Search Results (35)

Background/Objectives: Advances in nanopore sequencing have opened new avenues for studying DNA methylation at single-base resolution, yet their application in epigenetic ageing research remains underdeveloped. Methods: We present a novel framework that leverages the unique capabilities of nanopore sequencing to profile and interpret age-associated methylation patterns in native DNA. Results: Unlike conventional array-based approaches, long reads sequencing captures full CpG context, accommodates diverse and repetitive genomic regions, removes bisulfite conversion steps, and is compatible to the latest reference genome. Conclusions: This work establishes nanopore sequencing as a powerful tool for next-generation epigenetic ageing studies, offering a scalable and biologically rich platform for anti-ageing interventions monitoring and longitudinal ageing studies. Full article

Background: The identification of body fluids collected from crime scenes is crucial for determining the type and nature of assaults and for advancing the resolution of crimes. Objectives: The primary aim of this study was to investigate tissue-specific DNA methylation markers that can effectively distinguish buccal samples from blood, semen, and vaginal epithelial tissue. Methods: We screened various markers and selected four genomic locations for further analysis. Genomic DNA was extracted from tissue samples, followed by bisulfite conversion, locus-specific polymerase chain reaction (PCR) amplification, and pyrosequencing. Results: Four loci—cg-9652652, cg-11536474, cg-3867465, and cg-10122865—along with several adjacent CpG sites, were found to be hypermethylated in buccal samples compared to other tissue types. The difference in DNA methylation of buccal samples was statistically significant (p < 0.0001) compared to other tissues, indicating the potential usefulness of these loci for forensic tissue identification. Two additional studies were conducted: (a) a species specificity study and (b) a mixture study involving two different tissue types. The species specificity study showed that the primers used in the assay were specific to primates and humans. They did not amplify five non-primate samples, while the two primate samples—chimpanzee and rhesus—provided usable methylation data. The mixture study involved DNA from two different tissues—buccal samples and semen—combined in varying proportions. The results showed a decrease in the overall percentage of DNA methylation at the locus cg-9652652 as well as five adjacent CpG sites when the amount of buccal cell DNA in the mixture was reduced. Conclusion: The specificity of the primers and the significant differences in percent DNA methylation between buccal cells and other tissues make these markers excellent candidates for forensic tissue identification. Full article

In vertebrates, vitamin K is a cofactor for the gamma-glutamyl carboxylase (GGCX) involved in the carboxylation of glutamic acid residues. During the vitamin K cycle, vitamin K is oxidised by GGCX, and then reduced by vitamin K epoxide reductase (VKOR), which is inhibited by the synthetic coumarin warfarin. GGCX and VKOR are present in Drosophila melanogaster, but the existence of a vitamin K cycle remains unproven. Semi-lethal concentrations (LC₅₀) of K₃, menadione sodium bisulfite (MSB), and warfarin to neutralise the negative effect of MSB were selected for the Drosophila cultivation medium. LC-MS analysis was used for vitamin K measurement in flies’ extracts. The EPR method and RT-PCR were used for ROS level measurement and gene transcription assessment, respectively. The LC₅₀ of MSB in the medium resulted in a more than 20-fold increase in endogenous K₂ in flies, demonstrating the mechanism of K₃-to-K₂ conversion. Administration of 1 mM warfarin in the medium with MSB completely neutralised its negative effect on viability. Developed flies had decreased K₂ level, confirming the existence of a vitamin K cycle, and both reduced ROS level and hsp22 gene transcription. The biochemical pathways affected by elevated K₂ concentrations involves both elements of the vitamin K cycle and the adaptive mitochondrial antioxidant system. Full article

To explore the impact of epigenetic modifications on egg-laying traits in geese, we employed genome-wide bisulfite sequencing (WGBS) to analyze DNA methylation patterns in pituitary tissues of high-(HYP) and low-yield (LYP) Sichuan White geese. We achieved high-quality sequencing data (mean 19.09 Gb raw reads, 15.49 Gb clean reads, 79.1% unique mapping rate) with a bisulfite conversion efficiency of 99.88%. Comparative analysis revealed 2394 differentially methylated regions (DMRs) and 422 differentially methylated genes (DMGs) between HYP and LYP groups. We identified five key differentially methylated candidate genes (BMPER, INHA, NMBR, NK3R, and DSG2) linked to egg-laying traits in Sichuan White geese. Integrated GO and KEGG enrichment analysis conducted to explore the role of regulatory networks of epigenetic modification on egg-laying traits in Sichuan White geese identified multiple metabolic pathways associated with egg-laying traits (promoting egg transport, ovulation, and yolk protein synthesis and secretion), thus providing a basis for subsequent functional verification. Full article

Background/Objectives: The purpose of the current study was to compare the methylation of five regions of the CpG island of MLH1 with the presence of microsatellite instability (MSI) in colorectal cancer (CRC) patients. Methods: The study analyzed 138 CRC tumor samples. DNA extraction was performed, followed by bisulfite conversion. MLH1 gene methylation was assessed by methylation-specific PCR (MS-PCR), and the resulting fragments were analyzed using polyacrylamide gels. MSI was evaluated using multiplex PCR, and the fragments were run through capillary electrophoresis. R studio (v4.4.1) and SPSS (v29.0) software were used for the statistical analysis, and values of p < 0.05 were considered statistically significant. Results: The study showed 75.4% unmethylated, 21% partially methylated, and 3.6% fully methylated samples, with region A frequently methylated. MSI was observed in 7.2% of cases (MSI-H: 5.8%, MSI-L: 1.4%). BAT-26 was the most unstable marker. A significant difference between MLH1 methylation and MSI-H (p < 0.01) was identified, but there was no relationship with specific MLH1 regions. Conclusions: No differences were identified when analyzing specific methylation regions in relation to MSI. This study is the first to describe MSI frequency in Mexican patients regardless of age. Full article

Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed. Methods: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity. Results: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker. Full article

(1) Background: Cervical cancer, caused mainly by high-risk Human Papillomavirus (hrHPV), is a significant global health issue. While a Pap smear remains a reliable method for early detection, identifying new biomarkers to stratify the risk is crucial. For this purpose, extensive research has been conducted on detecting DNA methylation. (2) Methods: This cross-sectional study aimed to assess the expression levels of EIF4G3 and SF3B1 in precursor lesions and cervical tumor tissues through qRT-PCR and evaluate the methylation status of their promoters through bisulfite conversion. (3) Results: Both genes showed similar mRNA expression patterns, with the highest levels observed in squamous cell carcinoma (SCC) samples (p < 0.0001). Additionally, methylation analysis indicated increased percentages in the control group for both factors. Notably, the expression levels of both genes were inversely correlated with promoter methylation (EIF4G3—p = 0.0016; SF3B1—p < 0.0001). (4) Conclusions: Regarding the methylation pattern for both genes, we observe a decreasing trend from NILM to SCC patients. Therefore, we concluded that the decrease in methylation at the promoter level for both genes could be an indicator of abnormal cytology. Full article

The use of non-invasive liquid biopsy-based cell-free DNA (cfDNA) analysis is an emerging method of cancer detection and intervention. Different analytical methodologies are used to investigate cfDNA characteristics, resulting in costly and long analysis processes needed for combining different data. This study investigates the possibility of using cfDNA data converted for methylation analysis for combining the cfDNA fragment size with copy number variation (CNV) in the context of early colorectal cancer detection. Specifically, we focused on comparing enzymatically and bisulfite-converted data for evaluating cfDNA fragments belonging to chromosome 18. Chromosome 18 is often reported to be deleted in colorectal cancer. We used counts of short and medium cfDNA fragments of chromosome 18 and trained a linear model (LDA) on a set of 2959 regions to predict early-stage (I–IIA) colorectal cancer on an independent test set. In total, 87.5% sensitivity and 92% specificity were obtained on the enzymatically converted libraries. Repeating the same workflow on bisulfite-converted data yielded lower accuracy results with 58.3% sensitivity, implying that enzymatic conversion preserves the cancer fragmentation footprint in whole genome data better than bisulfite conversion. These results could serve as a promising new avenue for the early detection of colorectal cancer using fragmentation and methylation approaches on the same datasets. Full article

There is still much to learn about the epigenetic mechanisms controlling gene expression during carcinogenesis. When researching aberrant DNA methylation, active proliferative tumor cells from head and neck squamous cell cancer (HNSCC) can be used as a model. The aim of the study was to investigate the methylation status of CDKN1, CDKN2A, MYC, Smad3, SP1, and UBC genes in tumor tissue (control-normal tissue) in 50 patients (37 men and 13 women) with HPV-negative HNSCC. Methods: Bisulfite conversion methods and methyl-sensitive analysis of high-resolution melting curves were used to quantify the methylation of genes. In all patients and across various subgroups (tongue carcinoma, laryngeal and other types of carcinomas T2, T3, T4 status; age before and after 50 years; smoking and non-smoking), there are consistent differences in the methylation levels in the SP1 gene in tumor DNA compared to normal. Results: The methylation of the SP1 gene in tumor DNA suppresses its expression, hinders HNSCC cell proliferation regulation, and could be a molecular indicator of malignant cell growth. The study of DNA methylation of various genes involved in carcinogenesis is promising because hypermethylated promoters can serve as potential biomarkers of disease. Full article

Recent advancements in forensic genetics have facilitated the extraction of additional characteristics from unidentified samples. This study delves into the predictive potential of a five-gene (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) methylation rate analysis for human age estimation using buccal swabs collected from 60 Italian volunteers. The methylation levels of specific CpG sites in the five genes were analyzed through bisulfite conversion, single-base extension, and capillary electrophoresis. A multivariate linear regression model was crafted on the training set, then the test set was employed to validate the predictive model. The multivariate predictive model revealed a mean absolute deviation of 3.49 years in the test set of our sample. While limitations include a modest sample size, the study provides valuable insights into the potential of buccal swab-based age prediction, aiding in criminal investigations where accurate age determination is crucial. Our results also highlight that it is necessary to investigate the effectiveness of predictive models specific to biological tissues and individual populations, since models already proven effective for other populations or different tissues did not show the same effectiveness in our study. Full article

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive histological type of cancer in this location. Distant metastases are present in approximately 30% of patients at the time of first examination. Therefore, the ability to predict the occurrence of metastases in patients at early stages of the disease is an urgent task aimed at personalized treatment. Samples of tumor and paired histologically normal kidney tissue from patients with metastatic and non-metastatic ccRCC were studied. Gene expression was analyzed using real-time PCR. The level of gene methylation was evaluated using bisulfite conversion followed by quantitative methylation-specific PCR. Two groups of genes were analyzed in this study. The first group includes genes whose expression is significantly reduced during metastasis: CA9, NDUFA4L2, EGLN3, and BHLHE41 (p < 0.001, ROC analysis). The second group includes microRNA genes: MIR125B-1, MIR137, MIR375, MIR193A, and MIR34B/C, whose increased methylation levels are associated with the development of distant metastases (p = 0.002 to <0.001, ROC analysis). Based on the data obtained, a combined panel of genes was formed to identify patients whose tumors have a high metastatic potential. The panel can estimate the probability of metastasis with an accuracy of up to 92%. Full article

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as “possibly carcinogenic”; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied. Full article

In forensic genetics, the identification of an individual is often carried out by comparing unknown DNA profiles obtained in a case against databases or references. When no match is found, investigators need new tools in order to obtain additional leads. The latest technical advances now make it possible to predict externally visible characteristics. With this objective, predicting the age of an individual through DNA methylation analysis remains one of the last challenges. The prediction models have to account for the specific constraints of this field, including tissue specificity and DNA availability (i.e., low DNA amounts or low-quality DNA). Jung and colleagues have recently produced models from blood, saliva and buccal cells by using a single base extension sequencing method. With the goal of evaluating these models in our own analytical conditions, saliva and buccal cell samples from 115 French individuals between the ages of 0 and 88 years old were collected and analyzed. After having determined the optimal analysis conditions, including the DNA quantity for bisulfite conversion (75 ng), some differences were highlighted in the measured methylation rates between the two studies. Despite these discrepancies, the prediction performance levels remain very similar, our study showing mean absolute errors of 3.5 years, 3.9 years and 3.2 years, respectively, for the saliva, buccal swab and multitissue model, with limitations observed for the oldest and youngest individuals. Furthermore, we propose the use of a prediction interval with an error dispersion and correct prediction rate at ±5 years and ±10 years, respectively. Full article

DNA methylation is one of the epigenetic marks which has been studied intensively in recent years for age predicting purposes in the forensic area. In order to integrate age prediction into routine forensic workflow, the purpose of this study was to standardize and optimize a DNA methylation-based protocol tailored to the Italian context. A previously published protocol and age-predictive method was implemented for the analysis of 84 blood samples originating from Central Italy. The study here presented is based on the Single Base Extension method, considering five genes: ELOVL2, FHL2, KLF14, C1orf132, now identified as MIR29B2C, and TRIM59. The precise and specific steps consist of DNA extraction and quantification, bisulfite conversion, amplification of converted DNA, first purification, single base extension, second purification, capillary electrophoresis, and analysis of the results to train and test the tool. The prediction error obtained, expressed as mean absolute deviation, showed a value of 3.12 years in the training set and 3.01 years in the test set. Given that population-based differences in DNA methylation patterns have been previously reported in the literature, it would be useful to further improve the study implementing additional samples representative of the entire Italian population. Full article

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue. Full article