Screening for genomic fragments that are methylated specifically in colorectal carcinoma with a methylated MLH1 promoter

Koji Koinuma 1, 2, Ruri Kaneda 1, Minoru Toyota 3, Yoshihiro Yamashita 1, Shuji Takada 1, Young Lim Choi 1, Tomoaki Wada 1, Masaki Okada 2, Fumio Konishi 4, Hideo Nagai 2 and Hiroyuki Mano 1, 5, *

1 Division of Functional Genomics and 2 Department of Surgery, Jichi Medical School, Tochigi 329-0498, Japan, 3 Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Hokkaido 060-8556, Japan, 4 Department of Surgery, Omiya Medical Center of Jichi Medical School, Saitama 330-8503, Japan and 5 CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan

* To whom correspondence should be addressed. Tel: +81 285 58 7449; Fax: +81 285 44 7322; E-mail: hmano{at}jichi.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA–RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.

Abbreviations: CRC, colorectal carcinoma; COBRA, combined bisulfite restriction analysis; CIMP, CpG island methylator phenotype; EGF, epidermal growth factor; GDF, growth-differentiation factor; MSI, microsatellite instability; MMR, mismatch repair; PCR, polymerase chain reaction; TGF-ß, transforming growth factor-ß


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Colorectal carcinoma (CRC) is one of the leading causes of cancer death in humans. Evidence indicates the existence of two major types of genomic instability in CRCs: chromosomal instability and microsatellite instability (MSI) (1). Whereas chromosomal instability is associated with an abnormal DNA content (such as aneuploidy), inactivation of the tumor suppressor gene TP53, and activation of oncogenes (2), MSI is associated with defects in the DNA mismatch repair (MMR) system that result in frameshift mutations in microsatellite repeats, and thereby affect the structure of genes containing such repeats (3).

Although germ-line mutations of MMR genes have been detected in the genome of individuals with hereditary non-polyposis colorectal cancer (46), many sporadic CRCs positive for MSI are associated with epigenetic silencing of non-mutated MMR genes (7,8). MSI-positive CRCs are characterized by specific clinicopathologic features and gene mutations. Such tumors occur with a higher frequency in women than in men, develop in the right side of the colon, and manifest a mucinous or poorly differentiated histopathology. Many of the CpG dinucleotides within the promoter region of the MMR gene MLH1 are methylated in MSI-positive CRCs (9,10), and the BRAF gene frequently contains activating mutations in these cancers (1113). Some genomic fragments have been found to be methylated specifically in such CRCs (7), and an entity of CRC with a CpG island methylator phenotype (CIMP) has been proposed (14). However, the profiles of genes and genomic fragments that become methylated in CRC specimens positive for MLH1 methylation have remained uncharacterized.

With the use of methylated CpG island amplification coupled with representational difference analysis (MCA–RDA) (15), we have now performed a global screening of pooled genomic DNA from CRC specimens positive or negative for MLH1 methylation in order to identify differentially methylated genomic fragments. With this approach, we identified hundreds of CpG islands whose methylation was specific to CRCs with a methylated MLH1 promoter.


    Materials and methods
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 Materials and methods
 Results
 Discussion
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Tumor specimens and cell lines
Tumor specimens were obtained from patients with sporadic CRC who underwent surgical treatment in Jichi Medical School Hospital. Informed consent was obtained from each patient, and the study was approved by the ethics committee of Jichi Medical School. Normal portion of colon tissue was excised from a region >5 cm distant from the cancerous region in every case. Genomic DNA was extracted with the use of a QIAmp DNA Mini kit (Qiagen, Valencia, CA). The MSI status of each tumor was determined on the basis of analysis of nine microsatellite repeat loci as previously described (8). The methylation status of the MLH1 promoter was also examined in each sample (11).

Colon carcinoma cell lines (Caco-2, HCT116, SW480) were obtained from American Type Culture Collection (Manassas, VA). Caco2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 2 mM L-glutamine. HCT116 and SW480 cells were maintained in McCoy's 5A medium (Invitrogen) and Leibovitz's L-15 medium (Invitrogen), both supplemented with 10% FBS, respectively.

MCA–RDA
Genomic DNA from four tumor specimens positive for MLH1 methylation was mixed and used as a ‘tester’ sample, whereas that from four specimens negative for MLH1 methylation was used as a ‘driver.’ MCA–RDA was performed with the two pooled DNA samples as previously described (15). In brief, both the tester and the driver DNA samples (5 µg of each) were digested first with the SmaI endonuclease (New England Biolabs, Beverly, MA) and then with XmaI (New England Biolabs), and the resulting fragments were ligated to the RMCA adapter (15). Amplification of methylated CpG islands was achieved by polymerase chain reaction (PCR) with the RMCA24 primer. The amplified fragments of the tester-DNA and driver-DNA were digested with XmaI and SmaI, respectively. The tester amplicons were then ligated to the JMCA adapter and subjected to annealing with an excess amount of the driver amplicons. PCR with the JMCA24 primer then amplified only the tester-specific amplicons. Another round of amplification was performed with the NMCA adapter and the NMCA24 primer (15). The final products were digested with XmaI and ligated into XmaI-digested pBlueScript (Stratagene, La Jolla, CA) for nucleotide sequencing.

Combined bisulfite restriction analysis (COBRA)
The methylation status of isolated clones was tested by the COBRA method (16). The genomic DNA was denatured, incubated for 16 h at 55°C in 3.1 M sodium bisulfite, and then subjected to PCR to amplify CpG islands. The PCR products were digested with a restriction endonuclease, and the resulting fragments were fractionated by polyacrylamide gel electrophoresis (PAGE). The gel was stained with SYBR Green I (Takara Bio, Shiga, Japan) and scanned with an LAS3000 imaging system (Fuji Film, Tokyo, Japan). Genomic fragments were determined to be positive for CpG methylation if ≥10% of the PCR products were cleaved by the restriction endonuclease. The PCR primers and endonucleases used for COBRA are shown in the Supplementary Table online.

For bisulfite sequencing of the BMP3 promoter, genomic DNA isolated from cancer specimens or cell lines was treated with sodium bisulfite (11) and then subjected to PCR with the primers 5'-AGTTAGAGAGYGAAAGAATTAAG-3' and 5'-ATACAACRAAATAACRACCAACC-3'. The PCR product was ligated into pGEMT-easy (Promega, Madison, WI).

Real-time reverse transcription-polymerase chain reaction (RT–PCR)
Total RNA was prepared from tester or driver samples with an RNeasy Mini column (Qiagen), treated with RNase-free DNase (Qiagen), and subjected to reverse transcription with PowerScript reverse transcriptase (BD Biosciences Clontech, San Jose, CA) and an oligo(dT) primer. Portions of the resulting cDNAs were subjected to PCR with a QuantiTect SYBR Green PCR kit (Qiagen). The amplification protocol comprised incubations at 94°C for 15 s, 63°C (64°C for KIT cDNA) for 30 s, and 72°C for 60 s. Incorporation of the SYBR Green dye into PCR products was monitored in real time with an ABI PRISM 7900HT sequence detection system (PE Applied Biosystems, Foster City, CA), thereby allowing determination of the threshold cycle (CT) at which exponential amplification of PCR products begins. The CT values for DNA molecules corresponding to the ß-actin gene (ACTB) and to genomic fragments of interest were used to calculate the abundance of the latter relative to that of the former. The oligonucleotide primers for PCR were 5'-CCATCATGAAGTGTGACGTGG-3' and 5'-GTCCGCCTAGAAGCATTTGCG-3' for ACTB, 5'-AAGTCAACTCCTTGGCCATCTGT-3' and 5'-TGGAAAAGGTAACCTCCTCTTTGG-3' for the bone morphogenetic protein 3 gene (BMP3), and 5'-TGACGTCTGGTCCTATGGGATTT-3' and 5'-TACATTTCAGCAGGTGCGTGTTC-3' for KIT.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
MCA–RDA screening
A total of 249 cases with CRC were examined for their MSI status as well as for methylation in the promoter region of MLH1. The majority (n = 213) of the tumor specimens from these patients were negative for MSI, while the rest (n = 36) were MSI-positive. Also, most of them (n = 226) were shown to have no methylation within the MLH1 promoter (unmethylated group), whereas the remainder (n = 23) had a methylated promoter (methylated group) (11). Altogether 16 patients were positive for MSI, but did not have a methylated MLH1 promoter. On the other hand, three patients were negative for MSI despite the presence of the methylated MLH1 promoter. The characteristics of the patients examined in the present study are summarized in Table I.


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Table I. Patient characteristics

 
To isolate genes that were specifically methylated in the methylated group, we selected specimens from four men of each group: ID nos 2, 17, 20 and 77 (mean age, 70.0 years) from the methylated group, and ID nos 1, 8, 13 and 31 (mean age, 73.0 years) from the unmethylated group. We have excluded female subjects from the initial screening, since an intense methylation of one X chromosome in female cells may have yielded a large number of pseudopositive clones, methylation status of which may not have linked to the clinical classes, but to lyonization.

Equal amounts of genomic DNA isolated from the four selected tumor specimens of each group were mixed and subjected to MCA–RDA analysis, with the pooled DNA of the methylated group as the tester and that of the unmethylated group as the driver. A total of 384 clones were randomly picked up from the resultant MCA–RDA products, and digestion of the purified plasmid DNA with restriction endonucleases revealed that 294 out of the 384 clones carried the insert fragments. Nucleotide sequencing of such 294 clones indicated that 209 of the clones were found to contain CpG islands. Screening of human genome databases (http://www.ncbi.nlm.nih.gov/BLAST/ and http://genome.ucsc.edu/cgi-bin/hgBlat) with these sequences revealed that 186 of them were localized within or in close proximity to characterized or uncharacterized human genes (112 independent genes).

Candidate genes for differential methylation
The GenBank accession numbers and annotation information for the 112 genes identified by MCA–RDA are shown in Table II. Multiple clones were isolated for a single gene in 33 instances, whereas only one MCA–RDA product was obtained for the remaining genes. The genes listed in Table II were thus candidates for genes whose CpG islands are methylated in a manner dependent on MLH1 methylation.


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Table II. Genes identified by MCA–RDA with CRCs positive or negative for MLH1 methylation

 
We then examined the methylation status of the isolated fragments mapped to the promoter regions. First, we tried to amplify individually by PCR all 70 fragments mapped to the promoter regions in Table II, and could successfully amplify 35 fragments from the pooled DNAs used in MCR–RDA. By using COBRA, their CpG methylation status was assessed among the samples used in MCR–RDA. As indicated in Table II, 28 fragments out of 35 were preferentially methylated in the tester DNA, while 7 of them were not.

The methylation status of such 28 fragments was further tested in clinical specimens that had not been used for the initial screening. This test set included eight cancer specimens positive for MLH1 methylation and the paired normal colon tissue as well as eight cancer specimens negative for MLH1 methylation (Table I).

The methylation status of each genomic fragment in the clinical specimens is shown color-coded in Figure 1; fragments with a methylation level of ≥10% as determined by COBRA are indicated in red, whereas those with a methylation level of <10% are shown in blue. Most genomic fragments were extensively methylated in most or all of the cancer specimens positive for MLH1 methylation, but not in those negative for MLH1 methylation. The difference in CpG methylation for the MCA–RDA products between the methylated and unmethylated groups of patients was thus confirmed in a distinct test set of CRC specimens.



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Fig. 1. Gene methylation profiles of CRC specimens. Twenty-eight clones were randomly chosen from the MCA–RDA products of the selected study specimens, and their methylation status (plus that of MLH1) was determined by COBRA in CRC specimens positive for the methylation of the MLH1 promoter (n = 8), their paired normal colon tissue samples, and CRC specimens negative for MLH1 methylation (n = 8). Each column represents a clinical specimen (ID numbers are shown), and each row indicates a gene corresponding to an MCA–RDA product. Red box, methylated gene; blue box, unmethylated gene; white box, not examined.

 
A more detailed inspection of the data in Figure 1, however, indicates that the MCA–RDA products can be separated into three subgroups on the basis of their methylation profiles. The genomic fragments in the first group (MIG2 to NPHS2 in Figure 1) were also methylated in ≥25% of the paired normal colon tissue samples. The fragment corresponding to MIG2, for instance, was methylated in all of the MLH1 methylation-positive cancer specimens and the respective normal tissue. Methylation of these genomic regions thus probably occurred in each patient before the development of CRC and might be related to the aging process.

The genomic fragments in the second group (BMP3 to DPYSL3) were not methylated in normal colon tissue but were methylated in ≥25% of cancer specimens that were negative for MLH1 methylation. The methylation of these fragments thus appeared to be specific to the cancerous state with a slightly increased prevalence among MLH1 methylation-positive CRC.

The fragments in the third group (MGC29643 to IMAGE5728979) were methylated in <25% both of normal specimens and of cancer specimens negative for MLH1 methylation. The methylation of these genes thus appears to be regulated in concert with that of MLH1.

Analysis of BMP3
Among the genomic clones analyzed, we first focused on that corresponding to BMP3. BMP3 is a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins that also includes TGF-ß1, TGF-ß2, TGF-ß3, Mullerian inhibitory substance, BMP2A, BMP2B, BMP6, growth-differentiation factor (GDF) 5, GDF6 and GDF7 (17,18). Members of this protein superfamily exert inhibitory effects on various human cancers through activation of their cognate receptors and SMAD proteins (19,20). BMP2, for instance, induces both the activation of the p38 isoform of mitogen-activated protein kinase and apoptosis in medulloblastoma cells (21). Although little is known of the physiological functions of BMP3, it is possible that this protein also possesses antitumor activity and that its expression is epigenetically regulated in cancer cells. Interestingly, Dai et al. (22) have recently reported that BMP3 promoter is methylated frequently (~50%) in non-small-cell lung carcinoma, which many imply that dysfunction of BMP3 may be commonly involved in the carcinogenesis of a wide range of human tumors.

The COBRA assay revealed that the MCA–RDA clone corresponding to the promoter region of BMP3 was methylated in CRC specimens that were positive or negative for MLH1 methylation (Figures 1 and 2A). Further, as shown in Figure 2B, detailed analysis of the methylation status of the BMP3 promoter by sequencing of DNA fragments after sodium bisulfite treatment revealed extensive hemi- or biallelic methylation of the promoter in CRC specimens positive for MLH1 methylation (ID nos 225, 318 and 481) but not in one negative for MLH1 methylation (ID no. 249). CpG methylation throughout the promoter fragment was also evident in CRC cell lines positive (HCT116) or negative (Caco2) for MLH1 methylation, but not in the MLH1 methylation-negative line SW480. Together with the COBRA data in Figure 1, these results suggest that the promoter region of BMP3 is methylated in all clinical specimens and cell lines positive for methylation of the MLH1 promoter as well as in some specimens and cell lines negative for MLH1 methylation.



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Fig. 2. Methylation status of the BMP3 promoter and BMP3 expression in CRC specimens. (A) The methylation status of the promoter region of BMP3 in the indicated clinical specimens was examined by COBRA. Sensitivity of PCR products to digestion with TaiI is indicative of methylation of the CpG island examined. (B) Genomic DNA of the indicated clinical specimens and CRC cell lines (Caco2, HCT116 and SW480) was treated with sodium bisulfite, after which the BMP3 promoter region was amplified by PCR and sequenced. Closed and open circles indicate methylated and unmethylated CpG islands, respectively. The nucleotide positions of the CpG islands (numbered relative to the transcriptional start site) are indicated at the bottom, and the TaiI digestion site for COBRA in (A) is shown by the arrow. (C) The level of expression of BMP3 relative to that of ACTB in clinical specimens was determined by quantitative RT–PCR. (D) HCT116 cells were incubated for 72 h with or without 0.2 µM of 5'-azacytidine, and were then subjected to RT–PCR analysis for determination of the amount of BMP3 mRNA relative to that of ACTB mRNA.

 
We then examined whether the epigenetic changes in the BMP3 promoter affected its transcriptional activity. Quantitative real-time RT–PCR analysis revealed that BMP3 was transcriptionally silent in CRC specimens positive for MLH1 methylation (Figure 2C), in which the BMP3 promoter was also extensively methylated. In contrast, BMP3 mRNA was abundant in the paired normal colon tissue samples. Although BMP3 expression was detected in some CRC specimens negative for MLH1 methylation, the level of expression was greatly reduced compared with that in normal colon tissue. These data thus indicate that BMP3 transcription is suppressed in most CRCs.

In order to directly examine the relationship between promoter methylation and gene silencing of BMP3, HCT116 cells were incubated for 3 days with 5'-azacytidine, an inhibitor of de novo methylation of genomic DNA. Interestingly, treatment with 5'-azacytidine markedly induced the amount of BMP3 mRNA in the cells (Figure 2D) and demethylation of its promoter region as well (data not shown). Therefore, extensive methylation of the BMP3 promoter region should be directly linked to the suppression of its transcription.

Analysis of KIT
KIT encodes a receptor tyrosine kinase for stem cell factor. Point mutations in KIT that increase the kinase activity of the encoded protein have been identified in human gastrointestinal stromal tumors (23), suggestive of a causative role for KIT in these tumors. The expression and activation status of KIT in CRCs have been unclear, however (24,25). We therefore analyzed the methylation status of the KIT promoter region in our samples.

Methylation of the KIT promoter was highly restricted to CRC specimens positive for MLH1 methylation (Figures 1 and 3A). However, the abundance of KIT mRNA did not necessarily match the methylation status of the KIT promoter. Despite extensive methylation of the promoter in one CRC sample (ID no.77), for instance, the amount of KIT mRNA was relatively high (KIT/ACTB mRNA ratio = 4.78 x 10–5), indicating that promoter methylation might not be a major determinant of transcriptional activity. It is possible, however, that our COBRA analysis revealed the methylation status of a CpG site that is not important for KIT transcription.



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Fig. 3. Methylation status of the KIT promoter and KIT expression in CRC specimens. (A) The methylation status of the promoter region of KIT in the indicated clinical specimens was examined by COBRA. Sensitivity of PCR products to digestion with HhaI is indicative of methylation of the CpG island examined. (B) The level of expression of KIT relative to that of ACTB in clinical specimens was determined by quantitative RT–PCR.

 
The mean expression level of KIT in the MLH1 methylation-positive CRC specimens [KIT/ACTB mRNA ratio, 1.72 x 10–5 ± 3.02 x 10–5 (mean ± SD)] was significantly lower than that in normal colon tissue (6.03 x 10–5 ± 6.29 x 10–5; P = 0.038, Student's t-test). The level of KIT expression in CRCs negative for MLH1 methylation (2.92 x 10–5 ± 1.61 x 10–5) was also lower than that in normal colon tissue, but this difference was not significant (P = 0.123). It is therefore likely that KIT is not overexpressed in CRCs.


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 Abstract
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 Materials and methods
 Results
 Discussion
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We have screened for genomic fragments whose CpG islands are selectively methylated in CRC specimens positive for methylation of the MLH1 promoter region. We could readily identify hundreds of genomic fragments with CpG islands that were expected to be differentially methylated between CRCs with or without MLH1 methylation. Twenty-eight such clones (Table II) were indeed proved to be preferentially methylated in the four CRC specimens positive for MLH1 methylation compared with the four samples negative for MLH1 methylation, both of which were used in the original MCA–RDA screening (data not shown).

To verify the selective methylation of these clones, we performed COBRA with a different set of specimens including eight CRCs with MLH1 methylation and their paired normal tissue samples as well as eight CRCs without MLH1 methylation. Although all the 28 clones examined were preferentially methylated in the CRC specimens positive for MLH1 methylation, their methylation profiles among the specimens were not identical, indicating that all CpG methylation observed in MSI-positive CRCs was not specific to this subtype of tumor.

The methylation of certain genomic fragments (14 out of 28 clones examined), however, was highly specific to CRCs that manifested MLH1 methylation. Almost 50% of the genes were thus methylated in a parallel manner to the CpG methylation of the MLH1 gene, indicating that a subset of genes is specifically methylated in a subset of CRCs. Our data thus support the existence of CIMP-positive CRCs (14), while it would be mandatory for the better characterization of CIMP-positive tumors to further collect co-methylated genes and to define precisely the hallmark genes for the identification of CIMP (26). It would be interesting to examine whether such clearly defined CIMP is associated with certain clinical manifestations.

Genes corresponding to the co-methylated genomic fragments in our assay included those whose function relates to cell proliferation or differentiation. The predicted structure of NELL2, for example, contains epidermal growth factor (EGF)-like repeats (27), which are present in diverse proteins involved in regulation of the cell cycle, cell proliferation, and developmental processes. NTAK is a member of the EGF family of proteins and is a ligand and activator of ErbB protein tyrosine kinases (28). In addition, GDF7 is a member of the TGF-ß superfamily (18), and TCF7L1 is highly homologous to TCF1 which is a target gene of the Wnt-ß-catenin signaling pathway (29), and which plays an important role in CRC carcinogenesis. Aberrant epigenetic regulation of these genes may thus contribute to the pathogenesis or clinical features of CRCs positive for MLH1 methylation.

The MCA–RDA method thus proved to be highly effective for the identification of differentially methylated genes among fresh clinical specimens. Given the high fidelity of this approach, it is likely that a large number of genes (or genomic fragments) are methylated in CRCs in concert with methylation of the MLH1 promoter. Our study provides a basis for further characterization of the molecular pathogenesis of CRCs classified as MSI. Together with the results of other studies (7,30), it also suggests the possibility of development of a stratification scheme for CRCs based on genome methylation profile.


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Supplementary material can be found at: http://www.carcin.oxfordjournals.org/


    Acknowledgments
 
This study was supported by a Grant-in-Aid for Third-Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor, and Welfare of Japan, and by a grant for ‘High-Tech Research Center’ Project for Private Universities: Matching Fund Subsidy (2002–2006) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Conflict of Interest Statement: None declared.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received April 29, 2005; revised July 10, 2005; accepted July 12, 2005.





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