A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

Calcium signalling-dependent mitochondrial dysfunction and bioenergetics regulation in respiratory chain Complex II deficiency.

Despite advanced knowledge on the genetic basis of oxidative phosphorylation-related diseases, the molecular and/or cellular determinants for tissue-specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring before cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca(2+) signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (Δψ(mit)) loss, [ATP] reduction and increased reactive oxygen species production. Cytosolic and mitochondrial Ca(2+) overload are linked to increased endoplasmic reticulum (ER) Ca(2+) leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca(2+)](mit) is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca(2+)] activated on the one hand a compensatory Ca(2+)-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary function for Ca(2+) signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency.

Liver hepatoblastoma and multiple OXPHOS deficiency in the follow-up of a patient with methylmalonic aciduria.

A boy who was diagnosed with methylmalonic aciduria (MMA) at the age of 10 days developed persistent hepatomegaly and raised transaminases from the age of 4 years. He was subsequently diagnosed with Leigh syndrome and required a kidney transplantation for end-stage renal failure. A massive hepatoblastoma led to his death by the age of 11 years. Methylmalonyl-CoA mutase activity was undetectable on both cultured skin fibroblasts and kidney biopsy and multiple respiratory chain deficiency was demonstrated in the kidney. Mitochondrial dysfunction and/or post-transplant immunosuppressive therapy should be considered as a possible cause of liver cancer in this patient.

Mitochondrial oxidative phosphorylation: pitfalls and tips in measuring and interpreting enzyme activities.

Mitochondrial oxidative phosphorylation (OXPHOS) is fundamental in all aspects of cellular life in aerobic cells and organisms. It is therefore not surprising that a variety of diseases have been attributed to dysfunction of the OXPHOS enzymes. Assessment of OXPHOS in human samples has proved to be a difficult task over years, even when relying on well-established methods. The complexity and the flexibility of the mitochondrial organization in cells account for a large part in the difficulties encountered in assessing OXPHOS activity. Nevertheless, a careful and detailed analysis of OXPHOS enzyme activity in cells or biopsy samples from patients at risk provides diagnosis of potential OXPHOS deficiency. Problems inherent in the use of human material, mostly the small size of the samples to be analysed, are difficult to resolve. However, cautious handling of these samples permits reasonable confidence to be reached in the interpretation of the data.

Respiratory chain deficiency in Alpers syndrome.

Alpers syndrome is a progressive encephalopathy of early onset, characterized by rapid and severe developmental delay, intractable seizures and liver involvement in a previously healthy child. Here, we report on respiratory chain enzyme deficiency in the liver of four unrelated children presenting with epileptic encephalopathy and liver involvement diagnosed as Alpers syndrome. Interestingly, oxidative phosphorylation in skeletal muscle was normal in 4/4 and blood and CSF lactate in 3/4 patients. Liver involvement had a late clinical onset in patients with previously isolated epileptic encephalopathy. Based on these observations, we suggest 1. to give consideration to respiratory chain deficiency in the diagnosis of severe epileptic encephalopathy in childhood, even when no clinical or biological evidence of liver involvement or lactic acidosis is noted, and 2. to investigate the respiratory chain in a needle biopsy of the liver in children with epileptic encephalopathy prior to valproate administration if biochemical indications for respiratory chain disease or hepatic disturbance are noted, as this drug is believed to occasionally trigger hepatic failure and fatal outcome.

Determination of enzyme activities for prenatal diagnosis of respiratory chain deficiency.

Genetic counselling and prenatal diagnosis are major issues of mitochondrial respiratory chain deficiency, especially as these conditions are largely untreatable. In the absence of known mitochondrial or nuclear gene mutations, measurement of respiratory chain enzyme activities represents the only possibility to prevent recurrence of the disease in affected families. We carried out enzymatic prenatal diagnosis in 21 pregnancies from 10 unrelated couples using uncultured choriocytes and/or amniocytes. Twelve babies were born and are healthy, seven pregnancies were discontinued early on because of an enzyme deficiency detected prenatally. In two cases, a fetus which appeared normal after early and/or late prenatal diagnosis, turned out to be affected. We conclude that a deficient enzyme activity is indicative of recurrence, but a normal result at 10 weeks of gestation does not give conclusive evidence as to the outcome of the pregnancy. We therefore suggest the following procedure: (1) a choriocentesis or an amniocentesis in early pregnancy when the proband expresses the disease in cultured skin fibroblasts; (2) a second amniocentesis at 28 weeks' gestation should be offered to avoid false negative results due to a possible late expression of the disease, in combination with: (3) a careful and repeated ultrasound survey for detection of growth failure in the third trimester; (4) prenatal diagnosis should not be performed in case of late onset clinical symptoms in the proband; and (5) parents should be aware of the possibility of false negative results. Prenatal diagnosis should not be proposed for a complex I deficiency as this enzyme activity cannot be accurately measured in fetal cells.

Quinone-responsive multiple respiratory-chain dysfunction due to widespread coenzyme Q10 deficiency.

BACKGROUND: The respiratory-chain deficiencies are a broad group of largely untreatable diseases. Among them, coenzyme Q10 (ubiquinone) deficiency constitutes a subclass that deserves early and accurate diagnosis. METHODS: We assessed respiratory-chain function in two siblings with severe encephalomyopathy and renal failure. We used high-performance liquid chromatography analyses, combined with radiolabelling experiments, to quantify cellular coenzyme Q10 content. Clinical follow-up and detailed biochemical investigations of respiratory chain activity were carried out over the 3 years of oral quinone administration. FINDINGS: Deficiency of coenzyme Q10-dependent respiratory-chain activities was identified in muscle biopsy, circulating lymphocytes, and cultured skin fibroblasts. Undetectable coenzyme Q10 and results of radiolabelling experiments in cultured fibroblasts supported the diagnosis of widespread coenzyme Q10 deficiency. Stimulation of respiration and fibroblast enzyme activities by exogenous quinones in vitro prompted us to treat the patients with oral ubidecarenone (5 mg/kg daily), which resulted in a substantial improvement of their condition over 3 years of therapy. INTERPRETATION: Particular attention should be paid to multiple quinone-responsive respiratory-chain enzyme deficiency because this rare disorder can be successfully treated by oral ubidecarenone.

Cerebral white matter disease in children may be caused by mitochondrial respiratory chain deficiency.

Several mitochondrial diseases are known to occasionally involve the cerebral white matter, namely Leigh syndrome, Kearns-Sayre syndrome, and MELAS syndrome, but in these cases the major finding is alteration in the basal ganglia and brainstem. Here we report on severe diffuse white matter involvement and respiratory chain enzyme deficiency or mitochondrial DNA rearrangement in 5 unrelated families. It is interesting that white matter lesions were the only abnormal neuroradiologic feature in 3 of the 5 families, and multiple small cyst-like white matter lesions were found in 2 of 5 probands. Respiratory chain deficiency should be considered in the diagnosis of severe white matter involvement in childhood.

Respiratory chain deficiency presenting as recurrent myoglobinuria in childhood.

Myoglobinuria is an abnormal urinary excretion of myoglobin due to an acute destruction of skeletal muscle fibres. Several metabolic diseases are known to account for myoglobinuria including defects of glycolysis and fatty acid oxidation. Here, we report on respiratory chain enzyme deficiency in three unrelated children with recurrent episodes of myoglobinuria and muscle weakness (complex I: one patient, complex IV: two patients). All three patients had generalized hyporeflexia during attacks, a feature which is not commonly reported in other causes of rhabdomyolysis. Studying respiratory chain enzyme activities in cultured skin fibroblasts might help diagnosing this condition, especially when acute rhabdomyolysis precludes skeletal muscle biopsy during and immediately after episodes of myoglobinuria.

The neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G) triggers muscle ATPase deficiency and hypocitrullinaemia.

UNLABELLED: Based on the study of three unrelated families, we report what we believe to be the first in vivo evidence of muscle ATPase deficiency in individuals carrying the neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G). Since plasma citrulline was consistently low in 4/5 patients, we suggest that the NARP mutation caused complex V deficiency in the small intestine as well, thus reducing the availability of mitochondrial ATP required for citrulline synthesis. CONCLUSION: We suggest giving consideration to hypocitrullinaemia as a hallmark of the neurogenic weakness, ataxia and retinitis pigmentosa syndrome mutation and more generally of impaired oxidative phosphorylation in the small intestine in vivo.

Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia.

Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs. Hypertrophic cardiomyopathy is observed in most FRDA patients. The gene associated with the disease has been mapped to chromosome 9q13 (ref. 3) and encodes a 210-amino-acid protein, frataxin. FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles. The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport. Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients. Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well. Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast. The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals. Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder.

Nicotinamide adenine dinucleotides permeate through mitochondrial membranes in human Epstein-Barr virus-transformed lymphocytes.

Human cultured cells are widely used for the investigation of respiratory chain disorders. Oxidative properties are generally investigated by means of polarographic studies carried out on detergent-permeabilized cells. By studying the oxidative properties of Epstein- Barr virus-transformed B lymphocytes, we found that the respiration was significantly decreased after 3-4 days of cell culture. Simultaneously, we observed decreased NAD(+)-dependent oxidations (malate, glutamate, pyruvate) that became dependent upon the addition of exogenous NAD+. The effect of NAD+ was shown to be related to an influx of catalytic amount of NAD+ into the mitochondrial matrix. A full ability to oxidize NAD(+)-dependent substrates was restored less than 2 h after a change of the culture medium. These observations suggested: (a) the occurrence of fluxes of catalytic amounts of NAD+ through the mitochondrial inner membrane in human cells; (b) an early control of mitochondrial metabolism by matrix NAD+ content in cells grown under limiting growth conditions; (c) the possible confusion between complex I deficiency and a decrease content of matrix NAD+ when using human cultured cells.

Reconstruction of the centrosome cycle from cryoelectron micrographs.

The absence of detailed in vitro studies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

Fluxes of nicotinamide adenine dinucleotides through mitochondrial membranes in human cultured cells.

We report on the loss of mitochondrial nicotinamide adenine dinucleotides in human cultured cells along with cell culture and acidification of the culture medium. This was established both by the direct measurement of the decrease in the mitochondrial NAD content and by the alteration of the oxidative properties of the mitochondria. In situ, this loss could be reversed in less than 2 h by changing the culture medium or by readjusting the pH of the medium at physiological pH values. By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD+-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD+ into the mitochondrial matrix. These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. They also point to the possible confusion between a loss of mitochondrial NAD and a defect of respiratory chain complex I in the context of screening procedures for respiratory chain disorder in human.

Clinical presentation of mitochondrial disorders in childhood.

Respiratory-chain deficiencies have long been regarded as neuromuscular diseases. In fact, oxidative phosphorylation, i.e. adenosine triphosphate (ATP) synthesis by the respiratory chain, does not occur only in the neuromuscular system. Indeed, a number of non-neuromuscular organs and tissues are dependent upon mitochondrial energy supply. For this reason, a respiratory chain deficiency can theoretically give rise to any symptom, in any organ or tissue, at any age and with any mode of inheritance, owing to the twofold genetic origin of respiratory enzymes (nuclear DNA and mitochondrial DNA, mtDNA). In recent years, it has become increasingly clear that genetic defects of oxidative phosphorylation account for a large variety of clinical symptoms in childhood. Among 100 patients with respiratory-chain deficiencies identified in our centre, 56% presented with a non-neuromuscular symptom and 44% were referred for a neuromuscular problem. It appears that the diagnosis of a respiratory-chain deficiency is difficult initially when only one symptom is present. In contrast, this diagnosis is easier to consider when two seemingly unrelated symptoms are observed.

Spectrum of mitochondrial DNA rearrangements in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome (MIM 557000) is a disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a defect of oxidative phosphorylation associated with deletions of the mitochondrial DNA. We present here a series of 21 cases (including 15 unreported patients) with Pearson syndrome and describe mitochondrial DNA deletions as consistent features in this syndrome. Nine patients presented the same 4.9 kb deletion, while other patients presented different deletions ranging in size from 9 to 14 kb between tRNACyst and the D-loop. Direct repeats (4-13 bp) were consistently present in the wild-type mtDNA at the boundaries of the deletions. Deletion-dimers, deletion-multimers or duplications were observed in association with deletions. Duplications were identified both in patients who died of their Pearson syndrome and in the ones who survived and developed Kearns-Sayre syndrome, suggesting that no correlation could be made between the clinical severity and the type, size or location of the rearrangements.

Endomyocardial biopsies for early detection of mitochondrial disorders in hypertrophic cardiomyopathies.

Considering the high proportion of unexplained hypertrophic cardiomyopathies on the one hand and the occurrence of cardiomyopathies in several mitochondrial disorders on the other, we hypothesized that isolated hypertrophic cardiomyopathies in infancy could occasionally be the result of defects of oxidative phosphorylation. By means of a scaled-down technique, we were able to investigate oxidative phosphorylation on minute amounts of endomyocardial tissue (1 mg) in three patients with concentric hypertrophic cardiomyopathy (shortening fraction in diameter, 18% to 27%; normal mean +/- 1 SD, 33 +/- 3%) and in control subjects. Although the absolute respiratory chain enzyme activities in the endomyocardial biopsy specimens of the patients were within the low normal range, the determination of the activity ratios allowed us to ascribe hypertrophic cardiomyopathies to respiratory chain enzyme abnormalities in all three cases (complex I, two cases; multiple enzyme deficiency, one case). The respiratory chain enzyme activity ratios, which are normally constant irrespective of the tissue tested, were markedly abnormal in all three patients (cytochrome c oxidase/reduced nicotinamide-adenine dinucleotide cytochrome c reductase, 4.6 to 10.4; normal mean +/- 1 SD, 2.9 +/- 0.5). We conclude that mitochondrial disorders should be regarded as potential causes of hypertrophic cardiomyopathy in early infancy. Because cardiac catheterization is routinely performed for hemodynamic investigation of cardiomyopathies, we suggest that endomyocardial biopsies be considered as a tool for early detection of mitochondrial cardiomyopathies, especially in hypertrophic forms of the disease.

Fate and expression of the deleted mitochondrial DNA differ between human heteroplasmic skin fibroblast and Epstein-Barr virus-transformed lymphocyte cultures.

We report on the variant phenotypic expression of mitochondrial genotypes in cultured skin fibroblasts and Epstein-Barr virus-transformed lymphocyte cultures from a patient with Pearson syndrome (McKusick no. 260560). Both cell types harbored a heteroplasmic population of normal and deleted mtDNA molecules. The deletion encompassed five tRNA genes and seven genes encoding subunits of cytochrome c oxidase, complex I, and ATPase. Patient skin fibroblasts and lymphocytes harbored 60 and 80% of deleted mtDNA molecules, respectively, and initially displayed defective respiratory chain activities. In both cases, there was a progressive recovery of respiratory chain activities during in vitro cell proliferation. In cultured skin fibroblasts, the loss of the deleted mtDNA molecules accounted for the recovery of normal respiratory chain activities. These features were prevented by allowing respiratory chain-deficient cells to grow in the presence of uridine (200 microM). In Epstein-Barr virus-transformed lymphocytes containing 60% of deleted mtDNA, the recovery of respiratory chain activities was attributable to an increase in the mtRNA translation efficiency rather than to an increased content in mtDNA or mtRNA. The present study suggests that the variant cellular responses to abnormal mitochondrial genotypes might contribute to the tissue-specific expression of mitochondrial disorders in vivo.

Investigation of respiratory chain activity in human heart.

Although disorders of oxidative phosphorylation have long been regarded as neuromuscular diseases only, they can actually give rise to any symptom specifically affecting any organ or tissue, particularly in childhood. The early diagnosis of such a condition may thus require the specific assessment of the mitochondrial function in the organ or tissue shown to be clinically involved. A method is presented allowing such an early detection of respiratory chain defects in human heart. Respiratory chain enzyme studies were carried out using endomyocardial biopsies, less than 2 mg fresh weight. Enzyme activities measured in the endomyocardial biopsies were compared with those obtained using other sampling methods (surgical and postmortem microsamples). A comparison of the respiratory chain enzyme activities in heart and other human tissues is also presented. It was found that (i) the activities of respiratory chain complexes in human heart were similar with any sampling method; (ii) these activities were high compared to other human tissues, allowing the use of heart microsamples for enzyme measurements; (iii) the activity ratios between complexes of the respiratory chain were similar in heart and other human tissues or cells as well, allowing us to confidently characterize potential mitochondrial defects and to compare their expression in different tissues or cells. The value of such investigations on endomyocardial biopsies is illustrated in the case of two patients affected with mitochondrial cardiomyopathy and is discussed in regard to the tissue-specific nature of mitochondrial diseases.