Objective: Mitochondrial disorders can present with a wide variety of clinical signs and symptoms and the number of genes known to be envolved is still increasing.

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.

A 13-year-old girl with non-familial exercise intolerance, muscle pain and lactic acidaemia underwent a muscle biopsy for suspected mitochondrial disease. Muscle morphology showed 25% ragged-red fibres and 80% COX-negative staining. Enzymatic activities of mitochondrially co-encoded respiratory chain enzymes (complexes I, III, and IV) were decreased in muscle but normal in cultured skin fibroblasts. mtDNA analysis revealed the presence of the 7497G>A mutation in the tRNASer(UCN) gene, homoplasmic in skeletal muscle and 90% in leukocytes. Analysis of the mother's mtDNA showed 10% heteroplasmy in blood. It may be concluded that the 7497G>A mutation is associated with a muscle-only disease presentation for which high levels of mutated mtDNA are required. Exercise intolerance and muscle pain in otherwise normal children warrants further mitochondrial evaluation.

We describe a boy presenting at the end of the first year of life with severely delayed motor development and only mild mental retardation. Neurological examination revealed axial hypotonia, mild ataxia and pyramidal signs. Elevated lactate and protein in cerebrospinal fluid were the most prominent laboratory abnormalities. Brain MRI showed severe supratentorial white matter changes. Cerebellar white matter appeared normal whereas the signal of the atrophic cerebellar cortex was markedly increased. In vivo 1H-magnetic resonance spectroscopy of the parietooccipital white matter region showed a distinct resonance of lactate. By means of biochemical analysis of respiratory chain enzymes in fibroblasts, the diagnosis of an isolated complex I deficiency could be established in our patient.

A 31-year-old woman had encephalopathy, growth retardation, infantilism, ataxia, deafness, lactic acidosis, and increased signals of caudate and putamen on brain magnetic resonance imaging. Muscle biochemistry showed succinate:cytochrome c oxidoreductase (complex II-III) deficiency. Both clinical and biochemical abnormalities improved remarkably with coenzyme Q10 supplementation. Clinically, when taking 300mg coenzyme Q10 per day, she resumed walking, gained weight, underwent puberty, and grew 20cm between 24 and 29 years of age. Coenzyme Q10 was markedly decreased in cerebrospinal fluid, muscle, lymphoblasts, and fibroblasts, suggesting the diagnosis of primary coenzyme Q10 deficiency. An older sister has similar clinical course and biochemical abnormalities. These findings suggest that coenzyme Q10 deficiency can present as adult Leigh's syndrome.

A frequent etiology of congenital lactic acidosis is disturbed mitochondrial energy metabolism. Affected children generally present with neurologic symptoms, such as myopathy and epilepsy. Parents who have lost a child to mitochondrial disease often ask for prenatal diagnosis in subsequent pregnancies. The large number of possible mitochondrial or nuclear DNA mutations often makes the molecular defect unknown. In these cases, prenatal diagnosis rests solely on biochemical analysis. Here we report a possible pitfall in prenatal diagnosis of mitochondriopathies by biochemical methods that might occur despite all precautions. It is illustrated by a patient with isolated mitochondrial complex I deficiency and her family in the light of a new mutation (632C→T) in 1 of the 36 nuclear encoded genes of complex I ( NDUFV1 ). The girl (II.1 in Fig. 1A⇓ ) was the first child of healthy Caucasian first-degree cousins. Postnatally she showed acrocyanosis, muscular hypotonia, and a pendular nystagmus. Fundoscopy revealed bitemporal retinal depigmentation. The latencies of the visual evoked potentials were pathologically increased. Lactic acidosis (pH 7.19) was noted, with a plasma lactate concentration of 24.1 mmol/L (reference interval, 0.5–2.2 mmol/L), a lactate-to-pyruvate ratio of 57 (reference values <20), plasma alanine of 893 μmol/L (reference interval, 40–500 μmol/L), urine α-ketoglutaric acid of 1852 mmol/mol creatinine (reference interval, 159 ± 137 mmol/mol creatinine), urine lactate of 1713 mmol/mol creatinine (reference interval, 234 ± 165 mmol/mol creatinine), and cerebrospinal fluid lactate of 9.6 mmol/L (reference values <2 mmol/L). Cranial ultrasound and magnetic resonance imaging results were normal. Muscle histology revealed intracytoplasmic accumulation of glycogen. Mitochondria were ultrastructurally normal on electron microscopy. We measured the respiratory chain complex I, II+III, and IV activities in a fresh muscle biopsy specimen and in cultured fibroblasts according to standard procedures (1)(see the data supplement available with the online version of this Technical Brief, at …

We report on a 25-year-old patient with isolated mitochondrial complex III deficiency and a new heteroplasmic mutation (T14849C) in the cytochrome b gene. He suffered from septo-optic dysplasia, retinitis pigmentosa, exercise intolerance, hypertrophic cardiomyopathy, and rhabdomyolysis. A HESX1 mutation was excluded as a cause of his septo-optic dysplasia. Low alpha-tocopherol concentrations in his muscles and an elevated urinary leukotriene E(4) excretion indicate increased production of reactive oxygen species.

Human complex I is built up and regulated by genes encoded by the mitochondrial DNA (mtDNA) as well as the nuclear DNA (nDNA). In recent years, attention mainly focused on the relation between complex I deficiency and mtDNA mutations. However, a high percentage of consanguinity and an autosomal-recessive mode of inheritance observed within our patient group as well as the absence of common mtDNA mutations make a nuclear genetic cause likely. The NDUFS2 protein is part of complex I of many pro- and eukaryotes. The nuclear gene coding for this protein is therefore an important candidate for mutational detection studies in enzymatic complex I deficient patients. Screening of patient NDUFS2 cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with direct DNA sequencing revealed three missense mutations resulting in the substitution of conserved amino acids in three families.

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.

BACKGROUND Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

We present the cDNA sequence of the human mitochondrial acyl carrier protein NDUFAB1, a nuclear-encoded subunit of complex I of the mitochondrial respiratory chain. We obtained the NDUFAB1 cDNA using the cDNA sequence of the bovine mitochondrial acyl carrier protein. The human cDNA contains two putative translation initiation codons. The human NDUFAB1 protein contains a phosphopantetheine attachment site (DLGLDSLDQVEIIMAM), unique for acyl carrier proteins, and an EF-hand calcium binding domain (DIDAEKLMCPQEI). Transcripts of this gene are found in a wide range of human tissues. The highest expression levels were observed, in descending order, in adult heart, skeletal muscle and fetal heart. We subjected NDUFAB1 fibroblast cDNA of 20 patients with an isolated enzymatic complex I deficiency to mutational detection. No mutations in the NDUFAB1 open reading frame were observed. Future studies will answer whether mutations in the NDUFAB1 promoter or transcription elements are responsible for the observed complex I deficiency.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.

During the last two decades we performed biochemical investigations in more than one thousand muscle biopsies obtained from patients suspected to suffer from a mitochondrial (encephalo)myopathy. In several hundreds of them evidence was obtained for the presence of a disturbance in the mitochondrial energy generating system. Muscle samples from these patients clearly exhibited reduced oxidation rates of substrates, like pyruvate, malate and succinate. Besides, production rates of ATP and CrP from these substrates were reduced too. In nearly 75% of these patients measurement of the activities of the complexes I–IV of the respiratory chain and of the pyruvate dehydrogenase complex, revealed a deficiency of one or more of these complexes to a highly varying degree. However, in the remaining 25% of these patients no clear-cut defect in the aforementioned complexes could be established. From this observation it can be concluded that other defects must account for the established defect in the mitochondrial energy generation in muscle. Earlier we focussed our attention on the possible deficiency of mitochondrial creatine kinase as a cause of the disturbed energy production. However, investigation of more than one hundred of muscle samples with such a disturbed oxidative capacity of mitochondria did not reveal a patient with a MiCK deficiency. Another so-called post-respiratory chain enzyme which might be involved in the pathogenesis of mitochondrial myopathies concerns mitochondrial ATPase (complex V). At present only a few patients with an ATPase deficiency have been described. These observations prompted us to look at other possible causes of a disturbed mitochondrial energy generation. We speculated that malfunctioning of transporting systems in the mitochondrial inner or outer membrane might be the primary cause of the biochemical aberrations in some of these patients. At the moment at least 15 mitochondrial carriers have been identified. Some of these carriers are present only in a limited number of tissues. In this presentation special attention will be paid to those carriers which are directly involved in the process of oxidative phosphorylation. Among these the ATP/ADP translocator (ANT), the phosphate carrier (PiC), the pyruvate carrier and the 2-oxoglutarate carrier will be considered. Besides, alterations of the voltage-dependent anion channel (VDAC) and the malate-aspartate shuttle will be discussed as a possible cause of mitochondrial myopathies.

We report the cDNA cloning, chromosomal localization, and a mutation in the human nuclear gene encoding the 18-kD (AQDQ) subunit of the mitochondrial respiratory chain complex I. The cDNA has an open reading frame of 175 amino acids and codes for a protein with a molecular mass of 23.2 kD. Its gene was mapped to chromosome 5. A homozygous 5-bp duplication, destroying a consensus phosphorylation site, in the 18-kD cDNA was found in a complex I-deficient patient. The patient showed normal muscle morphology and a remarkably nonspecific fatal progressive phenotype without increased lactate concentrations in body fluids. The child's parents were heterozygous for the mutation. In 19 other complex I-deficient patients, no mutations were found in the 18-kD gene.

Journal of Inherited Metabolic DiseaseVolume 21, Issue 3 p. 210-215 Article The X-chromosomal NDUFA1 gene of complex I in mitochondrial encephalomyopathies: Tissue expression and mutation detection J. Loeffen, J. LoeffenSearch for more papers by this authorR. Smeets, R. SmeetsSearch for more papers by this authorJ. Smeitink, J. SmeitinkSearch for more papers by this authorW. Ruitenbeek, W. RuitenbeekSearch for more papers by this authorA. Janssen, A. JanssenSearch for more papers by this authorE. Mariman, E. MarimanSearch for more papers by this authorR. Sengers, R. SengersSearch for more papers by this authorF. Trijbels, F. TrijbelsSearch for more papers by this authorL. van den Heuvel, L. van den HeuvelSearch for more papers by this author J. Loeffen, J. LoeffenSearch for more papers by this authorR. Smeets, R. SmeetsSearch for more papers by this authorJ. Smeitink, J. SmeitinkSearch for more papers by this authorW. Ruitenbeek, W. RuitenbeekSearch for more papers by this authorA. Janssen, A. JanssenSearch for more papers by this authorE. Mariman, E. MarimanSearch for more papers by this authorR. Sengers, R. SengersSearch for more papers by this authorF. Trijbels, F. TrijbelsSearch for more papers by this authorL. van den Heuvel, L. van den HeuvelSearch for more papers by this author First published: 01 June 1998 https://doi.org/10.1023/A:1005339332062Citations: 10AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1Ali ST, Duncan AMV, Schappert K et al (1993) Chromosomal localisation of the human gene encoding the 51–kDa subunit of mitochondrial complex I (NDUFV1) to 11q13. Genomics, 18: 435–439. 2Bentlage HACM, Wendel U, Schagger H (1996) Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts, but with combined complex I, and IV deficiencies in muscle. Neurology, 47: 243–248. 3De Coo R, Buddiger P, Smeets H et al (1995) Molecular cloning, and characterization of the active human mitochondrial NADH: ubiquinone oxidoreductase 24 kDa gene (NDUFV2), and its pseudogene. Genomics, 26: 461–466. 4Gu JZ, Lin X, Wells DE (1996) The human B22 subunit of the NADH-ubiquinone oxidoreductase maps to the region of chromosome 8 in Branchio oto-renal syndrome. Genomics, 35: 6–10. 5Hatefi Y (1985) The mitochondrial electron transport, and oxidative phosphorylation system. Annu Rev Biochem, 54: 1015–1069. 6Hattori N, Suzuki H, Wang Y et al (1995) Structural organisation, and chromosomal localization of the human nuclear gene (NDUFV2) for the 24–kDa iron-sulfur subunit of complex I in mitochondrial respiratory chain. Biochem Biophys Res Commun, 216: 771–777. 7Hyslop SJ, Duncan AMV, Pitkanen S, Robinson BH (1996) Assignment of the PSST subunit gene of human mitochondrial complex I to chromosome 19p13. Genomics, 37: 375–380. 8Orstavik KH, Skjorten F, Hellebostad M, Haga P, Langslet A (1993) Possible X-linked congenital mitochondrial cardiomyopathy in three families. J Med Genet, 30: 269–272. 9Pata I, Tensing K, Metspalu A (1997) A human cDNA encoding the homologue of NADH: ubiquinone oxidoreductase subunit B13. Biochim Biophys Acta, 1350 (2): 115–118. 10Ploos van Amstel JK, Bergman AJ, Beurdenvan EA et al (1996) Hereditary tyrosinemia type 1: novel missense, nonsense, and splice consensus mutations in the human fumarylacetoacetate hydrolase gene: variability of the genotype—phenotype relationship. Hum Genet, 97: 51–59. 11Procaccio V, Depetris D, Soularue P, Matei MG, Lunardi J, Issartel J (1997) cDNA sequence, and chromosomal localisation of the NDUFS8 human gene coding for the 23 kDa subunit of the mitochondrial complex I. Biochim Biophys Acta, 1351: 37–41. 12Robinson BH (1993) Lacticacidemia. Biochim Biophys Acta, 1182: 231–244. 13(1989) J Sambrook, EF Fritsch, I Maniatis, eds. Molecular Cloning: A L aboratory Manual, 2nd edn.. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 14Walker JE (1992) The NADH: ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys, 25: 253–324. 15Zeviani M, Taroni F (1994) Mitochondrial diseases. Baillière's Clin Neurol, 3 (2): 315–334. 16Zhuchenko O, Wehnert M, Baily J, Sun ZS, Lee CC (1996) Isolation, mapping, and genomic structure of an X-linked gene for a subunit of human mitochondrial complex I. Genomics, 37: 281–288. Citing Literature Volume21, Issue3June 1998Pages 210-215 ReferencesRelatedInformation

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain can be fragmented in a flavoprotein (FP), iron-sulfur protein (IP), and hydrophobic protein (HP) subfraction. The IP subfraction is hypothesized to be significant, since it contains important prosthetic groups highly conserved among species. We cloned the cDNA of three remaining human NADH:ubiquinone oxidoreductase subunits of this IP fraction: the NDUFS2 (49 kDa), NDUFS3 (30 kDa), and NDUFS6 (13 kDa) subunits. All presented cDNAs include the complete open reading frame (ORF), which consist of 1392, 795, and 375 base pairs, coding for 463, 264, and 124 amino acids, respectively. The latter show 96, 90, and 83% homology with the corresponding bovine translation products. The 3′ untranslated regions (UTR) are complete in all three cDNAs. Polymerase chain reaction performed with DNA isolated from somatic human–rodent cell hybrids containing defined human chromosomes as template gave a human-specific signal which mapped the NDUFS2 and NDUFS3 subunits to chromosomes 1 and 11, respectively. In the case of the NDUFS6 subunit a pseudogene may be present since signals were seen in the lanes containing chromosomes 5 and 6. The NDUFS2 contains a highly conserved protein kinase C phosphorylation site and the NDUFS3 subunit contains a highly conserved casein kinase II phosphorylation site which make them strong candidates for future mutation detection studies in enzymatic complex I-deficient patients.

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The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC in various human tissues. Furthermore, mutation analysis was performed for a CAC-deficient infant. Direct sequencing of the patient's cDNA revealed a homozygous cytosine nucleotide insertion. This insertion provokes a frameshift and an extension of the open reading frame with 23 novel codons. This is the first report documenting a mutation, in the CAC cDNA, responsible for mitochondrial beta-oxidation impairment.