Specific CpG Sites Research Articles

Effects of endocrine disrupting chemicals, blood metabolome, and epigenetics on breast cancer risk: A multi-dimensional mendelian randomization study.

Current research on the relationship between environmental endocrine disrupting chemicals (EDCs) and breast cancer remains insufficient, with limited evidence and inconsistent conclusions. Mendelian randomization (MR) is a robust method for establishing causality, as it reduces biases from confounding factors and reverse causation. This study uses MR to investigate the effects of three types of EDCs, including bisphenols, parabens, and phthalates, on the risk of overall breast cancer and its subtypes-Luminal A, Luminal B, triple negative, human epidermal growth factor receptor 2-enriched, and estrogen receptor-positive/negative. The study also examines the 1400 blood metabolome as potential mediators and explores EDCs-associated DNA methylation changes as potential factors, with a focus on European populations. Our results shows that n-butyl paraben (n-BuP) is positively associated with Luminal A, mono-methyl phthalate is negatively associated with Luminal B, and mono-iso-butyl phthalate (MiBP) is positively associated with triple negative breast cancer (TNBC). Mediation analysis reveals that blood metabolites, such as caffeic acid sulfate and the caffeine-to-paraxanthine ratio, mediate the effect of n-BuP on Luminal A, while methylsuccinate mediate the effect of MiBP on TNBC. Epigenetic analysis shows associations between EDCs exposure-related DNA methylation changes at specific CpG sites (cg26325335, cg08537847, cg27454300) and different breast cancer risks. These findings not only suggest potential biomarkers for early detection and intervention but also underscore the imperative for further research to rigorously validate these associations.

Methylation sites of human papillomavirus 16 as potential biomarkers for cervical cancer progression

ObjectiveTo investigate the methylation levels at 13 specific sites of the human papillomavirus 16 (HPV16) L1 gene as potential biomarkers for the diagnosis of cervical cancer.MethodsSamples were collected from the gynecological outpatient and inpatient departments of the Xinjiang Uygur Autonomous Region People’s Hospital. A total of 107 women participated in this study, including 54 with cervical cancer (32 Uygur, 22 Han) and 53 with cervical inflammation (32 Uygur, 21 Han). Methylation analysis was performed using pyrosequencing to quantitatively assess methylation levels at specified CpG sites within the HPV16 L1 gene.ResultsHigh methylation levels were predominantly observed at sites 5927, 5963 and 6367 in cervical cancer cells compared with inflammatory cells. Methylation patterns exhibited no significant differences between the Han and Uygur ethnic groups but correlated with viral load and age within each group. Receiver operating characteristic curve analyses of these methylation sites indicated high diagnostic accuracy in distinguishing between high-grade lesions and less severe conditions.ConclusionsMethylation of specific CpG sites in the HPV16 L1 gene holds promise as a biomarker for cervical cancer progression. The gene locus at position 6367 has important features in the methylation pattern of cervical cancer, and high accuracy shown in diagnosis make it a potential biomarker for early diagnosis of cervical cancer.

Longitudinal DNA methylation profiles in saliva of offspring from mothers with gestational diabetes: associations with early childhood growth patterns

BackgroundThe prevalence of obesity and type 2 diabetes mellitus (T2DM) is rising globally, particularly among children exposed to adverse intrauterine environments, such as those associated with gestational diabetes mellitus (GDM). Epigenetic modifications, specifically DNA methylation, have emerged as mechanisms by which early environmental exposures can predispose offspring to metabolic diseases. This study aimed to investigate DNA methylation differences in children born to mothers with GDM compared to non-GDM mothers, using saliva samples, and to assess the association of these epigenetic patterns with early growth measurements.MethodsThis study analyzed saliva DNA methylation patterns in 30 children (15 born to GDM mothers and 15 to non-GDM mothers) from the EPIDG cohort. Samples were collected at two time points: 8–10 weeks postpartum and at one year of age. Epigenome-wide analysis of over 850,000 CpG sites was conducted using the Illumina Methylation EPIC Bead Chip. Differential methylation positions (DMPs) were identified with the limma package, using a significance threshold of p < 0.01 and delta β ≥ 5%. Correlation analysis examined associations between methylation and growth variables (weight, height, BMI and annual growth) using Spearman tests.ResultsWe identified 6,968 DMPs at the postpartum stage and 5,132 after one year, with 50 sites remaining differentially methylated over time, 16 of which maintained consistent methylation directionality. Functional analysis linked several of these DMPs to genes involved in inflammation and metabolic processes, including CYTH3 and FARP2, both implicated in growth and metabolic pathways. Significant correlations were found between specific CpG sites and growth-related variables such as weight, head circumference, height, and BMI.ConclusionsThis study’s longitudinal design reveals stable DNA methylation patterns in saliva samples that differentiate GDM-exposed children from controls across the first year of life, highlighting the feasibility of saliva as a minimally invasive biomarker source. The persistence of these epigenetic signatures underscores their potential as early indicators of metabolic risk, offering valuable insights into the long-term impact of maternal GDM on child health. Although the use of saliva offers a practical and non-invasive tool for pediatric epigenetic research, further studies are necessary to validate these findings in larger populations.

CYP24A1 DNA Methylation in Colorectal Cancer as Potential Prognostic and Predictive Markers.

The DNA methylation of CYP24A1 can regulate its gene expression and may play a role in the occurrence and progression of colorectal cancer (CRC). However, the association between CYP24A1 DNA methylation and the prognosis of CRC patients has not yet been reported. In this study, differential methylation analysis was conducted in both blood and tissue cohorts, and differential expression analysis was performed in the tissue cohort with in vitro validation. GO and KEGG enrichment analyses were performed on CYP24A1-related genes. A correlation between CYP24A1 promoter methylation and its gene expression was explored. Kaplan-Meier survival and Cox regression analyses were performed to investigate the impact of CYP24A1 DNA methylation on the prognosis of CRC patients. Prognostic risk scores were constructed for survival prediction. Immune infiltration analysis was also conducted. Our results showed that the hypermethylation of cg02712555 in tumor tissues (hazard ratio, 0.48; 95% confidence interval, 0.24-0.94; p = 0.032) and CpG site 41 in peripheral leukocytes (HR, 0.35; 95%CI, 0.14-0.84; p = 0.019) were both associated with decreased overall mortality in CRC patients. Prognostic risk scores showed robust predictive capabilities of these two CpG loci for the prognosis of CRC patients. CYP24A1 hypermethylation was positively correlated with infiltration levels of activated CD4 + T cells, activated CD8 + T cells, activated B cells, activated dendritic cells, and macrophages. Taken together, our findings indicate that the methylation levels of specific CpG sites within the CYP24A1 promoter region in blood leukocytes and tumors are potential prognostic and predictive markers for overall survival in CRC patients.

Hypomethylation of the low-density lipoprotein receptor class A domain containing 4 gene in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease linked to epigenetic changes, particularly DNA methylation. While LDLRAD4 has been implicated in RA through GWAS, its role in RA via methylation remains unclear. To investigate LDLRAD4 methylation patterns in RA and evaluate its potential as a diagnostic and inflammatory biomarker. We assessed DNA methylation at specific CpG sites within LDLRAD4 in 150 RA patients and 150 healthy controls. Clinical data, including disease duration and inflammatory markers, were collected. RA patients showed significant hypomethylation of LDLRAD4, especially in the LDLRAD4-43 and LDLRAD4-44 regions. ROC analysis yielded an AUC of 0.841, indicating strong diagnostic potential. Methylation levels correlated negatively with ESR, CRP and DAS28 in the RF+/CCP- subgroup. LDLRAD4 DNA present hypomethylation in rheumatoid arthritis, and methylation levels are correlated with inflammatory indicator, possibly via TGF-β signaling. Further research is needed to explore its therapeutic potential.

Peripheral Blood ABCG1 Gene DNA Methylation: Mediating the Relationship between Dietary Intake of Methyl Donor Nutrients and Stroke Risk

Dysregulation of methyl donor nutrients interferes with DNA methylation and is associated with neurological diseases. ABCG1 gene regulates cholesterol to HDL-C, maintains lipid homeostasis, and has been linked to both methyl nutrition and neurological risks. The aim was to investigate whether there is an effect of ABCG1 DNA methylation on the relationship between intake of methyl donor nutrients and the risk of stroke occurrence. We hypothesize that the intake of methyl donor nutrients may influence stroke occurrence by modulating the methylation status of ABCG1. This study utilized a case-control design and selected 52 stroke patients along with 52 healthy controls from Northwest China. Dietary information was collected using a FFQ, and methylation levels were measured at 29 CpG sites of the ABCG1 gene. A significant linear trend was found between dietary intake of the methyl donor nutrient choline and CpG_19.20 methylation of the ABCG1 gene (β = -0.037, P = 0.033). Additionally, a significant association was observed between CpG_19.20 methylation and the risk of stroke (OR 2.325, 95% CI 1.210 - 4.466). Mediation analysis revealed that choline intake indirectly influenced stroke occurrence through its effect on CpG_19.20 methylation levels in the ABCG1 gene (β = -0.015, SE = 0.013, 95% CI = [-0.053, -0.001]). We found that DNA methylation at specific CpG sites of the peripheral blood ABCG1 gene mediates the association between dietary methyl donor nutrient intake and stroke risk in an adult population from Northwest China. New insights are provided on the prevention and treatment of stroke.

Biological Age Acceleration and Colonic Polyps in Persons under Age 50.

Epigenetic clocks can quantify DNA methylation by measuring the methylation levels at specific sites in the genome, which correlate with biological age (BA). Accelerated aging, where BA exceeds chronologic age, has been studied in relation to cancer development, but its utility in cancer prevention remains unclear. Accelerated aging holds promise as a tool to explain the increase in early-onset colorectal cancer (EOCRC). We investigate the association of accelerated aging and the presence of preneoplastic polyps (PNP) in the colon, defined as tubular adenomas and sessile serrated adenomas. In this study of persons under age 50 undergoing colonoscopy, we used peripheral blood samples to determine BA and age acceleration metrics. Age acceleration was determined by interrogating DNA methylation at specific CpG sites across the genome, which has been shown to correlate with age. We then conducted logistic regression analyses to evaluate the association between age acceleration and PNPs. In total, 51 patient samples were evaluated. We found that that the odds of harboring a PNP are 16% higher with 1 year of accelerated aging, as measured by GrimAge. However, the strongest risk factor for PNPs remained male sex. This represents one of the first studies to explore accelerated aging and PNP in patients under the age of 50. A risk-stratified approach to EOCRC screening would minimize unnecessary colonoscopies and minimize healthcare burden while addressing the increase in EOCRC. Our findings suggest that BA calculations with peripheral blood collections could be an important component of such a risk model. Prevention Relevance: Understanding the association of accelerated aging and colorectal PNPs presents an opportunity to develop a risk-stratified approach to colorectal cancer screening in young persons.

Evaluation of ALKBH2 and ALKBH3 gene regulation in patients with adult T-cell leukemia/lymphoma

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) is an oncogenic virus that causes malignant adult T-cell leukemia/lymphoma (ATL). Patients infected with HTLV-1 are considered HTLV-1 carriers, and a small proportion of patients progress to life-threatening ATL after a long asymptomatic phase. No antiviral agent or preventive vaccine specific for HTLV-1 infection is established in current situation. For development of countermeasures to combat HTLV-1 infection and ATL, it is essential to expand our knowledge about their pathogenesis. Recently, AlkB homolog (ALKBH) family have been shown to participate in the oncogenesis of various cancer types.MethodsTo investigate the potential role of ALKBH family members in the pathogenesis of ATL, we analyzed their gene expression dynamics in HTLV-1-infected T-cell lines and peripheral blood mononuclear cell-derived clinical specimens obtained from asymptomatic HTLV-1 carriers and patients with acute-type ATL. Epigenetic analysis was performed to dissect the mechanisms of ALKBH3 gene regulation using cultivated cells and a public dataset.ResultsThe mRNA expression levels of ALKBH2 and ALKBH3 were significantly or suggestively decreased in asymptomatic HTLV-1 carriers, but reverted in acute-type ATL patients, correlating with HTLV-1 basic leucine zipper factor gene expression. Intriguingly, the pre-mRNA expression of ALKBH2 and ALKBH3 was significantly suppressed in patients infected with HTLV-1, but not in healthy controls. Epigenetic analysis was performed to dissect the mechanisms of ALKBH3 gene regulation. In vitro analysis suggested a possible relationship between DNA methylation and ALKBH3 gene expression. Investigation of a public dataset revealed that specific CpG sites exhibited characteristically regulated methylation states in HTLV-1-infected T-cell subsets.ConclusionWe discovered dynamically regulated patterns of ALKBH2 and ALKBH3 gene expression in patients infected with HTLV-1, and specific CpG sites epigenetically regulated by HTLV-1 infection. This study provides novel insights into HTLV-1 infection and contributes to the elucidation of ATL pathogenesis.

Potential role of TWIST1 and its methylation in bladder urothelial carcinoma.

Bladder urothelial carcinoma (BLCA), like other cancers, is strongly associated with genetic and epigenetic changes. TWIST1 is an epithelial-mesenchymal transition (EMT) promoter that has been linked to the development of many malignancies. It is still unclear, however, what role TWIST1 plays in BLCA, and the relationship between TWIST1 transcript levels and its promoter methylation and immune infiltration has been reported even less. This study aimed to reveal the potential role of TWIST1 promoter methylation-related changes in BLCA. Transcriptional expression data of TWIST1 in BLCA were acquired from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and University of Alabama at Birmingham (UALCAN) databases. TWIST1 methylation levels and prognosis were sourced from Gene Expression Profiling Interactive Analysis 2 (GEPIA2), LinkedOmics, cBio Cancer Genomics Portal (c-BioPortal), MethSurv, and DNA Methylation Information Visualization Database (DNMIVD) databases. The methylation status of the BLCA-associated TWIST1 in preoperative and postoperative urinary exfoliated cells was subsequently analyzed using methylation-specific real-time fluorescence polymerase chain reaction, with validation of accuracy through pyrophosphate sequencing. Finally, from the Gene Set Cancer Analysis (GSCA) and Tumor-Immune System Interaction Database (TISIDB) databases, we obtained the association between TWIST1 transcript expression and DNA methylation and cancer immune infiltration and immunolabelling. Our study demonstrated that TWIST1 expression was down-regulated in BLCA, which was negatively correlated with DNA methylation. The association between TWIST1 promoter hypermethylation and the progression, staging, grading, and recurrence of BLCA is highly significant. Furthermore, we revealed that hypermethylation of both the preoperative and postoperative TWIST1 promoters is useful as a biomarker for monitoring BLCA recurrence, particularly when considering the methylation status of specific CpG sites. Additionally, we observed that TWIST1 expression, promoter methylation, and immune infiltration immunoreactive markers correlated significantly in BLCA. We propose that TWIST1 holds great promise as a diagnostic and therapeutic target for BLCA, with the potential to influence tumor progression and patient prognosis through the regulation of immune cell infiltration. We hope these findings contribute valuable insights to the field of BLCA research.

Tissue-Specific DNA Methylation Changes in CD8+ T Cells During Chronic Simian Immunodeficiency Virus Infection of Infant Rhesus Macaques.

Robust CD8+ T cell responses are critical for the control of HIV infection in both adults and children. Our understanding of the mechanisms driving these responses is based largely on studies of cells circulating in peripheral blood in adults, but the regulation of CD8+ T cell responses in tissue sites is poorly understood, particularly in pediatric infections. DNA methylation is an epigenetic modification that regulates gene transcription. Hypermethylated gene promoters are associated with transcriptional silencing and, conversely, hypomethylated promoters indicate gene activation. In this study, we evaluated DNA methylation signatures of CD8+ T cells isolated from several different anatomic compartments during pediatric AIDS-virus infection by utilizing the SIVmac239/251 infected infant rhesus macaque model. We performed a stepwise methylation analysis starting with total cellular DNA, to immunomodulatory cytokine promoters, to specific CpG sites within the cytokine promoters in CD8+ T cells isolated from peripheral blood, lymph nodes, and intestinal tissue during the chronic phase of infection. Tissue-specific methylation patterns were determined for transcriptionally active promoters of key immunomodulatory cytokines: interferon gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFα). In this study, we observed tissue-specific differences in CD8+ T cell modulation by DNA methylation in SIV-infected infant macaques, highlighting the importance of evaluating cells from both blood and tissues to obtain a complete picture of CD8+ T cell regulation during pediatric HIV infection.

Sex-specific DNA methylation differences in Amyotrophic lateral sclerosis.

Sex is an important covariate in all genetic and epigenetic research due to its role in the incidence, progression and outcome of many phenotypic characteristics and human diseases. Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a sex bias towards higher incidence in males. Here, we report for the first time a blood-based epigenome-wide association study meta-analysis in 9274 individuals after stringent quality control (5529 males and 3975 females). We identified a total of 226 ALS saDMPs (sex-associated DMPs) annotated to a total of 159 unique genes. These ALS saDMPs were depleted at transposable elements yet significantly enriched at enhancers and slightly enriched at 3'UTRs. These ALS saDMPs were enriched for transcription factor motifs such as ESR1 and REST. Moreover, we identified an additional 10 genes associated with ALS saDMPs through chromatin loop interactions, suggesting a potential regulatory role for these saDMPs on distant genes. Furthermore, we investigated the relationship between DNA methylation at specific CpG sites and overall survival in ALS using Cox proportional hazards models. We identified two ALS saDMPs, cg14380013 and cg06729676, that showed significant associations with survival. Overall, our study reports a reliable catalogue of sex-associated ALS saDMPs in ALS and elucidates several characteristics of these sites using a large-scale dataset. This resource will benefit future studies aiming to investigate the role of sex in the incidence, progression and risk for ALS.

Longitudinal multimodal profiling of IDH-wildtype glioblastoma reveals the molecular evolution and cellular phenotypes underlying prognostically different treatment responses.

Despite recent advances in the biology of IDH-wildtype glioblastoma, it remains a devastating disease with median survival of less than 2years. However, the molecular underpinnings of the heterogeneous response to the current standard-of-care treatment regimen consisting of maximal safe resection, adjuvant radiation, and chemotherapy with temozolomide remain unknown. Comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent glioblastoma specimens from 106 patients was performed to investigate the molecular evolution and cellular phenotypes underlying differential treatment responses. While TERT promoter mutation and CDKN2A homozygous deletion were early events during gliomagenesis shared by initial and recurrent tumors, most other recurrent genetic alterations (eg, EGFR, PTEN, and NF1) were commonly private to initial or recurrent tumors indicating acquisition later during clonal evolution. Furthermore, glioblastomas exhibited heterogeneous epigenomic evolution with subsets becoming more globally hypermethylated, hypomethylated, or remaining stable. Glioblastoma that underwent sarcomatous transformation had shorter interval to recurrence and were significantly enriched in NF1, TP53, and RB1 alterations and the mesenchymal epigenetic class. Patients who developed somatic hypermutation following temozolomide treatment had significantly longer interval to disease recurrence and prolonged overall survival, and increased methylation at 4 specific CpG sites in the promoter region of MGMT was significantly associated with this development of hypermutation. Finally, an epigenomic evolution signature incorporating change in DNA methylation levels across 347 critical CpG sites was developed that significantly correlated with clinical outcomes. Glioblastoma undergoes heterogeneous genetic, epigenetic, and cellular evolution that underlies prognostically different treatment responses.

Abstract A035: A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA

Abstract Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics with methylation emerging as a reliable biomarker for low levels of circulating tumor DNA (ctDNA). There is a rising interest in sensitive, low-cost liquid biopsy assays to detect low copies of hypermethylated ctDNA in an excess background of non-cancer-associated hypomethylated cell- free DNA (cfDNA). Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of low abundance methylated fragments. qMSP approaches have previously been developed but are predominantly designed to target a specific CpG site. To date we are not aware of any qMSP approaches that target pan-cancer-associated methylation patterns across multiple CpG sites. Harbinger Health has developed a qMSP method with locked nucleic acid (LNA) blockers that target methylation haplotypes, allowing for multiplex enrichment and quantification of cancer-specific methylation patterns. Using targeted bisulfite sequencing, we identified ten pan-cancer-informative CpG-dense regions which showed consistent contiguous elevated methylation states in cancer cfDNA and low levels of methylation in non-cancer cfDNA. We designed qMSP assays for these regions and LNA blockers which bind to unmethylated target CpG sites, outcompeting the hydrolysis probe through a high Tm differential. This prevented non-specific probe hybridization. The qMSP-LNA assays were evaluated in simplex and multiplex using cell line DNA in a titration of hypermethylated “ctDNA” spiked into hypomethylated “cfDNA” to simulate varying ctDNA levels, and then validated with cancer and non-cancer cfDNA samples at 5 ng input, equivalent to 1450 haploid copies. The percentage of methylated DNA was estimated by the ratio of methylated copies (qMSP-LNA) to total copies (internal reference), which were quantified by standard curves. The qMSP-LNA assays detected low abundance methylated DNA fragments down to 23 target copies. LNA blockers completely depleted background signal originating from unmethylated DNA with high specificity, showing no inhibition of methylated DNA detection. When tested in cfDNA samples which varied in conversion efficiency, discordant methylation states, and methylation abundance, the assays reproducibly amplified and detected highly methylated fragments which correspond to cancer signal. Harbinger Health has developed qMSP-LNA assays for contiguous CpG biomarkers which differentiate cancer and non-cancer samples from only ten pan-cancer-informative regions. The qMSP-LNA method detected low abundance fragments with specific methylation patterns with high specificity and sensitivity. The assay reproducibly detected methylated DNA in cfDNA samples that represent a clinically relevant range of methylation signal and will be further validated in a larger cohort of cancer and non-cancer cfDNA samples. Due to the high specificity and sensitivity, this cost-effective PCR method may provide utility and improve access for early cancer detection and monitoring. Citation Format: Emily Neaga, Caitlin Gilley, Sarah Falotico, Mayur Gurnani, Zhenyu Zhang, Jocelyn Charlton, Miguel Williams, Anthony Shuber. A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A035.

Abstract 4137257: Hepatocyte growth factor -mediated Angiogenesis supersession in Hyperhomocysteinemia

Background: Homocysteine (Hcy) is a well-established cardiovascular risk factor. Elevated homocysteine, or hyperhomocysteinemia (HHcy), inhibit the growth of endothelial cells and stimulate vascular smooth muscle cell proliferation. However, the mechanisms of HHcy disrupts angiogenesis remain unclear. Here we examine the molecular pathways involved in HHcy-impaired angiogenesis. Method: We generated Tg-hCBS+ Cbs-/- mice by crossing Cbs knockout mice and Tg-hCBS mice where human CBS cDNA was regulated by a zinc-inducible metallothionein promoter. The model exhibited severe HHcy (＞115 μmol/L). We conducted both in vivo and in vitro experiments, including vasculogenesis assays of retinal and heart tissues, aortic ring assay, and tube formation. Retinas were stained with Isolectin B4 and imaged using a confocal microscope. The Human Angiogenesis RT 2 Profiler™ PCR Array was used to profile 84 key angiogenesis-related genes. Homocysteine metabolites in Cbs mice were measured by HPLC. ChIP-seq data were analyzed using Chip Atlas. Additionally, 91 patients went through coronary angiography were enrolled from the Second Affiliated Hospital of Nanchang University. Participants with CTFC, greater than 27 frames per second were identified as having slow coronary arteries flow (SCF). Results: HHcy caused SCF in patients and impaired vasculogenesis in the retinas of newborn and 12-week-old Tg-hCBS Cbs-/- mice. Capillary density in the hearts of adult Tg-hCBS Cbs-/- mice was reduced. HHcy suppressed tube formation and aortic ring microvessel growth in a concentration-dependent manner. The PCR array revealed significant gene expression changes in Hcy treated human aortic endothelial cells. Notably, genes encoding hepatocyte growth factor (HGF), collagen type IV alpha 3, epiregulin, interferons, and leukocyte cell-derived chemotaxin 1 were downregulated by at least four-fold. In an acute myocardial infarction model, HGF expression was further reduced, and HGF treatment rescued Hcy-inhibited tube formation and microvessel growth. Hcy and S-adenosylhomocysteine levels were negatively correlated with plasma HGF levels. ChIP-seq data identified homocysteine-responsive hypomethylation in ETF, TFII, FOXP3, RXRa, and c-Jun Hcy-hypomethylation Box (-2701/0). Conclusion: HHcy impair angiogenesis via HGF inhibition, which has a rescue effect. Hypomethylation caused by Hcy affects the methylation status of specific CpG sites within the Hcy-hypomethylation box of the HGF gene.

PATH-30. CLINICAL OUTCOMES AND PREDICTIVE BIOMARKERS FOR IDH-WILDTYPE GLIOBLASTOMAS DEVELOPING HYPERMUTATION FOLLOWING TEMOZOLOMIDE TREATMENT

Abstract Current standard of care for IDH-wildtype glioblastoma (GBM) includes maximal safe resection, adjuvant radiation, and chemotherapy with the alkylating agent temozolomide. A subset of gliomas treated with temozolomide develop somatic hypermutation with a predominance of C&amp;gt;T transitions due to the alkylation effects on DNA. However, the frequency of occurrence, clinical ramifications, and predictive biomarkers for developing temozolomide-induced hypermutation (TMZ-HM) in GBM are unknown. Here we performed comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent GBM from 106 patients. Seven patients (7%) developed hypermutation at time of first recurrence, while an additional five patients (5%) developed hypermutation at a subsequent recurrence. Patients who developed TMZ-HM had a significantly longer interval between initial surgery and first recurrence (27.8 vs. 9.9 months, p&amp;lt;0.001). In contrast to IDH-mutant gliomas where TMZ-HM is associated with poor prognosis, those patients with IDH-wildtype GBM who developed TMZ-HM had significantly longer overall survival from both initial surgery (67.9 vs. 20.3 months, p&amp;lt;0.001) and from time of recurrence after development of hypermutation (30.9 vs. 9.5 months, p=0.001). There was no difference in oncogenic alteration frequency in initial GBM that subsequently developed TMZ-HM. However, 4 specific CpG sites out of 12 total interrogated sites in the promoter region of MGMT (a DNA repair enzyme) and 5 specific CpG sites in the promoter region of KCNQ1DN (a negative regulator of c-Myc) were significantly more hypermethylated in those GBM that developed TMZ-HM. We then generated a weighted risk score across these CpG sites that can be used to prospectively identify GBM likely to develop TMZ-HM associated with favorable survival. In summary, patients with GBM that develop TMZ-HM follow a more favorable clinical course, and specific MGMT and KCNQ1DN promoter hypermethylation patterns could serve as a biomarker to identify these patients who may potentially benefit from extended alkylating chemotherapy treatment.

The impact of site-specific DNA methylation in KCNJ11 promoter on type 2 diabetes

AimsThis study explores the correlation between site-specific methylation levels of the KCNJ11 promoter and type 2 diabetes mellitus (T2DM), analyzing potential molecular mechanisms. MethodsThirty patients newly diagnosed with T2DM and 30 healthy controls were selected to determine the CpG methylation levels in the promoter region of the KCNJ11 gene using the bisulfite assay. The online software JASPAR was used to predict transcription factors binding to differentially methylated sites. Key transcription factors were further validated through quantitative PCR (q-PCR) and chromatin immunoprecipitation followed by PCR (ChIP-PCR). ResultsMethylation at multiple CpG sites within the KCNJ11 gene promoter was generally reduced in newly diagnosed T2DM patients compared with healthy individuals. The methylation status of CpG-471, a site crucial for the binding of the transcription factor TCF12, emerged as potentially influential in T2DM pathogenesis. This reduction in methylation at CpG-471 may enhance TCF12 binding, thereby altering KCNJ11 expression. ConclusionHypomethylation of specific CpG sites in the promoter region of the KCNJ11 gene in patients with incipient T2DM potentially contributes to the disease's pathogenesis. This hypomethylation may influence TCF12 binding, with potential regulatory effects on KCNJ11 expression and pancreatic beta-cell function, though further studies are needed to confirm the exact mechanisms involved.

Maternal e-cigarette exposure alters DNA methylome, site-specific CpG and CH methylation, and transcriptomic signatures in the neonatal brain

Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development, potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis. Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure, underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life.

Integrating Multi-Omics Data to Uncover Prostate Tissue DNA Methylation Biomarkers and Target Genes for Prostate Cancer Risk.

Previous studies have indicated that specific CpG sites may be linked to the risk of prostate cancer (PCa) by regulating the expression of PCa target genes. However, most existing studies aim to identify DNA methylation (DNAm) biomarkers through blood tissue genetic instruments, which impedes the identification of relevant biomarkers in prostate tissue. To identify PCa risk-associated CpG sites in prostate tissue, we established genetic prediction models of DNAm levels using data from normal prostate samples in the GTEx (N = 108) and assessed associations between genetically predicted DNAm in prostate and PCa risk by studying 122,188 cases and 604,640 controls. We observed significant associations for 3879 CpG sites, including 926 at novel genomic loci. Among them, DNAm levels of 80 CpG sites located at novel loci are significantly associated with expression levels of 45 neighboring genes in normal prostate tissue. Of these genes, 11 further exhibit significant associations with PCa risk for their predicted expression levels in prostate tissue. Intriguingly, a total of 31 CpG sites demonstrate consistent association patterns across the methylation-gene expression-PCa risk pathway. Our findings suggest that specific CpG sites may be related to PCa risk by modulating the expression of nearby target genes.

Epigenetic Age Estimation by Detecting DNA Methylation Status in Buccal Swabs.

The analysis of DNA methylation (DNAm) levels at specific CpG sites represents one of the most promising molecular techniques for estimating an individual's age. To date, a considerable number of studies have reported the development of age prediction models on the basis of DNAm in body fluids, with only a few utilizing buccal swabs. The objective of this study was to identify age-dependent methylation CpG sites in three different genes (HOXC4, TRIM59, and ELOVL2) in buccal swab samples from the Chinese Han population. A total of 461 buccal swabs, with an age range of 0.4-80.8 years, were divided into a training set (n=325) and a validation set (n=136). Samples were analyzed by pyrosequencing in order to identify age-related genes with correlation coefficient. A random forest regression model was ultimately proposed, including eight CpGs in three genes, with a mean absolute error (MAE) of 2.119 years. The model performs independent validation set with an MAE of 4.391 years. Our findings illustrate that buccal swabs present a suitable alternative to biological traces for age prediction based on DNAm pattern using pyrosequencing and random forest regression, offering the additional advantage of being collected noninvasively.

Applicability of epigenetic age models to next-generation methylation arrays

BackgroundEpigenetic clocks are mathematical models used to estimate epigenetic age based on DNA methylation at specific CpG sites. As new methylation microarrays are developed and older models discontinued, existing epigenetic clocks might become obsolete. Here, we explored the effects of the changes introduced in the new EPICv2 DNA methylation array on existing epigenetic clocks.MethodsWe tested the performance of four epigenetic clocks on the probeset of the EPICv2 array using a dataset of 10,835 samples. We developed a new epigenetic age prediction model compatible across the 450 k, EPICv1, and EPICv2 microarrays and validated it on 2095 samples. We estimated technical noise and intra-subject variation using two datasets with repeated sampling. We used data from (i) cancer survivors who had undergone different therapies, (ii) breast cancer patients and controls, and (iii) an exercise-based interventional study, to test the ability of our model to detect alterations in epigenetic age acceleration in response to theoretically antiaging interventions.ResultsThe results of the four epiclocks tested are significantly distorted by the EPICv2 probeset, causing an average difference of up to 25 years. Our new model produced highly accurate chronological age predictions, comparable to a state-of-the-art epiclock. The model reported the lowest epigenetic age acceleration in normal populations, as well as the lowest variation across technical replicates and repeated samples from the same subjects. Finally, our model reproduced previous results of increased epigenetic age acceleration in cancer patients and in survivors treated with radiation therapy, and no changes from exercise-based interventions.ConclusionExisting epigenetic clocks require updates for full EPICv2 compatibility. Our new model translates the capabilities of state-of-the-art epigenetic clocks to the EPICv2 platform and is cross-compatible with older microarrays. The characterization of epigenetic age prediction variation provides useful metrics to contextualize the relevance of epigenetic age alterations. The analysis of data from subjects influenced by radiation, cancer, and exercise-based interventions shows that despite being good predictors of chronological age, neither a pathological state like breast cancer, a hazardous environmental factor (radiation), nor exercise (a beneficial intervention) caused significant changes in the values of the “epigenetic age” determined by these first-generation models.