Global DNA Methylation Levels Research Articles

Maternal non-compliance with recommended folic acid supplement use alters global DNA methylation in cord blood of newborns: A cohort study

Background & aimsPrenatal folate exposure may alter epigenetic marks in the offspring. We aimed to evaluate associations between prenatal exposure to folic acid (FA) in preconception and in utero with cord blood DNA methylation in long interspersed nuclear element 1 (LINE-1) and Alu short interspersed nuclear elements (SINEs) as markers of global DNA methylation levels. MethodsData come from 325 mother-child pairs participating in the Nutrition in Early Life and Asthma (NELA) birth cohort (2015-2018). Pregnant women were asked about supplement use, including brand name and dose, one month before pregnancy (preconception) and through the trimesters of pregnancy. Maternal dietary folate intake was assessed using a validated food frequency questionnaire with additional questions for FA supplement use. Folate serum levels were measured in mothers at 24 weeks of gestation and in cord blood of newborns. DNA methylation was quantitatively assessed by bisulfite pyrosequencing on 5 LINE-1 and 3 Alu different elements. Associations were estimated using multivariable linear regression models. ResultsA reduction in methylation levels of LINE-1 in newborns was associated with the use of FA supplements below the recommended doses (<400 ug/day) during preconception (-0.50; 95% CI: -0.91, -0.09; P=0.016), and from preconception up to 12 weeks of gestation (-0.48; 95% CI: -0.88, -0.08; P=0.018). Maternal use of FA supplements above the tolerable upper intake level of 1000 ug/day from preconception until 12 weeks of gestation was also related to lower methylation in LINE-1 at birth (-0.77; 95% CI: -1.52, -0.02; P=0.044). Neither FA supplement use after 12 weeks of gestation nor maternal total folate intake (diet plus supplements) were associated with global DNA methylation levels at birth. ConclusionsMaternal non-compliance with the use of FA supplement recommendations from preconception up to 12 weeks of gestation reduces offspring global DNA methylation levels at birth.

DNA methylation profile and expression of the phyllagrin gene in patients with atopic dermatitis: case-control study

Background. Atopic dermatitis (AD) is one of the most common inflammatory skin diseases, affecting up to 20.0% of children and 2.0–8.0% of adults worldwide. Patients with atopic dermatitis usually have dry skin and itching, they are at higher risk of developing asthma, as well as allergic rhinitis. Features of the clinical course of AD are associated with both interaction of genes and influence of environmental factors. Studying epigenetic mechanisms could provide understanding of the most likely mechanisms by which environmental factors influence gene expression and contribute to the development of AD. The aim of the study is to conduct a comprehensive molecular genetic analysis (assessment of the level of global DNA methylation and expression of the filaggrin gene (FLG) in patients with moderate to severe AD. Material and methods: The case-control study was conducted from January 2022, through June 2022. A total of 32 patients with AD and 6 healthy volunteers were recruited. The level of global DNA methylation in the blood of patients with AD and healthy individuals was assessed. The FLG expression was measured by polymerase chain reaction in the leukocyte fraction of blood. A three-stage cycle is used during PCR. To determine the expression level of the FLG gene, total RNA was isolated, followed by a reverse transcription, amplification, and detection reaction. Results. A hypermethylation of the genome in patients with AD is 2.3 times higher than the methylation of genome in controls (p = 0.003),it was also found that the expression level of the FLG gene (exon 3, position 7–8, and exon 1) in the leukocyte fraction of blood in patients with AD did not significantly differ from the control group (p = 0.41). Conclusion. The current study found that the epigenome of AD patients differs from healthy individuals. The study of pathogenetic mechanisms is the basis for the development of new methods of targeted therapy for this socially significant dermatosis.

Vpr driving DNA methylation variation of CD4 + T cells in HIV-1 infection

BackgroundDespite the existence of available therapeutic interventions for HIV-1, this virus remains a significant global threat, leading to substantial morbidity and mortality. Within HIV-1-infected cells, the accessory viral protein r (Vpr) exerts control over diverse biological processes, including cell cycle progression, DNA repair, and apoptosis. The regulation of gene expression through DNA methylation plays a crucial role in physiological processes, exerting its influence without altering the underlying DNA sequence. However, a thorough examination of the impact of Vpr on DNA methylation in human CD4 + T cells has not been conducted.MethodsIn this study, we employed base-resolution whole-genome bisulfite sequencing (WGBS), real-time quantitative RCR and western blot to explore the effect of Vpr on DNA methylation of host cells under HIV-1 infection.ResultsWe observed that HIV-1 infection leads to elevated levels of global DNA methylation in primary CD4 + T cells. Specifically, Vpr induces significant modifications in DNA methylation patterns, particularly affecting regions within promoters and gene bodies. These alterations notably influence genes related to immune-related pathways and olfactory receptor activity. Moreover, Vpr demonstrates a distinct ability to diminish the levels of methylation in histone genes.ConclusionsThese findings emphasize the significant involvement of Vpr in regulating transcription through the modulation of DNA methylation patterns. Together, the results of this investigation will considerably enhance our understanding of the influence of HIV-1 Vpr on the DNA methylation of host cells, offer potential avenues for the development of more effective treatments.

Non-CG DNA methylation marks the transition from pupa to adult in Helicoverpa armigera.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

Global DNA Methylation Level in Tumour and Margin Samples in Relation to Human Papilloma Virus and Epstein-Barr Virus in Patients with Oropharyngeal and Oral Squamous Cell Carcinomas.

Aberrant DNA methylation is a common epigenetic modification in cancers, including oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinoma (OSCC). Therefore, the analysis of methylation levels appears necessary to improve cancer therapy and prognosis. The enzyme-linked immunosorbent assay (ELISA) was used to analyse global DNA methylation levels in OPSCC and OSCC tumours and the margin samples after DNA isolation. HPV detection was conducted by hybridisation using GenoFlow HPV Array Test Kits (DiagCor Bioscience Inc., Hong Kong, China). EBV detection was performed using real-time PCR with an EBV PCR Kit (EBV/ISEX/100, GeneProof, Brno, Czech Republic). OPSCC tumour samples obtained from women showed lower global DNA methylation levels than those from men (1.3% vs. 3.5%, p = 0.049). The margin samples from OPSCC patients with HPV and EBV coinfection showed global DNA methylation lower than those without coinfection (p = 0.042). G3 tumours from OSCC patients had significantly lower levels of global DNA methylation than G2 tumours (0.98% ± 0.74% vs. 3.77% ± 4.97%, p = 0.010). Additionally, tumours from HPV-positive OSCC patients had significantly lower global DNA methylation levels than those from HPV-negative patients (p = 0.013). In the margin samples, we observed a significant negative correlation between global DNA methylation and the N stage of OSCC patients (rS = -0.33, p = 0.039). HPV-positive OPSCC patients had higher global DNA methylation levels than HPV-positive OSCC patients (p = 0.015). We confirmed that methylation could be changed in relation to viral factors, such as HPV and EBV, as well as clinical and demographical parameters.

The association analysis of DNA methylation and transcriptomics identified BpCYCD3;2 as a participant in influencing cell division in autotetraploid birch (Betula pendula) leaves

Polyploidization plays a crucial role in plant breeding and genetic improvement. Although the phenomenon of polyploidization affecting the area and number of plant epidermal pavement cells is well described, the underlying mechanism behind this phenomenon is still largely unknown. In this study, we found that the leaves of autotetraploid birch (Betula pendula) stopped cell division earlier and had a larger cell area. In addition, compared to diploids, tetraploids have a smaller stomatal density and fewer stomatal numbers. Genome-wide DNA methylation analysis revealed no significant difference in global DNA methylation levels between diploids and tetraploids. A total of 9154 differential methylation regions (DMRs) were identified between diploids and tetraploids, with CHH-type DMRs accounting for 91.73% of all types of DMRs. Further research has found that there are a total of 2105 differentially methylated genes (DMEGs) with CHH-type DMRs in birch. The GO functional enrichment results of DMEGs showed that differentially methylated genes were mainly involved in terms such as cellular process and metabolic process. The analysis of differentially methylated genes and differentially expressed genes suggests that hyper-methylation in the promoter region may inhibit the gene expression level of BpCYCD3;2 in tetraploids. To investigate the function of BpCYCD3;2 in birch, we obtained overexpression and repressed expression lines of BpCYCD3;2 through genetic transformation. The morphogenesis of both BpCYCD3;2-OE and BpCYCD3;2-RE lines was not affected. However, low expression of BpCYCD3;2 can lead to inhibition of cell division in leaves, and this inhibition of cell proliferation can be compensated for by an increase in cell size. Additionally, we found that the number and density of stomata in the BpCYCD3;2-RE lines were significantly reduced, consistent with the tetraploid. These data indicate that changes in cell division ability and stomatal changes in tetraploid birch can be partially attributed to low expression of the BpCYCD3;2 gene, which may be related to hyper-methylation in its promoter region. These results will provide new insights into the mechanism by which polyploidization affects plant development.

Abstract 3485: Global DNA methylation level is associated with the sensitivity of cytotoxic and senotherapeutic drugs in gastrointestinal tumors

Abstract Background: Gastrointestinal cancer has become a severe burden around the world. Marker-guided treatment and clinical trials with sensitive anti-tumor drugs are critical for improving cancer survival. The decreasing DNA methylation levels of repetitive elements, such as LINE-1 and ALU, have been identified as the common biomarkers in cancer and aging. However, it remains unclear whether the methylation levels of repetitive elements can imply the drug sensitivity in gastrointestinal cancer. We investigated the association of LINE-1 and ALU methylations with the sensitivity of cytotoxic and senotherapeutic drugs in gastric cancer (GC) and colorectal cancer (CRC). Methods: The profiles of DNA methylation and gene expression were obtained from GC and CRC patients from the TCGA cohorts. Then, we calculated the averaging methylation level of two repetitive elements (LINE-1 and ALU) to quantify the global methylation levels by using the probes targeting the repetitive elements in 450K methylation array. The sensitivity of anti-tumor drugs, including the cytotoxic (oxaliplatin and fluorouracil) and five senotherapeutic agents (Dasatinib, Nutlin-3a, Navitoclax, Rapamycin, ABT737), were inferred with Oncopredict R package based on the gene expression profile. The Pearson analysis was conducted to test the correlation between DNA methylation and drug sensitivity. Results: The ALU methylation was associated with the sensitivities of cytotoxic (oxaliplatin: r = 0.171, P = 0.001; fluorouracil: r = 0.158, P = 0.002) and senotherapeutic agents (Dasatinib: r = 0.162, P = 0.002, Nutlin-3a: r = 0.109, P = 0.037, Navitoclax: r = 0.007, P = 0.931, Rapamycin: r = 0.144, P = 0.006, ABT737: r = 0.054, P = 0.523) in GC, while the association of LINE-1 methylation with drug sensitivities was observed in less drugs with weak correlation, including the cytotoxic (oxaliplatin: r = 0.132, P = 0.013; fluorouracil: r = 0.122, P = 0.019) and senotherapeutic agents (Dasatinib: r = 0.129, P = 0.014, Nutlin-3a: r = 0.109, P = 0.037, Rapamycin: r = 0.110, P = 0.034). In CRC, there was no correlation between DNA methylation levels of repetitive elements and drug sensitivity except Dasatinib (ALU: r = -0.107, P = 0.043). Conclusion: DNA methylation of repetitive elements could serve as a biomarker for cytotoxic and senotherapeutic drugs in GC. In addition, our findings suggested the promising value of ALU and LINE-1 methylation-guided chemotherapy with senotherapeutics in GC. Keywords: global DNA methylation, repetitive elements, gastrointestinal cancer, drug sensitivity, senotherapeutics Citation Format: Kaixin Lin, Xiaolin Wang, Yanxin Luo, Huichuan Yu. Global DNA methylation level is associated with the sensitivity of cytotoxic and senotherapeutic drugs in gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3485.

Abstract 7028: Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States. Health disparities in men diagnosed with prostate cancer are observed between patients of African and those of European ancestry. To elucidate molecular mechanisms behind prostate tumorigenesis and racial disparities, researchers have identified genetic alterations such as gene fusions, deletions, and mutations that occur at varying frequencies among different ethnic groups. However, results are inconsistent across studies, suggesting that racial disparities may be multifactorial. Our approach to further understand the mechanisms underlying racial disparities is to compare DNA methylation profiles between cancers from different ethnic groups using formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue samples. For this study, FFPE prostate cancer specimens from 32 Black men and 30 age-matched White men were obtained in the form of tissue sections on glass slides. To profile DNA methylation in these samples, tumor and normal tissue regions (in a subset of cases) were outlined on a hematoxylin &amp; eosin (H&amp;E) reference slide and the respective regions were macro-dissected off the corresponding unstained slides from the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to normalize DNA yield. Next, we compared magnetic bead and column based DNA extraction methods. We evaluated them on their relative DNA yield per tissue size, and identified that the bead-based extraction method had both higher and more consistent DNA yield. In total, we used 95 unique tissue regions to profile global DNA methylation levels using Illumina Epic array. We found that a subset of samples harbored an aberrant DNA methylation pattern. Therefore, we extracted DNA from additional FFPE tissue slides using the bead-based method and repeated the assay with a larger amount of DNA, successfully rescuing the majority of the aberrant samples. Next, we will evaluate associations between DNA methylation levels in different ethnic groups, clinicopathological features, and follow-up data to help identify potential DNA methylation biomarkers and therapeutic targets linked to prostate cancer from different ethnicities. Citation Format: Colton Duran Stensrud, Leonardo Gonzalez-Smith, Claire Stevens, Huan Cao, Jenaye Mack, Olumide Arigbede, Daniel Weisenberger, Sarah G. Buxbaum, Sara M. Falzarano, Suhn Rhie. Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7028.

Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells.

A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.

Biomarkers of Depression among Adolescent Girls: BDNF and Epigenetics.

Alterations in brain-derived neurotrophic factor (BDNF) expression have been suggested to mediate the influence of environmental factors on the emergence of depression through epigenetic modifications. However, research on this subject in the developmental population is lacking and the pathophysiology of adolescent depression remains unclear. We aimed to investigate the alterations in BDNF expression and global DNA methylation in depression among adolescent girls. Thirty female inpatients with the initial diagnosis of depression were assessed before and after the period of antidepressant treatment and compared with thirty age-matched healthy controls. The assessment involved BDNF and proBDNF serum levels, the BDNF gene exon IV promoter methylation, and global DNA methylation. The methylation level in the BDNF gene exon IV promoter was significantly lower in the studied group compared with the control and correlated negatively with the severity of depression. The test distinguished the studied group from the controls with a sensitivity of 37% and specificity of 90%. The differences were no longer present after the period of antidepressant treatment. No differences in the global DNA methylation, BDNF, and proBDNF levels were found. We concluded that decreased methylation in the BDNF exon IV promoter could be considered as a biomarker of a depression state among adolescent girls.

Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific β-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., β-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low β-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Global DNA Methylation Levels Viz-a-Viz Genetic and Biochemical Variations in One Carbon Metabolic Pathway: An Exploratory Study from North India.

Despite the importance of one carbon metabolic pathway (OCMP) in modulating the DNA methylation process, only a few population-based studies have explored their relationship among healthy individuals. This study aimed to understand the variations in global DNA methylation levels with respect to selected genetic (CBS 844ins68, MTRR A66G, MTR A2756G, and MTHFR C677T polymorphisms) and biochemical (folate, vitamin B12, and homocysteine) markers associated with OCMP among healthy North Indian adults. The study has been conducted among 1095 individuals of either sex (69.5% females), aged 30-75 years. A sample of 5 mL of blood was collected from each participant. Homocysteine, folate, and vitamin B12 levels were determined using the chemiluminescence technique. Restriction digestion was performed for genotyping MTRR A66G, MTR A2756G, and MTHFR C677T polymorphisms and allele-specific PCR amplification for CBS 844ins68 polymorphism. Global DNA methylation levels were analyzed using ELISA-based colorimetric technique. Of the selected genetic and biochemical markers, the mutant MTRR A66G allele was positively associated with global DNA methylation levels. Further, advanced age was inversely associated with methylation levels. MTRR 66GG genotype group was hypermethylated than other genotypes in folate replete and vitamin B12 deficient group (a condition prevalent among vegetarians), suggesting that the G allele may be more efficient than the wild-type allele in such conditions. Global DNA methylation levels appeared to be more influenced by genetic than biochemical factors. MTRR 66G allele may have a selective advantage in vitamin B12 deficient conditions. Further research should be undertaken to understand how genetics affects epigenetic processes.

CDCA7-associated global aberrant DNA hypomethylation translates to localized, tissue-specific transcriptional responses.

Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.

Investigating the effects of valproic acid on placental epigenetic modifications and development in the CD-1 mouse model

Gestational exposure to the anticonvulsant drug valproic acid (VPA) is associated with congenital malformations and neurodevelopmental disorders through its action as a histone deacetylase inhibitor. VPA can elicit placental toxicity and affect placental growth and development. The objective of this study was to evaluate the impact of maternal exposure to VPA on the mouse placenta following exposure on gestational day (GD) 13 since previous studies have shown that mice exposed at this time during gestation give birth to offspring with an autism spectrum disorder-like phenotype. We exposed CD-1 dams to a teratogenic dose (600 mg/kg) of VPA or saline on GD13 and assessed fetoplacental growth and development on GD18. We evaluated epigenetic modifications, including acetylated histone H4 (H4ac), methylated H3K4 (H3K4me2) using immunohistochemistry, and global DNA methylation in the placenta at 1, 3, and 24 h following maternal exposure on GD13. In utero exposure to VPA on GD13 significantly decreased placental weight and increased fetal resorptions. Moreover, VPA significantly increased the staining intensity of histone H4 acetylation and H3K4 di-methylation across the placenta at 1 and 3 h post maternal dose. Our results also demonstrate that VPA significantly decreased global DNA methylation levels in placental tissue. These results show that gestational exposure to VPA interferes with placental growth and elicits epigenetic modifications, which may play a vital role in VPA-induced developmental toxicity.

The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single-base resolution methylation profiles of the Pacific oyster, including female-to-male (FMa-to-FMb) and male-to-female (MFa-to-MFb) sex reversal, as well as sex non-reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during female-to-male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male-to-female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox-like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non-reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.

Exploring the influence of DNA methylation and single nucleotide polymorphisms of the Myostatin gene on growth traits in the hybrid grouper (Epinephelus fuscoguttatus (female) × Epinephelus polyphekadion (male)).

Investigations into the correlation between growth characteristics and DNA methylation levels, along with genetic variations, can provide fundamental insights to enhance growth performance in groupers. The Myostatin (mstn) gene plays a vital role in regulating skeletal muscle development and growth. This study scrutinized the DNA methylation levels of the mstn gene across hybrid groupers (E. fuscoguttatus (♀) × E. polyphekadion (♂)) and their parental species, to evaluate its impact on growth attributes in grouper fish. The nucleotide sequence of the mstn gene was directly sequenced in the hybrid grouper, exhibiting different growth performance to identify the single nucleotide polymorphisms (SNPs) of the mstn gene and explore their correlation with growth characteristics. The findings revealed no significant differences in global DNA methylation levels within muscle tissue among the hybrid grouper and parents. However, significant differences in DNA methylation sites were discovered between the hybrid grouper and E. polyphekadion at sites 824 and 1521 (located at exon 2 and intron 2, respectively), and between E. fuscoguttatus and E. polyphekadion at site 1521. These variations could potentially influence the mRNA expression of the mstn gene. The study also identified that SNP g.1003 T > C in exon 2 of the mstn gene was significantly associated with various growth traits including body weight, total length, body length, head length, caudal peduncle height, and body height (p < 0.01). Specimens with the TT genotype at site 1003 demonstrated superior growth performance compared to those with the TC genotype. Furthermore, microstructural analyses of muscle tissue showed that the average area and diameter of muscle fibers in TT genotype individuals were significantly greater than those in TC genotype individuals. Therefore, this research provides robust evidence linking the DNA methylation level and polymorphisms of the mstn gene with growth traits, which could be beneficial for grouper breeding programs.

Improved prognostic stratification of patients with isocitrate dehydrogenase-mutant astrocytoma

Prognostic factors and standards of care for astrocytoma, isocitrate dehydrogenase (IDH)-mutant, CNS WHO grade 4, remain poorly defined. Here we sought to explore disease characteristics, prognostic markers, and outcome in patients with this newly defined tumor type. We determined molecular biomarkers and assembled clinical and outcome data in patients with IDH-mutant astrocytomas confirmed by central pathology review. Patients were identified in the German Glioma Network cohort study; additional cohorts of patients with CNS WHO grade 4 tumors were identified retrospectively at two sites. In total, 258 patients with IDH-mutant astrocytomas (114 CNS WHO grade 2, 73 CNS WHO grade 3, 71 CNS WHO grade 4) were studied. The median age at diagnosis was similar for all grades. Karnofsky performance status at diagnosis inversely correlated with CNS WHO grade (p < 0.001). Despite more intensive treatment upfront with higher grade, CNS WHO grade was strongly prognostic: median overall survival was not reached for grade 2 (median follow-up 10.4 years), 8.1 years (95% CI 5.4–10.8) for grade 3, and 4.7 years (95% CI 3.4–6.0) for grade 4. Among patients with CNS WHO grade 4 astrocytoma, median overall survival was 5.5 years (95% CI 4.3–6.7) without (n = 58) versus 1.8 years (95% CI 0–4.1) with (n = 12) homozygous CDKN2A deletion. Lower levels of global DNA methylation as detected by LINE-1 methylation analysis were strongly associated with CNS WHO grade 4 (p < 0.001) and poor outcome. MGMT promoter methylation status was not prognostic for overall survival. Histomolecular stratification based on CNS WHO grade, LINE-1 methylation level, and CDKN2A status revealed four subgroups of patients with significantly different outcomes. In conclusion, CNS WHO grade, global DNA methylation status, and CDKN2A homozygous deletion are prognostic in patients with IDH-mutant astrocytoma. Combination of these parameters allows for improved prediction of outcome. These data aid in designing upcoming trials using IDH inhibitors.

Association between Tissue Polypeptide Specific Antigen and Vascular Endothelial Growth Factor in Colorectal Cancer Patients and their Relation with P53 Expression and Global DNA Methylation

Background: Colorectal cancer is a high risk disease with rapidly progression medical problems and high mortality rate. Tissue polypeptide specific antigen can be classified as biomarker candidates in colorectal cancer and other kinds of cancer. Vascular endothelial-derived growth factor has a curial role in the formation of new blood vessels. DNA methylation may decrease invasiveness of cancer. Objectives: This study was designed to measure the potential role of some serological biomarkers in the progression of colorectal cancer as well as their relations to P53 expression, global 5-methylcytosine. Patients and Methods: This study involved of 60 patients with colorectal cancer who attended Oncology Teaching Hospital in Baghdad, Iraq and compared with 30 apparently healthy volunteers. All participants were checked for serological biomarker levels using an enzyme-linked immunosorbent assay. Also, DNA and RNA extracted and the levels of 5-methylcytosine and P53 expression were evaluated. The SPSS version 24.0 program was used for all statistical analysis done in this study. Results: The analysis of serological biomarker data has shown that the mean of tissue polypeptide specific antigen and vascular endothelial growth factor was significantly higher for patients with colorectal cancer, while it was lower for control subjects (356.4 ± 16.9, 139.75 ± 7.31) and (1127.82±65.41, 388.93±35.86), respectively. Also, a significant positive relation between tissue polypeptide specific antigen and vascular endothelial growth factor in the tumor area was significantly associated with control (P≤ 0.001). However, there was significant correlation in the levels of P53 expression and global DNA methylation between studied groups as well as tissue polypeptide specific antigen and vascular endothelial growth factor (P ≥ 0.05). Conclusion: The serological biomarkers include tissue polypeptide specific antigen and vascular endothelial growth factor and they play a critical role in the development of colorectal cancer. Serum vascular endothelial growth factor is useful in distinguishing colorectal cancer from healthy controls, whereas tissue polypeptide specific antigen is an independent prognostic factor for colorectal cancer proliferation. Received: Mar. 2023 Accepted: Jun 2023 Published: Oct.2023

Elevated temperature triggers increase in global DNA methylation, 5-methylcytosine expression levels, apoptosis and NOx levels in the gonads of Atlantic sea urchin.

Global warming is one of the greatest threats to living organisms. Among them, marine invertebrates are severely impacted on reproductive fitness by rising seawater surface temperatures due to climate change (e.g., massive heat waves). In this study, we used highly sensitive radioimmunoassay, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), in situ TUNEL assay, luminescence assay, and colorimetric assay techniques to investigate the impacts of high temperatures on global DNA methylation, cellular apoptosis, and nitrative stress in gonads of Atlantic sea urchin (Arbacia punctulata, a commercially important species). Young adult sea urchins were exposed to 24, 28, and 32°C for one week in a controlled laboratory setting. High temperatures (28 and 32°C) markedly increased global DNA methylation (around 1.1-1.5-fold in testes and~1.7-fold in ovaries) and 5-methylcytosine (5-mC) levels in gonads (around 2.7- to ~5.1-fold in ovaries and~3.5- to ~6.2-fold in testes) compared with controls (24°C). The number of apoptotic nuclei in gonads was much higher in high-temperature groups. The caspase activity also increased significantly (P<0.05) in gonads in high-temperature groups. Nitrate/nitrites (NOx, a biomarker of reactive nitrogen species) levels were increased around 2.6- to ~5.2-fold in testes and~1.9- to ~3.8-fold in ovaries in high-temperature groups. Collectively, these outcomes indicate that high temperatures drastically induce global DNA methylation, 5-mC expression levels, cellular apoptosis, and NOx levels in the gonads of Atlantic sea urchin.

A comprehensive characterisation of phaeochromocytoma and paraganglioma tumours through histone protein profiling, DNA methylation and transcriptomic analysis genome wide

BackgroundPhaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours. Pathogenic variants have been identified in more than 15 susceptibility genes; associated tumours are grouped into three Clusters, reinforced by their transcriptional profiles. Cluster 1A PPGLs have pathogenic variants affecting enzymes of the tricarboxylic acid cycle, including succinate dehydrogenase. Within inherited PPGLs, these are the most common. PPGL tumours are known to undergo epigenetic reprograming, and here, we report on global histone post-translational modifications and DNA methylation levels, alongside clinical phenotypes.ResultsOut of the 25 histone post-translational modifications examined, Cluster 1A PPGLs were distinguished from other tumours by a decrease in hyper-acetylated peptides and an increase in H3K4me2. DNA methylation was compared between tumours from individuals who developed metastatic disease versus those that did not. The majority of differentially methylated sites identified tended to be completely methylated or unmethylated in non-metastatic tumours, with low inter-sample variance. Metastatic tumours by contrast consistently had an intermediate DNA methylation state, including the ephrin receptor EPHA4 and its ligand EFNA3. Gene expression analyses performed to identify genes involved in metastatic tumour behaviour pin-pointed a number of genes previously described as mis-regulated in Cluster 1A tumours, as well as highlighting the tumour suppressor RGS22 and the pituitary tumour-transforming gene PTTG1.ConclusionsCombined transcriptomic and DNA methylation analyses revealed aberrant pathways, including ones that could be implicated in metastatic phenotypes and, for the first time, we report a decrease in hyper-acetylated histone marks in Cluster 1 PPGLs.