Variation In Methylation Patterns Research Articles

Abstract 7003: Microfluidic platform for DNA methylation profiling towards early detection of ovarian cancer

Abstract We present a microfluidic platform for molecule-by-molecule detection of heterogeneous epigenetic patterns of rare tumor-derived DNA by highly parallelized digital melt. The platform digitizes DNA molecules in a 4-module, 40,000 1-nL array microfluidic device and observes sequence-dependent fluorescence changes during temperature ramping. The platform is applied to quantify DNA methylation heterogeneity of a biomarker panel within Pap specimens in a small cohort of healthy and ovarian cancer patients, and demonstrates an accuracy of 0.9. DNA hypermethylation of tumor-suppressor genes is an epigenetic phenomena that occurs early in tumorigenesis. Variable methylation patterns are observed in ovarian cancer precursor tissue, and trace amounts of ovarian-tumor-derived DNA can be found in Pap specimens, albeit at low frequencies (<0.01%). This suggests that methylation profiles of DNA extracted from routinely-obtained Pap specimens could contain early cancer biomarkers. However, due to the scarcity of these molecules, current techniques, such as sequencing and digital PCR (dPCR), have technical limitations precluding their ability to profile methylation patterns reliably. Sequencing, although comprehensive, has limited sensitivity, undermining its utility for routine detection of fractions below 0.1%. dPCR achieves high sensitivity, but is limited to known sequences, thus cannot provide comprehensive analysis of molecular heterogeneity. To overcome these limitations, we developed a technique called digital high resolution melt (dHRM), a thermodynamics-based approach to detecting molecular variability by observing the sequence-specific release of a DNA-intercalating dye under a thermal ramp. dHRM can discriminate methylation patterns on CpG-by-CpG basis and detect methylated epialleles at frequencies as low as 0.00005%. Here, DNA from Pap specimens was loaded on a microfluidic chip that digitizes rare target molecules into individual reaction chambers. A thermal-optical platform was developed to perform parallelized dPCR and dHRM. Methylation patterns of a biomarker panel were analyzed to produce a probability score that the Pap specimen contained tumor-derived DNA. 12 healthy and 12 ovarian cancer Pap specimens were assessed for methylation heterogeneity of 9 genes. Optimal methylation density thresholds were determined, and data above each threshold was combined into a single score for each possible combination of biomarkers. In this small cohort, methylation heterogeneity analysis of just 2 loci produced an area under the receiver-operator characteristics curve (AUC) of 0.9. This highly sensitive methylation profiling technology demonstrates promising utility towards early cancer detection. Future work aims to expand the testing cohort for ovarian cancer detection and permit further studies on the impact of methylation heterogeneity on early cancer development. Citation Format: Chrissy O'Hersey, Yang Zhao, Thomas R. Pisanic, Tian-Li Wang, Ie-Ming Shih, Tza-Huei Jeff Wang. Microfluidic platform for DNA methylation profiling towards early detection of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7003.

Cold exposure impacts DNA methylation patterns in cattle sperm.

DNA methylation is influenced by various exogenous factors such as nutrition, temperature, toxicants, and stress. Bulls from the Pacific Northwest region of the United States and other northern areas are exposed to extreme cold temperatures during winter. However, the effects of cold exposure on the methylation patterns of bovine sperm remain unclear. To address, DNA methylation profiles of sperm collected during late spring and winter from the same bulls were analyzed using whole genome bisulfite sequencing (WGBS). Bismark (0.22.3) were used for mapping the WGBS reads and R Bioconductor package DSS was used for differential methylation analysis. Cold exposure induced 3,163 differentially methylated cytosines (DMCs) with methylation difference ≥10% and a q-value < 0.05. We identified 438 differentially methylated regions (DMRs) with q-value < 0.05, which overlapped with 186 unique genes. We also identified eight unique differentially methylated genes (DMGs) (Pax6, Macf1, Mest, Ubqln1, Smg9, Ctnnb1, Lsm4, and Peg10) involved in embryonic development, and nine unique DMGs (Prmt6, Nipal1, C21h15orf40, Slc37a3, Fam210a, Raly, Rgs3, Lmbr1, and Gan) involved in osteogenesis. Peg10 and Mest, two paternally expressed imprinted genes, exhibited >50% higher methylation. The differential methylation patterns of six distinct DMRs: Peg10, Smg9 and Mest related to embryonic development and Lmbr1, C21h15orf40 and Prtm6 related to osteogenesis, were assessed by methylation-specific PCR (MS-PCR), which confirmed the existence of variable methylation patterns in those locations across the two seasons. In summary, cold exposure induces differential DNA methylation patterns in genes that appear to affect embryonic development and osteogenesis in the offspring. Our findings suggest the importance of replicating the results of the current study with a larger sample size and exploring the potential of these changes in affecting offspring development.

Estimating genome-wide DNA methylation heterogeneity with methylation patterns

BackgroundIn a heterogeneous population of cells, individual cells can behave differently and respond variably to the environment. This cellular diversity can be assessed by measuring DNA methylation patterns. The loci with variable methylation patterns are informative of cellular heterogeneity and may serve as biomarkers of diseases and developmental progression. Cell-to-cell methylation heterogeneity can be evaluated through single-cell methylomes or computational techniques for pooled cells. However, the feasibility and performance of these approaches to precisely estimate methylation heterogeneity require further assessment.ResultsHere, we proposed model-based methods adopted from a mathematical framework originally from biodiversity, to estimate genome-wide DNA methylation heterogeneity. We evaluated the performance of our models and the existing methods with feature comparison, and tested on both synthetic datasets and real data. Overall, our methods have demonstrated advantages over others because of their better correlation with the actual heterogeneity. We also demonstrated that methylation heterogeneity offers an additional layer of biological information distinct from the conventional methylation level. In the case studies, we showed that distinct profiles of methylation heterogeneity in CG and non-CG methylation can predict the regulatory roles between genomic elements in Arabidopsis. This opens up a new direction for plant epigenomics. Finally, we demonstrated that our score might be able to identify loci in human cancer samples as putative biomarkers for early cancer detection.ConclusionsWe adopted the mathematical framework from biodiversity into three model-based methods for analyzing genome-wide DNA methylation heterogeneity to monitor cellular heterogeneity. Our methods, namely MeH, have been implemented, evaluated with existing methods, and are open to the research community.

DNA methylation profile of single in vitro matured bovine oocytes.

Somatic cell nuclear transfer (SCNT) is commercially used despite incomplete nuclear reprogramming of the somatic cell nucleus by the enucleated oocyte compromising its efficiency. Oocyte selection is a key factor in increasing this efficiency as its cytoplasm reprograms the differentiated cell. In this study, we adapted a methodology to characterize epialleles in potential epigenetic markers in single in vitro matured oocytes. Characterization of the regions that control the expression of imprinted genes, X-chromosome inactivation, and satellite I DNA (IGF2, ICR-H19, XIST, RepA, and SAT1) showed methylated and unmethylated alleles in the imprinted genes IGF2 and ICR-H19 while XIST-DMR1 and RepA showed hypermethylated alleles. There was great variation in methylation patterns for candidate regions which may be related to oocyte quality. Moreover, the identification of different epialleles in the same oocyte suggests that, at least for those loci, the epigenome of the metaphase plate and polar body is different. The single-cell bisulfite polymerase chain reaction technique can be used to improve the precision of selecting the best oocytes for SCNT procedures, thereby increasing its efficiency.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

Abstract 781: DNA methylation patterns between aggressive and indolent prostate tumors from Puerto Rican men

Abstract Background: In 2020, approximately 191,930 new prostate cancer (PCa) cases and 33,330 deaths are estimated in the US only. Hispanic/Latinos (H/L) are the second largest racial/ethnic group in the US. A previous study reported that prostate cancer-specific mortality rate among Puerto Rican men with prostate cancer is higher than ones of non-Hispanic Whites and non-Hispanic Blacks. The aims of this study are to: (1) assess the methylation patterns between aggressive and indolent PCa, (2) to study the methylation status of DNA repair genes between these groups, and (3) to assess the ancestry proportions of the study participants. Variations in methylation patterns will be detected between study groups. Methods: Archived formalin fixed paraffin-embedded (FFPE) prostate tumors (n=24) were collected through the Puerto Rico Biobank. Tumor specimens were classified as aggressive (n=11) and indolent (n=13) cases based on the Gleason scores (6+7(3+4) vs. 7(4+3)+8+9). Tumor areas and adjacent normal tissue were annotated on the H&amp;E slides by a pathologist and extracted by macro-dissection. The Illumina 850K DNA methylation platform was used to measure DNA methylation patterns. The raw data was processed using minfi and normalized. Amount of missing values, histogram of β-values, principal component analysis (PCA) was to assess quality and to detect outliers. Results: A PCA model clearly separated the normal samples from the tumor samples. One of the most significantly differently methylated genes was AOX1. Further comparisons using TCGA PRAD data base revealed the predictable negative correlation between the gene expression and the average β-values. Regarding the PCa aggressiveness, 23 probes were identified as potential candidates using a p&amp;lt;0.001 and a Δβ-value of 0.2 as cut-off. These probes where located in six hypermethylated genes; RREB1, FAM71F2, JMJD1C, COL5A3, RAE1, GABRQ, and eleven hypomethylated; COL9A2, FAM179A, SLC17A2, PDE10A, PLEKHS1, TNNI2, OR51A4, RNF169, SPNS2, ADAMTSL5, and CYP4F12. Of the 179 candidate DNA repair genes. We found two significant differentially methylated DNA repair genes: JMJD1C and RNF169 between groups. The results from the ancestry proportions for African, European, and Indigenous American were: 24.1%, 64.2%, and 9% respectively. Conclusion: The identification of DNA methylation patterns related to PCa in Puerto Rican (PR) H/L men along with specific patterns related to aggressiveness and DNA repair constitutes a pivotal effort on the understanding of PCa disparities in this population. This constitutes a unique effort to provide an overview regarding methylation and ancestry patterns in PR H/L men. Acknowledgements; supported by U54 CA163071 and U54 CA163068-07 Citation Format: Jong Y. Park, Gilberto Ruiz-Deya, Jarline Encarnacion, Juile Dutil, Liang Wang, Carmen Ortiz Sanchez, Ryan Putney, Anders Berglund, Youngchul Kim, Jaime Matta. DNA methylation patterns between aggressive and indolent prostate tumors from Puerto Rican men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 781.

The assessment of epigenetic diversity, differentiation, and structure in the 'Fuji' mutation line implicates roles of epigenetic modification in the occurrence of different mutant groups as well as spontaneous mutants.

The ‘Fuji’ line includes many varieties with a similar genetic background and consistent inducement factors with epigenetic occurrence, thus it may be considered an ideal candidate for epigenetic research. In this study, 91 bud mutations of ‘Fuji’ apple were used as the test materials. Using the genetic variation within ‘Fuji’ as the control, the characteristics of epigenetic variation at different levels in both varieties and mutant groups were examined. The results showed that: (1) the global genomic DNA methylation level of the 91 bud mutants of ‘Fuji’ ranged from 29.120%-45.084%, with an average of 35.910%. Internal cytosine methylation was the main DNA methylation pattern. Regarding the variation of methylation patterns of ‘Fuji’ mutants, the vast majority of loci maintained the original methylation pattern existed in ‘Fuji’. CHG methylation variation was the main type of variation; (2) the variation in methylation patterns between the mutant groups was greater than that of methylation levels. Among these patterns, the variation in CHG methylation patterns (including CHG hypermethylation and CHG demethylation) was expected to be dominant. The observed variation in methylation levels was more important in the Color mutant group; however, the variation in methylation patterns was more obvious in both the early maturation and Spur mutant groups. Moreover, the range of variation in the Early-maturation group was much wider than that in the Spur mutant group; (3) epigenetic diversity and genetic diversity were both low between the mutant groups. In the ‘Fuji’ mutant groups, there was few correlation between genetic and epigenetic variation, and epigenetic differentiation resulted in more loci with moderate or greater differentiation; (4) the purifying selection seemed to play a major role in the differentiation of different groups of ‘Fuji’ mutants (65.618%), but epigenetic diversity selection still occurred at nearly 35% of loci. Sixteen epigenetic outlier loci were detected.

Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse

We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-lines. The general decline of transcript abundance of marker loci was detected in passage 11 which is the sign of dedifferentiation of cultivated chondrocytes in prolonged monolayer culture. Passages 13 and 14 were characterized by the upregulation of a number of genes, possibly due to the heterogeneity of developed cell lines at this stage of the culture. Instead, gradual increase of methylation percent at particular CpG sites of RUNX3 locus was associated with the growing number of passage. This finding led us to the conclusion that epigenetic alterations better describe the stage of cultivated chondrocytes.

Stage-specific differential DNA methylation data analysis during human erythropoiesis in chromosome 16

Previous studies have generated controversial findings regarding the correlation between DNA methylation in the human genome and gene expression. Some reports have indicated that promoter methylation is negatively correlated with gene expression levels; however, in some cases, a poor or positive correlation was reported. Most previous findings were based on general trends observed with whole-genome data analysis. Here, we present a novel chromosome-specific statistical analysis design of empirical Bayes differential tests for five phases of erythroid development. To better understand the common methylation patterns of differentially methylated regions (DMRs) during specific stages, we defined differential phases for each CpG locus, based on a maximum log2 fold change. Analyzing hypermethylated and hypomethylated CpG loci separately showed variations in methylation patterns during erythropoiesis in the gene body, promoter and enhancer regions. Hypomethylated DMRs showed stronger associations with erythroid-specific enhancers at the differentiation start phase and with exons in the intermediate phase. To investigate the hypomethylated DMRs further, transcription factor binding site-enrichment analysis was conducted. This analysis highlighted novel transcription factors during each differentiation stage that were not detected by previous differential methylation data analysis. In contrast, hypermethylated DMRs showed a consistent methylation pattern over the different genomic regions. Thus, a closer examination of DNA methylation patterns in a single chromosome during each developmental stage can contribute to verify the association nature between gene expression and DNA methylation.

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well-known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome-wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation-sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms.

Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

BackgroundDNA methylation, with a cryptic role in genome stability, gene transcription and expression, is involved in the drought response process in plants, but the complex regulatory mechanism is still largely unknown.ResultsHere, we performed whole-genome bisulfite sequencing (WGBS) and identified long non-coding RNAs on cotton leaves under drought stress and re-watering treatments. We obtained 31,223 and 30,997 differentially methylated regions (representing 2.48% of the genome) after drought stress and re-watering treatments, respectively. Our data also showed that three sequence contexts, including mCpG, mCHG, mCHH, all presented a hyper-methylation pattern under drought stress and were nearly restored to normal levels after the re-watering treatment. Among all the methylation variations, asymmetric CHH methylation was the most consistent with external environments, suggesting that methylation/demethylation in a CHH context may constitute a novel epigenetic modification in response to drought stress. Combined with the targets of long non-coding RNAs, we found that long non-coding RNAs may mediate variations in methylation patterns by splicing into microRNAs. Furthermore, the many hormone-related genes with methylation variations suggested that plant hormones might be a potential mechanism in the drought response.ConclusionsFuture crop-improvement strategies may benefit by taking into account not only the DNA genetic variations in cotton varieties but also the epigenetic modifications of the genome.

Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics

Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming.

89 METHYLATION STATUS OF IGF2/H19 DMR3 REGION AFFECTS IN VITRO BLASTOCYST PRODUCTION IN GOAT (CAPRA HIRCUS)

Parthenogenesis has been observed in lower animals but no known instance has been reported in mammals because both maternal and paternal genomes are a fundamental prerequisite for embryogenesis. A major reason for developmental failure of uniparental zygotes is expression of certain genes in a parent-of-origin-specific manner, i.e. genomic imprinting of genes. Out of many imprinted genes identified so far, IGF2/H19 have been extensively studied and known to play an important role in fetal and placental development. Gene IGF2 is expressed by the paternal allele, H19 is transcribed from the maternal allele, and the reciprocal expression of both genes is regulated by the DMR3 region placed upstream of the H19 gene. In the present study we compared the methylation status of IGF2/H19 DMR in parthenogenetic activated (PA) and IVF goat (Capra hircus) blastocyst through bisulphite sequencing. For this, immature oocytes of usable quality were subjected to in vitro maturation and subsequently used for embryo production through parthenogenesis (n = 993) (by calcium ionophore and 6-DMAP activation) and IVF (n = 1096). It was found that embryo production rate at all the embryonic stages (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) was significantly higher (P &lt; 0.05) in parthenogenesis (74.66 ± 3.35%, 61.90 ± 2.73%, 47.83 ± 2.95%, 38.13 ± 5.28%, and 21.11 ± 2.51%, respectively) as compared with IVF (55.21 ± 2.02%, 38.12 ± 2.48%, 28.53 ± 1.67%, 21.57 ± 1.59%, and 8.23 ± 1.02%, respectively). When blastocysts (n = 6 each) were subjected to TUNEL, it was found that PA blastocyst showed significantly higher (P &lt; 0.05) total cell number (217.83 ± 18.80 v. 159.67 ± 13.94) and significantly low (P &lt; 0.05) apoptotic index (2.04 ± 0.25 v. 4.03 ± 0.29) as compared with IVF blastocysts. For the methylation pattern study, we analysed 17 CpG sites on the DMR3 region of the IGF2/H19 gene. Variable methylation pattern was observed within these CpG sites in different clones (n = 15) of PA and IVF blastocyst. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (P &lt; 0.05) in PA blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively), which suggests higher expression of IGF2 in parthenotes. The result suggests IGF2 might play different roles in different species; the same expression pattern of IGF2 is observed in ovine, but a contrary result is found in porcine species. Our results signify the hypermethylation of IGF2/H19 DMR3, which leads to higher expression of IGF2 to support embryonic development at the blastocyst stage. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

DNA methylation-based variation between human populations.

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells.

Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns of HOX genes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer.

Mo1947 Detection of Variable Methylation Patterns Improves Sensitivity of a Colorectal Cancer Blood Test

Mo1947 Detection of Variable Methylation Patterns Improves Sensitivity of a Colorectal Cancer Blood Test

Nonparametric Bayesian clustering to detect bipolar methylated genomic loci

BackgroundWith recent development in sequencing technology, a large number of genome-wide DNA methylation studies have generated massive amounts of bisulfite sequencing data. The analysis of DNA methylation patterns helps researchers understand epigenetic regulatory mechanisms. Highly variable methylation patterns reflect stochastic fluctuations in DNA methylation, whereas well-structured methylation patterns imply deterministic methylation events. Among these methylation patterns, bipolar patterns are important as they may originate from allele-specific methylation (ASM) or cell-specific methylation (CSM).ResultsUtilizing nonparametric Bayesian clustering followed by hypothesis testing, we have developed a novel statistical approach to identify bipolar methylated genomic regions in bisulfite sequencing data. Simulation studies demonstrate that the proposed method achieves good performance in terms of specificity and sensitivity. We used the method to analyze data from mouse brain and human blood methylomes. The bipolar methylated segments detected are found highly consistent with the differentially methylated regions identified by using purified cell subsets.ConclusionsBipolar DNA methylation often indicates epigenetic heterogeneity caused by ASM or CSM. With allele-specific events filtered out or appropriately taken into account, our proposed approach sheds light on the identification of cell-specific genes/pathways under strong epigenetic control in a heterogeneous cell population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0439-2) contains supplementary material, which is available to authorized users.

A comparative analysis of methylome profiles of Campylobacter jejuni sheep abortion isolate and gastroenteric strains using PacBio data.

Campylobacter jejuni is a leading cause of human gastrointestinal disease and small ruminant abortions in the United States. The recent emergence of a highly virulent, tetracycline-resistant C. jejuni subsp. jejuni sheep abortion clone (clone SA) in the United States, and that strain's association with human disease, has resulted in a heightened awareness of the zoonotic potential of this organism. Pacific Biosciences' Single Molecule, Real-Time sequencing technology was used to explore the variation in the genome-wide methylation patterns of the abortifacient clone SA (IA3902) and phenotypically distinct gastrointestinal-specific C. jejuni strains (NCTC 11168 and 81-176). Several notable differences were discovered that distinguished the methylome of IA3902 from that of 11168 and 81-176: identification of motifs novel to IA3902, genome-specific hypo- and hypermethylated regions, strain level variability in genes methylated, and differences in the types of methylation motifs present in each strain. These observations suggest a possible role of methylation in the contrasting disease presentations of these three C. jejuni strains. In addition, the methylation profiles between IA3902 and a luxS mutant were explored to determine if variations in methylation patterns could be identified that might explain the role of LuxS-dependent methyl recycling in IA3902 abortifacient potential.

Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer–testis (CT-X) antigens

BackgroundL1CAM was originally identified as an adhesion molecule involved in neural development. In many human carcinomas L1CAM is over-expressed and is associated with a bad prognosis. We previously reported that L1CAM was absent in the vast majority of endometrioid endometrial carcinomas (ECs) (type 1) but was strongly expressed in the more aggressive serous and clear-cell ECs (termed type 2). The differential regulation of L1CAM in ECs is not well understood. Recent evidence suggests that it can be regulated by epigenetic mechanisms. Here we investigated the role of DNA-methylation of the L1CAM promoter for expression. We also studied the relationship to cancer testis (CT-X) antigens that co-localize with L1CAM on chromosome Xq28, a region that is often activated in human tumors.MethodsWe used EC cell lines and primary tumor tissues for our analysis. For expression analysis we employed RT-PCR and Western blotting. DNA-Methylation of the L1CAM promoter was determined after bisulfite conversation and DNA sequencing. Tumor tissues were examined by immunohistochemical (IHC) staining.ResultsWe demonstrate that the treatment of L1CAM low/negative expressing EC cell lines with 5′-Azacytidine (5-AzaC) or knock-down of DNMT1 (DNA methyltransferase 1) as well as the HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) up-regulated L1CAM at the mRNA and protein level. The L1CAM gene has two promoter regions with two distinct CpG islands. We observed that the expression of L1CAM correlated with hypermethylation in promoter 1 and 5-AzaC treatment affected the DNA-methylation pattern in this region. The CT-X antigens NY-ESO-1, MAGE-A3 and MAGE-A4 were also strongly up-regulated by 5-AzaC or knock-down of DNMT1 but did not respond to treatment with TSA. Primary EC tumor tissues showed a variable methylation pattern of the L1CAM promoter. No striking differences in promoter methylation were observed between tumor areas with L1CAM expression and those without expression.ConclusionsL1CAM expression correlated with methylation of the L1CAM promoter in EC cell lines. In negative cell lines L1CAM expression is up-regulated by epigenetic mechanism. Although genes localized on Xq28 are often re-expressed by human tumors, L1CAM and CT-X antigens show distinct regulation in response to HADC inhibitors and 5-AzaC.