Maya Kappil

Genome-wide DNA methylation profiles in hepatocellular carcinoma†‡

Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA before HCC diagnosis. To identify, with a genome‐wide approach, additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent nontumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays (Illumina, Inc., San Diego, CA) that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor, compared to nontumor tissues. Array data were validated with pyrosequencing in a subset of five of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37%‐63% of cases had detectable hypermethylated DNA (≥5% methylation) for these five genes individually. At least one of these genes was hypermethylated in 87% of the cases, suggesting that measurement of DNA methylation in plasma samples is feasible. Conclusion: The panel of methylated genes indentified in the current study will be further tested in a large cohort of prospectively collected samples to determine their utility as early biomarkers of HCC. (HEPATOLOGY 2012;55:1799–1810)

Global methylation profiles in DNA from different blood cell types

DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [3H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [3H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [3H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). . Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.

Association between arsenic exposure from drinking water and plasma levels of soluble cell adhesion molecules.

Background Epidemiologic studies of cardiovascular disease risk factors and appropriate biomarkers in populations exposed to a wide range of arsenic levels are a public health research priority. Objective We investigated the relationship between inorganic arsenic exposure from drinking water and plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule-1 (sVCAM-1), both markers of endothelial dysfunction and vascular inflammation, in an arsenic-exposed population in Araihazar, Bangladesh. Methods The study participants included 115 individuals with arsenic-related skin lesions participating in a 2 × 2 randomized, placebo-controlled, double-blind trial of vitamin E and selenium supplementation. Arsenic exposure status and plasma levels of sICAM-1 and sVCAM-1 were assessed at baseline and after 6 months of follow-up. Results Baseline well arsenic, a long-term measure of arsenic exposure, was positively associated with baseline levels of both sICAM-1 and sVCAM-1 and with changes in the two markers over time. At baseline, for every 1-μg/L increase in well arsenic there was an increase of 0.10 ng/mL [95% confidence interval (CI), 0.00–0.20] and 0.33 ng/mL (95% CI, 0.15–0.51) in plasma sICAM-1 and sVCAM-1, respectively. Every 1-μg/L increase in well arsenic was associated with a rise of 0.11 ng/mL (95% CI, 0.01–0.22) and 0.17 ng/mL (95% CI, 0.00–0.35) in sICAM-1 and sVCAM-1 from baseline to follow-up, respectively, in spite of recent changes in urinary arsenic as well as vitamin E and selenium supplementation during the study period. Conclusions The findings indicate an effect of chronic arsenic exposure from drinking water on vascular inflammation that persists over time and also suggest a potential mechanism underlying the association between arsenic exposure and cardiovascular disease.

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma.

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.

Global DNA methylation levels in girls with and without a family history of breast cancer

Lower levels of global DNA methylation in white blood cell (WBC) DNA have been associated with adult cancers. It is unknown whether individuals with a family history of cancer also have lower levels of global DNA methylation early in life. We examined global DNA methylation in WBC (measured in three repetitive elements, LINE1, Sat2 and Alu, by MethyLight and in LINE1 by pyrosequencing) in 51 girls ages 6-17. Compared to girls without a family history of breast cancer, methylation levels were lower for all assays in girls with a family history of breast cancer, and statistically significantly lower for Alu and LINE1 pyrosequencing. After adjusting for age, body mass index (BMI), and Tanner stage, only methylation in Alu was associated with family history of breast cancer. If these findings are replicated in larger studies, they suggest that lower levels of global WBC DNA methylation observed later in life in adults with cancer may also be present early in life in children with a family history of cancer.

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [3H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [3H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.

Biologic and epigenetic impact of commuting to work by car or using public transportation: a case-control study.

BACKGROUND AND AIMS Commuting by public transportation (PT) entails more physical activity and energy expenditure than by cars, but its biologic consequences are unknown. METHODS In 2009-2010, we randomly sampled New York adults, usually commuting either by car (n=79) or PT (n=101). Measures comprised diet and physical activity questionnaires, weight and height, white blood cell (WBC) count, C reactive protein, (CRP) gene-specific methylation (IL-6), and global genomic DNA methylation (LINE-1 methylation). RESULTS Compared to the 101 PT commuters, the 79 car drivers were about 9 years older, 2 kg/m(2) heavier, more often non-Hispanic whites, and ate more fruits and more meats. The 2005 guidelines for physical activity were met by more car drivers than PT users (78.5% vs. 65.0%). There were no differences in median levels of CRP (car vs. PT: 0.6 vs. 0.5mg/dl), mean levels of WBC (car vs. PT: 6.7 vs. 6.5 cells/mm(3)), LINE-1 methylation (car vs. PT: 78.0% vs. 78.3%), and promoter methylation of IL-6 (car vs. PT: 56.1% vs. 58.0%). CONCLUSIONS PT users were younger and lighter than car drivers, but their commute mode did not translate into a lower inflammatory response or a higher DNA methylation, maybe because, overall, car drivers were more physically active.

Serum Oxidized Protein and Prostate Cancer Risk within the Prostate Cancer Prevention Trial

To evaluate the role of oxidative stress in prostate cancer risk, we analyzed serum levels of protein carbonyl groups in 1,808 prostate cancer cases and 1,805 controls, nested in the Prostate Cancer Prevention Trial, a randomized, placebo-controlled trial that found finasteride decreased prostate cancer risk. There were no significant differences in protein carbonyl levels in baseline samples between those later diagnosed with prostate cancer and those without at the end of study biopsy. Adjusted odds ratios and 95% confidence intervals (95% CI) for the 4th quartile of protein carbonyl level for the combined, placebo, and finasteride arms were 1.03 (95% CI, 0.85-1.24), 0.88 (95% CI, 0.69-1.12), and 1.27 (95% CI, 0.94-1.71), respectively. There were no significant associations between carbonyl level and risk when analyzing high-grade and low-grade disease separately, nor did finasteride affect protein oxidation levels. The results of this large nested case-control study do not support the hypothesis that oxidative stress, at least as measured by protein carbonyl level, plays a role in prostate cancer. Cancer Prev Res; 3(4); 478–83. ©2010 AACR.