Difference In Global DNA Methylation Research Articles

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2'-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2.

DNA Methylation as a Biomarker for Monitoring Disease Outcome in Patients with Hypovitaminosis and Neurological Disorders.

DNA methylation remains an under-recognized diagnostic biomarker for several diseases, including neurodegenerative disorders. In this study, we examined differences in global DNA methylation (5mC) levels in serum samples from patients during the initial- and the follow-up visits. Each patient underwent a blood analysis and neuropsychological assessments. The analysis of 5mC levels revealed two categories of patients; Group A who, during the follow-up, had increased 5mC levels, and Group B who had decreased 5mC levels. Patients with low Fe-, folate-, and vitamin B12- levels during the initial visit showed increased levels of 5mC after treatment when assessed during the follow-up. During the follow-up, 5mC levels in Group A patients increased after treatment for hypovitaminosis with the nutraceutical compounds Animon Complex and MineraXin Plus. 5mC levels were maintained during the follow-up in Group A patients treated for neurological disorders with the bioproducts AtreMorine and NeoBrainine. There was a positive correlation between 5mC levels and MMSE scores, and an inverse correlation between 5mC and ADAS-Cog scores. This expected correlation was observed in Group A patients only. Our study appears to indicate that 5mC has a diagnostic value as a biomarker across different pathologies.

Epigenetic and Phenotypic Responses to Experimental Climate Change of Native and Invasive Carpobrotus edulis.

Despite the recent discoveries on how DNA methylation could help plants to adapt to changing environments, the relationship between epigenetics and climate change or invasion in new areas is still poorly known. Here, we investigated, through a field experiment, how the new expected climate scenarios for Southern Europe, i.e., increased temperature and decreased rainfall, might affect global DNA methylation in relation to phenotypic variation in individuals of clonal plant, Carpobrotus edulis, from its native (Southern African) and invaded (northwestern Iberian Peninsula) area. Our results showed that changes in temperature and rainfall induced phenotypic but not global DNA methylation differences among plants, and the climatic effects were similar for plants coming from the native or invaded areas. The individuals from the Iberian Peninsula showed higher levels of global methylation than their native counterparts from South Africa. We also observed differences between natives and invasive phenotypes in traits related to the pattern of biomass partitioning and to the strategies for water uptake and use and found an epigenetic contribution to phenotypic changes in some leaf traits, especially on the nitrogen isotopic composition. We conclude that the increased temperature and decreased rainfall projected for Southern Europe during the course of the twenty-first century may foster phenotypic changes in C. edulis, possibly endowing this species with a higher ability to successful cope the rapid environmental shifts. The epigenetic and phenotypic divergence that we observed between native and invasive plants suggests an intraspecific functional variation during the process of invasion. This result could indicate that phenotypic plasticity and global DNA methylation are related to the colonization of new habitats. Our findings reinforce the importance of epigenetic plasticity on rapid adaptation of invasive clonal plants.

Transgenerational effects of polyethylene microplastic fragments containing benzophenone-3 additive in Daphnia magna

Maternal exposure to microplastics (MPs) plays an important role in the fitness of unexposed progeny. In this study, the transgenerational effects of polyethylene MP fragments (17.35 ± 5.50 µm) containing benzophenone‐3 (BP-3; 2.85 ± 0.16% w/w) on chronic toxicity (21 d) in Daphnia magna were investigated across four generations. Only D. magna in the F0 generation was exposed to MP fragments, MP/BP-3 fragments, and BP-3 leachate to identify the transgenerational effect in the F3 generation. The mortality of D. magna induced by MP and MP/BP-3 fragments was recovered in the F3 generation, but somatic growth and reproduction significantly decreased compared to the control. Additionally, reproduction of D. magna exposed to BP-3 leachate significantly decreased in the F3 generation. These findings confirmed the transgenerational effects of MP fragment and BP-3 additive on D. magna. Particularly, the adverse effect on D. magna reproduction seemed to be cumulative across four generations for MP/BP-3 fragments, while it was an acclimation trend for BP-3 leachate. However, there was no significant difference in global DNA methylation in D. magna across four generations, thus requiring a gene-specific DNA methylation study to identify different epigenetic transgenerational inheritance.

Epigenetic MRI: Noninvasive imaging of DNA methylation in the brain

Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here, we present a method, epigenetic MRI (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene-expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C-labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.

Upper Aerodigestive Tract Squamous Cell Carcinomas Show Distinct Overall DNA Methylation Profiles and Different Molecular Mechanisms behind WNT Signaling Disruption.

Simple SummarySquamous cell carcinomas of the upper aerodigestive tract are highly incident, lethal, and share the same epithelial lining of origin, risk factors and genetic alterations. However, their biological and clinical behaviors differ, having an impact on patient survival. This study aimed at identifying the main DNA methylation differences between these tumors, giving an overview of the main genomic regions affected, whether DNA methylation gains or losses are more common, the impact on gene expression and the signaling pathways affected. This knowledge will help identifying potential site-specific biomarkers as well as shedding light on whether epigenetic mechanisms explain, at least in part, the diverse behavior of upper aerodigestive tract tumors.Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

Deregulation of imprinted genes expression and epigenetic regulators in placental tissue from intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a fetal growth complication that can be caused by ineffective nutrient transfer from the mother to the fetus via the placenta. Abnormal placental development and function have been correlated with abnormal expression of imprinted genes, which are regulated by epigenetic modifications at imprinting control regions (ICRs). In this study, we analyzed the expression of imprinted genes known to be involved in fetal growth and epigenetic regulators involved in DNA methylation, as well as DNA methylation at the KvDMR1 imprinting control region and global levels of DNA hydroxymethylation, in IUGR cases. Expression levels of imprinted genes and epigenetic regulators were analyzed in term placental samples from 21 IUGR cases and 9 non-IUGR (control) samples, by RT-qPCR. Additionally, KvDMR1 methylation was analyzed by bisulfite sequencing and combined bisulfite restriction analysis (COBRA) techniques. Moreover, global DNA methylation and hydroxymethylation levels were also measured. We observed increased expression of PHLDA2, CDKN1C, and PEG10 imprinted genes and of DNMT1, DNMT3A, DNMT3B, and TET3 epigenetic regulators in IUGR placentas. No differences in methylation levels at the KvDMR1 were observed between the IUGR and control groups; similarly, no differences in global DNA methylation and hydromethylation were detected. Our study shows that deregulation of epigenetic mechanisms, namely increased expression of imprinted genes and epigenetic regulators, might be associated with IUGR etiology. Therefore, this study adds knowledge to the molecular mechanisms underlying IUGR, which may contribute to novel prediction tools and future therapeutic options for the management of IUGR pregnancies.

Glutathione Protects the Developing Heart from Defects and Global DNA Hypomethylation Induced by Prenatal Alcohol Exposure.

Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C2 H5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant." Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts. GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues.

Global DNA methylation levels in white blood cells of patients with chronic heroin use disorder. A prospective study.

Increasing scientific evidence shows the significant role of epigenetic mechanisms in drug use disorder, abstinence and relapse. Studies on human subjects are limited compared to those on animals, for various reasons such as poly-substance abuse, high drop-out rate and technical difficulties. Our goal was to evaluate whether a monitored abstinence period of 21 days could induce changes in global DNA methylation in chronic heroin users. In the current study, we present data on global DNA methylation on a set of 18 male patients with chronic heroin use disorder, carefully selected based on inclusion and exclusion criteria, who were hospitalized and closely monitored during a 21-day detoxification program, one of the few where no opioid agonist is administered. The participants were sampled twice, once upon enrolment to the program and once upon completion. According to our results, no difference in global DNA methylation was detected between samples collected upon enrolment and samples collected upon completion of the program. The findings of this study do not rule out the possibility that the 21-day abstinence period was not long enough to observe changes in global DNA methylation, or that abstinence induced site-specific methylation changes (but not global changes), that certainly merit further evaluation.

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.

Aberrant DNA methylation and miRNAs in coronary artery diseases and stroke: a systematic review.

Coronary artery disease (CAD) and ischemic stroke are the two most predominant forms of cardiovascular diseases (CVDs) caused by genetic, epigenetic and environmental risk factors. Although studies on the impact of 'epigenetics' in CVDs is not new, its effects are increasingly being realized as a key regulatory determinant that may drive predisposition, pathophysiology and therapeutic outcome. The most widely studied epigenetic risk factors are regulated by DNA methylation and miRNA expression. To keep pace with growing developments and discoveries, a comprehensive review was performed using Pubmed, Science Direct and Scopus databases to highlight the role of DNA methylation and miRNAs in CAD and stroke subjects. Network analysis was performed using ClueGO software and miRTargetLink database. We identified 32 studies of DNA methylation on CAD and stroke, of which, 6 studies showed differences in global DNA methylation, 10 studies reported the genome-wide difference in DNA methylation and 16 studies demonstrated altered DNA methylation at 14 candidate loci. The network analysis showed positive regulation of nitric oxide biosynthetic process, homocysteine metabolic process and negative regulation of lipid storage. About, 155 miRNAs were associated with CAD, stroke and related phenotypes in 83 studies. Interestingly, mir-223 hypomethylation and altered expression were associated with cerebral infarction and stroke. The target prediction for 18 common miRNAs between CAD and stroke showed strong interaction with SP3 and SP1 genes. This systematic review addresses the present knowledge on DNA methylation and miRNAs in CAD and stroke, whose abnormal regulation has been implicated in etiology or progression of the diseases.

Whole genome bisulfite sequencing of Down syndrome brain reveals regional DNA hypermethylation and novel disorder insights

ABSTRACTDown Syndrome (DS) is the most common genetic cause of intellectual disability, in which an extra copy of human chromosome 21 (HSA21) affects regional DNA methylation profiles across the genome. Although DNA methylation has been previously examined at select regulatory regions across the genome in a variety of DS tissues and cells, differentially methylated regions (DMRs) have yet to be examined in an unbiased sequencing-based approach. Here, we present the first analysis of DMRs from whole genome bisulfite sequencing (WGBS) data of human DS and matched control brain, specifically frontal cortex. While no global differences in DNA methylation were observed, we identified 3,152 DS-DMRs across the entire genome, the majority of which were hypermethylated in DS. DS-DMRs were significantly enriched at CpG islands and de-enriched at specific gene body and regulatory regions. Functionally, the hypermethylated DS-DMRs were enriched for one-carbon metabolism, membrane transport, and glutamatergic synaptic signalling, while the hypomethylated DMRs were enriched for proline isomerization, glial immune response, and apoptosis. Furthermore, in a cross-tissue comparison to previous studies of DNA methylation from diverse DS tissues and reference epigenomes, hypermethylated DS-DMRs showed a strong cross-tissue concordance, while a more tissue-specific pattern was observed for the hypomethylated DS-DMRs. Overall, this approach highlights that low-coverage WGBS of clinical samples can identify epigenetic alterations to known biological pathways, which are potentially relevant to therapeutic treatments and include metabolic pathways. These results also provide new insights into the genome-wide effects of genetic alterations on DNA methylation profiles indicative of altered neurodevelopment and brain function.

LINE-1 DNA methylation and congenital heart defects in Down syndrome

DNA methylation is a key epigenetic mechanism that plays a significant role in regulating gene activity during cardiac development. Congenital heart defects (CHD) are one of the most common abnormalities occurring in 40% -60% of cases with Down syndrome (DS). The main aim of this study was to establish the association of long interspersed nucleotide element-1 (LINE-1) DNA methylation in children with DS and the presence of CHD. The LINE-1 DNA methylation was investigated in peripheral blood lymphocytes on a sample of 42 people with DS by quantification of LINE-1 methylation using the MethyLight method. No significant differences in global DNA methylation were found according to the presence of CHD (P=1.000), but values of LINE-1 DNA methylation were significantly influenced by gender (R2=19.1%; P=0.025). Significant probability of 19.1% was found in women with DS who had lower LINE-1 DNA methylation values than DS male . Gender was a statistically significant predictor of LINE-1 DNA methylation, although the difference was not statistically significant, female subjects had lower LINE-1 DNA methylation values (P=0.068). Further research will clarify the role of lower LINE-1 DNA methylation in the formation of CHD among DS females.

Integrated Molecular Characterization of Testicular Germ Cell Tumors

SUMMARYWe studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance—KIT, KRAS, and NRAS—exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas.

X Chromosome Dosage Influences DNA Methylation Dynamics during Reprogramming to Mouse iPSCs.

SummaryA dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.

Global and gene-specific DNA methylation and hydroxymethylation in human skin exposed and not exposed to sun radiation.

Backgroundepigenomes can be influenced by environmental factors leading to the development of diseases.ObjectiveTo investigate the influence of sun exposure on global DNA methylation and hydroxymethylation status and at specific sites of the miR-9-1, miR-9-3 and MTHFR genes in skin samples of subjects with no history of skin diseases.MethodsSkin samples were obtained by punch on sun-exposed and sun-protected arm areas from 24 corpses of 16-89 years of age. Genomic DNA was extracted from skin samples that were ranked according to Fitzpatrick's criteria as light, moderate, and dark brown. Global DNA methylation and hydroxymethylation and DNA methylation analyses at specific sites were performed using ELISA and MSP, respectively.ResultsNo significant differences in global DNA methylation and hydroxymethylation levels were found among the skin areas, skin types, or age. However, gender-related differences were detected, where women showed higher methylation levels. Global DNA methylation levels were higher than hydroxymethylation levels, and the levels of these DNA modifications correlated in skin tissue. For specific sites, no differences among the areas were detected. Additional analyses showed no differences in the methylation status when age, gender, and skin type were considered; however, the methylation status of the miR-9-1 gene seems to be gender related.Study limitationsthere was no separation of dermis and epidermis and low sample size.Conclusionsun exposure does not induce changes in the DNA methylation and hydroxymethylation status or in miR-9-1, miR-9-3 and MTHFR genes for the studied skin types.

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS.

Differences in global DNA methylation of testicular seminoma are not associated with changes in histone modifications, clinical prognosis, BRAF mutations or gene expression

Testicular germ cell tumours of young adults are comprised of a heterogeneous group of non-seminomas and a homogeneous group of seminomas. While the majority of seminomas retain a hypo-methylated genome, a small fraction displays a highly methylated genome, resembling hyper-methylated non-seminomas. It is well established from e.g. melanoma, colorectal and thyroid cancer that a methylated phenotype can be correlated to prognosis and can be related to BRAF mutations. In the present study we investigated the global methylation level in 67 seminomas and classified them as hypo-methylated, intermediate, patchy and hyper-methylated, respectively. A selected subset representing each level of DNA methylation and the TCam2 seminoma cell line were subsequently analysed for a range of other epigenetic marks (6 histone marks and 5-hydroxymethylcytosine), the presence of the BRAF V600E de novo mutation, differences in the transcriptome and finally correlated to the clinical outcome. We did not identify any histone marks or hydroxymethylation levels that correlated with the methylation level of the genome. Some histone marks, however, showed a great variation while others were found at the same level in all the investigated seminomas. We did not identify any tumours with the BRAF V600E mutation and transcriptome analysis revealed no significant differences between hypo- and hyper-methylated seminomas. Similarly, no obvious differences in the clinical manifestation of the patients representing hypo- or hyper-methylated seminomas were identified. The level of DNA methylation in testicular seminomas consequently seems secondary to the manifestation of the tumour phenotype.

Influence of smoking on methylation and hydroxymethylation levels in global DNA and specific sites of KRT14, KRT19, MIR-9-3 and MIR-137 genes of oral mucosa

ObjectiveThe aim of this study was to investigate the influence of smoking on the methylation and hydroxymethylation status of global DNA and specific sites of KRT14, KRT19, miR-9-3 and miR-137 genes in the healthy oral mucosa. MethodsSamples of oral epithelial cells were collected using mouthwash from a population of former and current smokers and those who had never smoked. Genomic DNA was extracted, and global DNA methylation and hydroxymethylation was performed using an ELISA-based technique; DNA methylation at specific sites was performed using Methylation-Specific PCR (MSP) (KRT14, miR-9-3 and miR-137) or Methylation-Sensitive Restriction Enzymes (MSRE) (KRT19). K14 and K19 protein expression was analysed by immunohistochemistry. ResultsHigher levels of global DNA methylation were found in current smokers with over 15 years of consumption (p=0.04), but no differences were found in relation to global DNA hydroxymethylation. No differences in global DNA methylation and hydroxymethylation levels were found in relation to age or gender. Global DNA methylation was higher than the hydroxymethylation level (p<0.001) but they were not correlated in the oral mucosa. For specific sites, miR-9-3 hypomethylation was detected in current smokers (p<0.001). Additional analysis showed no difference in the methylation status when age, gender, period of consumption or amount of cigarettes were considered for any of the studied genes. K19 expression was higher in current smokers in comparison to former smokers and those who had never smoked (p<0.05). ConclusionWe concluded that smoking habits were capable of inducing changes in global DNA methylation, miR-9-3 methylation status and K19 expression.