Peripheral Blood Methylation Research Articles

Shared genetic control of expression and methylation in peripheral blood.

BackgroundExpression QTLs and epigenetic marks are often employed to provide an insight into the possible biological mechanisms behind GWAS hits. A substantial proportion of the variation in gene expression and DNA methylation is known to be under genetic control. We address the proportion of genetic control that is shared between these two genomic features.ResultsAn exhaustive search for pairwise phenotypic correlations between gene expression and DNA methylation in samples from human blood (n = 610) was performed. Of the 5 × 109 possible pairwise tests, 0.36 % passed Bonferroni corrected p-value cutoff of 9.9 × 10-12. We determined that the correlation structure between probe pairs was largely due to blood cell type specificity of the expression and methylation probes. Upon adjustment of the expression and methylation values for observed blood cellular composition (n = 422), the number of probe pairs which survived Bonferroni correction reduced by more than 5400 fold. Of the 614 correlated probe pairs located on the same chromosome, 75 % share at least one methylation and expression QTL at nominal 10-5p-value cutoff. Those probe pairs are located within 1Mbp window from each other and have a mean of absolute value of genetic correlation equal to 0.69, further demonstrating the high degree of shared genetic control.ConclusionsOverall, this study demonstrates notable genetic covariance between DNA methylation and gene expression and reaffirms the importance of correcting for cell-counts in studies on non-homogeneous tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2498-4) contains supplementary material, which is available to authorized users.

New Insights Into Intellectual Disability Caused by Mutations in a Chromatin Regulator

New Insights Into Intellectual Disability Caused by Mutations in a Chromatin Regulator

Abstract P1-08-04: Sources of polycyclic aromatic hydrocarbons associated with gene-specific promoter methylation in women with breast cancer

Abstract Tobacco smoke, diet, and indoor and outdoor air pollution, all major sources of polycyclic aromatic hydrocarbons (PAHs), have been associated with breast cancer incidence. Aberrant methylation may be an early event in carcinogenesis, but whether PAHs influence the epigenome is unclear. Few studies have evaluated whether PAHs are associated with methylation, particularly in breast tumors where methylation changes are particularly relevant. In a population-based case-control study, we measured promoter methylation of 13 breast cancer-related genes in breast tumor tissue (n=765-851 cases) and global methylation in peripheral blood (1,055 cases/1,101 controls). PAH sources (current active smoking, residential environmental tobacco smoke (ETS), vehicular traffic, synthetic log burning, and grilled/smoked meat intake) were evaluated separately. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). When comparing methylated versus unmethylated genes, synthetic log use was associated with increased ORs for CDH1 (OR=2.28, 95%CI=1.07-4.83), HIN1 (OR=2.11, 95%CI=1.32-3.38) and RARβ methylation (OR=1.82, 95%CI=1.18-2.83) and decreased ORs for BRCA1 methylation (OR=0.44, 95%CI=0.30-0.65). Residential ETS was associated with decreased ORs for ESR1 (OR=0.74, 95%CI=0.56-0.99) and CCND2 methylation (OR=0.65, 95%CI=0.44-0.96). Current smoking and vehicular traffic were associated with decreased ORs for DAPK (OR=0.53, 95%CI=0.28-0.99) and increased ORs for TWIST1 methylation (OR=2.79, 95%CI=1.24-6.30), respectively. In controls, synthetic log use was inversely associated with LINE-1 methylation (OR=0.60, 95%CI=0.42-0.87). PAH sources were associated with hypo- and hypermethylation at multiple promoter regions in breast tumors and LINE-1 hypomethylation in blood of controls. Methylation may be a potential biologic mechanism for the association between PAHs and breast cancer incidence. Citation Format: White AJ, Chen J, McCullough LE, Xu X, Cho YH, Conway K, Beyea J, Stellman SD, Steck SE, Mordukhovich I, Eng SM, Terry MB, Engel LS, Hatch M, Neugut AI, Hibshoosh H, Santella RM, Gammon MD. Sources of polycyclic aromatic hydrocarbons associated with gene-specific promoter methylation in women with breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-08-04.

Global measures of peripheral blood-derived DNA methylation as a risk factor in the development of mature B-cell neoplasms.

To examine whether peripheral blood methylation is associated with risk of developing mature B-cell neoplasms (MBCNs). We conducted a case-control study nested within a large prospective cohort. Peripheral blood was collected from healthy participants. Cases of MBCN were identified by linkage to cancer registries. Methylation was measured using the Infinium(®) HumanMethylation450. During a median of 10.6-year follow-up, 438 MBCN cases were evaluated. Global hypomethylation was associated with increased risk of MBCN (odds ratio: 2.27, [95% CI: 1.59-3.25]). Within high CpG promoter regions, hypermethylation was associated with increased risk (odds ratio: 1.76 [95% CI: 1.25-2.48]). Promoter hypermethylation was observed in HOXA9 and CDH1 genes. Aberrant global DNA methylation is detectable in peripheral blood collected years before diagnosis and is associated with increased risk of MBCN, suggesting changes to DNA methylation are an early event in MBCN development.

Sources of polycyclic aromatic hydrocarbons are associated with gene-specific promoter methylation in women with breast cancer

BackgroundTobacco smoke, diet and indoor/outdoor air pollution, all major sources of polycyclic aromatic hydrocarbons (PAHs), have been associated with breast cancer. Aberrant methylation may be an early event in carcinogenesis, but whether PAHs influence the epigenome is unclear, particularly in breast tissue where methylation may be most relevant. We aimed to evaluate the role of methylation in the association between PAHs and breast cancer. MethodsIn a population-based case-control study, we measured promoter methylation of 13 breast cancer-related genes in breast tumor tissue (n=765–851 cases) and global methylation in peripheral blood (1055 cases/1101 controls). PAH sources (current active smoking, residential environmental tobacco smoke (ETS), vehicular traffic, synthetic log burning, and grilled/smoked meat intake) were evaluated separately. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). ResultsWhen comparing methylated versus unmethylated genes, synthetic log use was associated with increased ORs for CDH1 (OR=2.26, 95%CI=1.06–4.79), HIN1 (OR=2.14, 95%CI=1.34–3.42) and RARβ (OR=1.80, 95%CI=1.16–2.78) and decreased ORs for BRCA1 (OR=0.44, 95%CI=0.30–0.66). Residential ETS was associated with decreased ORs for ESR1 (OR=0.74, 95%CI=0.56–0.99) and CCND2 methylation (OR=0.65, 95%CI=0.44–0.96). Current smoking and vehicular traffic were associated with decreased ORs for DAPK (OR=0.53, 95%CI=0.28–0.99) and increased ORs for TWIST1 methylation (OR=2.79, 95%CI=1.24–6.30), respectively. In controls, synthetic log use was inversely associated with LINE-1 (OR=0.59, 95%CI=0.41–0.86). DiscussionPAH sources were associated with hypo- and hypermethylation at multiple promoter regions in breast tumors and LINE-1 hypomethylation in blood of controls. Methylation may be a potential biologic mechanism for the associations between PAHs and breast cancer incidence.

Results of First in Human (FIH) Phase 1 Pharmacokinetic (PK) Guided Dose-Escalation Study of ASTX727, a Combination of the Oral Cytidine Deaminase Inhibitor (CDAi) E7727 with Oral Decitabine in Subjects with Myelodysplastic Syndromes (MDS)

Results of First in Human (FIH) Phase 1 Pharmacokinetic (PK) Guided Dose-Escalation Study of ASTX727, a Combination of the Oral Cytidine Deaminase Inhibitor (CDAi) E7727 with Oral Decitabine in Subjects with Myelodysplastic Syndromes (MDS)

Epigenome-wide association study reveals decreased average methylation levels years before breast cancer diagnosis.

BackgroundInterest in the potential of DNA methylation in peripheral blood as a biomarker of cancer risk is increasing. We aimed to assess whether epigenome-wide DNA methylation measured in peripheral blood samples obtained before onset of the disease is associated with increased risk of breast cancer. We report on three independent prospective nested case-control studies from the European Prospective Investigation into Cancer and Nutrition (EPIC-Italy; n = 162 matched case-control pairs), the Norwegian Women and Cancer study (NOWAC; n = 168 matched pairs), and the Breakthrough Generations Study (BGS; n = 548 matched pairs). We used the Illumina 450k array to measure methylation in the EPIC and NOWAC cohorts. Whole-genome bisulphite sequencing (WGBS) was performed on the BGS cohort using pooled DNA samples, combined to reach 50× coverage across ~16 million CpG sites in the genome including 450k array CpG sites. Mean β values over all probes were calculated as a measurement for epigenome-wide methylation.ResultsIn EPIC, we found that high epigenome-wide methylation was associated with lower risk of breast cancer (odds ratio (OR) per 1 SD = 0.61, 95 % confidence interval (CI) 0.47–0.80; −0.2 % average difference in epigenome-wide methylation for cases and controls). Specifically, this was observed in gene bodies (OR = 0.51, 95 % CI 0.38–0.69) but not in gene promoters (OR = 0.92, 95 % CI 0.64–1.32). The association was not replicated in NOWAC (OR = 1.03 95 % CI 0.81–1.30). The reasons for heterogeneity across studies are unclear. However, data from the BGS cohort was consistent with epigenome-wide hypomethylation in breast cancer cases across the overlapping 450k probe sites (difference in average epigenome-wide methylation in case and control DNA pools = −0.2 %).ConclusionsWe conclude that epigenome-wide hypomethylation of DNA from pre-diagnostic blood samples may be predictive of breast cancer risk and may thus be useful as a clinical biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0104-2) contains supplementary material, which is available to authorized users.

Abstract 2953: Genomic DNA hypomethylation as an independent risk factor for renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is the most lethal genitourinary cancer. Cigarette smoking, hypertension and obesity are the major risk factors for RCC; however, much of the etiology of this disease remains to be elucidated. Genomic DNA hypomethylation is a hallmark of most cancer genomes, promoting genomic instability and cell transformation. In the present study, we sought to determine whether global DNA methylation in peripheral blood is associated with risk of RCC. A case control study consisting of 889 RCC cases and an equal number of age, gender, and ethnicity-matched controls was conducted. Genomic DNA was isolated from peripheral blood and global DNA methylation was measured as 5-mC% content using an antibody-based immune assay. Logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) for the association between DNA methylation level and the risk of RCC. The median age was 59.29±10.42 for cases and 59.39±10.30 for controls. Over two-thirds (67% in both cases and controls) were males, about half (50.2% in cases and 52.2% in controls) were never smokers, the vast majority (84.8% in both cases and controls) were Caucasian. BMI, physical activity, hypertension and family history of cancer were significantly different between cases and controls. RCC cases are more likely than controls to have high blood pressure level (p = 5.81E-15), higher BMI (p = 0.011), low physical activity (p = 1.48E-20), and a family history of cancer (other than kidney cancer) (p = 5.42E-07). The median methylation level in controls was 3.97, significantly higher than the median level of 3.64 in cases (p<0.001 Wilcoxon rank-sum test). In multivariate logistic regression analysis adjusted by age, gender, ethnicity, smoking status, and hypertension, individuals in the lowest tertile (T1) and middle tertile (T2) of 5-mC% had higher risks of RCC with ORs of 1.42 (95%CI 1.12-1.81) and 1.14 (95% CI 0.89-1.45), respectively, compared to individuals in the highest tertile (T3) of methylation (p for trend 0.004). When stratified by RCC risk factors, associations between hypomethylation and increased RCC risks were stronger among males (OR 1.64, 95%CI 1.21-2.21), younger individuals (OR 1.54, 95%CI 1.10-2.15), never-smokers (OR 1.61, 95%CI 1.14-2.28) and African-Americans (OR 2.75, 95%CI 0.75-10.08). These findings suggest for the first time that global DNA hypomethylation in peripheral blood is independently associated with RCC risk. The results may provide better understanding of the etiology of RCC tumorigenesis and in combination with modifiable risk factors and other potential biomarkers, may help identify high risk individuals for screening and early detection of RCC. This work was supported in part by the National Institutes of Health (grant R01 CA170298) and the Center for Translational and Public Health Genomics, Duncan Family Institute for Cancer Prevention, The University of Texas M D Anderson Cancer Center. Citation Format: Julia Mendoza Perez, Jian Gu, Nizar M. Tannir, Surena Matin, Jose A. Karam, David W. Chang, Luis A. Herrera, Christopher G. Wood, Xifeng Wu. Genomic DNA hypomethylation as an independent risk factor for renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2015-2953

DNA Methylation Patterns in Peripheral Blood of Pregnant Women With Group B Streptococcus Colonization.

The primary risk factor for neonatal Group B streptococcus (GBS) infection, which is the leading cause of infectious neonatal morbidity and mortality, is maternal colonization. However, no definitive maternal risk factors for GBS colonization have been identified and no systematic efforts have been made to prevent maternal colonization. The purpose of this exploratory secondary analysis was to evaluate genome-wide DNA methylation patterns in maternal peripheral blood early in pregnancy for association with GBS colonization status in the third trimester. Genome-wide DNA methylation was analyzed from 18 nulliparous GBS-positive and -negative women (n = 9/group) recruited for a previous study. No statistically significant differences in baseline characteristics or DNA methylation in peripheral blood were identified between GBS-positive and -negative women in early pregnancy. The results suggest that DNA methylation patterns in peripheral blood are not associated with risk for GBS colonization.

What is the latest evidence for nutritional management of patients undergoing treatment for cancer?

s / Journal of Nutrition & Intermediary Metabolism 1 (2014) 1e55 2 the more common cancers and contributed data to studies of less common cancers. While many associations evaluated were not strong our findings contribute to the overall evidence on which national and international recommendations are based. We have also added to the understanding of associations between diet and cancer by examining differences in associations between different cancer subgroups, such as by aggressiveness for prostate cancer or hormone receptor subtype for breast cancer. One area where reasonably consistent associations were found was for measures of body size. On the basis of this a TV campaign promoting waist circumferences of under 100 cm for men and 85 cm for women (6) was developed. New topics of research in this area include studies we are undertaking investigating whether DNA methylation in peripheral blood can predict cancer risk, and whether methylation could be a mechanism linking diet with cancer. We are also working with colleagues in the US to look at associations between a dietary inflammatory score and different cancer outcomes. Despite what we already know, the recent Australian Health Survey (7) indicates that Australians are not following recommendations that could help reduce cancer risk. Among people aged 18 years or older, 63% were obese or overweight, 48% met the Australian guidelines for fruit consumption (2 or more serves per day for adults), 8% met the guidelines for vegetable consumption (5 or more serves per day), around 20% consumed more than 2 standard drinks per day and 67% of those aged 15 years or older were sedentary or performed only a low level of exercise. Further research may refine recommendations and understanding of mechanisms bywhich diet canmodify cancer risk, but epidemiologists need toworkwith others to bring the benefits of this to the population in general. Funding source(s): NHMRC.

Elevation of peripheral BDNF promoter methylation links to the risk of Alzheimer's disease.

Brain derived neurotrophic factor (BDNF) has been known to play an important role in various mental disorders or diseases such as Alzheimer's disease (AD). The aim of our study was to assess whether BDNF promoter methylation in peripheral blood was able to predict the risk of AD. A total of 44 AD patients and 62 age- and gender-matched controls were recruited in the current case-control study. Using the bisulphite pyrosequencing technology, we evaluated four CpG sites in the promoter of the BDNF. Our results showed that BDNF methylation was significantly higher in AD cases than in the controls (CpG1: p = 10.021; CpG2: p = 0.002; CpG3: p = 0.007; CpG4: p = 0.005; average methylation: p = 0.004). In addition, BDNF promoter methylation was shown to be significantly correlated with the levels of alkaline phosphatase (ALP), glucose, Lp(a), ApoE and ApoA in males (ALP: r = −0.308, p = 0.042; glucose: r = −0.383, p = 0.010; Lp(a): r = 0.333, p = 0.027; ApoE: r = −0.345, p = 0.032;), ApoA levels in females (r = 0.362, p = 0.033), and C Reactive Protein (CRP) levels in both genders (males: r = −0.373, p = 0.016; females: r = −0.399, p = 0.021). Our work suggested that peripheral BDNF promoter methylation might be a diagnostic marker of AD risk, although its underlying function remains to be elaborated in the future.

P.1.a.026 Methylation of the promoter of brain-derived neurotrophic factor exon IV and antidepressant response in major depression

Tobacco smoke, diet and indoor/outdoor air pollution, all major sources of polycyclic aromatic hydrocarbons (PAHs), have been associated with breast cancer. Aberrant methylation may be an early event in carcinogenesis, but whether PAHs influence the epigenome is unclear, particularly in breast tissue where methylation may be most relevant. We aimed to evaluate the role of methylation in the association between PAHs and breast cancer.In a population-based case-control study, we measured promoter methylation of 13 breast cancer-related genes in breast tumor tissue (n=765–851 cases) and global methylation in peripheral blood (1055 cases/1101 controls). PAH sources (current active smoking, residential environmental tobacco smoke (ETS), vehicular traffic, synthetic log burning, and grilled/smoked meat intake) were evaluated separately. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs).When comparing methylated versus unmethylated genes, synthetic log use was associated with increased ORs for CDH1 (OR=2.26, 95%CI=1.06–4.79), HIN1 (OR=2.14, 95%CI=1.34–3.42) and RARβ (OR=1.80, 95%CI=1.16–2.78) and decreased ORs for BRCA1 (OR=0.44, 95%CI=0.30–0.66). Residential ETS was associated with decreased ORs for ESR1 (OR=0.74, 95%CI=0.56–0.99) and CCND2 methylation (OR=0.65, 95%CI=0.44–0.96). Current smoking and vehicular traffic were associated with decreased ORs for DAPK (OR=0.53, 95%CI=0.28–0.99) and increased ORs for TWIST1 methylation (OR=2.79, 95%CI=1.24–6.30), respectively. In controls, synthetic log use was inversely associated with LINE-1 (OR=0.59, 95%CI=0.41–0.86).PAH sources were associated with hypo- and hypermethylation at multiple promoter regions in breast tumors and LINE-1 hypomethylation in blood of controls. Methylation may be a potential biologic mechanism for the associations between PAHs and breast cancer incidence.

DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood.

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC.

DNA methylation array analyses to identify HYAL2 methylation in peripheral blood as a marker for the detection of early breast cancer.

26 Background: Early detection is crucial to improve the survival rate and quality of life of breast cancer (BC) patients. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. We aim to identify BC-associated DNA methylation signatures in peripheral blood. Methods: We identified a BC-associated differentially methylated locus by genome-wide investigation using Illumina 27K Methylation Assay. Two validation studies and replications in leucocytes and T cells were carried out using MassARRAY. The RNA expression levels were measured by real-time PCR. Results: The methylation level of CpG site cg27091787 in hyaluronoglucosaminidase 2 gene (HYAL2) in peripheral blood was significantly lower in BC cases than in controls (discovery round, 72 BC case and 24 controls, p = 2.61 × 10-9 adjusted for cell-type proportions; first validation round,338 BC case and 507 controls, p &lt; 0.0001; second validation round,189 BC case and 189 controls, p &lt; 0.0001). Compared to the highest quartile, the lowest quartile of cg27091787 methylation was associated with a more than 40-fold increased risk of BC (p &lt; 0.0001). In addition to cg27091787, the decreased methylation of a 650 bp CpG island shore of HYAL2 was also associated with BC. Moreover, the expression and methylation of HYAL2 in leucocytes were inversely correlated (p = 0.006). To note, the BC-associated decreased HYAL2 methylation was replicated in T cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early stage BC cases from healthy controls (area under curve (AUC) = 0.88), and was also robust for the detection of BC in younger women (AUC = 0.87). Conclusions: Validating the epigenome-wide study by independent cohorts, we have revealed a strong association between decreased HYAL2 methylation in peripheral blood and BC. Our results have given an answer to the debate on the origin of BC-associated differential methylation in blood, which is not only because of the change of cell type proportions, but more importantly due to altered methylation in specific blood cell fragments, like in T cells. And thus, we provide a promising blood-based marker for the detection of early BC.

Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state.

Early-life stress (ELS) is known to be associated with an increased risk of neuropsychiatric and cardiometabolic disease in later life. One of the potential mechanisms underpinning this is through effects on the epigenome, particularly changes in DNA methylation. Using a well-phenotyped cohort of 83 men from the Helsinki Birth Cohort Study, who experienced ELS in the form of separation from their parents during childhood, and a group of 83 matched controls, we performed a genome-wide analysis of DNA methylation in peripheral blood. We found no differences in DNA methylation between men who were separated from their families and non-separated men; however, we did identify differences in DNA methylation in association with the development of at least mild depressive symptoms over the subsequent 5–10 years. Notably, hypomethylation was identified at a number of genes with roles in brain development and/or function in association with depressive symptoms. Pathway analysis revealed an enrichment of DNA methylation changes in pathways associated with development and morphogenesis, DNA and transcription factor binding and programmed cell death. Our results support the concept that DNA methylation differences may be important in the pathogenesis of psychiatric disease.

Aberrant PTPRO methylation in tumor tissues as a potential biomarker that predicts clinical outcomes in breast cancer patients.

BackgroundAberrant hypermethylation of gene promoter regions is a primary mechanism by which tumor suppressor genes become inactivated in breast cancer. Epigenetic inactivation of the protein tyrosine phosphatase receptor-type O gene (PTPRO) has been described in several types of cancer.ResultsWe screened primary breast cancer tissues for PTPRO promoter hypermethylation and assessed potential associations with pathological features and patient outcome. We also evaluated its potential as a breast cancer biomarker. PTPRO methylation was observed in 53 of 98 (54%) breast cancer tissues but not in adjacent normal tissue. Among matched peripheral blood samples from breast cancer patients, 33 of 98 (34%) exhibited methylated PTPRO in plasma. In contrast, no methylated PTPRO was observed in normal peripheral blood from 30 healthy individuals. PTPRO methylation was positively associated with lymph node involvement (P = 0.014), poorly differentiated histology (P = 0.037), depth of invasion (P = 0.004), and HER2 amplification (P = 0.001). Multivariate analysis indicated that aberrant PTPRO methylation could serve as an independent predictor for overall survival hazard ratio (HR): 2.7; 95% CI: 1.1-6.2; P = 0.023), especially for patients with HER2-positive (hazard ratio (HR): 7.5; 95% CI: 1.8-31.3; P = 0.006), but not in ER + and PR + subpopulation. In addition, demethylation induced by 5-azacytidine led to gene reactivation in PTPRO-methylated and -silenced breast cancer cell lines.ConclusionsHere, we report that tumor PTPRO methylation is a strong prognostic factor in breast cancer. Methylation of PTPRO silences its expression and plays an important role in breast carcinogenesis. The data we present here may provide insight into the development of novel therapies for breast cancer treatment. Additionally, detection of PTPRO methylation in peripheral blood of breast cancer patients may provide a noninvasive means to diagnose and monitor the disease.

A-clustering: a novel method for the detection of co-regulated methylation regions, and regions associated with exposure

DNA methylation is a heritable modifiable chemical process that affects gene transcription and is associated with other molecular markers (e.g. gene expression) and biomarkers (e.g. cancer or other diseases). Current technology measures methylation in hundred of thousands, or millions of CpG sites throughout the genome. It is evident that neighboring CpG sites are often highly correlated with each other, and current literature suggests that clusters of adjacent CpG sites are co-regulated. We develop the Adjacent Site Clustering (A-clustering) algorithm to detect sets of neighboring CpG sites that are correlated with each other. To detect methylation regions associated with exposure, we propose an analysis pipeline for high-dimensional methylation data in which CpG sites within regions identified by A-clustering are modeled as multivariate responses to environmental exposure using a generalized estimating equation approach that assumes exposure equally affects all sites in the cluster. We develop a correlation preserving simulation scheme, and study the proposed methodology via simulations. We study the clusters detected by the algorithm on high dimensional dataset of peripheral blood methylation of pesticide applicators. We provide the R package Aclust that efficiently implements the A-clustering and the analysis pipeline, and produces analysis reports. The package is found on http://www.hsph.harvard.edu/tamar-sofer/packages/ tsofer@hsph.harvard.edu

Abstract 3642: Profiles of DNA methylation in peripheral blood are associated with bladder cancer survival.

Abstract Bladder cancer is a common solid tumor, with greater than 71,000 cases diagnosed in the United States last year, and greater than 14,000 deaths attributable to this disease. In addition to this mortality, the morbidity of this disease can be great, as the non-invasive form of this disease, making up &amp;gt;75% of the incidence, recurs frequently, and therefore requires frequent invasive monitoring through cystoscopy at a great cost to the health care system. There are critical gaps in our understanding of the predictors of its recurrence and survival, and identifying molecular alterations associated with these outcomes is essential for improving patient morbidity and the care of patients with this disease. We have shown that profiles of DNA methylation in peripheral blood are associated with human bladder cancer and have recently demonstrated that much, but not all, of the variation in DNA methylation is attributable to disease-associated changes in the underlying proportion of immune cells in the peripheral blood sample. We also have devised a novel statistical strategy that utilizes DNA methylation profiles to quantify the proportion of blood cell subtypes in archival DNA. This is able to disentangle the variability in DNA methylation profiles attributable to blood cell population differences from epigenetic variation arising within a cell population. We sought to determine if variation in DNA methylation identified in peripheral blood, reflective, in part, of altered white blood cell proportions, is associated with bladder cancer recurrence and survival in a population-based study of incident bladder cancers in New Hampshire, USA. Illumina Infinium 27K DNA methylation arrays were performed on whole peripheral blood derived DNA samples from 223 cases of bladder cancer, having a median follow-up time of approximately 13 years. Array data was subjected to quality control procedures and normalization using the ComBAT methodology to account for plate/BeadChip effects. Fitting a series of Cox proportional hazards models, stratified by histology (carcinoma in-situ, low-grade non-invasive, high-grade non-invasive, and invasive) and controlled for patient age and gender revealed 6,873 high-confidence (q &amp;lt; 0.05) associations with bladder cancer specific death as the endpoint. In a principle components analysis, the first principal component, accounting for a striking 46% of the variability in methylation, was significantly associated with bladder cancer specific death. Ongoing analyses are examining how changes to underlying cell proportions mediate the relationship between DNA methylation and patient survival. These data suggest that peripheral blood DNA methylation profiles proximal to the time of diagnosis are strongly prospectively predictive of bladder cancer specific survival and suggest that these biomarkers may have important use as non-invasive prognostic tools worthy of continued study. Citation Format: Devin C. Koestler, Megan A. Murphy, Eugene A. Houseman, Margaret R. Karagas, Karl T. Kelsey, Carmen J. Marsit. Profiles of DNA methylation in peripheral blood are associated with bladder cancer survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2013-3642

Abstract MP32: Epigenome-Wide DNA Methylation Profiling in a CVD Cohort: The ARIC Study

DNA methylation is mitotically heritable modification in chromatin structure that impacts transcriptional control of genes and cellular function. Recent technological advances provide opportunities to systematically interrogate variation in DNA methylation across the genome in large epidemiologic studies. However, unlike inherited changes to the genetic sequence, variation in site-specific methylation varies by tissue, stage of development, disease state, and may be affected by gender, aging and exposure to environmental factors. As a result, there is likely a greater threat of confounding in epigenome-wide methylation studies compared to genome-wide association studies of SNPs. The Illumina Infinium HumanMethylation450 BeadChip was used to measure DNA methylation in peripheral blood obtained from African American participants from the Jackson, Mississippi and Forsyth County, North Carolina field centers of the Atherosclerosis Risk in Communities (ARIC) Study, a population-based cohort of middle-aged men and women. After excluding outlier samples and CpG sites using quality control filters, we analyzed 473,687 sites in 2873 subjects who were between 47-71 years of age at the time of DNA collection. We used linear regression with robust standard errors to examine cross-sectional associations of demographic factors with the beta value, an estimate of the average methylation level at each locus, and applied a Bonferroni correction to account for multiple testing. In univariate analysis, 91% of sites on the X chromosome and 10% of sites on the autosomes exhibited statistically significant gender differences in methylation level (p&lt;1x10-7). Average methylation was higher in women than men for most of the significant sites (63% and 89% on the X chromosome and autosomes, respectively). Percent European ancestry estimated from ancestry informative markers was significantly associated with methylation level at 4% of sites. Age was also significantly associated with methylation at 4% of sites; average methylation was lower in older subjects compared to younger subjects for the majority (58%) of these sites. As we begin to implement epigenome-wide studies of DNA methylation and CVD outcomes, these results indicate that such studies will require careful consideration of adjustment techniques to avoid confounding by gender, age, and other covariates.

LINE-1 methylation in peripheral blood and the risk of melanoma in melanoma-prone families with and without CDKN2A mutations

Cutaneous malignant melanoma (CMM) is an etiologically heterogenous disease with genetic, environmental (sun exposure), and host (pigmentation/nevi) factors and their interactions contributing to risk. Recently, epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. We thus examined whether global methylation was associated with CMM risk in individuals from melanoma-prone families with and without CDKN2A germline mutations. We measured global DNA methylation using bisulfite pyrosequencing at four CpG sites of the long interspersed nucleotide element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from individuals in 64 melanoma-prone families including 114 CMM cases (45 CDKN2A-positive and 69 CDKN2A-negative) and 121 unaffected individuals (31 CDKN2A-positive and 90 CDKN2A-negative). We used unconditional logistic regression to evaluate the association between CMM status and LINE-1 methylation levels, adjusting for age at blood draw and accounting for familial correlation in the variance. We found that male sex was significantly associated with higher overall LINE-1 methylation (P=0.0001). However, the overall and site-specific levels of LINE-1 methylation did not vary significantly by CMM status (overall odds ratio: 1.57, 95% confidence interval: 0.84-2.95, P=0.16; comparing lowest to highest or reference methylation group). Similar results were obtained when CDKN2A-positive and CDKN2A-negative families were analyzed separately. Our findings did not support a significant association between constitutional LINE-1 methylation in PBMCs and risk of CMM in melanoma-prone families with or without CDKN2A mutations.