Long Interspersed Nucleotide Element-1 Methylation Research Articles

Association between Genetic Instability andHelicobacter pyloriInfection in Gastric Epithelial Dysplasia

Background. In gastric carcinogenesis, changes of DNA methylation appear to be an early molecular event, and the genome-wide methylation state is closely correlated with the level of long interspersed nucleotide element-1 (LINE-1) methylation. In this study, we measured LINE-1 methylation level according to genetic instability and evaluated the effect of Helicobacter pylori infection on genetic instability in gastric epithelial dysplasia. Methods. Total 100 tissue samples of gastric epithelial dysplasia were analyzed. Seven loci that linked to tumor suppressor genes were used to identify significant structural chromosomal aberrations. Microsatellite status was investigated for two different microsatellite marker loci (BAT25 and BAT26). Also, we measured LINE-1 methylation level by combined bisulfite restriction analysis (COBRA-LINE-1) method. Results. There were no significant differences of LINE-1 methylation level according to chromosomal/microsatellite instability and H. pylori state. In the dysplastic lesions with H. pylori infection, LINE-1 methylation level of MSI lesion was significantly lower than that of microsatellite stable (MSS) lesion (40.23 ± 4.47 versus 43.90 ± 4.81%, P < 0.01). Conclusions. In gastric epithelial dysplasia with H. pylori infection, MSI is correlated with reduced LINE-1 methylation level. Coexistence of H. pylori infection and MSI might be a driving force of gastric carcinogenesis.

Abstract A84: Reduced LINE-1 methylation in peripheral blood mononuclear cells is associated with risk of melanoma in families segregating CDKN2A germline mutations

Abstract Background: CDKN2A is a major susceptibility gene for familial melanoma, but its incomplete penetrance suggests that other factors may modify the effect of this gene in melanoma-prone families. Epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. In addition, genetic background has the potential to influence epigenetic processes through cis- or trans-regulatory variation. Therefore, we hypothesize that the CDKN2A carrier mutation state may be an important factor influencing global DNA methylation in predisposed individuals and that aberrant methylation may be an indicator of modified risk in individuals from melanoma-prone families segregating CDKN2A germline mutations. Methods: We measured the overall level of genomic DNA methylation using bisulfite pyrosequencing at four CpG sites (CpG1, CpG2, CpG3 and CpG4) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from 42 CDKN2A mutation positive cases (20 males and 22 females) and 73 controls (29 CDKN2A carrier controls [9 males and 20 females] and 44 non-carrier controls [19 males and 25 females]) in 26 families with CDKN2A mutations. LINE-1 methylation was reported as the average (of all 4 CpGs) and as site-specific percentage methylation. Odds' ratios (OR) and P-values were obtained by comparing melanoma cases to controls using unconditional logistic regression with adjustment for family correlation (using the generalized estimating equations [GEE] approach), age, sex, number of moles and with melanoma status as the outcome variable. Percentage LINE-1 methylation was categorized using tertiles with the highest tertile as the reference. Results: Male gender was significantly associated with higher average LINE-1 methylation (P=&amp;lt;0.001) and methylation at all CpG sites (P=&amp;lt;0.03). In contrast, age, solar injury, dysplastic nevi (DN) and number of moles were not associated with average LINE-1 methylation. Although mean levels of LINE-1 methylation were similar in melanoma cases, carrier controls, and non-carrier controls, after adjusting for family correlation, age, sex and number of moles, low average LINE-1 methylation (tertile 1) was significantly associated with increased risk of melanoma (OR=3.87, 95% confidence interval [CI]: 1.11–13.5, P=0.033, compared to all controls; OR=1.83, 95% CI: 1.09–11.7, P=0.035, compared to carrier controls; and OR=3.43, 95% CI: 1.17–10.0, P= 0.024, compared to non-carrier controls). This association was observed for most CpG sites with the most significant association seen for site 3 and risk of melanoma compared to all controls (OR=5.18, 95% CI: 1.54–17.4, P=0.008). We observed no significant association between average or CpG site-specific LINE-1 methylation and CDKN2A mutation status among controls although the comparison was limited by small sample size. Conclusions: Melanoma cases from families segregating CDKN2A germline mutations have significantly lower LINE-1 global methylation in their PBMCs compared to controls. Changes in LINE-1 methylation in PBMCs from CDKN2A mutation positive cases may reflect a potential genetic background influence on global DNA methylation in these individuals. Further studies are needed to clarify these preliminary observations. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A84.

Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients

BackgroundThe prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients.MethodsOverall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis.ResultsHypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01).ConclusionLINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

Abstract 295: Detection of hypomethylation of LINE1 on peripheral white blood cells and plasma DNA in US hepatocellular carcinoma patients

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is increasing in the United States. Epigenetic changes, including genomic DNA hypomethylation, have been implicated in cancer development by inducing chromosomal instability. In the present study, methylation of long interspersed nucleotide element-1(LINE1), a surrogate marker of global hypomethylation, in peripheral white blood cells and plasma DNA in 47 US HCC patients was detected by pyrosequencing. Correlations between LINE1 methylation, and clinical data and other biomarkers were also analyzed. Methylation of LINE1 in WBC DNA is statistically significantly correlated with that in plasma DNA (R=0.32, p=0.04). WBC DNA global hypomethylation was significantly decreased in subjects with hepatitis C (HCV) infection (p=0.05). Plasma folate levels were not correlated with plasma DNA LINE1 methylation (R=0.13, p=0.41) or with WBC LINE1 methylation (R=−0.19, p=0.24). However, WBC folate levels were statistically significantly associated with patients’ survival. HCC patients who survived at least 3 months after diagnosis had lower WBC folate compared to patients who died within 3 months (p=0.004). We are currently expanding the HCC cohort and also analyzing biomarkers of chemical exposure (polycyclic aromatic hydrocarbons and aflatoxin B1) to investigate associations with methylation changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2011-295

Predictors and Health Consequences of Epigenetic Changes Associated with Excess Body Weight in Women of Child-bearing Age

Abstract Background: Epigenetic alterations occurring during pregnancy have recently emerged as important factors for developmental programming of the fetus leading to obesity-related diseases in children. However, the role of excess body weight (EBW) in the modification of epigenetic patterns or its health consequences during child-bearing age is largely unknown. Because a lower degree of DNA methylation of long interspersed nucleotide element-1 (LINE-1) in PBMCs was shown to be associated with a higher risk of developing obesity-related diseases, for example cancer, the purpose of this study was to (1) evaluate the influence of indicators of obesity (BMI, WC, and % body fat) on PBMC LINE-1 methylation, (2) determine the predictors of PBMC LINE-1 methylation, and (3) determine the influence of PBMC LINE-1 methylation on biomarkers of obesity-related diseases. Methods: The study population consisted of 470 child-bearing age women. We quantified the degree of PBMC LINE-1 methylation by pyrosequencing. Folate concentrations were measured using a microbiological assay. The degree of LINE-1 methylation (&amp;gt; median vs. ≤ median) was the dependent variable in logistic models that specified BMI (&amp;gt;25 vs. ≤ 25), WC (&amp;gt;88 cm vs. ≤ 88 cm), or % body fat (&amp;gt;33% vs. ≤ 33%) separately as the independent predictors of primary interest, adjusting for other relevant variables. The predictors and determinants of lower LINE-1 methylation were evaluated among women with EBW. Results: Women with higher BMI, WC, or % body fat were 2.0, 1.9, and 1.8 times more likely to have lower LINE-1 methylation, respectively (P = 0.003, 0.005, and 0.01). The predictors and determinants of lower LINE-1 methylation yielded similar patterns with all three indicators of obesity. The following results are based on models run with BMI as the indicator for EBW. Women with higher plasma folate concentrations were less likely to have lower LINE-1 methylation (OR = 0.54, P = 0.0009). Higher LINE-1 methylation was associated with lower insulin resistance as indicated by HOMA (OR = 0.50, P = 0.02). Conclusions: EBW-associated lower LINE-1 methylation in women of child-bearing age appears to have significant, and potentially transgenerational, health consequences. Higher folate status may exert beneficial effects on obesity-related health outcomes.

Ischemic Heart Disease and Stroke in Relation to Blood DNA Methylation

Epigenetic features such as DNA hypomethylation have been associated with conditions related to cardiovascular risk. We evaluated whether lower blood DNA methylation in heavily methylated repetitive sequences predicts the risk of ischemic heart disease and stroke. We quantified blood DNA methylation of Long Interspersed Nucleotide Element-1 (LINE-1) repetitive elements through PCR-pyrosequencing in 712 elderly individuals from the Boston-area Normative Aging Study. We estimated risk-factor adjusted relative risks (RRs) for ischemic heart disease and stroke at baseline (242 prevalent cases), and during follow-up (44 new cases; median follow-up, 63 months), as well as subsequent mortality from ischemic heart disease (86 deaths; median follow-up, 75 months). Blood LINE-1 hypomethylation was associated with baseline ischemic heart disease (RR = 2.1 [95% confidence interval = 1.2-4.0] for lowest vs. highest methylation quartile) and for stroke (2.5 [0.9-7.5]). Among participants free of baseline disease, individuals with methylation below the median also had higher risk of developing ischemic heart disease (4.0 [1.8-8.9]) or stroke (5.7 [0.8-39.5]). In the entire cohort, persons with methylation below the median had higher mortality from ischemic heart disease (3.3 [1.3-8.4]) and stroke (2.8 [0.6-14.3]). Total mortality was also increased (2.0 [1.2-3.3]). These results were confirmed in additional regression models using LINE-1 methylation as a continuous variable. Subjects with prevalent IHD and stroke exhibited lower LINE-1 methylation. In longitudinal analyses, persons with lower LINE-1 methylation were at higher risk for incident ischemic heart disease and stroke, and for total mortality.

Relation between hypomethylation of long interspersed nucleotide elements and risk of neural tube defects

Impaired one-carbon metabolism is thought to be associated with the risk of neural tube defects (NTDs); however, the role of methylation in NTDs remains unclear. Long interspersed nucleotide element-1 (LINE-1) constitutes 17-25% of the human genome. LINE-1 hypomethylation correlates with global DNA methylation levels in cancerous cells, but limited information is available on LINE-1 methylation in NTDs. We determined whether LINE-1 methylation patterns were associated with neural tube development and the possible relations between DNA methylation and key maternal metabolites involved in folate-mediated one-carbon metabolism. Global methylation, maternal plasma folic acid, vitamin B-12, and total homocysteine (tHcy) concentrations were assessed in 48 NTD and 49 control samples by immunoassay, and LINE-1 methylation levels were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Methylation levels of genomic DNA and LINE-1 decreased significantly in the neural tissue of NTD samples. The risk of NTDs increased with decreasing levels of LINE-1 methylation, with an odds ratio of 5.246 (95% CI: 1.519, 18.124; P = 0.009) for the lowest quartile (methylation level < or = 57.94%) compared with the highest quartile (methylation level > or = 60.94%). Compared with control subjects, case subjects had lower maternal plasma concentrations of vitamin B-12. Hypomethylation of LINE-1 and genomic DNA was associated with an increased risk of NTDs. Functional insufficiency of maternal plasma vitamin B-12 was associated with NTDs, although no significant correlation could be established between maternal folic acid, vitamin B, tHcy, and LINE-1 methylation.

LINE‐1 methylation difference between ameloblastoma and keratocystic odontogenic tumor

Global hypomethylation is a common epigenetic event in cancer. Keratocystic odontogenic tumor (KCOT) and ameloblastoma are different tumors but posses the same tissue in origin. Here, we investigated long interspersed nuclear element-1 (LINE-1 or L1) methylation status between ameloblastoma and KCOT. We studied the methylation levels of the long interspersed nucleotide element-1 (LINE-1) in ameloblastoma and KCOT. After collecting ameloblastoma cells and epithelium lining cells of KCOT by laser capture microdissection from paraffin embedded tissue, combined bisulfite restriction analysis of LINE-1 (COBRALINE-1) was performed to measure LINE-1 methylation levels. The LINE-1 methylation level in KCOT (53.16 +/- 12.03%) was higher than that in ameloblastoma (36.90 +/- 16.52%), with a statistical significance of P = 0.001. The ranges of LINE-1 methylation of both lesions were not associated with either age or sex. We found LINE-1 hypomethylation levels between ameloblastoma and KCOT are different. Therefore, global methylations between these tumors are processed differently.

Dietary folate, alcohol and B vitamins in relation to LINE-1 hypomethylation in colon cancer

Background and aimsAlthough critical for methylation reactions, how dietary folate and B vitamins affect global DNA methylation level in colorectal cancers is currently unknown. Long interspersed nucleotide element-1 (LINE-1) is...

CpG island hypermethylation and repetitive DNA hypomethylation in premalignant lesion of extrahepatic cholangiocarcinoma

Biliary intraepithelial neoplasia (BilIN) is the premalignant lesion of extrahepatic cholangiocarcinoma (EHC), and there are no published data regarding epigenetic changes throughout disease progression from normal biliary epithelia to BilIN to EHC. The objective of this study was to identify the occurrence of CpG island hypermethylation and repetitive DNA hypomethylation in BilIN. A total of 50 EHCs, 31 BilINs, and 31 normal cystic duct samples were analyzed for their methylation status in seven genes and two repetitive DNA elements. The number of methylated genes increased with disease progression (normal bile duct, 0.6; BilIN, 2.0; EHC, 3.6; P < 0.001). The methylation level of examined genes was significantly higher in BilIN than in normal samples (TMEFF2, HOXA1, NEUROG1, and RUNX3, P < 0.05) and in EHC than in BilIN samples (TMEFF2, HOXA1, NEUROG1, RUNX3, RASSF1A, and APC, P < 0.05). Long interspersed nucleotide element-1 (LINE-1) and juxtacentromeric satellite 2 (SAT2) methylation levels were markedly lower in EHC than in normal duct and BilIN samples, and BilIN samples showed a decrease of SAT2 methylation levels but no decrease of LINE-1 methylation levels compared to normal samples. These findings suggest that most of cancer-specific CpG island hypermethylation occur in the stage of BilIN and that CpG island hypermethylation seems to occur earlier than repetitive DNA element hypomethylation.

A Cohort Study of p27 Localization in Colon Cancer, Body Mass Index, and Patient Survival

Energy balance and the AKT pathway are important in colorectal cancer development and regulate p27 (cyclin-dependent kinase inhibitor-1B/CDKN1B/KIP1), which plays a role in preventing cell cycle progression. However, little is known on the clinical outcome or prognostic significance of p27 alterations in relation to patient body mass index (BMI). Among 630 colon cancers (stage I-IV) in two prospective cohort studies, we detected p27 alterations (cytoplasmic p27 localization or p27 loss) in 500 tumors (79%) by immunohistochemistry. The remaining 130 (21%) tumors were "p27-nuclear+." Cox proportional hazard models computed hazard ratios (HR) of deaths, adjusted for patient and tumoral characteristics, including p53, p21, cyclin D1, KRAS, BRAF, PIK3CA, cyclooxygenase-2, fatty acid synthase (FASN), beta-catenin, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and long interspersed nucleotide element-1 (LINE-1) hypomethylation. Compared with p27-nuclear+ patients, p27-altered patients experienced low colon cancer-specific [adjusted HR, 0.63; 95% confidence interval (95% CI), 0.42-0.94] and overall mortality (adjusted HR, 0.70; 95% CI, 0.51-0.95), independent of FASN, MSI, CIMP, LINE-1 methylation, and other potential confounders. The effect of p27 alteration on overall mortality significantly differed by BMI (P(interaction) = 0.013); adjusted HR (p27-altered versus p27-nuclear+ tumors) was 0.28 (95% CI, 0.13-0.59) for BMI >or=30 kg/m(2), 0.67 (95% CI, 0.40-1.14) for BMI 25 to 29 kg/m(2), and 0.91 (95% CI, 0.57-1.46) for BMI <25 kg/m(2). Obesity was associated with inferior overall survival among p27-nuclear+ cases (adjusted HR, 3.07; 95% CI, 1.49-6.32; versus nonobese cases), but not among p27-altered cases (adjusted HR, 1.08). In conclusion, p27 alterations in colon cancer are associated with superior prognosis. Adverse prognostic effect of obesity seems limited to patients with nuclear p27 expression, suggesting a host-tumor interaction.

A Cohort Study of Tumoral LINE-1 Hypomethylation and Prognosis in Colon Cancer

Genome-wide DNA hypomethylation plays has an important role in genomic instability and colorectal carcinogenesis. However, the relationship between cellular DNA methylation level and patient outcome remains uncertain. Using 643 colon cancers in two independent prospective cohorts, we quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) elements using pyrosequencing, which is a good indicator of global DNA methylation level. We used Cox proportional hazard models to calculate hazard ratios (HRs) of colon cancer-specific and overall mortality, adjusting for patient and tumoral features, including CpG island methylator phenotype (CIMP). Statistical tests were two-sided. LINE-1 hypomethylation was linearly associated with a statistically significant increase in colon cancer-specific mortality (for a 30% decrease in LINE-1 methylation: multivariable HR = 2.37, 95% confidence interval [CI] = 1.42 to 3.94; P(trend) < .001) and overall mortality (multivariable HR = 1.85, 95% CI = 1.25 to 2.75; P(trend) = .002). The association was consistent across the two independent cohorts and strata of clinical and molecular characteristics, including sex, age, tumor location, stage, and CIMP, microsatellite instability, KRAS, BRAF, p53, and chromosomal instability status. In conclusion, tumoral LINE-1 hypomethylation is independently associated with shorter survival among colon cancer patients.

LINE‐1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer

The CpG island methylator phenotype (CIMP) with widespread promoter CpG island methylation is a phenotype in colorectal cancer, associated with microsatellite instability (MSI) and BRAF mutation. Genome-wide hypomethylation may also play an important role in genomic instability. However, the relation between global DNA methylation level and methylation in individual CpG islands remains uncertain. Utilizing 869 population-based colorectal cancers, we measured long interspersed nucleotide element-1 (LINE-1) methylation level by Pyrosequencing, which correlates with global DNA methylation level. We quantified DNA methylation in 8 CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) by real-time PCR (MethyLight technology). LINE-1 methylation levels in tumors were approximately normally distributed (mean, 61.4%; median, 62.3%; standard deviation, 9.6%). Among the 869 tumors, 128 (15%) were classified as CIMP-high (>or=6/8 methylated promoters). The mean LINE-1 methylation level was higher in CIMP-high tumors (65.1%, p < 0.0001) than non-CIMP-high tumors (60.7%), and higher in MSI-high tumors (64.7%, p < 0.0001) than non-MSI-high tumors (60.7%). When tumors were stratified by MSI/CIMP status, compared to non-MSI-high non-CIMP-high tumors (mean LINE-1 methylation level, 60.4%), the mean LINE-1 methylation level was higher in MSI-high CIMP-high (64.8%, p < 0.0001), MSI-high non-CIMP-high (64.6%, p = 0.03) and non-MSI-high CIMP-high tumors (66.1%, p = 0.0003). In addition, 18q loss of heterozygosity in non-MSI-high tumors was correlated with LINE-1 hypomethylation (p = 0.004). In conclusion, both CIMP-high and MSI-high are inversely associated with LINE-1 hypomethylation, suggesting that CIMP/MSI and genomic hypomethylation may represent different pathways to colorectal cancer. Our data also support a possible link between global hypomethylation and chromosomal instability.