Clinical and molecular features of mitochondrial DNA depletion syndromes.

Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-gammaA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype-phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.

Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy.

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A-->G3260 transition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho degrees). The presence and amount of the mutant G3260 vs the wild-type A3260 were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I- and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A-->G3260 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations.

Period of Hyperbaric Oxygen Delivery Leads to Different Degrees of Hepatic Ischemia/Reperfusion Injury in Rats.

BACKGROUND: This study evaluated the morphology of the rat liver when hyperbaric oxygen (HBO) was used at various stages of ischemia and reperfusion. METHODS: Thirty-two male Wistar rats, subjected to 30 minutes of hepatic ischemia and 30 minutes of reperfusion, were randomly assigned as follows: GIR (n = 8), control without HBO; GHBO/I (n = 8), in which HBO was applied only during ischemia; GHBO/R (n = 8), HBO only during reperfusion; and GHBO/IR (n = 8), HBO during both ischemia and reperfusion. Feasibility scores of hepatocytes were determined by assessing 8 items related to liver injury. RESULTS: The histologic injury score of the hepatic specimens was significantly lower in the GHBO/I group (79.0 ± 0.1) compared with the GIR group (135.0 ± 0.1). HBO was not effective when applied during reperfusion (GHBO/R, 151.3 ± 0.1) or during the ischemia plus reperfusion period (GHBO/IR, 131.0 ± 0.1). The sum was significantly higher (P < .05) in HBO-treated animals during the reperfusion period (ie, in the GHBO/R group compared with any of the other groups). CONCLUSIONS: A favorable effect was obtained when HBO was administered early during ischemia. HBO given in later periods of reperfusion was associated with a more severe sum index percentage of liver damage.

Clinical and molecular findings in children with complex I deficiency.

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.

A MERRF/MELAS overlap syndrome associated with a new point mutation in the mitochondrial DNA tRNA(Lys) gene.

Several members of a three-generation kindred from Sardinia were affected by a maternally inherited syndrome characterized by features of both myoclonus epilepsy with ragged-red fibers (MERRF) and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). Clinically, symptoms such as myoclonus epilepsy, neural deafness and ataxia were variably associated with stroke-like episodes and/or migrainous attacks. Morphologically, numerous MELAS-associated SDH-stained vessels were observed in muscle biopsies, either alone or in combination with ragged-red fibers, the morphological hallmark of MERRF. Sequence analysis of the mtDNA tRNA genes revealed the presence of a single, heteroplasmic T-->C transition at nt 8356, in the region of the tRNA(Lys) gene corresponding to the T-psi-C stem. The T-->C(8356) transition was exclusively found in the maternal lineage of our family, and the relative amount of the mutant mtDNA species in muscle was correlated with the severity of the clinical presentation. Therefore, we propose that the T-->C(8356) transition is responsible for the mitochondrial encephalomyopathy found in our family, and must be added to the expanding list of the pathogenetically relevant mutations of human mtDNA.

Primary carnitine deficiency: heterozygote and intrafamilial phenotypic variation.

Two boys from different families had primary carnitine deficiency: one had cardiomyopathy and myopathy, and the other had hypoglycemia and myopathy but no cardiomyopathy. Uptake of carnitine by cultured fibroblasts was negligible in both patients. Vmax for carnitine transport was reduced to 50% of controls' value in the parents and one brother (who had hypertrophic cardiomyopathy) of the first patient. A brother of the second non-cardiopathic patient died at an early age with autopsy findings of a dilated cardiomyopathy and low cardiac carnitine. Autosomal recessive primary carnitine deficiency can express a variable phenotype in different families as well as within the same family. Heterozygotes can manifest heart involvement.

Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.

A 7-month-old boy died in a demented state after a clinical history characterized by generalized seizures, psychomotor deterioration, and fumaric aciduria. We found a marked deficiency of both mitochondrial and cytosolic fumarases in skeletal muscle, brain, cerebellum, heart, kidney, liver, and cultured fibroblasts. Fumarase activities were 30 to 50% compared with controls in both mitochondria and cytosol from cultured fibroblasts of the parents. Antifumarase cross-reacting material was present in negligible amounts in the patient's tissues. Our data indicate that this disease is an autosomal recessive encephalopathy, due to a single mutation affecting the gene encoding both forms of the enzyme.

Epileptic phenotypes associated with mitochondrial disorders.

OBJECTIVE: To define the clinical and EEG features of the epileptic syndromes occurring in adult and infantile mitochondrial encephalopathies (ME). METHODS: Thirty-one patients with recurrent and apparently unprovoked seizures associated with primary ME were included in the study. Diagnosis of ME was based on the recognition of a morphologic, biochemical, or molecular defect. RESULTS: Epileptic seizures were the first recognized symptom in 53% of the patients. Many adults (43%) and most infants (70%) had nontypical ME phenotypes. Partial seizures, mainly with elementary motor symptoms, and focal or multifocal EEG epileptiform activities characterized the epileptic presentation in 71% of the patients. Generalized myoclonic seizures were an early and consistent symptom only in the five patients with an A8344G mitochondrial DNA point mutation with classic myoclonus epilepsy with ragged red fibers (MERRF) syndrome or "overlapping" characteristics. Photoparoxysmal EEG responses were observed not only in patients with typical MERRF, but also in adult patients with ME with lactic acidosis and strokelike episodes (MELAS), or overlapping phenotypes, and in one child with Leigh syndrome. CONCLUSIONS: Epilepsy is an important sign in the early presentation of ME and may be the most apparent neurologic sign of nontypical ME, often leading to the diagnostic workup. Except for those with an A8344G mitochondrial DNA point mutation, most of our patients had partial seizures or EEG signs indicating a focal origin.

Neuromuscular syndrome associated with the 3291T-->C mutation of mitochondrial DNA: a second case.

A mutation was found in an Italian child affecting the gene encoding the mitochondrial transfer RNA for leucine (codon UUR). This mutation (3291T-->C) had previously been reported in a single Japanese patient. In contrast with the original patient, who suffered from early-onset mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), our patient presented an apparently isolated mild myopathy. Mutational analysis in the proband and her family showed that the mutation was heteroplasmic, and that its relative amount was positively correlated with the severity of the phenotype. These findings lead to the definitive confirmation that the 3291T-->C is indeed pathogenic. As commonly found in mitochondrial-DNA related disorders, also for this mutation different clinical manifestations can be associated with the same genetic abnormality.

A novel mutation (8342G-->A) in the mitochondrial tRNA(Lys) gene associated with progressive external ophthalmoplegia and myoclonus.

We describe a patient who suffered from impaired ocular motility from age 10 years and at 16 years developed ptosis, proximal weakness and progressive fatigability. At 35 years she developed massive myoclonic jerks, and head and distal tremor. A muscle biopsy showed a high percentage of cytochrome c oxidase negative fibers but no ragged-red fibers. A novel heteroplasmic mutation (8342G-->A) was found in the mitochondrial transfer RNA(Lys) gene by single-strand conformation polymorphism screening, followed by sequence and restriction fragment length polymorphism analysis. Approximately 80% of muscle mitochondrial DNA (mtDNA) harbored the mutation, while the mutation was absent in lymphocyte DNA of the proband, as well as of her mother, daughter and a maternal aunt. However, the pathogenicity of the mutation was confirmed by restriction fragment length polymorphism analysis of single muscle fibers, which revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase negative over cytochrome c oxidase positive fibers.

Ultrastructural analysis of extraocular muscle in chronic progressive external ophthalmoplegia.

Extraocular muscles are primarily involved in many mitochondrial diseases, but no reports exist regarding the morphological appearance of the muscles in cases of long-standing ocular myopathies. For this reason, muscle samples obtained from surgery in a sporadic case of chronic progressive external ophthalmoplegia (CPEO) were used for ultrastructural investigation and molecular analysis of mitochondrial DNA. Genetic testing revealed a heteroplasmic macrodeletion of about 5.0 kilobases in length, localized between the 9570- and 14619-base pair regions. Electron microscopy revealed focal areas of both disruption and abnormality of mitochondria in only some of the muscle fibers, producing "selective vacuolization." This ultrastructural pattern was highly selective and limited to some extraocular muscle fibers, sparing all the others. The "selective damage" observed in this case of CPEO resembles that case occurring in another mitochondrial disease, Leber hereditary optic neuropathy, where damage occurs only in the papillomacular bundle of the retina, sparing peripheral axons. It is possible that some anatomical and physiological factors play a leading role in both Leber hereditary optic neuropathy and ocular myopathies. The ultrastructural aspect herein observed needs to be further investigated to better understand whether a particular muscle fiber type is the target of mitochondrial impairment in CPEO.

Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: a morphological, biochemical and molecular-genetic study.

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.

Papillitis as an onset sign of Leber's hereditary optic neuropathy: a case report.

Leber's hereditary optic neuropathy is a maternally transmitted disease resulting from a point mutation in mitochondrial (mt) DNA. In this report we describe a case of Leber's disease with typical clinical findings but atypical ophthalmoscopic presentation. A 14-year-old boy developed severe loss of vision acuity in the left eye, with only partial recovery, followed 4 months later by the same symptoms in the right eye. Fundoscopic examination showed hyperemic papilla on the right eye and optic disc pallor on the left eye. Polymerase chain reaction analysis of lymphocytic mt-DNA revealed a point mutation at 11778. Leber's disease should be considered in young patients (not always male) with sudden visual loss and simple papillary involvement at fundoscopic examination but without the typical telangiectatic microangiopathy.

Synthesis of sulfur-linked analogues of nigerose, laminarabiose, laminaratriose, gentiobiose, gentiotriose, and laminaran trisaccharide Y.

Sulfur-linked analogues of 3-O-alpha-D-glucopyranosyl-D-glucose (nigerose), 3-O-beta-D-glucopyranosyl-D-glucose (laminarabiose), 6-O-beta-D-glucopyranosyl-D-glucose (gentiobiose), O-beta-D-glucopyranosyl-(1-->3)-O-beta-D-glucopyranosyl-(1-->3)-D-glucos e (laminaratriose), O-beta-D-glucopyranosyl)-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-D-gluco se (gentiotriose) and 3,6-di-O-beta-D-glucopyranosyl-D-glucose (laminaran trisaccharide Y), namely, respectively, 3-thionigerose (6), 3-thiolaminarabiose (11), 6-thiogentiobiose (21), 3I,3II-dithiolaminaratriose (16), 6I,6II-dithiogentiotriose (29) and 3I,6I-dithiolaminaran trisaccharide Y (37) have been conveniently prepared by SN2 reactions of the corresponding anomer of D-glucopyranose 1-thiolate with suitably activated monosaccharide derivatives in N,N-dimethylformamide (for 6 and 21) or in tetrahydrofuran in the presence of a crown ether (for 11). A sequence involving the reaction of non-anomeric thiolates with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide was alternatively used for the preparation of 11 and 21 but proved less satisfactory. The preparation of thiotrisaccharides 16, 29, and 37 involved a mixed approach.