Methylation Of Certain Genes Research Articles

Mediation effect of JAK2 methylation on the association between sitting time and abdominal obesity in rural adults.

Sitting time may affect health by altering the methylation of certain genes. This research aimed to estimate the association of sitting time with abdominal obesity and the role of Janus kinase 2 (JAK2) methylation in the association among rural adults. A total of 1062 rural adults from the Henan Rural Cohort Study were included. Whole blood was used to extract genomic DNA. JAK2 DNA methylation level was assessed by MethylTargetTM. The logistic regression model was utilized to assess the association of sitting time with abdominal obesity, and the possible effect of JAK2 DNA methylation on the association were conducted by using mediation analyses. Average time of sitting of participants was 7.28 ± 3.37 h/d. For per 1 h increment in sitting time, the odd ratio (OR) and 95% confidence interval (CI) of abdominal obesity was 1.153 (1.095, 1.214) after controlling potential risk factors. Simultaneously, the methylation levels of Chr9: 4985407 site and Chr9: 4985238-4985455 region were negatively correlated with abdominal obesity (OR: 0.549, 95% CI: 0.394, 0.765; OR: 0.189, 95% CI: 0.056, 0.640, respectively). Moreover, Chr9: 4985407 site and Chr9: 4985238-4985455 region methylation levels mediated the association of sitting time with abdominal obesity, and the indirect effects account for 6.78% and 4.24%, respectively. Longer sitting time was positively correlated with abdominal obesity in the rural population, and methylation level of JAK2 may be an underlying mediation of the effect.

Identification of novel hypermethylated or hypomethylated CpG sites and genes associated with anthracycline-induced cardiomyopathy

Anthracycline-induced cardiomyopathy is a leading cause of late morbidity in childhood cancer survivors. Aberrant DNA methylation plays a role in de novo cardiovascular disease. Epigenetic processes could play a role in anthracycline-induced cardiomyopathy but remain unstudied. We sought to examine if genome-wide differential methylation at ‘CpG’ sites in peripheral blood DNA is associated with anthracycline-induced cardiomyopathy. This report used participants from a matched case–control study; 52 non-Hispanic White, anthracycline-exposed childhood cancer survivors with cardiomyopathy were matched 1:1 with 52 survivors with no cardiomyopathy. Paired ChAMP (Chip Analysis Methylation Pipeline) with integrated reference-based deconvolution of adult peripheral blood DNA methylation was used to analyze data from Illumina HumanMethylation EPIC BeadChip arrays. An epigenome-wide association study (EWAS) was performed, and the model was adjusted for GrimAge, sex, interaction terms of age at enrollment, chest radiation, age at diagnosis squared, and cardiovascular risk factors (CVRFs: diabetes, hypertension, dyslipidemia). Prioritized genes were functionally validated by gene knockout in human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 technology. DNA-methylation EPIC array analyses identified 32 differentially methylated probes (DMP: 15 hyper-methylated and 17 hypo-methylated probes) that overlap with 23 genes and 9 intergenic regions. Three hundred and fifty-four differential methylated regions (DMRs) were also identified. Several of these genes are associated with cardiac dysfunction. Knockout of genes EXO6CB, FCHSD2, NIPAL2, and SYNPO2 in hiPSC-CMs increased sensitivity to doxorubicin. In addition, EWAS analysis identified hypo-methylation of probe ‘cg15939386’ in gene RORA to be significantly associated with anthracycline-induced cardiomyopathy. In this genome-wide DNA methylation profile study, we observed significant differences in DNA methylation at the CpG level between anthracycline-exposed childhood cancer survivors with and without cardiomyopathy, implicating differential DNA methylation of certain genes could play a role in pathogenesis of anthracycline-induced cardiomyopathy.

DNA methylation mediated genetic risk in severe acne in a young men population.

Acne is a chronic inflammatory skin disease that affects the pilosebaceous follicle and is influenced by heredity, hormones, inflammation, and the environment. At present, the recognized pathogenesis mainly includes four categories: excessive sebum secretion, excessive Cutibacterium acnes proliferation, excessive keratinization of sebaceous glands in hair follicles, and inflammatory mechanisms. Previous studies have found that DNA methylation is closely related to some chronic inflammatory skin diseases, and there is evidence that DNA methylation is controlled by genetic factors, making us want to know the relationship between DNA methylation, genetic variation and acne. In our previous study, we performed genome-wide DNA methylation analysis in peripheral blood samples from 44 patients with severe acne and 44 unaffected normal subjects, and identified 23 differentially methylated probes (DMPs). In this study, we identified single nucleotide polymorphisms (SNPs) associated with severe acne by genome-wide association analysis in these 88 samples. To test the association between SNPs and DMPs, we conducted DNA methylation quantitative trait loci (methQTL) analysis. Next, causal inference testing (CIT) was used to determine whether genetic variation influences DNA methylation, which impacts disease phenotypes. We found 38,269 SNPs associated with severe acne. By methQTL analysis, we obtained 24 SNP-CpG pairs that reached the threshold (FDR < 0.05), which included 7 unique CpGs and 22 unique methQTL SNPs. After CIT analysis, we found that 11 out of 24 pairs of SNP-CpG showed a weakened SNP effect after adjustment for methylation, indicating a methylation-mediated relationship between SNPs and severe acne. These 11 SNP-CpG pairs consist of four unique CpG sites and 11 SNPs, of which three CpG sites, cg03020863, cg20652636, and cg19964325, are located on the gene body of PDGFD, the intron of SH2D6, and the 5'UTR of the IL1R1 gene, respectively. During this study, the DNA methylation of certain genes was found to be influenced by genetic factors and mediated the risk of severe acne in a young Chinese male population, providing a new perspective on the pathogenesis of severe acne.

Rescuing effects of periostin in advanced glycation end-products (AGEs) caused osteogenic and oxidative damage through AGE receptor mediation and DNA methylation of the CALCA promoter

An in vitro model was established to simulate a diabetes-type environment by treating human periodontal stem cells with advanced glycation end-products (AGEs). Periostin (POSTN) plays a crucial role in maintaining the integrity of periodontal tissues. However, the role of POSTN in human periodontal stem cells stimulated by AGEs remains unknown. Diabetes mellitus is considered a metabolic disease, and DNA methylation of CpG islands is a biomarker of metabolic syndromes. Diabetes has been found to be closely related to the DNA methylation of certain genes. Here, we investigated the protective mechanism and effect of POSTN on osteogenesis and oxidative stress in the AGE environment, and further explored the CpG island methylation of specific genes potentially mediated by POSTN. The optimal concentration of AGEs was screened using CCK8. AGEs were found to contribute to oxidative stress. Conversely, reactive oxygen species production and malondialdehyde and superoxide activity indicated that the AGE + POSTN group decreased oxidative injury. According to an alkaline phosphatase assay, Alizarin Red S staining, and the expression of key genes and proteins involved in osteogenesis, POSTN mitigated the inhibitory effects of AGE on cell proliferation and osteogenic differentiation potential during osteogenic differentiation. In contrast, the growth and osteogenesis of human periodontal stem cells were notably suppressed by POSTN knockdown. Bisulfite sequencing PCR was used to evaluate the DNA methylation status. Moreover, AGE elevated the expression of DNA methyltransferas 1 (DNMT1) and inhibited the activation of CALAL promoter methylation, which was rescued by the addition of POSTN and 5-Azacytidine (5-AZA). In conclusion, POSTN attenuated the AGE-induced inhibition of osteogenesis in periodontal ligament stem cells by reducing AGE receptor levels and DNA methylation of the calcitonin-related polypeptide α (CALCA) promoter. Thus, POSTN is a promising candidate for dental bone regeneration, representing a novel therapeutic agent for diabetic patients. The mechanism underlying these processes may provide new insights into novel therapeutic targets for improving abnormal bone metabolism in patients with diabetes.

Analysis of Epigenetic Changes in Vitamin D Pathway Genes in Rheumatoid Arthritis Patients.

.Background:Rheumatoid arthritis (RA) is an autoimmune inflammatory disease with complex etiopathogenesis launched by multiple risk factors, including epigenetic alterations. RA is possibly linked to vitamin D that is epigenetically active and may alter DNA methylation of certain genes. Therefore, the study aimed to evaluate the relationship between DNA methylation status of vitamin D signaling pathway genes (VDR, CYP24A1, CYP2R1), vitamin D level and associations with RA.Materials and Methods:Totally 76 participants (35 RA patients and 41 healthy controls) were enrolled from a case-control vitamin D and VDR gene polymorphisms study regarding age and vitamin D concentration. CpG islands in promoter regions of the VDR, CYP24A1, CYP2R1 genes were chosen for DNA methylation analysis by means of pyrosequencing. Chemiluminescent microplate immunoassay was used to assess 25(OH)D serum levels. RA clinical data, i.e. the disease activity score C-reactive protein 28 (DAS28 – CRP) as well as patient-reported outcome questionnaires were recorded.Results:The study showed similar methylation pattern in the promoter regions of vitamin D pathway genes in RA and control group with p>0.05 (VDR gene 2.39% vs. 2.48%, CYP24A1 gene 16.02% vs. 15.17% and CYP2R1 2.53% vs. 2.41%). CYP24A1 methylation intensity was significantly higher in compare to methylation intensity of VDR and CYP2R1 genes in both groups (p<0.0001). A tendency of higher vitamin D concentration in cases having methylated VDR (57.57±28.93 vs. 47.40±29.88 nmol/l), CYP24A1 (53.23±26.22 vs. 48.23±34.41 nmol/l) and CYP2R1 (60.41±30.73 vs. 44.54±27.63 nmol/l) genes and a positive correlation between VDR, CYP2R1 methylation intensity and vitamin D level in RA affected participants was revealed (p>0.05). A significantly higher CYP24A1 methylation intensity (p=0.0104) was detected in blood cells of vitamin D deficient (<50 nmol/l) RA patients vs. vitamin D deficient controls.Conclusions:Our data suggests some indirect associations between DNA methylation status of vitamin D pathway genes and vitamin D level in RA.

Comparative methylation and RNA-seq expression analysis in CpG context to identify genes involved in Backfat vs. Liver diversification in Nanchukmacdon Pig

BackgroundDNA methylation and demethylation at CpG islands is one of the main regulatory factors that allow cells to respond to different stimuli. These regulatory mechanisms help in developing tissue without affecting the genomic composition or undergoing selection. Liver and backfat play important roles in regulating lipid metabolism and control various pathways involved in reproductive performance, meat quality, and immunity. Genes inside these tissue store a plethora of information and an understanding of these genes is required to enhance tissue characteristics in the future generation.ResultsA total of 16 CpG islands were identified, and they were involved in differentially methylation regions (DMRs) as well as differentially expressed genes (DEGs) of liver and backfat tissue samples. The genes C7orf50, ACTB and MLC1 in backfat and TNNT3, SIX2, SDK1, CLSTN3, LTBP4, CFAP74, SLC22A23, FOXC1, GMDS, GSC, GATA4, SEMA5A and HOXA5 in the liver, were categorized as differentially-methylated. Subsequently, Motif analysis for DMRs was performed to understand the role of the methylated motif for tissue-specific differentiation. Gene ontology studies revealed association with collagen fibril organization, the Bone Morphogenetic Proteins (BMP) signaling pathway in backfat and cholesterol biosynthesis, bile acid and bile salt transport, and immunity-related pathways in methylated genes expressed in the liver.ConclusionsIn this study, to understand the role of genes in the differentiation process, we have performed whole-genome bisulfite sequencing (WGBS) and RNA-seq analysis of Nanchukmacdon pigs. Methylation and motif analysis reveals the critical role of CpG islands and transcriptional factors binding site (TFBS) in guiding the differential patterns. Our findings could help in understanding how methylation of certain genes plays an important role and can be used as biomarkers to study tissue specific characteristics.

BIOLOGICAL AGE: METHODOLOGY FOR DETERMINING

The analytical review is devoted to the methodology for quantifying age-related changes, namely, the determination of biological age (BA). In contrast to the chronological age (CA), which is the same for all people born at the same time, BA can vary significantly and characterizes the rate of aging. Diagnostics of accelerated aging is of great clinical importance, as it allows predicting the development of age-related pathology. In addition, the BA calculation is used to assess various effects on the body, including geroprotectors. To calculate BA, a set of biomarkers of aging (BMA) is used. The most accurate method for determining BA is by measuring the methylation of certain genes (Horvath clock). However, this method is not available for clinical practice yet. Therefore, the development of BA models based on clinical BMS remains relevant. In addition, there is a need to assess the degree of age-related changes at the level of individual functional systems of the body (functional age). The list of BMA available for use in clinical practice is given in this review. The requirements are given that allow using the indicator of age-related changes as BMA. The main methods for calculating BV are multiple linear regression (MLR), principal component analysis (PCA), Klemera and Doubal method (KDM), and neural network analysis. The criteria for assessing the quality of the BA model are described in this review. The MLR method, due to its simplicity, is most often used in clinical practice, however, it has a significant drawback, namely, the systematic error in calculating age, which is most pronounced in the extreme age groups. The article provides a method for correcting this error developed by the authors. The methodology of using MLR to obtain a formula for determining BA from a dataset BMA is described in detail. The theoretical foundations of the use of neural network algorithms for assessing the rate of aging are presented. The use of neural networks for assessing BA has shown their high efficiency by all criteria, in particular, the absence of a systematic error inherent in MLR-based methods. Keywords: biological age, biomarkers of aging, calculation methods.

Computational Insights into Molecular Adsorption Characteristics of Methylated DNA on Graphene Oxide for Multicancer Early Detection.

DNA methylation is an epigenetic modification involving the transfer of a methyl group to cytosine residues of a DNA molecule. Altered DNA methylation of certain genes is associated with several diseases including cancer. Nanomaterials, such as graphene oxide (GO), offer great potential as sensing elements for methylated DNA (mDNA) detection due to their distinct properties. Understanding molecular interactions between mDNA and GO can make provision for developing a universal cancer screening test. Molecular dynamics (MD) simulation and density functional theory (DFT) calculation have been employed for investigating their detailed macro- and microscale interactions. Based upon the MD simulation, different adsorption levels of methylated and unmethylated DNAs on GO were represented by a contacting surface area (CSA), which depends on surrounding conditions (in water or a MgCl2 solution). In water, the CSAs of the methylated and unmethylated single-stranded DNA (ssDNA) were ≈13 and ≈5 nm2, respectively, representing more preferable adsorption on GO for the methylated ssDNA. In the presence of divalent ions (Mg2+), the CSAs of both methylated and unmethylated DNA molecules were ≈8 nm2, suggesting that there was no significant difference in adsorption in a saline solution. To reveal the electrical property of GO covered by either methylated or unmethylated DNA, its electronic structure was investigated by the DFT calculation. The energy gaps of pristine graphene (pG) and GO adsorbed by 5-methylcytosine (5mC) were 1.6 and 12.9 meV, respectively, while cytosine adsorption resulted in lower energy gaps (1.2 meV for pG and 9.5 meV for GO). When comparing methylated DNA-covered GO with that covered with unmethylated DNA, remarkable differences in electrical conductivity, which were caused by the electronic structure of GO, were observed. These findings will provide a new route for an efficient detection method of DNA methylation, which can further be used to develop a universal cancer test.

Sex-Specific Age-Related Changes in Methylation of Certain Genes.

The aim of the study was to conduct a functional analysis of sex-specific age-related changes in DNA methylation.Materials and MethodsThe study used a GSE87571 methylation dataset obtained from the blood DNA of 729 individuals aged 14 to 94 using the Illumina Infinium HumanMethylation450K BeadChip (USA). Gene ontology analysis was performed for 3 groups of genes (females, males, and duplicates) using the PANTHER database. The DAVID platform was used to perform KEGG metabolic pathway analysis.ResultsThe studies revealed unique for males and females changes in methylation of CpG sites, associated with certain metabolic processes. It was demonstrated that most of the CpG sites, for which methylation changes with age were revealed in both sexes, are associated with the genes responsible for the development and functioning of the nervous system. In males, unique age-related methylation changes affect CpG sites associated with changes in the immune system and lipid metabolism. In females, most CpGs are associated with changes involved in transcription and translation processes. Analysis of biological functions by KEGG revealed that a unique process associated with age-related changes in methylation of the glutamatergic system is typical for males. In females, unique biological processes with age-related changes include genes responsible for the development of diabetes and genes associated with cAMP signaling cascades (KEGG:04024).ConclusionOur studies reveal fundamental features of sex-dependent changes in methylation of CpG sites with variance increasing, which may indicate differences in age-related changes.

Kidney and epigenetic mechanisms of salt-sensitive hypertension.

Dietary salt intake increases blood pressure (BP) but the salt sensitivity of BP differs between individuals. The interplay of ageing, genetics and environmental factors, including malnutrition and stress, contributes to BP salt sensitivity. In adults, obesity is often associated with salt-sensitive hypertension. The children of women who experience malnutrition during pregnancy are at increased risk of developing obesity, diabetes and salt-sensitive hypertension as adults. Similarly, the offspring of mice that are fed a low-protein diet during pregnancy develop salt-sensitive hypertension in association with aberrant DNA methylation of the gene encoding type1A angiotensin II receptor (AT1AR) in the hypothalamus, leading to upregulation of hypothalamic AT1AR and renal sympathetic overactivity. Ageing is also associated with salt-sensitive hypertension. In aged mice, promoter methylation leads to reduced kidney production of the anti-ageing factor Klotho and a decrease in circulating soluble Klotho. In the setting of Klotho deficiency, salt-induced activation of the vascular Wnt5a-RhoA pathway leads to ageing-associated salt-sensitive hypertension, potentially as a result of reduced renal blood flow and increased peripheral resistance. Thus, kidney mechanisms and aberrant DNA methylation of certain genes are involved in the development of salt-sensitive hypertension during fetal development and old age. Three distinct paradigms of epigenetic memory operate on different timescales in prenatal malnutrition, obesity and ageing.

Regulation of DNA methyltransferase gene expression by short peptides in nicotiana tabacum regenerants

Methylation of cytosine DNA residues is the most studied and stable of all epigenetic modifications. Methylation of eukaryotic DNA is carried out by DNA methyltransferases. DNA methylation is an active mechanism for controlling gene transcription and is usually associated with prolonged silencing of DNA. The effect of peptides AEDG and AEDL on the growth of calluses of tobacco (<i>Nicotiana tabacum</i>) at low concentrations (10⁻⁷ M) is similar to the effect of phytohormones, has a regulatory character, and is possibly epigenetic in nature. Peptides increase the expression of DNA methyltransferase genes. One of the possible mechanisms of regulation of DNA methyltransferase genes by AEDG and AEDL is their ability to bind to free DNA regions at certain CNG sites, which are also methylation sites of plant cytosine methyltransferases. The AEDG peptide preferably binds to the CAG site and the AEDL peptide to the CTG site. By binding to the same sites as DNA methyltransferase, peptides block methylation sites, thereby reducing the level of DNA methylation. The specific binding of peptides to different sites that we discovered can be of great importance in gene regulation, since peptides with different structures can block different DNA regions for methylation of certain genes, thereby activating or silencing their expression.

An integrated methylation and gene expression microarray analysis reveals significant prognostic biomarkers in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a life-threatening disease with a poor prognosis. Although previous studies have reported that the methylation of certain genes is associated with the pathogenesis of OSCC, the methylation of genes that have relevance to OSCC progression is not clearly documented. The present study aimed to gain insights into the mechanisms underlying DNA methylation regulation associated with OSCC progression and to identify potential prognostic markers for OSCC treatment. DNA methylation dataset GSE41114 and gene expression dataset GSE74530 were downloaded from the Gene Expression Omnibus database. The global methylation status of OSCC tumor samples and normal control samples was determined, and differentially methylated genes (DMGs) in OSCC samples compared with control samples were identified. The mRNA expression data were then integrated to identify differentially expressed genes (DEGs) in OSCC samples compared with control samples. Overlapping genes between DEGs and DMGs were identified, and functional enrichment analysis was performed. In addition, survival analysis of the overlapping genes was performed to screen genes with prognostic significance in OSCC. A total of 40,115 differential methylation CpG sites spanning 3,360 DMGs were identified; CpG sites in the promoter, gene body and intergenic regions were generally highly hypermethylated or hypomethylated. Additionally, 508 DEGs in OSCC samples were identified, including 332 upregulated and 176 downregulated genes. A total of 82 overlapping genes between DEGs and DMGs were found, which were mainly involved in protein metabolism, regulation of the metabolic process and the immune system. Additionally, differential methylation or expression of several genes, including fibroblast activation protein α (FAP), interferon α inducible protein 27 (IFI27), laminin subunit γ2 (LAMC2), matrix metallopeptidase 1 (MMP1), serine peptidase inhibitor Kazal-type 5 (SPINK5) and zinc finger protein 662 (ZNF662), was significantly associated with the survival of OSCC patients, and their differential expression in OSCC patients was further confirmed by reverse transcription-quantitative polymerase chain reaction in OSCC and normal oral cell lines. Overall, FAP, IFI27, LAMC2, MMP1, SPINK5 and ZNF662 genes caused by epigenetic changes via DNA methylation may be associated with the development and progression of OSCC, and should be valuable OSCC therapeutic biomarkers.

PO-388 Epigenetic silencing of ZNF177 by MIR-877–3 p could be involved in cervical cancer progression

IntroductionBreast cancer (BC) is the most frequent neoplasia among women and one of the leading causes of cancer-related death worldwide. It is a clinically heterogeneous disease, with the triple negative (TN) as the most aggressive subtype. Epigenetic alterations are common molecular abnormalities in cancer, including changes in DNA methylation, microRNA profiling and post-translational modifications of histones. Aberrant DNA methylation has been recognised as one of the most common epigenetic modifications in BC. Methylation of certain genes is associated with pathological features, differential response to therapy or metastases during follow-up, being also a predictor of survival in BC; but little is known about its involvement in TN BC. Our objective was to identify the methylome of TN BC compared to that of non-neoplastic mammary tissue.Material and methodsA 450k-methylation array was performed to identify differentially methylated genes in 8 TN BC samples (T) and 5 non-neoplastic (N) tissues from reduction mammoplasties. For that, DNA was extracted, bisulphite-converted, whole-genome amplified and hybridised to Infinium HumanMethylation450 BeadChip array. The level of DNA methylation at each CpG locus (β-value) was calculated as M/(M+U), ranging from 0 (100% unmethylated, U) to 1 (100% methylated, M). Differentially expressed probes were obtained by limma t-test (False Discovery Rate<0.05). Those probes with delta β-value (Δβ)>0.2 and located in CpG islands in the 5´UTR, 1500–200 bp upstream the transcription start site or in the first exon, were considered. ADAM12, VWCE and TSPAN9 genes were selected for further validation by pyrosequencing in a series of 30 T and 20 n, using a PyroMark Q24 after PCR amplification. Protein expression was explored by immunohistochemistry.Results and discussionsA total of 700 hypermethylated and 349 hypomethylated probes in T compared to N, corresponding to 15 and 7 hyper- and hypomethylated genes, respectively, were found to fulfil our criteria. ADAM12, VWCE and TSPAN9 genes showed lower methylation levels in T than in N (p<0.05). The increased protein expression in T compared to N was confirmed, highlighting the causal role of the hypomethylation in the regulation of the expression of these genes. No association was found between these alterations and clinicopathological variables.ConclusionThis is the first description of ADAM12, VWCE and TSPAN9 hypomethylation and overexpression in TN BC compared to non-neoplastic mammary tissues.

PO-387 Differential gene methylation in triple negative breast cancer and non-tumoral mammary tissue

Introduction Breast cancer (BC) is the most frequent neoplasia among women and one of the leading causes of cancer-related death worldwide. It is a clinically heterogeneous disease, with the triple negative (TN) as the most aggressive subtype. Epigenetic alterations are common molecular abnormalities in cancer, including changes in DNA methylation, microRNA profiling and post-translational modifications of histones. Aberrant DNA methylation has been recognised as one of the most common epigenetic modifications in BC. Methylation of certain genes is associated with pathological features, differential response to therapy or metastases during follow-up, being also a predictor of survival in BC; but little is known about its involvement in TN BC. Our objective was to identify the methylome of TN BC compared to that of non-neoplastic mammary tissue. Material and methods A 450k-methylation array was performed to identify differentially methylated genes in 8 TN BC samples (T) and 5 non-neoplastic (N) tissues from reduction mammoplasties. For that, DNA was extracted, bisulphite-converted, whole-genome amplified and hybridised to Infinium HumanMethylation450 BeadChip array. The level of DNA methylation at each CpG locus (β-value) was calculated as M/(M+U), ranging from 0 (100% unmethylated, U) to 1 (100% methylated, M). Differentially expressed probes were obtained by limma t-test (False Discovery Rate 0.2 and located in CpG islands in the 5´UTR, 1500–200 bp upstream the transcription start site or in the first exon, were considered. ADAM12, VWCE and TSPAN9 genes were selected for further validation by pyrosequencing in a series of 30 T and 20 n, using a PyroMark Q24 after PCR amplification. Protein expression was explored by immunohistochemistry. Results and discussions A total of 700 hypermethylated and 349 hypomethylated probes in T compared to N, corresponding to 15 and 7 hyper- and hypomethylated genes, respectively, were found to fulfil our criteria. ADAM12, VWCE and TSPAN9 genes showed lower methylation levels in T than in N (p Conclusion This is the first description of ADAM12, VWCE and TSPAN9 hypomethylation and overexpression in TN BC compared to non-neoplastic mammary tissues.

Aberrant DNA Methylation in Chronic Myeloid Leukemia: Cell Fate Control, Prognosis, and Therapeutic Response.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy characterized by the expression of the BCR-ABL1 fusion gene with different chimeric transcripts. Despite the crucial impact of constitutively active tyrosine kinase in CML pathogenesis, aberrant DNA methylation of certain genes plays an important role in disease progression and the development of drug resistance. This article reviews recent findings relevant to the effect of DNA methylation pattern of regulatory genes on various cellular activities such as cell proliferation and survival, as well as cell-signaling molecules in CML. These data might contribute to defining the role of aberrant DNA methylation in disease initiation and progression. However, further studies are needed on the validation of specific aberrant methylation markers regarding the prognosis and prediction of response among the CML patients.

Aberrant promoter hypermethylation of p16, survivin, and retinoblastoma in gastric cancer.

Gastric cancer is common and can be found throughout the world. Notoriously like other cancers, gastric cancer is a consequence of cellular regulation disorder. Epigenetics, including many oncogenes or tumour suppressor genes, can provide some clues for this kind of disorder. DNA methylation, especially promoter methylation, which causes the silence/decrease of tumour related genes, has become a focus in various tumour types. The aim of this study was to investigate promoter methylation of certain genes: p16, survivin, retinoblastoma (Rb), all of which have been regarded as genes related to gastric cancer. To detect the promoter methylation of p16, survivin, and Rb genes, peripheral blood samples from 106 gastric cancer patients as well as 18 healthy individuals were collected for Methylation-Specific Polymerase Chain Reaction (MSP) analysis. According to the statistical analysis, positive methylation ratio of p16 was 72.6 % (77 cases), 6 % had methylated survivin (7 cases), and positive methylation ratio of Rb was only 17.9 % (19 cases). The differences of promoter methylation of these genes between the patients group and control group were observed: P (P16) = 5.097E-08, P (survivin) = 0.262, and P (Rb) = 0.187. In the control group, methylated p16 was found in only one case that also had a methylated Rb. However, Survivin could not be found methylated in control cases. In this study, promoter methylation was observed for the p16 gene, which was considered an early potential marker in gastric cancer. This data supports that further investigations should study Rb and survivin as candidate markers for gastric cancer (Tab. 2, Fig. 1, Ref. 35).

Epigenetic Modification in Oocyte and Preimplantation Embryonic Development

DNA methylation and histone modification are two of the most characterized epigenetic modifications. With advanced detecting techniques, particularly single-cell sequencing, we can dissect epigenomic patterns and their regulatory roles in the growth and differentiation of gametes and early embryos in animals and humans. Assisted reproductive technology (ART) procedures have been shown to influence the methylation of certain genes. Aberrant epigenetic regulation may cause several developmental disorders and clinical diseases. Here, we describe some concepts in epigenetics and review recent researches on DNA methylation and the histone modification profile and their regulatory roles during early embryo development. We also summarize the recent progress in understanding the imprinting disorders associated with ART procedures.

DNA methylation detection based on difference of base content

Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

Oxytocin Receptor Genetic and Epigenetic Variations: Association With Child Abuse and Adult Psychiatric Symptoms.

Childhood abuse can alter biological systems and increase risk for adult psychopathology. Epigenetic mechanisms, alterations in DNA structure that regulate the gene expression, are a potential mechanism underlying this risk. While abuse associates with methylation of certain genes, particularly those in the stress response system, no study to date has evaluated abuse and methylation of the oxytocin receptor (OXTR). However, studies support a role for OXTR in the link between abuse and adverse adult outcomes, showing that abuse can confer greater risk for psychiatric symptoms in those with specific OXTR genotypes. This study therefore sought to (a) assess the role of epigenetics in the link between abuse and psychopathology and (b) begin to integrate the genetic and epigenetic literature by exploring associations between OXTR genotypes and DNA CpG methylation. Data on 18 OXTR CpG sites, 44 single nucleotide polymorphisms, childhood abuse, and adult depression and anxiety symptoms were assessed in 393 African American adults (age = 41 ± 12.8 years). Overall, 68% of genotypes were associated with methylation of nearby CpG sites, with a subset surviving multiple test correction. Child abuse associated with higher methylation of two CpG sites yet did not survive correction or serve as a mediator of psychopathology. However, abuse interacted with CpG methylation to predict psychopathology. These findings suggest a role for OXTR in understanding the influence of early environments on adult psychiatric symptoms.

DNA methylation status of a distinctively different subset of genes is associated with each histologic Lauren classification subtype in early gastric carcinogenesis.

DNA methylation change is known to play a crucial role in early gastric carcinogenesis. The present study aimed to identify and validate the correlation between differentially methylated regions (DMRs) and the subtypes of early gastric cancers (EGCs). Illumina Infinium methylation assay (IIMA; 450K BeadChip kit) was performed on fresh tumor and non‑tumor tissues of 12 EGCs to screen the methylation status of 450,000 CpG sites. To evaluate the significance of DNA methylation in each histologic subtype, pyrosequencing assay (PA) was performed on 38 EGCs (18 intestinal-, 12 mixed- and 8 diffuse-type) using 12 genes selected from the screening. Between tumors of the intestinal-type (n=6), and diffuse- (n=4) plus mixed-types (n=2), 169 regions showed significant differences (intensity>3,000, Δβ>0.2) in IIMA. Hierarchical clustering using the 169 DMRs revealed distinct separation between the two groups. In PA using 12 selected genes from the IIMA results, the aberrant methylation statuses of DVL2 (p=0.0186) and ETS1 (p=0.0222) were significantly related to diffuse- and mixed-types rather than the intestinal-type, while C19orf35 (p=0.019) and CNRIP1 (p=0.0473) were related to the diffuse‑type rather than intestinal‑type, and GAL3ST2 (p=0.0158) and ITGA3 (p=0.0273) were related to the mixed-type rather than the other two types. The methylation of other genes, CLIP4, XKR6, CCDC57, MAML3 and SDC2, was related with age, tumor location, or Helicobacter infection rather than the histologic subtype. Aberrant DNA methylation of certain genes may be independently involved in each histologic subtype of EGC. Furthermore, mixed-type EGCs may be a distinctive histologic subtype based on the different subset of DMRs compared to those of other subtypes.