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1.  Comparison of the DNA methylation profiles of human peripheral blood cells and transformed B-lymphocytes 
Human genetics  2010;127(6):651-658.
Epidemiological studies of DNA methylation (DNAm) profiles may hold substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. Different cell types are likely to have different DNAm patterns. We investigate the DNAm differences between two types of biospecimens available in many genetic epidemiology studies. We compared DNAm patterns in two different DNA samples from each of 34 participants in the Genetic Epidemiology Network of Arteriopathy study (20 Caucasians and 14 African-Americans). One was extracted from peripheral blood cells (PBC) and the other from transformed B-lymphocytes (TBL). The genome-wide DNAm profiles were compared at over 27,000 genome-wide methylation sites. We found that 26 out of the 34 participants had correlation coefficients higher than 0.9 between methylation profiles of PBC and TBL. Although a high correlation was observed in the DNAm profile between PBC and TBL, we also observed variation across samples from different DNA resources and donors. Using principal component analysis of the DNAm profiles, the two sources of the DNA samples could be accurately predicted. We also identified 3,723 autosomal DNAm sites that had significantly different methylation statuses in PBC compared to TBL (Bonferroni corrected p value <0.05). Both PBC and TBL provide a rich resource for understanding the DNAm profiles in humans participating in epidemiologic studies. While the majority of DNAm findings in PBC and TBL may be consistent, caution must be used when interpreting results because of the possibility of cell type-specific methylation modification.
doi:10.1007/s00439-010-0810-y
PMCID: PMC2873107  PMID: 20238126
2.  Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus12 
Neoplasia (New York, N.Y.)  2012;14(1):29-33.
Aberrant DNA methylation (DNAm) is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm) profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA) and Barrett esophagus (BE, EA precursor). We performed genome-wide DNAm profiling in EA tissue DNA (n = 8) and matched serum DNA (n = 8), in serum DNA of BE (n = 10), and in healthy controls (n = 10) using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92) in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.
PMCID: PMC3281939  PMID: 22355271
3.  Gene-Specific DNA Methylation Association with Serum Levels of C-Reactive Protein in African Americans 
PLoS ONE  2013;8(8):e73480.
A more thorough understanding of the differences in DNA methylation (DNAm) profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP), an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.
doi:10.1371/journal.pone.0073480
PMCID: PMC3747126  PMID: 23977389
4.  Genome-Wide DNA Methylation Scan in Major Depressive Disorder 
PLoS ONE  2012;7(4):e34451.
While genome-wide association studies are ongoing to identify sequence variation influencing susceptibility to major depressive disorder (MDD), epigenetic marks, such as DNA methylation, which can be influenced by environment, might also play a role. Here we present the first genome-wide DNA methylation (DNAm) scan in MDD. We compared 39 postmortem frontal cortex MDD samples to 26 controls. DNA was hybridized to our Comprehensive High-throughput Arrays for Relative Methylation (CHARM) platform, covering 3.5 million CpGs. CHARM identified 224 candidate regions with DNAm differences >10%. These regions are highly enriched for neuronal growth and development genes. Ten of 17 regions for which validation was attempted showed true DNAm differences; the greatest were in PRIMA1, with 12–15% increased DNAm in MDD (p = 0.0002–0.0003), and a concomitant decrease in gene expression. These results must be considered pilot data, however, as we could only test replication in a small number of additional brain samples (n = 16), which showed no significant difference in PRIMA1. Because PRIMA1 anchors acetylcholinesterase in neuronal membranes, decreased expression could result in decreased enzyme function and increased cholinergic transmission, consistent with a role in MDD. We observed decreased immunoreactivity for acetylcholinesterase in MDD brain with increased PRIMA1 DNAm, non-significant at p = 0.08.
While we cannot draw firm conclusions about PRIMA1 DNAm in MDD, the involvement of neuronal development genes across the set showing differential methylation suggests a role for epigenetics in the illness. Further studies using limbic system brain regions might shed additional light on this role.
doi:10.1371/journal.pone.0034451
PMCID: PMC3325245  PMID: 22511943
5.  Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation 
Genome Medicine  2012;4(3):24.
Background
Recently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation.
Methods
We analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions.
Results
We observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively).
Conclusions
We demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application.
Trial registration
The ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821.
doi:10.1186/gm323
PMCID: PMC3446274  PMID: 22453031
6.  Age-associated epigenetic drift: implications, and a case of epigenetic thrift? 
Human Molecular Genetics  2013;22(R1):R7-R15.
It is now well established that the genomic landscape of DNA methylation (DNAm) gets altered as a function of age, a process we here call ‘epigenetic drift’. The biological, functional, clinical and evolutionary significance of this epigenetic drift, however, remains unclear. We here provide a brief review of epigenetic drift, focusing on the potential implications for ageing, stem cell biology and disease risk prediction. It has been demonstrated that epigenetic drift affects most of the genome, suggesting a global deregulation of DNAm patterns with age. A component of this drift is tissue-specific, allowing remarkably accurate age-predictive models to be constructed. Another component is tissue-independent, targeting stem cell differentiation pathways and affecting stem cells, which may explain the observed decline of stem cell function with age. Age-associated increases in DNAm target developmental genes, overlapping those associated with environmental disease risk factors and with disease itself, notably cancer. In particular, cancers and precursor cancer lesions exhibit aggravated age DNAm signatures. Epigenetic drift is also influenced by genetic factors. Thus, drift emerges as a promising biomarker for premature or biological ageing, and could potentially be used in geriatrics for disease risk prediction. Finally, we propose, in the context of human evolution, that epigenetic drift may represent a case of epigenetic thrift, or bet-hedging. In summary, this review demonstrates the growing importance of the ‘ageing epigenome’, with potentially far-reaching implications for understanding the effect of age on stem cell function and differentiation, as well as for disease prevention.
doi:10.1093/hmg/ddt375
PMCID: PMC3782071  PMID: 23918660
7.  Adaptation of the CHARM DNA methylation platform for the rat genome reveals novel brain region-specific differences 
Epigenetics  2011;6(11):1378-1390.
Comprehensive high-throughput arrays for relative methylation (CHARM) was recently developed as an experimental platform and analytic approach to assess DNA methylation (DNAm) at a genome-wide level. Its initial implementation was for human and mouse. We adapted it for rat and sought to examine DNAm differences across tissues and brain regions in this model organism. We extracted DNA from liver, spleen and three brain regions: cortex, hippocampus and hypothalamus from adult Sprague Dawley rats. DNA was digested with McrBC, and the resulting methyl-depleted fraction was hybridized to the rat CHARM array along with a mock-treated fraction. Differentially methylated regions (DMRs) between tissue types were detected using normalized methylation log-ratios. In validating 24 of the most significant DMRs by bisulfite pyrosequencing, we detected large mean differences in DNAm, ranging from 33–59%, among the most significant DMRs in the across-tissue comparisons. The comparable figures for the hippocampus vs. hypothalamus DMRs were 14–40%, for the cortex vs. hippocampus DMRs, 12–29%, and for the cortex vs. hypothalamus DMRs, 5–35%, with a correlation of r2 = 0.92 between the methylation differences in 24 DMRs predicted by CHARM and those validated by bisulfite pyrosequencing. Our adaptation of the CHARM array for the rat genome yielded highly robust results that demonstrate the value of this method in detecting substantial DNAm differences between tissues and across different brain regions. This platform should prove valuable in future studies aimed at examining DNAm differences in particular brain regions of rats exposed to environmental stimuli with potential epigenetic consequences.
doi:10.4161/epi.6.11.18072
PMCID: PMC3242812  PMID: 22048247
epigenetics; DNA methylation; methylation array; genome-wide; rat; brain
8.  Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array 
Background
Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.
Results
Probes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.
Conclusion
DNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.
doi:10.1186/1756-8935-6-4
PMCID: PMC3740789  PMID: 23452981
Infinium HumanMethylation450 BeadChip array; DNA methylation; non-specific probes; Polymorphic probes; CpG islands; Annotation; CpG enrichment; Tissue-specific DNA methylation; Repetitive elements; 450 k
9.  Epigenome-Wide Association Studies for common human diseases 
Nature reviews. Genetics  2011;12(8):529-541.
Despite the success of genome-wide association studies (GWAS) in identifying loci associated with common diseases, a significant proportion of the causality remains unexplained. Recent advances in genomic technologies have placed us in a position to initiate large-scale studies of human disease-associated epigenetic variation, specifically variation in DNA methylation (DNAm). Such Epigenome-Wide Association Studies (EWAS) present novel opportunities but also create new challenges that are not encountered in GWAS. We discuss EWAS study design, cohort and sample selections, statistical significance and power, confounding factors, and follow-up studies. We also discuss how integration of EWAS with GWAS can help to dissect complex GWAS haplotypes for functional analysis.
doi:10.1038/nrg3000
PMCID: PMC3508712  PMID: 21747404
Epigenomics; Disease Genetics; DNA Methylation; Epigenetics; Quantitative Trait
10.  Primitive Neuroectodermal Tumor in an Ovarian Cystic Teratoma: Natural Killer and Neuroblastoma Cell Analysis 
Case Reports in Oncology  2014;7(1):70-78.
In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patient's peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.
doi:10.1159/000357802
PMCID: PMC3934805  PMID: 24575020
Activating natural killer receptors; Natural killer cells; Neuroblastoma; Ovarian cancer
11.  Genome-wide methylation analysis of human colon cancer reveals similar hypo- and hypermethylation at conserved tissue-specific CpG island shores 
Nature genetics  2009;41(2):178-186.
Alterations in DNA methylation (DNAm) in cancer have been known for 25 years, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes1. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands2,3. Here we show that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands but in sequences up to 2 kb distant which we term “CpG island shores.” CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a surprising overlap (45-65%) of the location of colon cancer-related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling non-colon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, and they are consistent with the epigenetic progenitor model of cancer4, that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.
doi:10.1038/ng.298
PMCID: PMC2729128  PMID: 19151715
12.  Integrative genomic analysis identifies epigenetic marks that mediate genetic risk for epithelial ovarian cancer 
Background
Both genetic and epigenetic factors influence the development and progression of epithelial ovarian cancer (EOC). However, there is an incomplete understanding of the interrelationship between these factors and the extent to which they interact to impact disease risk. In the present study, we aimed to gain insight into this relationship by identifying DNA methylation marks that are candidate mediators of ovarian cancer genetic risk.
Methods
We used 214 cases and 214 age-matched controls from the Mayo Clinic Ovarian Cancer Study. Pretreatment, blood-derived DNA was profiled for genome-wide methylation (Illumina Infinium HumanMethylation27 BeadArray) and single nucleotide polymorphisms (SNPs, Illumina Infinium HD Human610-Quad BeadArray). The Causal Inference Test (CIT) was implemented to distinguish CpG sites that mediate genetic risk, from those that are consequential or independently acted on by genotype.
Results
Controlling for the estimated distribution of immune cells and other key covariates, our initial epigenome-wide association analysis revealed 1,993 significantly differentially methylated CpGs that between cases and controls (FDR, q < 0.05). The relationship between methylation and case-control status for these 1,993 CpGs was found to be highly consistent with the results of previously published, independent study that consisted of peripheral blood DNA methylation signatures in 131 pretreatment cases and 274 controls. Implementation of the CIT test revealed 17 CpG/SNP pairs, comprising 13 unique CpGs and 17 unique SNPs, which represent potential methylation-mediated relationships between genotype and EOC risk. Of these 13 CpGs, several are associated with immune related genes and genes that have been previously shown to exhibit altered expression in the context of cancer.
Conclusions
These findings provide additional insight into EOC etiology and may serve as novel biomarkers for EOC susceptibility.
doi:10.1186/1755-8794-7-8
PMCID: PMC3916313  PMID: 24479488
Integrative genomics; Ovarian cancer; Blood-based DNA methylation
13.  CD8 T cell defect of TNF-α and IL-2 in DNAM-1 deficient mice delays clearance in vivo of a persistent virus infection 
Virology  2012;429(2):163-170.
DNAM-1 gene-deficient (−/−) mice take significantly longer to clear an acute and persistent LCMV infection in vivo than DNAM-1 +/+ mice. During acute LCMV priming, at the single cell level, DNAM-1 −/− mice made significantly less cytoplasmic CD8 TNF-α and IL-2 but not IFN-γ than their DNAM-1 +/+ counterparts. Restimulated immune memory CD8 T cells from DNAM-1 −/− and DNAM-1 +/+ mice were equivalent in cytolytic activity against LCMV-infected target cells but DNAM-1 −/− CD8 T cells had significant reductions in TNF-α and IL-2 that were associated on adoptive transfer with the inability to terminate the persistent viral infection.
doi:10.1016/j.virol.2012.04.006
PMCID: PMC3571684  PMID: 22579352
DNAM-1 deficiency; Acute LCMV infection; Persistent LCMV infection; T cell defects
14.  DNA Methylation Array Analysis Identifies Profiles of Blood-Derived DNA Methylation Associated With Bladder Cancer 
Journal of Clinical Oncology  2011;29(9):1133-1139.
Purpose
Epigenetic alterations in tissues targeted for cancer play a causal role in carcinogenesis. Changes in DNA methylation in nontarget tissues, specifically peripheral blood, can also affect risk of malignant disease. We sought to identify specific profiles of DNA methylation in peripheral blood that are associated with bladder cancer risk and therefore serve as an epigenetic marker of disease susceptibility.
Methods
We performed genome-wide DNA methylation profiling on participants involved in a population-based incident case-control study of bladder cancer.
Results
In a training set of 112 cases and 118 controls, we identified a panel of 9 CpG loci whose profile of DNA methylation was significantly associated with bladder cancer in a masked, independent testing series of 111 cases and 119 controls (P < .0001). Membership in three of the most methylated classes was associated with a 5.2-fold increased risk of bladder cancer (95% CI, 2.8 to 9.7), and a model that included the methylation classification, participant age, sex, smoking status, and family history of bladder cancer was a significant predictor of bladder cancer (area under the curve, 0.76; 95% CI, 0.70 to 0.82). CpG loci associated with bladder cancer and aging had neighboring sequences enriched for transcription-factor binding sites related to immune modulation and forkhead family members.
Conclusion
These results indicate that profiles of epigenetic states in blood are associated with risk of bladder cancer and signal the potential utility of epigenetic profiles in peripheral blood as novel markers of susceptibility to this and other malignancies.
doi:10.1200/JCO.2010.31.3577
PMCID: PMC3083868  PMID: 21343564
15.  Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity 
The Journal of Clinical Investigation  2011;121(9):3609-3622.
NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.
doi:10.1172/JCI45816
PMCID: PMC3171102  PMID: 21841316
16.  Hematopoietic Stem and Progenitor Cells Acquire Distinct DNA-Hypermethylation During in vitro Culture 
Scientific Reports  2013;3:3372.
Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34+ cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remained CD34+ - acquired significant DNA-hypermethylation. DNA-hypermethylation occurred particularly in up-stream promoter regions, shore-regions of CpG islands, binding sites for PU.1, HOXA5 and RUNX1, and it was reflected in differential gene expression and variant transcripts of DNMT3A. Low concentrations of DNAm inhibitors slightly increased the frequency of colony-forming unit initiating cells. Our results demonstrate that HPCs acquire DNA-hypermethylation at specific sites in the genome which is relevant for the rapid loss of stemness during in vitro manipulation.
doi:10.1038/srep03372
PMCID: PMC3842544  PMID: 24284763
17.  Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation? 
Mesenchymal stem cells change dramatically during culture expansion. Long-term culture has been suspected to evoke oncogenic transformation: overall, the genome appears to be relatively stable throughout culture but transient clonal aneuploidies have been observed. Oncogenic transformation does not necessarily entail growth advantage in vitro and, therefore, the available methods - such as karyotypic analysis or genomic profiling - cannot exclude this risk. On the other hand, long-term culture is associated with specific senescence-associated DNA methylation (SA-DNAm) changes, particularly in developmental genes. SA-DNAm changes are highly reproducible and can be used to monitor the state of senescence for quality control. Notably, neither telomere attrition nor SA-DNAm changes occur in pluripotent stem cells, which can evade the 'Hayflick limit'. Long-term culture of mesenchymal stem cells seems to involve a tightly regulated epigenetic program. These epigenetic modifications may counteract dominant clones, which are more prone to transformation.
doi:10.1186/scrt145
PMCID: PMC3580484  PMID: 23257053
18.  Investigation of human cationic antimicrobial protein-18 (hCAP-18), lactoferrin and CD163 as potential biomarkers for ovarian cancer 
Background
Epithelial ovarian cancer is one of the leading causes of gynaecological cancer morbidity and mortality in women. Early stage ovarian cancer is usually asymptomatic, therefore, is often first diagnosed when it is widely disseminated. Currently available diagnostics lack the requisite sensitivity and specificity to be implemented as community-based screening tests. The identification of additional biomarkers may improve the diagnostic efficiency of multivariate index assays. The aims of this study were to characterise and compare the ovarian tissue immunohistochemical localisation and plasma concentrations of three putative ovarian cancer biomarkers: human cationic antimicrobial protein-18 (hCAP-18); lactoferrin; and CD163 in normal healthy women and women with ovarian cancer.
Methods
In this case–control cohort study, ovarian tissue and blood samples were obtained from 164 women (73 controls, including 28 women with benign pelvic masses; 91 cancer, including 21 women with borderline tumours). Localisation of each antigen within the ovary was assessed by immunohistochemistry and serum concentrations determined by ELISA assays.
Results
Immunoreactive (ir) hCAP-18 and lactoferrin were identified in epithelial cells, while CD163 was predominately localised in stromal cells. Tissue ir CD163 increased significantly (P<0.05) with disease grade. Median plasma concentrations of soluble (s)CD163 were significantly greater in the cases (3220 ng/ml) than in controls (2488 ng/ml) (P< 0.01). Median plasma concentrations of hCAP-18 and lactoferrin were not significantly different between cases and controls. The classification efficiency of each biomarker (as determined by the area under the receiver operator characteristic curve; AUC) was: 0.67± 0.04; 0.62 ± 0.08 and 0.51 ± 0.07 for sCD163, hCAP-18 and lactoferrin, respectively. When the 3 biomarkers were modelled using stochastic gradient boosted logistic regression, the AUC increased to 0.95 ± 0.03.
Conclusions
The data obtained in this study establishes the localisation and concentrations of CD163, hCAP-18, and lactoferrin in ovarian tumours and peripheral blood. Individually, the 3 biomarkers display only modest diagnostic efficiency as assessed by AUC. When combined in a multivariate index assay, however, diagnostic efficiency increases significantly. As such, the utility of the biomarker panel, as an aid in the diagnosis of cancer in symptomatic women, is worthy of further investigation in a larger phase 2 biomarker trial.
doi:10.1186/1757-2215-6-5
PMCID: PMC3557177  PMID: 23339669
Ovarian cancer; hCAP-18; Lactoferrin; CD163
19.  Maternal Genome-Wide DNA Methylation Patterns and Congenital Heart Defects 
PLoS ONE  2011;6(1):e16506.
The majority of congenital heart defects (CHDs) are thought to result from the interaction between multiple genetic, epigenetic, environmental, and lifestyle factors. Epigenetic mechanisms are attractive targets in the study of complex diseases because they may be altered by environmental factors and dietary interventions. We conducted a population based, case-control study of genome-wide maternal DNA methylation to determine if alterations in gene-specific methylation were associated with CHDs. Using the Illumina Infinium Human Methylation27 BeadChip, we assessed maternal gene-specific methylation in over 27,000 CpG sites from DNA isolated from peripheral blood lymphocytes. Our study sample included 180 mothers with non-syndromic CHD-affected pregnancies (cases) and 187 mothers with unaffected pregnancies (controls). Using a multi-factorial statistical model, we observed differential methylation between cases and controls at multiple CpG sites, although no CpG site reached the most stringent level of genome-wide statistical significance. The majority of differentially methylated CpG sites were hypermethylated in cases and located within CpG islands. Gene Set Enrichment Analysis (GSEA) revealed that the genes of interest were enriched in multiple biological processes involved in fetal development. Associations with canonical pathways previously shown to be involved in fetal organogenesis were also observed. We present preliminary evidence that alterations in maternal DNA methylation may be associated with CHDs. Our results suggest that further studies involving maternal epigenetic patterns and CHDs are warranted. Multiple candidate processes and pathways for future study have been identified.
doi:10.1371/journal.pone.0016506
PMCID: PMC3031146  PMID: 21297937
20.  A measure of glucocorticoid load provided by DNA methylation of Fkbp5 in mice 
Psychopharmacology  2011;218(1):303-312.
Rationale
Given the contribution of cortisol dysregulation to neuropsychiatric and metabolic disorders, it is important to be able to accurately compute glucocorticoid burden, a measure of allostatic load. One major problem in calculating cortisol burden is that existing measures reflect cortisol exposure over a short duration and have not been proven to reliably quantify cortisol burden over weeks or months.
Method
We treated two cohorts of mice with corticosterone in the drinking water and determined the relationship between serial plasma corticosterone levels drawn over 4 weeks and the whole-blood DNA methylation (DNAm) changes in a specific glucocorticoid-sensitive gene, Fkbp5, determined at the end of the treatment period.
Results
We observed that the percent reduction in DNAm in the intron 1 region of Fkbp5 determined from a single blood draw strongly reflected average glucocorticoid burden generated weekly during the prior month of glucocorticoid exposure. There were also strong correlations in DNAm with glucocorticoid-induced end organ changes in spleen weight and visceral fat. We tested a subset of these animals for anxiety-like behavior in the elevated plus maze and found that DNAm in the blood also has predictive value in determining the behavioral consequences of glucocorticoid exposure.
Conclusion
A whole-blood assessment of Fkbp5 gene methylation is a biomarker that integrates 4 weeks of glucocorticoid exposure and may be a useful measure in states of excess exposure. It will be important to determine if Fkbp5 DNAm changes can also be a biomarker of glucocorticoid burden during chronic social stress.
doi:10.1007/s00213-011-2307-3
PMCID: PMC3918452  PMID: 21509501
DNA methylation; Fkbp5; Glucocorticoid Burden; Corticosterone; Epigenetics; Allostasis
21.  Systematic Evaluation of Candidate Blood Markers for Detecting Ovarian Cancer 
PLoS ONE  2008;3(7):e2633.
Background
Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.
Methods and Findings
We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.
Conclusions
By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.
doi:10.1371/journal.pone.0002633
PMCID: PMC2440813  PMID: 18612378
22.  Genome-Scale Screen for DNA Methylation-Based Detection Markers for Ovarian Cancer 
PLoS ONE  2011;6(12):e28141.
Background
The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer.
Methodology/Principal Findings
We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels.
We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients.
Conclusions/Significance
We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.
doi:10.1371/journal.pone.0028141
PMCID: PMC3233546  PMID: 22163280
23.  NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo 
The Journal of Clinical Investigation  2009;119(5):1251-1263.
NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell–mediated innate immunity to melanoma cells and provide a background to design NK cell–based immunotherapeutic strategies against melanoma and possibly other tumors.
doi:10.1172/JCI36022
PMCID: PMC2673866  PMID: 19349689
24.  Accelerated tumor growth in mice deficient in DNAM-1 receptor 
The Journal of Experimental Medicine  2008;205(13):2959-2964.
Since the identification of ligands for human and mouse DNAM-1, emerging evidence has suggested that DNAM-1 plays an important role in the T cell– and natural killer (NK) cell–mediated recognition and lysis of tumor cells. However, it remains undetermined whether DNAM-1 is involved in tumor immune surveillance in vivo. We addressed this question by using DNAM-1–deficient mice. DNAM-1–deficient cytotoxic T lymphocyte (CTL) and NK cells showed significantly less cytotoxic activity against DNAM-1 ligand-expressing tumors in vitro than wild-type (WT) cells. The methylcholanthrene (MCA)-induced fibrosarcoma cell line Meth A expressed the DNAM-1 ligand CD155, and DNAM-1–deficient mice showed increased tumor development and mortality after transplantation of Meth A cells. Moreover, the DNAM-1–deficient mice developed significantly more DNAM-1 ligand-expressing fibrosarcoma and papilloma cells in response to the chemical carcinogens MCA and 7,12-dimethylbenz[a]anthracene (DMBA), respectively, than did WT mice. These results indicate that DNAM-1 plays an important role in immune surveillance of tumor development.
doi:10.1084/jem.20081611
PMCID: PMC2605241  PMID: 19029379
25.  Peripheral blood DNA methylation profiles are indicative of head and neck squamous cell carcinoma 
Epigenetics  2012;7(3):291-299.
Head and neck cancer accounts for an estimated 47,560 new cases and 11,480 deaths annually in the United States, the majority of which are squamous cell carcinomas (HNSCC). The overall 5 year survival is approximately 60% and declines with increasing stage at diagnosis, indicating a need for non-invasive tests that facilitate the detection of early disease. DNA methylation is a stable epigenetic modification that is amenable to measurement and readily available in peripheral blood. We used a semi-supervised recursively partitioned mixture model (SS-RPMM) approach to identify novel blood DNA methylation markers of HNSCC using genome-wide methylation array data for peripheral blood samples from 92 HNSCC cases and 92 cancer-free control subjects. To assess the performance of the resultant markers, we constructed receiver operating characteristic (RJC) curves and calculated the corresponding area under the curve (AUC). Cases and controls were best differentiated by a methylation profile of six CpG loci (associated with FGD4, SERPINF1, WDR39, IL27, HYAL2 and PLEKHA6), with an AUC of 0.73 (95% CI: 0.62–0.82). After adjustment for subject age, gender, smoking, alcohol consumption and HPV16 serostatus, the AUC increased to 0.85 (95% CI: 0.76–0.92). We have identified a novel blood-based methylation profile that is indicative of HNSCC with a high degree of accuracy. This profile demonstrates the potential of DNA methylation measured in blood for development of non-invasive applications for detection of head and neck cancer.
doi:10.4161/epi.7.3.19134
PMCID: PMC3335952  PMID: 22430805
semi-supervised RPMM; HNSCC; epigenetics; biomarkers; Infinium; methylation array

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