Mol. Hum. Reprod. Advance Access originally published online on February 11, 2005
Molecular Human Reproduction 2005 11(3):223-228; doi:10.1093/molehr/gah152
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Transmission and prenatal diagnosis of the T9176C mitochondrial DNA mutation
1Department of Genetics and Cell Biology, Research Institute GROW, University of Maastricht, Maastricht and Department of 2Child Neurology, 3Clinical Genetics, 4Biochemistry, Erasmus MC—University Medical Center Rotterdam, Rotterdam and 5Department of Human Genetics, University Medical Center, St Radboud, Nijmegen, The Netherlands
6 To whom correspondence should be addressed at: Department of Genetics and Cell Biology, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Email: bert.smeets{at}molcelb.unimaas.nl
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Abstract |
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A family presented with three affected children with Leigh syndrome, a progressive neurodegenerative disorder. Analysis of the OXPHOS complexes in muscle of two affected patients showed an increase in activity of pyruvate dehydrogenase and a decrease of complex V activity. Mutation analysis revealed the T9176C mutation in the mtATPase 6 gene (OMIM 516060) and the mutation load was above 90% in the patients. Unaffected maternal relatives were tested for carrier-ship and one of them, with a mutation load of 55% in blood, was pregnant with her first child. The possibility of prenatal diagnosis was evaluated. The main problem was the lack of data on genotype–phenotype associations for the T9176C mutation and on variation of the mutation percentage in tissues and in time. Therefore, multiple tissues of affected and unaffected carriers were analysed. Eventually, prenatal diagnosis was offered with understanding by the couple that there could be considerable uncertainty in the interpretation of the results. Prenatal diagnosis was carried out twice on cultured and uncultured chorion villi and amniotic fluid cells. The result was a mutation percentage just below the assumed threshold of expression (90%). The couple decided to continue the pregnancy and an apparently healthy child was born with an as yet unclear prognosis. This is the first prenatal diagnosis for a carrier of the T9176C mutation. Prenatal diagnosis for this mutation is technically reliable, but the prognostic predictions are not straightforward.
Key words: Leigh syndrome/mtDNA/PGD/prenatal diagnosis
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Introduction |
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Leigh syndrome (OMIM 256000) or subacute necrotizing encephalomyopathy is a progressive neurodegenerative disorder with a poor prognosis and most of the patients die within a few years after age at onset (Rahman et al., 1996
). Symptoms, occurring in early infancy or childhood, are psychomotor developmental regression, optic atrophy, ophthalmoparesis, nystagmus, ataxia, dystonia, failure to thrive and respiratory abnormalities. Characteristics are bilateral necrotic lesions on magnetic resonance imaging (MRI) in basal ganglia and brainstem and lactic acidosis. Leigh syndrome is caused by functional or molecular defects in the enzyme complexes involved in the mitochondrial energy production, including pyruvate dehydrogenase and OXPHOS complexes I, II, IV and V (DiMauro and Tanji, 1997; Dahl, 1998; Tanji et al., 2001). The inheritance of Leigh syndrome is either autosomal recessive, autosomal dominant, X-linked or maternal, and defects in nuclear genes or mitochondrial DNA (mtDNA) can cause this disease.
In this paper, we describe a family with Leigh disease caused by a mutation in the ATPase 6 gene (OMIM 516060) at position 9176 of the mtDNA (Thyagarajan et al., 1993; Campos et al., 1997; Dionisi-Vici et al., 1998; Makino et al., 1998; Makino et al., 2000; Wilson et al., 2000; Akagi et al., 2002). The severe clinical manifestations of this disease occur at mutation percentages above 90% (Thyagarajan et al., 1993; Campos et al., 1997; Dionisi-Vici et al., 1998; Makino et al., 1998, 2000; Wilson et al., 2000; Akagi et al., 2002). The mutation is maternally transmitted, but the percentage of this mutation may vary widely between oocytes, making the transmission of the associated Leigh disease fairly unpredictable (Hauswirth and Laipis, 1982; Howell et al., 2000). Mitochondria go through a genetic bottleneck in oogenesis or early embryogenesis, which involves both reduction in the number of mitochondria and the mtDNA molecules to only a very few, that subsequently repopulate the oocyte. For point mutations in the mtDNA, three criteria have been proposed to allow reliable prenatal diagnosis (Poulton and Marchington, 2000; Poulton and Turnbull, 2000).
- A close association between the proportion of mutant: wild-type mtDNA (mutation load) and disease severity.
- A uniform distribution of mutant mtDNA in all tissues.
- No change in mutant load over time.
For most mtDNA mutations this information is lacking. Only a few mutations meet these criteria like the T8993G/C (associated with Leigh syndrome) and A8344G (associated with MERRF syndrome, i.e. myoclonic epilepsy and ragged-red fibres) mutation, but not the common A3243G mutation (associated with the MELAS syndrome, i.e. mitochondrial encephalopathy with lactic acidosis and stroke-like episodes). Here, we describe the segregation of the T9176C mutation in a family with Leigh syndrome and the development and implementation of a prenatal test for this mutation.
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Patients and methods |
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Patients
Case reports from a family with maternal Leigh syndrome
The index patient (III-2, Figure 1) first presented, at the age of 21 months, with a subacute onset of cerebellar ataxia and speech retardation. The pregnancy was complicated by toxicosis, but delivery and development during the first year were unremarkable. She walked at 12 months. At the age of 3 years, after a period with fever, she had an attack with loss of tone of her right arm and leg that lasted for 3 days. Clinical examination showed a further increased cerebellar ataxia with signs of dystonic posturing of the right arm and leg. She was a happy child and was apparently not impeded by the handicap. She spoke a few words only, but was able to express herself with gestures. At 4 years and 9 months of age she was admitted to hospital because of the suspicion of a metabolic derangement, after 4 weeks of stomachache, dyspnoea, and increasing fatigue and weakness. A sick girl was seen with irregular breathing and deep-set eyes. Apart from the cerebellar ataxia there were pyramidal tract signs with very easily elicited reflexes and extensor toe signs on both feet. MRI and computerized tomography of the brain showed extensive vaguely demarcated hypodensities in the cerebellar hemispheres, pons and mesencephalon. Supratentorially there were hypodense lesions in the caudate nuclei, the internal capsule and the basal ganglia. Peripheral and central cerebrospinal fluid (CSF) spaces were enlarged. Lesions could be compatible with infarctions in parts of the basilar and the internal carotid artery region. The clinical phenotype combined with lactic acidosis and CSF involvement made a mitochondrial encephalopathy or Leigh-like syndrome likely. The child's condition worsened and in the following 2 weeks she developed severe apnoeic and hypoventilation spells and became ventilator-dependent. She died from progressive brainstem dysfunction. She underwent a muscle biopsy a few hours before death, but there has been no post-mortem examination.
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The younger brother (III-3) and sister (III-4) of the index patient (III-2) developed a cerebellar ataxia with an onset at age 2 years. Their language development was slightly delayed. Both received, from age 4 and 2, respectively, daily vitamin B complex containing 100 mg vitamin B1, 2, 3, 5, 6, 50 mg vitamin E and 3 dd 330 mg carnitine. From the age of 3, after viral illnesses, attacks of one to several days occurred with loss of tone resulting in paraparesis losing ambulation, sighing, dyspnoea and dystonic movements. At the age of 4 years, the diagnosis of mitochondrial encephalomyopathy of the boy was confirmed by determination of the T9176C mutation in DNA from muscle and blood (van den Bosch et al., 2000
). Subsequently, DNA analysis in the other family members took place. The boy (III-3) was still ambulant at age 8, with a cerebellar ataxia, generalized mild dystonic movement disorder, a discrete pyramidal syndrome and speaks only a few words. His total IQ is 55. The electroencephalogram (EEG) showed a slight delay of the background pattern. The cerebellar hemispheres showed in the cortical and subcortical regions on MRI extensive abnormalities in the flair and T2 spin echo. A quadriceps muscle biopsy showed at electron microscopy an increase of lipid droplets and enlarged mitochondria. At the age of 6 years, the girl (III-4) had milder problems than her brother. She speaks in short, poorly articulated sentences. Her EEG showed a slight delay in background pattern. The MRI showed very discrete abnormalities in the cerebellar hemispheres. The mother of the index patient (II-2) has periodically stomachache, but no neurological abnormalities. None of the unaffected family members showed movement disorders or other neurological or psychiatric abnormalities. All numbered patients (Figure 1) were clinically examined except the grandmother of the index patient (I-1). She died due to gastrointestinal tumour.
Methods
Muscle biochemistry
Muscle biopsies were obtained from the index patient (quadriceps) and brother under generalized anaesthesia. Frozen muscle specimens were weighed and homogenised in 0.25 M sucrose, 10 mM HEPES and 1 mM EDTA, pH 7.1. The enzyme activities were assayed at 37 °C, unless indicated otherwise. Complex I, NADH co-enzyme Q reductase, was measured as the rotonone sensitive oxidation of NADH with decylubiquinone as electron acceptor (De Vries et al., 1996). Complex II and III, succinate cytochrome c reductase, was measured as the antimycin-sensitive reduction of cytochrome c in the presence of appropriate inhibitors at 25 °C (Scholte et al., 1995). Complex II, succinate co-enzyme Q reductase, was determined as the theonyl trifluoroacetone (2 mM) sensitive reduction of 2,6-dichlorophenolindophenol by succinate in the presence of decylubiquinone (Trounce et al., 1996). Complex III, ubiquinol cytochrome c reductase, was assayed as the antimycin-sensitive reduction of cytochrome c by decylubiquinol at 25 °C, in the presence of lauryl maltoside. Complex IV, cytochrome c oxidase, was measured as described (Cooperstein and Lazarow, 1951) at 25 °C, or with the detergent (Mayr and Sperl, 2000) Tween-20, at 37 °C. Complex V, ATP synthase, was determined as oligomycin-sensitive uncoupler stimulated Mg-ATPase by assay of ADP with pyruvate kinase, phosphoenolpyruvate, lactate dehydrogenase and NADH (Rustin et al., 1994). This reaction was started by sonication (Mayr and Sperl, 2000). Pyruvate dehydrogenase was assayed as in Arts et al. (1987) and citrate synthase (CS) according to Srere (1969). The other methods were as in Scholte et al. (1995).
DNA extraction
DNA was extracted from blood, cultured fibroblast cells, hair roots, muscle tissue, chorionic villi cells [chorionic villus sampling (CVS)] and amniotic fluid cells. DNA extraction from blood was done as described previously (Miller et al., 1988). DNA extraction from hair roots was performed using the Qiagen RNA/DNA minikit (Qiagen GmbH, Hilden, Germany). For the DNA extraction of the other samples the standard phenol/chloroform method was used (Sambrook et al., 1998).
Quantitative analysis of the T9176C mutation
The mtDNA was amplified using PCR with primers, corresponding to positions 09035–09055 of the L-strand and positions 09203–09177 of the H-strand. The latter primer contains a mismatch at location 09184–09186 to create a restriction site for the restriction enzyme BstXI in case of the T9176C mutation. The PCR was performed using 4 mM of each dNTP (Amersham Pharmacia Biotech AB, Uppsala, Sweden, 25 mM each), 15 mM MgCl2, 0.5 M NaCl, 0.1 M Tris–HCl, 50 ng per primer, 1 U Taq DNA polymerase (Life Technologies, Breda, The Netherlands) in a 25 µl volume. PCR conditions were 94 °C for 5 min, followed by 32 cycles at 92 °C for 1 min, 52 °C for 1 min and 72 °C for 45 s and a final step of 7 min at 72 °C (9600 Thermocycler, Applied Biosystems, Foster City, CA). In the last cycle, 50 fmol R6G labelled dUTP (Applied Biosystems, Foster City, CA) was added. The PCR product was purified with the Qiagen PCR purification kit (Qiagen GmbH, Hilden, Germany), digested with BstXI (Boehringer-Mannheim, Bayern, Germany) and separated by electrophoresis on a 4% non-denaturing polyacrylamide gel at 40 °C (ABI 377 Applied Biosystems, Foster City, CA). The wild-type fragment has a length of 169 bp and the mutated fragment is cleaved into fragments of 148 and 21 bp. The mutation percentage is determined by calculating the ratio between the area of the mutant peak (148 bp) and the total area of the mutant and normal peak (169 bp). The calculated mutation percentage is multiplied by 1.06 to correct for lower number of fluorescent labelled dUTPs that can be incorporated in the mutant peak. The experimental variation of this method is 3%.
Prenatal diagnosis
Data from the literature and family were used to determine if this mutation was suitable for prenatal diagnosis. CVS samples were obtained after 12 weeks of gestation and amniotic fluid after 17 weeks of gestation. Both samples were analysed for the T9176C mutation immediately and after a culture period of 2 weeks.
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Results |
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Clinical biochemistry
In the index patient (III-2), blood lactate was 3.2 mmol/l (normal <1.8 mmol/l) and pyruvate was as in healthy individuals (normal <160 µmol/l). Lactate 4.6 mmol/l (normal range 0.9–2.8 mmol/l) was also elevated in CSF. CSF pyruvate was 80 µmol/l. A muscle biopsy of the quadriceps muscle of the index patient (III-2) appeared normal. Histochemical staining of cytochrome c oxidase and succinate hydrogenase yielded normal results. Electron microscopy showed some irregularly formed fibrils with thickening of the Z-bands. One concentric laminated body was seen possibly derived from mitochondria. A muscle biopsy from patient III-3 also showed a normal morphology by routine histology and histochemistry. Electron microscopy showed enlarged intermyofibrillar mitochondria and increased lipid droplets. One small aggregate of elongated subsarcolemmal mitochondria was seen. The brother (III-3) of the index patient showed a blood lactate of 2.2 mmol/l and a pyruvate of 140 µmol/l. CSF lactate was 3.2 mmol/l. Blood lactate in the sister (III-4) of the index patient was 2.3 mmol/l and pyruvate 150 µmol/l, respectively.
Muscle biochemistry
Mitochondrial enzyme activities were strikingly abnormal with a severe decrease of complex V activity and an increase of pyruvate dehydrogenase activity. Complex V/CS ratio is 0.10 and 0.08 (normal 0.40) and pyruvate dehydrogenase/CS ratio is 0.10 and 0.09 (normal 0.03) for patient III-2 and III-3, respectively.
Mutation analysis and prenatal diagnosis
The identification of the T9176C mutation in muscle of the index patient was reported previously (van Den Bosch et al., 2000). The internal variation of the method used to determine the mutation percentages is ±3%. The mutation percentage in muscle was about 95% and in fibroblasts 90%. The same mutation was identified in a symptomatic brother and sister. The brother had a mutation percentage of about 93% in muscle and 94% in hair roots. The sister had a mutation percentage of about 92% in blood and 93% in hair roots (pedigree, Figure 1). A number of maternal relatives were tested showing either the absence or presence of the mutation (Figure 1). Mutation percentages appeared to be more constant among tissues in patients with the higher mutation ranges (>90%). One of the female family members (III-1) was 6 weeks pregnant at the time of investigation. The mutation load in blood and hair roots was about 55 and 57%. She was at risk of having severely affected offspring, although the exact risk was unknown.
We investigated whether this mutation would meet the criteria to allow reliable prenatal diagnosis (Poulton and Turnbull, 2000). Both data from the literature (Figure 2) and of the family (Figure 1) were used. No symptomatic patients have been reported with a mutation percentage below 90%, although the methods used vary in their precision. An estimate of the experimental variation is calculated, as it was not given in most references, and the number of patients is small (Figure 2). Only one healthy individual had a percentage between 80 and 90% and two between 70 and 80%, respectively. All patients developed symptoms between 0 and 8 years except for one patient with a mutation percentage of 96% who developed symptoms at the age of 29. Obviously, data on the distribution of the mutation among different tissues was mainly limited to patients, all of whom had >90% mutation load in every tissue analysed. Since no unaffected mutation carrier with less than 90% mutation has been followed over a longer period no firm conclusion is possible on either time-related changes in the mutation percentage or the onset of symptoms in carriers with subcritical mutation percentages. Based on these limited data and on the data of the T9176G (Carrozzo et al., 2001; Akagi et al., 2002) (which has a somewhat more malignant disease course and also a lower mutation threshold for clinical expression of about 70%) and two other mutations in the ATPase 6 gene (T89993G/C) [which have been studied in much more detail and for which reliable prenatal diagnosis is possible (Harding et al., 1992; Bartley et al., 1996; Ferlin et al., 1997; White et al., 1999a,b; Leshinsky-Silver et al., 2003)], we decided to offer prenatal diagnosis. The prenatal sampling was undertaken with the strict understanding by the couple, written in an informed consent, that there could be considerable uncertainty in the interpretation of the results given the scarcity of data available. Somewhat arbitrary, a mutation percentage in fetal cells above 70% was considered a high risk of being affected, a mutant load between 50 and 70% would be inconclusive and a mutant load of less than 50% would mean a high chance of being unaffected.
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Method A: Last cycle addition of [
-32P]dATP. Quantification by scanning in a Betascope 603 blot analyser (Thyagarajan et al., 1993
Method B: Ethidium bromide staining. Quantification using UV detection (Dionisi-Vici et al., 1998To gather more information on tissue variation of the mutation and the time factor, we decided to analyse both CVS sample (12 weeks of gestation) and amniotic fluid sample (17 weeks of gestation) directly and after a culturing period. The mutation percentage in CVS samples was about 87% and after culturing 85% (Figure 3A and B) and in amniotic fluid cells 88% and after culturing 86% (Figure 3C and D). The difference between cultured and uncultured cells is not statistically significant as the experimental variation was determined to be about 3%. Therefore, the fetus was diagnosed as at risk of becoming severely affected, although the mutation percentage was just below the level of the other three affected children. Despite these results, uncertainties and associated risks, the couple decided to continue the pregnancy. A healthy child was born at term after a normal pregnancy. The child did not show any abnormalities at the age of 1 year and for ethical reasons no further biochemical or genetic test will be performed as long as the child is healthy. It is important that the child remains under clinical control as the other affected children with a slightly higher mutation level did not show clinical signs of disease until the age of 2 years.
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Discussion |
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The segregation of point mutations in the mtDNA is not completely understood and the transmission of the mutation load is often unpredictable for heteroplasmic mutations. The family reported in this paper, with three affected children with Leigh disease and the T9176C mutation, is a clear illustration of this. Maternal relatives of the patients either do not carry the mutation or carry the mutation in varying percentages. They are all below the threshold of clinical expression, although in most cases no extensive neurological and biochemical examinations have been performed. In such families, only DNA studies can provide evidence for the carrier status of an individual and can identify those at risk of transmitting the disease. The exact estimate of the risk of having an affected child is not possible due to the genetic bottleneck for mtDNA mutations and the limited number of data available for this mutation (Thyagarajan et al., 1993
; Campos et al., 1997; Dionisi-Vici et al., 1998; Makino et al., 1998, 2000; Wilson et al., 2000). It has been proposed that oocyte sampling and testing could be an acceptable approach to determine the mutation load in individual oocytes and estimate the recurrence risk (Poulton and Marchington, 2002; Thorburn, 2004).
Because of these uncertainties, prenatal diagnosis, based on DNA analysis of the mtDNA mutations, is controversial. Until now seven prenatal tests were reported for the T8993G and T8993C mutations (Harding et al., 1992; Bartley et al., 1996; Ferlin et al., 1997; White et al., 1999a,b; Leshinsky-Silver et al., 2003). These mutations fulfil the criteria mentioned before and have well-established genotype–phenotype associations, based on sufficient number of families tested. Recurrence risks have been calculated dependent on the mutation load of the mother and a safe margin for the mutant load in case of prenatal diagnosis is established. Disease caused by the T8993C mutation is clinically less severe than the T8993G mutation. The probability of having severe symptoms of the T8993C and the T8993G mutations are low if mutation loads are below 80 and 60%, respectively.
The differences in recurrence risk for diseases caused by point mutations in the mtDNA and the potential pitfalls prompted a statement by a group of researchers, supported by the European NeuroMuscular Consortium, concerning prenatal options for carriers of the mtDNA mutations (Poulton and Marchington, 2000; Poulton and Turnbull, 2000). Sufficient data are available for only three mutations today to judge these criteria properly. For the T8993G/C and A8344G mutation prenatal diagnosis can be reliably performed, but for the A3243G mutation this is not possible. For other mutations this is still unknown and the families involved can only be counselled in general terms. Whether prenatal diagnosis will be an option for these families will depend on the frequency of the mutation and the ethical discussions on acceptable risks.
The T9176C mutation has been described only a few times in literature (Thyagarajan et al., 1993; Campos et al., 1997; Dionisi-Vici et al., 1998; Makino et al., 1998, 2000; Wilson et al., 2000). As the methods used are often not comparable and as usually no information is provided on detection level and experimental variation, it is difficult to draw general conclusions on the mutation threshold for clinical expression. Until now, severe symptoms were only reported for patients with mutation percentages above 90% in various tissues. As the T9176C mutation is located in the same gene as the T8993G/C mutation and shows some resemblance in clinical symptoms and progression of the disease, we considered this additional, though arguable, evidence that the T9176C mutation is also suitable for DNA-prenatal diagnosis. For the T9176C, also a T9176G variant has been found with somewhat more malignant progression similar to the T8993G mutation compared to the T8993C mutation (Carrozzo et al., 2000, 2001; Akagi et al., 2002). Given the high chance of an affected fetus or a borderline result it was advised not to offer prenatal diagnosis, but consider oocyte donation or PGD to carriers with a mutation percentage of more than 50% of the T8993G/C mutation (Poulton and Turnbull, 2000). Because the woman in our study was already pregnant it was not possible to discuss alternatives, but PGD will be offered for future pregnancies. The parents were informed about the uncertainties of borderline mutation percentages and the risk of an affected fetus and decided to continue with the prenatal diagnosis. The prenatal diagnosis revealed a percentage of 85–88% with a variation of 3%. Although there was a high likelihood that the child would be affected the parents decided to continue the pregnancy.
Assuming that the bottleneck occurs during oocyte development (Poulton and Marchington, 2002), PGD could be offered in case of mtDNA mutations as an alternative for conventional prenatal diagnosis (Thorburn and Dahl, 2001). For PGD, one or two blastomeres are removed from an 8-cellular embryo, obtained by IVF procedures and ICSI, and these blastomeres are tested for the specific mutation. Healthy embryos are transferred to the uterus. PGD is technically easier for mtDNA mutations than for nuclear genes, as the copy number is much higher. Protocols have been optimized at the single-cell level in our laboratory (L.J.Jacobs et al. in preparation). Data from heteroplasmic mice show that mutation percentages quantified in the biopsied cell are representative for the entire embryo (Dean et al., 2003). The same criteria apply for prenatal diagnosis as for PGD, but the main advantage of PGD is that no termination of pregnancy has to be considered in case of affected offspring. That these chances can be very high has been demonstrated in the oocytes of a carrier of the T8993G mutation (50% in blood), who had six oocytes with more than 95% mutation and one oocyte with no mutation at all (Blok et al., 1997), yielding a chance of more than 85% for offspring being affected. PGD can prevent multiple terminations of pregnancy in those cases.
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Acknowledgements |
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Dr P.A. van Doorn and Prof. Dr H. F. M Busch performed routine histology and histochemistry and electron microscopy.
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References |
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Submitted on October 20, 2004; accepted on January 7, 2005.
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