Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility, however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared to 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulfite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared to 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared to controls (p=0.0017) with the highest quartile of methylation associated with a three-fold increased risk of breast cancer (OR=3.20, 95% C.I.=1.78-5.86, p=0.000083). Increased methylation of this locus is associated with lower steady state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age-phenotype related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk.

Twin studies have provided the basis for genetic and epidemiological studies in human complex traits. As epigenetic factors can contribute to phenotypic outcomes, we conducted a DNA methylation analysis in white blood cells (WBC), buccal epithelial cells and gut biopsies of 114 monozygotic (MZ) twins as well as WBC and buccal epithelial cells of 80 dizygotic (DZ) twins using 12K CpG island microarrays. Here we provide the first annotation of epigenetic metastability of approximately 6,000 unique genomic regions in MZ twins. An intraclass correlation (ICC)-based comparison of matched MZ and DZ twins showed significantly higher epigenetic difference in buccal cells of DZ co-twins (P = 1.2 x 10(-294)). Although such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and animal studies favor the hypothesis that it is due to epigenomic differences in the zygotes, suggesting that molecular mechanisms of heritability may not be limited to DNA sequence differences.

noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. This chapter describes a method for profiling DNA methylation from small amounts of genomic-DNA starting material utilizing an efficient sodium bisulfite conversion method followed by whole-genome amplification (WGA). WGA is a useful method to overcome the problem of low initial amount of DNA and/or severe DNA degradation during conventional sodium bisulfite treatment in studies investigating DNA methylation. WGA is a relatively inexpensive process that can be optimized for high-throughput application and enables the thorough investigation of methylation at numerous genomic locations on samples for which DNA availability is low. Data from our lab has demonstrated that bisulfite-treated DNA amplified using WGA can be used for a range of downstream DNA methylation mapping procedures, including bisulfite-primer optimization, the sequencing of cloned PCR products, MS-SNuPE, and Pyrosequencing.

As the role for epigenetic signals in genome regulation becomes increasingly understood, the ability to accurately measure levels of DNA methylation at individual cytosines throughout the genome is becoming increasingly important. In contrast to traditional methods for the quantification of cytosine methylation, such as cloning and sequencing of PCR fragments amplified from sodium bisulfite-modified DNA, recent developments have created a fast and effective alternative called methylation-sensitive single nucleotide primer extension (Ms-SNuPE). The following protocol outlines the steps necessary to design and perform Ms-SNuPE experiments using the SNaPshot(R) chemistry and associated capillary electrophoresis platforms available through Applied Biosystems.

Traditionally, etiological research of schizophrenia has been focused on elucidating predisposing genes and environmental risk factors. While numerous putative environmental hazards have been suggested, inconsistencies and methodological limitations of epidemiological studies have made it difficult to identify even a single exogenous cause of schizophrenia. Furthermore, there is increasing evidence that environmental risk factors may not play as much of a significant role in schizophrenia as previously suspected. In this article, we argue that molecular epigenetic studies can overcome the complexities of traditional epidemiological studies and may become a productive line of research in understanding the nongenetic mechanisms of schizophrenia.

Attention-deficit hyperactivity disorder (ADHD) is a common childhood neurobehavioural disorder defined by symptoms of developmentally inappropriate inattention, impulsivity and hyperactivity. As is the norm for most psychiatric phenotypes, traditional aetiological studies have focused primarily on the interplay between genetic and environmental factors. It is likely that epigenetic factors, i.e., heritable, but reversible changes to genomic function that are independent of DNA sequence, are also important. It is known that epigenetic processes can be induced following exposure to a range of external factors, and thus provide a mechanism by which the environment can lead to long-term alterations in phenotype. In this article we hypothesise that epigenetic dysregulation may mediate the association observed between early-development environmental insults and ADHD. We propose that understanding the epigenetic processes involved in linking specific environmental pathogens to an increased risk for ADHD may offer new possibilities for preventative and therapeutic intervention.

Comprehensive analyses of the human epigenome may be of critical importance in understanding the molecular mechanisms of complex diseases, development, aging, tissue specificity, parental origin effects, and sex differences, among other systemic aspects of human biology. However, traditional DNA methylation methods allowed for screening of only relatively short DNA fragments. The advent of microarrays has provided new possibilities in DNA methylation analysis, because this technology is able to interrogate a very large number of loci in a highly parallel fashion. There are several permutations of the microarray application in DNA methylation profiling, and such include microarray analysis of bisulfite modified DNA and also the enriched unmethylated or hypermethylated DNA fractions using methylation-sensitive restriction enzymes or antibodies against methylated cytosines. The method described in detail here is based on the analysis of the enriched unmethylated DNA fraction, using a series of treatments with methylation-sensitive restriction enzymes, adaptor ligation, PCR amplification, and quantitative mapping of unmethylated DNA sequences using microarrays. The key advantages of this approach are the ability to investigate DNA methylation patterns using very small DNA amounts and relatively high informativeness in comparison to the other restriction-enzyme- based strategies for DNA methylation profiling [1].

Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.

Abstract DNA methylation differences between identical twins could account for phenotypic twin discordance of behavioral traits and diseases. High throughput epigenomic microarray profiling can be a strategy of choice for identification of epigenetic differences in phenotypically different monozygotic (MZ) twins. Epigenomic profiling of a pair of MZ twins with quantified measures of psychometric discordance identified several DNA methylation differences, some of which may have developmental and behavioral implications and are consistent with the contrasting psychometric profiles of the twins. In particular, differential methylation of CpG islands proximal to the homeobox DLX1 gene could modulate stress responses and risk taking behavior, and deserve further attention as a potential marker of aversion to danger. The epigenetic difference detected at DLX1 of approximately 1.2 fold change was used to evaluate experimental design issues such as the required numbers of technical replicates. It also enabled us to estimate the power this technique would have to detect a functionally relevant epigenetic difference given a range of 1 to 50 twin pairs. We found that use of epigenomic microarray profiling in a relatively small number (15-25) of phenotypically discordant twin pairs has sufficient power to detect 1.2 fold epigenetic changes.

blacksquare, square, filled Abstract Epigenetics is a new development in complex non-Mendelian disease, which may not only uncover etiologic and pathogenic mechanisms but may also provide the basis for the development of medications that would target the primary epigenetic causes of such diseases. Such epigenetic drugs would be novel, potentially possessing substantially higher therapeutic potential and much lower rate of adverse effects in comparison to current symptomatic treatments. A collection of epigenetic drugs already exist at various stages of development and, although their effectiveness has yet to be maximized, they show great promise in the treatment of cancer, psychiatric disorders, and other complex diseases. Here we present a review of the epigenetic theory of complex disease and an evaluation of current epigenetic therapies, as well as predictions of the future directions in this expanding field. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 48 is January 6, 2008. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.