Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis.

Genetically modified organisms (GMOs) have been continuously developed for their convenience and productivity. In the past three years, three new GM canola events (MON94100, LBFLFK, and NS-B50027-4) have been developed. To efficiently control these GM canola events, the detection methods were needed. Therefore, the multiplex PCR method combined with capillary electrophoresis was developed for three GM canola events. Ten GM canola, eighteen GM soybean, thirty-two GM maize, and ten non-GM crops were used to evaluate the specificity of the method. The detection limit of the multiplex PCR assay was determined to be 0.005 ng in the DNA mixture and 0.1% in the spiked sample. The aim of this study was to establish multiplex PCR coupled with capillary electrophoresis for the newly produced three GM canola events. The developed method is expected to contribute to monitor the commercially available GM canola events. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01377-z.

High Level of SARS-CoV-2 Infection in Young Population Is a Predictor for Peak Incidence.

South Korea adopted stringent preventive measures against Coronavirus virus disease 2019, resulting in three small and one large outbreaks until January 15, 2022. The fatality rate was 2.5-fold higher during peak transmission periods than in base periods. As new variants of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) are continuously emerging, the need for understanding their epidemic potential remains necessary. In South Korea, the epidemiologic data obtained from mass diagnostic testing enabled investigation of the true number of infected cases, exact incidence, and fatality numbers. Analysis found a similarity between estimated infection rates and confirmed cases. This suggested that the number of confirmed cases had an influence on the fatality rate as a quantitative parameter. The fatality rate decreased even as infection with SARS-CoV-2 variants rose. In comparative analysis, the confirmed cases in young people (ages 20-29) increased prior to every outbreak peak and marked the tipping point in infection spread. These results indicate that a high level of SARS-CoV-2 infection in young population drives peak incidence and mortality across all age groups.

Periodic Fluctuations in the Incidence of Gastrointestinal Cancer.

PURPOSE: Native stem cells can be periodically replaced during short and long epigenetic intervals. Cancer-prone new stem cells might bring about periodic (non-stochastic) carcinogenic events rather than stochastic events. We investigated the epigenetic non-stochastic carcinogenesis by analyzing regular fluctuations in lifelong cancer incidence. MATERIALS AND METHODS: Korean National Cancer Screening Program data were collected between 2009 and 2016. Non-linear and log-linear regression models were applied to comparatively evaluate non-stochastic and stochastic increases in cancer incidence. Prediction performances of regression models were measured by calculating the coefficient of determination, R2. RESULTS: The incidence of gastric and colorectal cancers fluctuated regularly during both short (8 years) and long (20 years) intervals in the non-linear regression model and increased stochastically in the log-linear regression model. In comparison between the 20-year interval fluctuation model and the stochastic model, R2 values were higher in the 20-year interval fluctuation model of men with gastric cancer (0.975 vs. 0.956), and in the stochastic model of men with colorectal cancer (0.862 vs. 0.877) and women with gastric cancer (0.837 vs. 0.890) and colorectal cancer (0.773 vs. 0.809). Men with gastric cancer showed a high R2 value (0.973) in the 8-year interval fluctuation model as well. CONCLUSION: Lifelong incidence of gastrointestinal cancer tended to fluctuate during short and long intervals, especially in men with gastric cancer, suggesting the influence of an epigenetic schedule.

Sequence analysis of the first B5 subgenogroup strain of enterovirus 71 isolated in Korea.

Enterovirus A71 (EV71), the main etiological agent of handfoot- mouth disease (HFMD), circulates in many areas of the world and has caused large epidemics since 1997, especially in the Asia-Pacific region. In this study, we determined the full-genome sequence of CMC718, a newly isolated EV71 strain in Korea. The CMC718 genome was 7,415 nucleotides in length and was confirmed by whole-genome phylogenetic analysis to belong to the B5 genotype. In particular, CMC718 demonstrated maximum identity with strain M988 of the B5 genotype and numerous amino acid variants were detected in the 3D domain of the viral protein P3, which is consistent with the mutation pattern of a B5 strain isolated in 2012-2013. Comparison of the CMC718 sequence with other EV71 reference strains confirmed the relationship and genetic variation of CMC718. Our study was a full-genome sequence analysis of the first EV71 strain of the B5 genotype isolated in South Korea. This information will be a valuable reference for the development of methods for the detection of recombinant viruses, the tracking of infections, and the diagnosis of EV71.

Gastric Mucosal Atrophy Impedes Housekeeping Gene Methylation in Gastric Cancer Patients.

PURPOSE: Helicobacter pylori infection induces phenotype-stabilizing methylation and promotes gastric mucosal atrophy that can inhibit CpG-island methylation. Relationship between the progression of gastric mucosal atrophy and the initiation of CpG-island methylation was analyzed to delineate epigenetic period for neoplastic transformation. MATERIALS AND METHODS: Normal-appearing gastric mucosa was biopsied from 110 H. pylori-positive controls, 95 H. pylori-negative controls, 99 gastric cancer patients, and 118 gastric dysplasia patients. Gastric atrophy was assessed using endoscopic-atrophic-border score. Methylation-variable sites of eight CpG-island genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR (MMP2, CDKN2A, RUNX2, and RUNX3) retroelements and stomach-specific TFF3 gene were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction. RESULTS: Mean ages of H. pylori-positive controls with mild, moderate, and severe atrophy were 51, 54, and 65 years and those of H. pylori-associated TFF3 overmethylation at the three atrophic levels (51, 58, and 63 years) tended to be periodic. Alu-adjacent overmethylation (50 years) was earlier than TFF3 overmethylation (58 years) in H. pylori-positive controls with moderate atrophy. Cancer patients with moderate atrophy showed late Alu-adjacent (58 years) overmethylation and frequent LTR-adjacent overmethylation. LTR-adjacent overmethylation was frequent in cancer (66 years) and dysplasia (68 years) patients with severe atrophy. CONCLUSION: Atrophic progression is associated with gastric cancer at moderate level by impeding the initiation of Alu-adjacent methylation. LTR-adjacent methylation is increased in cancer patients and subsequently in dysplasia patients.

Species-specific role of gene-adjacent retroelements in human and mouse gastric carcinogenesis.

Helicobacter pylori (HP) infection promotes the recruitment of bone marrow stem cells into chronic gastritis lesions. Some of these marrow stem cells can differentiate into gastric epithelial cells and neoplastic cells. We propose that HP-associated methylation could stabilize trans-differentiation of marrow-derived stem cells and that an unstable methylation status is associated with a risk of gastric cancer. Pathobiologic behavior of experimental mouse gastric cancer is mild compared to invasive and metastatic human gastric cancer. Differences in epigenetic stabilization of adult cell phenotypes between humans and mice could provide a foundation to explore the development of invasive and metastatic gastric cancer. Retroelements are highly repetitive sequences that play an essential role in the generation of species diversity. In this review, we analyzed retroelements adjacent to human and mouse housekeeping genes and proposed a possible epigenetic mechanism for HP-associated carcinogenesis.

DNA Methylation as Surrogate Marker For Gastric Cancer.

Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern.

Future climate data from RCP 4.5 and occurrence of malaria in Korea.

Since its reappearance at the Military Demarcation Line in 1993, malaria has been occurring annually in Korea. Malaria is regarded as a third grade nationally notifiable disease susceptible to climate change. The objective of this study is to quantify the effect of climatic factors on the occurrence of malaria in Korea and construct a malaria occurrence model for predicting the future trend of malaria under the influence of climate change. Using data from 2001-2011, the effect of time lag between malaria occurrence and mean temperature, relative humidity and total precipitation was investigated using spectral analysis. Also, a principal component regression model was constructed, considering multicollinearity. Future climate data, generated from RCP 4.5 climate change scenario and CNCM3 climate model, was applied to the constructed regression model to simulate future malaria occurrence and analyze the trend of occurrence. Results show an increase in the occurrence of malaria and the shortening of annual time of occurrence in the future.

Slow overmethylation of housekeeping genes in the body mucosa is associated with the risk for gastric cancer.

Helicobacter pylori infection increases age-related diverse overmethylation in gene-control regions, which increases the risk of gastric cancer. The H. pylori-associated overmethylation changes subsequently disappear when gastric atrophy and cancer develop. To identify cancer-risk epigenotypes, we traced dynamic methylation changes in the background mucosa of the stomach depending on the extent of gastric atrophy. Paired biopsy specimens were obtained from the noncancerous antrum and body mucosa of 102 patients with cancer and 114 H. pylori-positive and 112 H. pylori-negative controls. The grade of gastric atrophy was evaluated using the endoscopic atrophic border score. The methylation-variable sites at the CpG-island margins and near the transcriptional start sites lacking CpG islands were semiquantitatively analyzed by radioisotope-labeling methylation-specific PCR. We selected eight housekeeping genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR retroelements (MMP2, CDKN2A, RUNX2, and RUNX3) and eight stomach-specific genes (TFF2, PGC, ATP4B, TFF1, TFF3, GHRL, PGA, and ATP4A). Analysis of age-related methylation in the H. pylori-positive controls revealed slow overmethylation in the body and in the LTR-adjacent genes. A high-frequency overmethylation defined based on the slowly overmethylated genes was frequently observed in the body of patients with gastric cancer with open-type atrophy (OR, 12.7; 95% confidence interval, 3.2-49.8). The rapidly changing methylation of Alu-adjacent genes was barely increased in the antrum of patients with gastric cancer. Among diverse methylation changes associated with H. pylori infection, an increase in slowly changing methylation could serve as a cancer-risk marker.

Age-related methylation patterning of housekeeping genes and tissue-specific genes is distinct between the stomach antrum and body.

AIM: The methylation-variable sites around CpG islands are frequently overmethylated in Helicobacter pylori-infected stomachs. Age-related patterns of the overmethylation changes were compared between the fast-growing antrum cells and the slow-growing body cells. MATERIALS & METHODS: A total of 316 H. pylori-positive tissues and 380 H. pylori-negative tissues were obtained by endoscopic biopsy. The methylation-variable sites of ten housekeeping genes and nine tissue-specific genes were semiquantitatively analyzed, based on the ten-level classification of methylation-specific PCR intensity. The overmethylated genes were scored when their methylation levels were higher than an intermediate level of each gene common in the H. pylori-negative mucosa. RESULTS: The age-dependent methylation level of the inactive APC gene observed similarly in the antrum and the body was used as an age standard of methylation variation in a biopsy tissue. The overmethylation of housekeeping genes and stomach-specific genes rapidly increased to a high plateau frequency in the young-aged APC methylation cases (mean age: 43 years) in the H. pylori-positive antrum. In the H. pylori-positive body, most of the overmethylated housekeeping genes slowly increased to a peak frequency in the middle-aged APC methylation cases (mean age: 53 years). The housekeeping gene pairs showed high correlations (Spearman's correlation coefficient > 0.4) in both the antrum and the body. CONCLUSION: The overmethylation of housekeeping genes rapidly and slowly increased to a high frequency in concordance with a rapid and slow growth of epithelial cells in the H. pylori-infected stomach.

Epigenetic implication of gene-adjacent retroelements in Helicobacter pylori-infected adults.

A chronic inflammatory condition of gastric mucosa can facilitate the influx of new stem cells into the stomach. Epigenetic codes, such as DNA methylation, may be responsible for the stable maintenance of epigenetic phenotypes established in the new stomach-adapted stem cells. A number of hypotheses have been made for the role of CpG-island methylation, which is common in the Helicobacter pylori-infected stomach. However, they could not explain the plausible role of CpG-island methylation in the re-establishment of epigenetic phenotypes. These islands are highly repetitive sequences densely methylated throughout the human genome, the so-called parasitic retroelements, which expand a number of cDNA copies with reverse transcriptase. The densely methylated retroelements adjacent to the host genes can form the transitional-CpG sites around gene-control regions that are barely methylated. This review focuses on the putative role of transitional CpG methylation in the adaptive differentiation of new stem cells in the H. pylori-infected stomach.

The overmethylated genes in Helicobacter pylori-infected gastric mucosa are demethylated in gastric cancers.

BACKGROUND: The transitional-CpG sites between weakly methylated genes and densely methylated retroelements are overmethylated in the gastric mucosa infected with Helicobacter pylori (H. pylori) and they are undermethylated in the gastric cancers depending on the level of loss of heterozygosity (LOH) events. This study delineated the transitional-CpG methylation patterns of CpG-island-containing and -lacking genes in view of the retroelements. METHODS: The transitional-CpG sites of eight CpG-island-containing genes and six CpG-island-lacking genes were semi-quantitatively examined by performing radioisotope-labelling methylation-specific PCR under stringent conditions. The level of LOH in the gastric cancers was estimated using the 40 microsatellite markers on eight cancer-associated chromosomes. Each gene was scored as overmethylated or undermethylated based on an intermediate level of transitional-CpG methylation common in the H. pylori-negative gastric mucosa. RESULTS: The eight CpG-island genes examined were overmethylated depending on the proximity to the nearest retroelement in the H. pylori-positive gastric mucosa. The six CpG-island-lacking genes were similarly methylated in the H. pylori-positive and -negative gastric mucosa. In the gastric cancers, long transitional-CpG segments of the CpG-island genes distant from the retroelements remained overmethylated, whereas the overmethylation of short transitional-CpG segments close to the retroelements was not significant. Both the CpG-island-containing and -lacking genes tended to be decreasingly methylated in a LOH-level-dependent manner. CONCLUSIONS: The overmethylated genes under the influence of retroelement methylation in the H. pylori-infected stomach are demethylated in the gastric cancers influenced by LOH.

The gene-reduction effect of chromosomal losses detected in gastric cancers.

BACKGROUND: The level of loss of heterozygosity (LOH) that reduces a gene dose and exerts a cell-adverse effect is known to be a parameter for the genetic staging of gastric cancers. This study investigated if the cell-adverse effect induced with the gene reduction was a rate-limiting factor for the LOH events in two distinct histologic types of gastric cancers, the diffuse- and intestinal-types. METHODS: The pathologic specimens obtained from 145 gastric cancer patients were examined for the level of LOH using 40 microsatellite markers on eight cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q). RESULTS: Most of the cancer-associated chromosomes were found to belong to the gene-poor chromosomes and to contain a few stomach-specific genes that were highly expressed. A baseline-level LOH involving one or no chromosome was frequent in diffuse-type gastric cancers. The chromosome 17 containing a relatively high density of genes was commonly lost in intestinal-type cancers but not in diffuse-type cancers. A high-level LOH involving four or more chromosomes tended to be frequent in the gastric cancers with intestinal and mixed differentiation. Disease relapse was common for gastric cancers with high-level LOH through both the hematogenous (38%) and non-hematogenous (36%) routes, and for the baseline-level LOH cases through the non-hematogenous route (67%). CONCLUSIONS: The cell-adverse effect of gene reduction is more tolerated in intestinal-type gastric cancers than in diffuse-type cancers, and the loss of high-dose genes is associated with hematogenous metastasis.

DNA methylation patterns of ulcer-healing genes associated with the normal gastric mucosa of gastric cancers.

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.

DNA methylation and expression patterns of key tissue-specific genes in adult stem cells and stomach tissues.

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.

Chromosomal losses are associated with hypomethylation of the gene-control regions in the stomach with a low number of active genes.

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.

Transitional CpG methylation between promoters and retroelements of tissue-specific genes during human mesenchymal cell differentiation.

In general, methylation of the promoter regions is inversely correlated with gene expression. The transitional CpG area between the promoter-associated CpG islands and the nearby retroelements is often methylated in a tissue-specific manner. This study analyzed the relationship between gene expression and the methylation of the transitional CpGs in two human stromal cells derived from the bone marrow (BMSC) and adipose tissue (ATSC), both of which have a multilineage differentiation potential. The transitional CpGs of the osteoblast-specific (RUNX2 and BGLAP), adipocyte-specific (PPARgamma2), housekeeping (CDKN2A and MLH1), and mesenchyme-unrelated (RUNX3) genes were examined by methylation-specific PCR. The expression of each gene was measured using reverse-transcription PCR analysis. The RUNX2, BGLAP, and CDKN2A genes in the BMSC, and the PPARgamma2 gene in the ATSC exhibited hypomethylation of the transitional CpGs along with the strong expression. The CpG island of RUNX3 gene not expressed in both BMSC and ATSC was hypermethylated. Transitional hypomethylation of the MLH1 gene was accompanied by the higher expression in the BMSC than in the ATSC. The weakly methylated CpGs of the PPARgamma2 gene in the BMSC became hypomethylated along with the strong expression during the osteoblastic differentiation. There were no notable changes in the transitional methylation and expression of the genes other than PPARgamma2 after the differentiation. Therefore, the transitional methylation and gene expression established in mesenchymal cells tend to be consistently preserved under the induction of differentiation. Weak transitional methylation of the PPARgamma2 gene in the BMSC suggests a methylation-dependent mechanism underlying the adiopogenesis of bone marrow.

The 5'-end transitional CpGs between the CpG islands and retroelements are hypomethylated in association with loss of heterozygosity in gastric cancers.

BACKGROUND: A loss of heterozygosity (LOH) represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements. METHODS: The 5'-transitional area of all human genes (22,297) was measured according to the nearest retroelements to the transcription start sites. For 50 gastric cancer specimens, the level of LOH events on eight cancer-associated chromosomes was estimated using the microsatellite markers, and the 5'-transitional CpGs of 20 selected genes were examined by methylation analysis using the bisulfite-modified DNA. RESULTS: The extent of the transitional area was significantly shorter with the nearest Alu elements than with the nearest L1 and LTR elements, as well as in the extragenic regions containing a higher density of retroelements than in the intragenic regions. The CpG islands neighbouring a high density of Alu elements were consistently hypomethylated in both normal and tumor tissues. The 5'-transitional methylated CpG sites bordered by a low density of Alu elements or the L1 and LTR elements were hypomethylated more frequently in the high-level LOH cases than in the low-level LOH cases. CONCLUSION: The 5'-transitional methylated CpG sites not completely protected by the Alu elements were hypomethylated in association with LOH events in gastric cancers. This suggests that an irreversible unbalanced decrease in the genomic dose reduces the spread of L1 methylation in the 5'-end regions of genes.

The length of CpG islands is associated with the distribution of Alu and L1 retroelements.

Alu and L1 retroelements have been suggested to initiate the spread of CpG methylation. In this study, the spread of CpG methylation was estimated based on the distance between the CpG islands and the nearest retroelements. All human genes (23,116) were examined and the correlations between the length of the CpG islands and the distance and density of the confronting retroelements were examined using nonoverlapping 5-kb windows. There was a linear relationship between the length of the CpG islands and the density of the Alu elements and an inverse relationship between the CpG islands and the L1 elements located more distantly, suggesting a suppressive effect of the Alu's on the spread of L1 methylation. Methylation analysis of the transitional CpG sites between the CpG islands and the nearest retroelements upstream of 16 genes was then carried out using DNA preparations from 11 different human tissues. Methylation-variable transitional CpGs were observed for the selected genes and the different tissues.

Relationship between the extent of chromosomal losses and the pattern of CpG methylation in gastric carcinomas.

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression.