450k Array Research Articles

Differentially Methylated Regions in T Cells Identify Kidney Transplant Patients at Risk for De Novo Skin Cancer

Cutaneous squamous cell carcinoma (cSCC) occurs 65–200 times more in immunosuppressed organ transplant patients than in the general population. T cells, which are targeted by the given immunosuppressive drugs, are involved in anti-tumor immune surveillance and are functionally regulated by DNA methylation. Prior to kidney transplantation, we aim to discover differentially methylated regions (DMRs) in T cells involved in de novo post-transplant cSCC development. We matched 27 kidney transplant patients with a future de novo cSCC after transplantation to 27 kidney transplant patients without cSCC and studied genome-wide DNA methylation of T cells prior to transplantation. From 11 out of the 27 cSCC patients, the DNA methylation of T cells after transplantation was also examined to assess stability of the observed differences in DNA methylation. Raw methylation values obtained with the 450k array were confirmed with pyrosequencing. We found 16 DMRs between patients with a future cSCC and those who do not develop this complication after transplantation. The majority of the DMRs were located in regulatory genomic regions such as flanking bivalent transcription start sites and bivalent enhancer regions, and most of the DMRs contained CpG islands. Examples of genes annotated to the DMRs are ZNF577, coding for a zinc-finger protein, and FLOT1, coding for a protein involved in T cell migration. The longitudinal analysis revealed that DNA methylation of 9 DMRs changed significantly after transplantation. DNA methylation of 5 out of 16 DMRs was relatively stable, with a variation in beta-value lower than 0.05 for at least 50% of the CpG sites within that region. This is the first study demonstrating that DNA methylation of T cells from patients with a future de novo post-transplant cSCC is different from patients without cSCC. These results were obtained before transplantation, a clinically relevant time point for cSCC risk assessment. Several DNA methylation profiles remained relatively stable after transplantation, concluding that these are minimally affected by the transplantation and possibly have a lasting effect on post-transplant cSCC development.

Abstract LB-235: DNA methylation of individual repetitive elements in HCV infection-induced HCC

Abstract Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Hepatitis C virus (HCV) infection is a major risk factor for HCC in the US. HCV infection may cause cancer by suppressing host defense that controls the propagation of repetitive element (RE) sequences, including LINE-1 and Alu, the most representative REs in the human genome. DNA methylation of RE helps stabilize the genome, while reduced RE methylation, also referred as global hypomethylation, leads to genomic instability and is known as a hallmark of cancer. However, current available high-throughput technology for DNA methylation profiling only enables genomewide RE methylation study on a global scale. The role of individual RE methylation in cancer is largely understudied. In this study, using our recently developed and published machine learning algorithm to enhance the RE coverage of the Illumina methylation array platform, we examined the role of individual RE methylation in HCV-induced HCC across the entire genome. We conducted an epigenome-wide association study on 22 HCC, 20 cirrhotic, and 44 normal liver tissues. We included equal numbers of HCV-induced and alcohol-induced HCC/cirrhosis cases. The Illumina 450k array profiled 1939 REs and we computationally predicted 33184 REs; correlations between the overlapping 1541 profiled and predicted RE methylation were > 0.91. Sex, tumor differentiation, and tumor stage were similarly distributed across the HCV and alcohol groups. We adjusted for batch effects and used Benjamini-Hochberg adjusted false discovery rate (FDR) to account for multiple testing. Relative to normal liver tissue, we observed over twice as many differentially methylated (FDR < 0.05) REs in HCV-HCC compared to alcohol-HCC tissue (2911 vs 1304, 749 in common). We validated our results in The Cancer Genome Atlas (TCGA) (325 vs 129, 74 in common) with high correlation (r = 0.90). We also observed that 169 REs were significantly altered in HCV-cirrhosis tissue (compared to none in alcohol-cirrhosis tissue), of which, 17 REs in 13 genes, including PIK3CD, LY9, GCK, NLRC3, and PRKCZ, were consistently hypomethylated in both HCC and cirrhotic liver tissue induced by HCV (r = 0.87). Using TCGA RNA sequencing data, we found that these REs were located in gene body and their hypomethylation was associated with lower expression of their host genes (r > 0.3, p < 0.05). Gene Ontology Enrichment analysis showed that these genes were enriched in T-cell activation & differentiation and cytokine production (FDR < 0.05). Our findings indicate that HCV infection has a more extensive impact on the DNA methylation landscape in RE than alcohol consumption, and that HCV infection may also induce similar hypomethylation in cirrhotic liver. Hypomethylated RE may alter local chromatin environment and disrupt expression of genes involved in immune and inflammatory responses, both of which may promote HCC development. RE hypomethylation in specific genomic locus may serve as early detection biomarker of HCC in patients with HCV-induced cirrhosis. Citation Format: Yinan Zheng, Jimmy Ren, Ryan A. Hlady, Keith D. Robertson, Robert L. Murphy, Lewis R. Roberts, Lifang Hou. DNA methylation of individual repetitive elements in HCV infection-induced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-235.

Case-control meta-analysis of blood DNA methylation and autism spectrum disorder

BackgroundSeveral reports have suggested a role for epigenetic mechanisms in ASD etiology. Epigenome-wide association studies (EWAS) in autism spectrum disorder (ASD) may shed light on particular biological mechanisms. However, studies of ASD cases versus controls have been limited by post-mortem timing and severely small sample sizes. Reports from in-life sampling of blood or saliva have also been very limited in sample size and/or genomic coverage. We present the largest case-control EWAS for ASD to date, combining data from population-based case-control and case-sibling pair studies.MethodsDNA from 968 blood samples from children in the Study to Explore Early Development (SEED 1) was used to generate epigenome-wide array DNA methylation (DNAm) data at 485,512 CpG sites for 453 cases and 515 controls, using the Illumina 450K Beadchip. The Simons Simplex Collection (SSC) provided 450K array DNAm data on an additional 343 cases and their unaffected siblings. We performed EWAS meta-analysis across results from the two data sets, with adjustment for sex and surrogate variables that reflect major sources of biological variation and technical confounding such as cell type, batch, and ancestry. We compared top EWAS results to those from a previous brain-based analysis. We also tested for enrichment of ASD EWAS CpGs for being targets of meQTL associations using available SNP genotype data in the SEED sample.FindingsIn this meta-analysis of blood-based DNA from 796 cases and 858 controls, no single CpG met a Bonferroni discovery threshold of p < 1.12 × 10− 7. Seven CpGs showed differences at p < 1 × 10− 5 and 48 at 1 × 10− 4. Of the top 7, 5 showed brain-based ASD associations as well, often with larger effect sizes, and the top 48 overall showed modest concordance (r = 0.31) in direction of effect with cerebellum samples. Finally, we observed suggestive evidence for enrichment of CpG sites controlled by SNPs (meQTL targets) among the EWAS CpG hits, which was consistent across EWAS and meQTL discovery p value thresholds.ConclusionsNo single CpG site showed a large enough DNAm difference between cases and controls to achieve epigenome-wide significance in this sample size. However, our results suggest the potential to observe disease associations from blood-based samples. Among the seven sites achieving suggestive statistical significance, we observed consistent, and stronger, effects at the same sites among brain samples. Discovery-oriented EWAS for ASD using blood samples will likely need even larger samples and unified genetic data to further understand DNAm differences in ASD.

Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer

BackgroundCutaneous squamous cell carcinoma (cSCC) occurs 65–200 times more in immunosuppressed organ transplant patients than in the general population. T cells, which are targeted by the given immunosuppressive drugs, are involved in anti-tumor immune surveillance and are functionally regulated by DNA methylation. Prior to kidney transplantation, we aim to discover differentially methylated regions (DMRs) in T cells involved in de novo post-transplant cSCC development.MethodsWe matched 27 kidney transplant patients with a future de novo cSCC after transplantation to 27 kidney transplant patients without cSCC and studied genome-wide DNA methylation of T cells prior to transplantation. From 11 out of the 27 cSCC patients, the DNA methylation of T cells after transplantation was also examined to assess stability of the observed differences in DNA methylation. Raw methylation values obtained with the 450k array were confirmed with pyrosequencing.ResultsWe found 16 DMRs between patients with a future cSCC and those who do not develop this complication after transplantation. The majority of the DMRs were located in regulatory genomic regions such as flanking bivalent transcription start sites and bivalent enhancer regions, and most of the DMRs contained CpG islands. Examples of genes annotated to the DMRs are ZNF577, coding for a zinc-finger protein, and FLOT1, coding for a protein involved in T cell migration. The longitudinal analysis revealed that DNA methylation of 9 DMRs changed significantly after transplantation. DNA methylation of 5 out of 16 DMRs was relatively stable, with a variation in beta-value lower than 0.05 for at least 50% of the CpG sites within that region.ConclusionsThis is the first study demonstrating that DNA methylation of T cells from patients with a future de novo post-transplant cSCC is different from patients without cSCC. These results were obtained before transplantation, a clinically relevant time point for cSCC risk assessment. Several DNA methylation profiles remained relatively stable after transplantation, concluding that these are minimally affected by the transplantation and possibly have a lasting effect on post-transplant cSCC development.

Analysis of early changes in DNA methylation in synovial fibroblasts of RA patients before diagnosis

DNA methylation is an important epigenetic modification that is known to be altered in rheumatoid arthritis synovial fibroblasts (RASF). Here, we compared the status of promoter DNA methylation of SF from patients with very early RA with SF from patients with resolving arthritis, fully established RA and from non-arthritic patients. DNA was hybridized to Infinium Human methylation 450k and 850k arrays and differential methylated genes and pathways were identified. We could identify a significant number of CpG sites that differed between the SF of different disease stages, showing that epigenetic changes in SF occur early in RA development. Principal component analysis confirmed that the different groups of SF were separated according to their DNA methylation state. Furthermore, pathway analysis showed that important functional pathways were altered in both very early and late RASF. By focusing our analysis on CpG sites in CpG islands within promoters, we identified genes that have significant hypermethylated promoters in very early RASF. Our data show that changes in DNA methylation differ in RASF compared to other forms of arthritis and occur at a very early, clinically yet unspecific stage of disease. The identified differential methylated genes might become valuable prognostic biomarkers for RA development.

Global DNA methylation changes spanning puberty are near predicted estrogen-responsive genes and enriched for genes involved in endocrine and immune processes

BackgroundThe changes that occur during puberty have been implicated in susceptibility to a wide range of diseases later in life, many of which are characterized by sex-specific differences in prevalence. Both genetic and environmental factors have been associated with the onset or delay of puberty, and recent evidence has suggested a role for epigenetic changes in the initiation of puberty as well.ObjectiveTo identify global DNA methylation changes that arise across the window of puberty in girls and boys.MethodsGenome-wide DNA methylation levels were measured using the Infinium 450K array. We focused our studies on peripheral blood mononuclear cells (PBMCs) from 30 girls and 25 boys pre- and post-puberty (8 and 14 years, respectively), in whom puberty status was confirmed by Tanner staging.ResultsOur study revealed 347 differentially methylated probes (DMPs) in females and 50 DMPs in males between the ages of 8 and 14 years (FDR 5%). The female DMPs were in or near 312 unique genes, which were over-represented for having high affinity estrogen response elements (permutation P < 2.0 × 10−6), suggesting that some of the effects of estrogen signaling in puberty are modified through epigenetic mechanisms. Ingenuity Pathway Analysis (IPA) of the 312 genes near female puberty DMPs revealed significant networks enriched for immune and inflammatory responses as well as reproductive hormone signaling. Finally, analysis of gene expression in the female PBMCs collected at 14 years revealed modules of correlated transcripts that were enriched for immune and reproductive system functions, and include genes that are responsive to estrogen and androgen receptor signaling. The male DMPs were in or near 48 unique genes, which were enriched for adrenaline and noradrenaline biosynthesis (Enrichr P = 0.021), with no significant networks identified. Additionally, no modules were identified using post-puberty gene expression levels in males.ConclusionEpigenetic changes spanning the window of puberty in females may be responsive to or modify hormonal changes that occur during this time and potentially contribute to sex-specific differences in immune-mediated and endocrine diseases later in life.

Skeletal Muscle DNA Methylation Changes following Gastric Bypass in Women with Type 2 Diabetes

PURPOSE: Bariatric weight-loss surgery can resolve or ameliorate type 2 diabetes (T2D). The cellular and molecular adaptations driving this response remain largely unknown, but some evidence points to epigenetic changes in skeletal muscle following surgery. We assessed global skeletal muscle methylation patterns prior to and 1 year after Roux en Y gastric bypass surgery (RYGB) in women with and without T2D to determine if diabetes modifies the response of the skeletal muscle methylome to weight-loss surgery. METHODS: Global vastus lateralis methylation profiles were generated via Illumina 450k Arrays pre- and 1 yr post-RYGB in black adult females (N=12) with (D; n = 6, age = 51 ± 6 yr, BMI = 53.0 ± 5.8 kg/m2) and without (ND; n=6, 43 ± 6 yr, 51.0 ± 9.2 kg/m2) T2D. Clinical values for insulin, glucose, and HOMA were measured at each time point. RM ANCOVA (group*time with age covariate) assessed changes in skeletal muscle methylation profiles in Partek Genomics Suite. Resultant methylation probes were filtered at p<0.001 and uploaded into Ingenuity Pathway Analysis for biological interoperation. RESULTS: RYGB reduced BMI (P < 0.01; -62.6% ± 28.3) and HOMA (p = 0.01; -1.9 ± 2.4) 1 yr following surgery; with no differences between groups. ANCOVA detected interaction effects in 9016 methylation sites in 6059 known genes. Overall, 7541 methylation sites in 4557 genes were altered 1 yr following RYGB in ND as compared to 4056 methylation sites in 3245 genes in diabetics. Biological pathway analysis of genes with differential methylation in diabetics identified key metabolic signaling pathways such as AMPK Signaling (80 genes; p = 9.7 x 10-8), PI3K/AKT Signaling (52 genes; p = 2.7 x 10-7), and P70S6K Signaling (44 genes; 9.82 x 10-4). Our analysis also identified hypomethylation in the promoter region of key metabolic genes IRS1 (-4.8% methylated following surgery), SLC2A4 (GLUT4; -2.8% to -3.9%), and GSK3 (-3.4%) following surgery. CONCLUSION: Skeletal muscle DNA methylation 1 year following RYGB suggest epigenetic changes in key metabolic signaling genes that are modified by diabetes. Changes in gene and protein expression of these genes have previously been demonstrated with improved glucose homeostasis. These data identify DNA methylation patterns that may play an important role in diabetes resolution following surgery.

Hypomethylation of STAT1 and HLA-DRB1 in CD8+ T cells is associated with type-I interferon dependent activation of CD4+ T cells in systemic lupus erythematosus

Abstract Background Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by autoantibody and type I interferon (IFN) production. The goal of this study was to explore possible pathogenic roles of CD8+ T cells in lupus through characterizing DNA methylation changes. Methods Genome-wide DNA methylation of SLE and matched healthy control CD8+ T cells was measured using Infinium Human Methylation 450k arrays. Cell surface expression of HLA-DRB1 on CD8+ T cells with and without IFNα was measured by flow cytometry. Co-incubation of IFNα-treated CD8+ T cells with autologous naïve CD4+ T cells was performed. Results SLE CD8+ T cells had 188 hypomethylated CpG sites compared to healthy controls. Among the most demethylated were sites associated with HLA-DRB1 (Δβ = −0.33) and STAT1 (Δβ = −0.15). The proportion of CD8+ T cells expressing HLA-DRB1 was significantly higher in SLE compared to controls. Treatment with IFNα upregulated cell surface expression of HLA-DRB1 on CD8+ T cells of SLE patients but not healthy controls, as measured by median fluorescence intensity. Co-incubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to increased expression of the stimulation marker CD69 on CD4+ T cells in SLE patients, but not in healthy controls. There was a significant increase in STAT1 mRNA levels in both SLE patients and controls with IFNα treatment. Conclusion HLA-DRB1 and STAT1 loci are hypomethylated and epigenetically poised for overexpression in SLE CD8+ T cells in the presence of type-I interferon. IFNα-treated lupus CD8+ T cells stimulate autologous CD4+ T cells in vitro. These data suggest a possible pathogenic role for CD8+ T cells that is dependent upon a high type-I interferon environment in SLE patients.

Peripheral DNA methylation, cognitive decline and brain aging: pilot findings from the Whitehall II imaging study.

Aim:The present study investigated the link between peripheral DNA methylation (DNAm), cognitive impairment and brain aging.Methods:We tested the association between blood genome-wide DNAm profiles using the Illumina 450K arrays, cognitive dysfunction and brain MRI measures in selected participants of the Whitehall II imaging sub-study.Results:Eight differentially methylated regions were associated with cognitive impairment. Accelerated aging based on the Hannum epigenetic clock was associated with mean diffusivity and global fractional anisotropy. We also identified modules of co-methylated loci associated with white matter hyperintensities. These co-methylation modules were enriched among pathways relevant to β-amyloid processing and glutamatergic signaling.Conclusion:Our data support the notion that blood DNAm changes may have utility as a biomarker for cognitive dysfunction and brain aging.

GLI2 promoter hypermethylation in saliva of children with a respiratory allergy

BackgroundThe prevalence of respiratory allergy in children is increasing. Epigenetic DNA methylation changes are plausible underlying molecular mechanisms.ResultsSaliva samples collected in substudies of two longitudinal birth cohorts in Belgium (FLEHS1 & FLEHS2) were used to discover and confirm DNA methylation signatures that can differentiate individuals with respiratory allergy from healthy subjects. Genome-wide analysis with Illumina Methylation 450K BeadChips revealed 23 differentially methylated gene regions (DMRs) in saliva from 11y old allergic children (N=26) vs. controls (N=20) in FLEHS1. A subset of 7 DMRs was selected for confirmation by iPLEX MassArray analysis. First, iPLEX analysis was performed in the same 46 FLEHS1 samples for analytical confirmation of the findings obtained during the discovery phase. iPLEX results correlated significantly with the 450K array data (P <0.0001) and confirmed 4 out of the 7 DMRs. Aiming for additional biological confirmation, the 7 DMRs were analyzed using iPLEX in a substudy of an independent birth cohort (FLEHS2; N=19 cases vs. 20 controls, aged 5 years). One DMR in the GLI2 promoter region showed a consistent statistically significant hypermethylation in individuals with respiratory allergy across the two birth cohorts and technologies. In addition to its involvement in TGF-β signaling and T-helper differentiation, GLI2 has a regulating role in lung development.ConclusionGLI2 is considered an interesting candidate DNA methylation marker for respiratory allergy.

39.1 DNA METHYLATION OF IMMUNE CELLS IN PERSONS AT CLINICAL HIGH RISK FOR PSYCHOSIS

BackgroundA dysregulated immune system is implicated in the development of psychotic disorders. Persons with schizophrenia have altered levels of circulating immune cell signaling molecules (cytokines), and elevation of specific cytokines predict conversion to psychosis in persons at clinical high risk. Whether these peripheral signals are a causal or a secondary phenomenon is unclear. But, subpopulations of circulating immune cells do regulate the brain from meningeal and perivascular locations influencing cognition, mood, and behavior, and thus may be relevant to schizophrenia vulnerability. Hematopoietic stem cells in the bone marrow differentiate into cascading subtypes depending on signals from other organs, especially the brain. For example, a monocyte subpopulation emerges with repeated social defeat that establish the persistence of anxiety-like behaviors; blocking their release or inhibiting their attachment to brain vascular endothelium prevents the emergence of anxiety-like behaviors. In humans, a similar monocyte subpopulation is associated with social isolation and other adversities including low SES, chronic stress, and bereavement.MethodsThe North American Prodrome Longitudinal Study (NAPLS2) is an eight-site observational study of predictors and mechanisms of conversion to psychosis The full cohort includes 763 at clinical high risk (CHR) based on the Criteria of Prodromal State (COPS) and 279 demographically similar unaffected comparison (UC) subjects. Methylation of whole blood DNA collected in PAXgene tubes at baseline was analyzed with the Illumina 450k array in a subgroup of 59 subjects who converted to psychosis (CHR-C), 84 CHR subjects followed for 2 years who did not develop psychosis (CHR-NC) and 67 unaffected subjects (UC). Our analyses focused on methylation of promoter regions of genes, associated with gene expression. Classifier construction used Coarse Approximation Linear Function (CALF) with bootstrapping of 1000 random 80% subsets with replacement to determine statistical likelihood.ResultsWe found highly overlapping sets of differentially methylated promoter regions in CHR-C subjects compared to CHR-NC and to UC subjects. A set of 10 markers correctly classified CHR-C and CHR-NC subjects with high accuracy (AUC=0.94, 95% CI 0.89–0.98). Included was SIRT1, a gene that is upregulated with HSV reactivation.DiscussionCirculating immune cells excerpt powerful influences on mood, cognition and behavior. An obvious example is the experience of most human with “sickness syndrome”, characterized by apathy, avolition, and withdrawal, and triggered by immune-cell-released cytokines producing an adaptive, resource conserving, behavioral response. While at an early stage, our findings further implicate immune system dysregulation as a mechanism in the development of psychosis.

Genome-wide DNA methylation profiling in infants born to gestational diabetes mellitus

BackgroundOffspring exposed to gestational diabetes mellitus (GDM) are at a high risk for metabolic diseases. The mechanisms behind the association between offspring exposed to GDM in utero and an increased risk of health consequences later in life remain unclear. The aim of this study was to clarify the changes in methylation levels in the foetuses of women with GDM and to explore the possible mechanisms linking maternal GDM with a high risk of metabolic diseases in offspring later in life. MethodsA genome-wide comparative methylome analysis on the umbilical cord blood of infants born to 30 women with GDM and 33 women with normal pregnancy was performed using Infinium HumanMethylation 450 BeadChip assays. A quantitative methylation analysis of 18 CpG dinucleotides was verified in the validation umbilical cord blood samples from 102 newborns exposed to GDM and 103 newborns who experienced normal pregnancy by MassARRAY EpiTYPER. ResultsA total of 4485 differentially methylated sites (DMSs), including 2150 hypermethylated sites and 2335 hypomethylated sites, with a mean β-value difference of >0.05, were identified by the 450k array. Good agreement was observed between the massarray validation data and the 450k array data (R2 > 0.99; P < 0.0001). Thirty-seven CpGs (representing 20 genes) with a β-value difference of > 0.15 between the GDM and healthy groups were identified and showed potential as clinical biomarkers for GDM. “hsa04940: Type I diabetes mellitus” was the most significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, with a P-value = 3.20E−07 and 1.36E−02 in the hypermethylated and hypomethylated genepathway enrichment analyses, respectively. In the Gene Ontology (GO) pathway analyses, immune MHC (major histocompatibility complex)-related pathways and neuron development-related pathways were significantly enriched. ConclusionsOur results suggest that GDM has epigenetic effects on genes that are preferentially involved in the Type I diabetes mellitus pathway, immune MHC-related pathways and neuron development-related pathways, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming.

Evaluation of Promoter Methylation of RASSF1A and ATM in Peripheral Blood of Breast Cancer Patients and Healthy Control Individuals.

Breast cancer (BC) is the most common cancer among women and has high mortality rates. Early detection is supposed to be critical for the patient’s prognosis. In recent years, several studies have investigated global DNA methylation profiles and gene-specific DNA methylation in blood-based DNA to develop putative screening markers for cancer. However, most of the studies have not yet been validated. In our study, we analyzed the promoter methylation of RASSF1A and ATM in peripheral blood DNA of 229 sporadic patients and 151 healthy controls by the MassARRAY EpiTYPER assay. There were no significant differences in DNA methylation levels of RASSF1A and ATM between the sporadic BC cases and the healthy controls. Furthermore, we performed the Infinium HumanMethylation450 BeadChip (450K) array analysis using 48 sporadic BC cases and 48 healthy controls (cases and controls are the same from those of the MassARRAY EpiTYPER assay) and made a comparison with the published data. No significant differences were presented in DNA methylation levels of RASSF1A and ATM between the sporadic BC cases and the healthy controls. So far, the evidence for powerful blood-based methylation markers is still limited and the identified markers need to be further validated.

Adjusting for Batch Effects in DNA Methylation Microarray Data, a Lesson Learned.

It is well-known, but frequently overlooked, that low- and high-throughput molecular data may contain batch effects, i.e., systematic technical variation. Confounding of experimental batches with the variable(s) of interest is especially concerning, as a batch effect may then be interpreted as a biologically significant finding. An integral step toward reducing false discovery in molecular data analysis includes inspection for batch effects and accounting for this signal if present. In a 30-sample pilot Illumina Infinium HumanMethylation450 (450k array) experiment, we identified two sources of batch effects: row and chip. Here, we demonstrate two approaches taken to process the 450k data in which an R function, ComBat, was applied to adjust for the non-biological signal. In the “initial analysis,” the application of ComBat to an unbalanced study design resulted in 9,612 and 19,214 significant (FDR < 0.05) DNA methylation differences, despite none present prior to correction. Suspicious of this dramatic change, a “revised processing” included changes to our analysis as well as a greater number of samples, and successfully reduced batch effects without introducing false signal. Our work supports conclusions made by an article previously published in this journal: though the ultimate antidote to batch effects is thoughtful study design, every DNA methylation microarray analysis should inspect, assess and, if necessary, account for batch effects. The analysis experience presented here can serve as a reminder to the broader community to establish research questions a priori, ensure that they match with study design and encourage communication between technicians and analysts.

Demethylator phenotypes in acute myeloid leukemia.

Acute myeloid leukemia (AML) often harbors mutations in epigenetic regulators, and also has frequent DNA hypermethylation, including the presence of CpG island methylator phenotypes (CIMP). Although global hypomethylation is well-known in cancer, the question of whether distinct demethylator phenotypes (DMPs) exist remains unanswered. Using Illumina 450k arrays for 194 patients from The Cancer Genome Atlas we identified two distinct DMPs by hierarchical clustering: DMP.1 and DMP.2. DMP.1 cases harbored mutations in NPM1 (94%), FLT3 (71%) and DNMT3A (61%). Surprisingly, only 40% of patients with DNMT3A mutations were DMP.1, which has implications for mechanisms of transformation by this mutation. In contrast, DMP.2 AML was comprised of patients with t(8;21), inv(16), or t(15;17), suggesting common methylation defects connect these disparate rearrangements. RNA-seq revealed up-regulated genes functioning in immune response (DMP.1) and development (DMP.2). We confirmed these findings by integrating independent 450k datasets (236 additional cases), and found prognostic effects by DMP status, independent of age and cytogenetics. The existence of DMPs has implications for AML pathogenesis and may augment existing tools in risk stratification.

Occupational exposure to pesticides is associated with differential DNA methylation

ObjectivesOccupational pesticide exposure is associated with a wide range of diseases, including lung diseases, but it is largely unknown how pesticides influence airway disease pathogenesis. A potential mechanism might be...

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males.

This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males "controls" and 72 subfertile males "cases"). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene-related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p≤.00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer

ABSTRACTBreast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Genetically defined elevated homocysteine levels do not result in widespread changes of DNA methylation in leukocytes.

BackgroundDNA methylation is affected by the activities of the key enzymes and intermediate metabolites of the one-carbon pathway, one of which involves homocysteine. We investigated the effect of the well-known genetic variant associated with mildly elevated homocysteine: MTHFR 677C>T independently and in combination with other homocysteine-associated variants, on genome-wide leukocyte DNA-methylation.MethodsMethylation levels were assessed using Illumina 450k arrays on 9,894 individuals of European ancestry from 12 cohort studies. Linear-mixed-models were used to study the association of additive MTHFR 677C>T and genetic-risk score (GRS) based on 18 homocysteine-associated SNPs, with genome-wide methylation.ResultsMeta-analysis revealed that the MTHFR 677C>T variant was associated with 35 CpG sites in cis, and the GRS showed association with 113 CpG sites near the homocysteine-associated variants. Genome-wide analysis revealed that the MTHFR 677C>T variant was associated with 1 trans-CpG (nearest gene ZNF184), while the GRS model showed association with 5 significant trans-CpGs annotated to nearest genes PTF1A, MRPL55, CTDSP2, CRYM and FKBP5.ConclusionsOur results do not show widespread changes in DNA-methylation across the genome, and therefore do not support the hypothesis that mildly elevated homocysteine is associated with widespread methylation changes in leukocytes.

Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity.

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene-related amplicon (CpG1, p≤.003, CpG2, p≤.0001, CpG3, p≤.003 and CpG4, p≤.030) and CpG1 in CGβ gene-related amplicon (p≤.0001). Besides, a significant difference was found at two CpGs (CpG1, p≤.004 and CpG2, p≤.002) tested in the FAM189A1 gene-related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.