NDUFA9 point mutations cause a variable mitochondrial complex I assembly defect.

Mitochondrial respiratory chain complex I consists of 44 different subunits and contains 3 functional modules: the Q-, the N- and the P-module. NDUFA9 is a Q-module subunit required for complex I assembly or stability. However, its role in complex I biogenesis has not been studied in patient fibroblasts. So far, a single patient carrying an NDUFA9 variant with a severe neonatally fatal phenotype has been reported. Via exome sequencing, we identified a novel homozygous NDUFA9 missense variant in another patient with a milder phenotype including childhood-onset progressive generalized dystonia and axonal peripheral neuropathy. We performed complex I assembly analysis using primary skin fibroblasts of both patients. Reduced complex I abundance and an accumulation of Q-module subassemblies were present in both patients but more pronounced in the severe clinical phenotype patient. The latter displayed additional accumulation of P-module subassemblies, which was not present in the milder-phenotype patient. Lentiviral complementation of both patient fibroblast cell lines with wild-type NDUFA9 rescued complex I deficiency and the assembly defects. Our report further characterizes the phenotypic spectrum of NDUFA9 deficiency and demonstrates that the severity of the clinical phenotype correlates with the severity of the effects of the different NDUFA9 variants on complex I assembly.

Mutated PET117 causes complex IV deficiency and is associated with neurodevelopmental regression and medulla oblongata lesions.

The genetic basis of the many progressive, multi systemic, mitochondrial diseases that cause a lack of cellular ATP production is heterogeneous, with defects found both in the mitochondrial genome as well as in the nuclear genome. Many different mutations have been found in the genes encoding subunits of the enzyme complexes of the oxidative phosphorylation system. In addition, mutations in genes encoding proteins involved in the assembly of these complexes are known to cause mitochondrial disorders. Here we describe two sisters with a mitochondrial disease characterized by lesions in the medulla oblongata, as demonstrated by brain magnetic resonance imaging, and an isolated complex IV deficiency and reduced levels of individual complex IV subunits. Whole exome sequencing revealed a homozygous nonsense mutation resulting in a premature stop codon in the gene encoding Pet117, a small protein that has previously been predicted to be a complex IV assembly factor. PET117 has not been identified as a mitochondrial disease gene before. Lentiviral complementation of patient fibroblasts with wild-type PET117 restored the complex IV deficiency, proving that the gene defect is responsible for the complex IV deficiency in the patients, and indicating a pivotal role of this protein in the proper functioning of complex IV. Although previous studies had suggested a possible role of this protein in the insertion of copper into complex IV, studies in patient fibroblasts could not confirm this. This case presentation thus implicates mutations in PET117 as a novel cause of mitochondrial disease.

Socio-emotional Problems in Children with CDG.

BACKGROUND: Congenital disorders of glycosylation (CDG) form a group of inherited metabolic diseases. Although the clinical presentation shows extreme variability, the nervous system is frequently affected. Several parents of our patients diagnosed with CDG reported behavioral problems, including mood swings, depressive behavior, and anxiety. This raised the question whether patients with CDG have an increased risk for socio-emotional problems. METHODS: We evaluated 18 children with confirmed CDG. The Child Behavior Checklist (CBCL) was used to screen for socio-emotional problems. To determine the disease progression and severity in CDG, the Nijmegen Paediatric CDG Rating Scale (NPCRS) was used. RESULTS were compared to "norm scores" and to children with mitochondrial disorders and children with other chronic metabolic disorders with multisystem involvement. RESULTS: RESULTS showed a high prevalence of socio-emotional problems in children with CDG. Mean total scores, scores on withdrawn/depressed behavior, social problems, and somatic complaints were significantly increased. More than two thirds of our CDG patients have abnormal scores on CBCL. The mean score on social problems was significantly higher compared to our two control groups of patients with other chronic metabolic disorders. CONCLUSIONS: Patients with CDG have an increased risk of developing socio-emotional problems. A standard screening for psychological problems is recommended for the early detection of psychological problems in CDG patients.

Disturbed energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by creatine intake.

Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP-phosphocreatine phosphoryl exchange reaction mediated by creatine kinase. Given that the consequences of Cr depletion are incompletely understood, we assessed the morphological, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of l-arginine:glycine amidinotransferase (AGAT(-/-)), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hindlimb muscle of AGAT(-/-) mice. Compared with wild-type, the inorganic phosphate/ß-ATP ratio was increased fourfold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F(1)F(0)-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT(-/-) mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT(-/-) muscle fibres. Ischaemia resulted in exacerbation of the decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT(-/-) mice can be switched between Cr deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific creatine kinase deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the phosphocreatine-creatine kinase system.

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.

Depressive behaviour in children diagnosed with a mitochondrial disorder.

A higher incidence of depression has been described in adults with primary oxidative phosphorylation disease. We evaluated the psychological characteristics of eighteen non-retarded pediatric patients diagnosed with a disorder of the oxidative phosphorylation. We found significantly higher rate of withdrawn, depressive behaviour compared to population norm scores, to children with other types of inborn errors of metabolism and also in comparison to patients with Sotos syndrome. The occurrence of depressive behaviour showed no correlation with the degree of mitochondrial dysfunction. These findings support the hypothesis that mood disorders could be associated to abnormal cerebral energy metabolism.

Major depression in adolescent children consecutively diagnosed with mitochondrial disorder.

A higher incidence of major depression has been described in adults with a primary oxidative phosphorylation disease. Intriguingly however, not all patients carrying the same mutation develop symptoms of major depression, pointing out the significance of the interplay of genetic and non-genetic factors in the etiology. In a series of paediatric patients evaluated for mitochondrial dysfunction, out of 35 children with a biochemically and genetically confirmed mitochondrial disorder, we identified five cases presenting with major depression prior to the diagnosis. The patients were diagnosed respectively with mutations in MTTK, MTND1, POLG1, PDHA1 and the common 4977 bp mtDNA deletion. Besides cerebral lactic acidemia protein and glucose concentrations, immunoglobins, anti-gangliosides and neurotransmitters were normal. No significant difference could be confirmed in the disease progression or the quality of life, compared to the other, genetically confirmed mitochondrial patients. In three out of our five patients a significant stress life event was confirmed. We propose the abnormal central nervous system energy metabolism as the underlying cause of the mood disorder in our paediatric patients. Exploring the genetic etiology in children with mitochondrial dysfunction and depression is essential both for safe medication and adequate counselling.

Isolated deficiencies of OXPHOS complexes I and IV are identified accurately and quickly by simple enzyme activity immunocapture assays.

OXPHOS deficits are associated with most reported cases of inherited, degenerative and acquired mitochondrial disease. Traditional methods of measuring OXPHOS activities in patients provide valuable clinical information but require fifty to hundreds of milligrams of biopsy tissue samples in order to isolate mitochondria for analysis. We have worked to develop assays that require less sample and here report novel immunocapture assays (lateral flow dipstick immunoassays) to determine the activities of complexes I and IV, which are far and away the most commonly affected complexes in the class of OXPHOS diseases. These assays are extremely simple to perform, rapid (1-1.5 h) and reproducible with low intra-assay and inter-assay coefficients of variability (CVs) s (<10%). Importantly, there is no need to purify mitochondria as crude extracts of whole cells or tissues are suitable samples. Therefore, the assays allow use of samples obtained non-invasively such as cheek swabs and whole blood, which are not amenable to traditional mitochondrial purification and OXPHOS enzyme analysis. As a first step to assess clinical utility of these novel assays, they were used to screen a panel of cultured fibroblasts derived from patients with isolated deficiencies in complex I or IV caused by identified genetic defects. All patients (5/5) with isolated complex IV deficiencies were identified in this population. Similarly, almost all (22/24) patients with isolated complex I deficiencies were identified. We believe that this assay approach should find widespread utility in initial screening of patients suspected of having mitochondrial disease.

MR spectroscopy and serial magnetic resonance imaging in a patient with mitochondrial cystic leukoencephalopathy due to complex I deficiency and NDUFV1 mutations and mild clinical course.

We present clinical, magnetic resonance imaging and MR spectroscopic findings of a female patient, first admitted at the age of 9 months for regression of motor milestones and signs of mild spastic diplegia. Magnetic resonance imaging (MRI) demonstrated periventricular white matter abnormalities with sparing of the subcortical white matter. Subsequent MRIs, performed at the ages of 13 and 16 months, demonstrated progression of the white matter changes, progressive white matter rarefaction and cystic degeneration, and additional involvement of the corpus callosum; only the subcortical white matter remained spared. Proton MR spectroscopy revealed lactate elevation in the white matter. Blood lactate and lactate/pyruvate ratio were mildly elevated. Subsequent analysis of mitochondrial function in muscle tissue showed decreases in substrate oxidation and in ATP and CrP production rates. Complex I activity was seriously decreased, whereas mild decreases of complex II and IV activities were also noted. Analysis of the NDUFV1 gene revealed compound heterozygosity for two point mutations, each of them carried by one parent. The further clinical course of the patient was uphill; she slowly regained all previously lost motor milestones. In conclusion, diffuse white matter changes on MRI are compatible with mitochondrial encephalopathy and not necessarily associated with a severe clinical course.

Recurrent acute liver failure and mitochondriopathy in a case of Wolcott-Rallison syndrome.

A 10-year-old Arabic boy of consanguineous parents has suffered eight episodes of acute liver failure with haemolysis triggered by intercurrent febrile illnesses. The first crisis occurred at 9 months of age, after which diabetes mellitus developed. By the age of 6 years, short stature, mild myopathy and later skeletal epiphyseal dysplasia also became evident. His psychosocial development and educational achievements have remained within normal limits. While there were no clear biochemical indicators of a mitochondrial disorder, an almost complete deficiency of complex I of the respiratory chain was demonstrated in liver but not in fibroblast or muscle samples. Molecular analysis of the eukaryotic translation initiation factor 2alpha kinase gene (EIF2AK3) demonstrated a homozygous mutation, compatible with a diagnosis of Wolcott-Rallison syndrome (WRS). This patient's course adds a new perspective to the presentation of WRS caused by mutations in the EIF2AK3 gene linking it to mitochondrial disorders: recoverable and recurrent acute liver failure. The findings also illustrate the diagnostic difficulty of mitochondrial disease as it cannot be excluded by muscle or skin biopsy in patients presenting with liver disease. The case also further complicates the decision-making process for liver transplantation in cases of acute liver failure in the context of a possible mitochondrial disorder. Such patients may be more likely to recover spontaneously if a mitochondrial disorder underlies the liver failure, yet without neurological features liver transplantation remains an option.

A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

PURPOSE: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. METHODS: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing. Transmitochondrial cybrids were obtained by fusion of 143B206 TK(-) rho zero cells with patient-derived enucleated fibroblasts. Immunoblotting techniques were applied to study the complex V assembly. RESULTS: A homoplasmic nonsense mutation m.8529G-->A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNA. Immunoblotting after blue native polyacrylamide gel electrophoresis showed a lack of holocomplex V and increased amounts of mitochondrial ATP synthase subcomplexes. An in-gel activity assay of ATP hydrolysis showed activity of free F(1)-ATPase in the patient's muscle tissue and in the cybrid clones. CONCLUSION: We describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

Fatal hypertensive crisis as presentation of mitochondrial complex I deficiency.

Complex I deficiency is a frequent defect of the mitochondrial electron transport chain. We report on a 3-year-old boy, who rapidly deteriorated after sudden flushing and collapse. This fatal and unusual case was characterized by widely uncontrollable arterial hypertension. It might indicate that hypertensive crisis could be a potential manifestation of mitochondrial disorders.

Association of 3-methylglutaconic aciduria with sensori-neural deafness, encephalopathy, and Leigh-like syndrome (MEGDEL association) in four patients with a disorder of the oxidative phosphorylation.

In this paper, we describe a distinct clinical subtype of 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria is a group of different metabolic disorders biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. We performed biochemical and genetic investigations, including urine organic acid analysis, NMR spectroscopy, measurement of 3-methylglutaconyl-CoA hydratase activity, cardiolipin levels, OPA3 gene analysis and measurement of the oxidative phosphorylation in four female patients with 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria type I, Barth syndrome, and Costeff syndrome were excluded as the activity of 3-methylglutaconyl-CoA hydratase, the cardiolipin levels, and molecular analysis of the OPA3 gene, respectively, showed no abnormalities. The children presented with characteristic association of hearing loss and the neuro-radiological evidence of Leigh disease. They also had neonatal hypotonia, recurrent lactic acidemia, episodes with hypoglycemia and severe recurrent infections, feeding difficulties, failure to thrive, developmental delay, and progressive spasticity with extrapyramidal symptoms. Our patients were further biochemically characterized by a mitochondrial dysfunction and persistent urinary excretion of 3-methylglutaconic acid.

Metabolic capacity of the diaphragm in patients with COPD.

Chronic obstructive pulmonary disease (COPD) is associated with an increased load on the diaphragm. Chronic loading on skeletal muscles results in metabolic changes and fiber-type shifts. Therefore, we investigated whether the load on the human diaphragm imposed by COPD altered oxidative enzyme activity, glycogenolytic enzyme activity and mitochondrial energy generating capacity and efficiency. Biopsies of the diaphragm from COPD patients and control subjects were obtained and activities of L(+)3-hydroxyacylCoA-dehydrogenase (HADH, marker for beta-oxidation capacity) and phosphorylase (marker for glycogenolytic capacity) were measured spectrophotometrically. Mitochondrial energy generating capacity was measured by spectrophotometrical and radiochemical methods. Fiber-type distribution was determined electrophoretically. We found that HADH activity was increased with increasing severity of COPD (P=0.05). No change in glycogenolytic enzyme activity was observed. The activity of the mitochondrial respiratory chain complexes III and IV and oxidation of pyruvate was increased with increasing airflow obstruction. These results suggest that in COPD the diaphragm adapts to a higher workload by increasing the oxidative capacity and mitochondrial function.

Inhibitory effects of the beta-adrenergic receptor agonist zilpaterol on the LPS-induced production of TNF-alpha in vitro and in vivo.

In this study the anti-inflammatory properties of zilpaterol, a beta2-adrenergic receptor (AR) agonist specifically developed as a growth promoter in cattle were investigated. Although zilpaterol has a different structure compared with the beta2-AR agonists known to date, it was noted that it was able to bind to both the beta2-AR (Ki = 1.1 x 10(-6)) and the beta1-AR (Ki = 1.0 x 10(-5)). Using lipopolysaccharide (LPS)-exposed U937 macrophages, the production of cyclic adenosine-3',5'-cyclic monophosphate (cAMP) and tumor necrosis factor alpha (TNF-alpha) were investigated. Zilpaterol inhibited TNF-alpha release and induced intracellular cAMP levels in a dose-dependent manner. The inhibition of TNF-alpha release and induction of cAMP production was mainly mediated via the beta2-AR, as indicated by addition of beta1- and beta2-specific antagonists. The effects of zilpaterol were investigated in LPS-treated male Wistar rats after pretreatment with zilpaterol. Zilpaterol dosed at 500 microg/kg body weight reduced the TNF-alpha plasma levels. In conclusion, zilpaterol is a beta2-adrenergic agonist and an inhibitor of TNF-alpha production induced by LPS both in vivo and in vitro.

Mitochondrial dysfunction in a patient with Joubert syndrome.

Joubert syndrome is a genetically heterogeneous disorder. The diagnostic criteria include episodic hyperventilation, abnormal eye movements, psychomotor retardation, hypotonia, ataxia, and the characteristic neuro-imaging findings (molar-tooth sign). Many of these clinical features have been observed in new-borns with mitochondrial disorders as well. Congenital brain malformations, including cerebellar hypoplasia, have been described in pyruvate dehydrogenase deficiency. Malformations of the vermis and the cerebellar peduncles, with the lack of axonal decussations, however, are characteristic for Joubert syndrome but unique in patients with mitochondrial disorders. Here, we describe a child with Joubert syndrome presenting with primary lactic acidemia, decreased pyruvate oxidation rates, decreased ATP production, and a mildly decreased pyruvate dehydrogenase complex activity measured in a fresh muscle biopsy. Sequence analysis of the PDHc E1 alpha gene and the PDHX genes revealed no mutations. The patient received continuous feeding through a feeding tube for two years and showed a significant clinical improvement with a complete resolution of the chronic lactic acidemia. A second muscle biopsy revealed significantly decreased pyruvate oxidation rates and ATP production, but a normal pyruvate dehydrogenase complex activity. We suggest that the described mitochondrial dysfunction in our patient is secondary to an underlying mutation leading to Joubert syndrome.

Endotoxin-induced liver damage in rats is minimized by beta 2-adrenoceptor stimulation.

OBJECTIVE AND DESIGN: To investigate the effects of beta(2)-adrenoceptor (beta(2)-AR) stimulation on endotoxin-induced liver damage and systemic cytokine levels in rats. SUBJECTS: Standard male Wistar rats. TREATMENT: A disease-model of lipopolysaccharide (LPS)-induced acute systemic inflammation was used. The beta(2)-selective AR agonist clenbuterol was administered before, during, and after LPS-challenge to investigate its effects on the acute inflammatory response and associated liver-failure. METHODS: The following parameters have been measured in plasma: TNF alpha, IL-1 beta, IL-6, IL-10, AST, ALT, and Bilirubin. Liver histological examination was performed to look for changes in tissue morphology. RESULTS: Administration of clenbuterol (p.o.) one hour before, or intravenous at the same time as LPS-challenge resulted in a marked reduction of plasma levels of TNF alpha, IL-1 beta, and IL-6. A change both in plasma-level and in time-concentration profile of the anti-inflammatory cytokine IL-10 was found. Clenbuterol minimized LPS-induced liver damage, as represented by significantly lowered concentrations of several parameters for liver-failure (AST, ALT, Bilirubin), and improved hepatic tissue morphology. Clenbuterol administration after LPS challenge failed to inhibit TNF alpha-release but reduced liver-damage. Simultaneous use of the beta(2)-AR antagonist propranolol augmented LPS-induced liver failure, suggesting a role of endogenous adrenoceptor-agonists in prevention of organ-failure during systemic inflammation. CONCLUSIONS: The results indicate that a selective beta(2)-AR agonist might be used as an additional therapeutic agent in the clinic for the treatment of (acute) systemic inflammatory disorders in order to reduce or prevent subsequent liver failure.

Biochemical examination of fibroblasts in the diagnosis and research of oxidative phosphorylation (OXPHOS) defects.

The oxidative phosphorylation system (OXPHOS) is organized in five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. A fresh muscle biopsy is preferable to a frozen muscle biopsy because of the possibility to measure the overall capacity of the OXPHOS system. In about 25% of patients referred to our center for muscle biopsy, reduced substrate oxidation rates and ATP + creatine phosphate production rates were found without any defect in complex I-V and the pyruvate dehydrogenase complex. In a subset of patients it is necessary to investigate fibroblasts for diagnostic purposes. The indications for biochemical investigations in fibroblasts are: (a) If no muscle sample is available; (b) If prenatal diagnosis is required; (c) To clarify the results obtained in muscle tissue if no clear-cut diagnosis can be made; (d) If molecular-genetic investigations are required; (e) For research purposes. Fibroblasts are less suitable than fresh muscle for investigating respiratory chain disorders, for the following reasons: (i) A defect that is present in a muscle is not always expressed in fibroblasts. (ii) Exclusion of a defect in fibroblasts does not exclude the diagnosis with regard to muscle. (iii) A specific pattern of abnormalities demonstrated in fibroblasts may not be reflected in muscle tissue. (iv) Enzyme deficiencies found in muscle are generally more pronounced than in fibroblasts. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counseling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.