Hypermethylated Loci Research Articles

DNA methylation patterns in umbilical cord blood from infants of methadone maintained opioid dependent mothers

Methadone maintenance treatment for opioid dependent mothers is standard of care. Infants of methadone maintained opioid dependent (MMOD) mothers have better outcomes compared to infants of opioid dependent mothers without treatment. However, when compared to non-exposed infants, infants of MMOD mothers are associated with worse outcomes. We conducted a pilot study to examine genome wide differential DNA methylation using cord blood samples from sixteen term and near-term infants of MMOD and opioid naïve mothers, excluding Infants with chorioamnionitis. A total of 152 differentially methylated loci were identified at a difference > + 2, < − 2 and p-value < 0.05. There were 90 hypermethylated loci (59 annotated genes) and 62 hypomethylated loci (38 annotated genes) observed. The hypermethylated and hypomethylated DNA changes involved multiple genes, pathways and networks that may explain some of the changes seen in infants of MMOD mothers. Top hypermethylated and hypomethylated genes involved areas of cell growth, neurodevelopment, vision and xenobiotic metabolism functions. Our data may explain the role of key pathways and genes relevant to neonatal outcomes seen from methadone exposure in pregnancy. Functional studies on the identified pathways and genes could lead to improved understanding of the mechanisms and identify areas for intervention.

Clinical validation of Northstar response: A novel methylated ctDNA therapy response monitoring assay in patients with advanced gastrointestinal cancers.

e16000 Background: Circulating tumor DNA (ctDNA) offers a non-invasive method to comprehensively assess tumors. While most assays for therapy response monitoring rely on variant allele frequencies, methylated ctDNA has shown promise. However, accuracy has been limited by quantification precision. The CLIA-approved Northstar Response assay quantifies over 550 hyper-methylated loci and has demonstrated a 10-fold increase in detection signal over tissue-informed assays. Methods: We present an interim analysis from an ongoing prospective, observational study designed to assess the clinical validity of Northstar Response as a tool to monitor therapeutic response without prior tumor tissue. Our study aims to include 100 advanced GI cancer patients undergoing systemic therapies for advanced disease. Serial ctDNA measurements are collected at baseline, approximately every 30 days post-treatment initiation, and with imaging assessments. The primary endpoint is concordance between ctDNA and RECISTv1.1 criteria for disease response until progression or death. Results: Baseline ctDNA characteristics were examined from 44 patients, with 23 undergoing at least two longitudinal blood collections and subsequent imaging. The median age in this cohort was 65.5 years, with a male predominance (70.5%) and a majority identifying as White (79.5%). Cancers included esophageal (18.2%), hepatocellular (29.5%), biliary (15.9%) and colorectal (15.9%), among others. A majority (59.1%) had both primary and metastatic lesions. Baseline methylation scores ranged from 280 in biliary cancers to 100,000 in colorectal cancers. Those on second-line treatments had lower scores than first or third-line therapies. Methylation scores decreased in patients responding to treatment, indicating that these scores have the potential to serve as indicators of therapeutic efficacy. Conclusions: Baseline methylation scores differ across GI tumor types, disease burdens, and treatment lines, reflecting the complexity of using methylation as a biomarker. Northstar Response has shown initial promise in monitoring therapy response in GI cancers. More mature data, which will further elucidate the assay's clinical utility, will be presented at the meeting.

Nanoparticles loaded with the DNA methyltransferase inhibitor SGI-1027 decrease murine atherosclerosis and inflammation in cultured human macrophages

Aim: The DNA of the atheroma is hypermethylated relative to adjacent healthy vascular tissue. A significant portion of hypermethylated loci in the atheroma DNA map to genes related to macrophage function. Reversing macrophage DNA methylation to physiological levels by targeting DNA methyltransferase (DNMT) activity may therefore slow atherogenesis. Here, the anti-inflammatory and anti-atherogenic activity of macrophage-targeted DNMT inhibitor SGI-1027 were tested. Methods: SGI-1027 was encapsulated into human serum albumin (HSA) nanoparticle (HSANP) functionalized with the PP1 peptide, a macrophage scavenger receptor 1 ligand, fused to a FLAG epitope (S-HSANP-FLAGPP1). Results: Nanoparticle physico-chemical characteristics predicted good marginalization towards the vascular wall, although SGI-1027 encapsulation efficiency was relatively low (~23%). S-HSANP-FLAGPP1 were rapidly internalized compared to non-functionalized and, surprisingly, functionalized void controls, and induced a shift towards an anti-inflammatory profile of secreted cytokines in human THP-1 macrophages. S-HSANP-FLAGPP1 colonized the atheroma and induced a significant ~44% reduction of atherosclerosis burden in the aortic tree of apolipoprotein E (ApoE)-null mice compared to controls. A reduction in aortic root atherosclerosis was observed, although primarily induced by HSANP irrespective of loading or functionalization. No alteration of body weight, non-vascular tissue gross histology, plasma glucose, triglyceride or cholesterol were observed. HSA whether free or structured in nanoparticles, induced a 3–4-fold increase in high-density lipoprotein (HDL) compared to vehicle. Conclusions: Unexpectedly, effects that were likely non-epigenetic and induced by HSA per se were observed. HSANP loaded with SGI-1027 were anti-atherogenic but in an anatomical location-dependent fashion. SGI-1027 displayed a novel anti-inflammatory activity in non-proliferating THP-1 cells, implying that those effects are likely unrelated to DNMT inhibition. HSA elevated HDL per se, thus underlining a possible additional advantage of HSA-based nanocarriers.

Abstract C058: Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men

Abstract The incidence of penile squamous cell carcinoma (PSCC) in Puerto Rico is nearly three times higher when compared with other racial and ethnic groups in the United States. Mortality rate for PSCC is also significantly higher among Puerto Rican men when compared to the US population. Human papillomavirus (HPV) is a common virus, and its infection may increase the risk of developing PeCa. Despite the high incidence, the molecular mechanism of PSCC remains poorly understood. Our laboratory found that the prevalence of HPV in PSCC in Puerto Rican men is 49%. Due to the high rates of HPV infection and increased incidence of penile cancer in Puerto Rican men, there is a need to understand the pathogenesis of penile cancer. DNA Methylation is an epigenetic alteration that can be found in cancer cells, which can lead to changes in gene expression and cellular function. This study aims to analyze the DNA methylation patterns in HPV-positive and HPV-negative PSCC tissues from Puerto Rican men. DNA was extracted from twenty-four fresh PSCC tissue samples from Puerto Rican patients. HPV status and genotyping were determined using the DNA ELISA kit HPV SPF10 protocol. DNA was amplified and hybridized using PCR. We perform a DNA methylation assay using the Infinium Methylation EPIC v2.0 BeadChip Kit to identify and compare average DNA methylation levels (%) in the 9 HPV-positive and 15 HPV-negative penile cancer samples. Our results demonstrated 43 loci significantly different between HPV-positive and HPV-negative samples, with an adjusted p-value &amp;lt;0.1 (Benjamini-Hochberg method). Most of the hypermethylated loci were present in HPV-negative tumor samples. Additionally, we identified four methylated-differentially expressed genes: ANXA4, CACNA1C-AS1, UBXN2A, and DUOX1, which are found as uncharacterized proteins (open reading frames, ORFs) of the human genome with adjusted p-value &amp;lt;0.05. Studies have revealed that specific DNA methylation patterns could be linked with cancer development and progression. Identification of differentially methylated regions in HPV positive (HPV+) PSCC compared with HPV negative (HPV-) cases will provide much needed data to aid studies on early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Identification of differentially expressed genes and pathways in HPV positive (HPV+) PeCa compared with HPV negative (HPV-) cases will provide much needed data to aid studies on prevention, early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Citation Format: Yarelis M. Roque Reyes, María Sánchez-Vázquez, Josué Perez, Luis R. Llanos, Ingrid Montes, Jasmine Figueroa-Díaz, Carlos Rivera-Lopez, María Marcos, Antonio Puras, Magaly Martínez-Ferrer. Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican 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 C058.

G6PD Orchestrates Genome-Wide DNA Methylation and Gene Expression in the Vascular Wall.

Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and the de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of epigenetic writers and erasers, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. In the aorta of CRISPR-edited rats with the Mediterranean G6PD variant, we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Here, we documented higher expression of Dnmt1, Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aortas from G6PDS188F as compared to wild-type rats. Our results demonstrated that nitric oxide, which is generated in a G6PD-derived NADPH-dependent manner, increases TET and decreases DNMT activity. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveal that the G6PDS188F variant contributes to reducing large artery stiffness.

DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload

Left ventricular (LV) dilatation, a prominent risk factor for heart failure (HF), precedes functional deterioration and is used to stratify patients at risk for arrhythmias and cardiac mortality. Aberrant DNA methylation contributes to maladaptive cardiac remodeling and HF progression following pressure overload and ischemic cardiac insults. However, no study has examined cardiac DNA methylation upon exposure to volume overload (VO) despite being relatively common among HF patients. We carried out global methylome analysis of LV harvested at a decompensated HF stage following exposure to VO induced by aortocaval shunt. VO resulted in pathological cardiac remodeling, characterized by massive LV dilatation and contractile dysfunction at 16 weeks after shunt. Although methylated DNA was not markedly altered globally, 25 differentially methylated promoter regions (DMRs) were identified in shunt vs. sham hearts (20 hypermethylated and 5 hypomethylated regions). The validated hypermethylated loci in Junctophilin-2 (Jph2), Signal peptidase complex subunit 3 (Spcs3), Vesicle-associated membrane protein-associated protein B (Vapb), and Inositol polyphosphate multikinase (Ipmk) were associated with the respective downregulated expression and were consistently observed in dilated LV early after shunt at 1 week after shunt, before functional deterioration starts to manifest. These hypermethylated loci were also detected peripherally in the blood of the shunt mice. Altogether, we have identified conserved DMRs that could be novel epigenetic biomarkers in dilated LV upon VO exposure.

Application of Novel Breast Biospecimen Cell-Type Adjustment Identifies Shared DNA Methylation Alterations in Breast Tissue and Milk with Breast Cancer-Risk Factors.

DNA methylation patterning is cell-type-specific and altered DNA methylation is well established to occur early in breast carcinogenesis, affecting non-cancerous, histopathologically normal breast tissue. Previous work assessing risk factor-associated alterations to DNA methylation in breast tissue has been limited, with even less published research in breast milk, a noninvasively obtained biospecimen containing sloughed mammary epithelial cells that may identify early alterations indicative of cancer risk. Here, we present a novel library for the estimation of the cellular composition of breast tissue and milk and subsequent assessment of cell-type-independent alterations to DNA methylation associated with established breast cancer-risk factors in solid breast tissue (n = 95) and breast milk (n = 48) samples using genome-scale DNA methylation measures from the Illumina HumanMethylation450 array. We identified 772 hypermethylated CpGs (P < 0.01) associated with age consistent between breast tissue and breast milk samples. Age-associated hypermethylated CpG loci were significantly enriched for CpG island shore regions known to be important for regulating gene expression. Among the overlapping hypermethylated loci mapping to genes, a differentially methylated region was identified in the promoter region of SFRP2, a gene observed to undergo promoter hypermethylation in breast cancer. Our findings suggest the potential to identify epigenetic biomarkers of breast cancer risk in noninvasively obtained, tissue-specific breast milk specimens. This work demonstrates the potential of using breast milk as a noninvasive biomarker of breast cancer risk, improving our ability to detect early-stage disease and lowering the overall disease burden.

Vitamin C boosts DNA demethylation in TET2 germline mutation carriers

BackgroundAccurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation.MethodsIn a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members.ResultsWe show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals.ConclusionsThese results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations.Trial registration: This trial was registered at EudraCT with reference number of 2018-000155-41 (01.04.2019).Graphical

A DNA methylation atlas of normal human cell types

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

SARS-CoV-2 Covid-19 Infection During Pregnancy and Differential DNA Methylation in Human Cord Blood Cells From Term Neonates.

The global pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 18.4% of total Covid-19 cases were reported in children. Even though vertical transmission from mother to infant is likely to occur at a low rate, exposure to COVID-19 during fetal life may alter DNA methylation patterns with potential long-term effects. To determine if COVID-19 infection during pregnancy alters the DNA methylation patterns in umbilical cord blood cells from term infants and to identify potential pathways and genes affected by exposure to COVID-19 infection. Umbilical cord blood was collected from 8 infants exposed to COVID-19 during pregnancy and 8 control infants with no COVID-19 exposure. Genomic DNA was isolated from umbilical cord blood cells and genome-wide DNA methylation was performed using Illumina Methylation EPIC Array. 119 differentially methylated loci were identified at the FDR level of 0.20 (64 hypermethylated loci and 55 hypomethylated loci) in umbilical cord blood cells of COVID-19 exposed neonates compared to the control group. Important canonical pathways identified by Ingenuity Pathway Analysis (IPA) were related to stress response (corticotropin releasing hormone signaling, glucocorticoid receptor signaling, and oxytocin in brain signaling pathway), and cardiovascular disease and development (nitric oxide signaling in the cardiovascular system, apelin cardiomyocyte signaling pathways, factors promoting cardiogenesis, and renin-angiotensin signaling). The genes affected by the differential methylations were associated with cardiac, renal, hepatic, neurological diseases, developmental and immunological disorders. COVID-19 induces differential DNA methylation in umbilical cord blood cells. The differentially methylated genes may contribute to hepatic, renal, cardiac, developmental and immunological disorders in offspring born to mothers with COVID-19 infection during pregnancy, and their developmental regulation.

DNA methylome in pancreatic cancer identified novel promoter hyper-methylation in NPY and FAIM2 genes associated with poor prognosis in Indian patient cohort

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the leading cancers worldwide and has a poor survival, with a 5-year survival rate of only 8.5%. In this study we investigated altered DNA methylation associated with PDAC severity and prognosis.MethodsMethylome data, generated using 450 K bead array, was compared between paired PDAC and normal samples in the TCGA cohort (n = 9) and our Indian cohort (n = 7). The total Indian Cohort (n = 75) was split into cohort 1 (n = 7), cohort 2 (n = 22), cohort 3 (n = 26) and cohort 4 (n = 20).Validation of differential methylation (6 selected CpG loci) and associated gene expression for differentially methylated genes (10 selected gDMs) were carried out in separate validation cohorts, using MSP, RT-PCR and IHC correlations between methylation and gene expression were observed in TCGA, GTEx cohorts and in validation cohorts. Kaplan–Meier survival analysis was done to study differential prognosis, during 2–5 years of follow-up.ResultsWe identified 156 DMPs, mapped to 91 genes (gDMs), in PDAC; 68 (43.5%) DMPs were found to be differentially methylated both in TCGA cohort and our cohort, with significant concordance at hypo- and hyper-methylated loci. Enrichments of “regulation of ion transport”, “Interferon alpha/beta signalling”, “morphogenesis and development” and “transcriptional dysregulation” pathways were observed among 91 gDMs. Hyper-methylation of NPY and FAIM2 genes with down-regulated expression in PDAC, were significantly associated with poor prognosis in the Indian patient cohort.ConclusionsEthnic variations among populations may determine the altered epigenetic landscape in the PDAC patients of the Indian cohort. Our study identified novel differentially methylated genes (mainly NPY and FAIM2) and also validated the previously identified differentially methylated CpG sites associated with PDAC cancer patient’s survival. Comparative analysis of our data with TCGA and CPTAC cohorts showed that both NPY and FAIM2 hyper-methylation and down-regulations can be novel epigenetically regulated genes in the Indian patient population, statistically significantly associated with poor survival and advanced tumour stages.

H3K27me3 shapes DNA methylome by inhibiting UHRF1-mediated H3 ubiquitination

DNA methylation and histone lysine tri-methylation at H3K27 (H3K27me3) are two chromatin modifications for transcriptional gene silencing, which play important roles in diverse biological processes, including cell fate determination and cell lineage commitment. These two marks are largely mutually exclusive and target distinct sets of genes in the mammalian genome. However, how H3K27me3 shapes the DNA methylome remains elusive. Here, we report that the loss of H3K27me3 modification leads to increased DNA methylation at previously marked H3K27me3 sites, indicating that H3K27me3 negatively regulates DNA methylation. Genome-wide analysis of H3 ubiquitination, essential for recruitment and activation of DNA methyltransferase DNMT1, reveals the absence of H3 ubiquitination at H3K27me3 marked nucleosomes. Moreover, loss of H3K27me3 modification induces an increase in H3K18 ubiquitination at the corresponding hyper-methylated loci. Importantly, we show that H3K27me3 directly inhibits UHRF1-mediated H3 ubiquitination toward nucleosomes in a defined biochemical assay. Taken together, our findings reveal a general mechanism for H3K27me3-mediated shaping of the mammalian DNA methylome via modulation of H3 ubiquitination.

PD53-08 E-CIGARETTE EXPOSURE IN MICE PROMOTES CHANGES IN DNA METHYLATION OF THE BLADDER UROTHELIUM

You have accessJournal of UrologyCME1 May 2022PD53-08 E-CIGARETTE EXPOSURE IN MICE PROMOTES CHANGES IN DNA METHYLATION OF THE BLADDER UROTHELIUM Ibardo Zambrano, Ujjawal Manocha, Weida Gong, Yi-Hsuan Tsai, Manhua Zhu, Melissa Herman, Marc Bjurlin, and William Kim Ibardo ZambranoIbardo Zambrano More articles by this author , Ujjawal ManochaUjjawal Manocha More articles by this author , Weida GongWeida Gong More articles by this author , Yi-Hsuan TsaiYi-Hsuan Tsai More articles by this author , Manhua ZhuManhua Zhu More articles by this author , Melissa HermanMelissa Herman More articles by this author , Marc BjurlinMarc Bjurlin More articles by this author , and William KimWilliam Kim More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To date, no group has studied the epigenetic effects of e-cigarettes (EC) on the urothelium. DNA methylation changes appear to be one of the earliest events in bladder tumorigenesis and can be readily found in normal urothelium adjacent to bladder tumors. EC exposure causes urothelial hyperplasia in mice but not bladder tumors. We Vtherefore asked whether EC exposure results in early field alterations of DNA methylation in the bladder urothelium. METHODS: Mice (4 females, 4 males; 35 weeks old) were exposed to 12% nicotine in 1:1 polyethylene glycol: vegetable glycerol or vehicle daily: 3 hr sessions, 3 sec vape duration, 10 min interval between vapes. Plasma nicotine and cotinine serum concentrations were measured using LC-MS. Reduced-representation bisulfite sequencing (RRBS) was performed using DNA from microdissected urothelium harvested at 4 weeks (Zymo Research Kit). Number of loci covered by each sample was determined and data was filtered to only include loci with >10 reads. Differentially methylated loci were determined (p<0.01). Gene Set Enrichment Analysis was performed to contextualize the DNA methylation changes. RESULTS: No significant adverse events were observed in the mice during the exposure period to either vehicle or nicotine. Acute and repeated exposure resulted in significantly higher levels of nicotine and cotinine serum concentration in the nicotine vs. the vehicle group (Figure 1). No bladder tumors formed. RRBS analysis demonstrated that EC exposure resulted in hypermethylation of the promoters of 665 genes and hypomethylation of 285 genes after accounting for sex (p <0.05) Pathway analysis showed enrichment of MYC target genes in hypomethylated regions and enrichment of genes associated with oxidative phosphorylation, cilium assembly, and ciliopathies in hypermethylated loci. CONCLUSIONS: Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. vehicle control. EC metabolites in urine may result in aberrant methylation patterns in the bladder urothelium. Results are suggestive of a pre-malignant phenotype driven by EC exposure via stimulation of MYC targets and silencing of genes responsible for establishing primary cilia. Loss of primary cilia in epithelial cells has been associated with loss of cell polarity and increased cell migration. Source of Funding: NCI Geographical Management of Cancer Health Disparities Program (3P30-CA016086), UNC Lineberger Innovation Award 2020 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e912 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ibardo Zambrano More articles by this author Ujjawal Manocha More articles by this author Weida Gong More articles by this author Yi-Hsuan Tsai More articles by this author Manhua Zhu More articles by this author Melissa Herman More articles by this author Marc Bjurlin More articles by this author William Kim More articles by this author Expand All Advertisement PDF DownloadLoading ...

A pilot study that provides evidence of epigenetic changes among mother-child pairs living proximal to mining in the US.

Environmental exposures to chemicals can disrupt gene expression, and the effects could be mediated by methylation. This investigation focused on methylation of genes associated with exposure to metals. Mother–child pairs from three locations in Montana were recruited, and buccal cells were collected for genome-wide methylation assay. Four pairs were from Butte, where there is mining and a Superfund site, four pairs were from Anaconda with a Superfund site, and four pairs were from Missoula with neither a mine nor a Superfund site. Principal component analysis, linear mixed models, hierarchical clustering and heatmap, and gene set enrichment analysis were used to visualize the profiles, identify the top associated methylation loci, and investigate the involved pathways. Distinctly higher or lower methylation in samples from Butte were found at the top differentially methylated loci. The 200 genes harboring the most hypermethylated loci were significantly enriched in genes involved in actin cytoskeleton regulation, ABC transporters, leukocyte transendothelial migration, focal adhesion, and adherens junction, which plays a role in pathogenesis of disease, including autism spectrum disorders. This study lays a foundation for inquiry about genetic changes associated with environmental exposure to metals for people living in proximity to Superfund and open pit mining.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10653-022-01217-9.

Genome-Scale DNA Methylation Analysis Identifies Repeat Element Alterations that Modulate the Genomic Stability of Melanocytic Nevi

Acquired melanocytic nevi grow and persist in a stable form into adulthood. Using genome-wide methylation profiling, we evaluated 32 histopathologically and dermoscopically characterized nevi to identify the key epigenetic regulatory mechanisms involved in nevogenesis. Benign (69% globular and 31% nonspecific dermoscopic pattern) and dysplastic (95% reticular/nonspecific dermoscopic pattern) nevi were dissimilar, with only two shared differentially methylated loci. Benign nevi showed an increase in both genome-scale methylation and methylation of Alu/LINE-1 retrotransposable elements, a marker of genomic stability, as well as global methylation. In contrast, dysplastic nevi showed evidence for genomic instability through the hypomethylation of Alu/LINE-1 (Alu: P= 0.00019; LINE-1: P= 0.000035). Using dermoscopic classifications, reticular/nonspecific patterned nevi had 59,572 5'-C-phosphate-G-3' differentially methylated loci (Q < 0.05), whereas globular nevi had no significant differentially methylated loci. In reticular/nonspecific patterned nevi, the tumor suppressor PTEN had the greatest proportion of hypermethylated 5'-C-phosphate-G-3' loci in its promoter region than all other assayed gene promoters. The relative activity of reticular/nonspecific nevi was evidenced by 50,720 hypomethylated loci being enriched for accessible chromatin and 8,852 hypermethylated loci strongly enriched, for example, marks of active gene promoters, which suggests that gain of DNA methylation observed in these nevus types plays a role in gene regulation.

TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

Integrative Genomic Analysis of Adult Mixed Phenotype Acute Leukemia (MPAL)

IntroductionMixed phenotype acute leukemia (MPAL) is a rare subgroup of acute leukemia characterized by blasts presenting both myeloid and lymphoid lineages. Standard of care for MPAL is not defined and prognosis of MPAL is poor. Genetic basis for mixed immunophynotype and distinct molecular characteristics from other leukemia is not well understood.MethodsWe studied 31 bone marrow samples from previously untreated MPAL patients (median age 53) meeting WHO criteria. Genomic DNA/RNA from bone marrow samples were analyzed by comprehensive genomic analysis including targeted capture sequencing of 295 genes (median 393x coverage), RNA sequencing, and Infinium methylation EPIC array (Illumina). Copy number alterations (CNA) were analyzed from methylation array data using ChAMP algorithm. Mutational landscape of MPAL was compared with194 AML, 71 B-ALL, and 14 T-ALL cases sequenced with the same platform. CpG methylation pattern of MPAL was also compared with 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (data shared by Nordlund et al. Genome Biology. 2013).ResultsOf the 31 MPAL cases, 18 (58%) had myeloid-T and 13 (42%) had myeloid-B phenotype. Four (13%) cases had Philadelphia chromosome, one (3%) had 11q23 rearrangement, and 8 (26%) had complex karyotypes. Eighteen patients (62%) were treated with ALL regimen, 8 (28%) received AML regimen, and 3 (10%) received other therapy.Targeted sequencing revealed 86 driver mutations (55 SNVs and 31 indels) in 35 genes in 29 samples (94%). MPAL carried median 2 (range 0-6) mutations per case that was similar to AML (P = 0.79) and T-ALL (P = 0.92) but was significantly higher than B-ALL(P < 0.001). The most frequently mutated genes were DNMT3A , IDH2 , NOTCH1 , and RUNX1 in 7 (23%) cases each (Figure 1). Myeloid-T and myeloid-B showed distinct patterns of mutations, in which mutations in NOTCH pathway genes had strong trend of enrichment in myeloid-T (P = 0.06), whereas RUNX1 mutations were significantly enriched in myeloid-B (P = 0.02). When mutation landscape was compared among AML, B-ALL, T-ALL and MPAL, TP53 , NRAS , and DNMT3A mutations were shared across all leukemia. In contrast, NPM1 mutation was specific to AML and mutations in NOTCH1 , FBXW7 , and IL7R were specific to myeloid-T MPAL and T-ALL. CNA analysis showed that CDKN2B as a significantly deleted gene in MPAL.Unsupervised hierarchical clustering of promoter methylation revelaed two distinct clusters. This methylation cluster had significant correlation with immunophenotype (73% of cluster 1 was myeloid-B and 88% of cluster 2 was myeloid-T, P < 0.001). Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B (mean beta value 0.28 vs. 0.52 in myeloid-B vs. myeloid-T, P = 0.002). This difference in methylation was independent from IDH mutation. Molecular pathway analysis of differentially methylated probes (DMP) showed that T-cell receptor pathway, T cell differentiation, and activation pathway genes (CD3D , CD247 , LCK , PRF1 , RHOH , and PRKCQ ) were differentially methylated and were expressed between myeloid-B and myeloid-T. Furthermore, motif enrichment analysis of promoter DMP revealed that IRF8 and IRF4 binding motifs were significantly hyper-methylated in myeloid-T compared to myeloid-B. Consistent with this, RAG1 , RAG2 , CXCR4 and PAX5 , downstream targets of IRF4/IRF8 andessential genes in B-cell development, were differentially expressed between two phenotypes. RNA sequencing revealed two known translocations (NSD1-NUP98 , KMT2A-MLLT4 ) in addition to the novel translocations such as FOXP1-DNAJC15 , TNKS - LYZ , and NOP14 - PLEC in MPAL. We did not detect ZN384 fusions that have been reported in pediatric MPAL (Alexander et al. ASH 2016). Unsupervised hierarchical clustering of methylation pattern among MPAL, AML, B-ALL and T-ALL revealed that 41% and 59% of MPAL had AML-like and ALL-like methylation pattern, respectively (Figure 2). Patients who received therapy that matched with their methylation pattern had significantly better response to treatment than patients who received unmatched therapy (CR rate: 77% vs. 31%, P = 0.024).ConclusionMyeloid-T and myeloid-B MPAL showed distinct patterns of mutation, methylation and gene expressions. Methylation data could identify AML-like and ALL-like MPAL which corresponded to better response to therapy when matched therapy was given. These data suggest that molecularly guided therapy may be beneficial in the treatment of MPAL. [Display omitted] DisclosuresTakahashi:Symbio Pharmaceuticals: Consultancy. DiNardo:Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

A 10-gene-methylation-based signature for prognosis prediction of colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignant tumor of digestive tract which has high incidence and mortality rates. Accurate prognosis prediction of CRC patients is pivotal to reduce the mortality and disease burden. MethodsIn this study, we comprehensively analyzed the gene expression and methylation data of CRC samples from The Cancer Genome Atlas (TCGA). Differential expression genes (DEGs) and methylation CpGs (DMCs) in tumor tissues compared with adjacent normal tissues of CRC were first identified. Functional enrichment analysis of DEGs and DMCs was performed by Database for Annotation, Visualization and Integrated Discovery (DAVID). Spearman correlation analysis was used to screen DMCs that negatively correlated with gene expressions which were subsequently applied to sure independence screening (SIS) along with stepwise regression for screening optimal CpGs for CRC prognosis prediction model construction by Cox regression analysis. ResultsWe identified a total of 1774 DEGs (663 upregulated and 1111 downregulated) and 11,975 DMCs (7385 hypermethylated and 4590 hypomethylated) in CRC tumor samples compared with adjacent normal samples. The hypermethylated loci were mainly located on CpG island, while the hypomethylated loci were mainly located on N-shore. Spearman correlation analysis screened 321 DMCs that negatively correlated with expressions of their annotated genes. Cox regression model consist of 10 CpGs was finally established which could effectively stratified CRC patients that exhibited significantly different overall survival probability independent of age, gender, and pathological staging. ConclusionWe established a prognosis prediction model based on 10 methylation sites, which could evaluate the prognosis of CRC patients.

Copper stress induces zebrafish central neural system myelin defects via WNT/NOTCH-hoxb5b signaling and pou3f1/fam168a/fam168b DNA methylation

Unbalanced copper (Cu) homeostasis is associated with neurological development defects and diseases. However, the molecular mechanisms remain elusive. Here, central neural system (CNS) myelin defects and the down-regulated expression of WNT/NOTCH signaling and its down-stream mediator hoxb5b were observed in Cu2+ stressed zebrafish larvae. The loss/knockdown-of-function of hoxb5b phenocopied the myelin and axon defects observed in Cu2+ stressed embryos. Meanwhile, the activation of WNT/NOTCH signaling and ectopic expression of hoxb5b could rescue Cu induced myelin defects. Additionally, fam168b, similar to pou3f1/2, exhibited significant promoter hypermethylation and reduced expression in Cu2+ stressed embryos. The hypermethylated locus in fam168b promoter acted pivotally in its transcription, and the loss/knockdown of fam168b/pou3f1 also induced myelin defects. This study also demonstrated that fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis: Cu induced the down-regulated expression of the WNT&NOTCH-hoxb5b axis through the function of copper transporter cox17, coupled with the promoter methylation of genes fam168b/pou3f1, and its subsequent down-regulated expression through the function of another transporter atp7b, making joint contributions to myelin defects in embryos.

MicroRNA-30a-5pme: a novel diagnostic and prognostic biomarker for clear cell renal cell carcinoma in tissue and urine samples

BackgroundThe rising incidence of renal cell carcinomas (RCC) constitutes a significant challenge owing to risk of overtreatment. Because aberrant microRNA (miR) promoter methylation contributes to cancer development, we investigated whether altered miR-30a-5p expression associates with DNA promoter methylation and evaluated the usefulness as clear cell RCC (ccRCC) diagnostic and prognostic markers.MethodsGenome-wide methylome and RNA sequencing data from a set of ccRCC and normal tissue samples from The Cancer Genome Atlas (TCGA) database were integrated to identify candidate CpG loci involved in cancer onset. MiR-30a-5p expression and promoter methylation were quantitatively assessed by PCR in a tissue set (Cohort #1) and urine sets (Cohorts #2 and 3) from IPOPorto and Homburg University Hospital. Non-parametric tests were used for comparing continuous variables. MiR-30a-5p promoter methylation (miR-30a-5pme) performance as diagnostic (receiver operator characteristics [ROC] - validity estimates) and prognostic [metastasis-free (MFS) and disease-specific survival (DSS)] biomarker was further validated in urine samples from ccRCC patients by Kaplan Meier curves (with log rank) and both univariable and multivariable analysis.ResultsTwo significant hypermethylated CpG loci in TCGA ccRCC samples, correlating with miR-30a-5p transcriptional downregulation, were disclosed. MiR-30a-5pme in ccRCC tissues was confirmed in an independent patient’s cohort of IPOPorto and associated with shorter time to relapse. In urine samples, miR-30a-5pme levels identified cancer both in testing and validation cohorts, with 83% sensitivity/53% specificity and 63% sensitivity/67% specificity, respectively. Moreover, higher miR-30a-5pme levels independently predicted metastatic dissemination and survival.ConclusionTo the best of our knowledge, this is the first study validating the diagnostic and prognostic potential of miR-30a-5pme for ccRCC in urine samples, providing new insights for its clinical usefulness as non-invasive cancer biomarker.