Polymerase-γ (POLG) is a major human disease gene and may account for up to 25% of all mitochondrial diseases in the UK and in Italy. To date, >150 different pathogenic mutations have been described in POLG. Some mutations behave as both dominant and recessive alleles, but an autosomal recessive inheritance pattern is much more common. The most frequently detected pathogenic POLG mutation in the Caucasian population is c.1399G>A leading to a p.Ala467Thr missense mutation in the linker domain of the protein. Although many patients are homozygous for this mutation, clinical presentation is highly variable, ranging from childhood-onset Alpers-Huttenlocher syndrome to adult-onset sensory ataxic neuropathy dysarthria and ophthalmoparesis. The reasons for this are not clear, but familial clustering of phenotypes suggests that modifying factors may influence the clinical manifestation. In this study, we collected clinical, histological and biochemical data from 68 patients carrying the homozygous p.Ala467Thr mutation from eight diagnostic centres in Europe and the USA. We performed DNA analysis in 44 of these patients to search for a genetic modifier within POLG and flanking regions potentially involved in the regulation of gene expression, and extended our analysis to other genes affecting mitochondrial DNA maintenance (POLG2, PEO1 and ANT1). The clinical presentation included almost the entire phenotypic spectrum of all known POLG mutations. Interestingly, the clinical presentation was similar in siblings, implying a genetic basis for the phenotypic variability amongst homozygotes. However, the p.Ala467Thr allele was present on a shared haplotype in each affected individual, and there was no correlation between the clinical presentation and genetic variants in any of the analysed nuclear genes. Patients with mitochondrial DNA haplogroup U developed epilepsy significantly less frequently than patients with any other mitochondrial DNA haplotype. Epilepsy was reported significantly more frequently in females than in males, and also showed an association with one of the chromosomal markers defining the POLG haplotype. In conclusion, our clinical results show that the homozygous p.Ala467Thr POLG mutation does not cause discrete phenotypes, as previously suggested, but rather there is a continuum of clinical symptoms. Our results suggest that the mitochondrial DNA background plays an important role in modifying the disease phenotype but nuclear modifiers, epigenetic and environmental factors may also influence the severity of disease.

Mitochondrial neurogastrointestinal encephalomyopathy is a rare multisystemic autosomic recessive disorder characterized by: onset typically before the age of 30 years; ptosis; progressive external ophthalmoplegia; gastrointestinal dysmotility; cachexia; peripheral neuropathy; and leucoencephalopathy. The disease is caused by mutations in the TYMP gene encoding thymidine phosphorylasethymine phosphorylase. Anecdotal reports suggest that allogeneic haematopoetic stem cell transplantation may be beneficial for mitochondrial neurogastrointestinal encephalomyopathy, but is associated with a high mortality. After selecting patients who fulfilled the clinical criteria for mitochondrial neurogastrointestinal encephalomyopathy and had severe thymidine phosphorylase deficiency in the buffy coat (<10% of normal activity), we reviewed their medical records and laboratory studies. We identified 102 patients (50 females) with mitochondrial neurogastrointestinal encephalomyopathy and an average age of 32.4 years (range 11–59 years). We found 20 novel TYMP mutations. The average age-at-onset was 17.9 years (range 5 months to 35 years); however, the majority of patients reported the first symptoms before the age of 12 years. The patient distribution suggests a relatively high prevalence in Europeans, while the mutation distribution suggests founder effects for a few mutations, such as c.866A>G in Europe and c.518T>G in the Dominican Republic, that could guide genetic screening in each location. Although the sequence of clinical manifestations in the disease varied, half of the patients initially had gastrointestinal symptoms. We confirmed anecdotal reports of intra- and inter-familial clinical variability and absence of genotype–phenotype correlation in the disease, suggesting genetic modifiers, environmental factors or both contribute to disease manifestations. Acute medical events such as infections often provoked worsening of symptoms, suggesting that careful monitoring and early treatment of intercurrent illnesses may be beneficial. We observed endocrine/exocrine pancreatic insufficiency, which had not previously been reported. Kaplan–Meier analysis revealed significant mortality between the ages of 20 and 40 years due to infectious or metabolic complications. Despite increasing awareness of this illness, a high proportion of patients had been misdiagnosed. Early and accurate diagnosis of mitochondrial neurogastrointestinal encephalomyopathy, together with timely treatment of acute intercurrent illnesses, may retard disease progression and increase the number of patients eligible for allogeneic haematopoetic stem cell transplantation.

A 48-year-old man presented with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) syndrome and Kearns-Sayre syndrome (KSS): progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, bifascicular heart block, and ragged-red fibers. The m.3291T>C mutation in the tRNALeu(UUR) gene was found with 92% heteroplasmy in muscle. This mutation has been reported with MELAS, myopathy, and deafness with cognitive impairment. This is the first description with a MERRF/KSS syndrome.

Coenzyme Q10 (CoQ10) is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ10 biosynthesis. We observed a unimodal distribution of ROS production with CoQ10 deficiency: cells with <20% of CoQ10 and 50–70% of CoQ10 did not generate excess ROS while cells with 30–45% of CoQ10 showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ10 deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB), an analog of 4-hydroxybenzoate (4-HB) and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2) to induce a range of CoQ10 deficiencies. Our results support the concept that the degree of CoQ10 deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40–50% residual CoQ10 produces maximal oxidative stress and cell death.

CoQ10 deficiencies are clinically and genetically heterogeneous. This syndrome has been associated with five major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) cerebellar ataxia, (4) isolated myopathy, and (5) nephrotic syndrome. In a few patients, pathogenic mutations have been identified in genes involved in the biosynthesis of CoQ10 (primary CoQ10 deficiencies) or in genes not directly related to CoQ10 biosynthesis (secondary CoQ10 deficiencies). Respiratory chain defects, ROS production, and apoptosis variably contribute to the pathogenesis of primary CoQ10 deficiencies.

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2−/−). Although normal until postnatal day 8, Tk2−/− mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2−/− mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2−/− heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2−/− heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues.

Coenzyme Q (CoQ) is an essential component of the respiratory chain but also participates in other mitochondrial functions such as regulation of the transition pore and uncoupling proteins. Furthermore, this compound is a specific substrate for enzymes of the fatty acids β–oxidation pathway and pyrimidine nucleotide biosynthesis. Furthermore, CoQ is an antioxidant that acts in all cellular membranes and lipoproteins. A complex of at least ten nuclear (COQ) genes encoded proteins synthesizes CoQ but its regulation is unknown. Since 1989, a growing number of patients with multisystemic mitochondrial disorders and neuromuscular disorders showing deficiencies of CoQ have been identified. CoQ deficiency caused by muta-tion(s) in any of the COQ genes is designated primary deficiency. Other patients have displayed other genetic defects independent on the CoQ biosynthesis pathway, and are considered to have secondary deficiencies. This review updates the clinical and molecular aspects of both types of CoQ deficiencies and proposes new approaches to understanding their molecular bases.

Until even only a few years ago, the idea that effective therapies for human mitochondrial disorders resulting from dysfunction of the respiratory chain/oxidative phosphorylation system (OxPhos) could be developed was unimaginable. The obstacles to treating diseases caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), and which had the potential to affect nearly every organ system, seemed overwhelming. However, while clinically applicable therapies still remain largely in the future, the landscape has changed dramatically; we can now envision the possibility of treating some of these disorders. Among these are techniques to upregulate mitochondrial biogenesis, to enhance organellar fusion and fission, to “shift heteroplasmy,” and to eliminate the burden of mutant mtDNAs via cytoplasmic transfer.

Coenzyme Q10 (CoQ10) is an essential electron carrier in the mitochondrial respiratory chain and an important antioxidant. Deficiency of CoQ10 is a clinically and molecularly heterogeneous syndrome, which, to date, has been found to be autosomal recessive in inheritance and generally responsive to CoQ10 supplementation. CoQ10 deficiency has been associated with five major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) cerebellar ataxia, (4) isolated myopathy, and (5) nephrotic syndrome. In a few patients, pathogenic mutations have been identified in genes involved in the biosynthesis of CoQ10 (primary CoQ10 deficiencies) or in genes not directly related to CoQ10 biosynthesis (secondary CoQ10 deficiencies). Respiratory chain defects, ROS production, and apoptosis contribute to the pathogenesis of primary CoQ10 deficiencies. In vitro and in vivo studies are necessary to further understand the pathogenesis of the disease and to develop more effective therapies.

Metabolic myopathies comprise a clinically and etiologically diverse group of disorders caused by defects in cellular energy metabolism, including the breakdown of carbohydrates and fatty acids to generate adenosine triphosphate, predominantly through mitochondrial oxidative phosphorylation. Accordingly, the three main categories of metabolic myopathies are glycogen storage diseases, fatty acid oxidation defects, and mitochondrial disorders due to respiratory chain impairment. The wide clinical spectrum of metabolic myopathies ranges from severe infantile-onset multisystemic diseases to adult-onset isolated myopathies with exertional cramps. Diagnosing these diverse disorders often is challenging because clinical features such as recurrent myoglobinuria and exercise intolerance are common to all three types of metabolic myopathy. Nevertheless, distinct clinical manifestations are important to recognize as they can guide diagnostic testing and lead to the correct diagnosis. This article briefly reviews general clinical aspects of metabolic myopathies and highlights approaches to diagnosing the relatively more frequent subtypes (Fig. 1).

We describe a 62-year-old woman with chronic progressive external ophthalmoplegia (CPEO), multiple lipomas, diabetes mellitus, and a novel mitochondrial DNA (mtDNA) mutation at nucleotide 4302 (4302A>G) of the tRNAIle gene (MTTI). This is the first mutation at position 44 in the variable loop (V loop) of any mitochondrial tRNA.

The muscle biopsy revealed 10% ragged-red or ragged-blue fibers and 25% cytochrome c oxidase (COX)-deficient fibers.

No deletions or duplications were detected by Southern blot analysis. The 4302A>G transition was present only in the patient’s muscle and single-fiber analysis revealed significantly higher levels of the mutation in COX-deficient than in normal fibers. Like tRNALeu(UUR), tRNAIle appears to be a “hot spot” for mtDNA mutations causing CPEO.

Background

Coenzyme Q10 (CoQ10) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ10, coenzyme Q2 (CoQ2), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ10 deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ10 of patients treated with oral ubiquinone. CoQ10 supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ10 deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ10 in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ10 in the mitochondrial respiratory chain under conditions of CoQ10 deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ10 deficiencies should be treated with CoQ10 supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ2. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.

We sequenced all mitochondrial tRNA genes in a 61-year-old man with chronic progressive external ophthalmoplegia and mitochondrial myopathy but without mtDNA rearrangements, and identified a heteroplasmic m.3244G>A mutation in the tRNALeu (UUR) gene. This mutation had been previously associated with the MELAS phenotype, but not described in any detail.

The mutation load in muscle was 84% and COX-negative fibers harbored greater levels of mutant genomes than COX-positive fibers. The m.G3244G>A mutation affects a highly conserved nucleotide in the dihydrouridine loop and has been associated with a wobble modification deficiency of the mutant tRNA.

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP−/−UP−/−) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient's lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 ρ0 cells devoid of mtDNA restored cytochrome oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis.

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as ‘benign cytochrome c oxidase deficiency myopathy’ is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNAGlu mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNAGlu may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. Last year, we reported the first mutations in CoQ10 biosynthetic genes, COQ2, which encodes 4-parahydroxybenzoate: polyprenyl transferase; and PDSS2, which encodes subunit 2 of decaprenyl diphosphate synthase. However, the pathogenic mechanisms of primary CoQ10 deficiency have not been well characterized. In this study, we investigated the consequence of severe CoQ10 deficiency on bioenergetics, oxidative stress, and antioxidant defenses in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations. Defects in the first two committed steps of the CoQ10 biosynthetic pathway produce different biochemical alterations. PDSS2 mutant fibroblasts have 12% CoQ10 relative to control cells and markedly reduced ATP synthesis, but do not show increased reactive oxygen species (ROS) production, signs of oxidative stress, or increased antioxidant defense markers. In contrast, COQ2 mutant fibroblasts have 30% CoQ10 with partial defect in ATP synthesis, as well as significantly increased ROS production and oxidation of lipids and proteins. On the basis of a small number of cell lines, our results suggest that primary CoQ10 deficiencies cause variable defects of ATP synthesis and oxidative stress, which may explain the different clinical features and may lead to more rational therapeutic strategies.—Quinzii, C. M., López, L. C., Von-Moltke, J., Naini, A., Krishna, S., Schuelke, M., Salviati, L., Navas, P., DiMauro, S., Hirano, M. Respiratory chain dysfunction and oxidative stress correlate with severity of primary CoQ10 deficiency.

The Vma21p protein in yeast is an essential assembly chaperone for the vacuolar ATPase, the major proton pump of cellular membranes. In this issue, Ramachandran et al. (2009) report that mutations in the gene encoding the human homolog VMA21 cause the disease X-linked myopathy with excessive autophagy through an unexpected mechanism.

Sixteen Korean patients with Leigh syndrome were identified at the Seoul National University Children's Hospital in 2001-2006. Biochemical or molecular defects were identified in 14 patients (87.5%). Thirteen patients had respiratory chain enzyme defects; 9 had complex I deficiency, and 4 had combined defects of complex I + III + IV. Based on the biochemical defects, targeted genetic studies in 4 patients with complex I deficiency revealed two heteroplasmic mitochondrial DNA mutations in ND genes. One patient had the mitochondrial DNA T8993G point mutation. No mitochondrial DNA defects were identified in 11 (68.7%) of our LS patients, who probably have mutations in nuclear DNA. Although a limited study based in a single tertiary medical center, our findings suggest that isolated complex I deficiency may be the most common cause of Leigh syndrome in Korea.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2−/−) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2−/− animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Mutations in mitochondrial DNA (mtDNA) tRNA genes can be considered functionally recessive because they result in a clinical or biochemical phenotype only when the percentage of mutant molecules exceeds a critical threshold value, in the range of 70–90%. We report a novel mtDNA mutation that contradicts this rule, since it caused a severe multisystem disorder and respiratory chain (RC) deficiency even at low levels of heteroplasmy. We studied a 13-year-old boy with clinical, radiological and biochemical evidence of a mitochondrial disorder. We detected a novel heteroplasmic C>T mutation at nucleotide 5545 of mtDNA, which was present at unusually low levels (<25%) in affected tissues. The pathogenic threshold for the mutation in cybrids was between 4 and 8%, implying a dominant mechanism of action. The mutation affects the central base of the anticodon triplet of tRNATrp and it may alter the codon specificity of the affected tRNA. These findings introduce the concept of dominance in mitochondrial genetics and pose new diagnostic challenges, because such mutations may easily escape detection. Moreover, similar mutations arising stochastically and accumulating in a minority of mtDNA molecules during the aging process may severely impair RC function in cells.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease due to ECGF1 gene mutations causing thymidine phosphorylase (TP) deficiency. Analysis of post-mortem samples of five MNGIE patients and two controls, revealed TP activity in all control tissues, but not in MNGIE samples. Converse to TP activity, thymidine and deoxyuridine were absent in control samples, but present in all tissues of MNGIE patients. Concentrations of both nucleosides in the tissues were generally higher than those observed in plasma of MNGIE patients. Our observations indicate that in the absence of TP activity, tissues accumulate nucleosides, which are excreted into plasma.