Unique Associations of DNA Methylation Regions With 24-Hour Blood Pressure Phenotypes in Blacks

Background: Epigenetic marks (eg, DNA methylation) may capture the effect of gene-environment interactions. DNA methylation is involved in blood pressure (BP) regulation and hypertension development; however, no studies have evaluated its relationship with 24-hour BP phenotypes (daytime, nighttime, and 24-hour average BPs). Methods: We examined the association of whole blood DNA methylation with 24-hour BP phenotypes and clinic BPs in a discovery cohort of 281 Blacks using reduced representation bisulfite sequencing. We developed a deep and region-specific methylation sequencing method, Bisulfite ULtrapLEx Targeted Sequencing and utilized it to validate our findings in a separate validation cohort (n=117). Results: Analysis of 38 215 DNA methylation regions (MRs), derived from 1 549 368 CpG sites across the genome, identified up to 72 regions that were significantly associated with 24-hour BP phenotypes. No MR was significantly associated with clinic BP. Two to 3 MRs were significantly associated with various 24-hour BP phenotypes after adjustment for age, sex, and body mass index. Together, these MRs explained up to 16.5% of the variance of 24-hour average BP, while age, sex, and BMI explained up to 11.0% of the variance. Analysis of one of the MRs in an independent cohort using Bisulfite ULtrapLEx Targeted Sequencing confirmed its association with 24-hour average BP phenotype. Conclusions: We identified several MRs that explain a substantial portion of variances in 24-hour BP phenotypes, which might be excellent markers of cumulative effect of factors influencing 24-hour BP levels. The Bisulfite ULtrapLEx Targeted Sequencing workflow has potential to be suitable for clinical testing and population screenings on a large scale.

Methotrexate Treatment of Newly Diagnosed RA Patients Is Associated With DNA Methylation Differences at Genes Relevant for Disease Pathogenesis and Pharmacological Action

BackgroundMethotrexate (MTX) is the first line treatment of rheumatoid arthritis (RA), and methylation changes in bulk T cells have been reported after treatment with MTX. We have investigated cell-type specific DNA methylation changes across the genome in naïve and memory CD4+ T cells before and after MTX treatment of RA patients. DNA methylation profiles of newly diagnosed RA patients (N=9) were assessed by reduced representation bisulfite sequencing.ResultsWe found that MTX treatment significantly influenced DNA methylation levels at multiple CpG sites in both cell populations. Interestingly, we identified differentially methylated sites annotated to two genes; TRIM15 and SORC2, previously reported to predict treatment outcome in RA patients when measured in bulk T cells. Furthermore, several of the genes, including STAT3, annotated to the significant CpG sites are relevant for RA susceptibility or the action of MTX.ConclusionWe detected CpG sites that were associated with MTX treatment in CD4+ naïve and memory T cells isolated from RA patients. Several of these sites overlap genetic regions previously associated with RA risk and MTX treatment outcome.

Genome-Wide DNA Methylation and Hydroxymethylation Changes Revealed Epigenetic Regulation of Neuromodulation and Myelination in Yak Hypothalamus

Both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are important epigenetic modifications in neurodevelopment. However, there is little research examining the genome-wide patterns of 5mC and 5hmC in brain regions of animals under natural high-altitude conditions. We used oxidative reduced representation bisulfite sequencing (oxRRBS) to determine the 5mC and 5hmC sites in the brain, brainstem, cerebellum, and hypothalamus of yak and cattle. We reported the first map of genome-wide DNA methylation and hydroxymethylation in the brain, brainstem, cerebellum, and hypothalamus of yak (living at high altitudes) and cattle. Overall, we found striking differences in 5mC and 5hmC between the hypothalamus and other brain regions in both yak and cattle. Genome-wide profiling revealed that 5mC level decreased and 5hmC level increased in the hypothalamus than in other regions. Furthermore, we identified differentially methylated regions (DMRs) and differentially hydroxymethylated regions (DhMRs), most of which overlapped with each other. Interestingly, transcriptome results for these brain regions also showed distinctive gene levels in the hypothalamus. Finally, differentially expressed genes (DEGs) regulated by DMRs and DhMRs may play important roles in neuromodulation and myelination. Overall, our results suggested that mediation of 5mC and 5hmC on epigenetic regulation may broadly impact the development of hypothalamus and its biological functions.

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

Abstract P175: Discovery And Validation Of Unique Associations Of DNA Methylation Regions With 24-hour Blood Pressure Phenotypes In African Americans

DNA methylation, an epigenetic mark, may reflect the interactions between DNA, environment, and lifestyle. It has been implicated in the development and progression of hypertension, a risk factor for cardiovascular disease. We hypothesize that regions of DNA methylation in blood cells can explain 24h BP phenotypes in African Americans. We performed Reduced Representation Bisulfite Sequencing (RRBS) in a discovery cohort of 281 African Americans. Several DNA methylation regions (MRs) were significantly associated with continuously monitored 24-h, daytime, or nighttime SBP, DBP, PP, and MAP after adjustments for covariates age, sex, and body mass index (False Discovery Rate (FDR) = 0.013 - 0.050). Each of these MRs explained a substantial portion of 24h BP variance, ranging from 6.5% - 9.4%. After FDR adjustment, there were no MRs significantly associated with clinic BPs (FDR > 0.1374), calculated by the average of 4 resting measurements (2 per arm) by sphygmomanometer. To interrogate specific regions of DNA methylation, our lab developed a potentially clinically applicable, deep, and targeted methylation sequencing method called Bisulfite-Specific PCR ULtrapLEx Targeted Sequencing (BULLET-Seq), and tested it in two reference samples for three MRs of interest. BULLET-Seq is able to accurately quantify the 10% changes in the dilution series when methylation rates ranged from ~40% to 90% (a chr19 methylation region; R 2 = 0.95 - 0.97) and can modestly measure these changes when rates range from ~2% to 4% (a chr5 region, R 2 = 0.82), and is questionable when methylation rates are below 2% (a chr13 region, R 2 = 0.03 - 0.27). Validation of the chr19 MR in an independent cohort (n=117) was performed in a single BULLET-Seq run. After covariate adjustment, the chr19 region was significantly associated with 24h BPs (SBP, DBP, and MAP; FDR < 0.05), confirming the findings from the discovery cohort. The MR accounted for up to 1.75% of BP variance in the 24h phenotypes. In conclusion, the reported DNA MRs have potential to be excellent markers for the cumulative effect of factors that influence 24h BPs and the BULLET-Seq workflow can be applied in clinical and population settings to screen up to thousands of patients.

Intrauterine hyperglycemia impairs memory across two generations

Studies on humans and animals suggest associations between gestational diabetes mellitus (GDM) with increased susceptibility to develop neurological disorders in offspring. However, the molecular mechanisms underpinning the intergenerational effects remain unclear. Using a mouse model of diabetes during pregnancy, we found that intrauterine hyperglycemia exposure resulted in memory impairment in both the first filial (F1) males and the second filial (F2) males from the F1 male offspring. Transcriptome profiling of F1 and F2 hippocampi revealed that differentially expressed genes (DEGs) were enriched in neurodevelopment and synaptic plasticity. The reduced representation bisulfite sequencing (RRBS) of sperm in F1 adult males showed that the intrauterine hyperglycemia exposure caused altered methylated modification of F1 sperm, which is a potential epigenetic mechanism for the intergenerational neurocognitive effects of GDM.

Comparative Molecular Genomic Analyses of a Spontaneous Rhesus Macaque Model of Mismatch Repair-Deficient Colorectal Cancer

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C<G, p.Tyr343Ter). Our study incorporated a detailed molecular characterization of rhesus CRC for cross-comparison with human MMRd CRC. We performed PCR-based MSI testing, transcriptomic analysis, and reduced-representation bisulfite sequencing (RRBS) of rhesus CRC (n=41 samples) using next-generation sequencing (NGS). Systems biology pipelines were used for gene set enrichment analysis (GSEA) for pathway discovery, consensus molecular subtyping (CMS), and somatic mutation profiling. Overall, the majority of rhesus tumors displayed high levels of MSI (MSI-high) and differential gene expression profiles that were consistent with known deregulated pathways in human CRC. DNA methylation analysis exposed differentially methylated patterns among MSI-H, MSI-L (MSI-low)/MSS (MS-stable) and LS tumors with MLH1 predominantly inactivated among sporadic MSI-H CRCs. The findings from this study support the use of rhesus macaques as the preferred animal model to study carcinogenesis, develop immunotherapies and vaccines, and implement chemoprevention approaches pertinent to sporadic MSI-H and LS CRC in humans.

The DNA methylation landscape of multiple myeloma shows extensive inter- and intrapatient heterogeneity that fuels transcriptomic variability

Background Cancer evolution depends on epigenetic and genetic diversity. Historically, in multiple myeloma (MM), subclonal diversity and tumor evolution have been investigated mostly from a genetic perspective. Methods Here, we performed an analysis of 42 MM samples from 21 patients by using enhanced reduced representation bisulfite sequencing (eRRBS). We combined several metrics of epigenetic heterogeneity to analyze DNA methylation heterogeneity in MM patients. Results We show that MM is characterized by the continuous accumulation of stochastic methylation at the promoters of development-related genes. High combinatorial entropy change is associated with poor outcomes in our pilot study and depends predominantly on partially methylated domains (PMDs). These PMDs, which represent the major source of inter- and intrapatient DNA methylation heterogeneity in MM, are linked to other key epigenetic aberrations, such as CpG island (CGI)/transcription start site (TSS) hypermethylation and H3K27me3 redistribution as well as 3D organization alterations. In addition, transcriptome analysis revealed that intratumor methylation heterogeneity was associated with low-level expression and high variability. Conclusions We propose that disrupted DNA methylation in MM is responsible for high epigenetic and transcriptomic instability allowing tumor cells to adapt to environmental changes by tapping into a pool of evolutionary trajectories.

Nucleus Type-Specific DNA Methylomics Reveals Epigenetic “Memory” of Prior Adaptation in Skeletal Muscle

Using a mouse model of conditional and inducible in vivo fluorescent myonuclear labeling (HSA-GFP), sorting purification of nuclei, low-input reduced representation bisulfite sequencing (RRBS), and a translatable and reversible model of exercise (progressive weighted wheel running, PoWeR), we provide the first nucleus type-specific epigenetic information on skeletal muscle adaptation and detraining. Adult (&gt;4 month) HSA-GFP mice performed PoWeR for 8 weeks then detrained for 12 weeks; age-matched untrained mice were used to control for the long duration of the study. Myonuclei and interstitial nuclei from plantaris muscles were isolated for RRBS. Relative to untrained, PoWeR caused similar myonuclear CpG hypo- and hypermethylation of promoter regions and substantial hypomethylation in interstitial nuclear promoters. Over-representation analysis of promoters revealed a larger number of hyper- versus hypomethylated pathways in both nuclear populations after training and evidence for reciprocal regulation of methylation between nucleus types, with hypomethylation of promoter regions in Wnt signaling-related genes in myonuclei and hypermethylation in interstitial nuclei. After 12 weeks of detraining, promoter CpGs in documented muscle remodeling-associated genes and pathways that were differentially methylated immediately after PoWeR were persistently differentially methylated in myonuclei, along with long-term promoter hypomethylation in interstitial nuclei. No enduring gene expression changes in muscle tissue were observed using RNA-sequencing. Upon 4 weeks of retraining, mice that trained previously grew more at the whole muscle and fiber type-specific cellular level than training naïve mice, with no difference in myonuclear number. Muscle nuclei have a methylation epi-memory of prior training that may augment muscle adaptability to retraining.

Differential DNA methylation analysis reveals key genes in Chinese Qingyu and Landrace pigs

The Chinese Qingyu pig is a typical domestic fatty pig breed and an invaluable indigenous genetic resource in China. Compared with Landrace pig, Qingyu pig has unique meat characteristics, including muscle development, intramuscular fat, and other meat quality traits. At present, few studies have explored the epigenetic difference due to DNA methylation between Qingyu pig and Landrace pig. In this study, 30 Qingyu pigs and 31 Landrace pig were subjected to reduced representation bisulfite sequencing (RRBS). A genome wide differential DNA methylation analysis was conducted. Six genomic regions, including regions on sus scrofa chromosome (SSC) 1: 266.09-274.23Mb, SSC5:0.88-10.68Mb, SSC8:41.23-48.51Mb, SSC12:45.43-54.38Mb, SSC13:202.15-207.95Mb, and SSC14:126.43-139.85Mb, were regarded as key regions that may be associated with phenotypic differences between Qingyu pig and Landrace pig. Furthermore, according to the further analysis, 5 differential methylated genes (ADCY1, FUBP3, GRIN2B, KIT, and PIK3R6) were deemed as key candidate genes that might be associated with meat characteristics. Our findings provide new insights into the difference of DNA methylation between Qingyu pig and Landrace pig. The results enrich the epigenetic research of Chinese Qingyu pigs.