Karina Gonzalez

Significant differences in global genomic DNA methylation by gender and race/ethnicity in peripheral blood

Reduced levels of global DNA methylation are associated with genomic instability and are independent predictors of cancer risk. Little is known about the environmental determinants of global DNA methylation in peripheral blood. We examined the association between demographic and lifestyle factors and levels of global leukocyte DNA methylation in 161 cancer-free subjects enrolled in the North Texas Healthy Heart Study aged 45–75 years in 2008. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for diet, and bioelectrical impedance analysis and CT-scan for body composition. We measured genomic DNA methylation using bisulfite conversion of DNA and pyrosequencing for LINE-1. Body composition measures including body mass index, waist circumference, areas of subcutaneous fat and visceral fat, percent of fat mass and fat-free mass were not associated with global genomic DNA methylation after controlling the effect of age, gender and race/ethnicity. Instead, female gender was significantly associated with a reduced level of global methylation (β = -2.77, 95% CI: -4.33, -1.22). Compared to non-Hispanic whites, non-Hispanic blacks (β = -2.02, 95% CI: -3.55, -0.50) had significantly lower levels of global methylation. No association was found with age, cigarette smoking, alcohol drinking and dietary intake of nutrients in one-carbon metabolism. Global leukocyte DNA methylation differs by gender and race/ethnicity, suggesting these variables need to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite converted DNA and real time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45-75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26-30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤ 10 min/day (β=2.52, 95%CI: 0.70, 4.35) However, the association was attenuated and became statistically insignificant after multivariate adjustment (β=1.24, 95%CI:-0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β=1.50, 95%CI: -0.08, 3.08) that warrants further investigation.

Prenatal Smoke Exposure and Genomic DNA Methylation in a Multiethnic Birth Cohort

Background: Exposure to prenatal tobacco smoke (PTS) has been associated with a number of health outcomes in the offspring, including some childhood cancers. Lower levels of genomic DNA methylation have also been associated with several types of cancers. We investigated whether PTS was associated with global DNA methylation levels in the offspring. Methods: Our sample was drawn from a birth cohort of women born between 1959 and 1963 in New York City (n = 90). We measured methylation of repetitive elements (Sat2, Alu, LINE-1) from peripheral blood granulocytes. We combined prospectively collected data on PTS with adult epidemiologic data and blood samples collected in 2001 to 2007 (mean age, 43 years). We used linear regression to assess the association between PTS and repetitive element methylation. Results: Thirty-six percent of mothers smoked during pregnancy. We observed an inverse association between PTS and Sat2 methylation. This inverse association remained even after adjustment for potential mediators including child environmental tobacco smoke exposure, birth size, postnatal weight and height changes, and adult smoking status and alcohol intake (β = −0.22, 95% confidence interval = −0.40 to −0.03 for ever exposed to PTS vs. never exposed using models of log-transformed methylation levels). PTS exposure was not statistically significantly associated with LINE-1 or Alu methylation. Conclusions: PTS exposure, measured at the time of pregnancy and not retrospectively reported, was associated with a decrease in Sat2 methylation but not LINE-1 or Alu methylation. Impact: If replicated in larger studies, this study supports a persistent effect of PTS on DNA methylation levels, as measured by Sat2, in adulthood. Cancer Epidemiol Biomarkers Prev; 20(12); 2518–23. ©2011 AACR.

Early life socioeconomic factors and genomic DNA methylation in mid-life

Epigenetic modifications may be one mechanism linking early life factors, including parental socioeconomic status (SES), to adult onset disease risk. However, SES influences on DNA methylation patterns remain largely unknown. In a US birth cohort of women, we examined whether indicators of early life and adult SES were associated with white blood cell methylation of repetitive elements (Sat2, Alu and LINE-1) in adulthood. Low family income at birth was associated with higher Sat2 methylation (β = 19.7, 95% CI: 0.4, 39.0 for lowest vs. highest income quartile) and single parent family was associated with higher Alu methylation (β = 23.5, 95% CI: 2.6, 44.4), after adjusting for other early life factors. Lower adult education was associated with lower Sat2 methylation (β = -16.7, 95% CI: -29.0, -4.5). There were no associations between early life SES and LINE-1 methylation. Overall, our preliminary results suggest possible influences of SES across the life-course on genomic DNA methylation in adult women. However, these preliminary associations need to be replicated in larger prospective studies.

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.

DNA methylation changes correlate with Gleason score and tumor stage in prostate cancer.

DNA methylation, a widely used epigenetic mark, has been associated with many tumors. However, few studies have addressed the role of cell-free plasma DNA methylation in discriminating aggressive prostate cancer (PCa) from indolent cases. We conducted a case series and a case-control study among histologically confirmed stage II/III cases and matched controls recruited at Columbia University Medical Center. The aim of this study was to investigate whether plasma DNA methylation levels are appropriate surrogate biomarker of PCa tumor tissue levels and whether these markers are associated with worse clinicopathological tumor characteristics, which correlate with poorer prognosis. Quantitative pyrosequencing was used to detect methylation levels of p16 (CDKN4A), APC, GSTP1, and LINE-1 in 24 pairs of prostate tumor and adjacent tissues, as well as 27 plasma samples of PCa patients and 24 of controls. DNA methylation levels were significantly higher in tumor tissue than in adjacent nontumor tissue for p16 (CDKN4A), GSTP1, and APC; GSTP1 had a higher average percentage methylation in tumor tissue (38.9%) compared with p16 (CDKN4A) (5.9%) and APC (14.5%). GSTP1, p16 (CDKN4A), and APC methylation in tumor tissue was statistically significantly higher for cases with Gleason score ≥7 compared with those with Gleason score <7 [49.0% vs. 21.9% (p=0.01), 6.6% vs. 4.5% (p=0.04), and 19.1% vs. 7.4% (p=0.02), respectively]. Plasma LINE-1 methylation levels were higher in those with higher Gleason (67.6%) than in those with Gleasons below 7 (64.6%, p=0.03). Significant plasma-tissue correlations were observed for GSTP1 and LINE-1 methylation. These data, although preliminary, suggest that aberrant methylation may be a useful marker to identify PCa patients with clinically aggressive disease.

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.

Abstract 4936: Promoter methylation in prostate cancer: A pilot study

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Prostate cancer is a leading cause of cancer death among men in the United States. The current method used for the screening for prostate cancer, the prostate-specific antigen (PSA) test, has poor specificity and sensitivity. This has lead to the search for new biomarkers that can improve the prostate cancer screening, either by themselves or in combination with established markers. Epigenetic modifications, believed to occur early during tumorgenesis are one of the new measurements that could aid in improving prostate cancer screening. We conducted a case-control study to determine the association of promoter methylation of p16 INK4a and GSTP1 and prostate cancer risk. Twenty seven histologically confirmed prostate cancer patients were matched by age and ethnicity to twenty four controls. DNA was extracted from plasma, and percentage of DNA methylation was determined by pyrosequencing. We found that methylation levels were consistently higher in cases than in controls, but this difference was not statistically significant (p16 INK4a OR (95%CI)=2.38 (0.75-7.50); GSTP1 OR (95%CI)=2.57 (0.79-8.40). Methylation levels in these regions were also investigated in tumor and normal adjacent tissue samples from cases. Promoter regions of these genes are hypermethylated in prostatic tumors (7.2% for p16INK4a and 95.8% for GSTP1). Hypermethylation of both regions is statistically significantly associated with tumor status with OR p16INK4a =5.0, 95%CI: 1.3-19.1 and ORGSTP1=22.9, 95%CI: 2.7-198.6, respectively. The correlations of GSTP1 and p16INK4a methylation levels in matched tissue and plasma samples were also determined. Pearsons correlation coefficient was 0.46 for GSTP1 methylation when comparing tumor tissue and plasma (p=0.037). However, the tissue-plasma correlation for p16INK4a methylation levels was not statistically significant. Our samples size is small, but our results are encouraging suggesting that plasma DNA methylation levels at the promoter regions of p16INK4a and GSTP1 are relevant in the etiology of prostate cancer and may be useful as markers of the onset of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4936.

Abstract 3772: Lifestyle factors and global methylation in peripheral leukocyte DNA

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Global hypomethylation is associated with genomic instability and may mediate the association between environmental and lifestyle factors and cancer risk. We examined the association between lifestyle factors and levels of genomic DNA methylation in the peripheral blood leukocytes of 160 participants in the North Texas Healthy Heart Study aged 45-75. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for dietary data, bioelectrical impedance analysis (BIA) and CT-scan for body composition including fat mass % and areas of visceral and truncal subcutaneous adipose tissues at the L4L5 level. We measured genomic DNA methylation using bisulfite conversion of DNA and real time PCR (MethyLight) for SAT2-M1. The median level of global methylation was 61.3% in this cancer-free population. Male gender was positively associated with global hypomethylation (i.e. < median) (OR=3.7, 95%CI: 1.4-9.7). Compared to Hispanics (n=58), non-Hispanic whites (n=33) and non-Hispanics blacks (n=68) had a significantly higher level of global hypomethylation (OR=8.4, 95%CI: 2.4-29.6 and OR=4.1, 95%CI: 1.5-11.2 respectively). Although the area of L4L5 subcutaneous adipose tissue was positively associated with DNA methylation in the univariate model (342 cm2 among subjects with methylation < median vs. 395 cm2 among subjects with methylation ≥ median, p=0.04), none of the body composition measures were associated with DNA methylation in the multivariate model after adjusting for age, gender and ethnicity, nor were cigarette smoking, alcohol drinking, dietary folate intake and perceived stress. Although there was no association of lifestyle factors with genomic methylation in the peripheral leukocyte DNA, the strong association with gender and race/ethnicity indicates that interactions between these variables and lifestyle factors need to be explored in a larger sample before firm conclusions can be reached. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3772.

Abstract B125: Genomic methylation levels in white blood cell DNA among young girls

Lower levels of genomic DNA methylation in white blood cell DNA (WBC) have been observed in individuals with breast or other cancers. Genomic methylation in WBC has been also associated with risk factors for cancer in adulthood; data on the association with exposures earlier in life are limited. Using information from 31 girls 6 to 17 years of age from families enrolled in the Breast Cancer Family Registry, and 20 girls of similar age from families without breast cancer, we conducted a pilot study to examine whether genomic methylation in WBC is associated with selected breast cancer risk factors. Genomic methylation levels were measured by MethyLight for three repetitive elements ( LINE1, Sat2M1 and AluM2 ), the luminometric methylation assay (LUMA), and LINE1 ‐pyrosequencing. We found that age of menarche was inversely correlated with LINE1 MethyLight (r=−0.41, p=0.02) and pyrosequencing methylation (r=−0.48, p =0.01) levels. Girls with a body mass index (BMI, kg/m 2 ) between 18.5 and 24.9 had lower methylation of LINE1 pyrosequencing compared with girls with higher or lower BMI values. Genomic methylation levels, measured by LUMA, AluM2 MethyLight, and LINE1 pyrosequencing, differed between girls with and without a family history of breast cancer. These data, if replicated in larger samples, suggest that breast cancer risk factors may be associated with genomic methylation in WBC in early life. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B125.