Affect DNA Methylation Research Articles

Impact of maternal fructose intake on liver stem/progenitor cells in offspring: Insights into developmental origins of health and disease

AimsThe developmental origin of health and disease (DOHaD) theory postulates that poor nutrition during fetal life increases the risk of disease later in life. Excessive fructose intake has been associated with obesity, diabetes, and nonalcoholic fatty liver disease, and maternal fructose intake during pregnancy has been shown to affect offspring health. In this study, we investigated the effects of high maternal fructose intake on the liver stem/progenitor cells of offspring. Main methodA fructose-based DOHaD model was established using Sprague-Dawley rats. Small hepatocytes (SHs), which play an important role in liver development and regeneration, were isolated from the offspring of dams that were fed a high-fructose corn syrup (HFCS) diet. The gene expression and DNA methylation patterns were analyzed on postnatal day (PD) 21 and 60. Key findingsMaternal HFCS intake did not affect body weight or caloric intake, but differences in gene expression and DNA methylation patterns were observed in the SHs of offspring. Functional analysis revealed an association between metabolic processes and ion transport. SignificanceThese results suggest that maternal fructose intake affects DNA methylation and gene expression in the liver stem/progenitor cells of offspring. Furthermore, the prolonged retention of these changes in gene expression and DNA methylation in adulthood (PD 60) suggests that maternal fructose intake may exert lifelong effects. These findings provide insights into the DOHaD for liver-related disorders and highlight the importance of maternal nutrition for the health of the next generation.

ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming.

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.

Acupuncture increased the Number of Retrieved Oocytes in a Mouse Model of POR: The Involvement of DNA Methylation in the Oocytes

Background:: Poor ovarian response (POR) reduces the success rate of in vitro fertilization mainly because of fewer oocytes retrieved. Acupuncture (Ac) therapy can improve the number of retrieved oocytes in the controlled ovarian stimulation program. The role of Ac in the corresponding epigenetic mechanism of POR has not been studied. Objective:: This study was conducted to determine the effect of Ac on the number of retrieved oocytes and its role in DNA methylation in a mouse model of POR. Methods:: Forty C57BL/6N female mice with normal estrous cycles were randomly classified into 4 groups of 10 each: control (Con) group, Ac-Con group, POR group, and Ac-POR group. Mice in POR and Ac-POR groups received a gastric gavage of Tripterygium wilfordii polyglycoside suspension of 50 mg/kg-1 once a day for 14 consecutive days. Ac was applied at “Shenting” (DU 24), “Guanyuan” (CV 4), “Zusanli” (ST 36), and “Shenshu” (BL 23) in the Ac-POR group for 10 min per session, once a day for 14 consecutive days. All four groups were stimulated with pregnant mare serum gonadotropin and human chorionic gonadotropin, and the number of retrieved oocytes and proportion of mature oocytes were recorded. The DNA methylation level in a single mouse oocyte in each group was analyzed using single-cell genome-wide bisulfite sequencing (scBSseq), and key pathways were identified using GO and KEGG enrichment analyses. Results:: A dissecting microscope revealed that the Ac therapy improved the number of retrieved oocytes compared with the POR group (p < 0.05). ScBS-seq showed that there was no significant change in global DNA methylation levels between the POR model and control group mice. However, differences were primarily observed in the differentially methylated regions (DMRs) of each chromosome, and Ac decreased global DNA methylation. DMR analysis identified 13 genes that may be associated with Ac treatment. Cdk5rap2 and Igf1r, which mediate germ cell apoptosis, growth, and development, maybe most closely related to the Ac treatment of POR. KEGG analysis revealed that differentially expressed genes were mainly enriched in Wnt, GnRH, and calcium signaling pathways. The genes were closely related to the regulation of POR via Ac. Conclusion:: The results suggest that DNA methylation in oocytes is related to the development of POR and that the role of Ac in affecting DNA methylation in oocytes is associated with the Wnt, GnRH, and calcium signaling pathways as well as Cdk5rap2 and Igf1r in POR mice.

Rice requires a chromatin remodeler for Polymerase IV-small interfering RNA production and genomic immunity.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

Air pollutants in bronchoalveolar lavage fluid and pulmonary tuberculosis: A mediation analysis of gene-specific methylation

Particulate matter (PM) exposure could alter the risk of tuberculosis, but the underlying mechanism is still unclear. We enrolled 132 pulmonary tuberculosis (PTB) patients and 30 controls. Bronchoalveolar lavage fluid samples were collected from all participants to detect organochlorine pesticides, polycyclic aromatic hydrocarbons, metal elements, and DNA methylation of immunity-related genes. We observed that γ-HCH, Bap, Sr, Ag, and Sn were related to an increased risk of PTB, while Cu and Ba had a negative effect. IFN-γ, IL-17A, IL-2, and IL-23 had a higher level in the PTB group, while IL-4 was lower. The methylation of 18 CpG sites was statistically associated with PTB risk. The methylation at the IL-4_06_121 site showed a significant mediating role on γ-HCH, Sr, and Sn. Our study suggests that PM exposure can increase the risk of tuberculosis by affecting DNA methylation and cytokine expression.

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.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. While a plethora of studies indicate the relevance of affecting DNA methylation and histone acetylation, the influence on the mechanisms related to histone methylation is often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. However, recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (a H3K4/9 demethylase), or the effects on the global levels of histone methylation marks by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field still remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.

Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction affecting DNA methylation regulated transcription of miR-146a and miR-140-5p.

To explore whether electroacupuncture (EA) could alleviate osteoarthritis (OA) through affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p. Sixty male eight-week-old Sprague-Dawley rats were divided into three groups: normal group (normal healthy rats; no treatment), model group (OA rats; no treatment) and EA group (OA rats treated with EA). Safranin O staining and modified Mankin's score were performed to evaluate the histopathological alterations and degeneration of cartilage 8 weeks after 8 consecutive weeks of treatment. Quantitative real time polymerase chain reaction (qRT-PCR) assay was employed to evaluate the expression of miR-146a in the cartilage tissue and miR-140-5p in the synovium tissue, respectively. The bisulfite sequencing analysis and quantitative methylation specific PCR (qMSP) were used to analyze the status of methylation in the regulatory regions of miR-146a and miR-140-5p. Chromatin immunoprecipitation (ChIP) assay were performed to assess the binding of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (SMAD-3) in the regulatory regions of miR-146a and miR-140-5p. Western blot analysis was performed to detect the expressions of DNA Methyltransferase 1 (DMNT1), DNA Methyltransferase 3A (DMNT3A), and DNA Methyltransferase 3A (DMNT3b), NF-κB, SMAD3 levels. Our results showed that EA treatment significantly upregulated miR-146a and miR-140-5p expressions. qMSP analysis showed that EA significantly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium. Bisulfite DNA sequencing (BDS) and ChIP analysis showed that EA significantly increased binding affinity of SMAD3 and NF-kB on the hypermethylated miR-140 regulatory region and miR-146a promoter, respectively. Western Blot analysis demonstrated that EA also significantly decreased expressions of methylation related proteins- DMNT1, DMNT3a, and DMNT3b as well as NF-κB and SMAD3. Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) may alleviate OA affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.

The responsive mechanisms of DNA methylation and transcriptional regulation to acute hypoxia stress in HIF-1/VEGFA signal pathway of Japanese flounder (Paralichthys olivaceus)

The area of marine hypoxia is gradually expanding. This phenomenon seriously affects the normal life activities of a variety of marine organisms. Japanese flounder (Paralichthys olivaceus) is an important marine economic fish. However, its responses to hypoxia are still not very clear. Here, we conducted a hypoxia treatment (2.11 ± 0.11 mg/L) on Japanese flounder. Results showed that gill tissue morphology, macroscopically, was affected significantly by the hypoxia stress, especially gill lamellas. Molecularly, the HIF-1/VEGFA signal pathway, which was related to angiogenesis, was also significantly changed. In detail, expressions of hif-1α and vegfa genes, and methylation modified levels in DNA of hif-1α gene, changed significantly. The hif-1α expressions further were correlated negatively to its methylation levels, indicating that hypoxia may affect gene expression by acting on DNA methylation. In addition, we found that the CACGCA sequence, located in −1337 and −1317 sites, was the transcription factor HIF-1 binding sequence in vegfa gene. Furthermore, the −1337 site was more important for the transcriptional regulation. Therefore, it was concluded that hypoxia affected DNA methylation, regulated transcription of related factors, induced cascade amplification in signal pathways, and then influenced physiological morphology. These findings will enrich the environmental stress knowledge, and provide a reference for the breeding of teleost fishes in hypoxia tolerance.

DNA methylation cooperates with H3K9me2 at HCN4 promoter to regulate the differentiation of bone marrow mesenchymal stem cells into pacemaker-like cells.

Sick sinus syndrome (SSS) is a a life-threatening disease, and biological pacemakers derived from bone marrow mesenchymal stem cells (BMSCs) have practical clinical applications. Previous studies demonstrated that epigenetics plays an important role in the differentiation of BMSCs into pacemaker-like cells. However, the underlying mechanisms remain unclear. In the present study, we investigated the role of DNA methylation and histone methylation in pacemaker cells formation and found that changes in DNA and H3K9 methylation occur in the promoter region of the pacemaker cell-specific gene HCN4. In addition, the combined addition of methylation inhibitors was able to improve the efficiency of transduction of Tbx18 in inducing the differentiation of BMSCs into pacemaker-like cells. In vitro experiments have shown that inhibition of DNA methylation and H3K9 methylation can enhance the activity of the HCN4 promoter activity, and both can affect the binding of the transcription factor NKx2.5to the HCN4 promoter region. Further research on the interaction mechanism between DNA methylation and H3K9me2 in the HCN4 promoter region revealed that the two may be coupled, and that the methylesterase G9a and DNMT1 may directly interact to bind as a complex that affects DNA methylation and H3K9me2 regulation of HCN4 transcription. In conclusion, our studies suggest that the mutual coupling of DNA and H3K9 methylation plays a critical role in regulating the differentiation of BMSCs into pacemaker-like cells from the perspective of interactions between epigenetic modifications, and combined methylation is a promising strategy to optimise pacemaker-like cells for in vitro applications.

Nuclear localization of TET2 requires β-catenin activation and correlates with favourable prognosis in colorectal cancer.

Mutation-induced malfunction of ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is widely reported in haematological malignancies. However, the role of TET2 in solid cancers, including colorectal cancer (CRC), is unclear. Here, we found that TET2 malfunction in CRC is mostly due to decreased nuclear localization and that nuclear localization of TET2 is correlated with better survival of patients. To explore the underlying mechanisms, 14 immortalized solid tumour cell lines and 12 primary CRC cell lines were used. TET2 was mostly detected in the nucleus, and it induced significant DNA demethylation and suppressed cell growth by demethylating RORA and SPARC in cell lines like SW480. While in cell lines like SW620, TET2 was observed in the cytosol and did not affect DNA methylation or cell growth. Further examination with immunoprecipitation-mass spectrometry illustrated that β-catenin activation was indispensable for the nuclear localization and tumour suppression effects of TET2. In addition, the β-catenin pathway activator IM12 and the TET2 activator vitamin C were used simultaneously to enhance the effects of TET2 under low-expression conditions, and synergistic inhibitory effects on the growth of cancer were observed both in vitro and in vivo. Collectively, these data suggest that β-catenin-mediated nuclear localization of TET2 is an important therapeutic target for solid tumours.

Integration of methylation quantitative trait loci (mQTL) on dietary intake on DNA methylation levels: an example of n-3 PUFA and ABCA1 gene.

Epigenetic studies have reported relationships between dietary nutrient intake and methylation levels. However, genetic variants that may affect DNA methylation (DNAm) pattern, called methylation quantitative loci (mQTL), are usually overlooked in these analyses. We investigated whether mQTL change the relationship between dietary nutrient intake and leukocyte DNAm levels with an example of estimated fatty acid intake and ATP-binding cassette transporter A1 (ABCA1). A cross-sectional study on 231 participants (108 men, mean age: 62.7 y) without clinical history of cancer and no prescriptions for dyslipidemia. We measured leukocyte DNAm levels of 8 CpG sites within ABCA1 gene by pyrosequencing method and used mean methylation levels for statistical analysis. TaqMan assay was used for genotyping a genetic variant of ABCA1 (rs1800976). Dietary fatty acid intake was estimated with a validated food frequency questionnaire and adjusted for total energy intake by using residual methods. Mean ABCA1 DNAm levels were 5% lower with the number of minor alleles in rs1800976 (CC, 40.6%; CG, 35.9%; GG, 30.6%). Higher dietary n-3 PUFA intake was associated with lower ABCA1 DNAm levels (1st (ref) vs. 4th, β [95% CI]: -2.52 [-4.77, -0.28]). After controlling for rs180076, the association between dietary n-3 PUFA intake and ABCA1 DNAm levels was attenuated, but still showed an independent association (1st (ref) vs. 4th, β [95% CI]: -2.00 [-3.84, -0.18]). The interaction of mQTL and dietary n-3 PUFA intake on DNAm levels was not significant. This result suggested that dietary n-3 PUFA intake would be an independent predictor of DNAm levels in ABCA1 gene after adjusting for individual genetic background. Considering mQTL need to broaden into other genes and nutrients for deeper understanding of DNA methylation, which can contribute to personalized nutritional intervention.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua)

ABSTRACT After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway’s National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

Advances in DNA methylation and demethylation in medicinal plants: a review.

DNA methylation and demethylation are widely acknowledged epigenetic phenomena which can cause heritable and phenotypic changes in functional genes without changing the DNA sequence. They can thus affect phenotype formation in medicinal plants. However, a comprehensive review of the literature summarizing current research trends in this field is lacking. Thus, this review aims to provide an up-to-date summary of current methods for the detection of 5-mC DNA methylation, identification and analysis of DNA methyltransferases and demethyltransferases, and regulation of DNA methylation in medicinal plants. The data showed that polyploidy and environmental changes can affect DNA methylation levels in medicinal plants. Changes in DNA methylation can thus regulate plant morphogenesis, growth and development, and formation of secondary metabolites. Future research is required to explore the mechanisms by which DNA methylation regulates the accumulation of secondary metabolites in medicinal plants.

Influence of e-waste exposure on DNA damage and DNA methylation in people living near recycling sites.

The association between long-term exposure to e-waste and poor health is well established, but how e-waste exposure affects DNA methylation is understudied. In this study, we measured the DNA damage levels and the alternation of DNA methylation in peripheral blood mononuclear cells (PBMCs) collected from a population exposed to e-waste. The concentration of 28 PCB congeners in the blood samples of e-waste recycling workers was elevated than those of the reference group. DNA damage levels were significantly higher than that of samples from the reference group by detecting the SCGE, CA, and CBMN assays. Eventually, we found that the methylation level of 1233 gene loci was changed in the exposure group. Bioinformatic analysis of differential genes revealed that the hypermethylated genes were enriched in cell component movement and regulation of cell function, and hypomethylated genes were involved in the cellular metabolic process. Among the 30 genes we tested, 14 genes showed a negative correlation between methylation level and expression level. Therefore, e-waste exposure potentially increased the levels of DNA damage and alters DNA methylation, which would likely impact human health.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Methylation-based prediction of myelodysplastic syndrome survival outcomes.

7058 Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid malignancies associated with a myriad of deleterious outcomes and a 5-year relative survival of 37%. MDS risk stratification is key for optimal treatment decisions. DNA methylation is associated with MDS biology due to frequent somatic mutations in genes (i.e. TET2, DNMT3A, IDH1, IDH2, and WT1) that affect DNA methylation. We hypothesized that methylation-based markers may help stratify risk and improve IPSS-R (the Revised International Prognostic Scoring System). Here, we evaluated the potential of both Bone Marrow (BM) Whole-genome Bisulfite Sequencing (WGBS) and Serum-based Targeted Methylation (TM) Sequencing to predict survival of MDS patients. Methods: We conducted paired BM WGBS and Serum-based TM sequencing on a cohort of 127 patients with MDS (N = 104) and secondary AML (N = 23) treated at the Columbia University Medical Center. We categorized patients based on an overall survival of > 3 (“long”, N = 50) or < 3 years (“short”, N = 77). We then identified differentially methylated regions (DMRs) differing between long and short survivor groups, and analyzed the biological pathways and functions enriched in these regions. To assess survival association, we trained a random forest classifier on principal components (“methylation classifier”) on the methylation fractions from a subset of 96 Serum and 98 BM MDS patient samples to predict long vs. short survival, and compared this against a logistic regression IPSS-R model. We optimized the hyperparameters and assessed classifier performance using six-fold nested cross-validation. Finally, we conducted a multivariable cox regression with predicted scores from the methylation classifier, IPSS-R scores (recalculated at sample collection), age, and gender. Results: We identified a total of 7,742 DMRs in the BM WGBS and 14,093 DMRs in the Serum TM as significantly different between long and short survivor groups. Signaling pathways for calcium, cAMP, MAPK, Rap1, and PI3K-Akt were significantly enriched in DMRs. We also identified a previously-reported CpG island hypermethylation signature associated with AML progression in BM and Serum. The methylation classifier achieved a comparable AUROC compared to IPSS-R logistic regression for both BM WGBS (0.80 vs 0.78) and Serum TM (0.77 vs 0.73) samples. Multivariable Cox regression demonstrated that both predicted scores from the methylation classifier (Serum p =0.002; BM p =0.016) and IPSS-R scores (Serum p =0.003; BM p =0.004) are significant predictors of survival. Conclusions: We demonstrated methylation-based sequencing represents a promising new tool for MDS patient risk stratification. As IPSS-R relies on bone marrow aspirates, the Serum-based TM assay offers a less-invasive method for MDS risk stratification.

0608 PAX8/PAX8-AS1 DNA methylation levels are not associated with sleep duration in medicated patients with unexplained hypersomnolence

Abstract Introduction The biological basis of daytime sleepiness and excessive sleep duration in patients with unexplained hypersomnolence is unknown. However, a growing body of evidence suggests modulations in the epigenome coincide with objective measures of sleep, suggesting epigenetic mechanisms may be related to hypersomnolence. Previously, we identified significant correlations between saliva DNA methylation abundance at nine CpG sites located in the PAX8/PAX8-AS1 genes and total sleep time measured using polysomnography in unmedicated, clinical patients with unexplained hypersomnolence. Since psychotropic medications frequently affect clinical sleep testing and may also affect DNA methylation, here we attempted to replicate prior findings in a separate, medicated sample. Methods Forty-three medicated clinical patients with unexplained hypersomnolence were drawn from a larger study of a multimodal sleep assessment that included ad libitum overnight polysomnography. Pyrosequencing quantified DNA methylation at the previously identified CpG sites in PAX8/PAX8-AS1. Multivariate linear models, adjusting for age, sex, and body mass index (BMI), assessed relationships between total sleep time and DNA methylation at these prespecified CpG sites. Results Participants were young -to middle-aged (Mean Age = 32.2±10 years) and predominantly female (Percent Female = 95.3%), with a mean BMI of 27.8±5.4kg/m2. Epworth Sleepiness Scale (ESS) score was consistent with complaints of daytime sleepiness (Mean ESS =15.1±-4.3). Total sleep time on ad libitum polysomnography was 9.2±1.7 hours (range 5.6-12.8 hours). No significant associations between DNA methylation at the PAX8/PAX8-AS1 CpG sites and sleep duration were identified in adjusted models. Conclusion We were unable to replicate findings of an association between sleep duration and DNA methylation levels at specific PAX8/PAX8-AS1 CpG sites in medicated patients with unexplained hypersomnolence. Further research to clarify whether this is a result of medications affecting the DNA methylation levels at these CpG sites or previously identified associations between DNA methylation of PAX8/PAX8-AS1 and sleep length are spurious will require replication of prior findings in a novel unmedicated cohort and quantification of within-subjects effects of psychotropic medications on DNA methylation levels in genes of interest. Support (if any) AASM Foundation (229 SR-20 - Diversity Supplement Grant)

Abstract A49: Characterizing the order of mutation acquisition in acute myeloid leukemia using large-scale single-cell sequencing

Abstract Introduction: Acute myeloid leukemia (AML) has a poor prognosis, despite aggressive therapies and a recent expansion in the array of treatments. Treatment is often determined by mutations, which risk-stratify a patient’s leukemia or can identify mutations that serve as therapeutic targets. Although common mutations in AML have been extensively studied, less research has formally characterized the order by which mutations tend to occur and how this order relates to properties of the disease. Methods: We leveraged published, single-cell DNA sequencing data from three institutions to model the clonal evolution of AML. Sequencing had been performed using targeted sequencing panels covering 19 to 37 genes, and driver mutations were identified using conservative filters. Mutation trees were created using Single Cell Inference of Tumor Evolution (SCITE). Clonal evolution patterns, including across clinical timepoints, were identified and compared to patient characteristics, disease phenotype, and outcomes. The BeatAML dataset was leveraged to explore associations between mutation order and gene expression. Results: Using 835 driver mutations from 276 samples and 209 patients, we identified 223 total unique mutation patterns. Branched evolution primarily involves FLT3 and RAS pathway mutations, whereas certain mutation pairs, such as NPM1 and FLT3, always occurred linearly in the same clone. Although some mutation pairs, such as those related to DNA methylation versus the RAS pathway, tended to occur in a specific order, several cases exhibited atypical orderings. Early signaling gene mutations were associated with younger patient age and increased signaling mutation homozygosity while NRAS-first cases were associated with increased monocyte counts. NRAS-first cases were also associated with distinct gene expression patterns in the BeatAML dataset. Paired diagnosis and relapse samples revealed novel associations between mutations gained at relapse and their clonal context, and these analyses supported a relative fitness advantage for signaling mutations in clones containing mutations in NPM1 or genes affecting DNA methylation. Conclusions: Clonal evolution in AML can be reconstructed at scale using single-cell DNA sequencing data, and different mutation acquisition patterns are associated with distinct leukemia and patient characteristics, despite cases having similar co-mutation patterns. Citation Format: Matthew Schwede, Katharina Jahn, Linde A Miles, Robert L Bowman, Jack Kuipers, Troy M Robinson, Asiri Ediriwickrema, Andrew J Gentles, Ross Levine, Niko Beerenwinkel, Koichi Takahashi, Ravindra Majeti. Characterizing the order of mutation acquisition in acute myeloid leukemia using large-scale single-cell sequencing [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A49.

IGEM as a human iPS cell-based global epigenetic modulation detection assay provides throughput characterization of chemicals affecting DNA methylation

Chemical-induced dysregulation of DNA methylation during the fetal period is known to contribute to developmental disorders or increase the risk of certain diseases later in life. In this study, we developed an iGEM (iPS cell-based global epigenetic modulation) detection assay using human induced pluripotent stem (hiPS) cells that express a fluorescently labeled methyl-CpG-binding domain (MBD), which enables a high-throughput screening of epigenetic teratogens/mutagens. 135 chemicals with known cardiotoxicity and carcinogenicity were categorized according to the MBD signal intensity, which reflects the degree of nuclear spatial distribution/concentration of DNA methylation. Further biological characterization through machine-learning analysis that integrated genome-wide DNA methylation, gene expression profiling, and knowledge-based pathway analysis revealed that chemicals with hyperactive MBD signals strongly associated their effects on DNA methylation and expression of genes involved in cell cycle and development. These results demonstrated that our MBD-based integrated analytical system is a powerful framework for detecting epigenetic compounds and providing mechanism insights of pharmaceutical development for sustainable human health.