Differences In Methylation Levels Research Articles

Epigenetic differences in the tumor suppressor genes MLH1 and p16INK4a between Nepalese and Swedish patients with colorectal cancer

Abstract Objectives Colorectal cancer (CRC) is one of the most prevalent cancer types worldwide, exhibiting significant variance in incidence rates across different ethnicities and geographical regions. Notably, there is a rising incidence of CRC among younger adults, particularly evident in advanced stages, with a more pronounced trend observed in developing nations. Epigenetic alterations potentially play a role in the early onset of CRC and could elucidate interpopulation disparities. This study aimed to examine DNA methylation levels in the tumor suppressor genes MLH1 and p16INK4a, comparing Nepalese and Swedish patients with CRC. Methods Patients who underwent CRC surgery at Tribhuvan University Teaching Hospital, Nepal (n=39), and Sahlgrenska University Hospital, Sweden (n=39) were included. Demographic and clinicopathological data were analyzed, and pyrosequencing was employed to determine methylation levels in the MLH1 promoter region and the first exon of p16INK4a in tumor tissues and adjacent mucosa located 10 cm from the tumor site. Subsequently, methylation status was compared between Nepalese and Swedish patients and correlated with clinicopathological parameters. Results Nepalese and Swedish patients displayed equal levels of MLH1 and p16INK4a methylation in tumors, but Nepalese patients exhibited a significantly higher level of MLH1 methylation in mucosa compared to Swedish patients (p=0.0008). Moreover, a greater proportion of Nepalese patients showed MLH1 methylation in mucosa compared to Swedish patients (31 vs. 2.6 %). Aberrant methylation of p16INK4a was also observed in the mucosa of Nepalese patients, characterized by high methylation at specific sites rather than uniform methylation across CpG sites. There were no significant differences in methylation levels based on tumor location among Nepalese patients, whereas Swedish patients exhibited higher methylation in right- compared to left-sided colon tumors. Swedish patients showed an increase in p16INK4a methylation in tumors with advancing age. Conclusions Nepalese and Swedish patients displayed equal levels of MLH1 and p16INK4a methylation in tumors. In contrast, Nepalese patients had a higher level of MLH1 methylation as well as aberrant methylation of p16INK4a in mucosa compared to Swedish patients. These epigenetic differences may be linked to environmental and lifestyle factors. Ongoing research will further explore whether hypermethylation in the mucosa of Nepalese patients is associated with tumorigenesis and its potential utility in screening high-risk patients or predicting recurrence.

Low methylation marker levels among human papillomavirus-vaccinated women with cervical high-grade squamous intraepithelial lesions.

Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)-vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false-positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case-control study, 9242 women who received the three-dose HPV16/18-vaccine at ages 12-15 or 18 in a community-randomized trial were included. Subsequently, they were re-randomized for either frequent or infrequent cervical cancer screening trials. Over a 15-year post-vaccination follow-up until 2022, 17 high-grade squamous intraepithelial lesion (HSIL) and 15 low-grade (LSIL) cases were identified at the 25-year screening round, alongside 371 age and community-matched HPV16/18-vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host-cell genes (EPB41L3, FAM19A4, and miR124-2) was measured, along with HPV-genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV-genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host-cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value = .0001). HPV-vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV-vaccinated women and its implications for management.

DNA Methylation Analysis of Growth Differences between Upright and Inverted Cuttings of Populus yunnanensis.

DNA methylation is an important mechanism for epigenetic modifications that have been shown to be associated with responses to plant development. Previous studies found that inverted Populus yunnanensis cuttings were still viable and could develop into complete plants. However, the growth status of inverted cuttings was weaker than that of upright cuttings, and the sprouting time of inverted cuttings was later than that of upright cuttings. There is currently no research on DNA methylation patterns in inverted cuttings of Populus yunnanensis. In this study, we detected genome-wide methylation patterns of stem tips of Populus yunnanensis at the early growth stage and the rapid growth stage by Oxford Nanopore Technologies (ONT) methylation sequencing. We found that the methylation levels of CpG, CHG, CHH, and 6mA were 41.34%, 33.79%, 17.27%, and 12.90%, respectively, in the genome of inverted poplar cuttings, while the methylation levels of the four methylation types were higher in the genome of upright poplar cuttings than in inverted cuttings, 41.90%, 34.57%, 18.09%, and 14.11%, suggesting important roles for DNA methylation in poplar cells. In all comparison groups, CpG-type methylation genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were annotated to pathways associated with carbon metabolism, ribosome biogenesis in eukaryotes, glycolysis/gluconeogenesis, pyruvate metabolism, and mRNA detection pathways, suggesting that different biological processes are activated in upright and inverted cuttings. The results show that methylation genes are commonly present in the poplar genome, but only a few of them are involved in the regulation of expression in the growth and development of inverted cuttings. From this, we screened the DET2 gene for significant differences in methylation levels in upright or inverted cuttings. The DET2 gene is a key gene in the Brassinolide (BRs) synthesis pathway, and BRs have an important influence on the growth and development process of plants. These results provide important clues for studying DNA methylation patterns in P. yunnanensis.

Selective epigenetic alterations in RNF43 in pancreatic exocrine cells from high-fat-diet-induced obese mice; implications for pancreatic cancer

ObjectivePancreatic cancer (PC) originates and progresses with genetic mutations in various oncogenes and suppressor genes, notably KRAS, CDKN2A, TP53, and SMAD4, prevalent across diverse PC cells. In addition to genetic mutations/deletions, persistent exposure to high-risk factors, including obesity, induces whole-genome scale epigenetic alterations contributing to malignancy. However, the impact of obesity on DNA methylation in the presymptomatic stage, particularly in genes prone to PC mutation, remains uncharacterized.ResultsWe analyzed the methylation levels of 197 loci in six genes (KRAS, CDKN2A, TP53, SMAD4, GNAS and RNF43) using Illumina Mouse Methylation BeadChip array (280 K) data from pancreatic exocrine cells obtained from high-fat-diet (HFD) induced obese mice. Results revealed no significant differences in methylation levels in loci between HFD- and normal-fat-diet (NFD)-fed mice, except for RNF43, a negative regulator of Wnt signaling, which showed hypermethylation in three loci. These findings indicate that, in mouse pancreatic exocrine cells, high-fat dietary obesity induced aberrant DNA methylation in RNF43 but not in other frequently mutated PC-related genes.

Epigenetic clocks, sex markers and age-class diagnostics in three harvested large mammals.

The development of epigenetic clocks, or the DNA methylation-based inference of age, is an emerging tool for ageing in free ranging populations. In this study, we developed epigenetic clocks for three species of large mammals that are the focus of extensive management throughout their range in North America: white-tailed deer, black bear and mountain goat. We quantified differential DNA methylation patterns at over 30,000 cytosine-guanine sites (CpGs) from tissue samples of all three species (black bear n = 49; white-tailed deer n = 47; mountain goat n = 45). We used a penalized regression model (elastic net) to build explanatory (black bear r = .95; white-tailed deer r = .99; mountain goat r = .97) and robust (black bear Median Absolute Error or MAE = 1.33; white-tailed deer MAE = 0.29; mountain goat MAE = 0.61) models of age or clocks. We also characterized individual CpG sites within each species that demonstrated clear differences in methylation levels between age classes and sex, which can be used to develop a suite of accessible diagnostic markers. This tool has the potential to contribute to wildlife monitoring by providing easily obtainable representations of age structure in managed populations.

Significant heightened methylation levels of RUNX3 gene promoter in patients with systemic lupus erythematosus.

Researchers are actively investigating new diagnostic and prognostic biomarkers that offer improved sensitivity and specificity for systemic lupus erythematosus (SLE). One area of interest is DNA methylation changes. Previous studies have shown a connection between the RUNX3 gene dysfunction and SLE. In this study, the focus was on examining the methylation level of the RUNX3 promoter in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy individuals. A total of 80 individuals diagnosed with SLE from Iran, along with 77 healthy individuals, were included. The methylation levels of the RUNX3 gene in the extracted DNA were evaluated using the MethyQESD method. To determine the diagnostic effectiveness of the RUNX3 promoter methylation level, a receiver operating characteristic (ROC) curve was generated. The methylation of the RUNX3 promoter was found to be significantly higher in patients with SLE compared to healthy individuals (p < .001). This difference in methylation levels was observed between SLE patients and healthy individuals and between SLE patients with renal involvement and those without renal involvement (86.29 ± 10.30 vs 40.28 ± 24.21, p < .001). ROC analyses revealed that the methylation level of the RUNX3 promoter had a diagnostic power of 0.769 [95% CI (0.681-0.814)] for SLE. Additionally, there was a positive correlation between the RUNX3 methylation level and levels of creatinine and C4. The findings of this study emphasize the potential use of RUNX3 methylation levels in PBMCs of SLE patients as biomarkers for diagnosing the disease, predicting renal damage, and assessing disease activity.

Revealing distinct DNA methylation patterns in hepatic carcinoma through high-throughput sequencing

Abstract Objectives To study the relationship between DNA methylation and tumour development and provide experimental evidence for the personalized diagnosis and treatment of hepatic carcinoma. Methods The DNA of hepatic carcinoma tissue (Ca group) and adjacent normal tissue (T group) were extracted using the phenol-chloroform method and then treated with bisulfite. Twenty-five genes including 45 subtypes were amplified by PCR. The PCR products were sequenced via the Illumina 450k methylation array assay. The changes of methylated DNA performance were analysed through principal component analysis (PCA). Cluster analysis was used to evaluate the classification of methylated DNA regions. Haplotype abundance variation was tested for methylation differences. Statistical analysis was performed using the chi-square (χ2) test or Fisher’s exact test. Results Sequencing discoveries indicated CG-type methylation pervading all amplicons. However, CHG-type and CHH-type methylations were confined to only four amplicons (or nine subtypes). The methylation ratios of three specific amplicons (DAB2IP, PRDM14-1, Rab31-1) out of 45 amplicon subtypes in the Ca group significantly increased (over 10 %) compared to the T group (p&lt;0.05). Nineteen amplicons demonstrated minor distinction (methylation pattern variations between 1 and 10 %), with the remaining 23 amplicons showing only minimal disparities (under 1 %). PCA and cluster analysis unveiled a marked difference in methylation levels between cancerous and healthy tissues (p&lt;0.05). Conclusions The changes in haplotypes and methylation sites could serve as a biomarker for the clinical diagnosis of hepatic carcinoma. Methylation patterns might play an important role in the occurrence and development of hepatic carcinoma.

The regulation of plasma gelsolin by DNA methylation in ovarian cancer chemo-resistance

BackgroundOvarian cancer (OVCA) is the most lethal gynecologic cancer and chemoresistance remains a major hurdle to successful therapy and survival of OVCA patients. Plasma gelsolin (pGSN) is highly expressed in chemoresistant OVCA compared with their chemosensitive counterparts, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. In this study, we investigated the methylation role of Ten eleven translocation isoform-1 (TET1) in the regulation of differential pGSN expression and chemosensitivity in OVCA cells.MethodsChemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance (qPCR) as well as protein contents (Western blotting). To investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance, DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cells using specific inhibitors. DNA methylation was quantified using EpiTYPER MassARRAY system. Gain-and-loss-of-function assays were used to investigate the relationship between TET1 and pGSN in OVCA chemoresponsiveness.ResultsWe observed differential protein and mRNA expressions of pGSN and TET1 between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. We observed hypomethylation at pGSN promoter upstream region in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreased their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN but positively related to chemoresponsiveness of OVCA cells.ConclusionOur findings broaden our knowledge about the epigenetic regulation of pGSN in OVCA chemoresistance and reveal a novel potential target to re-sensitize resistant OVCA cells. This may provide a future therapeutic strategy to improve the overall OVCA patient survival.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN).For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10−8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity.In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

Validation of hypermethylated DNA regions found in colorectal cancers as potential aging-independent biomarkers of precancerous colorectal lesions

BackgroundWe previously identified 16,772 colorectal cancer-associated hypermethylated DNA regions that were also detectable in precancerous colorectal lesions (preCRCs) and unrelated to normal mucosal aging. We have now conducted a study to validate 990 of these differentially methylated DNA regions (DMRs) in a new series of preCRCs.MethodsWe used targeted bisulfite sequencing to validate these 990 potential biomarkers in 59 preCRC tissue samples (41 conventional adenomas, 18 sessile serrated lesions), each with a patient-matched normal mucosal sample. Based on differential DNA methylation tests, a panel of candidate DMRs was chosen on a subset of our cohort and then validated on the remaining part of our cohort and two publicly available datasets with respect to their stratifying potential between preCRCs and normal mucosa.ResultsStrong statistical significance for the difference in methylation levels was observed across the full set of 990 investigated DMRs. From these, a selected candidate panel of 30 DMRs correctly identified 58/59 tumors (area under the receiver operating curve: 0.998).ConclusionsThese validated DNA hypermethylation markers can be exploited to develop more accurate noninvasive colorectal tumor screening assays.

PRICKLE1 gene methylation and abnormal transcription in Chinese patients with ankylosing spondylitis

BackgroundAnkylosing spondylitis (AS) is a common inflammatory arthritis without a reliable biomarker. The role of methylation and mRNA expression of PRICKLE1 promoter in the pathogenesis of ankylosing spondylitis remains unclear. MethodsA two-stage case-control design was used to detect the characteristics of methyl group and transcriptome of PRICKLE1 gene in Ankylosing spondylitis. The methylation degree of PRICKLE1 gene promoter region was tested by phosphate-sequencing, and further analyzed whether there was significant difference in methylation level of PRICKLE1 gene. The expression levels of PRICKLE1 mRNA in 50 AS patients and 50 healthy controls were detected by real-time quantitative PCR (RT-qPCR). ResultsCompared with healthy control group, the intensity of methylation in 4 ponds of PRICKLE1 in patients with Ankylosing spondylitis was low, and the mRNA levels were overexpressed (P = 0.017). ROC results showed that the sensitivity of PRICKLE1 was 68.67% and specificity was 71.43%. ConclusionThere is a significant change in the concentration of serum PRICKLE1 mRNA​in patients with Ankylosing spondylitis, and the degree of gene methylation is significantly reduced, suggesting that PRICKLE1 gene maybe involved in the pathogenesis of Ankylosing spondylitis, which may be useful for predicting the occurrence of AS and finding new early screening indicators.

Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models.

Body fluid mixtures are complex biological samples that frequently occur in crime scenes, and can provide important clues for criminal case analysis. DNA methylation assay has been applied in the identification of human body fluids, and has exhibited excellent performance in predicting single-source body fluids. The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification, and accurately predict the mixture samples. In addition, the value of DNA methylation in the prediction of body fluid mixtures was further explored. In the present study, 420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system. Each kind of body fluid sample presented the specific methylation profiles of the 10 markers. Significant differences in methylation levels were observed between the mixtures and single body fluids. For all kinds of mixtures, the Spearman's correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions (1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1). Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components, based on the methylation levels of 10 markers. For the mixture prediction, Model-1 presented outstanding prediction accuracy, which reached up to 99.3% in 427 training samples, and had a remarkable accuracy of 100% in 243 independent test samples. For the mixture proportion prediction, Model-2 demonstrated an excellent accuracy of 98.8% in 252 training samples, and 98.2% in 168 independent test samples. The total prediction accuracy reached 99.3% for body fluid mixtures and 98.6% for the mixture proportions. These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

Association of DNA methylation signatures with cognitive performance among smokers and ex-smokers.

Alterations in DNA methylation profiles have been associated with cancer, and can be influenced by environmental factors such as smoking. A small but growing literature indicates there are reproducible and robust differences in methylation levels among smokers, never smokers, and ex-smokers. Here, we compared differences in salivary DNA methylation levels among current and ex-smokers (at least 2 years abstinent). Smokers (n=26) and ex-smokers (n=30) provided detailed smoking histories, completed the Paced Auditory Serial Addition Test (PASAT), and submitted a saliva sample. Whole-genome DNA methylation from saliva was performed, and ANCOVA models and a receiver operating characteristic (ROC) curve were used for the differences between groups and the performance of significant CpG sites. After controlling for race, age, and gender, smokers had significantly lower methylation levels than ex-smokers in two CpG sites: cg05575921 (AHRR) and cg21566642 (ALPPL2). Based on the ROC analyses, both CpGs had strong classification potentials (cg05575921 AUC=0.97 and cg21566642 AUC=0.93) in differentiating smoking status. Across all subjects, the percent methylation of cg05575921 (AHRR) and cg21566642 (ALPPL2) positively correlated with the length of the last quit attempt (r=0.65 and 0.64, respectively, p<0.001) and PASAT accuracy (r=0.29 and 0.30, respectively, p<0.05). In spite of the small sample size and preliminary research, our results replicate previously reported differences in AHRR hypomethylation among smokers. Furthermore, we show that the duration of smoking abstinence is associated with a recovery of methylation in ex-smokers, which may be linked to a reduced risk of smoking-associated diseases. The association with cognitive performance suggests that the hypomethylation of AHRR in saliva may reflect systemic exposure to cigarette-related toxicants that negatively affect cognitive performance, and should be validated in larger studies.

The Methylation Analysis of the Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR) Locus in GH-Secreting Pituitary Adenomas.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR-) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2'-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR- GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2'-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism.

The mechanisms underpinning anthocyanin accumulation in a red-skinned bud sport in pear (Pyrus ussuriensis)

In our study, we demonstrated that histone acetylation promotes anthocyanin accumulation in pears by affecting the expression of key genes. Color is an important trait of horticultural plants, and the anthocyanin content directly affects the nutritional value and commercial value of colored fruits. Therefore, it is important for fruit breeding to cultivate new varieties with bright colors. 'Nanhong' (NH) pear (Pyrus ussuriensis) is a bud sport cultivar of 'Nanguo' (NG) pear. The anthocyanin content in NH pear is significantly higher than that in NG pear, but the underlying molecular mechanism remains unclear. Here, we observed that the anthocyanin biosynthesis structural gene PuUFGT (UDP-glucose: flavonoids 3-O-glucosyltransferase) and an anthocyanin transporter gene PuGSTF6 (glutathione S-transferase) had significantly higher expression levels in NH than in NG pears during the late stages of fruit development. Meanwhile, the R2R3-MYB transcription factor PuMYB110a was also highly expressed in NH pears and could positively regulate the transcription of PuUFGT and PuGSTF6. Overexpression of PuMYB110a in pear increased the fruit anthocyanin content. In addition, despite no significant differences in methylation levels being found in the promoters of PuMYB110a, PuUFGT, and PuGSTF6 when comparing the two varieties, the histone acetylation levels of PuMYB110a were significantly higher in NH pear compared with those in NG pear. Our findings suggest a mechanism for anthocyanin accumulation in NH fruit.

Methylation of the glucocorticoid receptor gene (NR3C1) in dyads mother-child exposed to intimate partner violence in Cameroon: Association with anxiety symptoms.

The glucocorticoid receptor (GR), which is encoded by the NR3C1 (Nuclear Receptor Subfamily 3 Group C Member 1) gene plays an important role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis activity by providing feedback regulation which allows termination of the stress response. Little is known about epigenetic programming at the level of NGFI-A (nerve growth factor-inducible protein A) putative binding site (CpG) of the NR3C1 exon 1F in dyads mother-child exposed to intimate partner violence (IPV) more specifically in an unstudied region such as the sub-Saharan Africa where levels of violence are very high. Examine NR3C1 exon 1F methylation in response to IPV and possible association with cortisol concentration and mental health. We recruited 20 mother-child dyads exposed to IPV and a control group of 20 mother-child dyads not exposed to IPV. We administered self-reported questionnaires to measure mother's mental health and collected saliva samples for cortisol dosage and bisulfite sequencing of DNA methylation. Regarding the mothers, our results showed a significant difference in methylation level at CpG 16-21 sites of the NR3C1 exon 1F promoter region between the groups. In the exposed group as compared to the control group, there was a significant positive association between the level of methylation at CpG 16-21 sites and mother's mental health in particular anxiety symptoms. However, we did not find any significant correlation between methylation level and cortisol concentration. In children, we did not find any significant results. This study highlights a NGFI-A putative binding site (CpG 16-21) that is more methylated in mothers exposed to IPV and which may have the potential to confer vulnerability for psychopathologies.

Analysis of DNA methylation markers for tissue identification in individuals with different clinical phenotypes.

Deoxyribonucleic acid (DNA) methylation patterns can be used to identify the type of tissue or body fluid found at a crime scene. However, tissue-related methylation levels have not been analyzed in individuals with different illnesses and medical conditions in forensic-specific studies. The primary goal of this study was to investigate if certain clinical phenotypes can alter the methylation levels of CpG sites in genes involved in tissue typing. Four studies with focus on DNA methylation analysis on individuals with different clinical conditions were selected from the Gene Expression Omnibus database. Then, a list of 137 CpG sites was compiled for further investigation. Statistical tests were performed to compare the beta-values results obtained for the control groups and the individuals affected by medical conditions. For each study, CpG sites that presented significant statistical differences between patients and control group were identified and it was possible to notice that DNA methylation levels can be affected in sites with potential forensic use. Although the observed DNA methylation variation (less than 10% difference) in this study would likely not cause any issues in body fluid identification, the results are important to show that this type of analysis should be taken into consideration when investigating and further validating body fluid markers. The CpG sites identified in this study should be further investigated by future studies on body fluids identification, and due to the significant difference in methylation levels in samples from affected individuals, caution must be taken before including these sites in tissue identification investigations.

Sex differences in the intergenerational link between maternal and neonatal whole blood DNA methylation: a genome-wide analysis in 2 birth cohorts

BackgroundThe mother–child inheritance of DNA methylation (DNAm) variations could contribute to the inheritance of disease susceptibility across generations. However, no study has investigated patterns of mother–child associations in DNAm at the genome-wide scale. It remains unknown whether there are sex differences in mother–child DNAm associations.ResultsUsing genome-wide DNAm profiling data (721,331 DNAm sites, including 704,552 on autosomes and 16,779 on the X chromosome) of 396 mother–newborn pairs (54.5% male) from the Boston Birth Cohort, we found significant sex differences in mother–newborn correlations in genome-wide DNAm patterns (Spearman’s rho = 0.91–0.98; p = 4.0 × 10–8), with female newborns having stronger correlations. Sex differences in correlations were attenuated but remained significant after excluding X-chromosomal DNAm sites (Spearman’s rho = 0.91–0.98; p = 0.035). Moreover, 89,267 DNAm sites (12.4% of all analyzed, including 88,051 [12.5% of analyzed] autosomal and 1,216 [7.2% of analyzed] X-chromosomal sites) showed significant mother–newborn associations in methylation levels, and the top autosomal DNAm sites had high heritability than the genome-wide background (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.92). Additionally, significant interactions between newborn sex and methylation levels were observed for 11 X-chromosomal and 4 autosomal DNAm sites that were mapped to genes that have been associated with sex-specific disease/traits or early development (e.g., EFHC2, NXY, ADCYAP1R1, and BMP4). Finally, 18,769 DNAm sites (14,482 [77.2%] on the X chromosome) showed mother–newborn differences in methylation levels that were significantly associated with newborn sex, and the top autosomal DNAm sites had relatively small heritability (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.23). These DNAm sites were mapped to 2,532 autosomal genes and 978 X-chromosomal genes with significant enrichment in pathways involved in neurodegenerative and psychological diseases, development, neurophysiological process, immune response, and sex-specific cancers. Replication analysis in the Isle of Wight birth cohort yielded consistent results.ConclusionIn two independent birth cohorts, we demonstrated strong mother–newborn correlations in whole blood DNAm on both autosomes and ChrX, and such correlations vary substantially by sex. Future studies are needed to examine to what extent our findings contribute to developmental origins of pediatric and adult diseases with well-observed sex differences.

Methylome changes associated with functional movement/conversion disorder: Influence of biological sex and childhood abuse exposure

Epigenetic changes, such as DNA methylation (DNAm), may represent an important mechanism implicated in the etiopathogenesis of functional movement/conversion disorder (FMD). Here, we aimed to identify methylomic variations in a case-control cohort of FMD and to uncover specific epigenetic signatures associated with female sex and childhood abuse, two key risk factors for FMD and other functional neurological disorders.Genome-wide DNAm analysis was performed from peripheral blood in 57 patients with FMD and 47 healthy controls with and without childhood abuse. Using principal component analysis, we examined the association of principal components with FMD status in abused and non-abused individuals, in the entire study sample and in female subjects only. Next, we used enrichment pathway analysis to investigate the biological significance of DNAm changes and explored differences in methylation levels of genes annotated to the top enriched biological pathways shared across comparisons.We found that FMD was associated with DNAm variation across the genome and identified a common epigenetic ‘signature’ enriched for biological pathways implicated in chronic stress and chronic pain. However, methylation levels of genes included in the top two shared pathways hardly overlapped, suggesting that transcriptional profiles may differ as a function of childhood abuse exposure and sex among subjects with FMD.This study is unique in providing genome-wide evidence of DNAm changes in FMD and in indicating a potential mechanism linking childhood abuse exposure and female sex to differences in FMD pathophysiology. Future studies are needed to replicate our findings in independent cohorts.

Re-analysis of publicly available methylomes using signal detection yields new information

Cytosine methylation is an epigenetic mark that participates in regulation of gene expression and chromatin stability in plants. Advancements in whole genome sequencing technologies have enabled investigation of methylome dynamics under different conditions. However, the computational methods for analyzing bisulfite sequence data have not been unified. Contention remains in the correlation of differentially methylated positions with the investigated treatment and exclusion of noise, inherent to these stochastic datasets. The prevalent approaches apply Fisher’s exact test, logistic, or beta regression, followed by an arbitrary cut-off for differences in methylation levels. A different strategy, the MethylIT pipeline, utilizes signal detection to determine cut-off based on a fitted generalized gamma probability distribution of methylation divergence. Re-analysis of publicly available BS-seq data from two epigenetic studies in Arabidopsis and applying MethylIT revealed additional, previously unreported results. Methylome repatterning in response to phosphate starvation was confirmed to be tissue-specific and included phosphate assimilation genes in addition to sulfate metabolism genes not implicated in the original study. During seed germination plants undergo major methylome reprogramming and use of MethylIT allowed us to identify stage-specific gene networks. We surmise from these comparative studies that robust methylome experiments must account for data stochasticity to achieve meaningful functional analyses.