Human Molecular Genetics, 2002, Vol. 11, No. 16 1797-1805
© 2002 Oxford University Press
Metabolic consequences of a novel missense mutation of the mtDNA CO I gene
1Department of Epileptology, 2Department of Neurology and 3Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany, 4Department of Neurology, University of Magdeburg Medical Center, Leipziger Strasse 44, D-39120 Magdeburg, Germany, 5Institute of Clinical Chemistry and Pathobiochemistry, Medical Center of RWTH Aachen, Pauwelsstrasse 30, D-52072 Aachen, Germany and 6Institute of Human Genetics, Wilhelmstrasse 31, D-53111 Bonn, Germany
Received February 19, 2002; Accepted June 6, 2002
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ABSTRACT |
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We have identified a novel heteroplasmic C6489A missense mutation in the mitochondrial DNA (mtDNA) CO I gene encoding the cytochrome c oxidase (COX) subunit I in a 17-year-old girl with epilepsia partialis continua. This point mutation leads to an exchange of the highly conserved Leu196 to Ileu196. Muscle biopsy showed in single fibers decreased COX activity and lowered binding of COX antibodies, indicating decreased stability of the mutated enzyme. The analysis of blood mtDNA revealed about 30% mutant mtDNA in the patients blood but about 90% mutant mtDNA in the blood of two non-affected family members. Quantitative analysis of the mutation gene dose effect on COX activity on single muscle fiber level revealed a very high threshold—a COX deficiency was observed only in fibers containing >95% mutant mtDNA. In apparent contrast to this high mutation gene dose threshold, in vivo investigations of mitochondrial function in saponin-permeabilized muscle fibers of the index patient containing
90% mutated mtDNA showed decreased maximal rates of respiration and an increased sensitivity of fiber respiration to cyanide. This is due to a 2-fold increase of COX flux control on muscle fiber respiration and a 30% decrease of COX metabolic threshold, supporting the concept of tight COX control of oxidative phosphorylation in skeletal muscle. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Isolated deficiencies of cytochrome c oxidase (COX) are the most common respiratory chain defects in childhood with the clinically heterogeneous phenotypes of Leigh syndrome or fatal and benign infantile myopathies (1,2). For the majority of these patients, COX deficiency has being associated with mutations in several nuclear assembly genes of the enzyme: SURF1, SCO2 and COX10 (3). In very few patients, mutations in the mitochondrial COX genes have been identified so far: two stop codon mutations in CO I (4,5), an initiation codon mutation (6) and a missense mutation (7) in CO II, and different mutation types in CO III (8–11), whereas no mutations are known in nuclear genes encoding COX subunits. Here we describe a patient with epilepsia partialis continua in whom we identified a novel heteroplasmic mitochondrial DNA (mtDNA) missense mutation in the CO I gene causing COX deficiency. Since mitochondrial disorders with epileptic phenotypes have been previously associated exclusively with mitochondrial tRNA mutations (12), this is the first case of a therapy-resistant epilepsy syndrome associated with a mutation in a structural COX gene. This mutation showed a peculiar tissue distribution in the index patient and the non-affected relatives. In vivo investigations of mitochondrial function applying saponin-permeabilized muscle fibers indicate that high loads of this mutation, which affect COX protein levels, cause severe alterations of energy metabolism in tissues with high oxidative capacity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Morphological and biochemical studies
In the histology of the patient's muscle shown in Figure 1, COX-negative fibers were observed (asterisks in Fig. 1C). The COX-negative fibers had either normal or slightly diminished succinate dehydrogenase activity (A). Succinate dehydrogenase hyperreactive fibers or typical ‘ragged red’ fibers (Fig. 1B) were absent. Immunostaining of frozen muscle sections demonstrated almost complete absence of COX subunit I (Fig. 1E) and subunit II (Fig. 1D) protein in the COX-negative fibers. COX subunit IV protein was decreased but not completely absent (note the decreased punctuated pattern of the COX-negative fibers in Fig. 1F).
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In accordance with the decreased histological COX staining, we detected in the muscle homogenate an
40% lower total activity of cytochrome c oxidase. If the lowered activity of citrate synthase is additionally considered, then 30% lower specific COX activity was observed (Table 1). The activity of NADH : CoQ oxidoreductase (complex I of the respiratory chain) was not different from controls. Accordingly, the respiration of saponin-permeabilized muscle fibers with the COX-specific substrate combination TMPD+ascorbate was 40% decreased (Table 1). Using determinations of the cytochrome content, we observed in the muscle sample of the patient a decreased content of heme aa3 at an unchanged maximal turnover rate of COX.
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mtDNA studies
We excluded the presence of any known epilepsy-associated point mutations in the tRNA genes for lysine (13) and serine (14) and the presence of large-scale mtDNA rearrangements in the total genomic DNA extracted from the patient's muscle (data not shown). Direct sequence analysis revealed a homoplasmic C7028T transition as well as a 9 bp deletion including np8270–np8278. However, since these variants were previously found at polymorphic frequencies in the general population (MITOMAP: http://www.gen.emory.edu/mitomap.html), a disease-related role of these is most likely ruled out. Additionally, we identified a heteroplasmic mitochondrial C6489A point mutation both in DNA extracted from skeletal muscle and in DNA from peripheral blood cells (Fig. 2A). Since mtDNA mutations associated with diseases are usually found as a mixture of mutant and wild-type DNA, we quantified the degree of mtDNA heteroplasmy in skeletal muscle and blood obtained from the patient as well as in the blood samples obtained from her relatives. Results of a PCR-based restriction fragment length polymorphism (RFLP) analysis are presented in Figure 2B and C. In summary, we excluded the presence of the mtDNA C6489A mutation in the patient's father, while in her mother, we found a ratio of mutant to wild-type mtDNA of
90 : 10 (blood mtDNA). In the patient herself, we detected 29% mutant mtDNA in blood samples but 90% mutant mtDNA in skeletal muscle specimens. To determine the tissue distribution of the mutation in the non-affected mother and brother of the patient, we analyzed cellular subpopulations of blood samples (Table 2). We observed for both individuals a comparable distribution pattern of the mutation: the highest mutation load in platelets (95–96% mutant mtDNA), an intermediate mutation load in the granulocyte-containing lymphocyte fraction (90–92% mutant mtDNA) and the lowest mutation load in mononuclear cells (89–90% mutant mtDNA).
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Correlations of the mutation with the COX deficiency
To investigate whether the C6489A mutation correlated with COX deficiency, we determined the distribution of the COX deficiency and of the mutation load in single muscle fibers from the index case. A quantitative analysis of 50 randomly selected oxidative and glycolytic muscle fibers for the COX/SDH activity ratio is shown in Figure 3. In comparison with the identically processed control (Fig. 3: open bars), three populations of fibers were observed in the biopsy of the index patient (Fig. 3: filled bars): (i) fibers with normal COX/SDH ratios; (ii) fibers with slightly diminished COX/SDH ratios; (iii) a low percentage of fibers with very low COX/SDH ratios (COX-negative fibers). We analyzed these three populations of fibers for the presence of the mutation, and observed in fibers with control COX/SDH ratios (COX/SDH=3.11±0.22) between 9% and 40% of mutant alleles, in fibers with slightly lowered COX/SDH ratios [COX/SDH=2.81±0.14; with P<0.05 different from the first fiber population (unpaired two-tailed t-test)] between 75% and 93% mutant alleles, and in fibers with low COX/SDH ratios (COX/SDH=0.62±0.08) >95% of mutant alleles (Fig. 4).
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In vitro studies of mitochondrial function
To check possible metabolic consequences of the decreased COX activity in intact skeletal muscle, we investigated mitochondrial function in saponin-permeabilized muscle fibers. This preparation is suitable to study oxidative phosphorylation in skeletal muscle under conditions reflecting more the in vivo situation than isolated mitochondria (15,16). In accordance with the decreased COX activity, the maximal rates of fiber respiration with TMPD+ascorbate was lower (Table 1). In further experiments, we determined the sensitivity of mitochondrial respiration to cyanide, an irreversible inhibitor of cytochrome c oxidase. Typical titrations of glutamate+malate oxidizing control skeletal muscle fibers with cyanide are shown in Figure 5 (filled circles, solid curve). From this sigmoidal titration curve, the so-called flux control coefficient of cytochrome c oxidase was determined to be 0.26±0.04. This indicates an
2-fold excess capacity of COX in control human skeletal muscle (Fig. 6: solid curve, COXRmax(controls)=1.95). The cyanide titration of muscle fibers from the index patient revealed an almost-complete loss of the sigmoidal shape of the titration curve (Fig. 5: open circles, dashed curve). The increase in the flux control coefficient (0.51±0.18) points to a severe rate limitation of respiration flux by the lowered COX activity. On the other hand, the alteration of the titration curve cannot be attributed to a changed cyanide sensitivity of the enzyme. This information can be derived from the threshold plot in Figure 6 (open circles, dashed line) which clearly indicates a lowered excess capacity of COX in the muscle fibers of the patient (COXRmax(patient) =1.52). Accordingly, the Ki value for cyanide remained in the patient's skeletal muscle almost unchanged at 3.0±1.3 µM (the value determined was within the 2SD interval of cyanide Ki values from 12 control muscle samples: Ki=4.7±0.9 µM).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present report, we describe a heteroplasmic C6489A missense mutation in the mtDNA CO I gene in a patient with epilepsia partialis continua. In patients affected by mitochondrial disorders in this gene encoding COX subunit I, until now only two mutations have been described: a stop codon mutation in a patient with a multisystem disorder (4) and a stop codon mutation in a patient with recurrent myoglobinuria (5). Our patient is the first case of a therapy-resistant epilepsy syndrome due to a mutation in a structural COX gene.
Associated with the high loads of this mutation in the skeletal muscle of the index patient, we observed lowered activity of COX on applying independent methods: (i) the histochemical COX reaction of these muscle fibers was lowered; (ii) these muscle fibers had decreased immunostaining for COX I, II and IV; (iii) the activity of cytochrome c oxidase in the skeletal muscle homogenate was 40% lower; (iv) we observed a decreased oxygen consumption of saponin-permeabilized muscle fibers with N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD)+ascorbate, along with an increase in flux control of COX and a decreased excess capacity of COX. Furthermore, enzyme turnover calculations and cyanide titration experiments indicated no alteration of the catalytic properties of COX.
These biochemical data are compatible with the following putative molecular effect of the mutation. The C6489A point mutation leads to the exchange of a leucine residue at position 196 to isoleucine. This amino acid exchange does not alter hydrophobicity, but seems to have a structural effect on helix V of subunit I of cytochrome c oxidase, because, according to AGADIR (http://www.embl-heidelberg.de/ExternalInfo/serrano/agadir/agadir-start.html), the mutation decreases the percentage helix from 5.28% (wild type) to 4.11% (mutant). The hydrophobic inner membrane 
-helix V of subunit I has been shown to form the contact to helix III of COX subunit III of the COX complex (17). On the other hand, these structural data show that helix V is not involved in catalytic properties. It is therefore reasonable to assume that a possible spatial alteration of helix V of subunit I would decrease the stability of the entire COX enzyme complex, leading to a preferential degradation of mutated complexes, similar to what was reported previously for a stop-codon mutation in the COX I subunit (4) and a missense mutation in the COX II subunit (7). This mechanism could explain the observed decreased binding of COX I, II and IV antibodies. Furthermore, it explains the lowered heme aa3 content with unaltered kinetic properties of COX (identical turnover and unchanged Ki for cyanide). Moreover, a decreased stability of the mutated COX complexes would easily explain the observed high threshold for this mutation. Additionally, position 196 in helix V of subunit I is highly conserved, as shown by interspecies comparison (Table 3). Therefore, the reported C6489A point mutation fulfils the criteria that are generally applied to exclude a non-pathogenetic polymorphism of mtDNA: (i) the observed nucleotide change is absent at polymorphic frequencies (MITOMAP: http://www.gen.emory.edu/mitomap.html); (ii) it causes the exchange of an evolutionarily conserved amino acid; (iii) the mutation displays heteroplasmy; (iv) we observed a correlation between the percentage mutation and the COX activity in single muscle fibers.
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This mutation has, at the detected average mutation load of 90%, only an
40% effect on the maximal activity of COX in skeletal muscle. Previous reports using isolated mitochondria postulated a large ‘excess’ capacity of COX in skeletal muscle (18–21), implying phenotypic expression of a mutation only if
70% of the enzyme activity is lost. Using investigation of mitochondrial function in saponin-permeabilized fibers, we observed, however, a drop in maximal fiber respiration and a substantial increase in flux control of COX in our index patient as result of an only 40% decrease in enzyme activity. Our data are therefore in contradiction to findings proposing a large COX ‘excess’ capacity (18–21). Important problems of these studies with isolated mitochondria are the possible loss of essential metabolites during organelle isolation and the disruption of the normal interactions of mitochondria with cytoskeletal proteins (22,23). On the other hand, our result support previous data obtained for intact human cell lines (24–26), indicating also for skeletal muscle tight in vivo control of mitochondrial oxidative phosphorylation by cytochrome c oxidase. A similar situation might apply also for the exclusively oxidative neuronal tissue, explaining the epileptic phenotype of our patient. It remains to be discussed why the unaffected relatives of our patient (mother and brother) displayed higher degrees of the C6489A mutant in the blood than the index patient. As shown in our single-fiber experiments, >95% of mutated mtDNA is needed to detect a considerable COX deficiency. Assuming similar mutation load dependences for blood cell subpopulations, mutant loads <95% would be compatible with a still normal or only slightly reduced COX activity. Additionally, flux control and excess capacity of COX has been reported to be tissue-dependent (19). It is, therefore, reasonable to assume that subpopulations of blood cells that have no obligatory oxidative metabolism might be more resistant to a comparable mild COX activity drop than muscle fibers (27). In this context, it is interesting that for both unaffected relatives the highest mutation loads (close to the threshold level of 95%) were observed in predominantly glycolytic blood cell populations having a very short lifespan and high turnover: platelets and granulocytes. On the other hand, lymphocytes and monocytes with a much longer lifespan and a higher oxidative metabolism displayed the considerably lower mutation loads of 90%. Thus, our results show clearly that high loads of mitochondrial DNA mutation in the blood of unaffected individuals do not exclude its putative pathogenic role.
Summarizing, we have identified a novel heteroplasmic missense mutation in the mitochondrial CO I gene with an unusual tissue distribution in the index patient and her unaffected relatives. This mutation causes a mild cytochrome c oxidase deficiency in skeletal muscle that has considerable metabolic consequences in oxidative tissues. Our findings suggest a possible pathogenetic role of mutations in structural mtDNA genes in therapy-resistant forms of epilepsy.
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MATERIALS AND METHODS |
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Case report
The 17-year-old girl was born as the second child after an uneventful pregnancy. Family history was unremarkable for neurological disorders. During early childhood, pediatricians and parents noticed a mental and motorical delay with learning and speech difficulties. Unaided walking was possible within 2.5 years. Ictal episodes started at the age of 3 years and were characterized by prolonged left-sided motor seizures, which progressed to generalized tonic clonic seizures and were followed by a postictal left-sided hemiparesis. A cranial computer tomography was normal. At the age of 5 years, the child started to present myoclonic jerks, almost continuous, of the eyelids, and subsequently of the arms and legs. The frequency and the severity of these seizures were only temporarily modified by antiepileptic drug therapy with carbamazepine, and the patient received in addition 750 mg of valproate. Electroencephalograph (EEG) recordings revealed focal or diffuse epileptiform activities and marked background slowing. The ictal EEG discharges associated with partial motor seizures had frontocentral location, but generalized sharp wave paroxysms were also observed. A severe hepatic failure with lethargy occurred after 2 months of valproate therapy, prompting a therapeutic switch to lamotrigine followed by spontaneous functional recovery after a couple of weeks. At that time, repeated cerebral magnetic resonance imaging revealed cortical atrophy, which appeared more prominent on the right hemisphere, especially in the temporal and occipital region. Epilepsia partialis continua started at the age of 16.5 years by continuous myoclonic jerks repeated at short intervals and limited to the right hand. The frequency of these seizures and the severity of any generalized tonic clonic seizures were not modified by any antiepileptic drug therapy, including phenytoin, phenobarbitone, lamotrigine and benzodiazepines, which were taken in various combinations. The presence of slightly elevated liquor lactate levels (1.9 mM) and the history of valproate-induced hepatic failure prompted us to perform a skeletal muscle biopsy from the musculus vastus lateralis to look for a putative mitochondrial disorder. Because of recurrent status epilepticus, the patient was admitted to the intensive care unit and died shortly thereafter of respiratory failure at the age of 16.9 years.
The clinically unaffected mother and brother of the patient displayed no hematologic abnormalities. Normal control muscle specimens were obtained from 18 patients who underwent a muscle biopsy for diagnosis of neuromuscular symptoms but were ultimately deemed to be normal by means of combined clinical, electrophysiologic and histologic criteria.
Muscle histology and immunohistochemistry
Consecutive cryostat sections of muscle biopsy specimens (6 µm) were stained for Gomori's trichrome, succinate dehydrogenase (SDH), myosin ATPase and cytochrome c oxidase as described in (28). Quantitative single-fiber analysis of histochemical COX and SDH activities was performed using fiber-specific gray level determinations of 12-bit video images of identically stained adjacent sections. The video images were acquired with monochromatic illumination at 625 nm (for SDH, DIF 625 double interference filter) or at 450 nm (for COX, DIF 450 double interference filter) using an IX-70 microscope (Olympus, Tokyo) equipped with a 12-bit high resolution CCD camera (model Spot RT, Diagnostic Instruments, Burroughs, MI). The image analysis was performed using the MetaMorph software package (Universal Imaging, West Chester, PA). For the calculation of activity ratios, the individual average single-fiber gray-value readings were converted into absorbance values (
A) using the formula
A=log10 (gray value of background/gray value of fiber)
The linearity of the histochemical COX and SDH staining reactions (up to a 40 min developing time for COX and for SDH) was tested in control experiments. For immunohistochemistry, we used the mouse monoclonal antibodies against subunits I, II and IV of human cytochrome c oxidase (Molecular Probes, Eugene, OR). The immunochemical reaction was visualized with a Texas red-labeled anti-mouse secondary antibody (Dianova, Hamburg).
Enzyme activities
The activities of rotenone-sensitive NADH : CoQ1 oxidoreductase, cytochrome c oxidase and citrate synthase were measured spectrophotometrically as previously described (29). The heme aa3 content was determined as previously described (16).
Genetic analysis
Genomic DNA was extracted from 10 ml aliquots of EDTA-anticoagulated blood samples and from 30 mg skeletal muscle specimens using a salting-out method (30). After we excluded the presence of large-scale mtDNA rearrangements as well as the presence of the A3243G (MELAS) mutation, we amplified the mitchondrial genome from nucleotide position 5793 to 8380 and from 9169 to 10 116. The amplified fragments span the three mitochondrially encoded COX genes (CO I, CO II and CO III) and the tRNA genes for tyrosine, serine, aspartate, lysine and glycine. PCR fragments generated were subjected to direct sequence analysis carried out on an automatic sequence analyzer (ABI 377).
To determine the degree of heteroplasmy of the C6489A point mutation, we performed a PCR-based RFLP analysis assessing the amount of wild-type mtDNA using the mismatched primers 5'-GGGCCATCAATTTCATCACAACAA (forward) and 5'-CAGCAGCTAGGACTGGGAGAGATAGGT (reverse) by introducing a novel HphI restriction site in the case of the wild type, as well as the amount of mutant mtDNA using the primers 5'-GGGCCATCAATTTCATCACAACAA (forward) and 5'-CAGCAGCTAGGACTGGGAGAGATAGGC (reverse), giving rise to a novel DdeI restriction site in the case of the mutant mtDNA. After complete digestion, PCR products were run on a 6% polyacrylamide gel, and bands were visualized using a standard SYBR Green I (Molecular Probes, Eugene, OR) staining protocol. The proportion of wild-type and mutant mtDNA was estimated from the band intensities of restricted fragments using the Scion Image analyzing software. To verify the accuracy of the RFLP analysis we determined the proportions of mutant and wild-type mtDNA in mixtures consisting of different amounts of mutant and wild-type mtDNA PCR fragments subcloned in pCR2.1-TOPO (Invitrogen, Karlsruhe). The RFLP analysis (average ± SD of three independent PCR reactions) determined 1.9%±2.6% and 0% mutant DNA (HphI and DdeI digestion, respectively) in cases when only wild-type templates were present. When only mutant templates were present, we detected 100% and 97.4%±0.2% mutant DNA (HphI and DdeI digestion, respectively). Accordingly, we determined in a mixture of 25% mutant and 75% wild type 30.6%±5.5% and 21.4%±0.6%, in a 50% mixture 53.0%±2.1% and 46.7%±1.0%, in a 75% mixture 77.1%±2.1% and 71.1%±1.9%, in a 90% mixture 93.5%±1.5% and 86.3%±0.8% and in a 95% mixture 97.4%±0.4% and 90.7%±0.5% mutant DNA by HphI and DdeI digestion, respectively.
Single-fiber PCR analysis
The percentage of mutant mtDNA in individual fibers was correlated with single-fiber COX/SDH ratios determined as described above. For determination of the percentage of mutant mtDNA, 10 µm muscle sections were stained for COX activity and fixed in ethanol. From these sections, individual muscle fibers were selected for cutting and catapulting into a PCR tube using the PALM MicroBeam system operating with a nitrogen laser (PALM, Bernried, Germany). After proteinase K treatment, they were subjected to mismatch PCR amplification and restriction digestion, as described above, to determine the proportion of mutant mtDNA in single fibers.
Preparation of muscle fibers and fiber respiration studies
About 50 mg of biopsy tissue was used for isolation of saponin-permeabilized fibers. Bundles of muscle fibers containing usually two to four single fibers were isolated by mechanical dissection. The saponin treatment was performed by incubation of the fiber bundles in relaxing solution containing 50 µg/ml saponin as described in (15). The relaxing solution contained 10 mM Ca/CaEGTA buffer, with a free concentration of calcium of 0.1 µM, 20 mM imidazole, 20 mM taurine, 49 mM K-MES, 3 mM KH2PO4, 9.5 mM MgCl2, 5 mM ATP, 15 mM phosphocreatine, pH 7.1. The respiration measurements were performed as described in (15) in a medium consisting of 110 mM mannitol, 60 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, 0.5 mM Na2EDTA and 60 mM Tris–HCl, pH 7.4.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the University of Bonn (BONFOR) and from the Deutsche Forschungsgemeinschaft (Ku 911/11-1) to W.S.K. A.H. is supported by the German Volkswagenstiftung and by grants from the German genomic networks (BMBF).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +49 2282875744; Fax: +49 2282876294; Email: wolfram.kunz{at}ukb.uni-bonn.de
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