author will discuss some more specific treatments in situation events, but physical, occupational, respiratory, and speech therapies accelerate recovery from strokes and aid patients with other CNS problems.

Headaches and migraines are common. Severe headaches in individuals at risk for stroke-like episodes could indicate a stroke. Most conventional migraine interventions may be used (89).

Psychotropic drugs may be needed in patients with predominant or exclusive psychiatric symptoms.

Ophthalmology: ptosis, cataracts, optic atrophy, retinal disease. Regular screening for eye disease is important in mitochondrial disorders.

Ptosis, often with progressive external ophthalmoplegia, is very common and debilitating both functionally and psychologically. Frontalis suspension is the preferred form of surgery because it protects from corneal exposure (88).

Few therapies are available for optic atrophy and retinal disease, but attention to vision therapy and services for the blind are important to overall well-being. The European Commission has granted marketing authorization for Raxone (idebenone) to treat visual impairment in adolescent and adult patients with Leber hereditary optic neuropathy (62). Appropriate correction is also very helpful to minimize increased isolation due to vision loss.

Endocrinology: diabetes mellitus, adrenal insufficiency, growth hormone deficiency, hypothyroidism, parahypothyroidism, and short stature. Standard blood and urine tests should be used to screen for hormonal abnormalities every 1 to 2 years (89).

Diabetes mellitus should be treated by conventional means, including diet, sulfonylureas, and insulin, but metformin ought to be avoided because it has been associated with lactic acidosis. Hemoglobin A1C and fasting glucoses can be used to screen.

Adrenal insufficiency should be screened and treated through conventional means. Individuals with growth hormone deficiency can respond to growth hormone and are treated with the conventional method.

Screening and conventional treatment of parahypothyroidism help maintain bone density, although osteopenia can still occur in the absence of parahypothyroidism.

Cardiology: arrhythmia, cardiomyopathy, conduction defects, and heart block. Screening, including EKG and echocardiography, should be done with Holter monitoring in those at greatest risk (89).

In several mitochondrial disorders including Kearns-Sayre syndrome, conduction blocks dominate the cardiac picture, and timely placement of a pacemaker can be lifesaving. Arrhythmias also are treated in the conventional manner. Ablation can be used in arrhythmias that are responsive to this procedure (89).

Cardiomyopathy is common (89) and is often treated with the traditional method. Cardiac transplants have been done in cases of isolated cardiac disease (eg, Barth syndrome).

Gastroenterology: feeding difficulties, dysmotility, constipation, dysphagia, liver dysfunction, pancreatic insufficiency, failure to thrive, and pseudo-obstruction. Feeding difficulties in infants, children, and adults can be alleviated by drugs or surgical intervention, including percutaneous endoscopic gastrostomy or fundoplication. Some dysmotility is seen in a number of disorders and particular focus on trying to maintain a functional gut is important. Aggressive treatment of constipation is also helpful. Severe gastrointestinal dysmotility may necessitate parenteral nutrition in patients with MNGIE.

Liver dysfunction can occur in a number of mitochondrial disorders, and hepatotoxic drugs (eg, acetaminophen and valproic acid) should be avoided, especially in those diagnoses at greatest risk.

Diarrhea is not common (in the absence of overflow diarrhea from constipation), so exocrine pancreas dysfunction should be explored.

Hematology: pancytopenia, iron deficiency, and sideroblastic anemia. Complete blood count with differential should be considered annually (89).

Infants with Pearson syndrome and sideroblastic anemia may respond to repeated blood transfusion, although survivors often develop Kearns-Sayre syndrome later in life.

Mild anemia is common and only needs to be observed. Maximizing nutrition and feeding should be done. Iron, if deficient, should be replaced.

Nephrology: Fanconi syndrome, glomerular dysfunction, and tubulopathies. All should be screened by blood and urine tests (89).

Renal tubular acidosis and Fanconi syndrome require readjustment of the electrolytic balance. Primary CoQ10 deficiency is often associated with – or dominated by – nephropathy, and early CoQ10 supplementation may improve glomerular function and prevent neurologic complications. Other causes require electrolyte replacement and sometimes bicarbonate treatment.

Immunology: recurrent infections. Some immune dysfunction is probably due to the pancytopenia described in hematology, but this does not appear to explain the entirety of risk. There seems to be more infections and sepsis in those with mitochondrial disease than the general population (89).

Vaccines are considered generally safe and all should be vaccinated (89). Few patients prove the exception to this rule, but they should be addressed individually. With appropriate preventive care and adequate nutrition and fluids, we have successfully vaccinated those who have had difficulty in the past.

Nutrition. Adequate caloric and fluid delivery is important for medical management. In a population of individuals who may not be able to tolerate feeds easily due to dysmotility and who need adequate calories to maintain adequate growth and development, adequate calories should be provided by other means.

Some individuals with mitochondrial disorders that affect pyruvate do not tolerate dextrose as effectively as fats. Individuals with pyruvate dehydrogenase deficiency can more effectively utilize fats as a caloric source and so ketogenic diets are used (15). Others with mitochondrial disorders complicated by seizures may benefit as well, but the appropriate patient population is not well understood.

Numerous micronutrients, including vitamins, are utilized by a number of mitochondrial pathways (15). This concept becomes the basis of the “mitochondrial cocktail” of which components will be discussed below.

Situational interventions

Treatments of mitochondrial encephalopathy (historically called stroke-like episodes). Mitochondrial encephalopathy frequently presents as a stroke but does not demonstrate thrombosis and is not located within the watershed areas on MRI. Basic supportive therapy is important, including adequate calories.

In MELAS, there appears to be an underlying angiopathy with altered vascular contractility due to a deficiency of nitric oxide. L-citrulline and L-arginine are precursors of nitric oxide and, thus, IV arginine has been utilized to limit the extent of the stroke (65; 35; 33). These groups found that intravenous administration of L-arginine (0.5 g/kg) during the acute phase improved all stroke-like symptoms, whereas interictal oral administration (0.15 to 0.30 g/kg) diminished both frequency and severity of strokes (65; 66; 35; 33). Additional studies have looked at L-citrulline for interictal periods and have also shown potential benefit (34).

Acute illness. Individuals should have their underlying disease treated aggressively during acute illness with dextrose-containing fluids, except those who cannot tolerate dextrose due to a pyruvate metabolism disorder (90).

If infected with influenza responsive to antivirals, antivirals can be tried with caution because many antivirals can decrease mitochondrial function.

As with any other chronic disorder, prevention is the best policy, so avoidance of those ill, full vaccination, and hand washing should be used.

Pregnancy. Individuals are at risk for preeclampsia, preterm labor, poor labor progression, and gestational diabetes. They should receive calories during labor and postdelivery even if unable to eat.

Surgery. Adequate fluid hydration is important. Some individuals will require dextrose-containing fluids (others, such as those on ketogenic diets, will be less able to tolerate these) (90).

Individuals with mitochondrial disorders can have severe and adverse reactions to anesthesia including rapidly progressive white matter abnormalities and respiratory failure (23; 19; 24). There are several publications about anesthesia, but Niezgoda and Morgan have a good outline (86).

Medications to avoid: valproic acid, high-dose acetaminophen, statins, aminoglycosides, linezolid, tetracyclines, azithromycin, erythromycin, and metformin.

Directed therapy. The term implies tackling the root of the disease, ie, its etiology. Ultimately, this implies replacing the mutant DNA with wild-type DNA.

A way of, if not correcting, at least avoiding the cause of a disease includes genetic counseling and prenatal diagnosis. As detailed above, the rapid progress in our knowledge of the molecular defects underlying Mendelian mitochondrial disorders offers families, especially young families with fatal infantile conditions, the option of prenatal diagnosis (assuming the familial genetic variation(s) are known). Also available is preimplantation genetic diagnosis to those with a nuclear inherited mitochondrial disorder. This is an in vitro technique in which only embryos without the disorder are implanted after being checked for the familial mutation.

Unfortunately, prenatal diagnosis of most mtDNA-related diseases is more complicated, given that they are passed on through the maternal inherited mtDNA, which is influenced by heteroplasmy. Postimplantation diagnosis of mitochondrial disease is impeded by two factors: (1) the mutation load in amniocytes or chorionic villi does not necessarily reflect that of other fetal tissues and (2) mutation loads measured in prenatal samples may shift due to mitotic segregation. Thus, once pregnancy is established it is difficult to determine risk to the fetus.

Fortunately, there is good evidence that mutations in ATPase6 associated with NARP or maternally inherited Leigh syndrome do not undergo tissue- or age-related variations (09; 128).

Gene therapy for mitochondrial diseases due to mutations in nDNA faces the same hurdles as gene therapy for other Mendelian disorders. Gene therapy for mitochondrial disorders is complicated further in that mitochondrial disorders affect multiple organ systems requiring gene delivery to multiple organs.

Gene therapy for mtDNA-related diseases poses special problems because of polyplasmy and heteroplasmy. In addition, only two groups of investigators have been able to transfect DNA into mitochondria in a heritable manner (67). Of the many indirect strategies proposed, probably the most viable is heteroplasmic shifting, aimed at lowering the mutant mtDNA below the pathogenic threshold. Many different approaches have been tried, although their applicability to humans appears remote (107).

Another promising way of preventing mtDNA-related disorders is under study. Using this technique, a woman carrying an mtDNA mutation, such as the common and potentially devastating m.3243A>G MELAS change, could have her fertilized oocytes cleansed of the cytoplasm and most mitochondria in vitro. The naked pronucleus would then be transferred to a normal enucleated host oocyte and implanted in the woman’s uterus: the result of this procedure would be a mitochondrially normal child carrying the nuclear traits of both parents. This technology has been successful in monkeys (119) and is being tested in human oocytes both in the United Kingdom and in the United States (26; 91; 118). As technology advances, ethical discussions continue (48).

Another way of going to the root of the problem is stem cell therapy, which, for Mendelian disorders, offers real promise. For example, allogeneic stem cell transplantation in patients with MNGIE improves both clinical condition and nerve cell conduction. Biochemically, thymidine phosphorylase activity reaches mutation carrier levels in blood, where the concentrations of toxic compounds, thymidine and deoxyuridine, return to normal (51).

Deoxycytidine and deoxythymidine have been given to bypass a deficient pyrimidine salvage pathway for thymidine kinase 2 deficiency in monkeys and a single human patient (70). The intermediate bypass used here could be used in similar mitochondrial disorders.

Acting on pathogenesis

Removing noxious compounds. The most logical intervention in any inborn error of metabolism appears to be removing noxious compounds. In most mitochondrial encephalomyopathies, the obvious culprit is lactic acid. Bicarbonate therapy is common and almost “automatic” but should be used prudently (102). Dichloroacetate inhibits pyruvate dehydrogenase kinase, keeping pyruvate dehydrogenase in the dephosphorylated, active form, thus, favoring pyruvate metabolism and lactate oxidation. Although oral dichloroacetate was well tolerated in randomized studies of children with congenital lactic acidosis and heterogeneous mitochondrial diseases, it did not improve neurologic outcomes in patients with MELAS and was associated with evidence of peripheral neuropathy (57). In MNGIE, the toxic metabolites that accumulate in blood as a direct consequence of the thymidine phosphorylase defect are thymidine and deoxyuridine (115). Hemodialysis was only transiently effective in lowering blood levels, whereas the effect of allogeneic stem cell transplantation was more substantial and permanent, as discussed above.

Administration of electron acceptors. This is most effective in disorders due to primary defects of such acceptors, best exemplified by primary CoQ10 deficiencies. However, at least one child with Leigh syndrome and CoQ10 deficiency due to mutations in the PDSS2 gene did not respond to CoQ10 administration, possibly because therapy was started too late, because the dose was inadequate, or because the energy defect was too drastic (69). In patients with secondary CoQ10 deficiency, the response to supplementation is generally good but unpredictable and often variable. For example, patients with the myopathic form of glutaric aciduria type II due to electron transfer flavoprotein dehydrogenase deficiency may need both CoQ10 and riboflavin for optimal response (44). In patients with complex I deficiency due to mutations in ACAD9, a flavoprotein-containing protein, administration of riboflavin has been beneficial (43). Ubiquinol is the preferred form of CoQ10 due to its improved bio-availability, but much of the dosing has been determined with ubiquinone (05).

“Cocktails” of vitamins and cofactors. These are the most widely used therapy in clinical practice. Although there is little evidence of their benefit (15), vitamin and cofactor supplementation has been used based on the underlying pathophysiology, and with an intelligent approach, it may show some benefit (05).

In the hope of facilitating ATP production by a sluggish respiratory chain, electron flux is “boosted” by the addition of electron acceptors (CoQ10, vitamin C, vitamin K, succinate). Alternatively, we try to boost the synthesis of a naturally occurring high-phosphate compound, phosphocreatine by administering creatine. L-carnitine is prescribed because plasma-free carnitine tends to be lower and esterified carnitine higher than normal, probably reflecting a partial impairment of beta-oxidation. Folic acid deserves special mention because early observations had shown that it was decreased in the CSF of patients with Kearns-Sayre syndrome, and a study documented both clinical and neuroradiological improvement in a child after 1 year of monotherapy with 2.2 mg folinic acid/kg daily, suggesting that early and aggressive administration of this compound should be tried in this devastating condition (95).

Riboflavin and magnesium are both growing in terms of acceptance as therapies for migraines associated with mitochondrial disease (05).

A compound, a parabenzoquinone labeled EPI-743 is being tested in infants and children with Leigh syndrome (39; 75), but has not shown to be as promising as initially thought.

To improve ATP synthesis, creatine monohydrate supplementation has been used, but the only two randomized studies came to different conclusions, possibly due to the difference in muscle phosphocreatine concentration between the two groups (121; 61).

Scavenging excessive reactive oxygen species. This is probably the most common approach not just to mitochondrial diseases due to respiratory chain defects but also to late-onset neurodegenerative disorders, including amyotrophic lateral sclerosis, Parkinson disease, and Alzheimer disease, in which there is direct or indirect evidence of oxidative stress. Antioxidants used in clinical practice include vitamin E, CoQ10, idebenone, glutathione, and dihydrolipoate.

As mentioned above, CoQ10 is useful in primary CoQ10 deficiencies, but it is also widely prescribed to patients with respiratory chain disorders. Although anecdotal reports (too numerous to be cited here) are generally positive, we still lack a rigorous double-blind trial. However, clinical experience teaches that CoQ10 needs to be administered at high doses (no less than 300 mg daily in adults), which fortunately have shown to be well tolerated in numerous studies of large cohorts.

Importantly, initial studies of idebenone had suggested a beneficial effect only on the cardiopathic component of Friedreich ataxia, but a standardized study showed a dose-related beneficial effect also on the neurologic component of the disease (79; 109). Idebenone is now a recommended treatment of Leber hereditary optic neuropathy (18; 17).

Conclusion

The past 50 years have seen mitochondrial medicine develop beyond anyone’s wildest expectations, and the plot is still thickening. However, our understanding of pathogenesis leaves much to be desired, and progress in this area is indispensable for developing rational therapeutic strategies. The fervor of research in laboratories worldwide and the rise of national and international collaborative groups bode well for the future.