Search Results (48)

Background: Breast cancer susceptibility gene 1 (BRCA1) is a pivotal regulator of DNA repair, and its loss through germline mutations is strongly linked to the development of aggressive breast cancers with characteristic clinical and pathological features. Beyond genetic disruption, epigenetic silencing via promoter hypermethylation has emerged as a non-mutational mechanism of tumour suppressor inactivation and a potential biomarker for guiding therapeutic decisions. Here, we investigate BRCA1 promoter methylation, its impact on gene expression, and its association with clinicopathological features in a cohort of African women with breast cancer. Methods: Matched tumour and adjacent normal tissues from 27 Black African women with breast cancer were analysed for BRCA1 promoter methylation and gene expression using bisulfite pyrosequencing and quantitative real-time PCR. Associations with clinicopathological variables were assessed using Spearman’s correlation analyses. Results: Five CpG sites within the BRCA1 promoter were significantly hypermethylated in breast tumours compared with matched adjacent normal tissues and showed an inverse association with BRCA1 mRNA expression. Elevated promoter methylation was enriched in hormone receptor-negative and triple-negative breast cancer subtypes and was not influenced by neoadjuvant chemotherapy. BRCA1 promoter methylation occurred independently of BRCA1 mutational status. No significant associations were observed between BRCA1 methylation and age, body mass index, smoking status, or alcohol consumption. Conclusions: Our findings provide evidence of BRCA1 epigenetic silencing in breast tumours from African women, particularly within aggressive hormone receptor-negative subtypes. These results suggest that BRCA1 promoter methylation may represent a clinically informative biomarker for patient stratification and highlight the importance of validation in larger, population-representative cohorts before clinical translation. Full article

Studying epigenetic changes in cancer development can reveal the role of tumor suppressor genes and their regulation by DNA methylation. CpG islands, found in promoter regions, are of particular interest, as their hypermethylation can silence tumor suppressor gene expression. Here, we present a practical analysis pipeline for wet-lab biologists with the aim of identify novel epigenetically regulated tumor suppressors using freely available online tools. Bioinformatic platforms such as the R2 Genomics Analysis and Visualization Platform enable analysis of genomic organization, CpG islands, and regulatory elements. Differential methylation and gene expression analyses are based on datasets including TCGA, using tools such as MethSurv, TCGA Wanderer, and GEPIA2 to correlate DNA methylation with gene expression. This bioinformatic step is the basis for the tumor suppressor verification in the wet-lab. Using this pipeline, we identified CLDN10 and GJB2 as potential tumor suppressors in melanoma. Experimentally, our approach includes DNA methylation analysis based on DNA bisulfite conversion, combined bisulfite restriction analysis (CoBRA), pyrosequencing for specific CpG methylation quantification, and RT-PCR for RNA expression quantification. We verify these results in primary tumors, metastases, and cell line models. This approach supports efficient identification of novel epigenetically regulated tumor suppressors, providing practical research guidelines. Full article

Background: Protein prenylation is crucial for the function of hundreds of proteins. Aberrant protein prenylation can be caused by the aberrant expression of prenyltransferases (PTases), which has been reported for multiple cancer entities. The reasons for aberrant PTase expression in cancer have not yet been investigated. Methods: We analyzed CpG methylation within promoter-associated CpG islands of the PTase genes FNTA, FNTB, PGGT1B, and RABGGTA via bisulfite conversion and pyrosequencing to assess its role in PTase expression and gain deeper insight into the regulation of protein prenylation in cancer. We used DNA from three benign controls (whole blood samples, peripheral blood mononuclear cells, and HEK293) and 19 human cancer cell lines from various origins to assess DNA methylation within PTase gene promoter-associated CpG islands. For a subset of these cell lines, we measured mRNA expression via qPCR and correlated it with DNA methylation. Results: Methylation across all PTase genes ranged from 1.9 ± 0.9% to 11.4 ± 4.0% (mean methylation ± standard deviation) in benign cells, and 2.3 ± 1.0% to 16.0 ± 5.4% in cancer cells. DNA methylation and mRNA expression of PGGT1B correlated inversely (PCC = −0.75; p = 0.005). Conclusions: We saw no general differences between benign and malignant cells, but observed significant differences between non-malignant controls and multiple individual cancer cell lines regarding the methylation of PTase genes. This was prominently seen in PGGT1B in Caki-1 cells, raising the possibility that DNA methylation is involved in the dysregulation of PTase expression in cancer. Full article

Tail docking, serving as an important management intervention in animal husbandry, plays a significant role in regulating tail fat deposition and improving production performance and health status in fat-tailed sheep. This study systematically revealed the reprogramming effects of tail docking on the epigenetic landscape and transcriptome of fat-tailed sheep by integrating whole-genome bisulfite sequencing (WGBS) and RNA m6A methylated immunoprecipitation sequencing (MeRIP-seq). At the DNA level, the tail-docked group exhibited a pronounced trend of hypomethylation across multiple functional genomic regions, including promoters, exons, and introns. Differentially methylated regions (DMRs) were significantly enriched in pathways related to tissue development and stress response, such as the Hippo signaling pathway and adherens junctions. Pyrosequencing validation of the promoter region of the key gene DGAT1 further confirmed the reliability of the WGBS data. At the RNA level, RNA m6A modifications showed an overall up-regulated pattern: the tail-docked group displayed higher numbers of m6A peaks, greater total peak length, and increased genomic coverage compared to the control group, along with better overall prediction of modification sites. Genes associated with differential m6A peaks were closely related to processes such as stem cell pluripotency and cytoskeleton regulation. qPCR validation of several methylation-related enzyme genes (e.g., METTL3, FTO, YTHDF1) yielded results consistent with the sequencing trends. Through integrated analysis of DNA methylation and RNA methylation, we identified 143 genes with concurrent changes in methylation and mRNA expression, among which 41 genes were regulated by both DNA and RNA methylation. These genes were primarily enriched in the adherens junction pathway. Notably, two core genes CITED4 and ZNF644 showed significant changes across all three levels: DNA methylation, RNA methylation, and mRNA expression. This study systematically elucidates the epigenetic mechanism by which tail docking stress induces coordinated DNA hypo-methylation and RNA m6A hyper-methylation to regulate transcriptomic reprogramming in response to environmental intervention. The findings provide novel insights into the molecular basis of trait formation in livestock. Full article

Background/Objectives: Prematurity is a significant global health concern, often associated with neurodevelopmental challenges. Solute Carrier Family 6 Member 4 (SLC6A4), the gene encoding the serotonin transporter, a key component in serotonin reuptake in the synaptic cleft, plays a key role in stress response and neurodevelopment. Epigenetic regulation of stress-related genes, such as SLC6A4, influences neonatal stress adaptation and developmental outcomes. This study aimed to quantify and compare DNA methylation levels at 13 CpG sites in the promoter region of the SLC6A4 gene between preterm and term neonates at three time points. Methods: A cohort of 46 preterm infants and a cohort of 49 full-term infants were analyzed. Blood samples collected at birth (D0), the fifth day (D5), and the thirtieth day (D30) were used to analyze DNA methylation, using bisulfite conversion and pyrosequencing. Results: Significant differences in SLC6A4 methylation were observed. At D0, CpGs 12 and 13 showed higher methylation in preterm infants. CpG 9 showed lower methylation in preterm infants at D5. Extremely preterm infants had the highest values of methylation at the three time points. Longitudinal mixed-effects analysis revealed distinct temporal patterns between groups. Total and site-specific methylation at CpGs 2, 8, and 9 increased over time in full-term infants, while methylation remained stable over time in very preterm and extremely preterm infants. Conclusions: This study reveals significant differences in SLC6A4 methylation between very preterm, extremely preterm, and full-term infants, highlighting the impact of prematurity and early-life stress on the epigenome. These findings contribute to improving our understanding of the epigenetic mechanisms shaping neurodevelopment and stress adaptation in neonates. Full article

Mitochondria are essential organelles for cellular energy production, playing a central role in driving metabolic processes and supporting critical intracellular functions. Neurometabolic disorders encompass a wide variety of conditions characterized by mitochondrial dysfunction. Owing to their bacterial ancestry, mitochondria possess an independent genome consisting of a circular DNA molecule (mtDNA), which has been reported to be subject to methylation. However, the technical challenges in the detection of mtDNA methylation have led to debates on its existence. One of the concerns is that the compactness of mtDNA can lead to suboptimal bisulfite conversion, thereby causing mtDNA methylation overestimation. To address this, liquid chromatography tandem mass spectrometry (LC-MS/MS) offers a bisulfite-independent readout; however, this method requires mtDNA samples devoid of nuclear DNA (nDNA) contamination. To diminish nDNA contamination, we isolated mtDNA from the TRIzol RNA phase. Importantly, pyrosequencing showed no significant difference in the methylation levels of mtDNA isolated from the TRIzol RNA phase compared to those from the TRIzol DNA phase, or isolated via total genomic DNA (gDNA). Across different human cell lines, LC-MS/MS detected significantly lower global methylation levels for DNA isolated from the TRIzol RNA phase than those from the TRIzol DNA or gDNA isolation. Moreover, using mtDNA isolated from the TRIzol RNA phase, LC-MS/MS validated the enhanced mtDNA methylation in HepG2 transgenic cell lines expressing mitochondrial-targeted DNA methyltransferases (means of 2.89% and 2.03% for MCviPI and MSssI transgenic cell lines, respectively), compared to two negative control cell lines (1.36 and 1.39%). When applying it to clinically relevant material, LC-MS/MS demonstrated a significantly lower global methylation level for platelet DNA isolated from the TRIzol RNA phase (mean of 1.98%) compared to gDNA isolations (mean of 4.32%). Similar findings were confirmed in mouse brain tissue, in which a significantly lower methylation level was detected in DNA isolated from the TRIzol RNA phase (1.79%) compared to that from gDNA isolation (5.12%). In conclusion, isolating mtDNA from the TRIzol RNA phase holds significant potential in future studies, particularly for the quantification of mtDNA global methylation by LC-MS/MS, a technique that is independent of bisulfite conversion and bioinformatic analysis. Full article

Background and Objectives: Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Genetic and epigenetic changes, especially DNA methylation alterations, are key in CRC development. LINE-1 hypomethylation marks global DNA methylation loss and genomic instability, making it a potential early CRC biomarker. This study investigates the methylation status of LINE-1 in colorectal adenocarcinoma, precancerous lesions (tubular and serrated adenomas), and the surrounding normal mucosa, aiming to elucidate its role as an epigenetic marker in early colorectal tumorigenesis. Materials and Methods: Paired lesion and normal tissue samples from 66 patients were analyzed for LINE-1 methylation at three CpG sites using bisulfite pyrosequencing. Results: Adenocarcinomas and tubular adenomas showed significant hypomethylation, especially at loci A and B, while serrated adenomas exhibited no significant differences. Conclusions: LINE-1 hypomethylation is associated with colorectal tumorigenesis, with distinct patterns observed between tubular and serrated adenomas, indicating distinct pathways forming and progressing specific adenomas. These findings support the potential of LINE-1 methylation as an early epigenetic biomarker for CRC risk stratification and highlight the need for further research into its clinical utility. Full article

Purpose: Homocysteine (HCY) metabolism is regulated by the methionine cycle, which is essential for DNA methylation and is associated with the folate cycle. This study examines the alterations in DNA methylation signature including epigenetic age changes, measure cell-free DNA (cfDNA), and HCY concentrations, and identifies genetic markers that may influence homocysteine response following folic acid (FA) supplementation in individuals with hyperhomocysteinemia (HHC). Methods: Blood samples were obtained from 43 HHC patients undergoing FA supplementation. We quantified FA and HCY levels, separated plasma and white blood cell fractions, and evaluated global DNA methylation using LINE-1 bisulfite pyrosequencing. Biological age was determined using Illumina BeadArray technology, and whole-exome sequencing was performed to investigate the patients’ genetic backgrounds. Results: Following FA supplementation, cfDNA levels significantly decreased and correlated positively with HCY (r = 0.2375). Elevated average LINE-1 methylation of cfDNA and PBMC-origin DNA was observed, with mean relative changes of 1.9% for both sample types. Regarding HCY levels, we categorized patients based on their response to FA supplementation. FA responders showed decreased HCY from 15.7 ± 5.5 to 11 ± 2.9 µmol/L, while in FA non-responders, an opposite trend was detected. The average biological age was reduced by 2.6 years, with a notable reduction observed in 80% of non-responders and 48% of responders. Sequencing identified mutations in several genes related to the one-carbon cycle, including MTRR, CHAT, and MTHFD1, with strong correlations to the non-responder phenotypes found in genes like PRMT3, TYMS, DNMT3A, and HIF3A. Conclusions: FA supplementation influences the HCY level, as well as affects the cfDNA amount and the DNA methylation pattern. However, genetic factors may play a crucial role in mediating individual responses to folate intake, emphasizing the need for personalized approaches in managing hyperhomocysteinemia. 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

DNA methylation changes, especially hypermethylation of SOX1 and HOXA9, may serve as biomarkers for diagnosis and prognosis in non-small cell lung carcinoma (NSCLC). This study analyzed the methylation status of SOX1 and HOXA9 in 63 primary NSCLC tumor samples, corresponding normal lung tissues, and circulating blood, using bisulfite pyrosequencing. The relationship between methylation patterns and clinicopathologic features was also explored. SOX1 and HOXA9 promoter methylation levels were significantly higher in tumor tissues compared to normal lung tissues and blood samples. Histological subtypes influenced methylation patterns, with squamous cell carcinomas (SCC) showing higher hypermethylation rates at both loci compared to other NSCLC subtypes. HOXA9 hypermethylation was associated with advanced tumor stage (stages II and III). Gender and smoking status did not correlate with methylation status. These findings highlight the cancer-specific nature of SOX1 and HOXA9 hypermethylation in NSCLC. Further investigation into demographic and molecular factors influencing methylation could enhance the clinical utility of SOX1 and HOXA9 in NSCLC diagnosis and management. Full article

Recent studies show that patients with Alzheimer’s disease (AD) harbor specific methylation marks in the brain that, if accessible, could be used as epigenetic biomarkers. Liquid biopsy enables the study of circulating cell-free DNA (cfDNA) fragments originated from dead cells, including neurons affected by neurodegenerative processes. Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium^® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. Plasma pTau181 (Simoa) and cerebrospinal fluid (CSF) core biomarkers (Fujirebio) were also measured and correlated with differential methylation marks. Validation was performed with bisulfite pyrosequencing and bisulfite cloning sequencing. Epigenome-wide cfDNA analysis identified 102 DMPs associated with AD status. Most DMPs correlated with clinical cognitive and functional tests including 60% for Mini-Mental State Examination (MMSE) and 80% for Global Deterioration Scale (GDS), and with AD blood and CSF biomarkers. In silico functional analysis connected 30 DMPs to neurological processes, identifying key regulators such as SPTBN4 and APOE genes. Several DMRs were annotated to genes previously reported to harbor epigenetic brain changes in AD (HKR1, ZNF154, HOXA5, TRIM40, ATG16L2, ADAMST2) and were linked to APOE ε4 genotypes. Notably, a DMR in the HKR1 gene, previously shown to be hypermethylated in the AD hippocampus, was validated in cfDNA from an orthogonal perspective. These results support the feasibility of studying cfDNA to identify potential epigenetic biomarkers in AD. Thus, liquid biopsy could improve non-invasive AD diagnosis and aid personalized medicine by detecting epigenetic brain markers in blood. Full article

Background: Epigenetic alterations, including DNA methylation, play a crucial role in the development of oral squamous cell carcinoma (OSCC) by regulating the expression of tumor suppressor genes and oncogenes. This study investigated the methylation status of miR-124-3 and its role in OSCC progression. Methods: This study applied the Illumina Infinium MethylationEPIC BeadChip assay to profile >850,000 CpG sites in paired OSCC and normal tissues. The methylation data were validated by further analyzing the methylation level of miR-124-3 by using a bisulfite pyrosequencing assay. We investigated whether miR-124-3 acts as a tumor suppressor by establishing miR-124-3-overexpressing OSCC cells and subjecting them to cell proliferation, colony formation, and migration assays. Dual-luciferase reporter assay was used to validate the target genes of miR-124-3 in OSCC cells. Results: The Infinium MethylationEPIC BeadChip and bisulfite pyrosequencing assays consistently identified hypermethylation of miR-124-3 in OSCC tissues relative to normal oral tissues. It was especially notable that miR-124-3 methylation levels were markedly higher in late-stage tumors than in early-stage, and differed significantly between early-stage tumor and normal tissues, indicating that miR-124-3 methylation is an early event in OSCC development. Methylation of miR-124-3 contributes markedly to the downregulation of the gene, leading to the increased expression of its target gene, leucine-rich repeat-containing 1 (LRRC1), which is considered to be positively associated with cancer progression. Moreover, overexpression of miR-124-3 suppressed the proliferation and migration of OSCC cells, while silencing the expression of LRRC1 produced similar tumor-suppressive effects. Luciferase reporter assays confirmed that miR-124-3 directly targets the 3′ untranslated region of LRRC1 to downregulate LRRC1 expression. Conclusions: Hypermethylation-mediated downregulation of miR-124-3 results in increased LRRC1 expression, which drives OSCC progression. These findings highlight DNA methylation of miR-124-3 as a potential biomarker for the early detection of OSCC and a therapeutic target for OSCC treatments. Full article

Genetics and epigenetics are mechanisms proposed for explaining post-COVID-19 condition. This secondary analysis aimed to investigate if DNA methylation levels of the ACE2 promoter are different depending on the genotype of five COVID-19-related polymorphisms in individuals who had been previously hospitalized due to SARS-CoV-2 infection. We collected non-stimulated saliva samples from 279 (48.7% female, age: 56.0 ± 12.5 years) previously hospitalized COVID-19 survivors. The participants self-reported for the presence of post-COVID symptomatology that started after the infection and persisted at the time of the appointment. Three potential genotypes of ACE2 rs2285666 and rs2074192, TMPRSS2 rs12329760 and rs2070788, and ACE1 rs1799752 polymorphisms were identified from saliva samples. Further, methylation levels at five different locations (CpG) of dinucleotides in the ACE2 promoter were quantified using bisulfited pyrosequencing. Differences in the methylation percentage (%) of each CpG according to the genotype of the five polymorphisms were analyzed. Participants were evaluated up to 17.8 (SD: 5.2) months after hospital discharge. Eighty-eight percent (88.1%) of patients reported at least one post-COVID symptom (mean number of post-COVID symptoms: 3.0; SD: 1.9). Overall, we did not observe significant differences in the methylation levels of the ACE2 promoter according to the genotype of ACE2 rs2285666 and rs2074192, TMPRSS2 rs12329760 and rs2070788, or ACE1 rs1799752 single nucleoid polymorphisms. This study did not find an association between genetics (genotypes of five COVID-19-associated polymorphisms) and epigenetics (methylation levels of the ACE2 promoter) in a cohort of COVID-19 survivors with post-COVID-19 condition who were hospitalized during the first wave of the pandemic. Full article

It is known that SARS-CoV-2 can translocate via membrane ACE2 exopeptidase into the host cells, and thus hypomethylation of ACE2 possibly upregulates its expression, enhancing the risk of SARS-CoV-2 infection. This study investigated if DNA methylation levels of the ACE2 promoter are associated with the development of post-COVID-19 symptomatology in a cohort of COVID-19 survivors who had been previously hospitalized. Non-stimulated saliva samples were obtained from 279 (51.5 male, mean age: 56.5 ± 13.0 years old) COVID-19 survivors who were hospitalized during the first wave of the pandemic. A face-to-face interview in which patients described the presence of post-COVID-19 symptoms (defined as a symptom that started no later than three months after SARS-CoV-2 infection) that they suffered from to an experienced healthcare trainer was conducted. Methylation of five CpG dinucleotides in the ACE2 promoter was quantified using bisulfite pyrosequencing. The percentage of methylation (%) was associated with the presence of the following reported post-COVID-19 symptoms: fatigue, dyspnea at rest, dyspnea at exertion, brain fog, memory loss, concentration loss, or gastrointestinal problems. Participants were assessed a mean of 17.8 (SD: 5.3) months after hospitalization. At that time, 88.1% of the patients experienced at least one post-COVID-19 symptom (mean number for each patient: 3.0; SD: 1.9 post-COVID-19 symptoms). Dyspnea at exertion (67.3%), fatigue (62.3%), and memory loss (31.2%) were the most frequent post-COVID-19 symptoms in the sample. Overall, the analysis did not reveal any difference in the methylation of the ACE2 promoter in any of the CpG locations according to the presence or absence of fatigue, dyspnea at rest, dyspnea at exertion, memory loss, brain fog, concentration loss, and gastrointestinal problems. This study did not find an association between methylation of ACE2 promoter and the presence of post-COVID-19 fatigue, dyspnea, cognitive or gastrointestinal problems in previously hospitalized COVID-19 survivors. Full article

For several decades, cancers have demonstrably been one of the most frequent causes of death worldwide. In addition to genetic causes, cancer can also be caused by epigenetic gene modifications. Frequently, tumor suppressor genes are epigenetically inactivated due to hypermethylation of their CpG islands, actively contributing to tumorigenesis. Since CpG islands are usually localized near promoters, hypermethylation of the promoter can have a major impact on gene expression. In this study, the potential tumor suppressor gene Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) was examined for an epigenetic regulation and its gene inactivation in melanomas. A hypermethylation of the RIPK3 CpG island was detected by bisulfite pyrosequencing and was accompanied by a correlated loss of its expression. In addition, an increasing RIPK3 methylation rate was observed with increasing tumor stage of melanomas. For further epigenetic characterization of RIPK3, epigenetic modulation was performed using a modified CRISPR/dCas9 (CRISPRa activation) system targeting its DNA hypermethylation. We observed a reduced fitness of melanoma cells by (re-)expression and demethylation of the RIPK3 gene using the epigenetic editing-based method. The tumor suppressive function of RIPK3 was evident by phenotypic determination using fluorescence microscopy, flow cytometry and wound healing assay. Our data highlight the function of RIPK3 as an epigenetically regulated tumor suppressor in melanoma, allowing it to be classified as a biomarker. Full article