R.C.A. Sengers

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.

Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations

Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with ≥ two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh‐like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling. Hum Mutat 15:522–532, 2000.

Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi

Summary: Prenatal diagnosis for respiratory chain deficiencies is a complex procedure that requires a thorough diagnostic work-up of the index patient. This includes confirmation of the clinical and metabolic evaluations through histological and enzymatic examinations of tissue biopsies. Prenatal diagnosis currently relies on biochemical assays of respiratory chain complexes in chorionic villi or amniocytes and is possible by mutation analysis of nuclear genes in a limited but increasing proportion of cases. Based on a recent survey of prenatal diagnosis in families with complex I and complex IV deficiencies, performed at Nijmegen Centre for Mitochondrial Disorders (NCMD), prerequisites and strategies for performing prenatal diagnosis have been developed to increase reliability. Biochemical investigations in chorionic villi can be done reliably if the respiratory chain enzyme deficiency is expressed in both skeletal muscle and skin fibroblasts to rule out tissue specificity. No mitochondrial DNA defects must be suspected or established. The NCMD does not offer prenatal diagnosis until all the prerequisites have been confirmed. We expect prenatal diagnosis at the molecular level to become more feasible in time as the mutational spectrum broadens with advances in medical research.

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).

The human NADH:ubiquinone oxidoreductase NDUFS5 (15kDa) subunit: cDNA cloning, chromosomal localization, tissue distribution and the absence of mutations in isolated complex I-deficient patients

We have cloned the cDNA of the NDUFS5 subunit (15 kDa) of the human mitochondrial respiratory chain complex NADH:ubiquinone oxidoreductase (complex I). The open reading frame consists of 321 base-pairs, coding for 106 amino acids, with a calculated molecular mass of 12.5 kDa. There is an 81.0% identity with the bovine equivalent on cDNA level and 74.5% identity on amino acid basis. PCR analysis of rodent–human somatic cell hybrids revealed that the human NDUFS5 gene maps to chromosome 1. The NDUFS5 mRNA is expressed ubiquitously in human tissues, with a relative higher expression in human heart, skeletal muscle, liver, kidney and fetal heart. A mutation detection study of twenty isolated enzymatic complex I-deficient patients revealed no mutations, nor polymorphisms.

Oxidative Phosphorylation in Health and Disease

CONTENTS Foreword 1.The Human OXPHOS System: Structure,Function and Physiology Immo E.Scheffler.- Complexes of the Electron Transport Chain.- The ATP Synthase.- Regulation of Oxidative Phosphorylation.- Assembly of Electron Transport Complexes.- 2.Molecular Biology of the OXPHOS System Richard C.S carpulla.- mtDNA.- Mitochondrial Inheritance.- Replication,Transcription,RNA Processing.- Recombination and Repair.- Mitochondrial Translation System.- Bi-Genomic Expression of the Respiratory Chain.- 3.Clinical Diagnosis of Oxidative Phosphorylation Disorders Robert McFarland, Patrick F.Chinnery, Robert W.Taylor, Andrew M.Schaefer and Douglass M.Turnbull.- Epidemiology of Defects of Mitochondrial Oxidation.- Clinical Features of Patients with Defects of Mitochondrial Oxidation.- Investigation of Suspected Mitochondrial Disease.- 4.Contribution of Histopathological Examination to the Diagnosis of OXPHOS Disorders Martin Lammens and Henk ter Laak.- Muscle Biopsy Diagnosis.- Morphological Hallmarks for Diagnosis of OXPHOS Disorders.- Mitochondrial Changes in Muscle Biopsies without OXPHOS Disorder.- Muscle Biopsy in OXPHOS Disorders.- Pathological Findings in Other Organs.- 5.Biochemical Diagnosis of OXPHOS Disorders J.M.Frans Trijbels, Antoon J.M Janssen, Lambert P.van den Heuvel, Rob C.A.Sengers and Jan A.M.Smeitink.- Examination of Body Fluids.- Examination of Tissues.- Biochemical Diagnostic Investigations.- Frozen Muscle Samples.- Complex IV (Cytochrome c Oxidase).- Complex V.- Practical Guidelines for Biochemical Examinations of Muscle.- Investigation of Fibroblasts.- Residual Enzyme Activity.- 6.Mitochondrial DNA and OXPHOS Disorders Massimo Zeviani and Valerio Carelli.- General Background.- Mitochondrial Genetics.- Sequence and Gene Organization of mtDNA.- Mitochondrial Disorders Due to Mutations of mtDNA.- Mutations of mtDNA.- Large-Scale Rearrangements.- Point Mutations.- Heteroplasmic Point Mutations.- Other Syndromes.- Homoplasmic mtDNAMutations.- Other Homoplasmic Mutations.- Genetic Counseling.- 7.Nuclear DNA and Oxidative Phosphorylation Lambert P.van den Heuvel and Jan A.M.Smeitink.- Biochemistry and Molecular Biology of the OXPHOS System.- Nuclear DNA Mutations.- 8.Cell Biological Consequences of OXPHOS Disorders Werner J.H.Koopman, Henk-Jan Visch, Sjoerd Verkaart and Peter H.G.M.Willems.- Mitochondrial Function in the Living Cell.- Cellular Calcium Signalling.- Cellular Consequences of OXPHOS Deficiency.- 9.Animal Models of OXPHOS Disorders Nicole Hance and Nils-Goran Larsson.- A Drosophila Model of Mitochondrial Deafness.- Mouse Models of Nuclear DNA Mutations.- Manipulation of Mitochondrial Transcription Factor A Expression in Mice.- Transmitochondrial Mouse Models.- Defective Nuclear-Mitochondrial DNA Interactions Resulting in Hearing Loss.- 10.Therapeutic Options in OXPHOS Disorders Rob C.A.Sengers, J.M.Frans Trijbels, Carolien C.A.Boelen, Eva Morava and Jan A.M.Smeitink.- Therapeutic Approaches.- Practical Approaches.- Future Therapies.- Evaluation of Treatment.- 11.Prenatal Diagnostics in Oxidative Phosphorylation Disorders Antoon J.M.Janssen, Letitia E.M.Niers, Lambert P.van den Heuvel, Jan A.M.Smeitink, Rob C.A.Sengers and J.M.Frans Trijbels.- Prerequisites for Offering Prenatal Diagnosis in OXPHOS Disorders.- Tissues to Be Used for Prenatal Diagnosis in OXPHOS Disorders.- Methods for Prenatal Diagnosis in OXPHOS Disorders.- Results of Prenatal Diagnosis for OXPHOS Disorders in Our Center.- General Considerations.- 12.Future Developments in the Laboratory Diagnosis of OXPHOS Disorders David R.Thorburn.- In Vivo Assessment of OXPHOS Function.- Minimally Invasive Tissue Samples.- OXPHOS Function.- OXPHOS Constituents.- OXPHOS Genetics.- Prenatal Diagnosis &Prevention.-Index.

Therapeutic Options in OXPHOS Disorders

No curative treatment of OXPHOS disorders is currently available, despite great progress in our understanding of the molecular bases of these diseases. We review available and experimental therapeutic approaches that fall into the following categories:

Prenatal Diagnostics in Oxidative Phosphorylation Disorders

In this chapter we describe about 18 years of experience with prenatal diagnosis in oxidative phosphorylation (OXPHOS) diseases in our centre. We start diagnostics of OXPHOS disorders in patients with a mitochondrial (encephalo)myopathy by preference by measuring oxidation rates of pyruvate, malate and succinate and ATP production rates from oxidation of pyruvate in a “fresh” muscle biopsy. In the same biopsy activities of the mitochondrial respiratory chain enzymes complex-I, complex-II, complex-III and complex-IV are also measured. When decreased substrate oxidation rates and ATP production rates give indication for suspicion on a complex-V or a pyruvate dehydrogenase complex (PDHC) deficiency, activities of these enzymes are also measured. In frozen muscle biopsies we only can measure the respiratory chain enzymes. In which cases now can we offer prenatal diagnosis? In about 30% of the muscle biopsies with clearly decreased substrate oxidation rates and ATP production rates, all respiratory chain enzymes, complex-V and PDHC show normal activities. In these cases it is impossible at the moment to offer prenatal diagnosis. In the remainder of the biopsies with clearly reduced substrate oxidation- and ATP production rates, decreased activities are measured of one or more of the afore mentioned enzymes. The most frequendy occurring deficiencies in fresh as well as in frozen muscle biopsies are complex-I, complex-IV or combined deficiencies of these enzymes. The next step is to search if the deficiency is also expressed in cultured fibroblasts and to exclude a mtDNA mutation as a cause of the deficiency. If the deficiency is also expressed in cultured fibroblasts and mtDNA mutations have been excluded we are willing to offer prenatal diagnosis. This chapter is aggravated on prenatal diagnosis for complex-I, complex-IV or a combined deficiency of these enzymes because the majority of the total number of requests for prenatal diagnosis that reach us concerns pregnancies in families in which the index patient was suffering from a deficiency of one of these (or both) enzymes.

Biochemical Diagnosis of OXPHOS Disorders

In this chapter the biochemical diagnosis of OXPHOS disorders is presented. The laboratory investigations in suspected patients are started with the examination of body fluids. The most important metabolite to be measured is lactate, that is frequendy found to be elevated in blood, urine and cerebrospinal fluid of patients with OXPHOS disorders. The next step in the diagnostic procedure consists of the examination of tissues. The biochemical diagnostic investigations are preferably performed in muscle tissue because in most patients the defect is expressed in muscle. Biopsy material is preferred above autopsy material. Biochemical examination of a fresh muscle sample is to be preferred because mitochondria are intact in fresh muscle thus allowing measurement of the overall oxidative capacity of the mitochondria. In a frozen muscle sample only enzyme activities of the OXPHOS complexes can be measured. In the latter case patients with a disturbance in the oxidative phosphorylation not localized in one of the OXPHOS complexes remain undiagnosed. Practical guidelines for the biochemical examinations of muscle are provided. In certain circumstances it is necessary to examine also fibroblasts. This is an absolute prerequisite in case prenatal diagnosis is requested. The interpre-tation of the biochemical investigations is discussed with special emphasis on the observed residual enzyme activities.