Altered Methylation Research Articles

Systematic pan-cancer analysis of RNF186 with potential implications in progression and prognosis in human cancer

AimsCancer remains a significant global public health issue. There is growing proof that Ring Finger Protein 186 (RNF186) may play a function in pan-cancer, however, this has not yet been thoroughly determined. This study aims to analyze RNF186 with potential implications in progression and prognosis in human cancer. Materials and methodsA comprehensive bioinformatics approaches combined with experimental verification were used across 33 types of cancers in this study to conduct a pan-cancer investigation of RNF186 from the perspectives of gene expression, prognosis, genomic alterations, immunological markers, gene set, and function. Key findingsRNF186 is a valuable prognostic biomarker in several cancer types, especially breast invasive carcinoma (BRCA) and uterine corpus endometrial carcinoma (UCEC). The levels of RNF186 promoter methylation and genetic alterations may be responsible for some cancers' abnormal expression. Furthermore, RNF186 expression was determined to be associated with immune checkpoint genes. Analysis of RNF186-related genes revealed that proteasome and PI3K-AKT signaling pathway were primarily involved in the cellular function of RNF186. Additionally, our research first confirmed that RNF186 may function as an oncogene and contribute to cancer proliferation, migration and invasion in UCEC. In contrast, RNF186 may play an inhibitory role in BRCA progression. This function depends on the ligase activity of RNF186. SignificanceThis study suggests that RNF186 is a novel critical target for tumor progression in BRCA and UCEC. It reveals that RNF186 may be associated with tumor immunotherapy, which may provide an effective predictive evaluation of the prognosis of immunotherapy.

Identification of DNA methylation biomarkers for evaluating cardiovascular disease risk from epigenome profiles altered by low-dose ionizing radiation

BackgroundEnvironmental exposure, medical diagnostic and therapeutic applications, and industrial utilization of radionuclides have prompted a growing focus on the risks associated with low-dose radiation (< 100 mGy). Current evidence suggests that such radiation can induce epigenetic changes. Nevertheless, whether exposure to low-dose radiation can disrupt endothelial cell function at the molecular level is unclear. Because endothelial cells play crucial roles in cardiovascular health and disease, we aimed to investigate whether low-dose radiation could lead to differential DNA methylation patterns at the genomic level in endothelial cell (EC) lines.MethodsWe screened for changes in DNA methylation patterns in primary human aortic (HAECs) and coronary artery endothelial cells following exposure to low-dose ionizing radiation. Using a subset of genes altered via DNA methylation by low-dose irradiation, we performed gene ontology (GO) analysis to predict the possible biological network mediating the effect of low-dose radiation. In addition, we performed comprehensive validation using methylation and gene expression analyses, and ChIP assay to identify useful biomarkers among candidate genes for use in detecting low-dose radiation exposure in human primary normal ECs.ResultsLow-dose radiation is sufficient to induce global DNA methylation alterations in normal EC lines. GO analysis demonstrated that these hyper- or hypo-methylated genes were linked to diverse biological pathways. Our findings indicated a robust correlation between promoter hypermethylation and transcriptional downregulation of four genes (PGRMC1, UNC119B, RERE, and FNDC3B) in response to low-dose ionizing radiation in HAECs.ConclusionsBased on these findings, the identified genes can serve as potential DNA methylation biomarkers for the assessment of cardiovascular risk upon exposure to low-dose radiation.

Abstract IA023: Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer

Abstract Phenotypic plasticity is thought to assist immune evasion, metastasis, and therapeutic resistance in tumor evolution. Epigenetic mechanisms such as DNA methylation provide for a more easily acquired and flexible, yet semi-stable adaptation by the tumor cell than hardwired genetic changes. Cancer-associated gain of DNA methylation at promoter CpG islands or distal regulatory elements is associated with transcriptional silencing, whereas loss of DNA methylation is observed at late-replicating, lamina-attached regions of the genome, tracking with cumulative cell divisions. One of the most well-documented genomic features predisposing to DNA hypermethylation is Polycomb Repressive Complex (PRC) occupancy in precursor cells, consistent with an epigenetic block to cellular differentiation as an early or predisposing event in carcinogenesis. A central question is whether this epigenetic switch from reversible PRC repression to more stable DNA hypermethylation occurs before onset of clonal expansion, or whether it represents an ongoing process during tumor evolution. Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. We have developed a tumor processing pipeline to deeply and comprehensively map DNA methylation patterns in primary human colorectal cancer (CRC) and adjacent tissues, while preserving spatial information, enabling inference of the temporal order of molecular alterations. We employ a suite of technologies to map the spatial heterogeneity of DNA methylation patterns in primary human CRC. These include a deep single-cell whole-genome bisulfite sequencing technique that improves genomic coverage several-fold over existing published protocols, providing DNA methylation measurements at the majority of CpGs in the genome in individual tumor cells. We have also calibrated and validated an epigenetic clock that allows us to accurately measure the relative replicative history of individual cells. We infer phylogenetic relationships among subclones, and relate these to their spatial distribution in the primary tumor. We find that some tumors have high proportions of PRC-associated clonal hypermethylation, indicating that these hypermethylation events were already present prior to clonal expansion, possibly contributing to susceptibility to malignant transformation in the cell-of-origin, whereas other tumors appear to have active PRC-associated CpG island hypermethylation ongoing during tumor expansion, generating subclonal heterogeneity, which may influence post-therapeutic recurrence potential. Citation Format: Peter W. Laird, Nathan J. Spix, Hsiao-yun Milliron, Manpreet Kalkat, Emily Eugster, David Sokol, Emily Jung, Paula Nolte, Kelly K. Krzyzanowski, Toshinori Hinoue, Hui Shen. Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA023.

Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Investigating FGFR2 gene as a blood-based epigenetic biomarker in gastric cancer.

Gastric adenocarcinoma is a prevalent form of cancer that often remains undetected in its early stages due to the lack of specific symptoms. This delayed diagnosis leads to poor clinical outcomes, underscoring the need for an effective and non-invasive method for early detection. Recent advances in cancer epigenetics have led to the identification of biomarkers that have the potential to revolutionize the early detection and monitoring of this disease. One such promising biomarker is the methylation of the FGFR2 promoter. This study aims to measure the methylation levels of a specific CpG site in the FGFR2 promoter gene in DNA extracted from blood leukocytes from patients with intestinal metaplasia, gastric cancer, and healthy control. The CpG site of the FGFR2 gene promoter was identified in its control region. Methylation alteration of the selected FGFR2 CpG site was determined through the (methylation-sensitive restriction enzyme) MSRE-qPCR. Genomic DNA was extracted from one hundred twenty-five participants. The normal group had mean methylation levels of 93.23 ± 4.929%, while the IM group had a level of 69.85 ± 27.15%. In GC patients, the levels varied, with 25.96 ± 18.98% in the intestinal type and 28.30 ± 16.07% in the diffuse type. The methylation levels in the IM and GC patients were significantly lower than those in the normal control group. However, no significant difference was observed between the methylation status of the intestinal type of GC and the diffuse type. The Receiver operating characteristic (ROC) curve analysis showed that FGFR2 CpG methylation levels in GC patients compared to normal controls had a high sensitivity of 100% and specificity of 100%, with a cut-off of < 74.25%; when GC patients were compared to IM patients, the sensitivity was 85%, and the specificity was 80%, with a cut-off < 44.45%. The potential of the FGFR2 methylation status as a non-invasive biomarker lies in its ability to be detected in blood leukocytes, which makes it a promising tool for the early detection of intestinal metaplasia and gastric cancer. This could significantly improve the detection and management of these gastric conditions.

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.

Genome-wide DNA methylation and transcriptomic patterns of precancerous gastric cardia lesions.

Intestinal metaplasia (IM) and intraepithelial neoplasia (IEN) are considered precursors of gastric cardia cancer (GCC). Here, we investigated the histopathologic and molecular profiles of precancerous gastric cardia lesions (PGCLs) and biomarkers for risk stratification of gastric cardia IM. We conducted a hospital-based evaluation (n = 4578) for PGCL profiles in high-incidence and non-high-incidence regions for GCC in China. We next performed 850K methylation arrays (n = 42) and RNA-seq (n = 44) in tissues with PGCLs. We then examined the protein expression of candidate biomarker using immunohistochemistry. Of the 4578 participants, 791 were diagnosed with PGCLs (600 IM, 62 IM with IEN, and 129 IEN). We found that individuals from high-incidence regions (26.7%) were more likely to develop PGCLs than those from non-high-incidence areas (13.5%). DNA methylation and gene expression alterations, indicated by differentially methylated probes (DMPs) and differentially expressed genes (DEGs), exhibited a progressive increase from type I IM (DMP = 210, DEG = 24), type II IM (DMP = 3402, DEG = 129), to type III IM (DMP = 3735, DEG = 328), peaking in IEN (DMP = 47 373, DEG = 2278). Three DEGs with aberrant promoter methylation were identified, shared exclusively by type III IM and IEN. Of these DEGs, we found that OLFM4 expression appears in IMs and increases remarkably in IENs (P < .001). We highlight that type III IM and IEN share similar epigenetic and transcriptional features in gastric cardia and propose biomarkers with potential utility in risk prediction.

Association of GAL-8 promoter methylation levels with coronary plaque inflammation

Background and aimsCoronary heart disease (CHD) is a condition that carries a high risk of mortality and is associated with aging. CHD is characterized by the chronic inflammatory response of the coronary intima. Recent studies have shown that the methylation level of blood mononuclear cell DNA is closely associated with adverse events in CHD, but the roles and mechanisms of DNA methylation in CHD remain elusive. Methods and resultsIn this study, the DNA methylation status within the epigenome of human coronary tissue in the sudden coronary death (SCD) group and control (CON) group of coronary heart disease was analyzed using the Illumina® Infinium Methylation EPIC BeadChip (850 K chip), resulting in the identification of a total of 2553 differentially methylated genes (DMGs). The differentially methylated genes were then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and significant differential DNA methylation was found. Among the differentially hypomethylated genes were GAL-8, LTF, and RFPL3, while the highly methylated genes were TMEM9B, ANK3, and C6orF48. These genes were mainly enriched in 10 significantly enriched pathways, such as cell adhesion junctions, among which the differentially methylated gene GAL-8 was involved in inflammatory pathway signaling. For functional analysis of GAL-8, we first examined the differences in GAL-8 promoter methylation levels among different subgroups of human coronary tissue in the CON, CHD, and SCD groups using pyrophosphate sequencing. The results revealed reduced GAL-8 promoter methylation levels in the SCD group, while the difference between the CHD and CON groups was not statistically significant (P > 0.05). The reduced GAL-8 promoter methylation level was associated with upregulated GAL-8 expression, which led to increased expression of the inflammatory markers TNF-α, IL-1β, MCP-1, MIP-2, MMP-2, and MMP-9. This enhanced inflammatory response contributed to the accumulation of foam cells, thickening of the intima of human coronary arteries, and increased luminal stenosis, which promoted the occurrence of sudden coronary death. Next, we found that GAL-8 promoter methylation levels in PBMC were consistent with human coronary tissue. The unstable angina group (UAP) had significantly lower GAL-8 promoter methylation levels than stable angina (SAP) and healthy controls (CON) (P < 0.05), and there was a significant correlation between reduced GAL-8 promoter methylation levels and risk factors for coronary heart disease. These findings highlight the association between decreased GAL-8 promoter methylation and the presence of coronary heart disease risk factors. ROC curve analysis suggests that methylation of the GAL 8 promoter region is an independent risk factor for CHD. In conclusion, our study confirmed differential expression of GAL-8, LTF, MUC4D, TMEM9B, MYOM2, and ANK3 genes due to DNA methylation in the SCD group. We also established the consistency of GAL-8 promoter methylation alterations between human coronary tissue and patient peripheral blood monocytes. The decreased methylation level of the GAL-8 promoter may be related to the increased expression of GAL-8 and the coronary risk factors. ConclusionsAccordingly, we hypothesized that reduced levels of GAL-8 promoter methylation may be an independent risk factor for adverse events in coronary heart disease.

14-Week exercise training modifies the DNA methylation levels at gene sites in non-Alzheimer's disease women aged 50 to 70 years

Exercise training emerges as a key strategy in lifestyle modification, capable of reducing the risk of developing Alzheimer's disease (AD) due to risk factors such as age, family history, genetics and low level of education associated with AD. We aim to analyze the effect of a 14-week combined exercise training (CT) on the methylation of genes associated with AD in non-alzheimer's disease women. CT sessions lasted 60 min, occurring three times a week for 14 weeks. Forty non-Alzheimer's disease women aged 50 to 70 years (60.7 ± 4.1 years) with a mean height of 1.6 ± 0.1 m, mean weight of 73.12 ± 9.0 kg and a mean body mass index of 29.69 ± 3.5 kg/m2, underwent two physical assessments: pre and post the 14 weeks. DNA methylation assays utilized the EPIC Infinium Methylation BeadChip from Illumina. We observed that 14 weeks of CT led to reductions in systolic (p = 0.001) and diastolic (p = 0.017) blood pressure and improved motor skills post-intervention. Among 25 genes linked to AD, CT induced differentially methylated sites in 12 genes, predominantly showing hypomethylated sites (negative β values). Interestingly, despite hypomethylated sites, some genes exhibited hypermethylated sites (positive β values), such as ABCA7, BDNF, and WWOX. A 14-week CT regimen was adequate to induce differential methylation in 12 CE-related genes in healthy older women, alongside improvements in motor skills and blood pressure. In conclusion, this study suggest that combined training can be a strategy to improve physical fitness in older individuals, especially able to induce methylation alterations in genes sites related to development of AD. It is important to highlight that training should act as protective factor in older adults.

Altered DNA methylation within DNMT3A, AHRR, LTA/TNF loci mediates the effect of smoking on inflammatory bowel disease

This work aims to investigate how smoking exerts effect on the development of inflammatory bowel disease (IBD). A prospective cohort study and a Mendelian randomization study are first conducted to evaluate the association between smoking behaviors, smoking-related DNA methylation and the risks of Crohn’s disease (CD) and ulcerative colitis (UC). We then perform both genome-wide methylation analysis and co-localization analysis to validate the observed associations. Compared to never smoking, current and previous smoking habits are associated with increased CD (P = 7.09 × 10−10) and UC (P < 2 × 10−16) risk, respectively. DNA methylation alteration at cg17742416 [DNMT3A] is linked to both CD (P = 7.30 × 10−8) and UC (P = 1.04 × 10−4) risk, while cg03599224 [LTA/TNF] is associated with CD risk (P = 1.91 × 10−6), and cg14647125 [AHRR] and cg23916896 [AHRR] are linked to UC risk (P = 0.001 and 0.002, respectively). Our study identifies biological mechanisms and pathways involved in the effects of smoking on the pathogenesis of IBD.

The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single-base resolution methylation profiles of the Pacific oyster, including female-to-male (FMa-to-FMb) and male-to-female (MFa-to-MFb) sex reversal, as well as sex non-reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during female-to-male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male-to-female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox-like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non-reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.

Development of an epigenetic clock to predict visual age progression of human skin.

Aging is a complex process characterized by the gradual decline of physiological functions, leading to increased vulnerability to age-related diseases and reduced quality of life. Alterations in DNA methylation (DNAm) patterns have emerged as a fundamental characteristic of aged human skin, closely linked to the development of the well-known skin aging phenotype. These changes have been correlated with dysregulated gene expression and impaired tissue functionality. In particular, the skin, with its visible manifestations of aging, provides a unique model to study the aging process. Despite the importance of epigenetic age clocks in estimating biological age based on the correlation between methylation patterns and chronological age, a second-generation epigenetic age clock, which correlates DNAm patterns with a particular phenotype, specifically tailored to skin tissue is still lacking. In light of this gap, we aimed to develop a novel second-generation epigenetic age clock explicitly designed for skin tissue to facilitate a deeper understanding of the factors contributing to individual variations in age progression. To achieve this, we used methylation patterns from more than 370 female volunteers and developed the first skin-specific second-generation epigenetic age clock that accurately predicts the skin aging phenotype represented by wrinkle grade, visual facial age, and visual age progression, respectively. We then validated the performance of our clocks on independent datasets and demonstrated their broad applicability. In addition, we integrated gene expression and methylation data from independent studies to identify potential pathways contributing to skin age progression. Our results demonstrate that our epigenetic age clock, VisAgeX, specifically predicting visual age progression, not only captures known biological pathways associated with skin aging, but also adds novel pathways associated with skin aging.

Abstract B016: Developing diagnostic tools via mutational spectra analysis and multiomic plasma profiling from liquid biopsies to identify potential signatures in cancer in Trinidad and Tobago

Abstract Cancer, especially lung cancer is a leading cause of nosocomial deaths worldwide. Non-small cell lung cancer (NSCLC) has a five-year survival rate of only 24%. Early diagnoses markedly improves survival with personalized cancer therapies using non-invasive techniques for asymptomatic detection of mutational spectrum-related by genotyping NSCLC patients, even prior to symptoms. Analysis of conformational change in proteins due to mutations in oncogenes allow for mutation-specific treatment with specialized cancer drugs. In addition, tumor mutational burden (TMB) is a promising predictive biomarker for cancer immunotherapy as high TMB patients respond better to immune checkpoint inhibitors cancer drugs. Treatment planning for non-small cell lung cancer generally requires testing for EGFR, ALK, ROS1, BRAF, MET, RET and KRAS gene alterations or whole genome analysis to assess TMB. The Caribbean region remains disadvantaged by the lack of available genetic testing mainly due to accessibility. We propose the genotyping of 30 stage III-IV NSCLC patients via next-generation sequencing (NGS) to improve personalized therapies. Through the analysis of tumor-specific alterations amongst a complete mutation spectrum analysis, including single nucleotide variants (SNVs), insertions, deletions, copy number variations (CNVs), and methylation alterations, we would generate data to be used as an accurate form of cancer genotyping for diagnostic purposes. We intend to include these genes as biomarkers in a mutational spectrum analysis, to be collated into a predictive DNA assay to be used for the first time as a diagnostic/prognostic tool in a medically underserved community. We intend to also identify TMB-high patients without cost-prohibitive whole genome sequencing by focusing on DNA damage repair (DDR) genes. There is an increasing utilization of agnostic testing which involves the analysis of all potentially actionable genes across all tumor and cancer types. The invention of genomic-tailored specialized cancer treatments has resulted in a spectacular improvement in disease outcomes for a significant portion of cancer patients, which we hope to offer to improve clinical outcomes and prognosis in the racially diverse population of Trinidad and Tobago. Citation Format: Nicole Ramlachan, Samuel M. West. Developing diagnostic tools via mutational spectra analysis and multiomic plasma profiling from liquid biopsies to identify potential signatures in cancer in Trinidad and Tobago [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B016.

A Pregnancy and Childhood Epigenetics Consortium (PACE) meta-analysis highlights potential relationships between birth order and neonatal blood DNA methylation

Higher birth order is associated with altered risk of many disease states. Changes in placentation and exposures to in utero growth factors with successive pregnancies may impact later life disease risk via persistent DNA methylation alterations. We investigated birth order with Illumina DNA methylation array data in each of 16 birth cohorts (8164 newborns) with European, African, and Latino ancestries from the Pregnancy and Childhood Epigenetics Consortium. Meta-analyzed data demonstrated systematic DNA methylation variation in 341 CpGs (FDR adjusted P < 0.05) and 1107 regions. Forty CpGs were located within known quantitative trait loci for gene expression traits in blood, and trait enrichment analysis suggested a strong association with immune-related, transcriptional control, and blood pressure regulation phenotypes. Decreasing fertility rates worldwide with the concomitant increased proportion of first-born children highlights a potential reflection of birth order-related epigenomic states on changing disease incidence trends.

Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a prevalent hematologic malignancy in children, and methotrexate (MTX) is a widely employed curative treatment. Despite its common use, clinical resistance to MTX is frequently encountered. In this study, an MTX-resistant cell line (Reh-MTXR) was established through a stepwise selection process from the ALL cell line Reh. Comparative analysis revealed that Reh-MTXR cells exhibited resistance to MTX in contrast to the parental Reh cells. RNA-seq analysis identified an upregulation of ATP-binding cassette transporter G1 (ABCG1) in Reh-MTXR cells. Knockdown of ABCG1 in Reh-MTXR cells reversed the MTX-resistant phenotype, while overexpression of ABCG1 in Reh cells conferred resistance to MTX. Mechanistically, the heightened expression of ABCG1 accelerated MTX efflux, leading to a reduced accumulation of MTX polyglutamated metabolites. Notably, the ABCG1 inhibitor benzamil effectively sensitized Reh-MTXR cells to MTX treatment. Moreover, the observed upregulation of ABCG1 in Reh-MTXR cells was not induced by alterations in DNA methylation or histone acetylation. This study provides insight into the mechanistic basis of MTX resistance in ALL and also suggests a potential therapeutic approach for MTX-resistant ALL in the future.

Unveiling DNA methylation in Alzheimer's disease: a review of array-based human brain studies.

The intricacies of Alzheimer's disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms, particularly DNA methylation. This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer's disease neuropathology. The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer's disease progression. The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus. Notably, ANK1 hypermethylation, a protein implicated in neurofibrillary tangle formation, was recurrently identified in the entorhinal cortex. Further, the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3, RHBDF2, and MCF2L, potentially influencing neuroinflammatory processes. The complex role of BIN1 in late-onset Alzheimer's disease is underscored by its association with altered methylation patterns. Despite the disparities across studies, these findings highlight the intricate interplay between epigenetic modifications and Alzheimer's disease pathology. Future research efforts should address methodological variations, incorporate diverse cohorts, and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer's disease progression.

L-carnitine fails to rescue chemotherapy injured ovaries by epigenetic changes of transcription factors

Cyclophosphamide (CP), as an anti-cancer drug, is frequently used to treat various types of cancer. A decreased number of ovarian follicles impaired normal ovarian function, and subsequent premature ovarian failure (POF) presented as a side effect of cyclophosphamide usage. These events may eventually affect the fertility rate of individuals. The present study showed the effect of cyclophosphamide on ovarian reserves and the protective effect of L-carnitine (LC) as an antioxidant to prevent POF.To design the study, six to eight-week-old NMRI female mice were divided into three groups: control, cyclophosphamide (CP), and cyclophosphamide + L-carnitine (CP + LC). Mice received drugs intraperitoneally (IP) for 21 days. In the following 24 h after the last injection, both ovaries were used to evaluate the expression of Sohlh1 and Lhx8 genes by Real-time PCR. Furthermore, the alteration of Lhx8 promoter methylation was examined by Methylation-sensitive high-resolution melting analysis (MS-HRM).The present data showed the negative effect of CP on regulator genes of oogenesis including Sohlh1 and Lhx8. In addition, an examination of the epigenetic status of the Lhx8 gene showed a change in promoter methylation of this gene following cyclophosphamide injection. Although, L-carnitine is an effective antioxidant in relieving oxidative stress caused by cyclophosphamide and its damage, in the present study, however, the use of L-carnitine failed to protect the ovaries from changes caused by CP injection. So, using cyclophosphamide can alter the expression of folliculogenesis genes through its effects on epigenetic changes and may cause POF. The results of the present study showed that L-carnitine consumption can’t protect the ovaries against the adverse effects of CP.

Alterations in leukocyte DNA methylome are associated to immunosuppression in severe clinical phenotypes of septic patients.

Sepsis patients experience a complex interplay of host pro- and anti-inflammatory processes which compromise the clinical outcome. Despite considering the latest clinical and scientific research, our comprehension of the immunosuppressive events in septic episodes remains incomplete. Additionally, a lack of data exists regarding the role of epigenetics in modulating immunosuppression, subsequently impacting patient survival. To advance the current understanding of the mechanisms underlying immunosuppression, in this study we explored the dynamics of DNA methylation using the Infinium Methylation EPIC v1.0 BeadChip Kit in leukocytes from patients suffering from sepsis, septic shock, and critically ill patients as controls, within the first 24 h after admission in the Intensive Care Unit of a tertiary hospital. Employing two distinct analysis approaches (DMRcate and mCSEA) in comparing septic shock and critically ill patients, we identified 1,256 differentially methylated regions (DMRs) intricately linked to critical immune system pathways. The examination of the top 100 differentially methylated positions (DMPs) between septic shock and critically ill patients facilitated a clear demarcation among the three patient groups. Notably, the top 6,657 DMPs exhibited associations with organ dysfunction and lactate levels. Among the individual genes displaying significant differential methylation, IL10, TREM1, IL1B, and TNFAIP8 emerged with the most pronounced methylation alterations across the diverse patient groups when subjected to DNA bisulfite pyrosequencing analysis. These findings underscore the dynamic nature of DNA methylation profiles, highlighting the most pronounced alterations in patients with septic shock, and revealing their close association with the disease.

Tangshen formula improves diabetic nephropathy in STZ-induced diabetes rats fed with hyper-methionine by regulating the methylation status of kidney

BackgroundThe objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250–300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing.ResultsBoth the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes.ConclusionWhile HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

Exploring the potential of Paris polyphylla var. yunnanensis pollen manipulation in modifying seed dormancy.

Paris polyphylla var. yunnanensis, a well-known Chinese medicinal herb, shows a unique physiological trait characterized by the cyclic opening and closing of its anthers after pollen maturation. The aim of this study was to explore the implications of this phenomenon on breeding. RNA sequencing coupled with methylation sequencing was used to scrutinize and compare gene expression profiles and methylation alterations in pollen and seeds during anther opening and closing, along with cold exposure. Genes enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were examined to identify gene clusters susceptible to temperature-related methylation changes in both pollen and seeds. Four pollen treatment models, namely, normal control, "pollen protected from low temperatures," "pollen from just-opened anther," and "pollen from close-blocked anther," were used to produce corresponding seeds via artificial pollination. Subsequently, qRT-PCR was used to validate modifications in the expression patterns of marker genes in pollinated seeds under diverse treatment scenarios. Genes exhibiting significant differences in expression between anthers and normal tissues, along with gene regions linked to methylation variations attributed to low-temperature-treated pollen and seeds, were identified through transcriptomic analysis. Convergence was observed in three signaling pathways: oxidative phosphorylation (ko00190), plant hormone signal transduction (Ko04075), and zeatin biosynthesis (ko00908). Notably, gene clusters prone to temperature-induced methylation changes, such as NADH-ubiquinone oxidoreductase chain 5, plasma membrane ATPase 4, cytochrome c oxidase subunit 2, cis-zeatin O-glucosyltransferase, ABSCISIC ACID-INSENSITIVE 5-like protein 4, and indole-3-acetic acid-amido synthetase (IAAS), were identified. Evaluation using various pollen pollination models revealed altered expression patterns of five dormancy-regulating marker genes: IAAS, sucrose synthase (SUS), gibberellin 2-oxidase (GA2ox), ABA INSENSITIVE 2 (ABI2), and auxin-repressed protein (ARP), in seeds pollinated with pollen from close-blocked anthers, cold-protected pollen, and pollen from freshly opened anthers. The close-blocked anther treatment led to significantly upregulated expression of IAAS, SUS, GA2ox, and ABI2, whereas ARP expression decreased markedly, indicating a propensity toward prolonged seed dormancy. Conversely, in the low-temperature-protected anther model, SUS, ARP, GA2ox, and IAAS exhibited reduced expression levels, whereas the expression of ABI2 was upregulated, overall facilitating seed germination.