Identification of DNA methylation changes associated with human gastric cancer
© Park et al; licensee BioMed Central Ltd. 2011
Received: 11 August 2011
Accepted: 2 December 2011
Published: 2 December 2011
Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult.
We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay) in combination with a genome analyzer and a new normalization algorithm.
We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs), transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES) regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ) within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue.
Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.
Gastric cancer is the second leading cause of cancer deaths worldwide after lung cancer, resulting in more than 800,000 deaths worldwide every year . The current 5-year survival rate of individuals diagnosed with gastric cancer is only 20-30%, with this low rate being attributable to the fact that most cases are already in an advanced stage when diagnosed. As in all cancers, early detection remains the most promising approach for improving the survival rate. Hence, understanding the cause of tumorigenesis in human gastric tissue is essential.
Infection with H. pylori is a well-established and common cause of gastric cancer. However, alterations in various genetic factors are also important in increasing gastric cancer risk. It is well known that chromosomal instability originating from genetic factors such as microsatellite instability as well as KRAS and p53 mutations result in the development of tumors. Several genomic studies have identified germline mutations in specific genes [2–4] and disease susceptible loci [5, 6] for gastric cancer. Recent studies comparing gastric cancer and normal tissue have identified a number of genetic markers, including diagnostic markers [NF2, INHBA, SFRP4], prognostic markers [CD9, CDH17, PDCD6], and gastric cancer-associated genes [MUC13, CLDN1, Ki67 and CD34]. In addition, epigenetic mechanisms such as DNA methylation and histone modifications have been found to be important in regulating the expression of genes involved in the biology and disease of the gastrointestinal tract .
DNA methylation plays an essential role in eukaryotes and is associated with a number of key mechanisms including genomic imprinting, X chromosome inactivation, aging, and carcinogenesis. Alteration of DNA methylation in the genome is found in almost all types of cancer and can lead to changes in gene expression, such as over-expression of oncogenes and silencing of tumor suppressor genes during cancer development . Several studies have shown that accumulation of genetic and epigenetic alterations in gastric precancerous lesions may affect a large number of targets, such as DNA repair system components, tumor suppressors, oncogenes, cell cycle regulators, growth factors, and adhesion molecules [17–20]. However, these studies have been primarily focused on a few candidate genes or covered only a portion of the whole genome. Thus, accessing a global view of the epigenetic changes associated with cancer development has been difficult. In particular, understanding DNA methylation changes in the intragenic regions, CpG islands, intergenic regions, and repeat sequences remains limited. Consequently, there is great interest in genome-wide analysis of aberrant DNA methylation in these regions.
For comprehensive genome-scale profiling of DNA methylation in embryogenesis and carcinogenesis, high-resolution whole genome sequencing methods such as BS-seq [21–24], MeDIP-seq [25, 26], and MethylCap-seq [27–29] have been developed. Despite the rapid development of sequencing-based mapping technology, there is still a lack of comparative research, which is critical for clinical epigenomics studies, including those focused on cancer. Unlike microarray-based approaches, sequencing data are produced in a format that is not amenable to differential analysis, and the analysis workflow has not been standardized. Hence, computationally inexpensive normalization methods are needed to handle the computational burden of processing large-size, high-resolution sequencing data.
Here, we introduced a normalization algorithm, which takes into account the sample-specific total read density, the spatial distribution of CpG loci, and background sequencing bias. We then created a comprehensive whole-genome methylome of normal gastric tissue, gastric cancer tissue, and metastatic lymph nodes using the MethylCap-seq method and obtained detailed information on its perturbation during carcinogenesis and metastasis. This is readily applicable to a comparative analysis of methylomes and other types of epigenomic data, and it has particular implications for clinical epigenomics.
Gastric tissue samples
We obtained three snap-frozen gastric tumors and matched normal gastric tissue from Seoul National University College of Medicine for methylome study. Additionally, twenty-eight matched pairs of normal and tumor stomach tissues were obtained for further confirmation. All samples were obtained by endoscopic resection during examination of the patients who gave informed consent.
Methylated DNA recovery assay (MIRA)
Genomic DNA from 25 mg of gastric tissue was purified by using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). Genomic DNA samples from 3 individuals were pooled at the same concentration. MIRA was carried out as previously described [30–32]. Briefly, GST-tagged MBD2b and His-tagged MBD3L1 proteins were prepared as described. 15 ug of genomic DNA was fragmented to 100 ~ 500 bp by sonication and incubated with 28 ug of purified GST-MBD2b protein, 28 ug of His-MBD3L1 protein and 7 ug of JM110 Bacterial DNA for 6 hours. 30 ul of MagneGST beads (Promega, Madison, WI) preblocked with 7 ug of JM110 bacterial RNA were added and incubated at 4°C with rotating for 45 minutes in final 600 ul of MIRA binding reaction mixture. Beads were washed three times with 1 ml of washing buffer, and methylated fragments were eluted by incubation at RT for 5 minutes and then 56°C for 30 minutes with 30 ul of TE containing RNase A (100 ug, Qiagen) and Proteinase K (15 ug, Qiagen). Eluted DNA fragments were further purified by using Qiaquick PCR purification kits (Qiagen).
Illumina Genome Analyzer sequencing
We used 10 ng of eluted DNA for Illumina Genome Analyzer sequencing. Following ligation of a pair of Solexa adaptors, ligation products with the maximum insert size of 200 bp were gel purified on 2% agarose and subjected to PCR amplification. Cluster generation and 36 cycles of sequencing were performed following the manufacturer's instructions. We sequenced 120 ul of adaptor-ligated, size-fractionated DNA (2 ~ 4 pM) on the Illumina Genome Analyzer. Sequence tags were mapped to the human genome (UCSC hg18 database based on NCBI Build 36.1 assembly) using the Solexa Analysis Pipeline (version 0.3.0). Sequenced reads of 34 bp (excluding the first and last nucleotide) that passed quality control filters were used.
Data processing and MES calculation
We extended the 3' end of the 34-bp reads by 200 bp to cover DNA fragments bound by the MBD proteins. The readout was converted to browser extensible data (BED) files for visualization in the UCSC genome browser http://genome.ucsc.edu/. We counted overlapping sequence tags at 50 bp resolution. To find enriched genomic regions, the number of mapped reads in a sliding window of 1 kb was compared to the total number of reads or the background number of reads in the genome. As such, MES was calculated in two ways; one is as the log2 of (target read count/target size)/(total read count/genome size) and floored to zero, the other is as the log2 of (target read count/target size)/(background read count/background size) and floored to zero. To adjust for background sequencing bias, MESbg was calculated in the same manner for input sequencing without affinity purification and subtracted from MES.
Genomic positions of CGIs, promoters, transcript bodies, CDSs, and repetitive elements
All genomic positions of CGIs, transcripts and repeat elements were downloaded from the UCSC genome browser. A total of 27,639 CGIs (except randomly located CGIs) were predicted by the following criteria: GC content of 50% or greater, length greater than 200 bp, and ratio greater than 0.6 of observed number of CpG dinucleotides to the expected number . The NCBI mRNA reference sequences collection (RefSeq from release version 46; March 11, 2011) was used for identifying transcription units with the defined transcription start, end sites and CDS start, end sites. For promoters, we used the region 500 bp upstream ~ 500 bp downstream of the transcription start site. We obtained ~ 5 million repeat locations that had been determined by the RepeatMasker program based on the RepBase library of repeats.
Methylation level of genomic elements
The methylation level of a CGI, promoter, gene-body, and repeat element was estimated by means of MES overlapping each element. MES = 0 was used to define unmethylated elements. To measure hypermethylation or hypomethylation in cancer, we calculated the differential MESs as (Cancer MES - Normal MES). Differential MES > 1.0 was used as a threshold. To understand the functions of selected genes, we used the ontology classification of genes through the DAVID Functional Annotation Clustering tool http://david.abcc.ncifcrf.gov/.
Gene expression analysis
The microarray product used in this study was Codelink Human Whole Genome 55 K chip (GE Healthcare, USA). All experimental procedures including cRNA target preparation, hybridization, post-hybridization dye coupling were performed using vendor recommended protocols. The result files were imported into GeneSpring GX 7.3 (Agilent Technologies, USA) for filtering and basic statistical analysis. Among 55 K genes on the microarray, only the genes with present flags in at least 50% of samples were selected for subsequent analysis. The microarray data were deposited at the GEO http://www.ncbi.nlm.nih.gov/geo/ (accession number GSE33651).
MIRA and real-time qPCR
MIRA was performed on four additional individual samples. DNA was purified from the supernatant and monitored by real-time qPCR using Roche 480 machine. The sequences of used primers are presented in Additional file 1: Table S1.
Bisulfite treatment, methylation-specific PCR and pyrosequencing
We isolated the genomic DNA from individual sample by using a Qiagen DNeasy Tissue Kit (Qiagen). Bisulfite treatment was carried out using the EZ DNA methylation gold kit (Zymo research) according to the manufacturer's instructions. Bisulfite-treated DNA was stored at -80°C until further use. The primers used for MSP were designed using Methprimer , and are shown in Additional file 1: Table S1. PCR was performed with HotStarTaq Polymerase (Qiagen) and included an initial incubation at 95°C for 15 min, followed by 40 cycles of 95°C for 1 min, 59°C for 1 min and 72°C for 40 sec, followed by one cycle of 72°C for 10 minutes. MSP products were separated on 2% agarose gels and visualized by ETBR staining. The pyrosequencing reactions were automatically performed with a PSQ 96 system (Pyrosequencing AB) according to the manufacturer's instructions. Briefly, the biotinylated PCR product (50 ul) was purified by using streptavidin-sepharose beads (Amersham Biosciences). The purified product was loaded into the reagent cartridge with the enzyme, substrate and dNTP included in the PSQ96 SNP Reagent Kit (Pyrosequencing AB). The sequencing primers for pyrosequencing are shown in Additional file 1: Table S1.
Processing of MIRA-seq methylome data
We purified the methylated DNA enriched through MIRA (methylated CpG island recovery assay) and sequenced the DNA using next-generation sequencing. DNA methylation levels were determined using sequencing read counts of the corresponding regions, at 50 bp intervals, as described under Methods. We created DNA methylation maps for both normal and cancerous gastric tissues. For each sample, we obtained about 10 million sequence reads (Additional file 1: Table S2). Each methylome contained ~140 million CpG reads, covering ~48% of all genomic CpG sites excluding centromeres (Additional file 1: Table S3). The average coverage of CpG reads in each methylome was 4.5X. In support of the high sensitivity of MIRA, genomic segments containing only one CpG had higher read counts than those with no CpG (p value = 0), suggesting that single CpG changes could be resolved using MIRA. The average sequence reads increased in proportion to the number of CpGs within a 50-bp interval, and in fact, MIRA coverage was not low, even for regions of low CpG density (Additional file 2: Figure S1). Taken together, these results show that MIRA was successful in recovering a sufficient fraction of methylated regions. As for the accuracy of MIRA, ~99% of MIRA-captured fragments had at least one CpG site within their sequence, indicating a low false detection rate.
To measure enrichment of local methylation signals, we calculated methylation enrichment scores (MESs) by obtaining a read count in a given region and then performing normalization to control for the total read count (MESt) in the sample (global normalization) or the local read count (MESl) in a user-defined surrounding region (local normalization) (see Methods). This enables a direct comparison of independent samples with different read density. We then carried out a logarithmic transformation of the derived score. Along with having other mathematical merits, this provides the benefit of variance stabilization, particularly for high read counts, which are often coupled with high technical variations that may introduce significant bias in the data.
We assessed the statistical significance of the MES in two ways. Randomized MESs were generated numerically by permuting the genomic positions of our sequence reads. The background MES (MESbg) was experimentally obtained by sequencing the normal genome without affinity purification. As expected, the real data yielded markedly higher enrichment scores (Additional file 2: Figure S2). Notably, MESbg was higher than MES from randomized genomes, an indication that background sequences alone can create enrichment, probably due to chromatin accessibility and amplification bias. Consistent with recent reports , this illustrates the need for a proper calibration for inherent sequencing bias. Therefore, we normalized our MES with MESbg.
To find the optimal condition for normalization, we compared the statistical fitness of various normalization methods. Tag distribution along the genome can be modeled by the Poisson distribution [36, 37]. The goodness of fit was tested using the Kolmogorov-Smirnov test. In this test, a low D statistic indicates a good fit. While the Poisson model outperformed the Gaussian overall, the MES showed a better fit than raw read counts (Additional file 2: Figure S3), illustrating the rare event nature of the log-scaled read count measure. The normalized MESl calibrated by control sequencing (MESbg) yielded even better results than normalized MESt calibrated by control sequencing (MESbg).
Global and chromosomal views of DNA methylation
Generally, CGIs tend to remain methylation free in normal tissue. To analyze the high methylation patterns of CGIs, we checked the average MES distribution and found a slightly bimodal pattern (Figure 1C). About 66% (11,376/17,284) of CGIs in the left peak overlapped with a promoter (1 kb by our definition). In contrast, 13% (1,386/10,357) of CGIs in the right peak overlapped with a promoter, suggesting that most promoter-associated CGIs are unmethylated. In contrast to promoter-related CGIs, promoter-independent CGIs were heavily methylated (Figure 1C). Although most CGI-positive promoters were not methylated, CGI-negative promoters showed relatively high methylation levels (Figure 1D). We also checked the methylation level of promoters by CpG density as previously defined  (Additional file 3: Table S4). The methylation pattern of promoters was inversely related to CpG density (Additional file 2: Figure S4). On the other hand, CGI-containing gene bodies had higher methylation levels than those without CGIs (Figure 1D).
Human genome and normal sample information of genic and intergenic region
Human Genome Information
Normal Sample Information
Relative Enrichment Ratio
# of CpG
vs. CpG Count
Human genome and normal sample information of gene annotated regions
Human Genome Information
Normal Sample Information
Relative Enrichment Ratio
# of CpG
vs. CpG Count
Upstream 1 kb
Downstream 1 kb
Changes in DNA methylation patterns associated with gastric cancer
The region centered at the transcription start site showed completely different patterns depending on the presence of a CGI, reflecting the low methylation status of CGI-containing promoters (Figure 2C). We also found that, in cancerous tissue, remarkable hypermethylation of CGI-containing promoters occurs and that the density of CpGs is crucial for the increase in DNA methylation (Figure 2C). To further analyze whether 5' regions of genes were hypermethylated similarly to gene promoters, we checked the methylation pattern of the first exons. Interestingly, we found that the first exon was hypermethylated only when it was the 5'-end of a coding exon, but not when it was a 5' UTR exon (Figure 2D). These regions also contained high CpG density. Therefore, CGIs at the upstream regions of genes, the promoter, and the coding start appear to be the major targets of DNA hypermethylation in cancer.
Methylation pattern of CpG islands
Functional annotation clustering of genes with hypermethylated 5'CGIs
Annotation Cluster 1
Enrichment Score: 3.27
protein-DNA complex assembly
Annotation Cluster 2
Enrichment Score: 2.92
Annotation Cluster 3
Enrichment Score: 2.16
Homeobox, conserved site
Annotation Cluster 4
Enrichment Score: 2.02
brain-expressed X-linked protein
Brain expressed X-linked like protein
Annotation Cluster 5
Enrichment Score: 1.97
Annotation Cluster 6
Enrichment Score: 1.78
Basic helix-loop-helix dimerisation region bHLH
Annotation Cluster 7
Enrichment Score: 1.77
cell-substrate adherens junction
Annotation Cluster 8
Enrichment Score: 1.55
regulation of B cell proliferation
positive regulation of B cell activation
regulation of B cell activation
Annotation Cluster 9
Enrichment Score: 1.55
PIRSF500606:homeotic protein Hox D4
PIRSF002612:homeotic protein Hox A5/D4
Homeobox protein, antennapedia type
Annotation Cluster 10
Enrichment Score: 1.38
substrate specific channel activity
passive transmembrane transporter activity
DNA methylation of repetitive elements
In addition to the hypomethylation patterns of the SINE and LINE, a significant reduction in methylation was found in SVA (SINE-VNTR-Alus), satellites, and LTR. This is consistent with previous reports about cancer-specific hypomethylation . Alu elements are the most abundant class of repetitive elements in the human genome, with these elements having over one million copies and spanning over 30 lineages. AluS and AluY elements, which are younger subfamilies, were significantly hypomethylated when compared with older subfamilies, as previously reported  (Figure 4B). SVA elements, which have been extensively mobilized in the human genome, consist of a combination of sequences derived from other retroelements .
To understand if a correlation may exist between the degree of methylation changes and genomic location, we analyzed the methylation of repetitive elements based on their genomic location (i.e., promoter, gene body, or intergenic), even though it is unclear if repetitive elements participate in regulating gene expression (Figure 4C). We found that methylation changes were higher in gene-associated repetitive elements than in gene-independent repetitive elements. This suggests that a correlation exists between methylation changes in repetitive elements and the expression of adjacent genes.
Long-range epigenetic silencing (LRES) in gastric cancer
The LRES region around the 12q14 site spanned about 2.7 Mb and harbored about 21 genes. Among these genes was MDM2, which encodes a protein that is considered to be a negative regulator of p53 and a major regulator of cancer development. Promoters of genes in this LRES region displayed tumor-specific hypermethylation (p = 0.03, t-test) (Figure 5B). To determine whether genes in this LRES region show concordant gene silencing, we re-analyzed publicly available expression datasets (GSE27342) for differential gene expression in clinical samples. Consistent with previously reported expression patterns of LRES regions, the public data for this LRES region showed common gene suppression (p = 0.05, t-test) (Figure 5C).
To determine if the hypermethylation pattern of genes within this LRES region is commonly present in gastric cancer, we examined the methylation enrichment frequency of selected genes (Figure 5D). The methylation enrichment levels of cancer samples were over 2-fold higher than that of normal samples. However, one of the patients showed a low methylation level at several target sites (Additional file 2: Figure S9). Because the amplification of this region frequently occurs in many cancers, we examined the amplification frequency of several genes within these regions using real-time PCR. Intriguingly, we detected amplification of MDM2 (Additional file 2: Figure S10), suggesting that an interaction exists between DNA methylation and gene amplification. To examine the generality of MDM2 methylation, we used pyrosequencing to analyze the methylation level of a specific locus in an upstream region of MDM2. We analyzed MDM2 methylation in normal and cancer tissue samples from other 28 gastric cancer patients. Out of 28 independent samples, most patients showed higher MDM2 methylation (Figure 5E), while four samples showed decreased levels of methylation, along with gene amplification (Additional file 2: Figure S11). Therefore, the MDM2-containing region appears to be hypermethylated in cancer in general, but our results suggest that gene amplification in this region interferes with methylation. Therefore, except for cases with gene amplification, LRES across 12q14 appears to be a distinct epigenetic pattern associated with gastric cancer.
Methylation patterns in metastatic lymph nodes
Here we demonstrate a comprehensive methylation map of human gastric tissue at high resolution. Our data provide a global view of a mammalian methylome, along with several intriguing findings, some of which are novel and worth further investigation. First, we found that hypermethylation of CGIs in promoters is an important epigenetic feature that dictates gene expression changes in cancer. Second, hypermethylation of the 5'-end of coding exons arises in cancer and appears to play an important role in cancer progression. Third, cancer-induced methylation changes in younger repetitive elements and LRES have potential clinical implications in terms of early detection and therapeutic design.
Among the genes analyzed in this study was MDM2, which encodes an important negative regulator of p53. MDM2 and p53 are known to regulate one another through a feedback loop . MDM2 overexpression is frequently detected in many human cancers, suggesting that MDM2 overexpression may be one of the common features of tumorigenesis. In this study, we showed that the upstream region of MDM2 is hypermethylated in most cancer samples. We also found that, in some cancer samples, hypomethylation occurred along with MDM2 amplification at the same site, suggesting that there is major dysregulation of the MDM2-mediated pathway at both the genetic and epigenetic levels. This appears to cause aberrant early tumor cell development and subsequently cancer.
HOX genes cluster on chromosomes 2, 7, 12, and 17, and they are frequently inactivated by CpG hypermethylation in several cancers [40, 45, 46]. Accordingly, we found that many HOX genes were hypermethylated, indicating that HOX gene clusters may be general targets of epigenetic alterations during tumorigenesis. However, hypomethylation of a few HOX genes was also detected in gastric cancer. Therefore, the methylation of HOX genes may be regulated in a tissue-specific manner in cancer. In addition, the regulation of HOX expression is significantly correlated with histone modifications and interaction with Polycomb group genes [47, 48]. Our data point to another possible mode of regulating HOX gene expression-- DNA hypermethylation of the promoter region of histone genes such as H2B. Further investigation of histone genes might offer new insights for cancer studies.
DNA hypomethylation of repetitive elements as a major contributor to genome size is one of common features in cancer and the methylation changes in SINE, LINE, or LTR-retrotransposon with possess transcriptional activity are critical for cellular functions. A number of SINEs close to CpG islands retain a high proportion of CpG sites and frequently hypomethylated in cancer. Because younger Alu elements are usually closer to active chromatin regions , the hypomethylation of them has more biological significance than that of older Alu elements. Additionally, these hypomethylation of repeat elements, such as Alu and LINE1 might also affect the inactivation of X chromosome .
The findings in this study are based on an intuitive and efficient normalization method for comparative analysis. Unlike bisulfite sequencing, MIRA and MeDIP simulate the in vivo behavior of methyl-CpG binding domain (MBD) proteins, which recognize both the methylation level and concentration of individual CpG sites . For example, it has been shown that MBD binding is not sufficient for gene repression at low CpG densities, even when individual sites are highly methylated. In this case, the common practice is to use MeDIP or MIRA outputs as measures for the functional consequences of methylation of all CpGs in a given region [26, 30, 32, 38, 51–53]. However, a few studies have attempted to use the spatial density of CpGs to normalize the experimental readout of MeDIP-chip [25, 54]. Therefore, the MES normalization used here provides several advantages over other methods. First, using logarithmic transformation, we can scale down raw read counts four orders of magnitude to obtain MESs. This provides mathematical benefits such as variance stabilization. Second, MES normalization allows us to use the correct background distribution. As a probability function for the number of events in a given time interval, a Poisson distribution can be used to assess the statistical significance of read counts in a given genomic interval. However, we found that our MES index, particularly when normalized with local methods rather than raw read counts, better illustrates the nature of the Poisson event. Third and most importantly, it enables comparative analysis of independent samples. In the normalization step, direct subtraction of MESbg proves to be an efficient correction method for background sequencing.
Although the methods used here have clear advantages, the biological and technical limitations of these methods should also be mentioned. Since our methods are based on affinity purification, methylation changes and karyotypic alterations cannot be distinguished. However, this can be overcome by comparing normal and cancer genomes following measurement of background enrichment. Thus, this comparative analysis scheme should be of value for future clinical epigenomics investigations.
We have generated high resolution genome-wide map of human gastric cancer by MIRA-seq, and have found that 5' CGIs and the 5'-end of coding exons are hypermethylated. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes. We found novel long-range epigenetic silencing (LRES) regions and identified several hypermethylated genes (MDM2, DYRK2, and LYZ) within these regions. The methylation status of metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation is gradually increased in tumorigenesis. Our method is readily applicable to a comparative analysis of methylomes and other types of epigenomic data.
This work was supported by grants from the Korean Ministry of Science and Technology to Y.-J.K. (Epigenomic Research of Human Disease and Global Research Lab) and the WCU (World Class University) program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (MEST) of Korea (grants 20100020536 and R31-2010-000-10086-0).
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