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Functional Characterization of Novel Mutations in the Human Cytochrome B Gene

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Functional Characterization of Novel Mutations in the Human Cytochrome B Gene European Journal of Human Genetics (2001) 9, 510 ± 518 ã 2001 Nature Publishing Group All rights reserved 1018-4813/01 $15.00 www.nature.com/ejhg ARTICLE Functional characterization of novel mutations in the human cytochrome b gene FreÂdeÂric Legros1, Evi Chatzoglou1, Paule Frachon1,HeÂleÁne Ogier de Baulny3, Pascal LaforeÃt1, Claude Jardel2, Catherine Godinot4 and Anne LombeÁs*,1 1INSERM U523, Institut de Myologie, HoÃpital de la SalpeÃtrieÁre, 75651 Paris cedex 13, France; 2Biochimie B, HoÃpital de La SalpeÃtrieÁre, 75651 Paris cedex 13, France; 3Service de Neurologie, HoÃpital Robert DebreÂ, 75019 Paris; 4UMR5534 CGMC Universite Cl Bernard, Lyon I, France The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect. European Journal of Human Genetics (2001) 9, 510 ± 518. Keywords: Mitochondrial DNA; cytochrome b; complex III; human; mitochondrial diseases Introduction disease. Direct analysis of the very numerous candidate The human mitochondrial oxidative phosphorylation path- nuclear genes is not possible. On the other hand, the high way consists of 13 subunits encoded by mitochondrial DNA level of spontaneous mtDNA mutations2 is responsible for a (mtDNA) and at least 73 encoded by nuclear DNA. In wide diversity in the sequence of the mtDNA genes3,4 that addition, a very large number of nuclear genes are also renders difficult the functional evaluation of sequence indirectly involved in the respiratory chain function: variations in patients. chaperons, assembly factors, or enzymes involved in cofactor Ubiquinol : cytochrome c oxidoreductase (complex III or 1 metabolism. The first step in genetic investigations of cytochrome bc1 complex, EC 1.10.2.2) is the simplest human mitochondrial disorders is to determine which example of a respiratory complex with a double genetic genome, nuclear or mitochondrial, is responsible for the origin as it is made up of 11 subunits, only one of which, the cytochrome b, is encoded by the mtDNA. Cytochrome b forms, with cytochrome c1 and the Rieske iron-sulphur protein, the catalytic core of the cytochrome bc1 complex. *Correspondence: A LombeÁs, INSERM U523, Institut de Myologie, Significant changes in its amino acid sequence have HoÃpital de La SalpeÃtrieÁre, 75651 Paris cedex 13; France; E-mail: [email protected]. therefore a high probability to induce significant alteration Received 9 February 2001; revised 26 April 2001; accepted 2 May 2001 of the catalytic function of complex III. Defects of the Novel cytochrome b mutations F Legros et al 511 cytochrome bc1 complex are however rarely encountered in Patients and methods human mitochondrial disorders. They represented 2% of Patients the respiratory chain alterations in the patients from All investigations were performed after the patient or the Nijmegen (approximately 300)5 and 7% in the 157 patients parents of the patient had given written informed consent from Paris (Necker hospital).6 We have found a similar according to the rules in our Institution. The cytochrome b frequency (4%) as, over the past 7 years, we have diagnosed gene was analysed in an affected tissue from the patients and seven cases with isolated complex III in more than 160 in leukocytes from the controls. The ethnical background of patients with respiratory chain defects (Dr A LombeÁs, the patients and the controls is very diverse reflecting the unpublished results). cosmopolitan nature of the Paris urban area. Contrasting with the wealth of information provided A first group of seven patients had an isolated cytochrome by the analysis of cytochrome b mutants in yeast and bc1 complex defect. The patients' age at the onset of the bacteria,7±10 rare alterations of the human cytochrome b disease varied from birth to adolescence. The spectrum of gene have been associated with an overt isolated complex III affected organs was diverse: liver only (one patient), muscle defect5,11 ± 17 and their molecular consequences have only only (two patients) or multisystemic (four patients with brain been analysed in three patients.11,15,17,18 All these mutations involvement). Four patients had consanguineous parents were heteroplasmic ie they coexisted with a residual amount and two of them had similarly affected siblings. All patients of wild-type mtDNA. Half of them led to a truncated had a significant and isolated complex III defect in muscle cytochrome b protein. The deleterious potential of the other (five patients) or liver (two patients). The defect could be mutations has been ascertained on their absence in controls, demonstrated in both muscle and skin fibroblasts in two on their skewed tissue distribution in patients and/or on patients with a generalised disease. the phylogenetic conservation of the modified amino acid. A second group of 14 patients had a mitochondrial disorder On the other hand, homoplasmic mutations of the human without an isolated complex III defect. They were included in cytochrome b gene, ie mutations present in 100% of the study because a genetic alteration of mtDNA was the mtDNA molecules, have been considered deleterious considered to be a likely cause of their disease for several of but they have not been associated with a defective com- the following reasons: they had a typical mitochondrial plex III and their pathogenicity was only deduced from accumulation in muscle with ragged-red fibres (seven indirect criteria such as the phylogenetic conservation of the patients); analyses of their respiratory chain disclosed mild modified amino acid and control population screening.19 ± 21 and combined alterations of the respiratory complexes (eight In addition, numerous silent cytochrome b sequence variants patients); similarly affected relatives were noted in the family have been described and added as polymorphisms to the of seven patients but the familial pedigrees were not MITOMAP database of human mtDNA.4 Their identification sufficient to assess the mode of inheritance. The patients' as polymorphisms may however be considered disputable as phenotypes were diverse in the age at onset of the disease they were often described in patients referred for a suspicion (from birth to 40 years of age) and in the spectrum of affected of a mitochondrial disease and who may have had variable organs: muscle was affected in 11/14 patients and the disease defect of their complex III activity.22 was generalised in 9/14 patients. In order to evaluate the role of the cytochrome b gene in The control group comprised 146 healthy adult subjects patients with a mitochondrial disease, we undertook the who volunteered to participate in the genetic analysis of analysis of the human cytochrome b gene in 21 patients, autosomal dominant diseases. All these subjects were deemed seven with an overt isolated complex III defect and 14 to be free of any abnormal symptoms. without this enzymatic defect but with a disease pointing to an mtDNA dysfunction.23 We also determined the Mitochondrial DNA analyses sequence variability of the cytochrome b gene in a group Total DNA was extracted from muscle, liver, blood or cultured of 146 controls. Numerous mutations, several of which skin fibroblasts and Southern blot analysis was performed on were new mutations, were found both in the patients and muscle and liver total DNA as described by Moraes et al.24 the control group, thus demonstrating the high variability of the human cytochrome b sequence. Evaluation of the Cytochrome b gene analysis The patients' cytochrome b functional relevance of the sequence variants was first gene was sequenced in two overlapping fragments amplified based on indirect criteria such as the nature of the by PCR (Table 1). Direct sequencing of the PCR fragments was mutation, its frequency in controls, or the phylogenetic performed using dye-terminator cycling sequencing on an conservation of the mutated amino acid. When appro- automated sequencer according to the manufacturer (Abi priate, the mtDNA haplotype, the heteroplasmic state of Prism 377 from Perkin-Elmer Applied Biosystems). the mutation,
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