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Abstract Background As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, r s = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, r s = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, r s = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (p HBG2 = 0.011, r s = 0.480/p ZBTB7A = 0.026, r s = 0.427) and showed prognostic value for PFS (p ZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8). Conclusions Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action. Graphical abstract

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8). Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action. [ABSTRACT FROM AUTHOR]

Abstract Background The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250–300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. Results Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. Conclusion While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

Background: The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250–300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. Results: Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. Conclusion: While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation. [ABSTRACT FROM AUTHOR]

DNA methylation (DNAm) in mammals is mostly examined within the context of CpG dinucleotides. Non-CpG DNAm is also widespread across the human genome, but the functional relevance, tissue-specific disposition, and inter-individual variability has not been widely studied. Our aim was to examine non-CpG DNAm in the wider methylome across multiple tissues from the same individuals to better understand non-CpG DNAm distribution within different tissues and individuals and in relation to known genomic regulatory features. DNA methylation in umbilical cord and cord blood at birth, and peripheral venous blood at age 12–13 y from 20 individuals from the Southampton Women’s Survey cohort was assessed by Agilent SureSelect methyl-seq. Hierarchical cluster analysis (HCA) was performed on CpG and non-CpG sites and stratified by specific cytosine environment. Analysis of tissue and inter-individual variation was then conducted in a second dataset of 12 samples: eight muscle tissues, and four aliquots of cord blood pooled from two individuals. HCA using methylated non-CpG sites showed different clustering patterns specific to the three base-pair triplicate (CNN) sequence. Analysis of CAC sites with non-zero methylation showed that samples clustered first by tissue type, then by individual (as observed for CpG methylation), while analysis using non-zero methylation at CAT sites showed samples grouped predominantly by individual. These clustering patterns were validated in an independent dataset using cord blood and muscle tissue. This research suggests that CAC methylation can have tissue-specific patterns, and that individual effects, either genetic or unmeasured environmental factors, can influence CAT methylation.

DNA methylation (DNAm) in mammals is mostly examined within the context of CpG dinucleotides. Non-CpG DNAm is also widespread across the human genome, but the functional relevance, tissue-specific disposition, and inter-individual variability has not been widely studied. Our aim was to examine non-CpG DNAm in the wider methylome across multiple tissues from the same individuals to better understand non-CpG DNAm distribution within different tissues and individuals and in relation to known genomic regulatory features. DNA methylation in umbilical cord and cord blood at birth, and peripheral venous blood at age 12–13 y from 20 individuals from the Southampton Women's Survey cohort was assessed by Agilent SureSelect methyl-seq. Hierarchical cluster analysis (HCA) was performed on CpG and non-CpG sites and stratified by specific cytosine environment. Analysis of tissue and inter-individual variation was then conducted in a second dataset of 12 samples: eight muscle tissues, and four aliquots of cord blood pooled from two individuals. HCA using methylated non-CpG sites showed different clustering patterns specific to the three base-pair triplicate (CNN) sequence. Analysis of CAC sites with non-zero methylation showed that samples clustered first by tissue type, then by individual (as observed for CpG methylation), while analysis using non-zero methylation at CAT sites showed samples grouped predominantly by individual. These clustering patterns were validated in an independent dataset using cord blood and muscle tissue. This research suggests that CAC methylation can have tissue-specific patterns, and that individual effects, either genetic or unmeasured environmental factors, can influence CAT methylation. [ABSTRACT FROM AUTHOR]

Background and objectives: Cancer initiation and progression could influenced by both genetic and epigenetic events revealing of the overlap between epigenetic and genetic alteration can give important insights into cancer biology. Methods and results: In this experiment ISL1, MGMT, DMNT3b genes were candidate to investigate both methylation status and expression profile by using methylation-specific PCR and real time PCR in 40 breast cancer patients, respectively, also we have assessed relation of the promoter methylation status and expression variation of the target genes. The mean level of methylation of ISL1 and MGMT in tumor tissues were significantly greater than normal tissues. In Contrast, DMNT3b gene was showed lower mean level of methylation in tumor tissue compared to normal tissues, however, this was not statistically significant. Relative expression analysis was displayed a significant reduction in expression level of ISL1 and MGMT in tumor tissues. Furthermore, there was a meaningful association between down expression of ISL1 with histological grade, Her2 and ER status. Moreover, MGMT down expression was significantly associated with tumor sizes. Any remarkable relation was not observed between DMNT3b expression level and clinic pathological features. At the end, significant relation between methylation status and expression level has been revealed. Conclusions: In this study all observed results were exactly in line with the results were obtained from articles which were based on the methylation research and illustrate that the real-time PCR and methylation methods are in correlated with each other, furthermore, selected genes are capable to use as a potential biomarkers, however, more research on extended cases are needed. [ABSTRACT FROM AUTHOR]

DNA methylation is one of the most studied epigenetic modifications that has applications ranging from transcriptional regulation to aging, and can be assessed by bisulfite sequencing (BS-seq) or enzymatic methyl sequencing (EM-seq) at single base-pair resolution. The permutations of methylation statuses given by aligned reads reflect the methylation patterns of individual cells. These patterns at specific genomic locations are sought to be indicative of cellular heterogeneity within a cellular population, which are predictive of developments and diseases; therefore, methylation heterogeneity has potentials in early detection of these changes. Computational methods have been developed to assess methylation heterogeneity using methylation patterns formed by four consecutive CpGs, but the nature of shotgun sequencing often give partially observed patterns, which makes very limited data available for downstream analysis. While many programs are developed to impute genome-wide methylation levels, currently there is only one method developed for recovering partially observed methylation patterns; however, the program needs lots of data to train and cannot be used directly; therefore, we developed a probabilistic-based imputation method that uses information from neighbouring sites to recover partially observed methylation patterns speedily. It is demonstrated to allow for the evaluation of methylation heterogeneity at 15% more regions genome-wide with high accuracy for data with moderate depth. To make it more user-friendly we also provide a computational pipeline for genome-screening, which can be used in both evaluating methylation levels and profiling methylation patterns genomewide for all cytosine contexts, which is the first of its kind. Our method allows for accurate estimation of methylation levels and makes evaluating methylation heterogeneity available for much more data with reasonable coverage, which has important implications in using methylation heterogeneity for monitoring changes within the cellular populations that were impossible to detect for the assessment of development and diseases.

The synthesis and crystal structures of a series of 6-arylfuvlenes (fulvene is 5-methylidenecyclopenta-1,3-diene) with varying methylation patterns on the 6-phenyl substituent are reported, namely 6-(3-methylphenyl)-1,3-diphenylfulvene (C25H20), 6-(4-methylphenyl)-1,3-diphenylfulvene (C25H20), 6-mesityl-3-diphenylfulvene (C27H24) and 6-(2,3,4,5,6-pentamethylphenyl)-1,3-diphenylfulvene (C29H28). The bond lengths are typical of those observed in related fulvenes. A network of C—H...π ring interactions consolidates the packing in each structure.

Background: Aberrant methylation plays an essential role in early cancer development. In this study, we investigated methylation patterns in lung squamous cell carcinoma (LUSC) and matched non-tumor tissue and plasma samples to evaluate the potential of these patterns in the diagnosis of LUSC., Methods: The study group included 49 patients with stage I-III LUSC. We collected resected tumor tissue, paired peritumoral tissue, distant normal tissue, and corresponding plasma samples. A bespoke lung cancer bisulfite sequencing panel was used to profile the methylation level. Another 48 healthy volunteers provided control plasma samples., Results: Peritumoral and distant normal tissues presented similar methylation signatures, distinct from those in tumor tissue samples. A comparison of methylation profiles led to the identification of 871 tumor-specific differentially methylated blocks, including 847 hypermethylated and 24 hypomethylated blocks (adjusted P value <0.05). All top-ranked blocks were tumor-related. Tissue samples were analyzed for field cancerization to identify progressively aggravating aberrant methylations during tumor initiation and development. The analysis revealed that 221 blocks presented a stepwise increase in methylation levels, while seven blocks presented a stepwise decrease in methylation pattern as the sampling drew nearer to the tumor. The malignant contaminated ratio (MCR) confirmed the presence of distinct methylation patterns between tumor and peritumoral tissue samples. We then constructed a diagnostic panel using a combined diagnostic score of cell-free DNA (cfDNA) that showed high sensitivity and specificity. The healthy controls had a significantly lower combined diagnostic score (cd-score) than LUSC patients. Additionally, based on the methylation profiles, LUSC could be classified into two subgroups, C1 and C2. The methylation profile of the C2 group was not distinct from the healthy controls, which had a significantly lower cd-score than did the C1 group., Conclusions: LUSC-specific methylation patterns could potentially discriminate between peritumoral tissue, distant normal tumor tissue, and tumor tissues. This preliminary study also supported the potential utility of cfDNA methylation analysis in diagnosing LUSC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-1827/coif). J.J. reports support from BMS, Roche, and MSD for consulting or advisory roles; support from Takeda for travel, accommodation, and expenses; and support from Pfizer, Novartis, and MSD for speakers bureau. The other authors have no conflicts of interest to declare., (2024 Journal of Thoracic Disease. All rights reserved.)

Plant cell biofactories represent a promising solution to the increasing demand for plant-derived compounds, but there are still limiting factors that prevent optimal production, including the loss of yield during in vitro maintenance. Our results reveal a clear correlation between genomic methylation levels and a progressive decline in taxane production in Taxus spp. cell cultures. A comparative study of two cell lines, one 10 years old and low productive and the other new and high productive, revealed important differences in appearance, growth, taxane accumulation and expression levels of several taxane biosynthetic genes. Differences in taxane content and gene expression profile indicate an altered pathway regulation and that the BAPT gene, located in the center of the expression network of taxane biosynthetic genes, is active in a potentially flux-limiting step. The methylation patterns of the BAPT gene were studied in both cell lines by bisulfite sequencing, which revealed high rates of CHH methylated cytosines on the core promoter. Using a bioinformatics approach, this hotspot was identified as a Y-patch promoter element. The Y-patch may play a key role in the epigenetic regulation of the taxane biosynthetic pathway, which would open up novel genetic engineering strategies toward stable and high productivity. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, representing the third leading cause of cancer-related deaths. HCC genetic characterization at the tumor level has been recently completed, highlighting how a number of genes are frequently mutated in this pathology. Actionable somatic mutations found in a HCC tumor may represent targets for innovative drugs as well as prognostic/predictive markers. Nonetheless, surgical or bioptic tissue is hardly accessible in HCC and a single tumor sample is poorly representative of the tumor genetic heterogeneity. In this context, analyzing the circulating cell-free DNA (ccfDNA) and its tumor-derived fraction (ctDNA) could represent a promising strategy of liquid biopsy. Recent data suggested that the fluctuation of the ccfDNA quantity in the plasma of HCC patients could anticipate the detection of tumor progression. The presence of somatic mutations in p53 signaling, Wnt/β-catenin, chromatin remodeling, response to oxidative stress and telomerase maintenance pathways can also be studied in ccfDNA bypassing the need to perform a tumor biopsy. The profiling of ccfDNA fragmentation and the methylation pattern could further improve the clinical management of HCC patients. Performing a dynamic monitoring in the course of systemic treatment with sorafenib or regorafenib is a possible way to provide insights into the resistance mechanism, and to identify predictive and prognostic genetic alterations, helping the clinicians in terms of treatment decision making. This review will discuss the most recent literature data about the use of ccfDNA to monitor and improve the treatment of HCC.

Key message: The relationships between transcription and methylation were revealed in Arabidopsis thaliana NB-LRR-encoding genes in wild type (Col-0) and different mutants.Abstract: Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins constitute a large family that plays predominant roles in disease resistance. However, the regulation of NB-LRR-encoding genes at the transcriptional level is still poorly understood. Recently, DNA cytosine methylation in eukaryotes has been described as serving an important function in regulating gene expression. Here, we analysed the DNA methylation patterns of NB-LRR-encoding genes in Arabidopsis thaliana in samples from a wild type (Col-0) and ago4, met1, cmt3, drm1/2, and ddm1 mutants. Our results revealed that the vast majority of the NB-LRR-encoding genes in Col-0 were methylated, and the DNA methylation occurred predominantly in the CG sequence context. Moreover, DNA methylation was widely distributed in both the promoters and the bodies of most NB-LRR-encoding genes. Our results also showed that the loss of AGO4, MET1, CMT3, DRM1/2 or DDM1 functions generally led to decreased cytosine methylation in the NB-LRR-encoding genes. Analysis of the available transcriptome data from the wild type and the met1, cmt3, drm1/2 and ddm1 mutants revealed that differences in the transcription levels between the wild type and mutants were statistically significant for 63 of the NB-LRR-encoding genes. Of these genes, 38 were significantly upregulated, and the other 25 were significantly downregulated. Some NB-LRR-encoding genes with differential expression levels, which were revealed by the mRNA-Seq data, were confirmed to be significantly upregulated or downregulated in the mutants compared to the wild type by using quantitative RT-PCR. These data suggest that some Arabidopsis NB-LRR-encoding genes are likely to be regulated by altered DNA methylation patterns. [ABSTRACT FROM AUTHOR]

Pod1 encodes a Class II bHLH transcription factor involved in the development of a number of tissues such as gonad, spleen, lungs and heart. However, to date, little is known about its function in teleosts. In this study, we cloned and characterized Pod1 gene from Cynoglossus semilaevis . This gene contains three exons and two introns, with the full-length cDNA of 918 nucleotides that encodes a 183 amino acid protein with a conserved bHLH domain. Realtime quantitative PCR revealed that Pod1 was predominantly expressed in the testes of C. semilaevis . In different stages of testes development, Pod1 expression was undetectable up to 120 days after hatching (dah), and then increased at 210 dah and 1 year after hatching (yah). Furthermore, in situ hybridization (ISH) analysis revealed that Pod1 was mainly localized in the germ cells of testes, but was not detected in ovarian cells; which suggested its possible functions in spermatogenesis of C. semilaevis . The methylation profile analysis of Pod1 genomic sequence in the gonads showed that the differences in their putative promoter regions of Pod1 among ovary, male and pseudo-male testes were not obvious. Thus, further research might be needed to evaluate whether Pod1 expression is regulated by epigenetic level. [ABSTRACT FROM AUTHOR]

The tongue is the most common and aggressive site for tumors in the oral cavity. These tumors are usually located in the lateral border of the tongue and are often related to the use of tobacco and alcohol. Clinical management of these tumors is predominantly based on anatomic location and TNM classification. The identification of molecular signatures with ability to explain the different outcomes observed in these patients is of paramount importance to guide and help their management. Case presentation : we herein describe an 88-year-old woman diagnosed with synchronous bilateral tongue carcinoma. This woman did not present the traditional risk factors related to oral cancer—alcohol, tobacco, or presence of human papiloma virus (HPV). Both tumors were classified by a pathologist as pT2. This patient was submitted to surgery, 6 months later was diagnosed with cervical metastasis and in the following 2 months died. Copy number alterations and methylation status of these 2 simultaneous tumors were analyzed using array comparative genomic hybridization, multiplex ligation-dependent probe amplification, and methylation specific multiplex ligation-dependent probe amplification. In conclusion , in both tumors we identified several molecular traits usually found among oral cavity tumors and some of those have been associated with clinical outcome, reinforcing their importance to accurately establish biomarkers with clinical applicability. Specific genomic and epigenetic signatures for each of these 2 tumors were also observed allowing their molecular discrimination. The tumor of the right side of the tongue exhibited more copy number gains than the tumor of the left side. In the left side tumor less and smaller copy number alterations and more methylated genes were observed, which could be indicative of an early phase of tumor development. This case shows the molecular heterogeneity of oral cavity tumors even in the same patient and anatomic site, which could be the key to explain the different outcomes of oral tumor patients. [ABSTRACT FROM AUTHOR]

In the United States, schools offer special education services to children who are diagnosed with a learning or neurodevelopmental disorder and have difficulty meeting their learning goals. Pediatricians may play a key role in helping children access special education services. The number of children ages 6-21 in the United States receiving special education services increased 10.4% from 2006 to 2021. Children receiving special education services under the autism category increased 242% during the same period. The demand for special education services for children under the developmental delay and other health impaired categories increased by 184% and 83% respectively. Although student enrollment in American schools has remained stable since 2006, the percentage distribution of children receiving special education services nearly tripled for the autism category and quadrupled for the developmental delay category by 2021. Allowable heavy metal residues remain persistent in the American food supply due to food ingredient manufacturing processes. Numerous clinical trial data indicate heavy metal exposures and poor diet are the primary epigenetic factors responsible for the autism and attention deficit hyperactivity disorder epidemics. Dietary heavy metal exposures, especially inorganic mercury and lead may impact gene behavior across generations. In 2021, the United States Congress found heavy metal residues problematic in the American food supply but took no legislative action. Mandatory health warning labels on select foods may be the only way to reduce dietary heavy metal exposures and improve child learning across generations., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

DNA methylation is studied extensively for its relations with several biological processes such as transcriptional regulation. While methylation levels are usually estimated per cytosine or genomic region, additional information on methylation heterogeneity can be obtained when considering stretches of successive cytosines on the same reads; however, the majority of methylomes suffer from low coverage of genomic regions with sequencing depths enough for accurate estimation of methylation heterogeneity using existing methods. Here we describe a probabilistic-based imputation method that makes use of methylation information from neighboring sites to recover partially observed methylation patterns. Our method and software are proven to be faster and more accurate among all evaluated. Ultimately, our method allows for a more streamlined monitoring of epigenetic changes within cellular populations and their putative role in disease., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The detection of semen can assist in reconstructing the events of a sexual assault and impact the outcome of legal dispositions. Many methods currently are used for detecting the presence of semen, but they all have limitations with regards to specificity, sample degradation/consumption, stability of biomolecule assayed, and/or incompatibility with downstream individual identification assays. DNA is routinely collected at sexual assault crime scenes and is widely used for individual identification. The DNA also carries methylation patterns that are tissue specific. To date, however, assays designed to exploit methylation patterns suffer from complex chemistries and unwieldy analyses. DSI-Semen™ kit uses a novel approach involving CpG methylation-sensitive restriction endonuclease digestion coupled to a multiplexed polymerase chain reaction (PCR) to generate an amplicon profile that makes it possible to determine whether the tissue source of a DNA sample was semen or non-semen. The assay returned an appropriate positive result for semen with neat semen, semen stains, and semen/non-semen tissue mixtures. The assay is robust and reliable, with a positive result for semen given as little as 31 pg of template DNA input. Low levels of semen were detected in mixtures of semen and other body fluids. UV-exposed samples and those in the presence of limited concentrations of known PCR inhibitors were typeable. The DSI-Semen™ kit provides a reliable tool for the determination of DNA being derived from semen. [ABSTRACT FROM AUTHOR]

Background: CD14 is a pattern-recognition receptor for environmental LPS, and engagement of the CD14-LPS complex activates innate host defense mechanisms. Single nucleotide polymorphisms (SNPs) in the CD14 gene have been associated with soluble CD14 (sCD14) levels, but inconsistencies between studies suggest the presence of regulatory mechanisms hitherto not well understood. Objective: We sought to investigate possible associations between CD14 SNPs and sCD14 levels at different time points in childhood (at birth [cord blood] and 2 and 10 years) and to explore whether these associations were related to CD14 gene methylation. Methods: Four SNPs, rs2569191 (−1145GA), rs5744455 (−550CT or −651CT), rs2569190 (−159CT or −260CT), and rs4914 in CD14 were genotyped in 762 children from the Environmental and Childhood Asthma study. Genotype frequencies were analyzed for association with sCD14 levels in 660 babies, 346 children at age 2 years, and 360 children at age 10 years. In a subgroup of 157 children with DNA available at both 2 and 10 years of age, CD14 methylation patterns were determined and analyzed against detected CD14 gene–sCD14 associations. Results: rs2569191, rs5744455, and rs2569190 were associated with sCD14 levels at birth and 2 years, but only rs5744455 was associated with sCD14 levels at 10 years. CD14 methylation increased significantly from age 2 to 10 years, and the level of methylation was inversely correlated with sCD14 levels at 10 years. Conclusion: The reduced effect of CD14 polymorphisms on sCD14 levels from early to late childhood paralleled a small but significant increase in CD14 methylation during the same period. [Copyright &y& Elsevier]

Abstract: The positive surgical margins are associated with postsurgical recurrence in colorectal carcinoma patients, and molecular margin analysis is considered to be more sensitive in detecting preneoplastic lesions than is conventional histological margin examination. Here, we developed a microarray and established six calibration curves for hMLH1 gene methylation patterns analysis in 20 colorectal resected margin specimens and corresponding tumor tissue specimens as well as four normal tissue specimens. The results indicated that a moderate methylation level (8–42%) was found in 20 surgical margin tissues, extensive methylation (25–58%) was detected in 20 tumor tissues, and little or no methylation was observed in normal tissues. Of the six paired probes, the average methylation levels in 20 tumor tissues were 60, 35, 43, 53, 38, and 27%, whereas the average methylation levels of the six paired probes in 20 surgical margin tissues were 43, 16, 24, 28, 21, and 11%. Thus, this study demonstrated the feasibility of this assay for molecular assessment use. In addition, it will contribute significant information to our understanding of CpG island methylation for cancer diagnosis and postoperative recurrence. [Copyright &y& Elsevier]

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.Oncogene (2005) 24, 1625-1633. doi:10.1038/sj.onc.1208398 Published online 17 January 2005 [ABSTRACT FROM AUTHOR]

: ObjectivesAberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16Ink4a gene.: Design and methodsWe selected a 336 bp segment of the 5′ untranslated region and the first exon of the p16Ink4a gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites.: ResultsMethylation patterns of human p16Ink4a gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments.: ConclusionsThe methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites. [Copyright &y& Elsevier]

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development. [ABSTRACT FROM AUTHOR]

DNA methylation is one of the most studied epigenetic modifications that has applications ranging from transcriptional regulation to aging, and can be assessed by bisulfite sequencing (BS-seq) or enzymatic methyl sequencing (EM-seq) at single base-pair resolution. The permutations of methylation statuses given by aligned reads reflect the methylation patterns of individual cells. These patterns at specific genomic locations are sought to be indicative of cellular heterogeneity within a cellular population, which are predictive of developments and diseases; therefore, methylation heterogeneity has potentials in early detection of these changes. Computational methods have been developed to assess methylation heterogeneity using methylation patterns formed by four consecutive CpGs, but the nature of shotgun sequencing often give partially observed patterns, which makes very limited data available for downstream analysis. While many programs are developed to impute genome-wide methylation levels, currently there is only one method developed for recovering partially observed methylation patterns; however, the program needs lots of data to train and cannot be used directly; therefore, we developed a probabilistic-based imputation method that uses information from neighbouring sites to recover partially observed methylation patterns speedily. It is demonstrated to allow for the evaluation of methylation heterogeneity at 15% more regions genome-wide with high accuracy for data with moderate depth. To make it more user-friendly we also provide a computational pipeline for genome-screening, which can be used in both evaluating methylation levels and profiling methylation patterns genomewide for all cytosine contexts, which is the first of its kind. Our method allows for accurate estimation of methylation levels and makes evaluating methylation heterogeneity available for much more data with reasonable coverage, which has important implications in using methylation heterogeneity for monitoring changes within the cellular populations that were impossible to detect for the assessment of development and diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chang, Yen and Chen.)

Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, representing the third leading cause of cancer-related deaths. HCC genetic characterization at the tumor level has been recently completed, highlighting how a number of genes are frequently mutated in this pathology. Actionable somatic mutations found in a HCC tumor may represent targets for innovative drugs as well as prognostic/predictive markers. Nonetheless, surgical or bioptic tissue is hardly accessible in HCC and a single tumor sample is poorly representative of the tumor genetic heterogeneity. In this context, analyzing the circulating cell-free DNA (ccfDNA) and its tumor-derived fraction (ctDNA) could represent a promising strategy of liquid biopsy. Recent data suggested that the fluctuation of the ccfDNA quantity in the plasma of HCC patients could anticipate the detection of tumor progression. The presence of somatic mutations in p53 signaling, Wnt/β-catenin, chromatin remodeling, response to oxidative stress and telomerase maintenance pathways can also be studied in ccfDNA bypassing the need to perform a tumor biopsy. The profiling of ccfDNA fragmentation and the methylation pattern could further improve the clinical management of HCC patients. Performing a dynamic monitoring in the course of systemic treatment with sorafenib or regorafenib is a possible way to provide insights into the resistance mechanism, and to identify predictive and prognostic genetic alterations, helping the clinicians in terms of treatment decision making. This review will discuss the most recent literature data about the use of ccfDNA to monitor and improve the treatment of HCC. [ABSTRACT FROM AUTHOR]

Rationale: In breast cancer, high intratumor DNA methylation heterogeneity can lead to drug-resistant, metastasis and poor prognosis of tumors, which increases the complexity of cancer diagnosis and treatment. However, most studies are limited to average DNA methylation level of individual CpGs and ignore heterogeneous DNA methylation patterns of cell subpopulations within the tumor. Thus, quantifying the variability in DNA methylation pattern in sequencing reads is valuable for understanding intratumor heterogeneity. Methods: We performed Reduced Representation Bisulfite Sequencing and RNA sequencing for tumor core and tumor periphery regions within one breast tumor. By developing a method named "epialleJS" based on Jensen-Shannon divergence, we detected the differential epialleles between tumor core and tumor periphery (CPDEs). We then explored the correlation between intratumor methylation heterogeneity and hypoxic microenvironment in TCGA breast cancer cohort. Results: More than 70% of CPDEs had higher epipolymorphism in tumor core than tumor periphery, and these CPDEs had lower methylation in tumor core. The CPDEs with lower methylation in tumor core may associate with hypoxic tumor microenvironment. Moreover, we identified a signature of five hypoxia-related DNA methylation markers which can predict the prognosis of breast cancer patients, including a CpG site cg15190451 in gene SLC16A5 . Furthermore, immunohistochemical analysis confirmed that the expression of SLC16A5 was associated with clinicopathological characteristics and survival of breast cancer patients. Conclusions: The analysis of intratumor DNA methylation heterogeneity based on epialleles reveals that disordered methylation patterns in tumor core are associated with hypoxic microenvironment, which provides a framework for understanding biological heterogeneous behavior and guidance for developing effective treatment schemes for breast cancer patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Among women, breast cancer (BC) is not only the most common cancer worldwide but also the leading cause of cancer death. Only 5-10% of breast cancer cases are attributed to inherited mutations (BRCA1, BRCA2, and other breast cancer susceptibility genes). Breast cancer incidence has been rising particularly in young women who are not eligible for mammography, and it has been acting as a burden especially in developing countries that lack screening and awareness programs. For this reason, research has shifted to use minimally invasive liquid biopsies especially blood-based biomarkers with potential value for breast cancer risk prediction and early detection. This mini-review will tackle the different blood-based biomarkers focusing mainly on circulating miRNA, circulating proteins, cell-free nucleic acids, methylation patterns, and exosomes. It also introduces the potential opportunities for the clinical use of these blood-based biomarkers for breast cancer risk prediction., (Copyright © 2020 Nassar, Chamandi, Tfaily, Zgheib and Nasr.)

Background: Long interspersed nuclear elements (LINE‐1) sequences constitute a substantial portion of the human genome, and their methylation often correlating with global genomic methylation. Previous studies have highlighted the feasibility of using LINE‐1 methylation to discriminate tumors from healthy tissues. However, most studies are based on only a few specific LINE‐1 CpG sites. Methods: Herein, we have performed a systematic fine‐scale analysis of methylation at 14 CpGs located in the 5′‐region of consensus LINE‐1, in bladder, colon, prostate, and gastric tumor tissues using a global degenerate approach. Results: Our results reveal variable methylation levels between different CpGs, as well as some tissue‐specific differences. Trends toward hypomethylation were observed in all tumors types to certain degrees, showing statistically significance in bladder and prostate tumors. Our data points toward the presence of unique LINE‐1 DNA methylation patterns for each tumor type and tissue, indicating that not the same CpGs will be informative for testing in all tumor types. Conclusion: This study provides an accurate guide that will help to design further assays that could avoid artifacts and explain the variability of obtained LINE‐1 methylation values between different studies. [ABSTRACT FROM AUTHOR]

DNA methylation patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying methylated CpG sites. We compared the methylated CpG analysis of two different methods 1) massively parallel sequencing (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY ® System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY ® was very difficult to achieve. We tested the denaturation and conversion times of the Premium Bisulfite kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq. [ABSTRACT FROM AUTHOR]

Background: Hypomethylation of long interspersed element (LINE)-1 has been observed in tumorigenesis when using degenerate assays, which provide an average across all repeats. However, it is unknown whether individual LINE-1 loci or different CpGs within one specific LINE-1 promoter are equally affected by methylation changes. Conceivably, studying methylation changes at specific LINE-1 may be more informative than global assays for cancer diagnostics. Therefore, with the aim of mapping methylation at individual LINE-1 loci at single-CpG resolution and exploring the diagnostic potential of individual LINE-1 locus methylation, we analyzed methylation at 11 loci by pyrosequencing, next-generation bisulfite sequencing as well as global LINE-1 methylation in bladder, colon, pancreas, prostate, and stomach cancers compared to paired normal tissues and in blood samples from some of the patients compared to healthy donors., Results: Most (72/80) tumor samples harbored significant methylation changes at at least one locus. Notably, our data revealed not only the expected hypomethylation but also hypermethylation at some loci. Specific CpGs within the LINE-1 consensus sequence appeared preferentially hypomethylated suggesting that these could act as seeds for hypomethylation. In silico analysis revealed that these CpG sites more likely faced the histones in the nucleosome. Multivariate logistic regression analysis did not reveal a significant clinical advantage of locus-specific methylation markers over global methylation markers in distinguishing tumors from normal tissues., Conclusions: Methylation changes at individual LINE-1 loci are heterogeneous, whereas specific CpGs within the consensus sequence appear to be more prone to hypomethylation. With a broader selection of loci, locus-specific LINE-1 methylation could become a tool for tumor detection.