Mol. Hum. Reprod. Advance Access originally published online on June 24, 2008
Molecular Human Reproduction 2008 14(7):405-412; doi:10.1093/molehr/gan034
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CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation
1Department of Embryology and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 2Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 3Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 4Laboratory for Experimental Surgery, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 5Centre for Outcomes Research, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium
6 Correspondence address. Centre for Medical Genetics, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. Tel: +32-24774635; Fax: +32-24778680; E-mail: karen.sermon{at}uzbrussel.be
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Abstract |
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Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.
Key words: human embryonic stem cells/myotonic dystrophy type 1/DMPK/triplet repeat instability
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Introduction |
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Since the derivation of the first human embryonic stem cell (hESC) line by Thomson et al. (1998), hESC are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body, holding important promises in regenerative medicine. hESC are derived from the pluripotent inner cell mass (ICM) of a blastocyst and can therefore represent an important tool to study early human developmental biology (Brivanlou et al., 2003). Additionally, specific genetic disease processes can be studied by generating new hESC lines from embryos diagnosed as carrier of (a) mutation(s) or a chromosomal abnormality during preimplantation genetic diagnosis (Pickering et al., 2003). Several groups have reported the derivation of hESC lines carrying genetic mutations responsible for monogenic diseases such as cystic fibrosis, Huntington's disease (HD) and myotonic dystrophy type 1 (DM1) (Pickering et al., 2005; Mateizel et al., 2006; Verlinsky et al., 2006).
DM1 is a neuromuscular disorder, caused by an unstable CTG repeat in the 3' untranslated region of the DMPK gene (Aslanidis et al., 1992; Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). This CTG repeat is polymorphic in the population ranging from 3 to 37 repeats in non-affected individuals. DM1 patients with the adult-onset disease exhibit from at least 50 repeats up to hundreds of repeats, whereas infants born with congenital DM1 have often up to several thousands of repeats.
Expanded CTG repeats are highly unstable in somatic cells depending on the patients' age, repeat number and tissues examined (Wong et al., 1995). Also, in germ cells, a high degree of instability is detected. In male gametes, smaller repeats are highly unstable tending to enlarge significantly during spermatogenesis (Jansen et al., 1994; Martorell et al., 2001), whereas in female gametes, mainly larger repeats show a high instability. This explains why the congenital form of the disease nearly always occurs after female transmission. The timing of the intergenerational instability of repeats remains unclear. The existence of enlarged DM1 CTG repeats in (im)mature oocytes is evidence for a prezygotic event during female gametogenesis (De Temmerman et al., 2004; Dean et al., 2006). Additionally, Yoon et al. (2003) detected enlarged CAG repeats in spermatogonia of HD patients, even in spermatogonia that had not completed the first meiotic divisions, which indicates that repeat instability in male gametogenesis is not meiosis-dependent. In DM1 preimplantation embryos, from zygotes to blastocysts, no mosaicism was detected (De Temmerman et al., 2004) also suggesting that the intergenerational repeat instability occurs during gametogenesis.
The mechanisms causing triplet repeat instability remain to be elucidated. It became evident from mouse models that cell proliferation and DNA replication are not sufficient to explain repeat instability (Gomes-Pereira et al., 2001), that mismatch repair enzymes play a role in somatic mosaicism of the CTG repeat (van den Broek et al., 2002; Savouret et al., 2003; Gomes-Pereira et al., 2004) and that cis and/or trans factors influence the repeat stability differently in different tissues (Cleary and Pearson, 2003; Gomes-Pereira et al., 2004). These observations led to the hypothesis that DNA replication as well as recombination and repair, either separately or in combination and at different stages in human development, are responsible mechanisms for repeat instability (Pearson et al., 2005).
The derivation of a hESC line carrying an enlarged CTG expansion in the DM1 locus opens new possibilities for research into triplet repeat instability. VUB03_DM1 was derived from an affected blastocyst that presented an expanded CTG fragment of 470 repeats, whereas the affected female donor had 120 repeats (Mateizel et al., 2006). In this report, we describe the peculiar behaviour of the CTG expansion in this affected hESC line and compare this with the stable behaviour in two healthy hESC lines VUB01 and VUB04_CF.
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Materials and Methods |
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Culture of hESC
VUB03_DM1 and two control lines VUB01 and VUB04_CF were derived and propagated as described by Mateizel et al. (2006). VUB04_CF is compound heterozygous for the F508del mutation and the 5T variant that can lead to congenital absence of the vas deferens (CBAVD) in males, but never to a full-blown cystic fibrosis phenotype.
The cells were grown on mouse embryonic fibroblasts feeder layers and cultured at 37°C in 10% CO2. A standard hESC medium was used containing Knockout-Serum Replacement (KO SR, Invitrogen, Carlsbad, USA) and 4 ng/ml human recombinant basic Fibroblast Growth Factor (bFGF; Invitrogen). The colonies were passaged every 4–5 days. To avoid contamination with spontaneously differentiated hESC, undifferentiated areas of the colony were manually dissected into clumps (Fig. 1A and B). These were carefully removed from the feeder layer and passaged as a pool of clumps to freshly prepared 6 cm2 culture dishes.
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The doubling time of VUB03_DM1 was calculated by a standard doubling assessment protocol (Cowan et al., 2004) and karyotyping was carried out using the standard G-banding method in at least 20 metaphases (Gosden et al., 1992). The expression of the hESC-markers POU5F1 (previously known as OCT4), NANOG, SOX2, REX1, NPM1, LIN28 and GDF3 was tested by RT–PCR (35 cycles) as previously described by Mateizel et al. (2006) on cDNA of VUB03_DM1 at passage 93.
Repeat instability
Small amount PCR
At least three colonies of a given passage of VUB03_DM1 and VUB01 were mechanically cut into small clumps of undifferentiated cells. Each clump was individually removed from the feeder layer and transferred to a marked well of a 4-well dish, filled with standard hESC culture medium. By mechanical disruption, the small clump could be divided in single cells and aggregates of cells (±10 cells). For each analysed colony, at least three samples were collected, consisting of a group of single cells (±1–5 cells) or an aggregate of cells. Each sample was collected using methodology developed for standard PGD protocols (Goossens et al., 2003). Briefly, the groups of single cells or aggregates were washed in buffer and were picked up using a mouth-controlled, finely drawn glass capillary, after which they were blindly transferred to a 0.2 ml PCR tube containing alkaline lysis buffer (200 mM KOH, 50 mM DTT), and further analysed by a specific long PCR protocol to amplify expanded CTG repeats (De Temmerman et al., 2004). The PCR mix (total volume of 50 µl) contained: 0.3 µM primers DM101 and DM102 (Brook et al., 1992), 500 µM of each dNTP (DNA Polymerisation Mix, Amersham Pharmacia Biotech, Roosendaal, The Netherlands), 1x Expand Long Template (ELT) PCR buffer 2 provided by the manufacturer (Roche Diagnostics, Vilvoorde, Belgium), 2.5% dimethylsulphoxide (DMSO, Sigma Aldrich, St Louis, USA), 2% Tween 20 (Sigma Aldrich) and 1.4 U DNA polymerase from the ELT kit (Roche Diagnostics). The PCR programme used consisted of a first denaturation step at 95°C for 5 min, followed by 10 cycles at 95°C for 30 s, 65°C for 30 s and 68°C for 45 s, followed by 30 cycles at 95°C for 30 s, 65°C for 30 s and 68°C for 45 s, but this time with an increase of 20 s per cycle for the elongation step at 68°C.
The PCR products were separated by a denaturing agarose gel electrophoresis on a 1.5% agarose gel and transferred to a positively charged nylon membrane. After hybridization with a DIG-labelled oligoprimer (CAG)5- probe, the repeat sizes could be determined in comparison to the molecular size markers VI and VII (Roche Diagnostics) and the PCR products obtained from the genomic DNA of the affected embryo donor.
To determine the exact size of healthy alleles, labelled PCR products were analysed by capillary electrophoresis on an ABI 3130XL Genetic Analyser (Applied Biosystems, Foster City, USA) as small differences in repeat size are difficult to determine by agarose gel electrophoresis. The accuracy of sizing is ± 1 repeat in the normal allele size range.
Small pool PCR
Cells of VUB03_DM1, VUB01 and VUB04_CF at a given passage were collected from the feeder layer by collagenase type IV (Sigma Aldrich). This collection technique implies that undifferentiated as well as differentiated cells are collected. Special care was taken to analyse only those cultures with a low differentiation rate as viewed with the inverted microscope (Mateizel et al., 2006).
DNA was extracted from the cell pellet with the Dneasy tissue Extraction Kit (Qiagen, Hilden, Germany) and a DNA titration series was used to determine the concentration necessary to have 1–5 genome equivalents amplified per reaction (Monckton et al., 1995).
At least 30 dilutions of a given passage were amplified with the long PCR protocol and analysed as described in the small amount PCR (SA PCR) protocol.
Southern blot analysis
DNA of VUB03_DM1, VUB01 and VUB04_CF at certain passages was obtained as previously described for small pool PCR (SP PCR). After digestion of 10 µg DNA with EcoRI and SacI (Roche Diagnostics), the fragments were visualized with the Southern blot technique using a DIG-labelled probe that was generated with the PCR DIG Probe Synthesis Kit (Roche Diagnostics). This probe was generated with forward primer 5'-TAGGTGGGGACAGAC3' and reverse primer 5'-GGGATCACAGACCATTTCTTTCT-3' (NT_011109
[GenBank]
.15/Hs19_11266; positions 18541243-18541262 and 18541678-18541656).
Statistical analysis
Multiple regression analyses were conducted to explore the potential relationship between passage number (X, independent variable) and repeat size (Y, dependent variable). The magnitude of the strength of this relationship was quantified by the standardized regression coefficient (labelled Beta). All tests were two-tailed, and a P-value of <0.05 was considered to indicate statistical significance.
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Results |
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Culture of hESC
During the prolonged culture of VUB03_DM1 up to 150 passages or nearly 2 years, no significant alterations were observed in the morphological features considered characteristic of hESC: a compact colony structure (Fig. 1A and B), cells with a high nucleus-to-cytoplasm ratio and prominent nucleoli were present. The genetic stability of the line was re-assessed at passage 147 by karyotyping at least 20 metaphases which confirmed the previous normal 46,XX karyotype (Fig. 1C). RT–PCR of RNA extracted of cells at passage 93 (Fig. 1D) showed a clear expression of POU5F1 (previously known as OCT4), NANOG, SOX2, REX, NPM1, LIN28 and GDF3 as previously reported (Mateizel et al., 2006). The doubling time was assessed at passage 66 and was on average 24 h.
Repeat instability
Early passages
Because of the scarcity of the material at the lowest passages of this cell line, only groups of individual cells and small aggregates of cells could be analysed by the SA PCR technique. Good amplification rates, i.e. a significant proportion of samples showed amplification of the healthy allele (96%) and the affected allele (70%) of VUB03_DM1, were obtained for small groups of 1–5 cells and small aggregates of 
10 cells (Fig. 2). In both sample types, smaller and larger repeat sizes were amplified and detected, indicating that the amount of cells in the sample was probably not influencing the results (Fig. 3A). The amplification of the smaller healthy allele with 11 repeats was considered as an internal control for PCR amplification.
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In the first passage, i.e. in cells of colonies obtained from the first cuttings of the outgrowth of the embryo's ICM, only PCR fragments with repeat lengths of 470 were observed. At passage 10, a clear instability existed with the majority of cells having 470 repeats but also repeat enlargements and contractions were detected. A similar repeat distribution was detected at passage 18 and 21 but at passage 15, 17 and 27 mainly PCR fragments of larger repeat sizes were detected. Contractions into the range of 100 repeats were detected in all of these early passages.
Later passages
In later passages of VUB03_DM1, DNA could be extracted from whole culture dishes allowing another semi-quantitative analysis method to be used: SP PCR. A DNA dilution titration determined that the appropriate DNA concentration to amplify 1–5 genome equivalents was 150–200 pg.
Visual inspection of the results for both PCR techniques (Fig. 3) clearly show a high instability of the CTG repeat in the higher passages (from passage 30 on) of the hESC line. Stepwise increases of repeat numbers were detected in consecutive passages as well as a higher range of variability. Fragments of 470 repeats were rarely detected, whereas fragments with repeat sizes >1000 were clearly present in the later passages of VUB03_DM1. With the SP PCR (Fig. 3B), larger repeat sizes were more readily amplified in comparison to the SA PCR. Clear bands of 2100 repeats or 6400 bp were still detectable and probably represent the upper limit of our PCR system.
The variability in size of the CTG repeat was not only apparent in different passages but also between cells of the same colony (Fig. 2A). This intra-colony instability was apparent in all samples studied by SP PCR. The instability of the repeat was sometimes limited to only one or two repeat sizes in one colony, whereas a variety of repeat numbers were present in another colony of the same passage of VUB03_DM1.
Formal quantification of the relationship between repeat size and passage number using multiple regression analyses indicated that this relationship was statistically significant, with standardized regression coefficients of 0.327 (P < 0.001), 0.534 (P < 0.001) and 0.556 (P < 0.001) for the data presented in Fig. 3A–C, respectively. The positive signs of the standardized regression coefficients describe the direction of the relationship between repeat size (Y, dependent variable) and passage number (X, independent variable), i.e. repeat numbers are significantly higher in the higher passages.
In contrast to the instability of the affected allele, the healthy allele remained stable at 11 repeats over the whole examined culture period (Fig. 4).
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Southern blot analysis
The instability of the repeat in VUB03_DM1 was confirmed by the Southern blot analysis after digestion with SacI or EcoRI (Fig. 5A). Distinct bands were visible in the lower passages, whereas the prominent repeat number in every passage showed a stepwise increase. The prominent band became more diffuse after passage 60, indicating a larger variability in repeat size between cells of the same passage. In EcoRI-restricted DNA of VUB03_DM1 at passage 127, three smeared bands of
2500, 3000 and 3500 CTG repeats were prominent, whereas after SacI digestion, a faint band of 2300 repeats was detected at this passage. These were the largest repeat sizes detected after digestion.
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Normal hESC lines
The behaviour of the CTG repeat at the DM1 locus was also investigated in two normal hESC lines: VUB01 and VUB04_CF. For VUB01, the SA PCR assay was performed on groups of cells and small clumps at P38 and P40 representing early passages and P304 and P306 representing late passages (Fig. 2B). The repeat sizes were not only visualized after blotting, which is more sensitive for the larger alleles but does not allow sizing of smaller, normal alleles but also after fragment analysis on a DNA sequencer to size the repeat size accurately (data not shown). In all samples, the repeat remained stable at 3 and 9 repeats (Fig. 2B).
The SP PCR assay was performed on DNA dilutions of VUB01 at P31, P135, P177 and P222, and of VUB04_CF at P25, P66, P82, P117 and P144. At every passage, we found 3 and 9 repeats in the samples of VUB01 and 3 and 11 repeats in samples of VUB04_CF (Fig. 4).
Southern blotting also showed the absence of expanded repeat alleles for VUB01 at P31 and P222, and for VUB04_CF1 at P45 and P145 (Fig. 5B).
DNA samples of mouse embryonic feeder layers remained without any amplification, indicating that the primers were human-specific.
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Discussion |
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The derivation of hESC cell lines from embryos carrying a pathological mutation responsible for monogenic diseases provides us with a powerful in vitro tool to explore both the molecular mechanisms implicated in the disease and their treatment. The in vitro culture of hESC carrying an expanded CTG repeat in the 3' UTR of the DMPK gene showed a high instability of the repeat as the culture of VUB03_DM1 prolonged. On the contrary, two healthy lines VUB01 and VUB04_CF showed no instability of the repeats size after a prolonged culture.
To analyse the behaviour of the CTG repeat in VUB03_DM1, the same approach as for human gametes and preimplantation embryos was used (De Temmerman et al., 2004) picking up a restricted number of cells. Collecting a small number of cells, or aggregates of cells as a sample (SA PCR), allowed us to ensure the further culture of the line during the early passages. At later passages, enough cells could be obtained to extract DNA and analyse the behaviour of the CTG repeats additionally by SP PCR and Southern blot analysis. For both PCR approaches, the same specific and sensitive PCR system was used that was optimized to amplify long CTG tracts (De Temmerman et al., 2004). The use of a PCR system, even when optimized, is still restrictive in the number of repeats that can be amplified. This was exemplified by the results obtained for the highest passages, as in several samples, only the healthy alleles could be amplified. Both PCR techniques represent a semi-quantitative analysis method as the exact number of cells in each sample of the SA PCR could not be determined, although the number of genome-equivalents obtained for a certain dilution in the SP PCR could vary. Especially, the number of cells or genome-equivalents with the same CTG repeat number was not recognized by this procedure and was under-represented. The Southern blot analysis of restricted, extracted genomic DNA completes this data, but is less accurate in determining exact repeat lengths. This analysis clearly demonstrates the degree of instability in the consecutive passages with a stepwise increase of the prominent band and an apparent smearing of this band in the highest passages of VUB03_DM1.
hESC are derived from the pluripotent cells of the blastocysts and are believed to be related to these ICM cells before they differentiate into primitive ectoderm (Brook and Gardner, 1997). Previously, it was shown that the triplet repeat in the DMPK gene is stable from the zygote to embryos at the blastocyst stage (De Temmerman et al., 2004; Dean et al., 2006) and even in fetal tissue until approximately between the 13th and 16th week of pregnancy (Wöhrle et al., 1995; Martorell et al., 1997). This stability is highly contradictory to the observed instability of the CTG repeat in VUB03_DM1. An explanation for this peculiar behaviour of the repeat in hESC could be that the in vitro culture is influencing the stability of an already expanded repeat, as the repeat size remained stable in unaffected lines. In vitro cultures of fetal and adult fibroblasts as well as myoblasts displayed progressive expansions of the DM1 alleles with stepwise incremental changes in repeat numbers when the culture prolonged (Wöhrle et al., 1995). In clonal lymphoblastoid cell lines of DM1 patients, Khajavi et al. (2001) described ‘large jumps’ in repeat number that appeared to be products of clonal expansions of rare mutants. These subclones with longer repeats had a selective growth advantage over cells with smaller repeat sizes (Ashizawa et al., 1996), suggesting that DM1 cells with expanded alleles drive themselves to extinction through a process related to increased proliferation (Khajavi et al., 2001). A similar repeat behaviour was detected in VUB03_DM1, showing gradual stepwise increases in repeat lengths as the culture prolonged, whereas the level and range of variation increased with allele size. This repeat behaviour suggests that clonal events take place with cells with longer CTG tracts taking over the culture. However, VUB03_DM1 was cultured for more than 2 years showing no differences in proliferation rate or undifferentiated state compared with normal hESC lines.
The hypothesis that hESC are most related to ICM-derived cells was recently questioned, and another hypothesis was postulated that argues that hESC are more related to primordial germ cells (PGC) rather than to the ICM (Kehler et al., 2005; Zwaka and Thomson, 2005). This is evidenced by the fact that both cell types express the same markers, e.g. POU5F1 (OCT4), NANOG, FRAGILIS and STELLAR. This hypothesis would better explain the CTG repeat behaviour in the hESC as several publications report repeat instability in human diploid premeiotic and meiotic cells (Yoon et al., 2003; De Temmerman et al., 2004; Dean et al., 2006). In any case, more knowledge about the behaviour of the triplet repeat in early PGC is necessary but these cells are difficult to study due to their scarcity. More evidence sustaining the hypothesis of PGC-related hESC is also required.
At the molecular level, several mechanisms can be responsible for the high instability of expansions in our hESC line VUB03_DM1. Cells with high replication are subject to DNA replication errors, repair associated with replication and genome maintenance. Several publications suggest the involvement of the mismatch repair proteins Msh2, Msh3, Msh6 or Pms2 in repeat instability in transgenic mice (Manley et al., 1999; van den Broek et al., 2002; Savouret et al., 2003; Gomes-Pereira et al., 2004) and large scale expression micro-array data shows expression of these proteins in hESC (Skottman et al., 2005). Recently, Allegrucci et al. (2007) reported about a high distortion of the epigenetic status in six independent hESC lines. Most epigenetic changes arose in the earlier stages post-derivation but continued further in time as the culture prolonged. In this context, the methylation status of the CTG repeat and the surrounding sequence should be investigated thoroughly and correlated to the repeat instability in consecutive passages. The methylation status of the DMPK region and the activity of mismatch repair enzymes or DNA replication proteins in hESC will be the subject of a future study.
The stepwise increases of the CTG repeat in time models some aspects of repeat instability in vivo. Although the appearance of large contractions and expansions complicates the study of the mechanisms involved in repeat instability, hESC carrying expanded repeat tracts still represent valuable in vitro cell cultures of human, pluripotent cells for long-term studies of repeat instability.
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The research work was supported by grants from the Fund for Scientific Research, Flanders (FWO-Vlaanderen), a Concerted Research Action (GOA) and the Research Council (OZR) of the VUB, the Fund Alphonse and Jean Forton and Téléthon Belgium.
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We are grateful to the colleagues of the hESlab for the support and help in culturing the lines and especially to Ileana Mateizel for providing us with the RNA and the valuable discussions. We want to thank M. Urbina for the help with karyotyping and the colleagues of the IVF/PGD team for providing us with embryos.
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References |
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Submitted on July 27, 2007; resubmitted on May 20, 2008; accepted on May 23, 2008.
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