Methylation Capture Sequencing Research Articles

Wuzi Yanzong Decoction Ameliorates Oligoasthenozoospermia by Up-Regulating Methyltransferase and Increasing Spata, Bcl, and Pik3 Series Genes Methylation Level.

Wuzi Yanzong decoction (WZYZD) belongs to the traditional formula for treating male infertility caused by oligoasthenozoospermia (OLI). This research aims to elucidate the therapeutic substance basis and potential pharmacological mechanisms of WZYZD in treating OLI. A total of 52 chemical ingredients were identified from WZYZD. HE and TUNEL staining demonstrated that WZYZD can markedly alleviate OLI. Immunofluorescence analysis showed that WZYZD can significantly increase the expression levels of DNMT3 A, PIWIL1, SETDB1, and PRMT5. Methyl capture sequencing proved that WZYZD can markedly upregulate the methylated level of Spata, Bcl, and Pik3 series genes. Network pharmacology analysis proved that WZYZD can ameliorate OLI through BCL-2 and PI3 K-AKT signaling pathways. The immunofluorescence assay of BCL-2 and SPATA18 proved the aforementioned results. The potential mechanism of WZYZD in treating OLI mainly involved recruiting methyltransferase DNMT3 A, PIWIL1, PRMT5, and SETDB1 and increasing the methylation degree of Spata, Bcl, and Pik3 series genes.

Epigenetic regulation of HOXA3 and its impact on oral squamous cell carcinoma progression

ObjectiveThe role of homeobox A3 (HOXA3) in cancer progression is gaining prominence, however, to date, no studies have investigated its regulatory function in oral cancer. In this study, we explored the role of HOXA3 through epigenetic mechanisms. MethodsClinical samples were collected from 25 potentially malignant oral lesions and 50 oral squamous cell carcinoma (OSCC) patients, categorized into low-stage and high-stage tumors. The promoter activity of HOXA3 was determined through cloning and luciferase assays. CpG methylation patterns across the gene were identified using methyl-capture sequencing. Gene expression was analysed using RT‒qPCR. The Survminer R package was used to assess the clinical significance of 3′ UTR methylation associated with overall survival. RNA‒RNA interactions were analysed using RNAInter and TargetScan v8.0. ResultsHOXA3 expression was upregulated in dysplasia and downregulated in advanced cancer stages, showing an inverse correlation with promoter methylation, suggesting epigenetic regulation by DNA methylation. Hypermethylation of the 3′ UTR was associated with poor overall survival in advanced stages. Long non-coding RNAs and microRNAs may post-transcriptionally modulate HOXA3 in oral carcinogenesis. ConclusionCpG-specific hypermethylation in the 3′ UTR may serve as a potential biomarker in OSCC. Statement of Clinical RelevanceLocus-specific CpG hypermethylation in the 3′ UTR was significantly associated with poor overall survival and patient stratification based on OSCC progression.

The association between cumulative exposure to PM2.5 and DNA methylation measured using methyl-capture sequencing among COPD patients

BackgroundParticulate matter with a diameter of < 2.5 μm (PM2.5) influences gene regulation via DNA methylation; however, its precise mechanism of action remains unclear. Thus, this study aimed to examine the connection between personal PM2.5 exposure and DNA methylation in CpG islands as well as explore the associated gene pathways.MethodsA total of 95 male patients with chronic obstructive pulmonary disease (COPD) were enrolled in this study. PM2.5 concentrations were measured for 12 months, with individual exposure recorded for 24 h every 3 months. Mean indoor and estimated individual PM2.5 exposure levels were calculated for short-term (7 days), mid-term (35 days), and long-term (90 days). DNA methylation analysis was performed on the blood samples, which, after PCR amplification and hybridization, were finally sequenced using an Illumina NovaSeq 6000 system. Correlation between PM2.5 exposure and CpG methylation sites was confirmed via a mixed-effects model. Functional enrichment analysis was performed on unique CpG methylation sites associated with PM2.5 exposure to identify the relevant biological functions or pathways.ResultsThe number of CpG sites showing differential methylation was 36, 381, and 182 for the short-, mid-, and long-term indoor models, respectively, and 3, 98, and 28 for the short-, mid-, and long-term estimated exposure models, respectively. The representative genes were TMTC2 (p = 1.63 × 10-3, R2 = 0.656), GLRX3 (p = 1.46 × 10-3, R2 = 0.623), DCAF15 (p = 2.43 × 10-4, R2 = 0.623), CNOT6L (p = 1.46 × 10-4, R2 = 0.609), BSN (p = 2.21 × 10-5, R2 = 0.606), and SENP6 (p = 1.59 × 10-4, R2 = 0.604). Functional enrichment analysis demonstrated that the related genes were mostly associated with pathways related to synaptic transmission in neurodegenerative diseases and cancer.ConclusionA significant association was observed between PM2.5 exposure and DNA methylation upon short-term exposure, and the extent of DNA methylation was the highest upon mid-term exposure. Additionally, various pathways related to neurodegenerative diseases and cancer were associated with patients with COPD.ClinicalTrials.gov identifierNCT04878367.

Betaine alleviates spermatogenic cells apoptosis of oligoasthenozoospermia rat model by up-regulating methyltransferases and affecting DNA methylation

BackgroundOligoasthenozoospermia is the most common type of semen abnormality in male infertile patients. Betaine (BET) has been proved to have pharmacological effects on improving semen quality. BET also belongs to endogenous physiological active substances in the testis. However, the physiological function of BET in rat testis and its pharmacological mechanism against oligoasthenozoospermia remain unclear. PurposeThis research aims to prove the therapeutic effect and potential mechanism of BET on oligoasthenozoospermia rat model induced by Tripterygium wilfordii glycosides (TWGs). MethodsThe oligoasthenozoospermia rat model was established by a continuous gavage of TWGs (60 mg/kg) for 28 days. Negative control group, oligoasthenozoospermia group, positive drug group (levocarnitine, 300 mg/kg), and 200 mg/kg, 400 mg/kg, and 800 mg/kg BET groups were created for exploring the therapeutic effect of BET on the oligoasthenozoospermia rat model. The therapeutic effect was evaluated by HE and TUNEL staining. Immunofluorescence assay of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3, methylation capture sequencing, Pi-RNA sequencing, and molecular docking were used to elucidate potential pharmacological mechanisms. ResultsIt is proved that BET can significantly restore testicular pathological damage induced by TWGs, which also can significantly reverse the apoptosis of spermatogenic cells. The spermatogenic cell protein expression levels of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3 significantly decreased in oligoasthenozoospermia group. 400 mg/kg and 800 mg/kg BET groups can significantly increase expression level of the above-mentioned proteins. Methylation capture sequencing showed that BET can significantly increase the 5mC methylation level of Spata, Spag, and Specc spermatogenesis-related genes. Pi-RNA sequencing proved that the above-mentioned genes produce a large number of Pi-RNA under BET intervention. Pi-RNA can form complexes with PIWI proteins to participate in DNA methylation of target genes. Molecular docking indicated that BET may not directly act as substrate for methyltransferase and instead participates in DNA methylation by promoting the methionine cycle and increasing S-adenosylmethionine synthesis. ConclusionBET has a significant therapeutic effect on oligoasthenozoospermia rat model induced by TWPs. The mechanism mainly involves that BET can increase the methylation level of Spata, Specc, and Spag target genes through the PIWI/Pi-RNA pathway and up-regulation of methyltransferases (including DNA methyltransferases and histone methyltransferases).

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

DOP24 Multi-omic sequencing reveals distinctive gene expression and DNA methylation alterations as potential predictors of primary sclerosing cholangitis development in treatment-naïve paediatric Ulcerative Colitis

Abstract Background Primary sclerosing cholangitis (PSC) is a progressive choleostatic disease and up to 80% of patients also have ulcerative colitis (PSC-UC). This presents a clinical challenge owing to difficulty in diagnosis and increased risk for developing cancer. While several multifactorial processes including inflammation and microbial dysbiosis have been associated with PSC-UC pathogenesis, the precise molecular factors that regulate the phenotype of this disease subtype remain unknown. Methods We applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies derived from the DOCHAS study (GEN-193/11), to identify transcriptomic and epigenetic differences between treatment naïve paediatric UC (n=10), PSC-UC (n=10) and healthy controls (n=10) samples. Results Differential gene expression between UC and PSC-UC identified 9 up-regulated genes - ADMTS14, PNCK, NLRP3, SLC6A19, DLL1, FCGR2C, KLHL17, APOB, EHBP1L1 and 5 downregulated - SLC37A2, SLC14A2, RPL27, RPS25, SLC38A4 in PSC-UC relative to UC. Importantly, we show that expression of these genes was intricately regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (TFs) :AR, p53, JUND, CEBPA. Similarly, differential methylation analysis between PSC-UC and UC identified 22 differentially methylated regions (DMRs) relative to controls, where 5 were hypermethylated and 8 hypomethylated. Intriguingly, in general we show that these DMRs are largely localised in gene promoter regions as opposed to enhancers. Importantly, we show that these DMR’s identified between PSC-UC vs UC is enriched for binding sites for the TF: ASCL1, suggesting its activity likely is effected due to the altered methylation of its binding site in PSC-UC patients. Collectively, these results highlight the importance of TF’s in driving molecular differences between PSC-UC and UC paediatric patients in a treatment naïve setting. Conclusion In summary, for the first time this study provides a critical insight into the transcriptional differences between treatment naïve children with PSC-UC vs UC as well as highlights the intricate regulatory processes involving master transcriptional regulators, transcription factors and DNA methylation. These processes thus warrant further examination in larger cohorts to rationalise their role as diagnostic/therapeutic targets.

EBV DNA methylation profiles and its application in distinguishing nasopharyngeal carcinoma and nasal NK/T-cell lymphoma

BackgroundAs an oncovirus, EBV is associated with multiple cancers, including solid tumors and hematological malignancies. EBV methylation plays an important role in regulating tumor occurrence. However, the EBV methylation profiles in EBV-associated tumor tissues are poorly understood.ResultsIn this study, EBV methylation capture sequencing was conducted in several different tumor tissue samples, including NPC, EBVaGC, lung LELC and parotid LELC. Besides, EBV capture sequencing and following qMSP were performed on nasopharyngeal brushing samples from NPC and nasal NKTCL patients. Our results showed that the EBV genome among different types of tumors displayed specific methylation patterns. Among the four types of tumors from epithelial origin (NPC, EBVaGC, lung LELC and parotid LELC), the most significant differences were found between EBVaGC and the others. For example, in EBVaGC, all CpG sites within 1,44,189–1,45,136 bp of the EBV genome sequence on gene RPMS1 were hyper-methylated compared to the others. Differently, significant differences of EBV CpG sites, particularly those located on gene BILF2, were observed between NPC and nasal NKTCL patients in nasopharyngeal brushing samples. Further, the methylated level of BILF2 was further detected using qMSP, and a diagnostic model distinguishing NPC and nasal NKTCL was established. The AUC of the model was 0.9801 (95% CI 0.9524–1.0000), with the sensitivity and specificity of 98.81% (95% CI 93.63–99.94%) and 76.92% (95% CI 49.74–91.82%), respectively.ConclusionsOur study reveals more clues for further understanding the pathogenesis of EBV, and provides a possibility for distinguishing EBV-related tumor by detecting specific EBV CpG sites.

Hypermethylation of Bmp2 and Fgfr2 Promoter Regions in Bone Marrow Mesenchymal Stem Cells Leads to Bone Loss in Prematurely Aged Mice.

Osteoporosis is an age-related, systemic skeletal disease that poses a significant public health challenge in contemporary society. Development at the epigenetic level is emerging as an important pathogenic mechanism of osteoporosis. Despite indications of a robust association between DNA methylation and osteoporosis development, a comprehensive understanding of the specific role of DNA methylation in osteoporosis remains limited. In this study, significant bone loss was detected at the beginning of eight weeks of age in mouse models of premature aging (SHJHhr mice). We identified a notable upregulation of DNA methyltransferase 3b/3l (Dnmt3b/l) and downregulation of ten eleven translocation dioxygenase 1 (Tet1) in bone marrow mesenchymal stem cells (BMSCs) isolated from SHJHhr mice, along with an increase in the overall 5-methylcytosine (5mC) levels. Moreover, methylation capture sequencing revealed genomic hypermethylation in SHJHhr mice BMSCs. Integrated methylome and transcriptome analyses revealed several crucial methylated genes and networks that are potentially associated with osteoporosis development. Notably, elevated methylation levels of genes linked to the Wnt signaling pathway, particularly bone morphogenetic protein 2 (Bmp2) and fibroblast growth factor receptor (Fgfr2), appeared to compromise the osteogenic differentiation potential of BMSCs. Concurrently, DNA methyltransferase inhibitors attenuated the methylation of the promoter regions of Bmp2 and Fgfr2 and rescued the osteogenic differentiation potential of the BMSCs from SHJHhr mice. In summary, our study provides novel insights into the role of DNA methylation in the development of osteoporosis and suggests promising prospects for employing epigenetic interventions to manage osteoporosis.

Zhizi-Chuanxiong herb pair alleviates atherosclerosis progression in ApoE−/− mice by promoting the methylation of FGFR3 to inhibit MAPK/ERK-mediated apoptosis

Ethnopharmacological relevanceGardenia Fructus (Gardenia jasminoides Ellis, Zhizi) and Chuanxiong Rhizoma (Ligusticum chuanxiong Hort., Chuanxiong) are both traditional Chinese medicines with vascular protective effects, which help detoxify and activate blood, and are clinically used to treat atherosclerosis (AS). Previously, Zhizi-Chuanxiong showed good efficacy in attenuating AS progression in rabbits. However, its potential mechanism is yet unclear. Aim of the studyThis study aimed to investigate the mechanism of the Zhizi-Chuanxiong herb pair (ZCHP) in attenuating AS progression from the perspective of DNA methylation. Materials and methodsAn AS mouse model was developed with ApoE−/− mice fed a high-fat diet (HFD). The therapeutic effects and mechanisms of ZCHP in treating HFD-induced AS were identified using an automated biochemical analyzer, enzyme-linked immunosorbent assays, histopathology, methyl-capture sequencing (MC-seq), pyrosequencing, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), western blotting, and TUNEL staining. ResultsZCHP attenuated the development of AS by reducing lipid levels and enhancing the stability of plaques and via anti-inflammation. MC-seq and Kyoto Encyclopedia of Genes and Genomes analysis revealed that ZCHP corrected the expressions of both aberrant hypomethylated and hypermethylated genes, which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Protein–protein network interaction analysis and molecular docking showed that fibroblast growth factor 3 (FGFR3) and serine/threonine protein kinase (AKT1) were closely related to the MAPK signaling pathway among differentially methylated genes induced by ZCHP. Furthermore, pyrosequencing showed that ZCHP could induce FGFR3 hypermethylation and AKT1 hypomethylation in the promoter region, which was consistent with the MC-seq results. Molecular docking showed that the ZCHP was more tightly docked to FGFR3. Furthermore, RT-qPCR and western blotting showed that the mRNA and protein expression levels of FGFR3 decreased after treatment with ZCHP. Finally, western blotting showed that ZCHP suppressed the expression of phosphorylated MAPK and phosphorylated extracellular signal-regulated kinase (ERK), and TUNEL staining showed that ZCHP treatment could inhibit apoptosis in AS. ConclusionOur findings suggest that ZCHP can effectively attenuate AS progression by inhibiting MAPK/ERK signaling–mediated apoptosis via FGFR3 hypermethylation in the promoter region.

A DNA methylation signature for the prediction of tumour recurrence in stage II colorectal cancer.

A major challenge in stage II colorectal carcinoma is to identify patients with increased risk of recurrence. Biomarkers that distinguish patients with poor prognosis from patients without recurrence are currently lacking. This study aims to develop a robust DNA methylation classifier that allows the prediction of recurrence and chemotherapy benefit in patients with stage II colorectal cancer. We performed a genome-wide DNA methylation capture sequencing in 243 stage II colorectal carcinoma samples and identified a relapse-specific DNA methylation signature consisting of eight CpG sites. Two hundred and forty-three patients with stage II CRC were enrolled in this study. In order to select differential methylation sites among recurrence and non-recurrence stage II CRC samples, DNA methylation profiles of 62 tumour samples including 31 recurrence and 31 nonrecurrence samples were analysed using the Agilent SureSelectXT Human Methyl-Seq, a comprehensive target enrichment system to analyse CpG methylation. Pyrosequencing was applied to quantify the methylation level of candidate DNA methylation sites in 243 patients. Least absolute shrinkage and selection operator (LASSO) method was employed to build the disease recurrence prediction classifier. We identified a relapse-related DNA methylation signature consisting of eight CpG sites in stage II CRC by DNA methylation capture sequencing. The classifier showed significantly higher prognostic accuracy than any clinicopathological risk factors. The Kaplan-Meier survival curve showed an association of high-risk score with poor prognosis. In multivariate analysis, the signature was the most significant prognosis factor, with an HR of 2.80 (95% CI, 1.71-4.58, P < 0.001). The signature could identify patients who are suitable candidates for adjuvant chemotherapy. An eight-CpG DNA methylation signature is a reliable prognostic and predictive tool for disease recurrence in patients with stage II CRC.

Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway.

DNA methylation, including aberrant hypomethylation and hypermethylation, plays a significant role in atherosclerosis (AS); therefore, targeting the unbalanced methylation in AS is a potential treatment strategy. Gualou-xiebai herb pair (GXHP), a classic herb combination, have been used for the treatment of atherosclerotic-associated diseases in traditional Chinese medicine. However, the effects and underlying mechanism of GXHP on AS remain nebulous. In this study, the CCK-8 method was applied to determine the non-toxic treatment concentrations for GXHP. The formation of foam cells played a critical role in AS, so the foam cells model was established after RAW264.7 cells were treated with ox-LDL. The contents of total cholesterol (TC) and free cholesterol (FC) were determined by Gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was used to check the expressions of inflammatory factors including IL-1β, TNF-α, and VCAM-1. Methyl-capture sequencing (MC-seq) and RNA-seq were applied to observe the changes in genome-wide DNA methylation and gene expression, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze differentially methylated genes (DMGs) and differentially expressed genes (DEGs). The targeted signaling pathway was selected and verified using western blotting (WB). The results showed that the lipids and inflammatory factors in foam cells significantly increased. GXHP significantly reduced the expression of TC, FC, and inflammatory factors. MC-seq and RNA-seq showed that GXHP not only corrected the aberrant DNA hypermethylation, but also DNA hypomethylation, thus restored the aberrant DEGs in foam cells induced by ox-LDL. GXHP treatment may target the PI3K-Akt signaling pathway. GXHP reduced the protein levels of phosphorylated(p)-PI3K and p-AKT in foam cells. Our data suggest that treatment with GXHP showed protective effects against AS through the inhibition of DNA methylation mediated PI3K-AKT signaling pathway, suggesting GXHP as a novel methylation-based agent.

DOP80 Multi-omic sequencing reveals distinctive gene expression and DNA methylation alterations as potential predictors of primary sclerosing cholangitis development in treatment-naïve paediatric ulcerative colitis

Abstract Background Primary sclerosing cholangitis (PSC) is a progressive choleostatic disease and up to 80% of patients also have ulcerative colitis (PSC-UC). This presents a clinical challenge owing to difficulty in diagnosis and increased risk for developing cancer. While several multifactorial processes including inflammation and microbial dysbiosis have been associated with PSC-UC pathogenesis, the precise molecular factors that regulate the phenotype of this disease subtype remain unknown. Methods Here, we applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies to identify transcriptomic and epigenetic alterations differentiating UC from PSC-UC. Samples were taken from 3 groups of treatment-naïve children at diagnosis who participated in the DOCHAS study (GEN-193/11) - UC (n=10), PSC-UC (n=10) and healthy controls(n=10). Results Differential gene expression between UC and PSC-UC identified disease-associated genes that were either up- or down-regulated in UC or PSC relative to controls. Furthermore, expression of these genes was intricately regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (AR, p53, JUND, CEBPA). Importantly, gene expression comparison between UC vs PSC-UC revealed 4 up-regulated genes in PSC-UC (RAB31, TENM3, KLHL17 and COL7A1). Notably, RAB31 and TENM3 are also significantly up-regulated in gastrointestinal (GI) cancers. We also identified 4 down-regulated genes in PSC-UC (H3C15, SLC37A2, SLC14A2 and IL20RA). Differential methylation analysis between healthy control biopsies vs PSC-UC and UC demonstrated &amp;gt;1000 differentially methylated regions (DMRs, 5Kb), with majority of these sites displaying hypermethylation. Interestingly, PSC-UC vs UC analysis identified 53 regions and 59 DMRs that are hyper- and hypomethylated respectively in PSC-UC, with a large proportion of these DMRs located in gene promoters and putative enhancer regions and notably impacted gene expression of differentially expressed genes identified from the mRNAseq analysis. Conclusion Taken together, our study for the first time identifies distinct gene expression and DNA methylation alterations that differentiate UC from PSC-UC at diagnosis in treatment-naïve paediatric patients, some of which are associated with GI cancers. Their potential utility as predictive biomarkers of PSC development in UC or dysplasia risk in PSC-UC warrants further validation through larger patient cohorts.

349-OR: ADA President's Select Abstract: Circulating Level of DNA Methylation Near CPT1A and Long-Term Changes in Triglycerides in Response to Diet Interventions: The POUNDS Lost Trial

Background: Epigenome-wide association studies identified DNA methylation (DNAm) at several CpG sites of the carnitine palmitoyltransferase-1A (CPT1A) gene to be associated with triglycerides (TG) . However, little is known about whether the pre-treatment DNAm level at CPT1A is associated with changes in TG in response to weight-loss diet interventions. Methods: We included 673 white participants with overweight or obesity, who were randomly assigned to 1 of 4 diets varying in macronutrients components. Blood DNAm levels were profiled by a high-resolution methyl-capture sequencing at baseline. We defined the regional DNAm at CPT1A as the average methylation level over CpGs within 500 bp of the 3 TG-related DNAm sites (cg00574958, cg09737197, and cg17058475) , which were closely located. Two-year changes in TG were calculated. Linear regression models were used to examine the associations between baseline DNAm at CPT1A and 2-year changes in TG. Results: Higher regional DNAm level at CPT1A was associated with lower TG at baseline (β=-2.21, SE=1.01, p=0.029) . We found that dietary fat intake significantly modified the association between baseline DNAm at CPT1A and 2-year changes in TG, independent of concurrent weight-loss (p-int=0.018) . In the low-fat diet group, a higher regional DNAm level was associated with a greater reduction in TG at 2 years, after adjustment for age, sex, baseline body mass index, use of lipid-lowering medication, baseline TG level, and concurrent weight-loss (p=0.01) , whereas no such association was observed in the high-fat diet group (p=0.64) . A similar interaction pattern was also found between dietary fat and baseline DNAm at CPT1A on 2-year changes of TG in very-low-density lipoprotein without apolipoprotein C-III (p-int=0.01) . Conclusion: Our data indicate that participants with a higher regional DNAm level at CPT1A benefited more in long-term improvement in TG when assigned a low-fat weight-loss diet. Disclosure X.Li: None. X.Shao: None. Q.Xue: None. E.Grundberg: None. G.Bray: None. F.Sacks: None. L.Qi: None. Funding The study was supported by grants from the National Heart, Lung, and Blood Institute (HL071981, HL034594, HL126024) , the National Institute of Diabetes and Digestive and Kidney Diseases (DK115679, DK091718, DK100383) , the Fogarty International Center (TW010790) , and Tulane Research Centers of Excellence Awards. Xiang Li was the recipient of the American Heart Association Predoctoral Fellowship Award (19PRE34380036) .

P697 Examining the regulatory role of epigenetic and transcriptomic alterations in anti-TNFα treatment response and disease progression in patients with Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory condition characterized by significant morbidity and escalating economic costs. Effective patient management is severely hampered by a lack of unambiguous molecular biomarkers that can predict response to anti-TNFα treatment such and/or disease progression. While, both genetic and epigenetic factors have been reported to impact UC pathogenesis, their role in regulating disease progression and treatment response remains unclear. Methods Here, we applied methyl-capture sequencing and RNAsequencing to mucosal biopsies derived from clinical cohorts of patients with mild-to-moderate and severe UC with the aim of identifying various regulatory factors that orchestrate treatment response and disease severity in these patients. Results First, differential gene expression analysis between responders and non-responders to infliximab and vedolizumab (anti-TNFα) enabled identification of disease-associated genes that were either up- or down-regulated in responders to both these anti-TNFα agents. Next, upstream analysis of these differentially expressed genes revealed that both up- and downregulated genes in responders are intricately regulated by “master regulators” (MRs) including chemokine ligands and receptors such as FGFR2 and IL8 respectively. Moreover, we show that these MRs play a pivotal role in regulating gene expression through a complex signalling network mediated by transcription factors (TFs) such as FYN and ICAM1. These results for the first time provide evidence of impact of MRs and TFs on genes involved in differential response to anti-TNFα agents in patients with UC. In parallel, targeted DNA methylation profiling of mucosal biopsies derived from mild-to-moderate (n=85) and severe UC (n=33) patients enabled identification of a wide-spread DNA methylation alterations at regulatory regions such as promoters and enhancers. Moreover, we identified distinct significant methylation differences between UC patients who progress in their disease course leading to treatment with anti-TNFα/immunodulators agents vs. patients who are on 5’Asa maintenance treatment. Indeed, similar differences was also observed between responders and non-responders of anti-TNFαagents. Conclusion Taken together, our preliminary results for the first time highlight the regulatory role of master regulator-mediated signalling networks involving transcription factors that regulate gene expression underpinning response to anti-TNFa in UC patients. In parallel, these results identify specific DNA methylation alterations that impact treatment response and disease severity and therefore could ultimately enact as predictive biomarkers in UC.

Hypermethylation Effects of Yiqihuoxue Decoction in Diabetic Atherosclerosis Using Genome-Wide DNA Methylation Analyses.

PurposeTo investigate if a traditional Chinese medicine formulation, called “Yiqihuoxue” (YQHX), could improve diabetic atherosclerosis (DA) and explore potential mechanisms based on DNA methylation.MethodsApolipoprotein E-knockout mice were administered streptozotocin (50 mg/d, i.p.) for 5 days and fed a high-fat diet for 16 weeks. Mice were divided randomly into DA model, rosiglitazone, as well as low-, medium-, and high-dose YQHX groups. Ten healthy C57BL/6J mice were the control group. Serum levels of fasting insulin, blood glucose, homeostasis model-insulin resistance index (HOMA-IR), serum lipids, and inflammatory factors were analyzed after the final treatment. Aorta tissues were collected for staining (hematoxylin and eosin, and Oil red O). Genomic DNA was extracted for methyl-capture sequencing (MC-seq). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases were used to analyze differentially methylated genes. Pyrosequencing was used to verify MC-seq data.ResultsLow-dose and high-dose YQHX could reduce the HOMA-IR (P < 0.05). Low-dose YQHX reduced expression of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), TNF-α, andI L-6 in serum compared with that in the model group (P < 0.05). Medium-dose YQHX decoction inhibited the expression level of TNF-α (P < 0.05). High-dose YQHX decreased the expression level of IL-6 (P < 0.05). Staining also showed the anti-atherosclerosis effects of YQHX (P < 0.05). MC-seq revealed many abnormally hypermethylated and hypomethylated genes in DA mice compared with those in the control group. KEGG database analysis showed that the hypermethylated genes induced by YQHX treatment were related to pathways in cancer, Hippo signaling, and mitogen activated protein kinase. The network analysis suggested that the hypermethylated genes epidermal growth factor receptor(Egfr) and phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) induced by YQHX treatment had important roles in DA. Pyrosequencing revealed that YQHX treatment increased methylation of AKT1, Nr1h3 and Fabp4 significantly (P < 0.05).ConclusionYQHX decoction had positive treatment effects against DA, because it could regulate aberrant hypomethylation of DNA.

PD49-04 ACCURATE DIFFERENTIATION OF PATHOLOGICAL SUBTYPES OF RENAL TUMOUR BY APPLYING A MACHINE LEARNING MODEL TO EPIGENETIC MARKERS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (PD49)1 Sep 2021PD49-04 ACCURATE DIFFERENTIATION OF PATHOLOGICAL SUBTYPES OF RENAL TUMOUR BY APPLYING A MACHINE LEARNING MODEL TO EPIGENETIC MARKERS Sabrina Rossi, Izzy Newsham, Sara Pita, Gahee Park, Radolsaw Lach, Anne Babbage, Christopher Smith, Kevin Brennan, Hong Zheng, Tom Mitchell, Anne Warren, John Leppert, Olivier Gevaert, Grant Stewart, Charles Massie, and Shamith Samarajiwa Sabrina RossiSabrina Rossi More articles by this author , Izzy NewshamIzzy Newsham More articles by this author , Sara PitaSara Pita More articles by this author , Gahee ParkGahee Park More articles by this author , Radolsaw LachRadolsaw Lach More articles by this author , Anne BabbageAnne Babbage More articles by this author , Christopher SmithChristopher Smith More articles by this author , Kevin BrennanKevin Brennan More articles by this author , Hong ZhengHong Zheng More articles by this author , Tom MitchellTom Mitchell More articles by this author , Anne WarrenAnne Warren More articles by this author , John LeppertJohn Leppert More articles by this author , Olivier GevaertOlivier Gevaert More articles by this author , Grant StewartGrant Stewart More articles by this author , Charles MassieCharles Massie More articles by this author , and Shamith SamarajiwaShamith Samarajiwa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002071.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Small renal masses (SRM) represent a diagnostic challenge despite advances in imaging and renal biopsy. Approximately 20% of SRM removed at surgery are found to be benign post-operatively. DNA methylation is an early and stable event in tumorigenesis, which reveals cell ontology. We developed and validated a machine learning model, leveraging DNA methylation analysis in >1500 patient samples, to classify pathological subtypes of renal tumours and provide a platform to improve diagnosis. METHODS: Methylation was evaluated in gDNA from fresh frozen nephrectomy specimens, using the Illumina 450k microarray and EPIC Methyl Capture sequencing platforms. We combined our own dataset and TCGA data (N=approx 158000 markers). This dataset was divided into training and testing sets, using 4-fold cross validation, and used to train an XGBoost model to classify samples into one of five pathological subtypes. External validation was carried out on two independent publicly available datasets, from fresh frozen nephrectomy tissue (Brennan et al) and ex-vivo tissue biopsies (Chopra et al). RESULTS: The integrated training and testing set consisted of 1268 samples, including: 465 ccRCC, 303 pRCC, 101 chRCC, 73 oncocytomas and 329 adjacent normal kidney. The prediction accuracy in the testing set was 0.954; with high class-wise Area Under the Receiver Operating Characteristic Curves (ROC AUCs): 0.994 (ccRCC), 0.995 (pRCC), 0.982 (chRCC), 0.999 (oncocytoma), 1 (adjacent normal). The model was externally validated on 245 ex-vivo tissue biopsies from 100 renal tumours. The accuracy was 0.824 and average class-wise ROC AUCs were: 0.978 (ccRCC), 0.946 (pRCC), 0.994 (chRCC), 0.892 (oncocytoma), 0.969 (adjacent normal). Furthermore, an independent external validation was performed on 34 tissue samples, achieving average class-wise ROC AUCs: 1 (ccRCC), 0.904 (chRCC), 0.946 (oncocytoma), 0.976 (adjacent normal). In order to assess the impact of methylation heterogeneity on our classifier, the model was evaluated in 97 multi-region samples from 18 ccRCC patients. CONCLUSIONS: A machine learning model based on DNA methylation data can differentiate pathological subtypes of renal tumours with excellent accuracy, including external validation. The model may be applied to renal biopsy samples to improve the diagnostic pathway for renal tumours. This has the potential to reduce over-treatment by preventing unnecessary surgery, enhance patient QoL and minimize healthcare costs. Source of Funding: S.H. Rossi is funded by a Cancer Research UK Doctoral Research (PhD) fellowship © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e846-e846 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sabrina Rossi More articles by this author Izzy Newsham More articles by this author Sara Pita More articles by this author Gahee Park More articles by this author Radolsaw Lach More articles by this author Anne Babbage More articles by this author Christopher Smith More articles by this author Kevin Brennan More articles by this author Hong Zheng More articles by this author Tom Mitchell More articles by this author Anne Warren More articles by this author John Leppert More articles by this author Olivier Gevaert More articles by this author Grant Stewart More articles by this author Charles Massie More articles by this author Shamith Samarajiwa More articles by this author Expand All Advertisement Loading ...

HPV-16/18 E6-induced APOBEC3B expression associates with proliferation of cervical cancer cells and hypomethylation of Cyclin D1.

Oncogenic high-risk human papillomavirus (HR-HPV) infection causes a majority of cases of cervical cancer and pre-cancerous cervical lesions. However, the mechanisms underlying the direct evolution from HPV-16/18-infected epithelium to cervical intraepithelial neoplasia (CIN) III, which can progress to cervical cancer, remain poorly identified. Here, we performed RNA-seq after laser capture microdissection, and found that APOBEC3B was highly expressed in cervical cancer specimens compared with CIN III with HPV-16/18 infection. Furthermore, immunohistochemical analysis confirmed that high levels of APOBEC3B were correlated with lymph node metastasis in cervical cancer. Subsequent experiments revealed that HPV-16 E6 could upregulate APOBEC3B through direct binding to the promoter of APOBEC3B in cervical cancer cells. Silencing of APOBEC3B by stable short hairpin RNA-mediated knockdown reduced the proliferative capacity of Caski and HeLa cells in vitro and in vivo, but had only a small effect on the migration and invasion of two cervical cancer cell lines. Finally, we identified the changes in gene expression following APOBEC3B silencing in Caski cells by microarray, demonstrating a biological link between APOBEC3B and CCND1 in cervical cancer cells. Importantly, through methyl-capture sequencing and pyrosequencing, APOBEC3B was found to affect the levels of the downstream protein Cyclin D1 (which is encoded by the CCND1 gene) through hypomethylation of the CCND1 promoter. In conclusion, our study supports HPV-16 E6-induced APOBEC3B expression associates with proliferation of cervical cancer cells and hypomethylation of Cyclin D1. Thus, APOBEC3B may be a potential therapeutic target in human cervical cancer.

Comparison of methylation capture sequencing and Infinium MethylationEPIC array in peripheral blood mononuclear cells

BackgroundEpigenome-wide association studies (EWAS) have been widely applied to identify methylation CpG sites associated with human disease. To date, the Infinium MethylationEPIC array (EPIC) is commonly used for high-throughput DNA methylation profiling. However, the EPIC array covers only 30% of the human methylome. Methylation Capture bisulfite sequencing (MC-seq) captures target regions of methylome and has advantages of extensive coverage in the methylome at an affordable price.MethodsEpigenome-wide DNA methylation in four peripheral blood mononuclear cell samples was profiled by using SureSelectXT Methyl-Seq for MC-seq and EPIC platforms separately. CpG site-based reproducibility of MC-seq was assessed with DNA sample inputs ranging in quantity of high (> 1000 ng), medium (300–1000 ng), and low (150 ng–300 ng). To compare the performance of MC-seq and the EPIC arrays, we conducted a Pearson correlation and methylation value difference at each CpG site that was detected by both MC-seq and EPIC. We compared the percentage and counts in each CpG island and gene annotation between MC-seq and the EPIC array.ResultsAfter quality control, an average of 3,708,550 CpG sites per sample were detected by MC-seq with DNA quantity > 1000 ng. Reproducibility of DNA methylation in MC-seq-detected CpG sites was high among samples with high, medium, and low DNA inputs (r > 0.96). The EPIC array captured an average of 846,464 CpG sites per sample. Compared with the EPIC array, MC-seq detected more CpGs in coding regions and CpG islands. Among the 472,540 CpG sites captured by both platforms, methylation of a majority of CpG sites was highly correlated in the same sample (r: 0.98–0.99). However, methylation for a small proportion of CpGs (N = 235) differed significantly between the two platforms, with differences in beta values of greater than 0.5.ConclusionsOur results show that MC-seq is an efficient and reliable platform for methylome profiling with a broader coverage of the methylome than the array-based platform. Although methylation measurements in majority of CpGs are highly correlated, a number of CpG sites show large discrepancy between the two platforms, which warrants further investigation and needs cautious interpretation.

Abstract P5-01-18: A non-invasive diagnostic assay of early stage breast cancer

Abstract Objective: To develop a non-invasive diagnostic assay for breast cancer. Methods: Breast cancer patients and non-cancerous donors were recruited. Cancer or benign non-cancerous tissues and peripheral blood were collected. The study was designed to have a training group and a validation group. The training group included cancer tissue and blood samples from 6 patients with breast cancer and 34 healthy donors, and the validation group including 40 patients with breast cancer and 11 with benign breast disease. Methylation capture sequencing and exome capture sequencing with an oncology panel were performed on the tissue samples. Based on these result, a liquid phase capture assay was designed to cover coding sequences of 340 genes as well as over 1000 common SNPs and more than 2000 differentially methylated sites in breast cancer tissues. NGS was performed with cfDNA from patients with breast cancer and healthy donors. Mutation abundance measurement based on population genetic statistics, methylation haplotype analysis based on machine learning model, and Gaussian decomposition based on spatial localization of nucleosomes were developed to calculate cancer specific genetic and epigenetic features. Results: We identified a major epigenetic landscape shift of breast cancer tissues from normal tissues or benign non-cancerous hyperplasia. Breast cancer tissues processed specific methylated haplotypes those absent in benign noncancerous or normal breast tissues. Prevalent nucleosomal shifts across the genome in breast cancer were identified, suggesting possible scenario of a global epigenetic switch. Using Gaussian mixture model and non-negative matrix factorization, we found a large number of breast cancer specific nucleosome spatial localization signals carried by cfDNA. Based on the uncovered epigenetic features, we trained a machine learning computational model to identify patients with breast cancer from those with benign disease and healthy donors. The model predicts nucleosomal fraction of malignancy for breast cancer originated signal. With the model, we uncovered nucleosomal spatial distribution in cfDNA associated with tumor burden and staging. In a Her2-positive early stage breast cancer patient, the tumor specific shift of nucleosome in cfDNA on a germline pathogenic mutation of ATR became unmeasurable after treatment with trastuzumab. The model archieved 100% accuracy (6/6 positive, and 34/34 negative) in the training group consisting 6 cancer pateints, 3 of which were patients with early stage (T1) breast cancer. In the validation group, the sensitivity, specificity and accuracy were 0.810 (33/40), 1.000 (11/11) and 0.862 (44/51). Conclusions: In this study, we developed a new non-invasive diagnostic assay for breast cancer based on decomposition of nucleosome spatial distribution in cfDNA sequencing. The model showed satisfactory diagnostic sensitivity and specificity in distinguishing early stage breast cancer patients from benign, non-cancerous donor. Further large cohort evaluation is necessary to confirm their potential in clinical applications. Key Words: Breast cancer; cfDNA; cancer specific nucleosome prediction; machine learning Citation Format: Wanyan Tang, Xin Zhou, Haiwei Zhang, Chuang Ge, Huiqing Yu, Weiqi Nian, Yi Zhang. A non-invasive diagnostic assay of early stage breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-18.

Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism

Reprogramming of adipocyte function in obesity is implicated in metabolic disorders like type 2 diabetes. Here, we used the pig, an animal model sharing many physiological and pathophysiological similarities with humans, to perform in-depth epigenomic and transcriptomic characterization of pure adipocyte fractions. Using a combined DNA methylation capture sequencing and Reduced Representation bisulfite sequencing (RRBS) strategy in 11 lean and 12 obese pigs, we identified in 3529 differentially methylated regions (DMRs) located at close proximity to-, or within genes in the adipocytes. By sequencing of the transcriptome from the same fraction of isolated adipocytes, we identified 276 differentially expressed transcripts with at least one or more DMR. These transcripts were over-represented in gene pathways related to MAPK, metabolic and insulin signaling. Using a candidate gene approach, we further characterized 13 genes potentially regulated by DNA methylation and identified putative transcription factor binding sites that could be affected by the differential methylation in obesity. Our data constitute a valuable resource for further investigations aiming to delineate the epigenetic etiology of metabolic disorders.