Nuclear DNA origin of mitochondrial complex I deficiency in fatal infantile lactic acidosis evidenced by transnuclear complementation of cultured fibroblasts.

We have studied complex I (NADH-ubiquinone reductase) defects of the mitochondrial respiratory chain in 2 infants who died in the neonatal period from 2 different neurological forms of severe neonatal lactic acidosis. Specific and marked decrease in complex I activity was documented in muscle, liver, and cultured skin fibroblasts. Biochemical characterization and study of the genetic origin of this defect were performed using cultured fibroblasts. Immunodetection of 6 nuclear DNA-encoded (20, 23, 24, 30, 49, and 51 kDa) and 1 mitochondrial DNA-encoded (ND1) complex I subunits in fibroblast mitochondria revealed 2 distinct patterns. In 1 patient, complex I contained reduced amounts of the 24- and 51-kDa subunits and normal amounts of all the other investigated subunits. In the second patient, amounts of all the investigated subunits were severely decreased. The data suggest partial or extensive impairment of complex I assembly in both patients. Cell fusion experiments between 143B206 rho degrees cells, fully depleted of mitochondrial DNA, and fibroblasts from both patients led to phenotypic complementation of the complex I defects in mitochondria of the resulting cybrid cells. These results indicate that the complex I defects in the 2 reported cases are due to nuclear gene mutations.

Necrotizing encephalopathy and macrocephaly with mitochondrial complex I deficiency.

A neonate presented in the first weeks after birth with vomiting. He was unresponsive, with hypotonia, macrocephaly, and lactic acidosis. The cranial computed tomographic scan revealed a hypodense brain, with increased brain volume and extensive cerebral edema. He died at 6 weeks of age; postmortem examination revealed necrotizing encephalopathy with marked brain edema, spongiosis, thalamic necrosis, and basal ganglia calcifications. Enzyme studies of the mitochondrial respiratory chain revealed complex I deficiency in both muscle and liver.

Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis.

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.

Quantification of OXPHOS gene transcripts during muscle cell differentiation in patients with mitochondrial myopathies.

The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.

Metabolic studies in a patient with severe carnitine palmitoyltransferase type II deficiency.

Here we report on a patient with severe ("non-classic") carnitine palmitoyltransferase type II (CPT II) deficiency. Hypoglycemia prompted by an infectious episode and associated with non-ketotic dicarboxylic aciduria orientated diagnosis towards beta-oxidation deficiency disorders. Blood carnitine levels revealed a secondary carnitine deficiency that was responsive to oral L-carnitine supplementation. Blood acylcarnitine profiles were abnormal and included acetyl (C2:0), butyryl/isobutyryl (C4:0), isovaleryl/2-methylbutyryl (C5:0), hexanoyl (C6:0), myristoyl (C14:0), palmitoyl (C16:0), hexadecenoyl (C16:1), oleyl (C18:1) and stearoyl (C18:0) carnitine. In urine, excess excretion of dicarboxylylcarnitines, mainly dodecanedioylcarnitine, was noticed. Upon carnitine supplementation, C8 to C12 fatty acylcarnitines, with decanoylcarnitine as well as C10 to C14 dicarboxylylcarnitines being prominent, were observed in urine. Biochemical measurements disclosed a severe reduction of mitochondrial CPT II activity (7% of normal values). Correlations of metabolic findings in the patient and physiological roles of CPT II are briefly discussed.

Segregation of the G8993 mutant mitochondrial DNA through generations and embryonic tissues in a family at risk of Leigh syndrome.

We identified the T8993G mitochondrial mutation in a female infant who died of Leigh syndrome. The proportion of mutant mitochondrial DNA increased to near homoplasmy in three generations of the pedigree. A similarly high proportion of mutant mitochondrial DNA was found in the chorionic villi and in fetal tissues from a pregnancy interrupted because of the risk of Leigh syndrome. This study supports the concept that prenatal diagnosis can be used for Leigh syndrome with the T8993G mitochondrial DNA mutation.

Detection of mitochondrial DNA deletions by a screening procedure using the polymerase chain reaction.

We describe an accurate procedure for a rapid diagnosis of heteroplasmic mtDNA deletions based on the polymerase chain reaction (PCR). For a selective amplification of deleted mtDNA across the breakpoints of the deletion, we used seven combinations of primers surrounding the most common deleted region between the two origins of mtDNA replication. This procedure was performed on muscle biopsies of twenty patients harboring a single mtDNA deletion and one patient with multiple mtDNA deletions. The results were compared with Southern-blotting analysis. We conclude that this PCR procedure is a sensitive and convenient screening method for the detection of mtDNA deletions.

Identification of a large-scale mitochondrial deoxyribonucleic acid deletion in endocrinopathies and deafness: report of two unrelated cases with diabetes mellitus and adrenal insufficiency, respectively.

In recent years, a broad variety of chronic diseases have been related to different mitochondrial DNA (mtDNA) rearrangements. We have investigated two 16-yr-old unrelated girls with unexplained endocrine disorders for a mtDNA mutation. One initially presented with an adrenal crisis at the age of 4 yr. Complete adrenal insufficiency for nearly 15 yr was the main clinical manifestation, along with insiduous growth retardation and sensorineural hearing loss since age 6. The other girl presented with ketoacidosis at the age of 15 yr. She exhibited incomplete deafness since age 6 and poor growth. In both patients, brain magnetic resonance imaging abnormalities and raised cerebrospinal fluid protein concentration indicated mild leucodystrophy. Biopsy of skeletal muscle showed a mitochondrial dysfunction; molecular analysis using a PCR screening procedure revealed a 7.4 kb deletion of the mtDNA in skeletal muscle but not in leucocytes. Direct sequence analysis of the junctional regions showed that the deletion spanned 7.436 kb (nucleotide 8649 to nucleotide 16084). The relative amount of deleted mtDNA estimated by Southern blot analysis was 25 and 15%, respectively. No deletion was present in leukocytes obtained from the asymptomatic mothers. The presence of the same mutation in different patients with various endocrine conditions supports the view that the 7.4 kb mtDNA deletion should be considered as one of the candidate causes for phenotypically uncommon cases of endocrinopathies, specially in children with deafness. This is the first report of a mitochondrial disease with primary adrenocortical insufficiency as the clinical onset.

Expression of oxidative phosphorylation genes in muscle cell cultures from patients with mitochondrial myopathies.

The expression of several mitochondrial and nuclear genes involved in ATP production was examined in cells cultured from muscle biopsies of patients harboring mitochondrial pathologies. The transcript patterns in muscle cells from the patients affected by carnitine palmitoyl transferase II or 2-ketoglutarate dehydrogenase deficiencies were almost similar to control patterns. In the opposite, patterns were strikingly abnormal in all the other cell cultures from patients with defects in enzymatic complexes involved in oxidative phosphorylation: mitochondrial complex II and III deficiencies, two MELAS syndromes (myopathy, encephalopathy, lactic acidosis and stroke like episodes), a case of Kearns-Sayre syndrome and a case of chronic progressive external ophthalmoplegia. In cultured muscle cells from patients with mtDNA mutations, the percentage of mutated mtDNA was low as compared with those determined in the corresponding skeletal muscle biopsy. Moreover, the complex II defect resulting of a nuclear mutation was not expressed in the cell cultures. Thus, an undetermined transcriptional event, transmitted from muscle biopsies to cultured muscle cells, should be involved to account for such abnormal transcript patterns.

[MNGIE syndrome in 2 siblings]. / Le MNGIE syndrome: deux cas dans une même fratrie.

Two siblings (one man, one woman), presenting with diarrhea, severe weight loss peripheral neuropathy, ophthalmoparesis, asymptomatic leukoencephalopathy were diagnosed as a new cases of Mitochondrial Neuro Gastro Intestinal Encephalomyopathy syndrome (MNGIE). Hirano (1994) defined four criteria for the diagnostic: peripheral neuropathy, ophthalmoparesis, gastro intestinal dysmotility, muscle biopsy with histologic features of mitochondrial myopathy (ragged-red fibers, muscle fibers with increased succinate deshydrogenase stain or ultra structurally abnormal mitochondria). In a review of the literature, we found 31 cases with MNGIE. With our two cases, we study this group of 33 patients. First symptoms begin about 13.5 years with a median of 10 years and extremes for 1 to 32 years. The first signs are gastro intestinal symptoms (recurrent nausea, vomiting or diarrhea with intestinal dysmotility) in 22 cases, an ophthalmoparesia in 4 cases, intestinal and ocular signs in 1 case, gait ataxia or peripheral neuropathy in 3 cases, hearing loss in 1 case, gait ataxia or peripheral neuropathy in 3 cases, hearing loss in 1 case. During the evolution, besides the cardinal signs, the following features have been observed with a variable frequency: hearing loss, short stature, facial palsy, dysphonia, dysarthria, sweating, orthostatic hypotension, bladder dysfunction, hepatomegalia, The laboratory features are: abnormal Nerve Condition Studies/EMG compatible with a sensory motor neuropathy, lactic acidosis, mitochondrial respiratory chain defect (essentially complex IV deficiency, complex I deficiency or multiple complex defect), MRI leukodystrophy, elevated CSF protein, heart block, ragged-red fibers or increased SDH stain. The prognosis is poor, due to a severe weight loss bordering on cachexia 13 patients died with a mean age of 28.5 years (median 24 years, extreme 3 years to 51 years). The prognosis seems to be worsened by a young age of onset. The 33 patients belong to 19 families with 7 cases of consanguinity. 25 patients had a brother, a sister or a cousin affected. The study of these families is compatible with an autosomic recessive transmission, suggesting a pathology of the nuclear genomi, probably impliying the control of the mitochondrial DNA replication. In fact, in 13 cases, a study of the mt DNA was realized: multiple deletions were founded in 6 cases, multiples mutations in one case, unique mutation in 1 case. In 5 cases ther was no evidence of abnormality. These precise etiology and pathophysiologic significance of the mt DNA deletions, and the heterogeneity of the modifications of the mt DNA remain unknown. However, the possibility of various phenotypes for a same genotype or inversely is known in mitochondriopathies.

[MELAS syndrome with pure vascular manifestation?]. / Syndrome "MELAS" à expression vasculaire pure?

Two stroke-like episodes then a grand mal seizure occurred within nine years in a 42-year-old patient. Neuroradiological findings (CT-scan and MRI) led to the diagnosis of incomplete MELAS syndrome. MRI with T2-weighted images (TR: 1000ms; TE: 35ms) showed two small asignal lesions possibly resulting from hemosiderine. Metabolic studies are required to help decide on muscular biopsies. Histological findings are needed for diagnosis of this form of mitochondrial cytopathy with only stroke-like manifestations.

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies.

Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percentage of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA int he first patient's cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation.

A novel gly290asp mitochondrial cytochrome b mutation linked to a complex III deficiency in progressive exercise intolerance.

We have identified a new mitochondrial (mt) cytochrome b mutation in a 29-year-old man with progressive exercise muscle intolerance associated with a marked deficiency of complex III activity and a decreased amount of mitochondrial-encoded cytochrome b. This G to A transition at mtDNA position 15615 leads to the substitution (G290D) of a very highly conserved amino acid of cytochrome b during evolution. The mutant mtDNA was heteroplasmic (80% mutant) in patient muscle but was undetectable in blood from the patient and his healthy mother and sisters. A maternally inherited cytochrome b polymorphism was also identified in this patient. Molecular screening of 150 individuals showed that the G290D mutation associated with the described phenotype. We suggest that this molecular defect is the primary cause of the muscle disease in this patient.

Recurrent myoglobinuria due to carnitine palmitoyltransferase II deficiency: expression of the molecular phenotype in cultured muscle cells.

Carnitine palmitoyltransferase II deficiency (CPT II) is an autosomal recessive disorder and the most frequent cause of hereditary myoglobinuria. We report the case of a young man who presented a severe fever-induced episode of rhabdomyolysis and myoglobinuria resulting in acute renal failure. Cultured skeletal muscle cells have been used for the biochemical and molecular characterization of the defect in this patient. Immunoblot analysis revealed reduced steady-state level of CPT II protein. A PCR-based method detected the common Ser113Leu substitution only in one allele, suggesting that the patient is a compound heterozygote for this common mutation and a different as yet unidentified mutation.

Molecular histology of mitochondrial and nuclear transcripts in the muscle of patients harbouring a single mitochondrial DNA deletion.

The distribution of transcripts of mitochondrial and nuclear genes involved in oxidative phosphorylation and of the mitochondrial creatine kinase nuclear gene was examined, using in situ hybridisation, in the skeletal muscle of 11 patients harbouring a heteroplasmic mitochondrial DNA (mtDNA) single deletion. Levels of mRNAs transcribed from genes located within the deletions were not decreased, suggesting that the remaining wild-type mtDNA was still transcribed. Those muscle fibres with characteristic abnormal mitochondrial proliferation always showed overexpression of mRNAs and rRNAs transcribed from mitochondrial genes located outside the deletions. Interestingly, they also showed overexpression of the nuclear-encoded ATP synthase beta subunit mRNA, but not of mitochondrial creatine kinase mRNA. These observations lead to three proposals: (1) overexpression of mitochondrial transcripts within fibres harbouring mitochondrial proliferation, together with the apparently normal expression of the remaining wild-type mtDNA, is not related to decreased mitochondrial translation; (2) it is more probably related to an up-regulation mechanism which co-ordinates both mitochondrial and nuclear expression; and (3) this mechanism is restricted to transcripts directly involved in oxidative phosphorylation and to fibres with mitochondrial accumulation.

An abnormal exercise test response revealing a respiratory chain complex III deficiency.

A 29-year-old man with a progressive exertional muscle intolerance since childhood was referred for incremental exercise test on a bicycle ergometer. The response pattern suggested a mitochondrial myopathy: that is, a greatly reduced maximum oxygen consumption with appropriate heart rate increase and an anaerobic threshold point reached early. The metabolic investigation in plasma revealed an abnormal oxidoreduction status (hyperlactataemia and high lactate/pyruvate ratio) at rest and after a carbohydrate rich meal. The histochemical examination of a muscle biopsy revealed red granular deposits under the sarcolemma for all type 1 fibers. Oxypolarographic and enzymological studies of the mitochondrial respiratory chain in both isolated mitochondria and muscle homogenate demonstrated a marked deficiency of ubiquinol cytochrome c reductase (complex III) activity.

Fine mapping of randomly distributed multiple deletions of mitochondrial DNA in a case of chronic progressive external ophthalmoplegia.

Multiple deletions of mitochondrial DNA have been detected by Southern blotting in the skeletal muscle of a 42-year-old woman with chronic progressive external ophthalmoplegia. A PCR method, using several combinations of primers covering the whole mtDNA as well as sequence analysis, disclosed the wide spectrum of these multiple deletions differing in size, location and sequence at the breakpoint junction. Most involved the major region between the two replication origins. However, three deletions affected the minor region and lacked either the light strand origin of replication or the heavy strand promoter. These data suggest an impairment of mtDNA replication leading to illegitimate recombination and extensive damage of mtDNA.

Serum 5'-nucleotidase and alkaline phosphatase activities after high dose chemotherapy and bone marrow transplantation in cases of malignancy in children.

After bone marrow transplantation in children, it is essential to detect secondary liver diseases and hepatotoxic effects of immunosuppressive therapy. These can be revealed by cytolytic syndromes sometimes associated with cholestasis. It is therefore important to find an early and specific cholestasis enzymatic marker. A retrospective study of the changes in levels of biological parameters has been carried out in 13 children who underwent one or more bone marrow transplantations. During the 3 months following bone marrow transplantation, all patients developed liver injury characterized by an early and very elevated 5'-nucleotidase activity (sometimes more than 40 times the upper reference limit), a moderate increase in alkaline phosphatase activity, a variable increase in alanine aminotransferase activity and inconstant changes in total bilirubin levels. These results show that cytolytic syndrome and cholestasis are often associated with increases in 5'-nucleotidase and alkaline phosphatase activities. These increases are not correlated, probably due to the influence of therapy on the synthesis and release of both enzymes in the liver. 5'-nucleotidase seems to be the best marker for the detection and follow-up of liver disease in this patient group.