J.A.M. Smeitink

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.

Mitochondrial disease criteria Diagnostic applications in children

Background: Based on a previous prospective clinical and biochemical study, a consensus mitochondrial disease scoring system was established to facilitate the diagnosis in patients with a suspected mitochondrial disorder. Objective: To evaluate the specificity of the diagnostic system, we applied the mitochondrial disease score in 61 children with a multisystem disease and a suspected oxidative phosphorylation disorder who underwent a muscle biopsy and were consecutively diagnosed with a genetic mutation. Methods: We evaluated data of 44 children diagnosed with a disorder in oxidative phosphorylation, carrying a mutation in the mitochondrial or nuclear DNA. We compared them with 17 children who, based on the clinical and metabolic features, also had a muscle biopsy but were finally diagnosed with a nonmitochondrial multisystem disorder by further genetic analysis. Results: All children with a genetically established diagnosis of a primary oxidative phosphorylation disorder had a mitochondrial disease score above 6 (probable mitochondrial disorder), and 73% of the children had a score above 8 (definite mitochondrial disorder) at evaluation of the muscle biopsy. In the nonmitochondrial multisystem disorder group, the score was significantly lower, and no patients reached a score comparable with a definite respiratory chain disorder. Conclusions: The mitochondrial disease criteria system has a high specificity to distinguish between mitochondrial and other multisystem disorders. The method could also be applied in children with a suspected mitochondrial disorder, prior to performing a muscle biopsy.

A common point mutation in the tyrosine hydroxylase gene in autosomal recessive L-DOPA-responsive dystonia in the Dutch population.

Abstract This report concerns one new mutation in the tyrosine hydroxylase (TH) gene in three patients originating from three unrelated Dutch families with autosomal recessive L-DOPA-responsive dystonia (DRD). In this study, all exons of the TH gene were amplified by the polymerase chain reaction and subjected to analyses by single-strand conformation polymorphism. An aberrant migration pattern was observed for exon 6 of the TH gene in all patients. Direct sequencing of the coding region of exon 6 revealed the presence of one novel missense mutation. An a698g transition resulted in the substitution of the evolutionary conserved arginine 233 by a histidine (R233H). All patients were homozygous for the mutation. This new mutation in the TH gene was confirmed by restriction enzyme analysis with the restriction enzyme HhaI. Thus, a high proportion of defective TH alleles may be R233H in The Netherlands.

Protein Complexes in the Archaeon Methanothermobacter thermautotrophicus Analyzed by Blue Native/SDS-PAGE and Mass Spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Human mitochondrial transmembrane metabolite carriers: Tissue distribution and its implication for mitochondrial disorders

Mitochondrial transmembrane carrier deficiencies are a recently discovered group of disorders, belonging to the so-called mitochondriocytopathies. We examined the human tissue distribution of carriers which are involved in the process of oxidative phosphorylation (adenine nucleotide translocator, phosphate carrier, and voltage-dependent anion channel) and some mitochondrial substrate carriers (2-oxoglutarate carrier, carnitine-acylcarnitine carrier, and citrate carrier). The tissue distribution on mRNA level of mitochondrial transport proteins appears to be roughly in correlation with the dependence of these tissues on mitochondrial energy production capacity. In general the main mRNA expression of carriers involved in mitochondrial energy metabolism occurs in skeletal muscle and heart. Expression in liver and pancreas differs between carriers. Expression in brain, placenta, lung, and kidney is lower than in the other tissues. Western and Northern blotting experiments show a comparable HVDAC1 protein and mRNA distribution for the tested tissues. Patients studies showed that cultured skin fibroblasts may not be a reliable alternative for skeletal muscle in screening for human mitochondrial carrier defects.

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.

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.

Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I

Summary: A comparison of the clinical presentation, disease course and results of laboratory and imaging studies of all patients so far published with a NDUFS4 mutation are presented. This reveals marked clinical heterogeneity, even in patients with the same genotype.

Complex I assembly: a puzzling problem

Purpose of reviewDisturbances in the mitochondrial oxidative phosphorylation pathway most often lead to devastating disorders with a fatal outcome. Of these, complex I deficiency is the most frequently encountered. Recent characterization of the mitochondrial and nuclear DNA-encoded complex I subunits has allowed mutational analysis and reliable prenatal diagnosis. Nevertheless, complex-I-deficient patients without a mutation in any of the known subunits remain. It is assumed that these patients harbour defects in proteins involved in the assembly of this largest member of the oxidative phosphorylation complexes. This review describes current understanding of complex I assembly, new developments and future perspectives. Recent findingsThe first model of human complex I assembly has been proposed recently. New insights into supercomplex assembly and stability may help to explain combined deficiencies. Recent functional characterization of some of the 32 accessory subunits of the complex may link these subunits to complex I biogenesis and activity regulation. SummaryResearch on complex I assembly is increasing rapidly. However, comparison between theoretical and experimental models of complex I assembly is still problematic. The growing understanding of complex I assembly at the subunit and supercomplex level will clarify the picture in the future. The elucidation of complex I assembly, by combining patient data with new experimental methods, will facilitate the diagnosis of (and possibly therapy for) many uncharacterized mitochondrial disorders.

l-dopa–responsive infantile hypokinetic rigid parkinsonism due to tyrosine hydroxylase deficiency

Article abstract Tyrosine hydroxylase deficiency was confirmed biochemically and genetically in four unrelated Dutch patients. The patients have a hypokinetic-rigid parkinsonian syndrome with symptoms in early infancy (3 to 6 months of age). Only sporadic dystonic movements were seen. There was no diurnal fluctuation. All patients showed a rapid favorable response to low-dose l-dopa/carbidopa treatment. Motor performance improved but did not fully normalize. The patients have mild mental retardation.