Autopsied case with MERRF/MELAS overlap syndrome accompanied by stroke-like episodes localized to the precentral gyrus.

We present an autopsied case with A8344G-mutated myoclonus epilepsy with ragged red fibers (MERRF)/mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) overlap syndrome accompanied by stroke-like episodes localized to the precentral gyrus. A 16-year-old Japanese woman suddenly experienced repetitive consciousness disturbances with increased serum lactate and creatine kinase levels. Magnetic resonance imaging showed abnormal intensity of bilateral precentral gyrus. She was clinically diagnosed as having a mitochondrial disorder and the A8344G mutation was detected in mitochondrial DNA. At 17 years of age, she died from congestive heart failure secondary to a third episode of lactic acidosis. Neuropatho-logically, multifocal laminar necrosis, which is responsible for stroke-like episodes in MELAS, was seen in the frontal cortex including the precentral gyrus, but there was no neuronal loss and gliosis in the basal ganglia, cerebellum, and brainstem, which were compatible with MERRF. Hypertrophy of the vascular smooth muscle and choroidal epithelium were seen, and were strongly visualized by an anti-mitochondrial antibody. Skeletal muscles showed uneven muscular diameters, increased central nuclei, and ragged red fibers (RRFs). Decreased cytochrome c oxidase (COX) activity and strongly succinate dehydrogenase (SDH)-reactive blood vessels were also noted. Stroke-like episodes in MERRF/MELAS overlap syndrome are thought to be rare in the frontal cortex including the precentral gyrus. Only two cases of MERRF/MELAS overlap syndrome with A8344G mutation, including this case, have shown stroke-like episodes in the frontal lobes. Other than the A8344G mutation and frontal lobe involvement, they had a high degree of similarity in terms of presence of RRFs, gastrointestinal dysfunction, and lack of typical MERRF neuropathology. In conclusion, this is an important case describing the clinical spectrum associated with A8344G-mutated MERRF/MELAS overlap syndrome.

[Ultrastructural and clinical findings of mitochondrial encephalomyopathy:report of 27 cases].

Objective: To investigate the ultrastructural features of muscle in patients with mitochondrial encephalomyopathy for its diagnosis and differential diagnosis. Methods: The clinical data of 27 mitochondrial encephalomyopathy patients who underwent left or right biceps brachii muscle biopsy at Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University from July 2006 to August 2017 were analyzed retrospectively. The muscle biopsy specimens were examined underlight microscope and transmission electron microscope. Results: There were 27 patients (17 males, 10 females) with an age range of 12 to 62 years (mean 29 years). The age of onset ranged from 3 to 38 years. The course of disease ranged from 1 month to 24 years. Twenty-two cases presented with lactic acidosis and stroke-like episodes (MELAS) syndrome, four with myoclonic epilepsy with ragged red fibers (MERRF) syndrome, and one with chronic progressive paralysis of extraocular muscle (CPEO) syndrome. Skeletal muscle biopsy showed abundant ragged red fibers and strongly SDH-reactive vessel. Genetic studies showed 17 of 22 cases of MELAS syndrome had A3243G mutation, and the other 5 cases had no abnormality. A8344G mutation was found in 3 of 4 cases of MERRF syndrome. No single or multiple mtDNA mutations were found in the single case of CPEO. Transmission electron microscopy of all 27 cases showed diffuse proliferation of mitochondria between the myofibrils and beneath the sarcolemma, with increased spacing between muscle cells. Seven cases showed numerous glycogen and four showed subsarcolemmal lipid droplets, 13 cases showed unusual mitochondrial morphology, including mitochondrial electron-dense substances and paracrystal line inclusions ("parking lot" change)in eight cases. Conclusions: Transmission electron microscopy shows significant differences in ultrastructural pathological changes among different patients with mitochondrial encephalomyopathy. Some patients with mild clinical symptoms have increased mitochondrial number, increased metabolism of glycogen and lipid droplets, while others with severe clinical symptoms have abnormal mitochondrial morphology. Typical crystalloid inclusions are found in mitochondria, which are of great value in the diagnosis of this disease.

Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, ß30_31 and ß32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.

Pathology of skeletal muscle fibers and small blood vessels in MERRF syndrome: an ultrastructural study.

Our studies concerned skeletal muscle biopsy specimens from a patient with clinically suspected MERRF syndrome, confirmed by genetic tests showing the presence of point mutation in the m.8344A> G in the tRNALys gene. Ultrastructurally, extensive damage of mitochondria in skeletal muscle fibres was observed, including the presence of two types of mitochondrial inclusions. Mild damage of mitochondria was revealed in small blood vessels and the presence of calcium deposits in the vascular walls were observed. The differences in mitochondrial damage may be related to different origin and expenditure of biologically useful energy in these cells.

The Bacterial Protein CNF1 as a Potential Therapeutic Strategy against Mitochondrial Diseases: A Pilot Study.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

Characteristic cardiac phenotypes are detected by cardiovascular magnetic resonance in patients with different clinical phenotypes and genotypes of mitochondrial myopathy.

BACKGROUND: Mitochondrial myopathies (MM) are a heterogeneous group of inherited conditions resulting from a primary defect in the mitochondrial respiratory chain with consecutively impaired cellular energy metabolism. Small sized studies using mainly electrocardiography (ECG) and echocardiography have revealed cardiac abnormalities ranging from conduction abnormalities and arrhythmias to hypertrophic or dilated cardiomyopathy in these patients. Recently, characteristic patterns of cardiac involvement were documented by cardiovascular magnetic resonance (CMR) in patients with chronic progressive external ophthalmoplegia (CPEO)/Kearns-Sayre syndrome (KSS) and with mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS). The present study aimed to characterize the prevalence and pattern of cardiac abnormalities and to test the additional diagnostic value of CMR in this patient population. The hypothesis that different neuromuscular MM syndromes present with different cardiac disease phenotypes was evaluated. METHODS: Sixty-four MM patients (50 ± 15 years, 44% male) and 25 matched controls (52 ± 14 years, 36% male) prospectively underwent cardiac evaluations including CMR (comprising cine- and late-gadolinium-enhancement (LGE) imaging). Based on the neuromuscular phenotype and genotype, the patients were grouped: (a) CPEO/KSS (N = 33); (b) MELAS/-like (N = 11); c) myoclonic epilepsy with ragged-red fibers (MERRF) (N = 3) and d) other non-specific MM forms (N = 17). RESULTS: Among the 64 MM patients, 34 (53%) had at least one abnormal CMR finding: 18 (28%) demonstrated an impaired left ventricular ejection-fraction (LV-EF <60%), 14 (22%) had unexplained LV hypertrophy and 21 (33%) were LGE-positive. Compared to controls, MM patients showed significantly higher maximal wall thickness (10 ± 3 vs. 8 ± 2 mm, p = 0.005) and concentricity (LV mass to end-diastolic volume: 0.84 ± 0.27 vs. 0.67 ± 0.11, p < 0.0001) with frequent presence of non-ischemic LGE (30% vs. 0%, p = 0.001). CPEO/KSS showed a predominantly intramural pattern of LGE mostly confined to the basal LV inferolateral wall (8/10; 80%) in addition to a tendency toward concentric remodelling. MELAS/-like patients showed the highest frequency of cardiac disease (in 10/11 (91%)), a mostly concentric LV hypertrophy (6/9; 67%) with or without LV systolic dysfunction and a predominantly focal, patchy LGE equally distributed among LV segments (8/11; 73%). Patients with MERRF and non-specific MM had no particular findings. Pathological CMR findings indicating cardiac involvement were detected significantly more often than pathological ECG results or elevated cardiac serum biomarkers (34 (53%) vs. 18 (28%) vs. 21 (33%); p = 0.008). CONCLUSION: Cardiac involvement is a frequent finding in MM patients - and particularly present in KSS/CPEO as well as MELAS/-like patients. Despite a high variability in clinical presentation, CPEO/KSS patients typically show an intramural pattern of LGE in the basal inferolateral wall whereas MELAS patients are characterized by overt concentric hypertrophy and a rather unique, focally accentuated and diffusely distributed LGE.

Role of taurine in the pathologies of MELAS and MERRF.

Taurine is an abundant ß-amino acid that concentrates in the mitochondria, where it participates in the conjugation of tRNAs for leucine, lysine, glutamate and glutamine. The formation of 5-taurinomethyluridine-tRNA strengthens the interaction of the anticodon with the codon, thereby promoting the decoding of several codons, including those for AAG, UUG, CAG and GAG. By preventing these series of events, taurine deficiency appears to diminish the formation of 5-taurinomethyluridine and causes inefficient decoding for the mitochondrial codons of leucine, lysine, glutamate and glutamine. The resulting reduction in the biosynthesis of mitochondria-encoded proteins deprives the respiratory chain of subunits required for the assembly of respiratory chain complexes. Hence, taurine deficiency is associated with a reduction in oxygen consumption, an elevation in glycolysis and lactate production and a decline in ATP production. A similar sequence of events takes place in mitochondrial diseases MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fiber syndrome). In both diseases, mutations in their respective tRNAs interfere with the formation of 5-taurinomethyluridine in the wobble position. Hence, the taurine-deficient phenotype resembles the phenotypes of MELAS and MERRF.

MERRF/MELAS overlap syndrome due to the m.3291T>C mutation.

We report the case of a 19-year-old Chinese female harboring the m.3291T>C mutation in the MT-TL1 gene encoding the mitochondrial transfer RNA for leucine. She presented with a complex phenotype characterized by progressive cerebellar ataxia, frequent myoclonus seizures, recurrent stroke-like episodes, migraine-like headaches with nausea and vomiting, and elevated resting lactate blood level. It is known that the myoclonus epilepsy with ragged-red fibers (MERRF) is characterized by cerebellar ataxia and myoclonus epilepsy, while that the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is characterized by recurrent stroke-like episodes, migraine-like headaches, and elevated resting lactate blood level. So the patient's clinical manifestations suggest the presence of a MERRF/MELAS overlap syndrome. Muscle biopsy of the patient showed the presence of numerous scattered ragged-red fibers, some cytochrome c oxidase-deficient fibers, and several strongly succinate dehygrogenase-reactive vessels, suggestive of a mitochondrial disorder. Direct sequencing of the complete mitochondrial genome of the proband revealed no mutations other than the T-to-C transition at nucleotide position 3291. Restriction fragment length polymorphism analysis of the proband and her family revealed maternal inheritance of the mutation in a heteroplasmic manner. The analysis of aerobic respiration and glycolysis demonstrated that the fibroblasts from the patient had mitochondrial dysfunction. Our results suggest that the m.3291T>C is pathogenic. This study is the first to describe the m.3291T>C mutation in association with the MERRF/MELAS overlap syndrome.

[Digestive system disease as manifestation of the pleiotropic action of genes in mitochondrial dysfunction].

Defined involvement lesions of the digestive system of clinical manifestations of mitochondrial dysfunction associated with both point mutations and polymorphism of mitochondrial DNA. The nature of the clinical signs of mtDNA polymorphisms carriers--multi organical, a progressive, clinical polymorphism, genetic heterogeneity with predominant involvement of energotropic bodies (cerebrum, cordis, hepatic). Set individual nosological forms of mitochondrial dysfunctions--syndromes Leia, Leber, Cairns, Sarah, MERRF, MELAS, NARP, MNGIE confirmed by clinical and genetic, morphological, biochemical, enzymatic, molecular genetics methods. It was found that 84-88% of these syndromes involving the violation of the digestive system with varying degrees of injury. This damage will be the first in the complex chain signs recovery which determines the direction of early rehabilitation.

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases.

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

The 2-thiouridylase function of the human MTU1 (TRMU) enzyme is dispensable for mitochondrial translation.

MTU1 (TRMU) is a mitochondrial enzyme responsible for the 2-thiolation of the wobble U in tRNA(Lys), tRNA(Glu) and tRNA(Gln), a post-transcriptional modification believed to be important for accurate and efficient synthesis of the 13 respiratory chain subunits encoded by mtDNA. Mutations in MTU1 are associated with acute infantile liver failure, and this has been ascribed to a transient lack of cysteine, the sulfur donor for the thiouridylation reaction, resulting in a mitochondrial translation defect during early development. A mutation in tRNA(Lys) that causes myoclonic epilepsy with ragged-red fibers (MERRF) is also reported to prevent modification of the wobble U. Here we show that mitochondrial translation is unaffected in fibroblasts from an MTU1 patient, in which MTU1 is undetectable by immunoblotting, despite the severe reduction in the 2-thiolation of mitochondrial tRNA(Lys), tRNA(Glu) and tRNA(Gln). The only respiratory chain abnormality that we could observe in these cells was an accumulation of a Complex II assembly intermediate, which, however, did not affect the level of the fully assembled enzyme. The identical phenotype was observed by siRNA-mediated knockdown of MTU1 in HEK 293 cells. Further, the mitochondrial translation deficiencies present in myoblasts from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode and MERRF patients, which are associated with defects in post-transcriptional modification of mitochondrial tRNAs, did not worsen following knockdown of MTU1 in these cells. This study demonstrates that MTU1 is not required for mitochondrial translation at normal steady-state levels of tRNAs, and that it may possess an as yet uncharacterized function in another sulfur-trafficking pathway.

Treatment of human cells derived from MERRF syndrome by peptide-mediated mitochondrial delivery.

BACKGROUND AIMS: The feasibility of delivering mitochondria using the cell-penetrating peptide Pep-1 for the treatment of MERRF (myoclonic epilepsy with ragged red fibers) syndrome, which is caused by point mutations in the transfer RNA genes of mitochondrial DNA, is examined further using cellular models derived from patients with MERRF syndrome. METHODS: Homogenesis of mitochondria (wild-type mitochondria) isolated from normal donor cells with about 83.5% preserved activity were delivered into MERRF fibroblasts by Pep-1 conjugation (Pep-1-Mito). RESULTS: Delivered doses of 52.5 µg and 105 µg Pep-1-Mito had better delivered efficiency and mitochondrial biogenesis after 15 days of treatment. The recovery of mitochondrial function in deficient cells receiving 3 days of treatment with peptide-mediated mitochondrial delivery was comprehensively demonstrated by restoration of oxidative phosphorylation subunits (complex I, III and IV), mitochondrial membrane potential, adenosine triphosphate synthesis and reduction of reactive oxygen species production. The benefits of enhanced mitochondrial regulation depended on the function of foreign mitochondria and not the existence of mitochondrial DNA and can be maintained for at least 21 days with dramatically elongated mitochondrial morphology. In contrast to delivery of wild-type mitochondria, the specific regulation of Pep-1-Mito during MERRF syndrome progression in cells treated with mutant mitochondria was reflected by the opposite performance, with increase in reactive oxygen species production and matrix metalloproteinase activity. CONCLUSIONS: The present study further illustrates the feasibility of mitochondrial intervention therapy using the novel approach of peptide-mediated mitochondrial delivery and the benefit resulting from mitochondria-organelle manipulation.

Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.

Mitochondrila DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the form of point mutations or rearrangements. There is no effective treatment for patients with mtDNA mutations. In the majority of patients with mtDNA defects, both mutant and wild-type molecules are present in the same cell-a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent. Based on the presence of heteroplasmy and the recessive nature of these mutations, we believe it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach we synthesised peptide nucleic acids (PNAs) complementary to human mtDNA templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro replication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates. Furthermore, we have shown uptake of these PNAs into cultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with heteroplasmic mtDNA disorders.

Mitochondrial diseases and epilepsy.

The mitochondrial respiratory chain is the final common pathway for energy production. Defects affecting this pathway can give rise to disease that presents at any age and affects any tissue. However, irrespective of genetic defect, epilepsy is common and there is a significant risk of status epilepticus. This review summarizes our current understanding of the epilepsy that occurs in mitochondrial disease, focusing on three of the most common disorders: mitochondrial myopathy encephalopathy, lactic acidosis and stroke-like episodes (MELAS), myoclonus epilepsy and ragged-red fibers (MERRF), and polymerase gamma (POLG) related disease. In addition, we review the pathogenesis and possible treatment of these disorders.

A Novel MTTK Gene Variant m.8315A>C as a Cause of MERRF Syndrome

In this study, we report on a novel heteroplasmic pathogenic variant in mitochondrial DNA (mtDNA). The studied patient had myoclonus, epilepsy, muscle weakness, and hearing impairment and harbored a heteroplasmic m.8315A>C variant in the MTTK gene with a mutation load ranging from 71% to >96% in tested tissues. In muscle mitochondria, markedly decreased activities of respiratory chain complex I + III and complex IV were observed together with mildly reduced amounts of complex I and complex V (with the detection of V*- and free F1-subcomplexes) and a diminished level of complex IV holoenzyme. This pattern was previously seen in other MTTK pathogenic variants. The novel variant was not present in internal and publicly available control databases. Our report further expands the spectrum of MTTK variants associated with mitochondrial encephalopathies in adults.

MERRF/MELAS overlap syndrome: a double pathogenic mutation in mitochondrial tRNA genes.

BACKGROUND: Myoclonic epilepsy with ragged-red fibres (MERRF) and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are established phenotypes of mitochondrial encephalomyopathy. The m.8356T>C transition in the mitochondrial tRNA(Lys) gene is a pathogenic mutations of MERRF. The m.3243A>G transition in the mitochondrial tRNA(Leu) gene is detected in most MELAS patients. Although previous analyses of double mutations in mitochondrial DNA (mtDNA) were useful for discussing their nature, many unsolved questions remain. OBJECTIVE: To describe the clinical and genetic features of a family with the above mtDNA double-point mutations and discuss the role of double mtDNA mutations in diverse clinical features in the family. PATIENTS AND METHODS: The proband was a 23-year-old woman with MERRF harbouring m.8356T>C and m.3243A>G transitions in mitochondrial tRNA genes. We assessed clinical aspects of her and those of her three relatives and performed mutation analyses on their mtDNA. RESULTS: Phenotypes of the four patients were MERRF, MERRF/MELAS overlap syndrome and asymptomatic carrier. We hypothesise that the course of the phenotype of this family begins with MERRF and is followed by MELAS. This double mutation was heteroplasmic in blood of all four patients but with different rates in each patient, while m.8356T>C appeared homoplasmic and m.3243A>G was heteroplasmic in muscle of the two examined cases. No other mutations were detected in the total mtDNA sequence in this family. CONCLUSIONS: This is the first reported case of a double-point mutation in mtDNA, both of which were heteroplasmic and pathogenic for the established phenotypes.

Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import.

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA(Gln) into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA(CUG)(Gln) and tRNA(UUG)(Gln) species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA(Gln) import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA(Gln) species and a bacterial pre-tRNA(Asp) can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.

Clinical spectrum and prognostic factors of acute necrotizing encephalopathy in children.

This study was conducted to investigate the etiology, the clinical characteristics and prognosis of acute necrotizing encephalopathy (ANE) in Korean children. Six children (1 yr to 7 yr) patients with ANE were enrolled. They were diagnosed by clinical and radiological characteristics and their clinical data were retrospectively analyzed. In a search of clinically plausible causes, brain MRI in all patients, mitochondrial DNA studies for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) and myoclonus epilepsy and ragged red fibers (MERRF) in four patients, and genomic typing on HLA DRB/HLA DQB genes in three patients were performed. All had precedent illnesses and the main initial symptoms included mental change (83%), seizures (50%), and focal deficits (50%). MRI revealed increased T2 signal density in the bilateral thalami and/or the brainstem in all patients. Mitochodrial DNA studies for MELAS and MERRF were negative in those children and HLA-DRB1*1401, HLA-DRB3*0202, and HLA-DQB1*0502 seemed to be significant. A high dose steroid was given to all patients, which seemed to be partly effective except for 2 patients. In conclusion, ANE is relatively rare, but can result in serious neurological complication in children. Early detection and appropriate treatment may lead to a better neurological outcome.