Long Interspersed Nuclear Element-1 Methylation Levels Research Articles

LINE-1 hypomethylation, increased retrotransposition and tumor-specific insertion in upper gastrointestinal cancer.

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.

Biological age and diet: Measuring the impact of lifestyle on a 6CpG-epigenetic clock

BACKGROUND: Changes in DNA methylation along the life have been documented, and environmental exposures (including diet, physical activity and smoking) can accelerate or decelerate this process. The epigenetic clock estimates the biological age of an individual measuring methylation patterns in specific areas of its genome. Recently, a new epigenetic clock based on 6 CpGs has been proposed, with high potential to become an easy accessible tool able to measure the epigenetic age (EA) of an individual. OBJECTIVES: This study aims to validate the 6 CpG epigenetic clock comparing it with other biomarkers of aging such as telomere length (TL) and methylation in the long interspersed nuclear element-1 (LINE-1). The impact of lifestyle associated factors on these molecular marks has been evaluated. METHODS: 200 healthy participants having extreme dietary patterns (healthy vs western diet) were selected. Dietary intakes, body composition, physical activity level and smoking has been assessed. DNA extracted from whole blood was used to measure the 6CpG-EA, TL and LINE-1 methylation levels. RESULTS: 6CpG-EA was positively correlated with chronological age (r = 0.591; p = 7.2*10-20) and negatively with TL (r = –0.150; p = 0.040) and LINE-1 methylation (r = –0.240; p = 0.001). Despite the fact that no significant associations were detected with the overall diet quality (HEI), 6CpG-EA was correlated with dietary intakes of nutrients involved in the one-carbon (1 C) metabolism, especially in the western diet group. CONCLUSION: These results support the 6CpG epigenetic clock as an easy accessible tool to estimate biological age, in accordance with other molecular markers of aging, and suggest that EA can be modulated by micronutrients involved in the 1 C metabolism.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content

BackgroundHypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect.MethodsLINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40).ResultsSignificantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05).ConclusionOur results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Association between Serum Vitamin B12 and Global DNA Methylation in Colorectal Cancer Patients.

Vitamin B12 has been widely related to methionine metabolism, which is an essential component for biological methylation reactions, including DNA methylation. However, the relationship between vitamin B12 and DNA methylation is still controversial. In addition, there is increasing evidence for the association between vitamin B12 and the risk of colorectal cancer (CRC), although results of this association need to be assessed with caution. For this purpose, we hypothesized that serum vitamin B12 could be associated with global DNA methylation in the CRC context. To test this hypothesis, we studied the association between global DNA methylation through long interspersed nuclear element-1 (LINE1) in CRC patients under the 25th percentile of serum vitamin B12. We found that the high vitamin B12 group had low LINE1 methylation in both tumor area and peripheral blood mononuclear cells (PBMCs) than the low serum vitamin B12 group. LINE1 methylation levels were significantly lower in tumor area compared to the adjacent tumor-free area, only in the high vitamin B12 group. LINE1 methylation in visceral adipose tissue (VAT) and PBMCs were correlated with tumoral, inflammatory, and insulin metabolism markers. However, the interaction between LINE1 methylation and vitamin B12 levels was associated with neoadjuvant therapy in the regression analysis only in men, suggesting a beneficial relationship. In conclusion, our results reported an inverse association between DNA methylation and vitamin B12 in the CRC context, which suggests that vitamin B12 may be implicated in an epigenetic state or mediation in CRC.

DNA methylation and retinal degenerative diseases: at the crossroad between genes and diet

Retinal degenerative diseases are the leading causes of blindness and low vision among working-age and older adults worldwide, with 170 and 130 million individuals suffering from age-related macular degeneration (AMD) and diabetic retinopathy, respectively. Although several studies began to show benefits from dietary interventions against retinal degenerative disease, an integrated approach is needed to understand molecular mechanisms underpinning the protective or risky effect of dietary factors. A specific area of research that elucidates mechanisms involved in gene-diet interaction is the Nutri-epigenomics, the study of the impact of diet on gene expression by modulating epigenetic mechanisms. The present research investigated the role of DNA methylation – one of the most commonly analysed epigenetic mechanisms - in the pathophysiology of retinal degenerative diseases, by exploiting a multiple integrated approach. In vitro studies initially helped us to understand how pathological features of retinal degeneration (e.g. oxidative stress, inflammation and hyperglycaemia) modulated functions of enzymes involved in the methylation of Long Interspersed Nuclear Element 1 (LINE-1) sequences in retinal cells. We also proved that some nutrients (e.g. resveratrol and curcumin) might counteract these effects and restore DNA methylation level in retinal cells under oxidative, inflammatory and high glucose conditions. We further analysed whether LINE-1 methylation level differed between patients with AMD and controls without posterior segment eye diseases. Interestingly, we noted a significant difference between the two groups, with higher LINE-1 methylation level in blood samples from AMD patients. This evidence -albeit promising for biomarker discovery- requires confirmation by further large-size prospective studies taking into account different factors. Our research, in fact, also suggested that the risk of retinal degenerative diseases derives from the combination of genetic risk variants, clinical characteristics, environmental exposures and unhealthy lifestyles, which in turn are interrelated. Thus, it would be interesting to study how the exposome -the totality of exposures individuals experience over the course of life- might induce epigenetic mechanisms able to reduce or increase the risk for retinal degenerative diseases.

LINE-1 Hypomethylation Is Associated With Malignant Traits and Cell Proliferation in Lung Adenocarcinoma.

Long interspersed nuclear element-1 (LINE-1) methylation status is a marker for global DNA methylation. However, the relationship between LINE-1 methylation and the biology of lung adenocarcinoma remains unclear. Here, we aimed to examine the role of LINE-1 in lung cancer. LINE-1 methylation levels were quantified by bisulfite pyrosequencing of resected tumor specimens from 162 patients with lung adenocarcinoma. The relationships of LINE-1 methylation with clinicopathological factors, gene mutations, and Ki-67 immunoreactivity were investigated. LINE-1 hypomethylation was associated with tumor invasion and advanced stage. TP53 mutations were more frequently detected in the LINE-1 hypomethylation group than in the hypermethylation group. LINE-1 hypomethylation was associated with poor recurrence-free survival, high maximum standardized uptake value in positron-emission tomography, and high Ki-67 expression in tumors. LINE-1 hypomethylation was associated with high-grade malignancy and poor prognosis in lung adenocarcinoma, but was not related to driver mutations.

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression.

Long interspersed nuclear element-1 methylation status in the circulating DNA from blood of patients with malignant and chronic inflammatory lung diseases.

Along with other malignant diseases, lung cancer arises from the precancerous lung tissue state. Aberrant DNA methylation (hypermethylation of certain genes and hypomethylation of retrotransposons) is known as one of the driving forces of malignant cell transformation. Epigenetic changes were shown to be detectable in DNA, circulating in the blood (cirDNA) of cancer patients, indicating the possibility to use them as cancer markers. The current study is the first to compare the Long interspersed nuclear element-1 (LINE-1) methylation level in the blood from lung cancer patients before treatment versus different control groups as healthy subjects, patients with bronchitis and patients with chronic obstructive pulmonary disease (COPD). The concentration of LINE-1 methylated fragments, region 1 (LINE-1 methylated, LINE-1-met) was estimated by quantitative methyl-specific PCR. The total concentration of the circulating LINE-1 copies was measured by qPCR specific for LINE-1 region 2, which was selected due to its CpG methylation-independent sequence (LINE-1-Ind). Both LINE-1 methylation level and LINE-1 methylation index (LINE-1-met/LINE-1-Ind ratio) was decreased in lung cancer patients compared with the joint control group (healthy subjects + patients with bronchitis + COPD patients) (Mann-Whitney U-test, P = 0.016). We also found that the tendency of LINE-1 methylation index decreases in the cirDNA from lung cancer patients versus COPD patients (Mann-Whitney U-test, P = 0.07). Our data indicate that the quantitative analysis of the LINE-1 methylation level in the cirDNA is valuable for discrimination of lung cancer patients from patients with chronic inflammatory lung diseases.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́′UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-β1 (TGF-β1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-β1 and AhR pathways. Results show that 0.5 μM CPF and 0.005 μM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́′UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 μM HCB and 50 μM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 μM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-β1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.

LINE-1 Methylation in Chronic HBV Infected Patients: Association with HCC, Gender and MTHFR C677T Polymorphism.

Polymorphism of methylene tetrahydrofolate reductase (MTHFR) C677T has been reported to be associated with HBV-related hepatocellular carcinoma (HCC) risk. However, the underlying mechanism remains elusive. DNA methylation has been suggested to be associated with HCC onset. MTHFR is the key enzyme in folic acid metabolism, thus, it influences the production of the main donor of methyl groups for DNA methylation. This study aimed to determine the association of global DNA methylation with MTHFR C677T polymorphism in chronic HBV infected patients, as well as its association with HCC and gender. In all, 130 chronic hepatitis B (CHB) and 131 HBV-related HCC patients were enrolled in the study. The methylation level of long interspersed nuclear element-1 (LINE-1), the surrogate marker of global DNA methylation, and MTHFR C677T genotypes were determined. The HCC group showed significantly lower LINE-1 methylation than the CHB group (p = 0.016). Females were observed to have a markedly lower LINE-1 methylation level than males in both CHB and HCC groups (p = 0.000, p = 0.014, respectively). A significant relationship between MTHFR C677T polymorphism and LINE-1 methylation was observed in the CHB group (F = 5.985, p = 0.003). CT, TT, and CT + TT genotypes were significantly associated with lower LINE-1 methylation level compared with the CC genotype (p = 0.005, p = 0.018, p = 0.001, respectively). In addition, the association between MTHFR C677T and LINE-1 methylation was more significant in females (F= 5.036, p = 0.011) than in males (F = 3.083, p = 0.051); further, the association was significant in the subjects older than 60 years (F = 3.865, p = 0.028), but not in the subgroup aged less than 60 years (F = 2.496, p = 0.089). LINE-1 methylation level in chronic HBV infected patients was associated with the occurrence of HBV-related HCC, gender, and MTHFR C677T polymorphism.

Characterization of SIRT1/DNMTs Functions and LINE-1 Methylation in Patients with Age-Related Macular Degeneration.

Previous studies proposed the application of DNA methylation signatures as clinical biomarkers of age-related macular degeneration (AMD). However, the characterization of Long Interspersed Nuclear Element-1 (LINE-1) methylation levels—a surrogate marker of global DNA methylation—in AMD patients has not been investigated so far. In the present study, we first characterized DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions in blood samples of 40 AMD patients and 10 age- and sex-matched controls. Then, we evaluated whether changes in DNMTs functions were associated with different LINE-1 methylation levels in leukocyte DNA. We demonstrated that total DNMTs activity was 48% higher in AMD patients than in controls (p = 0.005). AMD patients also exhibited up-regulation of DNMT1 and DNMT3B expression (FC = 2.6; p = 0.003 and FC = 2.4; p = 0.018, respectively). In line with increased DNMTs functions, the LINE-1 methylation level was higher in AMD patients than in controls (mean = 69.10%; SE = 0.68 vs. mean = 65.73%; SE = 0.59; p = 0.020). All p-values were adjusted by Bonferroni correction. In AMD patients, LINE-1 methylation level was positively associated with total DNMTs activity (r = 0.694; p < 0.001), DNMT1 (r = 0.579; p < 0.001), and DNMT3B (r = 0.521; p = 0.001) expression. Our results encourage further large-size prospective research to understand the relationship between LINE-1 methylation and AMD aetiology, and its usefulness in the clinical setting.

Abstract 3231: Prospective study of genomic 5-methylcytosine and LINE-1 methylation levels of leukocyte DNA and colorectal cancer risk

Abstract Global hypomethylation in peripheral blood leukocyte DNA, measured by the reduction of genome-wide methylated cytosine (5-mC) or the long interspersed nuclear element-1 (LINE-1) level, has been associated with an increased risk of colorectal cancer, mainly in retrospective studies. Little is known as to how these global methylation markers relate in individuals and their associations with colorectal cancer risk in a prospective study setting. We studied leukocyte 5-mC and LINE-1 methylation levels in prospectively collected blood specimens from 404 cases and 528 controls who were free of colorectal cancer at blood collection from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Leukocyte 5-mC level was determined by an HPLC/Tandem Mass Spectrometry method and expressed as the relative amount of methyl- to total cytosine residues (%5-mC). LINE-1 methylation level was measured by McrBC digestion followed by real-time PCR of the undigested/unmethylated LINE1 promoter sequences. In our analysis, we excluded cases diagnosed within one year of blood draw to minimize the potential for reverse causation from prevalent disease. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) for tertiles of methylation using multivariable logistic regression, adjusting for age, sex, and other potential confounders. We observed a significant association between low LINE-1 methylation and colorectal cancer risk (1st vs. 3rd tertile: OR=1.63, 95% CI=1.10-2.41; P trend=0.01) and a near-significant association with low %5-mC methylation (OR=1.37, 95% CI=0.92-2.03; P trend=0.15). The two markers were not correlated (Spearman's r=0.01). Comparing subjects with low methylation levels (1st tertiles) at both markers with those having the highest methylation level (3rd tertile) at either marker, we observed a stronger significant association (OR=2.49, 95% CI=1.62-3.81; P trend=0.0001). Interestingly, we also found that the association between natural folate intake and colorectal cancer risk was modified by %5-mC or two markers combined (P interaction= 0.0003 and 0.02, respectively). Among individuals with the highest methylation level at either marker, high natural folate intake (3rd tertile) was associated with a reduced risk of colorectal cancer compared to low (1st tertile) folate intake (OR=0.49, 95% CI=0.25-0.96); no association was observed among individuals with lower levels of methylation. No significant risk modification was found for other suspected risk factors, such as smoking, obesity, and aspirin/ibuprofen use. This prospective investigation showed that reduced LINE-1 methylation in leukocyte DNA, especially in conjunction with reduced 5-mC methylation, was associated with increased colorectal cancer risk. Folate may protect against colorectal carcinogenesis through mechanisms involving adequate DNA methylation in the genome. Citation Format: Wen-Yi Huang, Mark P. Purdue, L. Joseph Su, Hormuzd A. Katki, Lee E. Moore, Srinivasan Yegnasubramanian, Sonja I. Berndt. Prospective study of genomic 5-methylcytosine and LINE-1 methylation levels of leukocyte DNA and colorectal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3231.

Gender-specific association of exposure to non-dioxin-like polychlorinated biphenyls during pregnancy with methylation levels of H19 and long interspersed nuclear element-1 in cord blood in the Hokkaido study.

Associations between prenatal exposure to polychlorinated biphenyls (PCBs) and reduced birth-size, and between DNA methylation of insulin-like growth factor-2 (IGF-2), H19 locus, and long interspersed nuclear element-1 (LINE-1) and reduced birth-size are well established. To date, however, studies on the associations between prenatal exposure to PCBs and alterations in methylation of IGF-2, H19, and LINE-1 are lacking. Thus, in this study, we examined these associations with infant-gender stratification. We performed a prospective birth cohort study using the Sapporo cohort from the previously described Hokkaido Birth Cohort Study on Environment and Children's Health conducted between 2002 and 2005 in Japan. In the final 169 study participants included in this study, we measured the concentrations of various non-dioxin-like PCBs in maternal blood during pregnancy using high-resolution gas chromatography/high-resolution mass spectrometry. IGF-2, H19 and LINE-1 methylation levels in cord blood were measured using the bisulfite pyrosequencing methods Finally, we assessed the associations between prenatal exposure to various PCBs and the gene methylation levels using multiple regression models stratified by infant gender. We observed a 0.017 (95% confidence interval [CI]: 0.003-0.031) increase in the log10-transformed H19 methylation levels (%) in cord blood for each ten-fold increase in the levels of decachlorinated biphenyls (decaCBs) in maternal blood among all infants. Similarly, a 0.005 (95% CI: 0.000-0.010) increase in the log10-transformed LINE-1 methylation levels (%) in cord blood was associated with each ten-fold increase in heptachlorinated biphenyls (heptaCBs) in maternal blood among all infants. In particular, we observed a dose-dependent association of the decaCB levels in maternal blood with the H19 methylation levels among female infants (P value for trend=0.040); likewise a dose-dependent association of heptaCB levels was observed with LINE-1 methylation levels among female infants (P value for trend=0.015). Moreover, these associations were only observed among infants of primiparous women. Our results suggest that the dose-dependent association between prenatal exposure to specific non-dioxin-like PCBs and increases in the H19 and LINE-1 methylation levels in cord blood might be more predominant in females than in males.

Global methylation correlates with clinical status in multiple sclerosis patients in the first year of IFNbeta treatment

The alteration of DNA methylation patterns are a key component of disease onset and/or progression. Our objective was to evaluate the differences in Long Interspersed Nuclear Element-1 (LINE-1) methylation levels, as a surrogate marker of global DNA methylation, between multiple sclerosis (MS) patients and healthy controls. In addition, we assessed the association of LINE-1 methylation with clinical disease activity in patients treated with IFNbeta (IFNβ). We found that individuals with high levels of LINE-1 methylation showed 6-fold increased risk of suffering MS. Additionally, treated MS patients who bear high LINE-1 methylation levels had an 11-fold increased risk of clinical activity. Moreover, a negative correlation between treatment duration and percentage of LINE-1 methylation, that was statistically significant exclusively in the group of patients without clinical activity, was observed. Our data suggest that in MS patients, a slight global DNA hypermethylation occurs that may be related to the pathophysiology of the disease. In addition, global DNA methylation levels could play a role as a biomarker for the differential clinical response to IFNβ.

Chronic exposure to arsenic, LINE-1 hypomethylation, and blood pressure: a cross-sectional study in Bangladesh

BackgroundChronic exposure to arsenic is associated with cancer and hypertension. Growing evidence suggests that altered methylation in long interspersed nuclear element-1 (LINE-1) is involved in many types of disorders, including cardiovascular disease. Here we evaluated the association between arsenic exposure and LINE-1 methylation levels, especially in relation to blood pressure (BP).MethodsA total of 236 subjects (175 from arsenic-endemic areas and 61 from a non-endemic area) in rural Bangladesh were recruited. The subjects’ arsenic exposure levels (i.e., drinking water, hair and nail arsenic concentrations) were measured by inductively coupled plasma mass spectroscopy. The subjects’ LINE-1 methylation levels were determined by pyrosequencing.ResultsThe average LINE-1 methylation levels of the subjects living in the arsenic-endemic areas were significantly (p < 0.01) lower than those of the subjects living in the non-endemic area. In a sex-stratified analysis, the arsenic exposure levels in female but not male subjects showed a significant inverse association with LINE-1 methylation levels before (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) and after (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) adjustment for age, body mass index and smoking. Analyses examining interactions among arsenic levels, BP and LINE-1 methylation showed that arsenic-related elevated levels of BP were associated with LINE-1 hypomethylation.ConclusionsOur findings demonstrated that chronic exposure to arsenic was inversely associated with LINE-1 methylation levels in blood leukocyte DNA and this was more pronounced in females than males; in addition, the decreased levels of LINE-1 methylation might be involved in the arsenic-induced elevation of BP.

Radiation-induced changes in DNA methylation and their relationship to chromosome aberrations in nuclear power plant workers

Purpose: We investigated the association between occupational radiation exposure and DNA methylation changes in nuclear power plant workers. We also evaluated whether radiation- induced DNA methylation alterations are associated with chromosome aberrations.Materials and methods: The study population included 170 radiation-exposed workers and 30 controls. We measured global, long interspersed nuclear element-1 (LINE-1), and satellite 2 methylation levels in blood leukocyte DNA. The analysis of chromosome aberrations was performed on peripheral lymphocytes.Results: Global DNA methylation levels were lower in radiation-exposed workers than in controls. The methylation levels were negatively associated with the recent 1.5-year radiation dose in a multiple linear regression model (β = − 0.0088, p ≤ 0.001); the levels increased proportionally with the total cumulative dose in radiation-exposed workers. LINE-1 methylation levels were higher in radiation-exposed workers than in controls and were significantly associated with the total cumulative radiation dose in a multiple linear regression model (β = − 0.031, p = 0.035). Global DNA methylation levels were also correlated with chromosome aberrations among workers. Workers with low global methylation levels had a higher frequency of chromosome aberrations than did subjects with high global methylation levels.Conclusion: Occupational exposure to low-dose radiation could affect DNA methylation levels, and the radiation-induced DNA methylation alterations may be associated with chromosome aberrations.

A cross-sectional study of global DNA methylation and risk of colorectal adenoma.

BackgroundThe methylation of DNA is recognized as a key epigenetic mechanism and evidence for its role in the development of several malignancies is accumulating. We evaluated the relationship between global methylation in DNA derived from normal appearing colon mucosal tissue and blood leukocytes, and colorectal adenoma risk.MethodsPatients, aged 40 to 65, scheduled for a screening colonoscopy were recruited. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. A fasting blood sample was also collected. The methylation status of LINE-1 (long interspersed nuclear element-1) repetitive sequences, as a surrogate measure of global methylation, was quantified in DNA extracted from normal colon mucosa and blood leukocytes. Statistical analysis of the relationship between global DNA methylation and adenoma risk was conducted on 317 participants, 108 subjects with at least one pathologically confirmed adenoma and 209 subjects with a normal colonoscopy.ResultsA statistically significant inverse relationship was observed between LINE-1 methylation in colon tissue DNA and adenoma risk for males and for both sexes combined for the lowest methylation quartile compared to the highest (adjusted ORs = 2.94 and 2.26 respectively). For blood, although the overall pattern of odds ratio estimates was towards an increase in risk for lower methylation quartiles compared to the highest methylation quartile, there were no statistically significant relationships observed. A moderate correlation was found between LINE-1 methylation levels measured in tissue and blood (Pearson correlation 0.36).ConclusionsWe observed that lower levels of LINE-1 DNA methylation in normal appearing background colon mucosa were associated with increased adenoma risk for males, and for both sexes combined. Though these findings provide some support for a relationship between LINE-1 DNA methylation in colon mucosal tissue and adenoma risk, large prospective cohort studies are needed to confirm results. Until such investigations are done, the clinical usefulness of LINE-1 methylation as a biomarker of increased adenoma risk is uncertain. Regardless, this study contributes to a better understanding of the role of global DNA methylation as an early event in CR carcinogenesis with implications for future etiologic research.

Hypomethylation of long interspersed nuclear element-1 (LINE-1) is associated with poor prognosis via activation of c-MET in hepatocellular carcinoma.

Long interspersed nuclear element-1 (LINE-1) methylation status, representing global DNA methylation levels, is associated with patient prognosis in several types of cancer. This study was designed to examine the prognostic significance of LINE-1 methylation in patients with hepatocellular carcinoma (HCC) and the possible mechanisms related to oncogene activation. Seventy-five HCC patients who underwent hepatectomy between 2006 and 2012 were enrolled in this study. Quantitative pyrosequencing was performed to quantify the methylation level of three CpG sites in the LINE-1 promoter. Clinicopathological variables and prognosis were compared between LINE-1 hypo- and hypermethylation groups. LINE-1-inserted c-MET (L1-MET) gene expression and its correlation with LINE-1 methylation levels also were analyzed. LINE-1 was significantly hypomethylated in tumor tissues compared with nontumor tissues (48.3 ± 12.2 % vs. 68.2 ± 2.0 %, respectively, p < 0.0001). LINE-1 hypomethylation was not associated with any clinicopathological factors in HCC patients, except sex (p < 0.05). However, patients with LINE-1 hypomethylation exhibited significantly poorer outcome, and multivariate analysis revealed that LINE-1 hypomethylation was an independent risk factor for overall survival (hazard ratio (HR) = 6.1, p = 0.031) and disease-free survival (HR = 2.34, p = 0.045). L1-MET expression was significantly higher in tumor tissues (p < 0.01). L1-MET expression levels were inversely correlated with LINE-1 methylation levels, and positively correlated with c-MET expression (p < 0.05). Furthermore, higher c-MET protein expression was observed in the LINE-1 hypomethylated tumor tissues compared with hypermethylated tumor tissues (p = 0.032). LINE-1 hypomethylation is significantly associated with poor prognosis in patients with HCC, possibly due to activation of c-MET expression.

Chromosomal instability associated with global DNA hypomethylation is associated with the initiation and progression of esophageal squamous cell carcinoma.

Global DNA hypomethylation is associated with increased chromosomal instability and plays an important role in tumorigenesis. The methylation status of the long interspersed nuclear element-1 (LINE-1) element is a useful surrogate marker for global DNA methylation. Although LINE-1 hypomethylation is recognized as a poor prognostic marker, the correlation of LINE-1 methylation level with tumor suppressor gene mutation, chromosomal instability, and clinical significance in esophageal squamous cell carcinoma (ESCC) remains unclear. Using resected tumor tissues and the corresponding normal esophageal mucosa from 105 patients with ESCC, bisulfite pyrosequencing analysis was performed to quantify the LINE-1 methylation levels. p53 mutations in exons two to ten were detected by polymerase chain reaction direct sequencing. Chromosomal instability was assessed by single nucleotide polymorphism array comparative genomic hybridization analysis. The LINE-1 methylation level of ESCC was significantly lower than matched normal mucosa. LINE-1 methylation levels of normal mucosa from the esophagus had a significant inverse correlation with both cigarette smoking and alcohol consumption of the study subjects. LINE-1 hypomethylation of ESCC was significantly associated with lymph node metastasis, lymphovascular invasion, the frequency of p53 mutation and poor survivability. The LINE-1 methylation levels in ESCC had a significant inverse association with the percentage of copy number alterations in the whole genome, mirroring chromosomal instability. Our results suggested that whole genome hypomethylation caused by chronic inflammation could initiate carcinogenesis of esophageal squamous cells through chromosomal instability. In addition, chromosomal instability associated with the global hypomethylation might correlate highly with the progression of ESCC.

Global long interspersed nuclear element 1 DNA methylation in a Colombian sample of patients with late-onset Alzheimer's disease.

Alterations in DNA methylation have implicated as an epigenetic event in the pathogenesis of late-onset Alzheimer's disease (LOAD). The objective of this work was to evaluate global DNA methylation levels for long interspersed nuclear element 1 (LINE-1) repetitive sequences in Colombian patients with LOAD and controls. The LINE-1 DNA methylation levels in peripheral blood samples from 28 Colombian patients with LOAD and 30 healthy participants were assessed using a methylation-sensitive high-resolution melting (MS-HRM) quantitative assay. We did not find differences in LINE-1 methylation levels between patients with Alzheimer's disease (AD; median 76.2%, interquartile range [IQR]: 69.8-81.9) and control participants (median 79.8%, IQR: 73.2-83.8; P = .3). Additional stratified analyses did not show differences in LINE-1 methylation levels for male or female patients versus controls nor for apolipoprotein E4 carriers and noncarriers. This is the first report of LINE-1 methylation levels in patients with LOAD using the cost-effective MS-HRM technique, and this is the first global DNA methylation study in Latin American patients with AD.