A.J.M. Janssen

Isolated complex I deficiency in children: clinical, biochemical and genetic aspects.

We retrospectively examined clinical and biochemical characteristics of 27 patients with isolated enzymatic complex I deficiency (established in cultured skin fibroblasts) in whom common pathogenic mtDNA point mutations and major rearrangements were absent. Clinical phenotypes present in this group are Leigh syndrome (n = 7), Leigh‐like syndrome (n = 6), fatal infantile lactic acidosis (n = 3), neonatal cardiomyopathy with lactic acidosis (n = 3), macrocephaly with progressive leukodystrophy (n = 2), and a residual group of unspecified encephalomyopathy (n = 6) subdivided into progressive (n = 4) and stable (n = 2) variants. Isolated complex I deficiency is one of the most frequently observed disturbance of the OXPHOS system. Respiratory chain enzyme assays performed in cultured fibroblasts and skeletal muscle tissue in general reveal similar results, but for complete diagnostics we recommend enzyme measurements performed in at least two different tissues to minimize the possibility of overlooking the enzymatic diagnosis. Lactate levels in blood and CSF and cerebral CT/MRI studies are highly informative, although normal findings do not exclude complex I deficiency. With the discovery of mutations in nuclear encoded complex I subunits, adequate pre‐ and postnatal counseling becomes available. Finally, considering information currently available, isolated complex I deficiency in children seems to be caused in the majority by mutations in nuclear DNA. Hum Mutat 15:123–134, 2000.

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 F1-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.

Some practical aspects of providing a diagnostic service for respiratory chain defects.

The oxidative phosphorylation system (OXPHOS) is organized into 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. Practical guidelines are presented to provide optimal conditions for performance of laboratory investigations and a reliable diagnosis. A fresh muscle biopsy is preferable to a frozen muscle sample because the overall capacity of the OXPHOS system can be measured in a fresh biopsy. In about 25% of patients referred for muscle biopsy to our centre, reduced substrate oxidation rates and ATP+creatine phosphate production rates were found without any defect in complexes I-V and the pyruvate dehydrogenase complex. Investigation of frozen muscle biopsy alone may lead to false-negative diagnoses in many patients. In some patients, it is necessary to investigate fibroblasts for prospective diagnostic purposes. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counselling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.

Clinical and biochemical characteristics in patients with a high mutant load of the mitochondrial T8993G/C mutations.

We retrospectively analyzed the clinical, histological, and biochemical data of 11 children, five of which carried the maternally‐inherited mitochondrial T8993C and six carrying the T8993G point mutations in the ATP synthase 6 gene. The percentage of heteroplasmy was 95% or higher in muscle and in blood. All patients had an early clinical presentation with muscle hypotonia, severe extrapyramidal dysfunction and Leigh disease demonstrated by the cranial MRI. A slower clinical progression and more frequent sensory‐neuronal involvement were noted in the patients carrying the T8993C mutation in a high mutation load in muscle and blood. No histological abnormality was found. In 9 out of 11 patients a decreased ATP production was detected, and complex V activity was deficient in all children. The activities of the respiratory enzyme complexes II and IV were normal, whereas an associated combined complex I and III deficiency were present in two patients. No obvious difference was found between the biochemical parameters of the two patient groups harboring different mutations in the same gene. No correlation was found between the degree of complex V enzyme deficiency and the severity of the phenotype. We confirmed an impaired assembly/stability of complex V in our patients. This is the first report of decreased activity and impaired assembly/stability of complex V in patients with T8993C mutations measured in muscle tissue.

The X-chromosomal NDUFA1 gene of complex I in mitochondrial encephalomyopathies: Tissue expression and mutation detection

electron transport chain consists of four protein enzyme complexes, of which The the NADH:ubiquinone oxidoreductase (complex I) is the largest. Complex I contains at least 41 subunits, 7 of which are encoded by the mitochondrial DNA (ND16 and ND4L) ; nuclear genes encode the remainder (HateÐ 1985 ; Walker 1992 ; Complex I catalyses the transfer of electrons from NADH to ubiRobinson 1993). quinone, which is coupled to the translocation of protons across the inner mitochondrial membrane. Patients described with a (partial) complex I deÐciency can generally be categorized into two major clinical phenotypes : an isolated myopathy and a multisystem disorder with predominantly encephalopathy. Respiratory chain defects may be inherited as autosomal, or X-linked Mendelian traits et al et al or in the case of certain muta(Orstavik 1993 ; Zhuchenko 1996), tions in mitochondrial DNA as maternal traits. To date, no mutations in a nuclearencoded subunit of complex I have been described. In our biochemically proven complex I-deÐcient patients as well as among the a†ected siblings, (the latter currently not all biochemically evaluated), we observed a strong male preponderance, suggestive of an X-linked inheritance. Recently, the NDUFA1 gene, one of the nuclear-encoded complex I genes, was isolated and mapped to chromosome Xq24 et al The NDUFA1 (Zhuchenko 1996). gene is composed of three exons, and spans about 5.0 kb of genomic DNA. It shows 80% homology with the bovine MWFE subunit of complex I. The knowledge of function of the human NDUFA1, and the bovine MWFE subunit is very limited. The bovine MWFE subunit is thought to be situated in the extrinsic membrane domain of complex I (Walker 1992).

Impaired ubiquitin-proteasome-mediated PGC-1α protein turnover and induced mitochondrial biogenesis secondary to complex-I deficiency.

Most eukaryotic cells depend on mitochondrial OXidative PHOSphorylation (OXPHOS) in their ATP supply. The cellular consequences of OXPHOS defects and the pathophysiological mechanisms in related disorders are incompletely understood. Using a quantitative proteomics approach we provide evidence that a genetic defect of complex‐I of the OXPHOS system may associate with transcriptional derangements of mitochondrial biogenesis through stabilization of the master transcriptional regulator PPARγ co‐activator 1α (PGC‐1α) protein. Chronic oxidative stress suppresses the gene expression of PGC‐1α but concomitant inhibition of the ubiquitin–proteasome system (UPS) can stabilize this co‐activator protein, thereby inducing its downstream metabolic gene expression programs. Thus, mitochondrial biogenesis, which lays at the heart of the homeostatic control of energy metabolism, can be deregulated by secondary impairments of the protein turnover machinery.

The first patient diagnosed with cytochrome c oxidase deficient Leigh syndrome: Progress report

SummaryMutations in SURF1, an assembly gene for cytochrome c oxidase (COX), the fourth complex of the oxidative phosphorylation system, are most frequently encountered in patients with COX deficiency. We describe a patient with Leigh syndrome harbouring a mutation in SURF1 who was reported decades ago with a tissue-specific cytochrome c oxidase deficiency.

Tall stature and progressive overweight in mitochondrial encephalopathy.

We describe two children carrying an inherited T899C mutation in the mitochondrial ATPase 6 gene with mild encephalopathy and normal postnatal growth followed by tall stature and obesity. No familial tall stature, endocrine anomaly or advanced skeletal age were present. Failure to thrive is a characteristic finding in most patients with a mitochondrial disease. Our observations suggest that children with encephalomyopathy, even in the presence of a significant clinical overgrowth, should be screened for a possible defect in oxidative phosphorylation.