In patients with proximal hypospadias, often no genetic cause is identified despite extensive genetic testing. Many genes involved in sex development encode transcription factors with strict timing and dosing of the gene products. We hypothesised that there might be recurrent differences in DNA methylation in boys with hypospadias and that these might differ between patients born small versus appropriate for gestational age. Genome-wide Methylated DNA sequencing (MeD-seq) was performed on 32bp LpnPI restriction enzyme fragments after RE-digestion in leucocytes from 16 XY boys with unexplained proximal hypospadias, one with an unexplained XX testicular disorder/difference of sex development (DSD) and twelve, healthy, sex- and age-matched controls. Five of seven differentially methylated regions (DMRs) between patients and XY controls were in the Long Intergenic Non-Protein Coding RNA 665 (LINC00665; CpG24525). Three patients showed hypermethylation of MAP3K1. Finally, no DMRs in XX testicular DSD associated genes were identified in the XX boy versus XX controls. In conclusion, we observed no recognizable epigenetic signature in 16 boys with XY proximal hypospadias and no difference between children born small versus appropriate for gestational age. Comparison to previous methylation studies in individuals with hypospadias did not show consistent findings, possibly due to the use of different inclusion criteria, tissues and methods.

BACKGROUND: DNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss.
RESULTS: The search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss.
CONCLUSIONS: Evidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research.

As an important epigenetic modification, DNA methylation is involved in many biological processes such as animal cell differentiation, embryonic development, genomic imprinting and sex chromosome inactivation. As DNA methylation sequencing becomes more sophisticated, it becomes possible to use it to solve more zoological problems. This paper reviews the characteristics of DNA methylation, with emphasis on the research and application of DNA methylation in poultry.

Cardiovascular disease (CVD) is the leading cause of death worldwide and considered one of the most environmentally driven diseases. The role of DNA methylation in response to the individual exposure for the development and progression of CVD is still poorly understood and a synthesis of the evidence is lacking.
A systematic review of articles examining measurements of DNA cytosine methylation in CVD was conducted in accordance with PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. The search yielded 5,563 articles from PubMed and CENTRAL databases. From 99 studies with a total of 87,827 individuals eligible for analysis, a database was created combining all CpG-, gene- and study-related information. It contains 74,580 unique CpG sites, of which 1452 CpG sites were mentioned in ≥ 2, and 441 CpG sites in ≥ 3 publications. Two sites were referenced in ≥ 6 publications: cg01656216 (near ZNF438) related to vascular disease and epigenetic age, and cg03636183 (near F2RL3) related to coronary heart disease, myocardial infarction, smoking and air pollution. Of 19,127 mapped genes, 5,807 were reported in ≥ 2 studies. Most frequently reported were TEAD1 (TEA Domain Transcription Factor 1) and PTPRN2 (Protein Tyrosine Phosphatase Receptor Type N2) in association with outcomes ranging from vascular to cardiac disease. Gene set enrichment analysis of 4,532 overlapping genes revealed enrichment for Gene Ontology molecular function \"DNA-binding transcription activator activity\" (q = 1.65 × 10-11) and biological processes \"skeletal system development\" (q = 1.89 × 10-23). Gene enrichment demonstrated that general CVD-related terms are shared, while \"heart\" and \"vasculature\" specific genes have more disease-specific terms as PR interval for \"heart\" or platelet distribution width for \"vasculature.\" STRING analysis revealed significant protein-protein interactions between the products of the differentially methylated genes (p = 0.003) suggesting that dysregulation of the protein interaction network could contribute to CVD. Overlaps with curated gene sets from the Molecular Signatures Database showed enrichment of genes in hemostasis (p = 2.9 × 10-6) and atherosclerosis (p = 4.9 × 10-4).
This review highlights the current state of knowledge on significant relationship between DNA methylation and CVD in humans. An open-access database has been compiled of reported CpG methylation sites, genes and pathways that may play an important role in this relationship.

BACKGROUND: Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). However, whether the influence of methyl donor intake is modified by polymorphisms in such epigenetic regulators is still unclear.
OBJECTIVE: To improve the current understanding of the molecular basis of CRC.
METHODS: A literature search in the Medline database, Reference Citation Analysis (https:// www.referencecitationanalysis.com/), and manual reference screening were performed to identify observational studies published from inception to May 2022.
RESULTS: A total of fourteen case-control studies and five cohort studies were identified. These studies included information on dietary methyl donors, dietary components that potentially modulate the bioavailability of methyl groups, genetic variants of methyl metabolizing enzymes, and/or markers of CpG island methylator phenotype and/or microsatellite instability, and their possible interactions on CRC risk.
CONCLUSIONS: Several studies have suggested interactions between methylenetetrahydrofolate reductase polymorphisms, methyl donor nutrients (such as folate) and alcohol on CRC risk. Moreover, vitamin B6, niacin, and alcohol may affect CRC risk through not only genetic but also epigenetic regulation. Identification of specific mechanisms in these interactions associated with CRC may assist in developing targeted prevention strategies for individuals at the highest risk of developing CRC.

The prediction of chronological age from methylation-based biomarkers represents one of the most promising applications in the field of forensic sciences. Age-prediction models developed so far are not easily applicable for forensic caseworkers. Among the several attempts to pursue this objective, the formulation of single-locus models might represent a good strategy. The present work aimed to develop an accurate single-locus model for age prediction exploiting ELOVL2, a gene for which epigenetic alterations are most highly correlated with age. We carried out a systematic review of different published pyrosequencing datasets in which methylation of the ELOVL2 promoter was analysed to formulate age prediction models. Nine of these, with available datasets involving 2298 participants, were selected. We found that irrespective of which model was adopted, a very strong relationship between ELOVL2 methylation levels and age exists. In particular, the model giving the best age-prediction accuracy was the gradient boosting regressor with a prediction error of about 5.5 years. The findings reported here strongly support the use of ELOVL2 for the formulation of a single-locus epigenetic model, but the inclusion of additional, non-redundant markers is a fundamental requirement to apply a molecular model to forensic applications with more robust results.

BACKGROUND: Gene silence via methylation of the CpG islands is cancer\'s most common epigenetic modification. Given the highly significant role of NIS in thyroid cancer (TC) differentiation, this cross-sectional study aimed to investigate the DNA methylation pattern in seven CpG islands (CpG1-7 including +846, +918, +929, +947, +953, +955, and +963, respectively) of the NIS promoter in patients diagnosed with papillary (PTC), follicular (FTC), and multinodular goiter (MNG). Additionally, a systematic review of the literature was conducted to compare our results with studies concerning methylation of the NIS gene promoter.
METHODS: Thyroid specimens from 64 patients met the eligibility criteria, consisting of 28 PTC, 9 FTC, and 27 benign MNG cases. The mRNA of NIS was tested by qRT-PCR. The bisulfite sequencing PCR (BSP) technique was performed to evaluate the promoter methylation pattern of the NIS gene. Sequencing results were received in chromatograph, FASTA, SEQ, and pdf formats and were analyzed using Chromas. The methylation percentage at each position and for each sample was calculated by mC/(mC+C) formula for all examined CpGs; following that, the methylation percentage was also calculated at each CpG site. Besides, a literature search was conducted without restricting publication dates. Nine studies met the eligibility criteria after removing duplicates, unrelated articles, and reviews.
RESULTS: NIS mRNA levels decreased in tumoral tissues of PTC (P = 0.04) and FTC (P = 0.03) patients compared to their matched non-tumoral ones. The methylation of NIS promoter was not common in PTC samples, but it was frequent in FTC (P < 0.05). Significant differences were observed in the methylation levels in the 4th(+ 947), 6th(+ 955), and 7th(+ 963) CpGs sites in the forward strand of NIS promoter between FTC and MNG tissues (76.34 ± 3.12 vs 40.43 ± 8.42, P = 0.004, 69.63 ± 3.03 vs 23.29 ± 6.84, P = 0.001 and 50.33 ± 5.65 vs 24 ± 6.89, P = 0.030, respectively). There was no significant correlation between the expression and methylation status of NIS in PTC and FTC tissues.
CONCLUSIONS: Perturbation in NIS promoter\'s methylation individually may have a potential utility in differentiating MNG and FTC tissues. The absence of a distinct methylation pattern implies the importance of other epigenetic processes, which may alter the production of NIS mRNA. In addition, according to the reversibility of DNA methylation, it is anticipated that the design of particular targeted demethylation medicines will lead to a novel cancer therapeutic strategy.

Several studies have demonstrated an association between multiple gene hypermethylation and gastric cancer. However, the intrinsic mechanisms remain elusive and highly debatable. To this end, our study aims to investigate the correlation between the methylation status of multiple gene promoters and gastric cancer.
PubMed, EMBASE, CNKI, WanFang, Cqvip, and Cochrane Library were queried from inception to May 2021, and the relationship between the methylation status of the CpG islands and gastric cancer risk was systematically assessed under the inclusion and exclusion criteria. The incidence of DNA methylation between tumor and non-tumor tissues was compared, and the clinicopathological significance of DNA methylation in gastric carcinoma was further evaluated. The odds ratio (OR) was estimated with a 95% confidence interval (CI), and forest plots were generated using the fixed-effects or random-effects model.
In total, 201 studies were enrolled, and a higher frequency of CpG islands methylation was identified in gastric cancer tissues than in non-neoplastic tissues. This suggests that aberrant polygene methylation might be associated with the initial onset and progression of gastric cancer.
This study sheds light on the significance of polygene methylation status in gastric cancer. The DNA methylation of these genes may serve as underlying epigenetic biomarkers, providing a promising molecular diagnostic approach for human gastric cancer clinical diagnosis. More large randomized trials are needed to confirm the findings.

Epigenetic alterations (including DNA methylation or miRNAs) influence oncogene/oncosuppressor gene expression without changing the DNA sequence. Prostate cancer (PC) displays a complex genetic and epigenetic regulation of cell-growth pathways and tumor progression. We performed a systematic literature review (following PRISMA guidelines) focused on the epigenetic regulation of PD-L1 expression in PC. In PC cell lines, CpG island methylation of the CD274 promoter negatively regulated PD-L1 expression. Histone modifiers also influence the PD-L1 transcription rate: the deletion or silencing of the histone modifiers MLL3/MML1 can positively regulate PD-L1 expression. Epigenetic drugs (EDs) may be promising in reprogramming tumor cells, reversing epigenetic modifications, and cancer immune evasion. EDs promoting a chromatin-inactive transcriptional state (such as bromodomain or p300/CBP inhibitors) downregulated PD-L1, while EDs favoring a chromatin-active state (i.e., histone deacetylase inhibitors) increased PD-L1 expression. miRNAs can regulate PD-L1 at a post-transcriptional level. miR-195/miR-16 were negatively associated with PD-L1 expression and positively correlated to longer biochemical recurrence-free survival; they also enhanced the radiotherapy efficacy in PC cell lines. miR-197 and miR-200a-c positively correlated to PD-L1 mRNA levels and inversely correlated to the methylation of PD-L1 promoter in a large series. miR-570, miR-34a and miR-513 may also be involved in epigenetic regulation.

Loss-of-function events in tumor suppressor genes (TSGs) contribute to the development and progression of cutaneous malignant melanoma (CMM). Epigenetic alterations are the major mechanisms of TSG inactivation, in particular, silencing by promoter CpG-island hypermethylation. TSGs are valuable tools in diagnosis and prognosis and, possibly, in future targeted therapy. The aim of this narrative review is to outline bona fide TSGs affected by promoter CpG-island hypermethylation and their functional role in the progression of CMM. We conducted a systematic literature review to identify studies providing evidence of bona fide TSGs by cell line or animal experiments. We performed a broad first search and a gene-specific second search, supplemented by reference checking. We included studies describing bona fide TSGs in CMM with promoter CpG-island hypermethylation in which inactivating mechanisms were reported. We extracted data about protein role, pathway, experiments conducted to meet the bona fide criteria and hallmarks of cancer acquired by TSG inactivation. A total of 24 studies were included, describing 24 bona fide TSGs silenced by promoter CpG-island hypermethylation in CMM. Their effect on cell proliferation, apoptosis, growth, senescence, angiogenesis, migration, invasion or metastasis is also described. These data give further insight into the role of TSGs in the progression of CMM.