Methylome Analysis Research Articles

Prospective Screening of Cancer Syndromes in Patients with Mesenchymal Tumors

Background: The etiology of most mesenchymal tumors is unknown, and knowledge about syndromes with an increased risk of tumors in bone or soft tissue is sparse. Methods: We present a prospective germline analysis of 312 patients with tumors suspected of being sarcomas at a tertiary sarcoma center. Germline and tumor whole genome sequencing, tumor transcriptome, and methylome analyses were performed. Results: Germline pathogenic or likely pathogenic variants associated with an increased risk of tumors were detected in 24 patients (8%), of which 11 (4%) harbored a detectable second hit in the tumor. Second hits were confirmed in genes with (NF1, RB1, TP53, EXT2, and SDHC) and without (ATM, CDC73, MLH1, MSH6, POLG, and KCNQ1) known association with mesenchymal tumor predisposition. Sarcomas from two Lynch syndrome patients showed mismatch repair deficiency, predicting a treatment response to immune checkpoint inhibitors (Level 1 biomarker according to the FDA (Federal Drug Administration) and ESMO (European Society for Medical Oncology)). None of the three CHEK2 carriers had a second hit in the tumor, suggesting a weak link to sarcoma. Conclusions: We conclude that second-hit analyses can be used in standard of care to identify syndrome-related tumors. This approach can help distinguish true manifestations of tumor syndromes from unrelated germline findings and enhance the understanding of germline predisposition in soft tissue tumors. Prospective screening using germline whole genome sequencing should be considered when comprehensive somatic sequencing is introduced into clinical practice.

TMOD-08. DETAILED GENERATION OF AN ORTHOTOPIC INTRACRANIAL GLIOBLASTOMA (GBM) PATIENT-DERIVED XENOGRAFT (PDX) MODEL FOR PRECLINICAL IMMUNOTHERAPEUTIC STUDIES

Abstract PURPOSE This study aimed to establish an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues expanded in a heterotopic subcutaneous PDX model for immunotherapeutic studies. The model is designed to closely replicate human GBM, enabling the exploration of its pathophysiology and the development of effective immunotherapies for this type of cancer. METHODS Glioblastoma tissues from 16 fresh and cryopreserved samples were used to establish a heterotopic subcutaneous PDX mouse model for tumor expansion. Successful engraftments were then used to create an orthotopic intracranial PDX mouse model. The tumor formation rates and durations were estimated in both models. Tumor fidelity to the original GBM tumors was confirmed through immunohistology (GFAP, P53, and Ki67) and genetic analysis (whole exome sequencing, RNA sequencing, and methylome analysis). The therapeutic effects of immunotherapy (allogenic NK cells) in combination with Avastin and Irinotecan were tested in the orthotopic intracranial PDX model. RESULTS The orthotopic intracranial GBM mouse model achieved 100% engraftment from successful tumor growth in the subcutaneous PDX model. The overall success rate was 81.25% (85.71% for cryopreserved tissues), with a median process duration of 26.5 weeks (19 weeks for the subcutaneous and 7.5 weeks for intracranial establishment). No significant differences in tumor formation were observed within one year for cryopreserved tissue in the subcutaneous model. Molecular and genetic analysis confirmed similar tumor characteristics between the intracranial PDX tumors and the original patient tumors. Additionally, NK cells combined with Avastin and Irinotecan demonstrated efficacy in this model. CONCLUSIONS We successfully established an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues, with no observed differences within one year. These models accurately mirror patients’ tumor characteristics, promising advancements in glioblastoma research and treatment, particularly for immunotherapy studies. Keywords: Glioblastoma (GBM), cryopreserved GBM tissues, patient-derived xenograft (PDX) model, NOD/SCID IL-12Rγnull (NSG) mice

Integrated Profiling Identifies Long-Term Molecular Consequences of Prenatal Dexamethasone Treatment in the Rat Brain-Potential Triggers of Depressive Phenotype and Cognitive Impairment.

Prenatal excess of glucocorticoids (GCs) is considered to be one of the highly impacting factors contributing to depression development. Although GCs are crucial for normal fetal development and their administration (mainly dexamethasone, DEX) is a life-saving procedure for those at risk of preterm delivery, exposure to excess levels of GCs during pregnancy can yield detrimental consequences. Therefore, we aimed to systematically investigate the brain molecular alterations triggered by prenatal DEX administration. We used a rat model of depression based on prenatal exposure to DEX and performed integrative multi-level methylomic, transcriptomic, and proteomic analyses of adult rats' brains (i.e., frontal cortex (FCx) and hippocampus (Hp)) to identify the outcomes of DEX action. Each of the investigated levels was significantly affected by DEX in the long-term manner. Particularly, we found 200 CpG islands to be differentially methylated in the FCx and 200 in the Hp of prenatally DEX-treated rats. Global transcriptomic analysis uncovered differential expression of transcripts mostly in FCx (271) and 1 in Hp, while proteomic study identified 146 differentially expressed proteins in FCx and 123 in Hp. Among the identified enriched molecular networks, we found altered pathways involved in synaptic plasticity (i.e., cAMP, calcium, and Wnt signaling pathways or tight junctions and adhesion molecules), which may contribute to cognitive impairment, observed in DEX-treated animals. Moreover, in the FCx, DEX administration in the prenatal period downregulates the expression of ribosome protein genes associated both with large and small ribosomal subunit assembly which can lead to a global decrease in translation and protein synthesis processes and, indirectly, alterations in the neurotransmission process.

CNSC-53. SHH ACTIVATION IN DEDIFFERENTIATION DURING TUMOR PROGRESSION IN MPNST

Abstract BACKGROUND Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previously, unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumors uncovered two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH-pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1-pathway activation). Further, single nuclear RNA sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. PURPOSE & HYPOTHESIS To examine whether the activation of the SHH pathway in MPNST-G1 cell lines induces overexpression of factors known to play canonical roles in early neural crest cell specification (TWIST1, SNAI2, PAX3, PAX6, SOX9, OTX2) and to investigate the role of Src activation on SHH pathway-induced dedifferentiation. We hypothesize that MPNST-G1 cells will exhibit SHH pathway activation, synergizing with Src to induce the overexpression of early neural crest cell (ENCC) transcription factors. METHODS SHH pathway activation, expression of ENCC transcription factors, and effects of inhibitors were analyzed in MPNST-G1 cells using RT-PCR, western blotting, Alamar Blue assay, Trypan Blue assay, and Soft Agar Transformation assay. RESULTS Compared to MPNST-G2 cells, MPNST-G1 cells displayed SHH-pathway activation, SMO-dependent activation of Src, and elevated expression of the ENCC transcription factors. Sonidegib (SMO inhibitor) and Dasatinib (Src inhibitor) were able to revert dedifferentiation. CONCLUSIONS The SHH-pathway cooperates with Src to promote expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options.

Integrative DNA methylome and transcriptome analysis illuminate leaf phenotype alterations in tetraploid citrus

Whole-genome duplication (WGD) in plants triggers profound morphological and physiological changes, with DNA modification being a key epigenetic factor that helps neo-polyploids overcome challenges and gain adaptive advantages. Tetraploids were previously mined from diploid citrus seedlings, showing enhanced environmental adaptability and potential as rootstocks. These tetraploids exhibited increased leaf and cell wall thickness compared to their diploid counterparts. To explore the impact of WGD, transcriptomic and whole-genome bisulfite sequencing (WGBS) were conducted on two pairs of citrus tetraploids and their diploid controls, revealing significant molecular changes. Notably, tetraploid citrus displayed lower CG methylation levels in gene and transposable element (TE) bodies relative to diploids. Differentially methylated genes (DMGs) between tetraploids and diploids were primarily associated with immune stress, organ development, metabolic pathways, and secondary metabolism. In Trifoliate orange (Poncirus trifoliata L. Raf.) and Ziyang Xiangcheng (Citrus junos Sieb. ex Tanaka), only 150 and 58 differentially expressed genes (DEGs) were identified, respectively, with enrichment in critical cellular processes such as cell wall synthesis, plastid development, and pathways modulating chloroplasts and plasma membranes. A total of 70 genes showed both differential methylation and expression, including ACN1, Nac036, and ASMT1, which are involved in stomatal development, leaf morphology, and melatonin synthesis, respectively, offering insight into the regulatory mechanisms of phenotype alterations after polyploidization. These findings reveal the epigenetic modifications in polyploid citrus and highlight the role of polyploidization-induced methylation in driving phenotypic changes.

Multiomic Analyses Reveal New Targets of Polycomb Repressor Complex 2 in Schwann Lineage Cells and Malignant Peripheral Nerve Sheath Tumors

Abstract Background Malignant peripheral nerve sheath tumors (MPNST) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic addback experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells has not been defined. We hypothesized PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1,327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog signaling in PRC2-deficient iHSCs. Functional studies confirm Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

Genome-wide DNA methylation profiling reveals a novel hypermethylated biomarker PRKCB in gastric cancer

Globally, gastric cancer (GC) ranks among the most prevalent forms of malignancy, posing a significant health burden. Epigenetic modifications, predominantly characterized by alterations in DNA methylation patterns, have been linked to a diverse array of neoplastic processes. Here, we undertake a comprehensive analysis of the DNA methylation signature in GC, with the aim to discover the potential diagnostic epigenetic biomarkers. Utilizing the Illumina 935 K BeadChip, we conducted a genome-wide exploration of DNA methylation patterns in four paired samples of GC tissues and adjacent non-cancerous counterparts. The bisulfite-pyrosequencing (n = 7) was employed to the quantification for methylated gene. The pubic databases including GWAS Catalog, TCGA and GEO were used. The immunohistochemistry and qRT-PCR analysis were performed. In contrast to adjacent tissues, GC tissues manifested pronounced hypermethylation patterns specifically within the promoter cytosine-phosphate-guanine (CpG) islands, indicating localized epigenetic alterations. DNA methylome analysis further revealed 4432 differentially-methylated probes (DMPs), with the gene PRKCB exhibited the most prominent average DNA methylation disparity (mean Δβ = 0.353). Pyrosequencing validation confirmed three DMPs within the PRKCB promoter (cg08406370, cg00735962, and cg18526361). Notably, the mean methylation levels of PRKCB were inversely correlated with mRNA expression levels in the GWAS Catalog. Furthermore, both mRNA and protein expression levels of PRKCB were significantly reduced in GCs when compared to their adjacent non-cancerous counterparts, verified by TCGA and GEO database. Our study reveals significant DNA methylation alterations in GC and emphasizes the pivotal role of PRKCB gene hypermethylation in conferring GC risk, which offers fresh perspectives for advancing diagnostic approaches and therapeutic strategies for GC.

Proximal and classic epithelioid sarcomas are distinct molecular entities defined by MYC/GATA3 and SOX17/endothelial markers, respectively

Epithelioid sarcoma (ES) is a rare tumor hallmarked by the loss of INI1/SMARCB1 expression. Apart from this alteration, little is known about the biology of ES. Despite recent advances in treatment, the prognosis of ES remains unsatisfactory. To elucidate the molecular underpinnings of ES, and to identify diagnostic biomarkers and potential therapeutic vulnerabilities, we performed an integrated omics profiling (RNA sequencing and methylation array) of 24 primary, untreated ESs. Transcriptome and methylome analysis identified two distinct molecular clusters that essentially corresponded to the morphologic variants of ES, classic ES (C-ES) and the more aggressive proximal ES (P-ES). The P-ES group was characterized by hyperactivation of GATA3 and MYC pathways, with extensive epigenetic rewiring associated with EZH2 overexpression. Both DNA methylation and gene expression analysis indicated a striking similarity with the “MYC subgroup” of ATRT, another SMARCB1-deficient tumor, implying a shared molecular background and potential therapeutic vulnerabilities. Conversely, the C-ES group exhibited an endothelial-like molecular profile, with expression of vascular genes and elevated pro-angiogenic SOX17 signaling. Immunohistochemistry validated the overexpression of the chromatin regulators GATA3 (9/12 vs. 0/16) and EZH2 (7/7 vs. 2/6) in P-ESs, and of the vascular factors SOX17 (8/8 vs. 1/10) and N-cadherin (5/9 vs 0/10) in C-ESs. Therefore, these molecules emerge as potential diagnostic tools to fill the gap represented by the lack of ES subtype-specific biomarkers. In summary, our study shows that P-ES and C-ES represent distinct molecular entities defined by MYC/GATA3 and SOX17/endothelial molecular traits, respectively. Besides providing insights into the biology of ES, our study pinpoints subtype-specific biomarkers and potential therapeutic vulnerabilities.

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies.

Genome-wide integrated DNA methylome and transcriptome analysis reveals a strong dysregulated myeloid component in the epigenetic landscape of Systemic Sclerosis.

Non-genetic factors influence Systemic Sclerosis (SSc) pathogenesis, underscoring epigenetics as a relevant contributor to the disease. We aimed to unravel DNA methylation abnormalities associated with SSc through an epigenome-wide association study (EWAS). We analyzed DNA methylation data from whole blood samples in 179 SSc patients and 241 unaffected individuals, to identify differentially methylated positions (DMPs) with a FDR<0.05. These results were further integrated with RNA-seq data from the same patients to assess their functional consequence. Additionally, we examined the impact of DNA methylation changes on transcription factors and analyzed the relationship between alterations of the methylation and gene expression profile and serum proteins levels. This analysis yielded 525 DMPs enriched in immune-related pathways, being leukocyte cell-cell adhesion the most significant (FDR=4.91x10-9), prioritizing integrins as they were exposed by integrating methylome and transcriptome data. Furthermore, through this integrative approach we observed an enrichment of neutrophil related pathways, highlighting this myeloid cell type as a relevant contributor in SSc pathogenesis. In addition, we uncovered novel profibrotic and proinflammatory mechanisms involved in the disease. Finally, the altered epigenetic and transcriptomic signature revealed an increased activity of CCAAT/enhancer-binding protein (CEBP) transcription factor family in SSc, which is crucial in the myeloid lineage development. Our findings uncover the impaired epigenetic regulation of the disease and its impact on gene expression, identifying new molecules for potential clinical applications and improving our understanding of SSc pathogenesis.

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

Genomic and epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse survival in lethal prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSIs) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood. We applied targeted cell-free DNA sequencing to 126 mCRPC patients from three academic cancer centers, and separately performed genome-wide cell-free DNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome-positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 mCRPC patients, respectively, to develop and validate a stem-like signature, which we inferred from cell-free DNA. Targeted cell-free DNA sequencing detected AR/enhancer alterations prior to first-line ARSIs which correlated with significantly worse PFS (p = 0.01; HR = 2.12) and OS (p = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (p < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cell-free DNA revealed enrichment for stemness-associated transcription factors in lethal mCRPC patients. The resulting stemness signature was then validated in a completely held-out cohort of 80 mCRPC patients profiled by tumor RNA sequencing. We analyzed a total of 220 mCRPC patients, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.

Histomorphologic spectrum of nodal marginal zone lymphoma as defined by its methylome.

Primary nodal marginal B-cell lymphoma (NMZL) is rare and histologically very variable. Its large-cell presentation is difficult to distinguish from nodal diffuse large B-cell lymphoma (nDLBCL) due to the absence of specific markers for nodal marginal zone lymphomas in general. Using a comprehensive cohort of NMZLs and a control cohort of nDLBCLs, we conducted a methylome analysis on subgroups of both. The methylomes were strikingly different between the cohorts but unexpectedly homogeneous within the NMZL cohort. This allowed us to describe the morphologic spectrum of NMZL in all its value ranges. The considerable overlap in growth pattern and cytology of NMZL with nDLBCL was explored morphometrically, leading to an operational tool for separating both by a simple measurement of cell size and nuclear size. This was integrated in a hierarchical approach, including a scoring system for the parameter growth pattern, follicular colonization, follicular dendritic network, IgD expression, and Ki-67 rate, and led to a proposal for a classifier that we present here. This methylome-based study extends the morphological spectrum of NMZL towards large cell morphology and offers a conventional way to distinguish it from nDLBCL.

Role of genetics and epigenetics in Graves' orbitopathy.

Objectives The pathogenesis of Graves' orbitopathy (GO) remains to be fully elucidated. Here we reviewed the role of genetics and epigenetics. Design We conducted a PubMed search with the following key words: Graves' orbitopathy, thyroid eye disease; or Graves' ophthalmopathy; or thyroid-associated ophthalmopathy; and: genetic, or epigenetic, or gene expression, or gene mutation, or gene variant, or gene polymorphism, or DNA methylation, or DNA acetylation. Articles in which whole DNA and/or RNA sequencing, proteome and methylome analysis were performed were chosen. Results The different prevalence of GO in the two sexes as well as racial differences suggest that genetics play a role in GO pathogenesis. In addition, the long-lasting phenotype of GO and of patient-derived orbital fibroblasts suggest a genetic or epigenetic mechanism. Although no genes have been found to confer a specific risk for GO, differential gene expression has been reported in orbital fibroblasts from GO patients vs control fibroblasts, suggesting that an epigenetic mechanism may be involved. In this regard, a different degree of DNA methylation, which affects gene expression, has been found between GO and control fibroblasts, which was confirmed by whole methylome analysis. Histone acetylation and deacetylation, which also affect gene expression, remain to be investigated. Conclusions Although pathogenetic gene variants have not been reported, epigenetic mechanisms elicited by an initial autoimmune insult seem to be needed for differential gene expression to occur and, thus, for GO to develop and persist over time.

Combined analysis of genome-wide DNA methylome and transcriptome reveals the first epigenetic-based antibiotic-resistance mechanism in Acinetobacter baumannii

The World Health Organization (WHO) has defined carbapenem-resistant Acinetobacter baumannii (A. baumannii) as a critical species for which new therapeutic strategies are needed. In this tragic context, the discovery of new antibiotics with innovative pharmacological targets is essential. Currently, limited information is available on the relationship between epigenetic changes of m5C/m6A DNA and drug resistance in A. baumannii. In this study, we used Oxford Nanopore sequencing to analyze m5C/m6A epigenetic marks and Illumina RNA-Seq to evaluate the transcriptome of 5 A. baumanni isolates with different resistance profiles. The data clearly demonstrated that the m5C pattern of resistant strains globally differs from the multi susceptible strain, with more than 600 hypo- and hypermethylated sites for each resistant strain. For m6A, less severe changes occurred, however, more than 40 hyper- and hypomethylated sites were still found between each resistant and susceptible strain. Combined whole-genome DNA methylome and transcriptome analysis revealed a greater involvement of m5C modification in drug resistance processes. This study provides a new understanding of the epigenetic regulation of antibiotic resistance in A. baumannii and has potential implications for future antibiotic resistance research.

O69 THE REGULATORY ROLE OF CIRCULAR RNAS IN RESPONSE TO RAAS INHIBITORS

Circular RNAs (circRNAs) are a class of non-coding RNA that may play an important role in disease progression and diagnosis. This study aimed to investigate the role of renal circRNAs in hypertension and to identify circRNAs pathways explaining chronic reset in blood pressure. Two groups of young spontaneously hypertensive rats (SHR) were used: rats treated with or without losartan (RAAS inhibitor, n = 18 in each group). Through established circRNA identification and quantification tools, we identified five significant circular RNA candidates (P &lt; 0.05), each aligning with differentially expressed and biologically significant miRNA. Furthermore, these candidate circRNAs aligned with expression pathways found in both treatment and control groups. In conclusion, this study identified several circRNAs which may play an important role in blood pressure regulation. It may advance our understanding of circRNAs in mediating losartan-induced blood pressure reduction. Future studies on validation, expression networking and methylome analysis may shed light on these circRNAs’ significance and identify potential therapeutic biomarkers.

Comprehensive Analysis of Methylome and Transcriptome to Identify Potential Genes Regulating Porcine Testis Development.

DNA methylation plays a critical role in regulating gene expression during testicular development. However, few studies report on candidate genes related to the DNA methylation regulation of porcine testicular development. This study examined the differentially expressed genes (DEGs) and their methylation levels in testicular tissues from pigs at 60 days of age (60 d) and 180 days of age (180 d) using RNA-Seq and whole genome bisulfite sequencing (WGBS). It was determined that DNA methylation primarily occurs in the cytosine-guanine (CG) context, and the analysis identified 106,282 differentially methylated regions (DMRs) corresponding to 12,385 differentially methylated genes (DMGs). Further integrated analysis of RNA-Seq and WGBS data revealed 1083 DMGs negatively correlated with the expression of DEGs. GO analysis showed that these genes were significantly enriched in spermatogenesis, germ cell development, and spermatid differentiation. The screening of enriched genes revealed that hyper-methylation repressed ADAM30, ADAM3A, DPY19L2, H2BC1, MAK, RPL10L, SPATA16, and YBX2, while hypo-methylation elevated CACNA1I, CADM1, CTNNB1, JAM2, and PAFAH1B3 expression. Additionally, the methylation status of the key genes ADAM3A, ADAM30, YBX2, JAM2, PAFAH1B3, and CTNNB1 was detected by bisulfite sequencing PCR (BSP). This study offers insights into the epigenetic regulation mechanisms underlying porcine testicular development.

Role of 6mA in the Regulation of Metabolic Biosynthesis in Sorghum Callus.

Plant cell culture technology helps to obtain natural plant-derived metabolites. The callus of sorghum, a prominent cereal crop, possesses various metabolites with potential health benefits. However, the epigenetic mechanism regulating metabolic biosynthetic capabilities in sorghum remains unknown. Therefore, we conducted N6-methyladenine (6mA) methylome analysis using transcriptome profiling and metabolome analysis to investigate the role of 6mA alterations in two calluses having different biosynthetic capacities, which were derived from immature sorghum embryos. Our findings indicate that the 6mA upregulation within gene bodies is crucial in transcriptional activity potentially mediated by the DNA demethylase SbALKBH1. Furthermore, 6mA was significantly enriched in genes involved in the biosynthesis of flavonoids and isoflavonoids. This could serve as a novel source of bioactive compounds for human health. Thus, 6mA could play an essential role in flavonoid biosynthesis in the sorghum callus.

Comprehensive Molecular Characterization of a Large Series of Calcified Chondroid Mesenchymal Neoplasms Widening Their Morphologic Spectrum.

Recently, FN1 fusions to receptor tyrosine kinase genes have been identified in soft tissue tumors with calcified chondroid matrix named calcifying chondroid mesenchymal neoplasms (CCMNs). We collected 33 cases of CCMN from the French network for soft tissue and bone tumors. We performed whole-exome RNA sequencing, expression analysis, and genome-wide DNA methylation profiling in 33, 30, and 20 cases of CCMN compared with a control group of tumors, including noncalcified tenosynovial giant cell tumor (TGCT). Among them, 15 cases showed morphologic overlap with soft tissue chondroma, 8 cases with tophaceous pseudogout, and 10 cases with chondroid TGCT. RNA-sequencing revealed a fusion of FN1 in 76% of cases (25/33) with different 5' partners, including most frequently FGFR2 (14 cases), TEK or FGFR1. Among CCMN associated with FGFR1 fusions, 2 cases had overexpression of FGF23 without tumor-induced osteomalacia. Four CCMN had PDGFRA::USP8 fusions; 3 of which had histologic features of TGCT and were located in the hip, foot, and temporomandibular joint (TMJ). All cases with FN1::TEK fusion were located at TMJ and had histologic features of TGCT with or without chondroid matrix. They formed a distinct cluster on unsupervised clustering analyses based on whole transcriptome and genome-wide methylome data. Our study confirms the high prevalence of FN1 fusions in CCMN. In addition, through transcriptome and methylome analyses, we have identified a novel subgroup of tumors located at the TMJ, exhibiting TGCT-like features and FN1::TEK fusions.

Methylome analysis of endothelial cells suggests new insights on sporadic brain arteriovenous malformation

Arteriovenous malformation of the brain (bAVM) is a vascular phenotype related to brain defective angiogenesis. Involved vessels show impaired expression of vascular differentiation markers resulting in the arteriolar to venule direct shunt. In order to clarify aberrant gene expression occurring in bAVM, here we describe results obtained by methylome analysis performed on endothelial cells (ECs) isolated from bAVM specimens, compared to human cerebral microvascular ECs. Results were validated by quantitative methylation-specific PCR and quantitative realtime-PCR. Differential methylation events occur in genes already linked to bAVM onset, as RBPJ and KRAS. However, among differentially methylated genes, we identified EPHB1 and several other loci involved in EC adhesion as well as in EC/vascular smooth muscle cell (VSMC) crosstalk, suggesting that only endothelial dysfunction might not be sufficient to trigger the bAVM phenotype. Moreover, aberrant methylation pattern was reported for many lncRNA genes targeting transcription factors expressed during neurovascular development. Among these, the YBX1 that was recently shown to target the arteridin coding gene. Finally, in addition to the conventional CpG methylation, we further considered the role of impaired CHG methylation, mainly occurring in brain at embryo stage. We showed as differentially CHG methylated genes are clustered in pathways related to EC homeostasis, as well as to VSMC-EC crosstalk, suggesting as impairment of this interaction plays a prominent role in loss of vascular differentiation, in bAVM phenotype.