Unique Methylation Research Articles

Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

A new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered: a clinico-radiological and histomolecular characterisation

Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumour type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumours. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogeneous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large paediatric and adult cohort of 29 tumours H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbour Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbours atypical radiological and histopathological profiles with calcification and/or a solid tumour component both for BRAFV600E and FGFR1MUT cases. The analyses of a H3.3-K27M BRAFV600E tumour at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis. Contrary to other DMG, these tumours occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype/phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management.

Abstract C060: DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations

Abstract African Americans (AAs) have among the highest incidence and mortality rates of colorectal cancer (CRC) in the US. They present with more right-sided, microsatellite stable tumors and are diagnosed at earlier ages than non-Hispanic Whites (NHW). While DNA methylation changes and their significance have been previously described in CRC, much less is known about the unique DNA methylation changes that occur in AA CRCs compared to NHW CRCs. In the current study, we analyzed DNA methylation changes in AA patients and compared those changes to data from NHWs. AA patients diagnosed with a microsatellite-stable colorectal tumor at the Yale New Health System were included in the study. A pathologist reviewed archival slides, and selected areas from tumor (n=160) and adjacent normal tissue (n=42) were cored. The median age of patients was 59 from 82 females and 78 males. Extracted DNA was bisulfite converted and tested using EPIC arrays. DNA methylation (450K) from NHW CRCs was also analyzed for 217 CRCs and 20 normal tissues obtained from TCGA (median age=70; 114 males, 103 females). Differentially methylated regions (DMRs) with |Db|>0.1, FDR corrected p-value <0.05 between tumor and normal tissue were identified using SeSAMe. We identified 4881 DMRs (57% hyper- and 44% hypomethylated) between AA CRC and normal tissue. Of these, 46% were in CpG Islands, 15% in CpG Shores, 4% in CpG shelves, and 36% in open sea. Most DMRs (71%) were in promoter regions, compared with 18% in gene bodies, 5% in enhancer regions, and 6% in intergenic regions. Next, we performed an additional DMR analysis using overlapping probes from the 450K and EPIC arrays to compare TCGA NHW with Yale AA data. We identified 2290 DMRs between AA CRC and normal tissue compared to 4657 in NHW. Of the 2290 AA DMRs, 1405 (61%) were common with NHW DMRs, 885 (39%) were unique to AAs. There were many overlapping pathways and genes involving metabolic functions, DNA damage response, immune response, and WNT signaling. However, the DMRs impacting these pathways differed between AA and NHW CRCs. Several AA-specific DMRs were associated with immune response and included LCK and GPSM3 genes. Hypermethylation of LCK, a tyrosine kinase involved in T-cell receptor signaling, was found in 60% of AA CRCs compared to 8% of NHW CRCs. LCK hypermethylation leads to CD4/8 T-cell inactivation and is linked to poor outcomes in lung cancer. We also observed AA-specific hypermethylation of GPSM3 in 60% of AA CRCs compared to 18% of NHW CRCs. GPSM3 is associated with CD4/8 T-cell infiltration. Lastly, promoter hypermethylation of 6 protocadherin genes was found in 59% of AAs compared to 20% NHWs. Protocadherins play a role in regulating WNT signaling and hypermethylation has been associated with poor outcomes in multiple cancers. Taken together, these results show AA-specific CRC methylation changes compared with NHW CRCs. Further work is warranted to investigate the potential functional and clinical implications of these methylation alterations, some of which may represent therapeutic targets and biomarkers. Citation Format: David N. Buckley, Rosa Xicola, Mikaeel Reger, Mary K. Yagle, Baris Kerimoglu, Curtis Thorne, Dante Bellomo, Megha Padi, Xavier Llor, Nathan Ellis, Bodour Salhia. DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C060.

Abstract C001: Effective visualization of methylation changes in prostate cancer among African American men

Abstract Epigenetic modification, involving changes in the methylation of genes, play a key role in the molecular changes seen in many cancers, with prostate cancer (PCa) being one of them. These changes include both hyper-methylation, where genes are silenced and hypo-methylation where genes are over-expressed in tumors. Changes of methylation have also been linked to both drug-resistance and immune-evasion. We will in this work present a visualization suite that we have developed in our analysis of an in-house cohort of adjacent normal and prostate tumor tissue samples from men with African American ancestry (AA). We demonstrate significant changes in immune synapse genes in PCa using a principal component model that describe the dysregulation of both co-stimulatory genes and immune checkpoint genes. We also demonstrate significant changes in STING and changes in immune cell content between adjacent normal and tumor tissue. Treg and B memory cells are increase in tumor tissue, while there is a lower amount of naïve CD4 T cells and naïve B cells. Furthermore, we show that hyper-methylation occurs in many PCa samples, with multiple immune genes being silenced. The degree of hyper-methylation is shown on both a global and a focal view, with Chromosome 17 being most affected by hyper-methylation and zooming in at a region including RARA, IGFBP4, STAT5A, CAVIN1, PLEKHH3, RND2;VAT1, TMEM106A, and DHX8;ETV4 genes. We also compare our results from African American patients to a European cohort, demonstrating an overall similarity in methylation changes but also identifies unique methylation of genes, including GLRX, IFFO1, RASSF1, GEFT that are hyper-methylated in our PCa AA cohort. Additionally, we identify different categories of genes based on their methylation changes across PCa AA samples with different Gleason score. Using a Sankey graph, we classify significant CpG-probes into 8 different categories, describing both hyper- and hypo-methylation and if the changes are seen as a continues change between the different grades area more threshold like. Key genes include SEPT9, CCND2, TMHLE, and SPARCL1 being hyper methylation in tumors with a higher Gleason score, while CDH4, PRDM16, CSMD1, and IFI16 show hypo-methylation in higher Gleason score tumors. We also see an increase of Treg immune cells in PCa samples with high Gleason score. In conclusion, the visualization presented in this study provides an effective way to better understand the epigenetic changes seen in PCa AA men and can potentially also lead to a better understanding of the mechanisms contributing to PCa disparities in the AA population. Citation Format: Anders Berglund, Ryan M. Putney, Kosj Yamoah, Hyun Park, Jong Y. Park. Effective visualization of methylation changes in prostate cancer among African American men [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C001.

Mucosal DNA methylome alteration in Crohn's disease: surgical and non-surgical groups.

Crohn's disease (CD) is characterized as a chronic, relapsing, and progressive disorder with a complex etiology involving interactions between host, microbiome, and the external environment. Genome wide association studies (GWAS) suggest several genetic variations in the diseased individuals but that explains only a small proportion of susceptibility to disease conditions. This indicates the possible role of epigenome which links environmental factors to the genetic variation in the disease etiology. The current study is focused on the DNA methylome evolution with disease progression. We performed Reduced Representation Bisulfite Sequencing (RRBS) to analyze differential DNA methylation in the diseased and healthy mucosal tissues of 2 different groups of CD patients: non-surgical and surgical, categorized based on the severity of disease and standard of care needed. Patients in both groups have unique DNA methylation signature compared to the healthy tissue. After removing single nucleotide polymorphisms (SNPs), 1,671 differentially methylated loci were found in the non-surgical and 3,334 in the surgical group of which only 206 were found overlapping in both groups. Furthermore, differential DNA methylation was noted in some of the GWAS associated genes implicated in CD. Also, functional enrichment analysis showed high representation of several key pathways where differential methylations were observed, and these can be implicated in CD pathogenesis. We identified specific DNA methylation patterns in the mucosal DNA of surgical and non-surgical CD patients which indicates evolution of the methylome as the disease progresses from initial to the advance stage. These unique patterns can be used as DNA methylation signatures to identify different stages of the disease.

PATH-07. GENOMIC AND IMMUNE ANALYSIS OF PRIMARY REPLICATION-REPAIR DEFICIENT (RRD) GLIOMAS REVEALS THREE SUBGROUPS WITH DISTINCT DRIVERS AND RESPONSE TO IMMUNOTHERAPY: AN IRRDC REPORT.

Abstract BACKGROUND Replication-repair deficiency (RRD) caused by germline/somatic defects in mismatch repair (MMRD) and/or polymerase-proofreading genes (PPD) drives 5-10% of high-grade gliomas in children, adolescents, and young adults (CAYA). Despite harbouring high mutation-burden (TMB), the basis of their heterogenous responses to immune-checkpoint inhibitors (ICI) is unknown. METHODS We performed multi-omic and immune analyses on human RRD-glioma specimens and developed murine models to investigate their differential responses to ICI. RESULTS RRD-gliomas in 202 CAYA-patients uniformly harbored high genomic microsatellite-instability and hypermutation (median TMB: 302-mutations/megabase), with specific MMRD mutational signatures contributing to frequent mutations in TP53 (88%), ATRX (83%), RAS/MAPK (77%) and IDH1/2 (17%). Strikingly, common pediatric mutations (K27M, G34R/V and BRAF;p.V600E) unrelated to MMRD signatures were absent, and a unique methylation profile distinct from non-RRD gliomas was observed. Molecularly, RRD-gliomas segregated into three genomic subgroups: RRD1 (MMRD+PPD; 56%), RRD2 (MMRD-only; 27%), and RRD3 (MMRD+IDH1/2; 17%). RRD1 included glioblastomas that developed early in germline CMMRD patients across the cerebral hemispheres and posterior-fossa, harbored extreme TMB (median: 420-mutations/megabase), balanced copy-number profiles, and immunogenic PPD signatures. RRD3 predominated in Lynch syndrome, presented in older children, were located in the forebrain, harbored lower TMB (median: 31-mutations/megabase) and demonstrated specific chromosomal gains/losses. Enrichment for immune-resistant JAK-STAT signalling in the RRD2/RRD3 transcriptome, and presence of high CD8+ T-cells in RRD1-glioma microenvironment, correlated with significantly improved 24-month post-ICI survival of 75% in RRD1 versus 43% and 33% for RRD2/3, respectively (p < 0.0001). Genetically engineered mouse models recapitulated the immuno-genomic phenotype and ICI-responses of the human RRD-subgroups, allowing successful preclinical testing of combinatorial checkpoint-inhibition strategies. Significant temporal and spatial heterogeneity linked to genomic instability and post-therapy immune-escape were observed. CONCLUSION Our data emphasizes an immediate need to distinguish RRD-glioma subgroups in clinical practice and translate our preclinical discoveries to subgroup-specific ICI-based combinatorial clinical trials.

PATH-65. INTEGRATING DNA METHYLATION AND SEQUENCING TECHNOLOGIES FOR ADVANCED BRAIN TUMOR DIAGNOSIS

Abstract DNA methylation has emerged as an indispensable tool in neuropathological diagnostics, improving the diagnosis of various brain tumor entities. At our institution, methylation arrays as well as DNA and RNA sequencing from Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks have become routine diagnostic practices for several years. In this study, we present an integration of DNA methylation analyses with targeted gene panel sequencing and RNA sequencing data in an extensive cohort comprising 5749 cases. The cohort encompassing both adult and pediatric patients with central nervous system (CNS) tumors. Only those cases conclusively classified by the Heidelberg brain tumor classifier (v12.5, cutoff > 0.9) and possessing available sequencing data were included in the analyses. The study includes several rare and less characterized brain tumor entities, unveiling frequent mutations and gene fusions that will improve the diagnostic process. Our study offers an unparalleled comprehensive overview of the prevalence of genetic alterations in brain tumors, precisely defined by their unique methylation classes. The profound implications of our results extend far beyond diagnosis alone, significantly augmenting the evaluation of targeted treatment options for patients undergoing DNA methylation analyses.

Clinico-biological refinement of BCL11B-related disorder and identification of an episignature: A series of 20 unreported individuals

PurposeBCL11B-related disorder (BCL11B-RD) arises from rare genetic variants within the BCL11B gene, resulting in a distinctive clinical spectrum encompassing syndromic neurodevelopmental disorder, with or without intellectual disability, associated with facial features and impaired immune function. This study presents an in-depth clinico-biological analysis of 20 newly reported individuals with BCL11B-RD, coupled with a characterization of genome-wide DNA methylation patterns of this genetic condition. MethodsThrough an international collaboration, clinical and molecular data from 20 individuals were systematically gathered, and a comparative analysis was conducted between this series and existing literature. We further scrutinized peripheral blood DNA methylation profile of individuals with BCL11B-RD, contrasting them with healthy controls and other neurodevelopmental disorders marked by established episignature. ResultsOur findings unveil rarely documented clinical manifestations, notably including Rubinstein-Taybi-like facial features, craniosynostosis, and autoimmune disorders, all manifesting within the realm of BCL11B-RD. We refine the intricacies of T cell compartment alterations of BCL11B-RD, revealing decreased levels naive CD4+ T cells and recent thymic emigrants while concurrently observing an elevated proportion of effector-memory expressing CD45RA CD8+ T cells (TEMRA). Finally, a distinct DNA methylation episignature exclusive to BCL11B-RD is unveiled. ConclusionThis study serves to enrich our comprehension of the clinico-biological landscape of BCL11B-RD, potentially furnishing a more precise framework for diagnosis and follow-up of individuals carrying pathogenic BCL11B variant. Moreover, the identification of a unique DNA methylation episignature offers a valuable diagnosis tool for BCL11B-RD, thereby facilitating routine clinical practice by empowering physicians to reevaluate variants of uncertain significance within the BCL11B gene.

The PWWP domain and the evolution of unique DNA methylation toolkits in Hymenoptera

DNMT3 in Hymenoptera has a unique duplication of the essential PWWP domain. Using GST-tagged PWWP fusion proteins and histone arrays we show that these domains have gained new properties and represent the first case of PWWP domains binding to H3K27 chromatin modifications, including H3K27me3, a key modification that is important during development. Phylogenetic analyses of 107 genomes indicate that the duplicated PWWP domains separated into two sister clades, and their distinct binding capacities are supported by 3D modeling. Other features of this unique DNA methylation system include variable copies, losses, and duplications of DNMT1 and DNMT3, and combinatorial generations of DNMT3 isoforms including variants missing the catalytic domain. Some of these losses and duplications of are found only in parasitic wasps. We discuss our findings in the context of the crosstalk between DNA methylation and histone methylation, and the expanded potential of epigenomic modifications in Hymenoptera to drive evolutionary novelties.

High-throughput Sequencing Reveals N6-methyladenosine-modified LncRNAs as Potential Biomarkers in Mice with Liver Fibrosis.

N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic RNA. Long noncoding RNAs (lncRNAs) are a new type of noncoding regulatory molecule with multiple cellular functions. Both are closely related to the occurrence and development of liver fibrosis (LF). However, the role of m6A-methylated lncRNAs in the progression of LF remains largely unknown. In this study, HE and Masson staining were used to observe pathological changes in the liver, m6A-modified RNA immunoprecipitation sequencing (m6A-seq) was performed to systematically evaluate the m6A modification level of lncRNAs in LF mice, meRIP-qPCR and RT-qPCR were used to detect the m6A methylation level and RNA expression level of the target lncRNAs. A total of 415 m6A peaks were detected in 313 lncRNAs in liver fibrosis tissues. There were 98 significantly different m6A peaks in LF, which were located on 84 lncRNAs, of which 45.2% of the lncRNA length was between 200-400 bp. At the same time, the first three chromosomes of these methylated lncRNAs were chromosomes 7, 5 and 1. RNA sequencing identified 154 differentially expressed lncRNAs in LF. The joint analysis of m6A-seq and RNA-seq found that there were three lncRNAs with significant changes in m6A methylation and RNA expression levels: lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586. Subsequently, the verification results showed that the m6A methylation levels of lncRNA H19 and lncRNA Gm17586 were significantly increased, while that of lncRNA Gm16023 was significantly decreased, and the RNA expression of three lncRNAs was significantly decreased. Through the establishment of a lncRNA-miRNA-mRNA regulatory network, the possible regulatory relationships of lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586 in LF were revealed. This study revealed the unique m6A methylation pattern of lncRNAs in LF mice, suggesting that the m6A methylation modification of lncRNAs is related to the occurrence and development of LF.

Liquid biopsy for early detection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal cancer globally. Over 90% of HCC cases arise in the context of liver cirrhosis, and the severity of the underlying liver disease or advanced tumor stage at diagnosis significantly limits treatment options. Early diagnosis is crucial, and all guidelines stress the importance of screening protocols for HCC early detection as a public health objective. As serum biomarkers are not optimal for early diagnosis, liquid biopsy has emerged as a promising tool for diagnosis, prognostication, and patients' stratification for personalized therapy in various solid tumors, including HCC. While circulating tumor cells (CTCs) are better suited for personalized therapy and prognosis, cell-free DNA (cfDNA) and extracellular vesicle-based technologies show potential for early diagnosis, HCC screening, and surveillance protocols. Evaluating the added value of liquid biopsy genetic and epigenetic biomarkers for HCC screening is a key goal in translational research. Somatic mutations commonly found in HCC can be investigated in cfDNA and plasma exosomes as genetic biomarkers. Unique methylation patterns in cfDNA or cfDNA fragmentome features have been suggested as innovative tools for early HCC detection. Likewise, extracellular vesicle cargo biomarkers such as miRNAs and long non-coding RNAs may serve as potential biomarkers for early HCC detection. This review will explore recent findings on the utility of liquid biopsy for early HCC diagnosis. Combining liquid biopsy methods with traditional serological biomarkers could improve the overall diagnostic accuracy for early HCC detection.

Epigenomic analysis reveals a unique DNA methylation program of metastasis-competent circulating tumor cells in colorectal cancer

Circulating tumor cells (CTCs) and epigenetic alterations are involved in the development of metastasis from solid tumors, such as colorectal cancer (CRC). The aim of this study was to characterize the DNA methylation profile of metastasis-competent CTCs in CRC. The DNA methylome of the human CRC-derived cell line CTC-MCC-41 was analyzed and compared with primary (HT29, Caco2, HCT116, RKO) and metastatic (SW620 and COLO205) CRC cells. The association between methylation and the transcriptional profile of CTC-MCC-41 was also evaluated. Differentially methylated CpGs were validated with pyrosequencing and qMSP. Compared to primary and metastatic CRC cells, the methylation profile of CTC-MCC-41 was globally different and characterized by a slight predominance of hypomethylated CpGs mainly distributed in CpG-poor regions. Promoter CpG islands and shore regions of CTC-MCC-41 displayed a unique methylation profile that was associated with the transcriptional program and relevant cancer pathways, mainly Wnt signaling. The epigenetic regulation of relevant genes in CTC-MCC-41 was validated. This study provides new insights into the epigenomic landscape of metastasis-competent CTCs, revealing biological information for metastasis development, as well as new potential biomarkers and therapeutic targets for CRC patients.

P20.10.B CHARACTERIZATION OF NEW PROGNOSTIC MARKERS IN A COHORT OF ADULT SPINAL EPENDYMOMAS

Abstract BACKGROUND Spinal ependymomas (SP-EPN) are group of primary central nervous system tumors recently re-classified by the WHO (CNS5 2021), located in spinal cord. SP-EPN are grade 2-3 tumors, found mainly in cervical and thoracic region, characterized mostly by NF2 mutations and classical histology: grade 3 tumors present also microvascular proliferation, necrosis and high mitotic rate. Recently, a novel subtype was discovered with a unique methylation profile (SP-EPN-MYCN) and characterized by the amplification of MYCN gene, grade 3 histology and a very aggressive behavior. Treatment of these tumors remains challenging: gross-total resection is not often achieved and radiation failed to give sacceptable results. Besides, no prognostic factors except the mitotic index and tumor location, are available, thus, new diagnostic and prognostic biomarkers would be very useful for their accurate classification and management. MATERIAL AND METHODS We have collected 170 patients operated at our Institute from 1997 to 2019 of spinal ependymomas located in the cervical and thoracic region, excluded conus/filum mixopapillary ones. All tumors were revised according to the new WHO CNS5 2021 classification. In a cohort of 50 grade 2/3 SP-EPN we have evaluated by immunohistochemistry the expression of MYCN, H3K27me3, CDK4 and EZH2: the latter is a component of the PRC2 complex, which regulates the histone H3 methylation at Lys27 (K27) and recently described as a transcription factor which controls the expression of MYCN and CDK4 genes. We have also evaluated the amplification of MYCN and CDK4 genes by Copy Number Variation Assay and MLPA techniques. Clinical data were collected and related to the results obtained. RESULTS We have found strong overexpression of MYCN only in grade 3 SP-EPN (3/50), while grade 2 tumors showed low or no expression; amplification of MYCN gene was similarly confirmed by CNV assay and MLPA only in grade 3. Interestingly, also CDK4 overexpression was found only in grade 3 ependymomas (1/50), but it is expressed oppositely to MYCN: in tumors were MYCN is overexpressed, CDK4 was completely absent. Amplification of CDK4 gene was confirmed only in those with CDK4 overexpression. These results were both related to an interesting overexpression of EZH2 only in grade 3 SP-EPN (4/50), while H3K27me3 is expressed in all grades without differences. Finally, in our study both MYCN and CDK4-amplified ependymomas were related to a very aggressive behaviour and poor outcome with high recurrence rate, dissemination and metastases. CONCLUSION In this study we confirmed the prognostic role of MYCN as indicated by WHO classification and we have showed that CDK4 and EZH2 overexpression could be considered as new promising prognostic and predictive factors in SP-EPN. Since many trials with EZH2 and CDK4 inhibitors have been set up in the last years, these analyses could suggest a new therapeutic approach.

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome.

U7 snRNP is a multisubunit endonuclease required for 3' end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B, and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50, and pICln known to methylate arginines in the carboxy-terminal regions of the Sm proteins B, D1, and D3 during the spliceosomal Sm ring assembly. Both biochemical and cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.

Genome-wide DNA methylation and transcriptome analyses reveal important epigenetic genes associated with valgus-varus deformity in broilers

ABSTRACT 1. Valgus-varus deformity (VVD) is a common leg bone problem in broilers that causes serious economic losses to the breeding industry. The genetic aetiology of VVD is not clear, which restricts the genetic control of VVD. 2. In this study, knee cartilage of 35-day-old VVD and normal broilers was sequenced by whole-genome bisulphite sequencing (WGBS). The unique whole-genome DNA methylation profile of VVD broilers was described, and the methylation data and transcription data were used for joint analysis. 3. The mean methylation level of the VVD group was greater than that in the normal group. A total of 4315 differentially methylated regions (DMRs) were detected from methylation data, with the highest DMR density on chromosomes 25, 27, 31 and 33. DMRs were mainly located in introns, which accounted for more than 60%, followed by promoter and exon regions. 4. A total of 2326 differentially methylated genes (DMGs) were identified from DMRs, including 1159 genes with upregulated DMRs, 936 genes with downregulated DMRs, and 231 genes with two types of DMRs. 5. The ESPL1 gene may be an important epigenetic gene of VVD. The methylation of particular CpG17, CpG18 and CpG19 sites in the promoter region of the ESPL1 gene may hinder the binding of transcription factors and promoters and increase the expression of ESPL1.

Identification of a New m6A Regulator-Related Methylation Signature for Predicting the Prognosis and Immune Microenvironment of Patients with Pancreatic Cancer.

Pancreatic cancer (PC) is a malignant tumor of the digestive system that has a bad prognosis. N6-methyladenosine (m6A) is involved in a wide variety of biological activities due to the fact that it is the most common form of mRNA modification in mammals. Numerous research has accumulated evidence suggesting that a malfunction in the regulation of m6A RNA modification is associated with various illnesses, including cancers. However, its implications in PC remain poorly characterized. The methylation data, level 3 RNA sequencing data, and clinical information of PC patients were all retrieved from the TCGA datasets. Genes associated with m6A RNA methylation were compiled from the existing body of research and made available for download from the m6Avar database. The LASSO Cox regression method was used to construct a 4-gene methylation signature, which was then used to classify all PC patients included in the TCGA dataset into either a low- or high-risk group. In this study, based on the set criteria of |cor| > 0.4 and p value < 0.05. A total of 3507 gene methylation were identified to be regulated by m6A regulators. Based on the univariate Cox regression analysis and identified 3507 gene methylation, 858 gene methylation was significantly associated with the patient's prognosis. The multivariate Cox regression analysis identified four gene methylation (PCSK6, HSP90AA1, TPM3, and TTLL6) to construct a prognosis model. Survival assays indicated that the patients in the high-risk group tend to have a worse prognosis. ROC curves showed that our prognosis signature had a good prediction ability on patient survival. Immune assays suggested a different immune infiltration pattern in patients with high- and low-risk scores. Moreover, we found that two immune-related genes, CTLA4 and TIGIT, were downregulated in high-risk patients. We generated a unique methylation signature that is related to m6A regulators and is capable of accurately predicting the prognosis for patients with PC. The findings might prove useful for therapeutic customization and the process of making medical decisions.

Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

DNA methylation in neurons is directly linked to neuronal genome regulation and maturation. Unlike other tissues, vertebrate neurons accumulate high levels of atypical DNA methylation in the CH sequence context (mCH) during early postnatal brain development. Here, we investigate to what extent neurons derived in vitro from both mouse and human pluripotent stem cells recapitulate in vivo DNA methylation patterns. While human ESC-derived neurons did not accumulate mCH in either 2D culture or 3D organoid models even after prolonged culture, cortical neurons derived from mouse ESCs acquired in vivo levels of mCH over a similar time period in both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition was coincident with a transient increase in Dnmt3a, preceded by the postmitotic marker Rbfox3 (NeuN), was enriched at the nuclear lamina, and negatively correlated with gene expression. We further found that methylation patterning subtly differed between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional noncell autonomous processes. Our findings show that mouse ESC-derived neurons, in contrast to those of humans, can recapitulate the unique DNA methylation landscape of adult neurons in vitro over experimentally tractable timeframes, which allows their use as a model system to study epigenome maturation over development.

Differential methylation pattern in pubertal girls associated with biochemical premature adrenarche

ABSTRACT Biochemical premature adrenarche is defined by elevated serum DHEAS [≥40 μg/dL] before age 8 y in girls. This condition is receiving more attention due to its association with obesity, hyperinsulinemia, dyslipidemia, and polycystic ovary syndrome. Nevertheless, the link between early androgen excess and these risk factors remains unknown. Epigenetic modifications, and specifically DNA methylation, have been associated with the initiation and progression of numerous disorders, including obesity and insulin resistance. The aim of this study was to determine if prepubertal androgen exposure is associated with a different methylation profile in pubertal girls. Eighty-six healthy girls were studied. At age 7 y, anthropometric measurements were begun and DHEAS levels were determined. Girls were classified into Low DHEAS (LD) [<42 μg/dL] and High DHEAS (HD) [≥42 μg/dL] groups. At Tanner stages 2 and 4 a DNA methylation microarray was performed to identify differentially methylated CpG positions (DMPs) between HD and LD groups. We observed a differential methylation pattern between pubertal girls with and without biochemical PA. Moreover, a set of DNA methylation markers, selected by the LASSO method, successfully distinguished between HD and LD girls regardless of Tanner stage. Additionally, a subset of these markers were significantly associated with glucose-related measures such as insulin level, HOMA-IR, and glycaemia. This pilot study provides evidence consistent with the hypothesis that high DHEAS concentration, or its hormonally active metabolites, may induce a unique blood methylation signature in pubertal girls, and that this methylation pattern is associated with altered glucose metabolism.

Abstract 3334: Epigenetic immune cell markers for CLL

Abstract CLL is a rare hematological cancer and is classified as low-malignancy non-Hodgkin lymphoma. While rare, it is the most frequent form of leukemia in Europe and US accounting for approximately a quarter of all leukemias. As it is a slowly growing tumor, its 5-year survival rate is high compared to faster developing B cell tumors and survival has further improved since introduction of Anti-CD20 Antibody therapies. However, overall the 5-year survival of rare cancers (49%) is significantly lower than for their common counterparts (63%), even though hematological cancers improved outcome over the last years. Due to the currently very late and often incidental diagnosis, some cases turn into aggressively progressing cancers prior to treatment and with concomitant poor outcome. Therefore, diagnosis of CLL and other B cell leukemias remains a challenge. To further improve outcome of CLL and other lymphatic leukemias earlier diagnosis is important. To this end, simple home testing could broaden access to diagnostic tools, but no current method would provide adequate remote initial screening of lymphatic leukemia in populations at risk, such as persons older than 65 years. Epigenetic qPCR has been shown to detect and quantify immune cell populations efficiently from liquid and dried blood samples (DBS). This system is based on specifically unmethylated regulatory elements in gene loci associated with certain cell types. These unique methylation patterns allow specific quantification of cells of interest via qPCR. Different epigenetic qPCR systems were developed for total B cells, naïve and memory B cells as well as IgM producing B cells. While these gene loci are fully unmethylated in the respective target cells, they are fully methylated in all relevant control cell types. The cell type specificity of these markers was shown using bisulfite specific sequencing using purified cells from healthy donors. Here, we investigated blood samples from healthy donors (n = 112) as well as patients at the time of their initial CLL diagnosis. The mean total B cell count of healthy donors at 5.7% was clearly lower than the 60.4% obtained for CLL patients. Similarly drastic differences were observed for naïve B (2.3% normal vs. 20.8% CLL) and IgM (3.3% vs. 49.5%) as well as memory B cells (1.6% vs. 32.6%). All differences were statistically significant. To investigate if these mean differences may also lead to differential (and potentially early) diagnosis, we designed receiver operator characteristics and contingency tables, both indicating near perfect segregation between CLL patients and healthy donors in Ig M, memory and all B cells. Our data indicate applicability of epigenetic immune cell counting as easy, potentially DBS- and home-based approach to test increase of immune cells in people potentially at risk of hematological cancers. Citation Format: Janine Jung, Jeannette Werner, Konstantin Schildknecht, Janika Schulze, Steffi Walter, Christoph Sachsenmaier, Deborah Phippard, Barbara Seliger, Claudia Wickenhauser, Sven Olek. Epigenetic immune cell markers for CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3334.

MP30-06 A UNIQUE EPIGENETIC-BASED TEST TO ACCURATELY PREDICT SUCCESSFUL TESE AND mTESE OUTCOMES

You have accessJournal of UrologyCME1 Apr 2023MP30-06 A UNIQUE EPIGENETIC-BASED TEST TO ACCURATELY PREDICT SUCCESSFUL TESE AND mTESE OUTCOMES Tim Jenkins, Ryan Barney, Kelaney Stalker, Ryan Miller, Kristin Brogaard, Jordan Kassab, and Larry Lipshultz Tim JenkinsTim Jenkins More articles by this author , Ryan BarneyRyan Barney More articles by this author , Kelaney StalkerKelaney Stalker More articles by this author , Ryan MillerRyan Miller More articles by this author , Kristin BrogaardKristin Brogaard More articles by this author , Jordan KassabJordan Kassab More articles by this author , and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Affecting 1% of men, non-obstructive azoospermia (NOA) is the absence of sperm in the ejaculate, and is the most severe form of male infertility. Currently, men with NOA have decreased chances of conceiving a child of their own. Despite recent progress in reproductive medicine, men with NOA have essentially one invasive and expensive procedure to isolate sperm (microdissection testicular sperm extraction or mTESE). Unfortunately, mTESE has a ∼50% failure rate. Here we present the use of an innovative DNA methylation-based technology to non-invasively predict the presence of sperm in the testicle of men with NOA. METHODS: At a single academic center biopsies (N=18) and azoospermic semen samples (N=5) were procured from consented men who had undergone a testicular aspirate, biopsy, or mTESE procedure. Utilizing new epigenetic technology, the biopsies and semen samples were analyzed for sperm-specific DNA. Specifically, these samples were analyzed for a sequence of DNA that can distinguish sperm cells from all other somatic cells via a unique methylation pattern. The quantity of sperm-specific DNA can be used to determine the absence (men with <0.25 ratio of sperm to somatic DNA) or presence (>0.5 ratio of sperm to somatic DNA) of sperm in the testicles. These ratios were defined for each sample and compared to clinical outcomes of the TESA, TESE or mTESE procedures. RESULTS: Out of the 18 biopsies, 13 had >0.5 ratios of sperm DNA to somatic cell DNA in the testicular biopsies. All of these men were found to have sperm in their biopsies as defined from the clinical notes. 3 of the 18 men had <0.25 ratios of sperm DNA to somatic cell DNA. Again, consistent with previous analysis, none of these men had sperm in their biopsies. The remaining 2 men had ratios between 0.26 – 0.49, with one having sperm in his biopsy and one without sperm. Of the 5 azoospermic semen samples with associated TESA results, 3 has ratios of <0.25 and all 3 had no sperm in the corresponding TESA samples. 2 semen samples had ratios of >0.5 and were confirmed to have sperm in the TESA sample. CONCLUSIONS: In the analysis of testicular biopsies and semen samples, the sperm-specific DNA methylation ratios of <0.25% or >0.5% were 100% predictive of either the absence or presence of sperm in the testicle. With these data the next steps of our work will focus on continued analysis of sperm-specific cfDNA in seminal plasma to predict the presence or absence of sperm prior to a testicular extraction procedure. The ultimate goal will be to develop a non-invasive and cost-effective way to avoid unnecessary surgical procedures. Source of Funding: Venture Capital © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e393 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tim Jenkins More articles by this author Ryan Barney More articles by this author Kelaney Stalker More articles by this author Ryan Miller More articles by this author Kristin Brogaard More articles by this author Jordan Kassab More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement PDF downloadLoading ...