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Research Notes: Mitochondrial DNA Depletion SyndromesAnnu Rev Med. 2007 Sep 24. Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17. Hum Mol Genet. 2007 Jun 15. Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients. Nat Genet. 2007 Jun. Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b(-/-) mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis. Diagn Mol Pathol. 2007 Jun. Human mitochondrial diseases are usually caused by dysfunction of mitochondrial DNA (mtDNA), particularly by point mutations, deletions, or depletions. In commonly used procedures for molecular diagnostics of mitochondrial dysfunction, one of the first steps is linearization of circular mitochondrial genomes with either BamHI or PvuII restriction endonulease, which cuts human mtDNA at a unique site. Here, we describe a case of false positive results, which suggested mtDNA depletion or a large deletion in a patient's tissue sample. More detailed analysis (mtDNA sequencing) revealed that these false positive results were caused by the presence of the 12753A>G substitution in the gene coding for NADH dehydrogenase subunit 5 (ND5). This substitution results in no change in amino acid sequence of the gene product but creates an additional PvuII site. Investigating a population of 200 patients not affected by mitochondrial diseases, we found an additional case of 12753A>G, and also another substitution, 12804T>C, which also results in no change in amino acid sequence of ND5 but creates an additional PvuII site. A few cases of 12753A>G and 12804T>C substitutions were found previously in Asian, American, African, and European populations (though they were not reported to date in the MITOMAP), but those samples were used in population studies and not tested for mtDNA deletion or depletion. Therefore, we present a cautionary report indicating that these mtDNA polymorphisms exist in various human populations (and thus, they are panethnic) and may cause false positive results of standard molecular analyses, including molecular diagnostics, of human mtDNA. J Pediatr. 2007 May. OBJECTIVE: To determine the actual incidence of mitochondrial DNA (mtDNA) depletion syndrome in multiple respiratory chain deficiency. STUDY DESIGN: We carried out a real-time polymerase chain reaction quantification of mtDNA in liver or muscle tissue of 100 children with unexplained multiple oxidative phosphorylation enzyme deficiency. RESULTS: A reduction of mtDNA copy number to <35% of control values was found in liver and/or muscle in half of the children (50/100). Most of these patients (32/50; 64%) presented with severe neonatal onset liver involvement; 7 (14%) had Alpers syndrome, and 11 (22%) exhibited various forms of neurologic involvement. Deoxyguanosine kinase or polymerase gamma (POLG) mutations could be identified in 11 of 32 patients with liver involvement, and POLG mutations were consistently found in all 7 patients with Alpers syndrome. Homozygous thymidine kinase 2 and MPV17 gene mutations were found in 2 patients. CONCLUSIONS: Our findings show that mtDNA depletion is a prevalent cause of multiple respiratory chain deficiency in infancy. Diabetes Res Clin Pract. 2007 Apr 24. Since the bioenergetic capacity of skeletal muscle mitochondria is decreased in type 2 diabetes and obesity, the reduction of mitochondrial DNA (mtDNA) content may be involved in the development of insulin resistance in skeletal muscle. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long-term treatment with ethidium bromide (EtBr). L6 GLUT4myc cells cultured with 0.2mug/ml EtBr (termed depleted cells) revealed a marked decrease in cellular mtDNA, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Interestingly, the expression of IRS-1 associated with insulin signaling was decreased by 90% in the depleted cells, and the insulin-stimulated phosphorylation of IRS-1 and Akt2/PKB were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reduction in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/PKB are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes. Neurologia. 2007 Apr. INTRODUCTION: Mitochondrial DNA depletion (mtDNA) is an highly heterogeneous condition characterized by a decreased number of mtDNA copies. CASE REPORT: The patient is a 22-month-old girl with generalized hypotonia, marked weakness, respiratory failure, arterial hypertension, hyperlactacidemia, hepatosplenomegaly and mild hypertransaminasemia without hepatic failure neither hypoketotic hypoglycemia. Electromyographic findings were consistent with neuromyopathy and muscle biopsy suggested a neurogenic atrophy. Electron microscopy revealed lipid droplets, subsarcolemmal accumulation of mitochondrias and glycogen granules. Respiratory chain enzime activities were normal. Genetic study in muscle showed mtDNA depletion, and the diagnosis of spinal muscular atrophy caused by survival motoneuron gene deletion was excluded. CONCLUSIONS: This case might be a novel phenotype of mtDNA depletion which could be named hepatomioneuropatyc [hepatomitoneuropathic?] form. A normal result of respiratory chain enzimes in muscle doesn't excluded mtDNA depletion. Biochem J. 2007 Mar 1. DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal. Eur J Pediatr. 2007 Mar. Failure to thrive, feeding difficulties, variable forms of infantile epilepsy or psychomotor developmental delay and hypotonia were the most frequent clinical disease presentations in eight children with combined oxidative phosphorylation enzyme complex deficiencies carrying mutations in the polymerase gamma (POLG1) gene. Five out of eight patients developed severe liver dysfunction during the course of the disease. Three of these patients fulfilled the disease criteria for Alpers syndrome. Most children showed deficiencies of respiratory chain enzyme complexes I and III, in combination with complex II, complex IV and/or PDHc in muscle, whereas in fibroblasts normal enzyme activities were measured. All children carried homozygous or compound heterozygous mutations in the POLG1 gene, including two novel mutations in association with mtDNA depletion. Conclusion. We suggest performing POLG1 mutation analysis in children with combined oxidative phosphorylation deficiencies in muscle, even if the clinical picture is not Alpers syndrome. Obesity (Silver Spring). 2006 Dec. OBJECTIVE: To investigate whether mitochondrial DNA (mtDNA) content may be associated with clinical features, anthropometric variables, and laboratory findings in both extremes of abnormal fetal growth: small and large size for gestational age. RESEARCH METHODS AND PROCEDURES: Eighty-eight pregnant women and their infants were included in a cross-sectional study. According to the offspring birthweight, normalized by sex and gestational age, there were 57 newborns with appropriate weight for gestational age (AGA) and 31 with abnormal weight for gestational age: 17 small for gestational age (SGA) and 14 large for gestational age (LGA). mtDNA quantification using nuclear DNA as a reference was measured by a real-time quantitative polymerase chain reaction method. RESULTS: The mothers' pregestational BMI was associated with the weight of their offspring: SGA infants had lean mothers (BMI, 21.4 +/- 0.7), and LGA infants had overweight mothers (BMI, 26.7 +/- 1.4) in comparison with AGA infants (BMI, 23.0 +/- 0.7) (p < 0.003). Newborn leptin levels were associated with birthweight after adjustment for sex and gestational age (SGA, 7.0 +/- 1.1 ng/mL; AGA, 15.2 +/- 1.6 ng/mL; and LGA, 25.6 +/- 4.1 ng/mL) (p < 0.002). Conversely, mtDNA/nuclear DNA ratio was significantly lower in both extremes of abnormal fetal growth, SGA (18 +/- 6) and LGA (9 +/- 2), at birth in comparison to AGA-weight infants (28 +/- 4) (p < 0.03). DISCUSSION: Our findings show that mtDNA content is decreased in newborns with abnormal weight in comparison with AGA infants. On the basis of a cumulative body of evidence, we speculate that mtDNA depletion is one of the putative links between abnormal fetal growth and metabolic and cardiovascular complications in later life. Pediatr Res. 2006 Sep. We present the clinical and laboratory features of a boy with a new syndrome of mitochondrial depletion syndrome and T cell immunodeficiency. The child suffered from severe recurrent infectious diseases, anemia, and thrombocytopenia. Clinically, he presented with severe psychomotor retardation, axial hypotonia, and a disturbed pain perception leading to debilitating biting of the thumb, lower lip, and tongue. Brain imaging showed hypoplasia of corpus callosum and an impaired myelinization of the temporo-occipital region with consecutive supratentorial hydrocephalus. Histologic examination of a skeletal muscle biopsy was normal. Biochemical investigation showed combined deficiency of respiratory chain complexes II+III and IV. MtDNA depletion was found by real-time PCR. No pathogenic mutations were identified in the TK2, SUCLA2, DGUOK, and ECGF1 genes. A heterozygous missense mutation was found in POLG1. The pathogenic relevance of this mutation is unclear. Interestingly, a lack of CD8(+) T lymphocytes as well as NK cells was also observed. The percentage of CD45RO-expressing cells was decreased in activated CD8(+) T lymphocytes. Activation of T lymphocytes via IL-2 was diminished. The occurrence of the immunologic deficiency in our patient with mtDNA depletion is a rare finding, implying that cells of the immune system might also be affected by mitochondrial disease. Arch Neurol. 2006 Aug. BACKGROUND: Mitochondrial DNA depletion syndrome is an autosomal recessive disorder characterized by decreased mitochondrial DNA copy numbers in affected tissues. It has been linked to 4 genes involved in deoxyribonucleotide triphosphate metabolism: thymidine kinase 2 (TK2), deoxyguanosine kinase (DGUOK), polymerase gamma (POLG), and SUCLA2, the gene encoding the beta-subunit of the adenosine diphosphate-forming succinyl coenzyme A synthetase ligase. OBJECTIVE: To highlight the variability in the clinical spectrum of TK2-related mitochondrial DNA depletion syndrome. DESIGN: Review of patients and the literature. SETTING: Tertiary care university. PATIENTS: Four patients with mitochondrial DNA depletion syndrome and mutations in the TK2 gene. MAIN OUTCOME MEASURES: Definition of clinical variability. RESULTS: Patient 1 had evidence of lower motoneuron disease and was initially diagnosed as having spinal muscular atrophy type 3. Patient 2, who is alive and ambulatory at age 9 years, presented at age 2 years with a slowly progressive mitochondrial myopathy. Patient 3 had a more severe myopathy, with onset in infancy and death at age 6 years of respiratory failure. Patient 4 had a rapidly progressive congenital myopathy with rigid spine syndrome and he died at age 19 months. CONCLUSION: The clinical spectrum of TK2 mutations is not limited to severe infantile myopathy with motor regression and early death but includes spinal muscular atrophy type 3-like presentation, rigid spine syndrome, and subacute myopathy without motor regression and with longer survival. Arch Neurol. 2006 Aug. BACKGROUND: Autosomal recessive mutations in deoxyguanosine kinase (DGUOK) have been identified in the hepatocerebral form of mitochondrial DNA (mtDNA) depletion syndrome. OBJECTIVES: To describe the clinical spectrum of DGUOK-related mtDNA depletion syndrome in 6 children and to summarize the literature. RESULTS: We identified pathogenic mutations in DGUOK in 6 children with the hepatocerebral form of mtDNA depletion syndrome. We describe the clinical, neuroradiologic, histologic, and genetic features in these children. All children showed severe hepatopathy, while involvement of other organs (skeletal muscle and brain) was variable. We identified 5 novel mutations (1 of them in 2 children) and 2 previously described mutations. Three different mutations affected the initial methionine, suggesting a mutational hot spot. One of our patients underwent liver transplantation; pathologic findings revealed (in addition to diffuse hepatopathy) a hepatocellular carcinoma, implying a possible link between mtDNA depletion syndrome and tumorigenesis. CONCLUSION: We studied 12 children with infantile hepatoencephalopathies and mtDNA depletion syndrome and found pathogenic DGUOK mutations in 6, suggesting that this gene defect is a frequent but not an exclusive cause of the hepatic form of mtDNA depletion syndrome. Eur J Hum Genet. 2006 Aug. ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening. Acta Neurol Belg. 2006 Jun. Mitochondrial DNA (mtDNA) is maternally inherited. After birth, secondary mtDNA defects can arise. MtDNA depletion is a reduction in the amount of mtDNA in particular tissues. Multiple deletions of mtDNA accumulate as somatic mutations in mainly postmitotic tissues. These disorders of mtDNA maintenance frequently show Mendelian inheritance. Positional cloning has identified several genes involved in the control of mtDNA stability. Recessive mutations in the genes ECGF1, dGK, TK2, SUCLA2 and POLG cause mtDNA depletion syndromes (MDS). Generally, MDS has infantile onset tissue specific features. Mutations in the genes ECGF1, ANT1, C10orf2 and POLG are associated with multiple mtDNA deletions. The nature of these mutations is dominant in ANT1, C10orf2 and POLG and recessive in ECGF1, C10orf2 and POLG. Mutations in these genes frequently cause progressive external ophthalmoplegia (PEO). However clinical heterogeneity results in different neurological syndromes with considerable overlap. The most common features are PEO, neuropathy, myopathy, ataxia, epilepsy and hepatopathy. Neurology. 2006 May 9. The authors sequenced POLG1, C10ORF2, and ANT1 in 38 sporadic progressive external ophthalmoplegia patients with multiple mitochondrial DNA (mtDNA) deletions. Causative mutations were identified in approximately 10% of cases, with two unrelated individuals harboring a novel premature stop codon mutation (1356T>G). None had a mutation in C10ORF2 or ANT1. In the majority of patients, the primary nuclear genetic defect is likely to affect other unknown genes important for mtDNA maintenance. Nat Genet. 2006 May. The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice. Pediatr Neurol. 2006 Mar. Mitochondrial deoxyribonucleic acid depletion syndromes are autosomal recessive disorders characterized by a reduction of the amount of mitochondrial deoxyribonucleic acid, which impairs the synthesis of respiratory chain complexes. Mutations in the deoxyguanosine kinase and polymerase gamma genes have been identified in hepatocerebral forms, whereas thymidine kinase 2 gene mutations have been found in patients with isolated myopathy, encephalomyopathy, or spinal muscular atrophy. Mutations in the gene encoding the beta subunit of the adenosine diphosphate-forming succinyl-coenzyme A synthetase have also been reported in a family. In this report, the clinical, molecular, morphologic, and biochemical features of five children from two independent families with an infantile encephalomyopathy are characterized. The affected children manifested muscle mitochondrial deoxyribonucleic acid depletion and three novel thymidine kinase 2 gene mutations. They consist of a homozygous substitution resulting in Ala to Val change at the highly conserved position 181 of thymidine kinase in the first family, and two heterozygous substitutions in the second family: a Cys to Trp change at residue 108 and a Leu to Pro change at residue 257 of the enzyme. Common clinical features associated with these TK2 mutations are a normal early developmental phase followed by psychomotor regression, encephalopathy often with epileptic seizures, and myopathy with features of a progressive dystrophic process. Arch Neurol. 2006 Jan. BACKGROUND: Both dominant and recessive mutations were reported in the gene encoding the mitochondrial (mt) DNA polymerase gamma (POLG) in patients with progressive external ophthalmoplegia (PEO). Phenotypes other than PEO were recently documented in patients with mutations in the POLG gene. OBJECTIVE: To screen patients with mitochondrial disease and multiple mtDNA deletions in muscle for mutations in the coding regions of the POLG, PEO1, and SLC25A4 genes. DESIGN: To identify the underlying molecular defect in a group of patients with multiple mtDNA deletions comparing their molecular genetic findings with those of healthy controls. PATIENTS: Twenty-four patients (16 men and 8 women) diagnosed with mitochondrial disease and having multiple mtDNA deletions in muscle by Southern blot analysis. Thirteen patients had PEO; 2 had PEO alone, 4 had PEO and myopathy, and 5 had PEO and multisystem involvement. Four patients had multisystem disease without PEO. The remaining 9 patients had isolated myopathy. DNA from 100 healthy individuals was also studied. RESULTS: No mutation was identified in the PEO1 or SLC25A4 genes. Nine POLG mutations were observed in 6 of 24 patients. Four novel mutations were detected and mapped in the linker region (M603L) and in the pol domain of the enzyme (R853W; D1184N; R1146C). Five patients with PEO had mutations: 2 were compound heterozygotes, 1 was homozygous, and another showed a mutation in a single allele. The remaining patient also showed a sole mutation and had an unusual phenotype lacking ocular involvement. CONCLUSIONS: POLG molecular defects were found in 25% of our patients with multiple mtDNA deletions and mitochondrial disease. The uncommon phenotype found in 1 of these patients stresses the clinical variability of patients harboring POLG mutations. Molecular studies in the POLG gene should be addressed in patients with mitochondrial disease, particularly in those with PEO, and multiple mtDNA deletions. Acta Biochim Pol. 2006. The influence of mutations in the mitochondrial DNA (mtDNA) on the bioenergetic metabolism of the cell is still poorly understood. Many of the mutations in the mtDNA affect the expression of the mitochondrial genome. Investigations on cells from patients are not easy, especially as the mitochondrial DNA is heteroplasmic and this state is changed in culture. Moreover, the nuclear background and the mitochondrial haplotype may affect the behaviour of cells. Transfer of patient mitochondria to rho zero cell lines is also not optimal as these cells in general have many nuclear changes which may also affect cell behaviour. Thus, we decided to use inhibitors of mitochondrial genome expression, such as thiamphenicol, ethidium bromide and dideoxycytidine to investigate the bioenergetic metabolism of HeLa cells. We found that oxidative phosphorylation and glycolysis participate equally in ATP production in HeLa cells and that decreased activity of the respiratory chain leads to increased glycolysis and the reduction of cell growth. Insufficient ATP production in the oxidative phosphorylation process was not compensated by increased proliferation of the mitochondria. However, we were able to show that there are some mechanisms compensating limited expression of the mitochondrial genome within the mitochondria. Experiments with dideoxycytidine revealed that 10-fold decrease of the mtDNA copy number resulted in almost normal activity of cytochrome c oxidase. We found that mtDNA depletion is compensated mostly on the level of RNA metabolism in the mitochondria. Thus, our results are in agreement with the hypothesis that transcription initiation rather than mtDNA copy number is a rate limiting factor for expression of the mitochondrial genome. Nucleic Acids Res. 2006. Mitochondrial transcription factor A (TFAM) is an abundant mitochondrial protein of the HMG superfamily, with various putative roles in mitochondrial DNA (mtDNA) metabolism. In this study we have investigated the effects on mtDNA replication of manipulating TFAM expression in cultured human cells. Mammalian mtDNA replication intermediates (RIs) fall into two classes, whose mechanistic relationship is not properly understood. One class is characterized by extensive RNA incorporation on the lagging strand, whereas the other has the structure of products of conventional, strand-coupled replication. TFAM overexpression increased the overall abundance of RIs and shifted them substantially towards those of the conventional, strand-coupled type. The shift was most pronounced in the rDNA region and at various replication pause sites and was accompanied by a drop in the relative amount of replication-termination intermediates, a substantial reduction in mitochondrial transcripts, mtDNA decatenation and progressive copy number depletion. TFAM overexpression could be partially phenocopied by treatment of cells with dideoxycytidine, suggesting that its effects are partially attributable to a decreased rate of fork progression. TFAM knockdown also resulted in mtDNA depletion, but RIs remained mainly of the ribosubstituted type, although termination intermediates were enhanced. We propose that TFAM influences the mode of mtDNA replication via its combined effects on different aspects of mtDNA metabolism. Mol Genet Metab. 2005 Dec. The activity of deoxyguanosine kinase (DGUOK), a mitochondrial enzyme involved in the anabolism of mitochondrial (mt) deoxyribonucleotides, governs the maintenance of the mtDNA. Deleterious mutations of the DGUOK gene are thus associated with mtDNA depletion and result in combined deficiencies of mtDNA-encoded respiratory chain enzymes. With the aim to estimate the prevalence of DGUOK mutations in a cohort of 30 patients with hepatocerebral disease and combined respiratory chain deficiencies, we studied the DGUOK gene and identified previously unreported mutations in five families. Two patients and their affected sibs, born to non-consanguineous parents, were homozygous for a missense mutation (M1T, and L250S, respectively). One patient presented a homozygous 4 pb insertion (796 insTGAT) and two other patients, and their affected sibs, were compound heterozygous (E165V/L266R and E211G/L266R, respectively). These findings allowed us to propose prenatal diagnosis in two families. In conclusion, we observed a high prevalence of DGUOK mutations (17%) in patients with hepatic involvement and combined respiratory chain deficiencies with hepatic involvement. Mutat Res. 2005 Oct 15. The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease. FEBS J. 2005 Oct. Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood. In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho-L929 and rho(0)143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up-regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF-1, NRF-2, CREB and PGC-1alpha) known to control the expression of numerous nuclear genes encoding mitochondrial proteins. Among these regulators, cyclic AMP-responsive element binding protein (CREB) activity was the only one to be increased in mtDNA-depleted cells. CREB activation mediated by a calcium-dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA-depleted cells that over-express CREB dominant negative mutants. Mitochondrial biogenesis in mtDNA-depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass. Despite a slight increase in mitochondrial mass in mtDNA-depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix-targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA-apoaequorin targeted to the mitochondria. Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA-depleted cells. Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA-depleted cells and involves a calcium-CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins. IUBMB Life. 2005 Sep. Mitochondrial disorders of oxidative phosphorylation (OXPHOS) comprise a growing list of potentially lethal diseases caused by mutations in either mitochondrial (mtDNA) or nuclear DNA (nDNA). Two such conditions, autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger's Syndrome, are associated with dysfunction of the heart and muscle-specific isoform of the adenine nucleotide translocase (ANT1), a nDNA gene product that facilitates transport of ATP and ADP across the inner mitochondrial membrane. AdPEO is a mtDNA deletion disorder broadly characterized by pathology involving the eyes, skeletal muscle, and central nervous system. In addition to ANT1, mutations in at least two other nuclear genes, twinkle and POLG, have been shown to cause mtDNA destabilization associated with adPEO. Senger's syndrome is an autosomal recessive condition characterized by congenital heart defects, abnormalities of skeletal muscle mitochondria, cataracts, and elevated circulatory levels of lactic acid. This syndrome is associated with severe depletion of ANT1, which may be the result of an as yet unidentified ANT1-specific transcriptional or translational processing error. ANT1 has also been associated with a third condition, autosomal dominant facioscapulohumeral muscular dystrophy (FSHD), an adult onset disorder characterized by variable muscle weakness in the face, feet, shoulders, and hips. FSHD patients possess specific DNA deletions on chromosome 4, which appear to cause derepression of several nearby genes, including ANT1. Early development of FSHD may involve mitochondrial dysfunction and increased oxidative stress, possibly associated with overexpression of ANT1. Neurology. 2005 Aug 9. The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome. Gene. 2005 Jul 18. Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a number of autosomal disorders classified as defects of nuclear-mitochondrial intergenomic signaling. The mendelian forms of progressive external ophthalmoplegia (PEO) are clinically and genetically heterogeneous disorders characterized by the accumulation of multiple deletions of mtDNA in postmitotic patient's tissues. Most of the autosomal dominant PEO (adPEO) families carry heterozygous mutations in either one of three genes: ANT1, Twinkle, and POLG1. Mutations in POLG1 can also cause autosomal recessive PEO (arPEO) and apparently sporadic cases. In addition, recessive POLG1 mutations are responsible for sensory-atactic neuropathy, dysarthria and ophthalmoplegia (SANDO), juvenile spino-cerebellar ataxia-epilepsy syndrome (SCAE) and Alpers-Huttenlocher hepatopathic poliodystrophy. Mutations in thymidine phosphorylase gene (TP) are linked to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder in which PEO is associated with gastrointestinal dysmotility and leukodystrophy. Finally, mitochondrial DNA depletion syndromes (MDS), defined by tissue-reduction in mtDNA copy number, have been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGUOK). Am J Hum Genet. 2005 Jun. The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome. J Biol Chem. 2005 Mar 18. Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes. Mol Genet Metab. 2005 Jan. Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development. J Acquir Immune Defic Syndr. 2004 Dec 15. Nucleoside analogue use is often related to mitochondrial DNA (mtDNA) depletion, but mitochondrial function is preserved in most asymptomatic patients. We determined whether homeostatic mechanisms are able to compensate for this mtDNA depletion in patients receiving stavudine plus didanosine (d4T + ddI), an antiretroviral combination with great in vitro and in vivo capacity to decrease mtDNA. We included 28 asymptomatic HIV-infected individuals: 17 subjects (cases) on a first-line antiretroviral regimen consisting of d4T + ddI as the nucleoside backbone plus nevirapine or nelfinavir for at least 6 months (mean: 16 +/- 8 months) and 11 naive subjects (controls). We assessed the following in peripheral blood mononuclear cells: mitochondrial mass by citrate synthase activity, mtDNA content by real-time polymerase chain reaction, cytochrome c oxidase subunit II (COX-II) expression by Western blot analysis, and COX activity by spectrophotometry. The mitochondrial mass and mtDNA content of cases decreased when compared with controls, whether normalized per cell or per mitochondrion. Conversely, COX-II expression and COX activity were similar in cases and controls. COX-II expression was constant and independent of the mtDNA content, whereas it was closely related to COX activity. We concluded that treatment with dd4T + ddI is associated with decreased mitochondrial mass and mtDNA content but that COX-II expression and COX activity remain unaltered. These data suggest that upregulatory transcriptional or posttranscriptional mechanisms compensate for mtDNA depletion caused by d4T + ddI before profound mtDNA depletion occurs. Nucleosides Nucleotides Nucleic Acids. 2004 Oct. Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools. Neuropediatrics. 2004 Aug. Mitochondrial dysfunction of the energy generating system was suggested in two infants with progressive infantile poliodystrophy characterised by hypotonia, refractory epilepsy, visual impairment, psychomotor retardation, profound brain atrophy, hepatopathy, and increased levels of lactate in blood and cerebrospinal fluid. Histochemical and electron microscopic analyses of liver biopsies revealed cytochrome c oxidase deficiency, microvesicular steatosis, and enormous multiplication of mitochondria of various sizes. In the first patient, the quantitative Southern blot analyses in tissues obtained at autopsy demonstrated reduced content of mtDNA in the liver, brain, and fibroblasts (11 %, 15 %, and 25 % of the mean values in controls) while a normal content of mtDNA was found in muscle and heart. In the second patient, a reduced content of mtDNA was found in the muscle, liver, and brain (15 %, 10 %, and 30 %, respectively, of the mean values in controls). Biochemical studies in the first patient revealed decreased activities of all respiratory chain complexes except complex II in isolated liver mitochondria and decreased amounts of respiratory chain complexes I, III, IV and ATP synthase in liver and frontal cortex, but not in muscle, heart, and fibroblasts. In conclusions, mtDNA depletion associated with Alpers syndrome may be tissue specific. Ann Neurol. 2004 May. Alpers' syndrome is a fatal neurogenetic disorder first described more than 70 years ago. It is an autosomal recessive, developmental mitochondrial DNA depletion disorder characterized by deficiency in mitochondrial DNA polymerase gamma (POLG) catalytic activity, refractory seizures, neurodegeneration, and liver disease. In two unrelated pedigrees of Alpers' syndrome, each affected child was found to carry a homozygous mutation in exon 17 of the POLG locus that led to a Glu873Stop mutation just upstream of the polymerase domain of the protein. In addition, each affected child was heterozygous for the G1681A mutation in exon 7 that led to an Ala467Thr substitution in POLG, within the linker region of the protein. Am J Physiol Cell Physiol. 2004 Apr. Defects in mitochondrial DNA (mtDNA) evoke distinctive responses in the nuclear genome, leading to altered mitochondrial biogenesis. We used C(2)C(12) cells depleted of mtDNA (rho(-) cells) and fibroblasts from a mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) patient to examine adaptations of the protein import machinery and transcription factors involved in mitochondrial biogenesis. In rho(-) cells, Tom20 and Tim23 protein levels were reduced by 25% and 59%, whereas mtHSP70 was induced by twofold relative to control cells. These changes were accompanied by a 21% increase in enhanced yellow fluorescent protein (EYFP) import into mitochondria in rho(-) cells (P < 0.05). In contrast, in MELAS cells mtHSP70 was elevated by 70%, whereas Tom20 and Tom34 protein levels were increased by 45% and 112% relative to control values. EYFP import was not altered in MELAS cells. In rho(-) cells, protein levels of the transcription factors nuclear respiratory factor-1 (NRF-1) and transcription factor A (Tfam) declined by 33% and 54%, whereas no change was observed for the coactivator peroxisome proliferator receptor-gamma coactivator-1alpha (PGC-1alpha). In contrast, Tfam was increased by 40% in MELAS cells. Rho(-) cells displayed reduced oxygen consumption (Vo(2)) and ATP levels, along with a twofold increase in lactate levels (P < 0.05). In electrically stimulated C(2)C(12) cells, 109%, 78%, 60%, and 67% increases were observed in mtDNA, Vo(2), cytochrome-c oxidase (COX) activity, and Tom34 levels, respectively (P < 0.05). Our findings suggest that compensatory adaptations occurred to maintain normal rates of protein import in response to mtDNA defects and support a role for contractile activity in reducing pathophysiology associated with mtDNA depletion. Because the expression of nuclear-encoded transcription factors and protein import machinery components was dependent on the type of mtDNA defect, these findings suggest involvement of distinct signaling cascades, each dependent on the type of mitochondrial defect, resulting in divergent changes in nuclear gene expression patterns. Arch Pediatr. 2004 Feb. Villous atrophy in an infant immediately suggests food intolerance. We report a case with an unusual cause. This female infant was first examined at 5 months for anorexia and failure to thrive. Intestinal biopsy showed total villous atrophy. A diet excluding gluten and cow milk proteins failed to improve her condition and the infant was hospitalized at 7 months for further investigations. The infant was hypotonic with a head lag. No other clinical sign was noted. Serum transaminases were increased 5- to 10-fold and CSF proteins concentration was increased. Metabolic investigations revealed hyperlactacidaemia and an increased lactate/pyruvate ratio during fasting and feeding, suggesting a mitochondrial cytopathy. Respiratory chain enzymatic activity measurements confirmed the diagnosis and showed severely decreased activities of complexes I, III and IV in both the liver and muscle. Molecular analysis demonstrated depletion of mitochondrial DNA in the liver (75%) and in muscle (97%). The infant was discharged under continuous enteral nutrition. Improvement was of short duration and the infant died at 1 year of age of massive hepatic failure. This is the first report of a mitochondrial DNA depletion with total villous atrophy and malabsorption as early clinical onset. A mitochondrial cytopathy should be considered in such conditions when food exclusion diets fail. J Pediatr. 2004 Jan. OBJECTIVES: To further characterize mtDNA defects associated with autistic features, especially the A3243G mtDNA mutation and mtDNA depletion. Study design. Five patients with autistic spectrum disorders and family histories of mitochondrial DNA diseases were studied. We performed mtDNA analysis in all patients and magnetic resonance spectroscopy in three. RESULTS: Three patients manifested isolated autistic spectrum features and two had additional neurologic symptoms. Two patients harbored the A3243G mutation. In two others, the A3243G mutation was not found in accessible tissues but was present in tissues from their mothers. The fifth patient had 72% mtDNA depletion in skeletal muscle. CONCLUSIONS: Autistic spectrum disorders with or without additional neurologic features can be early presentations of the A3243G mtDNA mutation and can be a prominent clinical manifestation of mtDNA depletion. Mitochondrial dysfunction should be considered in patients who have autistic features and associated neurologic findings or who have evidence of maternal inheritance. Biochem Biophys Res Commun. 2003 Oct 24. Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions. Mitochondrion. 2003 Aug. The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function. Arch Neurol. 2003 Jul. BACKGROUND: The mitochondrial DNA depletion syndrome is an autosomal recessive disorder of infancy or childhood characterized by decreased mitochondrial DNA copy number in affected tissues. Mutations in 2 genes involved in deoxyribonucleotide metabolism, the deoxyguanosine kinase gene (DGK) and the thymidine kinase 2 gene (TK2), have been related to this syndrome. OBJECTIVE: To describe 3 siblings with the myopathic form of mitochondrial DNA depletion syndrome and a homozygous mutation in the TK2 gene. PATIENTS AND METHODS: These children developed normally until 12 to 16 months of age, when they started showing difficulty walking, which rapidly progressed to severe limb weakness. They died of respiratory failure between the ages of 23 and 40 months. Histochemical and biochemical studies of respiratory chain complexes were performed in muscle biopsy specimens. The whole coding region of the TK2 gene was sequenced. RESULTS: Muscle biopsy showed ragged-red cytochrome-c oxidase-negative fibers. All affected siblings had markedly decreased activities of respiratory chain complexes. Southern blot analysis showed severe reduction of the mitochondrial DNA-nuclear DNA ratio in muscle biopsy specimens from all patients, indicating 80% to 90% mitochondrial DNA depletion. Sequencing of the TK2 gene showed a homozygous C-->T transition at nucleotide 228 in exon 5, which changes a threonine to a methionine at position 77 (T77M). CONCLUSIONS: These results document the importance of screening the TK2 gene in patients with myopathic mitochondrial DNA depletion syndrome and confirm that exon 5 is a "hot spot" for TK2 mutations. Clin Chem Lab Med. 2003 Jul. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). TP deficiency alters the metabolism of the nucleosides thymidine and deoxyuridine, which, in turn, produces abnormalities of mitochondrial DNA (mtDNA) including depletion, deletions, and point mutations. MNGIE is the best characterized of the expanding number of mitochondrial disorders caused by alterations in the metabolism of nucleosides/nucleotides. Because mitochondria contain their own machinery for nucleoside and nucleotide metabolism and have physically separate nucleotide pools, it is not surprising that disorders of these pathways cause human diseases. Other diseases in this group include mtDNA depletion syndromes caused by mutations on the nuclear genes encoding the mitochondrial thymidine kinase and deoxyguanosine kinase; autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA due to mutations in the genes encoding the muscle-isoform of mitochondrial ADP/ATP translocator; and mitochondrial DNA depletion due to toxicities of nucleoside analogues. Mutations in the deoxynucleotide carrier, a transporter of deoxynucleoside diphosphates, have been identified as a cause of congenital microcephaly. However, alterations of mtDNA have not yet been established in this disorder. Future studies are likely to reveal additional diseases and provide further insight into this new subject. Mol Genet Metab. 2003 May. Decreased mitochondrial thymidine kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency. Neurology. 2003 Apr 8. Mutations in the thymidine kinase 2 (TK2) gene cause a myopathic form of the mitochondrial DNA depletion syndrome (MDS). Here, the authors report the unusual clinical, biochemical, and molecular findings in a 14-year-old patient in whom pathogenic mutations were identified in the TK2 gene. This report extends the phenotypic expression of primary TK2 deficiency and suggests that factors other than TK2 may modify expression of the clinical phenotype in patients with MDS syndrome. Hum Mutat. 2003 Apr. Sixteen unrelated Southern European patients with the mitochondrial depletion syndrome (MDS) were analyzed for mutations in the TK2 and DGUOK genes. Three novel mutations were identified in TK2 (R183G, R254X, and 142insG). When we analyzed additional genes involved in the dNTPs pool, such as SLC25A19 (DNC) and NT5M (d-NT2), we did not detect mutations. The current study suggest that scanning the TK2, DGUOK, SLC25A19, and NT5M genes is likely to help about 10% of MDS families in terms of genetic counseling. Also, our findings indicate that genotype-phenotype correlations are not straightforward in MDS. Acta Neuropathol (Berl). 2003 Mar. Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3+/-7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome. J Biol Chem. 2003 Feb 28. Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed. J Inherit Metab Dis. 2003. Mitochondrial DNA (mtDNA) depletion refers to a quantitative defect in mtDNA and is heterogeneous with regard to causal genotypes and the associated clinical phenotypes. We report two unrelated infants with mtDNA depletion. A diagnosis of methylmalonic aciduria was initially raised in both on the basis of high urine methylmalonic acid and related organic acids and elevated propionylcarnitine and methylmalonylcarnitine. Carboxylase assay with skin fibroblasts revealed low propionyl-CoA and 3-methylcrotonyl-CoA carboxylase and normal pyruvate carboxylase activities. Quantitative Southern blot analysis of mitochondrial and nuclear DNA with muscle tissues revealed the patients' mtDNA to be depleted to 24% and 39% of normal controls. Our two patients showed multiple mitochondrial dysfunction including respiratory chain defects and deficiencies in the two nuclear DNA encoded carboxylases resulting in abnormal urine organic acids. To our knowledge, there is no obvious connection between the defective pathways other than their mitochondrial locations. These two cases may represent a new entity of mitochondrial disease that might be due to a defective common mechanism, such as assembly, maintenance and transport, affecting various mitochondrial enzymes and functions. Mitochondrial depletion should be considered in infants with atypical organic aciduria that may resemblemethylmalonicaciduria, propionicacidaemia, or 3-methylcrotonyl-CoA carboxylase deficiency. Neuromolecular Med. 2003. Over the last decade, many sporadic and familial cases have been reported with multiple deletions of mitochondrial DNA (mtDNA) in postmitotic tissues. Most patients suffer from progressive external ophthalmoplegia (PEO) and may have a nuclear gene defect that predisposes to the accumulation of mtDNA deletions. Recently, positional cloning has led to the discovery of mutations in four such nuclear genes. Some mutations are dominant and others recessive. In all autosomal mutations, defective mtDNA replication and/or repair are probably responsible for the generation of secondary mtDNA deletions. There are also data suggestive of a prominent pathogenic role for disturbed nucleotide metabolism. We here present a tentative genotype-phenotype correlation. Since clinical presentations are heterogeneous and overlap with different previously described clinical syndromes, we advocate the use of a genetic, instead of a clinical, classification of disorders with multiple mtDNA deletions. Mitochondrion. 2002 Nov. The presence of mtDNA abnormalities inherited as Mendelian traits indicates the existence of mutations in nuclear genes affecting the integrity of the mitochondrial genome. Two groups of nucleus-driven abnormalities have been described: qualitative alterations of mtDNA, i.e. multiple large-scale deletions of mtDNA, and quantitative decrease of the mtDNA copy number, i.e. tissue-specific depletion of mtDNA. Autosomal dominant or recessive (adPEO), progressive ophthalmoplegia and autosomal-recessive mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), are three neurodegenerative disorders associated with the coexistence of wild-type mtDNA with several deletion-containing mtDNA species. Heterozygous mutations of the genes encoding the muscle-heart isoform of the adenosine diphosphate/adenosine triphosphate mitochondrial translocator (ANT1), the main subunit of polymerase gamma (POLG1), and of the putative mtDNA helicase (Twinkle) have been found in adPEO families linked to three different loci, on chromosomes 4q34-35, 10q24, and 15q25, respectively. Mutations in the gene encoding thymidine phosphorylase have been identified in several MNGIE patients. Severe, tissue-specific depletion of mtDNA is the molecular hallmark of rapidly progressive hepatopathies or myopathies of infancy and childhood. Two genes, deoxyguanosine kinase and thymidine kinase type 2, both involved in the mitochondrion-specific salvage pathways of deoxynucleotide pools, have been associated with depletion syndromes in selected families. Neurology. 2002 Oct 22. BACKGROUND: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form. OBJECTIVES: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS. RESULTS: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls). CONCLUSIONS: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous. Exp Cell Res. 2002 Aug 1. It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells. J Mol Med. 2002 Jul. We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion. J Hepatol. 2002 May. Mitochondrial DNA depletion is a well established cause of severe liver failure in infancy. The autosomal inheritance of this quantitative mitochondrial DNA defect supports the involvement of a nuclear gene in the control of mitochondrial DNA level. We previously described a case of a 28-month-old child presenting with a progressive liver fibrosis due to a mitochondrial DNA depletion (85% at 12 months of age). As this syndrome was clinically liver-restricted, a liver transplant was initially discussed. We report the clinical, biochemical and molecular follow-up of this child, now 6 years old. The patient displayed a spontaneous gradual improvement of his liver function with continuous increment of clotting factor values since 32 months of age. A marked reduction of the previous extensive fibrosis was evidenced on a liver biopsy performed at 46 months of age associated with a dramatic decrease of the mitochondrial DNA depletion (35%). Consequently, an almost complete restoration of respiratory chain activities containing mitochondrial DNA-encoded subunits was observed. This is the first report of a revertant phenotype in liver mitochondrial DNA depletion syndrome. Acta Neurol Belg. 2002 Mar. Progressive external ophthalmoplegia (PEO) with secondary accumulation of multiple deletions of mitochondrial DNA (mtDNA) clinically resembles disorders due to primary mutations of mtDNA but follows a Mendelian inheritance pattern. The disorder belongs to an interesting group of diseases in which both the nuclear and the mitochondrial genome are involved in the pathology. Both autosomal dominant (adPEO) and recessive (arPEO) variants of this disorder occur. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or depletion of mtDNA. Recent reports of mutations in Thymidine Phosphorylase in MNGIE, and of mutations in adenine nucleotide translocator (ANT1), Twinkle and mitochondrial DNA polymerase gamma (POLG) in adPEO, have lead to new insights in the pathogenesis of these disorders of mtDNA maintenance. We also identified POLG mutations in two families with arPEO, which underlines the crucial role of the mtDNA replication machinery for mtDNA maintenance. Hum Pathol. 2002 Feb. A boy presented with lactic acidosis, hepatomegaly, hypoglycemia, generalised icterus, and muscle hypotonia in the first weeks of life. At the age of 2 months, neonatal giant cell hepatitis was diagnosed by light microscopy. Electron microscopy of the liver revealed an accumulation of abnormal mitochondria and steatosis. Skeletal muscle was normal on both light and electron microscopy. At the age of 5 months, the patient died of liver failure. Biochemical studies of the respiratory chain enzymes in muscle showed that cytochrome-c oxidase (complex IV) and succinate-cytochrome-c oxidoreductase (complex II + III) activities were (just) below the control range. When related to citrate synthase activity, however, complex IV and complex II + III activities were normal. Complex I activity was within the control range. The content of mitochondrial DNA (mtDNA) was severely reduced in the liver (17% to 18% of control values). Ultracytochemistry and immunocytochemistry of cytochrome-c oxidase demonstrated a mosaic pattern of normal and defective liver cells. In defective cells, a reduced amount of the mtDNA-encoded subunits II-III and the nuclear DNA-encoded subunits Vab was found. Cells of the biliary system were spared. Immunohistochemistry of mtDNA replication factors revealed normal expression of DNA polymerase gamma. The mitochondrial single-stranded binding protein (mtSSB) was absent in some abnormal hepatocytes, whereas the mitochondrial transcription factor A (mtTFA) was deficient in all abnormal hepatocytes. In conclusion, depletion of mtDNA may present as giant cell hepatitis. mtTFA and to a lesser degree mtSSB are reduced in mtDNA depletion of the liver and may, therefore, be of pathogenetic importance. The primary defect, however, is still unknown. J Inherit Metab Dis. 2002 Feb. Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome. Prenat Diagn. 2002 Jan. We describe two newborn sisters who presented in the third trimester with diminished fetal movements and skin edema, but with no other signs of hydrops fetalis. Within hours of birth, both developed profound lactic acidemia, followed by multi-organ failure. In muscle mitochondria, the activity of all enzymatic complexes that contain mitochondrial DNA (mtDNA)-encoded subunits was markedly decreased. Southern blot analysis revealed a profound reduction in the mtDNA/nuclear DNA ratio, implying mtDNA depletion. The prenatal identification of skin edema in two patients with mtDNA depletion, and its absence in a healthy sibling, suggest that skin edema should be regarded as a novel manifestation of mtDNA depletion. This finding shows that mtDNA depletion can present prenatally and, consequently, may aid the clinician in making a diagnosis, prenatally, of this genetic defect. Neuromuscul Disord. 2002 Jan. Cardiomyopathy associated with a mitochondrial DNA depletion syndrome is a rare condition. We report on a child with a hypertrophic cardiomyopathy and a mitochondrial depletion syndrome who was successfully treated by heart transplantation, given the tissue-specific nature of her mitochondrial disorder. Semin Cell Dev Biol. 2001 Dec. Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity. Nat Genet. 2001 Nov. Mitochondrial DNA (mtDNA)-depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools. Mitochondrion. 2001 Oct. We have previously described two cases of children with a liver mitochondrial DNA (mtDNA) depletion syndrome, characterised by a low ratio of mtDNA to nuclear DNA. Light microscopy performed on liver biopsy showed abnormal hepatocytes with a characteristic 'oncocytic' appearance, indicative of perturbed oxidative phosphorylation. The adenine nucleotide translocator (ANT), the last step in oxidative phosphorylation, catalyses the exchange of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) between the cytosol and mitochondria. The ANT2 gene, which is not normally expressed in human tissues, encodes an isoform preferentially expressed under conditions of glycolytic metabolism. ANT2 gene expression is regulated by a transcription factor involved in a molecular mechanism selecting for the import of glycolytic ATP into the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which are highly dependent on glycolysis for ATP synthesis. We postulated that, as a result of the defective oxidative phosphorylation observed in these patient biopsies, the ANT2 expression would be induced. We simultaneously quantified the mtDNA depletion and the ANT2 gene expression in liver biopsies from the two patients and six controls. ANT2 mRNA levels were significantly increased in the two patient liver biopsies. Moreover, in one patient, the liver mtDNA depletion was found to be partially reversed after less than 4 years and this reversion was coupled to a concomitant decrease of the ANT2 expression. These results suggest that dysfunction of oxidative phosphorylation could lead to a switch from mitochondrial to glycolytic ATP production, to restore tissue-specific energy requirements. Am J Physiol Endocrinol Metab. 2001 Jun. Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization. Ann Neurol. 2001 May. Through a report of 4 late-onset cases with mitochondrial DNA (mtDNA) depletion, we address the specificity of the clinical entities associated with a very low residual amount of mtDNA. Three of the patients met the diagnostic criteria of Kearns Sayre syndrome, which has never been associated with mtDNA depletion. The fourth patient had an isolated skeletal myopathy. Deleted mtDNA molecules were found by long-range polymerase chain reaction (PCR) only in the Kearns Sayre syndromes, which strengthens the clinical differences between the two types of patients. All patients had extremely low residual amounts of mtDNA as shown by Southern blot analysis. Using an original method based on competitive PCR, we were able to measure the number of mtDNA copies per diploid genome. These results demonstrated the severity of the depletion in the patients by comparison not only to normal controls but also to patients with mtDNA disorders. Despite the severity of the depletion, in situ hybridization using two mtDNA transcripts revealed a normal steady-state level of transcription. Such compensation provides clues to the striking contrast between the severity of mtDNA depletion and the late onset and slowly progressive disease. Am J Med Genet. 2001 Spring. Diseases caused by nuclear genes that affect mitochondrial DNA (mtDNA) stability are an interesting group of mitochondrial disorders, involving both cellular genomes. In these disorders, a primary nuclear gene defect causes secondary mtDNA loss or deletion formation, which leads to tissue dysfunction. Therefore, the diseases clinically resemble those caused by mtDNA mutations, but follow a Mendelian inheritance pattern. Several clinical entities associated with multiple mtDNA deletions have been characterized, the most frequently described being autosomal dominant progressive external ophthalmoplegia (adPEO). MtDNA depletion syndrome (MDS) is a severe disease of childhood, in which tissue-specific loss of mtDNA is seen. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or mtDNA depletion. Recent reports of thymidine phosphorylase mutations in MNGIE and adenine nucleotide translocator mutations in adPEO have given new insights into the mechanisms of mtDNA maintenance in mammals. The common mechanism underlying both of these gene defects could be disturbed mitochondrial nucleoside pools, the building blocks of mtDNA. Future studies on MNGIE and adPEO pathogenesis, and identification of additional gene defects in adPEO and MDS will provide further understanding about the mammalian mtDNA maintenance and the crosstalk between the nuclear and mitochondrial genomes. Brain Pathol. 2000 Jul. An expanding number of autosomal diseases has been associated with mitochondrial DNA (mtDNA) depletion and multiple deletions. These disorders have been classified as defects of intergenomic communication because mutations of the nuclear DNA are thought to disrupt the normal cross-talk that regulates the integrity and quantity of mtDNA. In 1989, autosomal dominant progressive external ophthalmoplegia with multiple deletions of mitochondrial DNA was the first of these disorders to be identified.Two years later, mtDNA depletion syndrome was initially reported in infants with severe hepatopathy or myopathy. The causes of these diseases are still unclear, but genetic linkage studies have identified three chromosomal loci for AD-PEO. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder associated with both mtDNA depletion and multiple deletions, is now known to be due to loss-of-function mutations in the gene encoding thymidine phosphorylase. Increased plasma thymidine levels in MNGIE patients suggest that imbalanced nucleoside and nucleotide pools in mitochondria may lead to impaired replication of mtDNA. Future research will certainly lead to the identification of additional genetic causes of intergenomic communication defects and will likely provide insight into the normal "dialogue" between the two genomes. Clin Sci (Lond). 1999 Aug. A wide variety of human diseases have been associated with defects in mitochondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be explained on the basis of impaired mitochondrial gene function alone, the role of altered nuclear gene expression must also be considered. In recent years, the experimental technique of depleting cells of mtDNA by culturing them with ethidium bromide has become a popular method of studying mitochondrial disorders. However, apart from depleting mtDNA, ethidium bromide may have many other intracellular and nuclear effects. The aim of the present study was to investigate the effects of ethidium bromide treatment on nuclear gene expression. A simian-virus-40-transformed human thyroid cell line was depleted of mtDNA by culture in ethidium bromide, and differential display reverse transcriptase-PCR (DDRT-PCR) was then employed to compare mRNA expression between wild-type, mtDNA-replete (rho(+)) and ethidium bromide-treated, mtDNA-depleted (rho(0)) cells. Expression of the majority of nuclear-encoded genes, including those for subunits involved in oxidative phosphorylation, remained unaffected by the treatment. Seven clones were found to be underexpressed; three of the clones showed significant similarity with sequences of the human genes encoding RNase L inhibitor, human tissue factor and ARCN1 (archain vesicle transport protein 1), a highly conserved species which is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression are not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate which of these changes are due to mtDNA depletion, ATP deficiency or other disparate effects of ethidium bromide exposure. Given that most genes appear unaffected, the results suggest that depleting cells of mtDNA by ethidium bromide treatment is a valuable approach for the study of mitochondrial mutations by cybrid techniques. Am J Pathol. 1999 Jul. Mitochondrial DNA depletion syndrome is an autosomal inherited disease associated with grossly reduced cellular levels of mitochondrial DNA in infancy. Most patients are born after a full and uncomplicated pregnancy, are normal at birth, but develop symptoms in the early neonatal period. These observations have led to the suggestion that the patients have a defect affecting the control of mitochondrial DNA copy number after birth. Using immunocytochemical techniques, we demonstrated that the disease is already expressed in amniotic fluid cells. Detection of mitochondrial DNA depletion in these fetal cells indicates that the defect may already be expressed early in embryological development. Eur J Biochem. 1999 May. Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells. J Hepatol. 1999 Jan. Few cases of infantile liver disease associated with mitochondrial DNA (mtDNA) depletion have been reported. Most of the patients died before 1 year of age of severe liver failure. We describe a new case, a 28-month-old child, presenting with cholestasis at age 2 months, complicated by progressive portal and lobular liver fibrosis. Growth and psychomotor development are undisturbed. There is no clinical evidence of either myopathy or neurological involvement. Metabolic investigation in plasma revealed an abnormal oxido-reduction status after fasting and after carbohydrate-rich meals. Light microscopy performed on liver biopsies revealed steatosis, abnormal hepatocytes with an "oncocytic" appearance and extensive fibrosis. Electron microscopic investigation showed an increased number of mitochondria with rare or enlarged cristae. Biochemical studies of liver biopsies showed that the respiratory chain activities containing mtDNA-encoded subunits were severely decreased (complexes I, III and IV). In contrast, the complex II activity was normal and the citrate synthase activity was greatly increased. Southern blotting analysis revealed that the ratio of mtDNA to nuclear DNA in liver was only 15% and 20% of the mean control value at ages 12 and 21 months, respectively. For this mtDNA depletion syndrome which is clinically expressed in the liver, a liver transplantation is discussed. Ann Neurol. 1999 Jan. Deficiency of mitochondrial DNA polymerase gamma activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase gamma was undetectable. Neuromuscul Disord. 1998 Dec. We studied 10 patients with a variable degree of mtDNA depletion in muscle. Seven patients showed a clear-cut myopathic pattern, while the three remaining had brain involvement. There was no relationship between age at onset and relative mtDNA copy number in muscle, but we found an apparent correlation between clinical severity and degree of muscle mtDNA depletion. Muscle morphology showed that mtDNA depletion was associated with mitochondrial proliferation and cytochrome c oxidase negative fibers. Biochemical studies revealed single or combined defects of mtDNA-dependent respiratory chain complexes. Our data indicate that patients with mtDNA depletion may have a more variable age at onset and clinical evolution and wider phenotype than previously thought. The diagnosis of this condition, so far regarded as rare, may have been overlooked to some extent. Muscle Nerve. 1998 Nov. We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. Neurology. 1998 Oct. We studied a 29-year-old woman with myopathy since childhood with evidence of mitochondrial DNA (mtDNA) depletion. Muscle biopsy sample showed cytochrome c oxidase (COX)-negative fibers. Biochemistry showed COX deficiency. Southern blot analysis showed 76% depletion of mtDNA as compared with controls. This patient's clinical course suggests that long survival is possible in some patients with mtDNA depletion. J Biol Chem. 1998 Sep 4. F1-ATPase assembly has been studied in human rho degrees cells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, rho degrees cells possessed normal levels of F1-ATPase alpha and beta subunits. This F1-ATPase was functional and azide- or aurovertin-sensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in rho degrees cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these rho degrees cells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced rho degrees cell growth and mitochondrial membrane potential. In conclusion, rho degrees cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP3- at a sufficient rate to maintain a rapid exchange with the glycolytic ATP4- by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway. Neurology. 1998 Jun. OBJECTIVE: We studied five new patients with mitochondrial DNA (mtDNA) depletion to better define the clinical spectrum of this disorder. BACKGROUND: mtDNA depletion has been associated with myopathy or hepatopathy, or both, in infants and young children. Involvement of the CNS and peripheral nervous system has not been clearly established. METHODS: We reviewed the clinical course and performed morphologic, biochemical, and genetic analyses of muscle samples from five patients. RESULTS: Age at onset ranged from 3 months to 5 years, and one patient survived until age 10 1/2 years. Two patients had laboratory and clinical features reminiscent of dystrophinopathy, two had evidence of brain involvement, and two had peripheral neuropathy. Muscle biopsy specimens in all patients showed abundant ragged-red fibers. Biochemistry showed cytochrome c oxidase deficiency in all patients tested and decreased activities of other respiratory chain complexes in some. CONCLUSIONS: Inheritance appeared to be autosomal recessive, suggesting that mutations in nuclear DNA are responsible for mtDNA depletion. mtDNA depletion should be considered in children with mitochondrial disorders of uncertain etiology, and criteria for diagnosis are proposed. J Inherit Metab Dis. 1998 Jun. The ratio of mtDNA and a nuclear reference gene was estimated by Southern blotting in the skeletal muscle DNA of a 3-year-old girl who suffered from congenital brain damage, focal epilepsy, hepatomegaly, malabsorption syndrome and severe myopathy. The signal ratio of mtDNA versus 18S rDNA was 22% of the mean value obtained from controls. No major deletions or insertions were found and the MERRF, MELAS and NARP mutations were ruled out. Mitochondrial DNA-encoded enzyme activities and mitochondrial respiration were reduced. The analysis of the NAD(P)H and flavoprotein redox states of intact fibres revealed the presence of mitochondrial dysfunction. In tissue sections a moderate elevation of type I and type II fibre diameter variation was detected, aberrant NADH- and succinate dehydrogenase staining and some ragged red fibres. This suggested that a mitochondrial disorder caused by a decrease in the amount of intact wild-type mtDNA was responsible for the severe myopathy. J Hepatol. 1998 Apr. BACKGROUND/AIMS: Liver failure in infancy can result from several disorders of the mitochondrial respiratory chain. In some patients, levels of mitochondrial DNA are markedly reduced, a phenomenon referred to as mitochondrial DNA depletion. To facilitate diagnosis of this condition, we have reviewed the clinical and pathological features in five patients with mitochondrial DNA depletion. METHODS: Cases were identified by preparing Southern blots of DNA from muscle and liver, hybridising with appropriate probes and quantifying mitochondrial DNA relative to nuclear DNA. RESULTS: All our patients with mitochondrial DNA depletion died of liver failure. Other problems included hypotonia, hypoglycaemia, neurological abnormalities (including Leigh syndrome) and cataracts. Liver histology showed geographic areas of fatty change, bile duct proliferation, collapse of liver architecture and fibrosis; some cells showed decreased cytochrome oxidase activity. Muscle from three patients showed mitochondrial proliferation, with loss of cytochrome oxidase activity in some fibres but not in others; in these cases, muscle mitochondrial DNA levels were less than 5% of the median control value. The remaining two patients (from a single pedigree) had normal muscle histology and histochemistry associated with less severe depletion of mitochondrial DNA in muscle. CONCLUSIONS: Liver failure is common in patients with mitochondrial DNA depletion. Associated clinical features often include neuromuscular disease. Liver and muscle histology can be helpful in making the diagnosis. Mitochondrial DNA levels should be measured whenever liver failure is thought to have resulted from respiratory chain disease. Diabetes. 1998 Mar. Mitochondrial dysfunction due to alterations in the mitochondrial genome (mtDNA) has recently attracted much attention, with the finding that mutations in the mitochondrially encoded proteins perturb cell function. Several disorders have been linked to such genetic changes, including a specific diabetic phenotype. Using ethidium bromide (EtBr) that intercalates into mtDNA, we have effectively eliminated functions under the control of mtDNA from the highly differentiated INS-1 insulin-secreting cell line. We have investigated the consequences on insulin secretion, mitochondrial enzyme activity, organelle structure, and membrane polarization in such cells (INS-1 rho0). Under these conditions, the mitochondrial membrane potential fails to hyperpolarize in response to either glucose or methylsuccinate. In agreement with this finding, the morphology of the mitochondria is altered in the presence of EtBr, sharing similarities with mitochondria in which the membrane potential has been collapsed with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). In addition, there is no effect of either nutrient secretagogue at the level of the plasma membrane potential, although the effect of the depolarizing agent KCl on membrane depolarization is completely preserved. Similarly, glucose and methylsuccinate fail to increase insulin secretion, whereas KCl is still effective. To test further the effects of mtDNA depletion on exocytosis, we permeabilized INS-1 cells with Staphylococcus aureus alpha-toxin, which forms small holes in the plasma membrane. In contrast to control cells, mitochondrial substrates were incapable of stimulating insulin secretion in mtDNA-deficient cells, emphasizing that the defect in secretion lies at the level of mitochondrial function rather than in the exocytotic process. The results indicate the paramount importance of the mitochondria in the downstream effects elicited by exposure to elevated concentrations of nutrient secretagogue. Biochim Biophys Acta. 1997 Dec 31. Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits. Hum Mol Genet. 1997 Jun. Depletion of mitochondrial DNA (mtDNA) appears to be an important cause of mitochondrial dysfunction in neonates and infants. We have identified another child in whom depletion of mtDNA was demonstrated in liver and serial skeletal muscle biopsies. A primary myoblast culture from the patient initially showed normal levels of mtDNA, but there was a progressive loss of mtDNA in later cell passages and clonal myoblast cell cultures, similar to that observed in the skeletal muscle tissue of the patient. Thus, these clonal myoblast cultures provide an in vitro model of the in vivo mtDNA dynamics. The levels of mitochondrial mRNAs for subunits I and II of cytochrome c oxidase declined with declining mtDNA levels, but the fall in mitochondrial transcript levels lagged behind that of the mtDNA levels. Levels of cytochrome c oxidase subunit I and II polypeptides, however, declined ahead of declining mtDNA levels. Immunocytochemistry showed that between individual cells of the clonal myoblast cultures, the expression of the mitochondrially encoded subunit I of cytochrome c oxidase was heterogeneous, suggesting variable levels of mtDNA. Transfer of patient mitochondria with residual mtDNA levels to control cells devoid of mtDNA (rho0 cells) led to restoration of mtDNA levels and, hence, suggests a nuclear involvement in the depletion. J Biol Chem. 1996 Oct 18. To determine whether mtDNA and mitochondrial respiratory function in pancreatic beta cells are necessary for the phenotypic expression of glucose-stimulated insulin secretion, we used a cultured mouse pancreatic beta cell line, MIN6, and two derivative lines, mtDNA knockout MIN6 (rho0 MIN6) and mtDNA repopulated cybrid MIN6. The MIN6 cells retain the property of glucose-stimulated insulin secretion, but their mtDNA knockout induced the loss of mitochondrial transcription, translation, and respiration activity, without inhibition of transcription of the insulin gene or loss of succinate dehydrogenase activity, indicating that the observed mitochondrial dysfunction in rho0 MIN6 cells was not due to a cytotoxic side effect derived from the mtDNA knockout. Moreover, the mtDNA depletion also inhibited both the glucose-stimulated increase in the intracellular free Ca2+ content and the elevation of insulin secretion. The possibility of the involvement of nuclear genome-encoded factors in this process was excluded by the observation that the missing sensitivity to extracellular glucose stimulation in rho0 MIN6 cells was restored reversibly by repopulation with foreign mtDNA and isolating cybrid MIN6 clones. Therefore, these findings provide unambiguous evidence for the involvement of the mitochondrial dysfunction induced by mtDNA impairment in developing pathogeneses of some forms of diabetes mellitus. J Pediatr. 1996 May. We describe a family in which three children of consanguineous parents died of hepatic failure before the age of 3 months. The first child had clinical symptoms of liver disease with hypoglycemia that were evident at birth. The second child was healthy and has normal development. The third child had severe liver dysfunction noted a few days after birth. Liver failure also developed in the fourth child soon after birth. Recently a mitochondrial disorder was considered as a possible cause. Deficiency of respiratory chain enzymes that contain polypeptides encoded by mitochondrial DNA (mtDNA) and depletion of mtDNA were found in the liver of the fourth child, but mitochondrial abnormalities were absent in muscle of the third child. The similarities in clinical presentation suggest that liver-specific depletion of mtDNA was the cause of the hepatic failure in all three children. We conclude that liver dysfunction with onset in the perinatal period can be caused by depletion of mtDNA. J Pediatr. 1996 May. An infant with feeding difficulties, hypotonia, lactic acidemia, and severe hypoketotic hypoglycemia died at the age of 7 months of liver disease. Electron microscopy revealed abnormal mitochondria. Biochemical studies of mitochondrial enzymes in liver showed a decreased activity of complexes I, III, and IV. Mitochondrial DNA (mtDNA) content was reduced in liver 7% of the mean value in control subjects) and in muscle (50%). In kidney, brain, and heart, the mtDNA content was normal. The liver-specific mtDNA depletion syndrome in this patient manifested itself with features of both a respiratory chain defect and a mitochondrial fatty acid oxidation defect. Syndromes involving depletion of mtDNA can be diagnosed only when the activity of the respiratory chain enzymes and the content of mtDNA are investigated in the most affected tissues. Pediatr Neurol. 1996 Apr. Mitochondrial DNA depletion is a quantitative disorder of mtDNA, characterized by tissue-specific reductions in mtDNA copy number, that presents in infancy or early childhood. It is most likely transmitted as an autosomal recessive trait, although about half of the described cases are sporadic. To estimate its prevalence we measured relative mtDNA copy number (mtDNA: 18S rDNA ratio) by Southern blot analysis in muscle biopsy samples from all children with compatible histories referred between 1983 and 1994. Of the 304 biopsies evaluated, 54 met the study criteria. We found 6 patients (2 male, 4 female) with mtDNA depletion (relative mtDNA copy number 7.9-33.2% of control). Their clinical course and findings were heterogeneous, however all but one manifested weakness, hypotonia, and developmental delay. Clinical severity was not obviously related to the degree of mtDNA depletion. No patient had ragged-red fibers, although 2 had a lipid storage myopathy. Immunofluorescence with antibodies to double-stranded DNA, COX IV, and COX II demonstrated homogeneously reduced reactivity to all three antibodies compared with control. mtDNA depletion may be a relatively common neurogenetic disorder of infancy and early childhood and should be considered in children with unexplained weakness, hypotonia, or developmental delay. J Neurol. 1995 Sep. A male infant, born from consanguineous parents, suffered from birth with a progressive neuromuscular disorder characterized by psychomotor delay, hypotonia, muscle weakness and wasting, deep-tendon areflexia and spastic posture. High levels of lactic acid in blood and cerebrospinal fluid suggested a mitochondrial respiratory chain defect. Muscle biopsy revealed ragged-red and cytochrome c oxidase-negative fibres, lipid accumulation and dystrophic changes. Multiple defects of respiratory complexes were detected in muscle homogenate, but cultured fibroblasts, myoblasts and myotubes were normal. Southern blot analysis showed markedly reduced levels of mitochondrial DNA (mtDNA) in muscle, while lymphocytes, fibroblasts and muscle precursor cells were normal. Neither depletion of mtDNA nor abnormalities of the respiratory complexes were observed in innervated muscle fibres cultured for as long as 4 months. No mutations were observed in two candidate nuclear genes, mtTFA and mtSSB, retro-transcribed, amplified and sequenced from the proband's mRNA. Sequence analysis of the mtDNA D-loop and of the origin of replication of the mtDNA light strand failed to identify potentially pathogenic mutations of these replicative elements in the proband's muscle mtDNA. Our findings indicate that mtDNA depletion is due to a nuclear encoded gene and suggest that the abnormality underlying defective mtDNA propagation must occur after muscle differentiation in vivo. Eur J Pediatr. 1995 Jul. We studied a 3-month-old girl who was admitted to hospital because of respiratory distress. The clinical course was characterized by a rapidly progressive generalized hypotonia with lactic acidosis and she died at 4 months of age. A muscle biopsy showed few ragged-red fibres and lack of histochemical cytochrome c oxidase reaction in all fibres. Enzyme activities of the respiratory chain complexes containing subunits encoded by the mitochondrial DNA (mtDNA) were markedly decreased. A quantitative Southern blot analysis revealed 99% depletion of mtDNA in muscle and normal amounts in blood. There was no family history and the dizygotic twin sister of the patient was no symptomatic. CONCLUSION: This new case confirms the rapidly fatal evolution associated with severe depletion of muscle mtDNA. Biochem J. 1995 Feb 1. We have characterized cultured skin fibroblasts from two siblings affected with a fatal mitochondrial disease caused by a nuclear genetic defect. Mitochondrial respiratory-chain function was severely decreased in these cells. Southern-blot analysis showed that the fibroblasts had reduced levels of mitochondrial DNA (mtDNA). The mtDNA was unstable and was eliminated from the cultured cells over many generations, generating the rho0 genotype. As the mtDNA level decreased, the cells became more dependent upon pyruvate and uridine for growth. Nuclear-encoded subunits of respiratory-chain complexes were synthesized and imported into the mitochondria of the mtDNA-depleted cells, albeit at reduced levels compared with the controls. Mitochondrial protein synthesis directed by the residual mtDNA indicated that the mtDNA was expressed and that the defect specifically involves the replication or maintenance of mtDNA. This is a unique example of a respiratory-deficient human cell line exhibiting defective mtDNA replication. J Inherit Metab Dis. 1995. Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with 35S[dCTP] and a reference nuclear DNA probe labelled with [32P]dCTP. The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by 'normal' quadriceps muscle. The clinical features to two of these patients were similar to earlier case reports of mtDNA depletion. In three patients the clinical course was relatively benign compared to cases that have previously been described. Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect. Biochem Biophys Res Commun. 1994 May 16. Low levels of mitochondrial transcription factor A (mtTFA) were found in muscle fibers with mitochondrial DNA (mtDNA) depletion in a child with fatal mitochondrial myopathy and also in a human cell line lacking mtDNA. High levels of this transcriptional activator were found in muscle fibers with mtDNA accumulation in two patients with single mtDNA deletions. This is the first example of a nuclear gene product that varies with the levels of mtDNA in humans. Since transcription is required for the replication of mtDNA, it is possible that mtTFA may be involved in the regulation of mtDNA replication. J Neurol Sci. 1994 May. Multiple deletions of mitochondrial DNA (mtDNA) were first identified in patients with mitochondrial encephalomyopathy with a clear mendelian inheritance. We found this genetic alteration in four atypical and sporadic cases of mitochondrial encephalomyopathy, characterized by RRF and partial COX deficiency. One patient was affected by essential hyperCPKemia, 1 by subacute onset flaccid tetraplegia and 2 by parkinsonism. Southern blot and PCR revealed mtDNA multiple deletions in muscle tissue of these patients. These findings indicate that these alterations are not confined to the families with mendelian transmission, but can be present in sporadic cases with heterogeneous phenotypic features. Am J Hum Genet. 1993 Sep. We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome. J Pediatr. 1992 Dec. A 3-month-old girl was admitted to the hospital because of hypotonia and frequent vomiting. She had severe metabolic acidosis and her liver function was abnormal. Hepatomegaly and rapidly progressive liver failure developed, and she died at 4 months of age. Two half-siblings from a different mother had died in infancy of an undiagnosed myopathy. The liver was fatty and hepatocytes were filled with large and small lipid droplets. Other tissues were morphologically normal. The respiratory chain enzymes containing subunits encoded by mitochondrial DNA were markedly decreased in liver, partially decreased in muscle, but normal in other tissues. Southern blot analysis showed 90% depletion of mitochondrial DNA in liver, 53% depletion in muscle, and normal amounts in other tissues. This is the second case of fatal infantile liver failure associated with mitochondrial DNA depletion. This pathogenetic mechanism should be considered in infants with multiple respiratory chain defects and variable tissue expression. J Clin Invest. 1992 Jul. Multiple deletions of mitochondrial DNA (mtDNA) have recently been reported in familial progressive external ophthalmoplegia (PEO), in a case of progressive encephalomyopathy, and in inherited recurrent myoglobinuria. The inheritance of familial PEO has been autosomal dominant, which indicates that a mutation in an unknown nuclear gene results in several mtDNA deletions of different sizes in these patients. We report a patient with autosomal dominant PEO, whose major clinical symptom, however, was severe retarded depression. The morphological analyses of the tissue samples derived from autopsy showed various abnormalities in the mitochondria in all the tissues studied. The activities of the respiratory chain enzymes encoded by mtDNA were remarkably reduced in the skeletal muscle. The mtDNA analyses confirmed that besides myopathy, this patient had a multisystem disorder with widespread distribution of multiple deletions of mtDNA. The highest percentage of mutated mtDNA was found in the brain, skeletal muscle and the heart, the relative quantity of mutated mtDNA correlating to the severity of the clinical symptoms. Brain Pathol. 1992 Apr. Multiple deletions of mitochondrial DNA (mtDNA) have recently been described in a number of patients with neurological disorders. Most cases have been clinically characterized by autosomal dominant inheritance, adult onset, and a slowly progressive course with external ophthalmoplegia and muscle weakness. Some patients have had evidence of central or peripheral nervous system involvement or episodes of myoglobinuria. Muscle biopsy findings include ragged-red fibres (RRF), muscle fibres with absent COX-activity and abundant abnormal mitochondria with paracrystalline inclusions. Biochemically, a generalized reduction in the activities of mtDNA-encoded enzymes is observed in skeletal muscle. Southern blotting or PCR analysis reveal multiple populations of deleted mtDNA. The deletions occur at multiple sites between the replication initiation sites, involving a large portion of mtDNA, and most deletions seem to be flanked by direct sequence repeats, shown to be "hot spots" in the case of single large deletions. Apparently, a defect in a nuclear gene results in multiple deletions of mtDNA. Both clinical, genetic and molecular genetic observations indicate heterogeneity of this new disease category, apparently based on a disturbance in the "cross-talk" between the nuclear and the mitochondrial genomes. J Neurol Sci. 1992 Mar. Three children displaying hypotonia, cardiac involvement and defects of the mitochondrial respiratory chain complexes are reported. The first case showed severe neonatal hypotonia, failure to thrive, hepatomegaly, dilation of the right cardiac cavities, profound lactic acidosis and amino aciduria. The boy died at the age of 7 weeks. In the second case hypotonia, severe cardiomyopathy, cyclic neutropenia, lactic acidosis and 3-methylglutaconic aciduria occurred. The boy died at the age of 27 months. The third case presented at the age of 16 months as an acute hypokinetic hypertrophic cardiomyopathy with transient hypotonia and mild lactic acidosis. Spontaneous clinical remission occurred. In all cases muscle biopsy was performed. Morphological studies failed to show ragged-red fibers but there was lipid storage myopathy and decreased cytochrome c oxidase activity. Biochemical studies confirmed the cytochrome c oxidase deficiency in muscle in all cases. It was associated with complex I III deficiency in case 1 and with severe deficits of all respiratory chain complexes in case 2. Post-mortem studies in case 1 indicated that complex IV was reduced in the liver but not in the heart and quantitative analysis of mtDNA revealed a depletion in muscle. Cases 1 and 2 shared some clinical features with fatal infantile myopathy associated with cytochrome c oxidase deficiency, while case 3 displayed a very unusual clinical presentation. The histochemical enzyme reaction of cytochrome c oxidase is useful for the diagnosis of mitochondrial myopathy because ragged-red fibers may be lacking. Finally, biochemical measurement of the different mitochondrial respiratory chain complexes is required because multiple defects are frequent and occasionally related to mtDNA depletion. Neurology. 1992 Jan. We have studied five children with mitochondrial myopathy manifesting within or soon after the first year of life. Muscle biopsies showed ragged-red fibers and decreased respiratory chain activity. All five patients had a severe decrease (2 to 34% of normal) in the amount of muscle mitochondrial DNA (mtDNA). The depletion of mtDNA correlated with absence of mtDNA-encoded translation products and with loss of cytochrome c oxidase enzyme activity in individual muscle fibers. This mitochondrial myopathy of childhood illustrates one phenotypic expression of a novel pathogenetic mechanism in mitochondrial diseases, the specific depletion of mtDNA in affected tissues. Am J Hum Genet. 1991 Mar. We studied two related infants with a fatal mitochondrial disease, affecting muscle in one and liver in the other. Quantitative analysis revealed a severe depletion of mtDNA in affected tissues. This genetic abnormality was also observed in muscle of an unrelated infant with myopathy and in muscle and kidney of a fourth child with myopathy and nephropathy. Biochemistry, immunohistochemistry, and in situ hybridization showed that the depletion of mtDNA in muscle fibers was correlated with a respiratory chain defect and with lack of mitochondrially translated proteins. Although the differential tissue involvement in these infants suggests mtDNA heteroplasmy, sequence analysis of mtDNA replication origins did not reveal any abnormality that could account for the low copy number. Am J Hum Genet. 1990 Dec. We studied several affected and one nonaffected individuals belonging to three unrelated pedigrees. The pathological trait was an autosomal dominant mitochondrial myopathy due to large-scale multiple deletions of the mitochondrial genome. Clinically, symptomatic patients had progressive external ophthalmoplegia, muscle weakness and wasting, sensorineural hypoacusia, and, in some cases, vestibular areflexia and tremor. The muscle biopsies of all patients examined showed ragged-red fibers, neurogenic changes, and a partially decreased histochemical reaction to cytochrome c oxidase. Multiple mtDNA heteroplasmy was detected in the patients by both Southern blot analysis and PCR amplification, whereas the unaffected individual had the normal homoplasmic hybridization pattern. These findings confirm and add further details to the existence of a new human disease - defined clinically as a mitochondrial myopathy, genetically as a Mendelian autosomal dominant trait, and molecularly by the accumulation of multiple, large-scale deletions of the mitochondrial genome - that is due to impaired nuclear control during mtDNA replication. Biochem Biophys Res Commun. 1990 Mar 16. Muscle mitochondrial DNA (mtDNA) from a patient with mitochondrial myopathy was examined by Southern blotting. Her family history suggests autosomal dominant inheritance of this disorder. In contrast to other cases of this myopathy that are associated with a compensatory increase of mitochondria, in this patient, the content of mtDNA was markedly decreased (15% of controls), whereas protein of the ATP synthase beta-subunit was not decreased appreciably as judged by Western blotting. In addition, the mitochondrial DNA had multiple deletions, which were located between the replication origins of the heavy strand and light strand. |