Tissue-specific DNA Methylation Research Articles

Integrating muti-omics data to identify tissue-specific DNA methylation biomarkers for cancer risk

The relationship between tissue-specific DNA methylation and cancer risk remains inadequately elucidated. Leveraging resources from the Genotype-Tissue Expression consortium, here we develop genetic models to predict DNA methylation at CpG sites across the genome for seven tissues and apply these models to genome-wide association study data of corresponding cancers, namely breast, colorectal, renal cell, lung, ovarian, prostate, and testicular germ cell cancers. At Bonferroni-corrected P < 0.05, we identify 4248 CpGs that are significantly associated with cancer risk, of which 95.4% (4052) are specific to a particular cancer type. Notably, 92 CpGs within 55 putative novel loci retain significant associations with cancer risk after conditioning on proximal signals identified by genome-wide association studies. Integrative multi-omics analyses reveal 854 CpG-gene-cancer trios, suggesting that DNA methylation at 309 distinct CpGs might influence cancer risk through regulating the expression of 205 unique cis-genes. These findings substantially advance our understanding of the interplay between genetics, epigenetics, and gene expression in cancer etiology.

A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells

Pathogenic variants in KANSL1 and 17q21.31 microdeletions are causative of Koolen-de Vries syndrome (KdVS), a neurodevelopmental syndrome with characteristic facial dysmorphia. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes have identifiable patterns of DNA methylation (DNAm) change: DNAm signatures or episignatures. Given the role of KANSL1 in histone acetylation, we tested whether variants underlying KdVS are associated with a DNAm signature. We profiled whole-blood DNAm for 13 individuals with KANSL1 variants, four individuals with 17q21.31 microdeletions, and 21 typically developing individuals, using Illumina’s Infinium EPIC array. In this study, we identified a robust DNAm signature of 456 significant CpG sites in 8 individuals with KdVS, a pattern independently validated in an additional 7 individuals with KdVS. We also demonstrate the diagnostic utility of the signature and classify two KANSL1 VUS as well as four variants in individuals with atypical clinical presentation. Lastly, we investigated tissue-specific DNAm changes in fibroblast cells from individuals with KdVS. Collectively, our findings contribute to the understanding of the epigenetic landscape related to KdVS and aid in the diagnosis and classification of variants in this structurally complex genomic region.

Chronic graft-versus-host disease detected by tissue-specific cell-free DNA methylation biomarkers.

Accurate detection of graft-versus-host disease (GVHD) is a major challenge in the management of patients undergoing hematopoietic stem cell transplantation (HCT). Here, we demonstrated the use of circulating cell-free DNA (cfDNA) for detection of tissue turnover and chronic GVHD (cGVHD) in specific organs. We established a cocktail of tissue-specific DNA methylation markers and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon, and specific immune cells in 101 patients undergoing HCT. Patients with active cGVHD showed elevated concentrations of cfDNA, as well as tissue-specific methylation markers that agreed with clinical scores. Strikingly, transplanted patients with no clinical symptoms had abnormally high levels of tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident clinical pathology. An integrative model taking into account total cfDNA concentration, monocyte/macrophage cfDNA levels and alanine transaminase was able to correctly identify GVHD with a specificity of 86% and precision of 89% (AUC of 0.8). cfDNA markers can be used for the detection of cGVHD, opening a window into underlying tissue dynamics in patients that receive allogeneic stem cell transplants. This work was supported by grants from the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation and the Helmsley Charitable Trust (to YD).

Chronic graft-versus-host disease is characterized by high levels and distinctive tissue-of-origin patterns of cell-free DNA

Chronic graft-versus-host disease (cGvHD) is a devastating complication of hematopoietic stem cell transplantation (HSCT). Effective early detection may improve the outcome of cGvHD. The potential utility of circulating cell-free DNA (cfDNA), a sensitive marker for tissue injury, in HSCT and cGvHD remains to be established. Here, cfDNA of prospectively collected plasma samples from HSCT recipients (including both cGvHD and non-cGvHD) and healthy control (HC) subjects were evaluated. Deconvolution methods utilizing tissue-specific DNA methylation signatures were used to determine cfDNA tissue-of-origin. cfDNA levels were significantly higher in HSCT recipients than HC and significantly higher in cGvHD than non-cGvHD. cGvHD was characterized by a high level of cfDNA from innate immune cells, heart, and liver. Non-hematologic tissue-derived cfDNA was significantly higher in cGvHD than non-cGvHD. cfDNA temporal dynamics and tissue-of-origin composition have distinctive features in patients with cGvHD, supporting further exploration of the utility of cfDNA in the study of cGvHD.

Grandmaternal allergen sensitization reprograms epigenetic and airway responses to allergen in second-generation offspring.

Asthma susceptibility is influenced by environmental, genetic, and epigenetic factors. DNA methylation is one form of epigenetic modification that regulates gene expression and is both inherited and modified by environmental exposures throughout life. Prenatal development is a particularly vulnerable time period during which exposure to maternal asthma increases asthma risk in offspring. How maternal asthma affects DNA methylation in offspring and what the consequences of differential methylation are in subsequent generations are not fully known. In this study, we tested the effects of grandmaternal house dust mite (HDM) allergen sensitization during pregnancy on airway physiology and inflammation in HDM-sensitized and challenged second-generation mice. We also tested the effects of grandmaternal HDM sensitization on tissue-specific DNA methylation in allergen-naïve and -sensitized second-generation mice. Descendants of both allergen- and vehicle-exposed grandmaternal founders exhibited airway hyperreactivity after HDM sensitization. However, grandmaternal allergen sensitization significantly potentiated airway hyperreactivity and altered the epigenomic trajectory in second-generation offspring after HDM sensitization compared with HDM-sensitized offspring from vehicle-exposed founders. As a result, biological processes and signaling pathways associated with epigenetic modifications were distinct between lineages. A targeted analysis of pathway-associated gene expression found that Smad3 was significantly dysregulated as a result of grandmaternal allergen sensitization. These data show that grandmaternal allergen exposure during pregnancy establishes a unique epigenetic trajectory that reprograms allergen responses in second-generation offspring and may contribute to asthma risk.NEW & NOTEWORTHY Asthma susceptibility is influenced by environmental, genetic, and epigenetic factors. This study shows that maternal allergen exposure during pregnancy promotes unique epigenetic trajectories in second-generation offspring at baseline and in response to allergen sensitization, which is associated with the potentiation of airway hyperreactivity. These effects are one mechanism by which maternal asthma may influence the inheritance of asthma risk.

Tissue-specific DNA methylation variability and its potential clinical value.

Complex diseases have multifactorial etiologies making actionable diagnostic biomarkers difficult to identify. Diagnostic research must expand beyond single or a handful of genetic or epigenetic targets for complex disease and explore a broader system of biological pathways. With the objective to develop a diagnostic tool designed to analyze a comprehensive network of epigenetic profiles in complex diseases, we used publicly available DNA methylation data from over 2,400 samples representing 20 cell types and various diseases. This tool, rather than detecting differentially methylated regions at specific genes, measures the intra-individual methylation variability within gene promoters to identify global shifts away from healthy regulatory states. To assess this new approach, we explored three distinct questions: 1) Are profiles of epigenetic variability tissue-specific? 2) Do diseased tissues exhibit altered epigenetic variability compared to normal tissue? 3) Can epigenetic variability be detected in complex disease? Unsupervised clustering established that global epigenetic variability in promoter regions is tissue-specific and promoter regions that are the most epigenetically stable in a specific tissue are associated with genes known to be essential for its function. Furthermore, analysis of epigenetic variability in these most stable regions distinguishes between diseased and normal tissue in multiple complex diseases. Finally, we demonstrate the clinical utility of this new tool in the assessment of a multifactorial condition, male infertility. We show that epigenetic variability in purified sperm is correlated with live birth outcomes in couples undergoing intrauterine insemination (IUI), a common fertility procedure. Men with the least epigenetically variable promoters were almost twice as likely to father a child than men with the greatest number of epigenetically variable promoters. Interestingly, no such difference was identified in men undergoing in vitro fertilization (IVF), another common fertility procedure, suggesting this as a treatment to overcome higher levels of epigenetic variability when trying to conceive.

Graft-Derived Cell-Free DNA Quantification following Liver Transplantation Using Tissue-Specific DNA Methylation and Donor-Specific Genotyping Techniques: An Orthogonal Comparison Study.

Background: Graft-derived cell-free DNA (gdcfDNA) analysis has shown promise as a non-invasive tool for monitoring organ health following solid organ transplantation. A number of gdcfDNA analysis techniques have been described; however, the majority rely on sequencing or prior genotyping to detect donor-recipient mis-matched genetic polymorphisms. Differentially methylated regions of DNA can be used to identify the tissue-of-origin of cell-free DNA (cfDNA) fragments. In this study, we aimed to directly compare the performance of gdcfDNA monitoring using graft-specific DNA methylation analysis and donor-recipient genotyping techniques in a pilot cohort of clinical samples from patients post-liver transplantation. Results: 7 patients were recruited prior to LT, 3 developed early, biopsy-proven TCMR in the first 6 weeks post-LT. gdcfDNA was successfully quantified in all samples using both approaches. There was a high level of technical correlation between results using the two techniques (Spearman testing, rs = 0.87, p < 0.0001). gdcfDNA levels quantified using the genotyping approach were significantly greater across all timepoints in comparison to the tissue-specific DNA methylation-based approach: e.g., day 1 post-LT median 31,350 copies/mL (IQR 6731-64,058) vs. 4133 copies/mL (IQR 1100-8422), respectively. Qualitative trends in gdcfDNA levels for each patient were concordant between the two assays. Acute TCMR was preceded by significant elevations in gdcfDNA as quantified by both techniques. Elevations in gdcfDNA, using both techniques, were suggestive of TCMR in this pilot study with a 6- and 3-day lead-time prior to histological diagnosis in patients 1 and 2. Conclusions: Both the graft-specific methylation and genotyping techniques successfully quantified gdcfDNA in patients post-LT with statistically significant concordance. A direct comparison of these two techniques is not only important from a technical perspective for orthogonal validation, but significantly adds weight to the evidence that gdcfDNA monitoring reflects the underlying biology. Both techniques identified LT recipients who developed acute TCMR, with several days lead-time in comparison to conventional diagnostic workflows. Whilst the two assays performed comparably, gdcfDNA monitoring based on graft-specific DNA methylation patterns in cfDNA offers major practical advantages over the donor-recipient genotyping, and hence enhances the potential to translate this emerging technology into clinical practice.

Abstract 6012: Comparison of pre-analytical methods for DNA methylation analysis of cfDNA

Abstract Liquid biopsies in the form of cell-free circulating tumor DNA (ctDNA) isolated from blood plasma or serum have shown great promise to be used as minimally-invasive markers in cancer patients providing an insight into the composition of both primary tumor and metastases, and an opportunity to assess clonal dynamics throughout the course of disease. CtDNA carries not only tumor- specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation patterns similar to the patterns in the primary tumor. Deregulation of tissue-specific DNA methylation patterns is an early event in carcinogenesis holding great promise to be exploited for early detection of cancer. However, in contrast to mutation detection, targeted DNA methylation sequencing of cfDNA remains a challenge due to low concentration and heavy fragmentation. The limiting step in detection of any analyte is the recovery of the DNA molecules. The pre-analytical steps in the DNA methylation detection pipeline include extraction of cfDNA molecules and their conversion using sodium bisulfite. Bisulfite conversion is a harsh chemical reaction that induces further fragmentation and degradation of already scarce cfDNA. In this study we compared commercial methods for cfDNA extraction and for bisulfite conversion to evaluate the yield, degradation and procedural loss. We compared five commercial kits for cfDNA extraction from blood plasma, three column-based (Qiagen’s QIAamp Circulating nucleic acids kit, Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification and Macherey-Nagel’s NucleoSpin® Plasma XS) and two based on magnetic beads cleanup (Bioo Scientific’s Next Prep magnetic Cell-Free Circulating DNA isolation kitand Applied Biosystems’ Magmax Cell-Free Circulating DNA isolation kit. The kits used for bisulfite conversion were Zymo EZ DNA Methylation Lightning with column based purification and with magnetic beads purification, Zymo EZ DNA Methylation Direct with column based purification and with magnetic bead purification, Qiagen Epitect Fast DNA Bisulfite and Diagenode Premium Bisulfite kits. Same samples were processed by each of the kits, Sample degradation was determined using Agilent Bioanalyzer dsDNA kit. Yield was evaluated by Picogreen, absolute qPCR for single-copy gene and for contamination of gDNA by a B-cell specific assay, while procedural loss was determined based on the recovery of a synthetic spike-in. Citation Format: Miljana Tanic, Oluwadunni Emiloju, Pawan Dhami, Stephan Beck. Comparison of pre-analytical methods for DNA methylation analysis of cfDNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6012.

Impacts of Eccentric Resistance Exercise on DNA Methylation of Candidate Genes for Inflammatory Cytokines in Skeletal Muscle and Leukocytes of Healthy Males.

Physical inactivity and a poor diet increase systemic inflammation, while chronic inflammation can be reduced through exercise and nutritional interventions. The mechanisms underlying the impacts of lifestyle interventions on inflammation remain to be fully explained; however, epigenetic modifications may be critical. The purpose of our study was to investigate the impacts of eccentric resistance exercise and fatty acid supplementation on DNA methylation and mRNA expression of TNF and IL6 in skeletal muscle and leukocytes. Eight non-resistance exercise-trained males completed three bouts of isokinetic eccentric contractions of the knee extensors. The first bout occurred at baseline, the second occurred following a three-week supplementation of either omega-3 polyunsaturated fatty acid or extra virgin olive oil and the final bout occurred after eight-weeks of eccentric resistance training and supplementation. Acute exercise decreased skeletal muscle TNF DNA methylation by 5% (p = 0.031), whereas IL6 DNA methylation increased by 3% (p = 0.01). Leukocyte DNA methylation was unchanged following exercise (p > 0.05); however, three hours post-exercise the TNF DNA methylation decreased by 2% (p = 0.004). In skeletal muscle, increased TNF and IL6 mRNA expression levels were identified immediately post-exercise (p < 0.027); however, the leukocyte mRNA expression was unchanged. Associations between DNA methylation and markers of exercise performance, inflammation and muscle damage were identified (p < 0.05). Acute eccentric resistance exercise is sufficient to induce tissue-specific DNA methylation modifications to TNF and IL6; however, neither eccentric training nor supplementation was sufficient to further modify the DNA methylation.

Probing prenatal bisphenol exposures and tissue-specific DNA methylation responses in cord blood, cord tissue, and placenta

The early-gestational fetal epigenome establishes the landscape for fetal development and is susceptible to disruption via environmental stressors including chemical exposures. Research has explored how cell- and tissue-type-specific epigenomic signatures contribute to human disease, but how the epigenome in each tissue comparatively responds to environmental exposures is largely unknown. This pilot study compared DNA methylation in four previously identified genes across matched cord blood (CB), cord tissue (CT), and placental (PL) samples from 28 mother-infant pairs in tthe Michigan Mother Infant Pairs study; evaluated association between prenatal exposure to bisphenols (BPA, BPF, and BPS) and DNA methylation (DNAm) by tissue type; compared epigenome-wide DNAm of CB and PL; and explored associations between prenatal bisphenol exposures and epigenome-wide DNAm in PL. Bisphenol concentrations were quantified in first-trimester maternal urine. DNAm was assessed at four genes via pyrosequencing in three tissues; epigenome-wide DNAm analysis via Infinium MethylationEPIC array was completed on CB and PL. Candidate gene analysis revealed tissue-specific differences across all genes. In adjusted linear regression, BPA and BPF were associated with DNAm across candidate genes in PL but not CB and CT. Epigenome-wide comparison of matched CB and PL DNAm revealed tissue-specific differences at most CpG sites and modest associations between maternal first-trimester bisphenol exposures and PL but not CB DNAm. These data endorse inclusion of a variety of tissues in prenatal exposure studies. Overlapping and divergent responses in CB, CT, and PL demonstrate their utility in combination to capture a fuller picture of the epigenetic effect of developmental exposures.

Predictable and stable epimutations induced during clonal plant propagation with embryonic transcription factor.

Clonal propagation is frequently used in commercial plant breeding and biotechnology programs because it minimizes genetic variation, yet it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have linked epigenetic modifications induced during regeneration with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes underpinning this process remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors- RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct founder tissues. We found that both the epi(genetic) status of the explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic landscape of clonal plants. Phenotypic variation in regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. For instance, regenerants were particularly affected by the inheritance of root-specific epigenetic imprints, which were associated with an increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal specific pathways underpinning the phenotypic and molecular variation that arise and accumulate in clonal plant populations.

Chronic Graft Versus Host Disease Detected By Tissue-Specific Cell-Free DNA Methylation Biomarkers

Accurate detection of graft versus host disease (GVHD) is a major challenge in the management of patients that undergo hematopoietic stem cell transplantation (HCT). Current diagnosis of chronic GVHD (cGVHD) in bone marrow transplant patients is based on inaccurate, operator-dependent clinical markers, and less often on biopsies. These methods are time consuming, costly, invasive and yield late-stage diagnoses that negatively affect morbidity and mortality. Here we demonstrate the use of circulating cell-free DNA (cfDNA) for detection of chronic GVHD and damage to specific organs. We established a cocktail of tissue-specific DNA methylation markers, and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon and specific immune cells in 203 samples from 82 HCT patients(Fig 1). Patients with active cGVHD show elevated concentration of cfDNA, as well as tissue-specific and immune cell driven methylation markers that agree with the 2014 National Institute of Health (NIH) clinical scores. Interestingly transplanted patients with no clinical symptoms show abnormally high levels of cfDNA and tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident pathology. We used machine learning to create a model, which can aid predicting the likelihood that a patient has cGVHD. We compared XG boost and random forest classifiers on our data set. Algorithms were tested on data that included pre-selected specific measures (cfDNA of skin, lung, liver, GI, neutrophils, monocyte, eosinophil, T lymphocytes, CD8+ cells, Treg cells and B lymphocytes, as well as white blood cell (WBC) counts, BIL, AST, ALT and ALP liver enzyme levels). The data was randomly divided to a training set consisting of 67% of the samples and a testing set consisting of the remaining 33%. The model was constructed based on the training set alone that was later verified on the testing set. As the XG boost model had both higher accuracy as well as more robust results by cross-validation, we applied XG boost for further analyses. A total of 179 samples were divided between the training and test set. One hundred nineteen samples were randomly chosen for training the machine learning classifier, while 60 samples were used solely for testing the model. Each sample was intended either for the training or test sets. The integrative model was able to correctly identify GVHD with a sensitivity of 91.49% and precision of 89%% (AUC of 0.9, Fig 2). Our study shows that tissue-specific DNA methylation patterns can serve as plasma biomarkers for detection of tissue damage in cGVHD. To our knowledge, this is the first report of potential tissue-specific cfDNA utility in the context of chronic GVHD. Larger cohorts will be needed to assess the prognostic potential of methylation-based biomarkers. Tissue-specific cfDNA markers can open a window into uncovering underlying tissue dynamics in patients who underwent allogeneic stem cell transplantation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery and validation of tissue-specific DNA methylation as noninvasive diagnostic markers for colorectal cancer

BackgroundNoninvasive diagnostic markers that are capable of distinguishing patients with colorectal cancer (CRC) from healthy individuals or patients with other cancer types are lacking. We report the discovery and validation of a panel of methylation-based markers that specifically detect CRC.MethodsThis was a large-scale discovery study based on publicly available datasets coupled with a validation study where multiple types of specimens from six cohorts with CRC, other cancer types, and healthy individuals were used to identify and validate the tissue-specific methylation patterns of CRC and assess their diagnostic performance.ResultsIn the discovery and validation cohort (N = 9307), ten hypermethylated CpG sites located in three genes, C20orf194, LIFR, and ZNF304, were identified as CRC-specific markers. Different analyses have suggested that these CpG sites are CRC-specific hypermethylated and play a role in transcriptional silencing of corresponding genes. A random forest model based on ten markers achieved high accuracy rates between 85.7 and 94.3% and AUCs between 0.941 and 0.970 in predicting CRC in three independent datasets and a low misclassification rate in ten other cancer types. In the in-house validation cohort (N = 354), these markers achieved consistent discriminative capabilities. In the cfDNA pilot cohort (N = 14), hypermethylation of these markers was observed in cfDNA samples from CRC patients. In the cfDNA validation cohort (N = 155), the two-gene panel yielded a sensitivity of 69.5%, specificity of 91.7%, and AUC of 0.806.ConclusionsHypermethylation of the ten CpG sites is a CRC-specific alteration in tissue and has the potential use as a noninvasive cfDNA marker to diagnose CRC.

Spontaneous and ART-induced large offspring syndrome: similarities and differences in DNA methylome

ABSTRACT Large/abnormal offspring syndrome (LOS/AOS) is a congenital overgrowth syndrome reported in ruminants produced by assisted reproduction (ART-LOS) which exhibit global disruption of the epigenome and transcriptome. LOS/AOS shares phenotypes and epigenotypes with the human congenital overgrowth condition Beckwith-Wiedemann syndrome. We have reported that LOS occurs spontaneously (SLOS); however, to date, no study has been conducted to determine if SLOS has the same methylome epimutations as ART-LOS. In this study, we performed whole-genome bisulphite sequencing to examine global DNA methylation in bovine SLOS and ART-LOS tissues. We observed unique patterns of global distribution of differentially methylated regions (DMRs) over different genomic contexts, such as promoters, CpG Islands, shores and shelves, as well as at repetitive sequences. In addition, we included data from two previous LOS studies to identify shared vulnerable genomic loci in LOS. Overall, we identified 320 genomic loci in LOS that have alterations in DNA methylation when compared to controls. Specifically, there are 25 highly vulnerable loci that could potentially serve as molecular markers for the diagnosis of LOS, including at the promoters of DMRT2 and TBX18, at the imprinted gene bodies of IGF2R, PRDM8, and BLCAP/NNAT, and at multiple CpG Islands. We also observed tissue-specific DNA methylation patterns between muscle and blood, and conservation of ART-induced DNA methylation changes between muscle and blood. We conclude that as ART-LOS, SLOS is an epigenetic condition. In addition, SLOS and ART-LOS share similarities in methylome epimutations.

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As is evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation, also control tissue-specific methylation patterns, with the most striking pattern observed in ovules where CLSY3 and CLSY4 control DNA methylation at loci with a highly conserved DNA motif. On a more global scale, we demonstrate that specific clsy mutants are sufficient to shift the epigenetic landscape between tissues. Together, these findings reveal substantial epigenetic diversity between tissues and assign these changes to specific CLSY proteins, elucidating how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

Risk-focused differences in molecular processes implicated in SARS-CoV-2 infection: corollaries in DNA methylation and gene expression

BackgroundUnderstanding the molecular basis of susceptibility factors to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global health imperative. It is well-established that males are more likely to acquire SARS-CoV-2 infection and exhibit more severe outcomes. Similarly, exposure to air pollutants and pre-existing respiratory chronic conditions, such as asthma and chronic obstructive respiratory disease (COPD) confer an increased risk to coronavirus disease 2019 (COVID-19).MethodsWe investigated molecular patterns associated with risk factors in 398 candidate genes relevant to COVID-19 biology. To accomplish this, we downloaded DNA methylation and gene expression data sets from publicly available repositories (GEO and GTEx Portal) and utilized data from an empirical controlled human exposure study conducted by our team.ResultsFirst, we observed sex-biased DNA methylation patterns in autosomal immune genes, such as NLRP2, TLE1, GPX1, and ARRB2 (FDR < 0.05, magnitude of DNA methylation difference Δβ > 0.05). Second, our analysis on the X-linked genes identified sex associated DNA methylation profiles in genes, such as ACE2, CA5B, and HS6ST2 (FDR < 0.05, Δβ > 0.05). These associations were observed across multiple respiratory tissues (lung, nasal epithelia, airway epithelia, and bronchoalveolar lavage) and in whole blood. Some of these genes, such as NLRP2 and CA5B, also exhibited sex-biased gene expression patterns. In addition, we found differential DNA methylation patterns by COVID-19 status for genes, such as NLRP2 and ACE2 in an exploratory analysis of an empirical data set reporting on human COVID-9 infections. Third, we identified modest DNA methylation changes in CpGs associated with PRIM2 and TATDN1 (FDR < 0.1, Δβ > 0.05) in response to particle-depleted diesel exhaust in bronchoalveolar lavage. Finally, we captured a DNA methylation signature associated with COPD diagnosis in a gene involved in nicotine dependence (COMT) (FDR < 0.1, Δβ > 0.05).ConclusionOur findings on sex differences might be of clinical relevance given that they revealed molecular associations of sex-biased differences in COVID-19. Specifically, our results hinted at a potentially exaggerated immune response in males linked to autosomal genes, such as NLRP2. In contrast, our findings at X-linked loci such as ACE2 suggested a potentially distinct DNA methylation pattern in females that may interact with its mRNA expression and inactivation status. We also found tissue-specific DNA methylation differences in response to particulate exposure potentially capturing a nitrogen dioxide (NO2) effect—a contributor to COVID-19 susceptibility. While we identified a molecular signature associated with COPD, all COPD-affected individuals were smokers, which may either reflect an association with the disease, smoking, or may highlight a compounded effect of these two risk factors in COVID-19. Overall, our findings point towards a molecular basis of variation in susceptibility factors that may partly explain disparities in the risk for SARS-CoV-2 infection.

Abstract 2087: Breast tissue-specific DNA methylation levels predicted by genetic variants in association with breast cancer

Abstract Aberrant DNA methylation (DNAm) contributes to the development of breast cancer. Both genetic and environmental factors influence DNAm levels in a tissue-specific fashion. Previously studies have reported that genetically predicted DNAm levels at CpGs were associated with breast cancer risk. However, the prediction models were built based on DNAm measured in blood, which may not be the most relevant tissue for breast cancer. In this study, we aim to build statistical models to predict breast tissue-specific DNAm levels using genetic data and DNAm sequencing data of normal breast tissue from healthy women (N=293). We applied these models to the GWAS data from the Breast Cancer Association Consortium that includes 46,785 breast cancer patients and 42,892 controls. We evaluated the association of genetically predicted breast tissue-specific DNAm levels at CpGs with breast cancer risk using MetaXcan. Due to our small sample size of normal breast tissue samples, we restricted our statistical models to those that were well-validated and highly correlated between predicted and measured DNAm levels (r≥0.6). We thus identified 45 CpGs that were significantly associated with breast cancer risk after correction for multiple testings, and many of these CpGs were also differentially methylation between breast tumor and normal tissue samples. Genes annotated to these CpG sites are enriched in Wnt signaling, cell cycle, and stem cell proliferation pathways, including known genes such as FGFR2, KLF4, CASC8, TERT, and EMBP1. We also identified novel putative DNAm markers for the development of breast cancer. Our study provides insight into tissue-specific genetic regulation of DNAm in normal breast tissue. Citation Format: Chunyan He, James Castle, Nan Lin, Jinpeng Liu, Chi Wang, Yunlong Liu. Breast tissue-specific DNA methylation levels predicted by genetic variants in association with breast cancer [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 2087.

Accelerated aging in normal breast tissue of women with breast cancer

BackgroundDNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean.MethodsWe generated whole-genome DNA methylation profiles (GSE160233) on purified epithelial cells and used publicly available Infinium HumanMethylation 450K array datasets (TCGA, GSE88883, GSE69914, GSE101961, and GSE74214) for discovery and validation.ResultsWe found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39/427) were outliers for DNA methylation from 6 DNA methylation datasets. We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) than in normal samples (15/228, 5.2%). Additionally, we found significant differences between the predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, we found that the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer.ConclusionsA subset of patients with breast cancer has severely altered epigenomes which are characterized by accelerated aging in their normal-appearing tissue. In the future, these DNA methylation sites should be studied further such as in cell-free DNA to determine their potential use as biomarkers for early detection of malignant transformation and preventive intervention in breast cancer.

Genetic and epigenetic interplay fine-tunes expression of TGFB1, encoding TGF-Β1, contributing to osteoarthritis risk

Genetic and epigenetic interplay fine-tunes expression of TGFB1, encoding TGF-Β1, contributing to osteoarthritis risk

Perinatal DEHP exposure induces sex- and tissue-specific DNA methylation changes in both juvenile and adult mice.

Di(2-ethylhexyl) phthalate (DEHP) is a type of phthalate plasticizer found in a variety of consumer products and poses a public health concern due to its metabolic and endocrine disruption activities. Dysregulation of epigenetic modifications, including DNA methylation, has been shown to be an important mechanism for the pathogenic effects of prenatal exposures, including phthalates. In this study, we used an established mouse model to study the effect of perinatal DEHP exposure on the DNA methylation profile in liver (a primary target tissue of DEHP) and blood (a common surrogate tissue) of both juvenile and adult mice. Despite exposure ceasing at 3 weeks of age (PND21), we identified thousands of sex-specific differential DNA methylation events in 5-month old mice, more than identified at PND21, both in blood and liver. Only a small number of these differentially methylated cytosines (DMCs) overlapped between the time points, or between tissues (i.e. liver and blood), indicating blood may not be an appropriate surrogate tissue to estimate the effects of DEHP exposure on liver DNA methylation. We detected sex-specific DMCs common between 3-week and 5-month samples, pointing to specific DNA methylation alterations that are consistent between weanling and adult mice. In summary, this is the first study to assess the genome-wide DNA methylation profiles in liver and blood at two different aged cohorts in response to perinatal DEHP exposure. Our findings cast light on the implications of using surrogate tissue instead of target tissue in human population-based studies and identify epigenetic biomarkers for DEHP exposure.