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: The plasma metabolome reflects the physiological state of various biological processes and can serve as a proxy for disease risk. Plasma metabolite variation, influenced by genetic and epigenetic mechanisms, can also affect the cellular microenvironment and blood cell epigenetics. The interplay between the plasma metabolome and the blood cell epigenome remains elusive. In this study, we performed an epigenome-wide association study (EWAS) of 1183 plasma metabolites in 693 participants from the LifeLines-DEEP cohort and investigated the causal relationships in DNA methylation-metabolite associations using bidirectional Mendelian randomization and mediation analysis.
RESULTS: After rigorously adjusting for potential confounders, including genetics, we identified five robust associations between two plasma metabolites (L-serine and glycine) and three CpG sites located in two independent genomic regions (cg14476101 and cg16246545 in PHGDH and cg02711608 in SLC1A5) at a false discovery rate of less than 0.05. Further analysis revealed a complex bidirectional relationship between plasma glycine/serine levels and DNA methylation. Moreover, we observed a strong mediating role of DNA methylation in the effect of glycine/serine on the expression of their metabolism/transport genes, with the proportion of the mediated effect ranging from 11.8 to 54.3%. This result was also replicated in an independent population-based cohort, the Rotterdam Study. To validate our findings, we conducted in vitro cell studies which confirmed the mediating role of DNA methylation in the regulation of PHGDH gene expression.
CONCLUSIONS: Our findings reveal a potential feedback mechanism in which glycine and serine regulate gene expression through DNA methylation.

Chronic kidney disease (CKD) impacts about 1 in 7 adults in the United States, but African Americans (AAs) carry a disproportionately higher burden of disease. Epigenetic modifications, such as DNA methylation at cytosine-phosphate-guanine (CpG) sites, have been linked to kidney function and may have clinical utility in predicting the risk of CKD. Given the dynamic relationship between the epigenome, environment, and disease, AAs may be especially sensitive to environment-driven methylation alterations. Moreover, risk models incorporating CpG methylation have been shown to predict disease across multiple racial groups. In this study, we developed a methylation risk score (MRS) for CKD in cohorts of AAs. We selected nine CpG sites that were previously reported to be associated with estimated glomerular filtration rate (eGFR) in epigenome-wide association studies to construct a MRS in the Hypertension Genetic Epidemiology Network (HyperGEN). In logistic mixed models, the MRS was significantly associated with prevalent CKD and was robust to multiple sensitivity analyses, including CKD risk factors. There was modest replication in validation cohorts. In summary, we demonstrated that an eGFR-based CpG score is an independent predictor of prevalent CKD, suggesting that MRS should be further investigated for clinical utility in evaluating CKD risk and progression.

Exposure to fluoride in early childhood has been associated with altered cognition, intelligence, attention, and neurobehavior. Fluoride-related neurodevelopment effects have been shown to vary by sex and very little is known about the mechanistic processes involved. There is limited research on how fluoride exposure impacts the epigenome, potentially leading to changes in DNA methylation of specific genes regulating key developmental processes. In the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS), urine samples were analyzed using a microdiffusion method to determine childhood urinary fluoride adjusted for specific gravity (CUFsg) concentrations. Whole blood DNA methylation was assessed using the Infinium MethylationEPIC BeadChip 850 k Array. In a cross-sectional analysis, we interrogated epigenome-wide DNA methylation at 775,141 CpG loci across the methylome in relation to CUFsg concentrations in 272 early adolescents at age 12 years. Among all participants, higher concentrations of CUF were associated with differential methylation of one CpG (p < 6 × 10-8) located in the gene body of GBF1 (cg25435255). Among females, higher concentrations of CUFsg were associated with differential methylation of 7 CpGs; only three CpGs were differentially methylated among males with no overlap of significant CpGs observed among females. Secondary analyses revealed several differentially methylated regions (DMRs) and CpG loci mapping to genes with key roles in psychiatric outcomes, social interaction, and cognition, as well as immunologic and metabolic phenotypes. While fluoride exposure may impact the epigenome during early adolescence, the functional consequences of these changes are unclear warranting further investigation.

Opioid use disorder (OUD) is a multifaceted condition influenced by sex, genetic and environmental factors that could be linked with epigenetic changes. Understanding how these factors interact is crucial to understand and address the development and progression of this disorder. Our aim was to elucidate different potential epigenetic and genetic mechanisms between women and men that correlate with OUD under real-world pain unit conditions. Associations between analgesic response and the DNA methylation level of the opioid mu receptor (OPRM1) gene (CpG sites 1-5 selected in the promoter region) were evaluated in 345 long opioid-treated chronic non cancer pain: cases with OUD (n = 67) and controls (without OUD, n = 278). Cases showed younger ages, low employment status and quality of life, but higher morphine equivalent daily dose and psychotropic use, compared to the controls. The patients with OUD showed a significant decrease in OPRM1 DNA methylation, which correlated with clinical outcomes like pain relief, depression and different adverse events. Significant differences were found at the five CpG sites studied for men, and exclusively in women for CpG site 3, in relation to OUD diagnosis. These findings support the importance of epigenetics and sex as biological variables to be considered toward efficient OUD understanding and therapy development.

BACKGROUND: Older patients are at risk for acute kidney injury and chronic kidney disease. Age-related increases in DNA methylation at CpG islands have been linked to aging-related diseases like cancer and cardiovascular disease, but the exact causal relationship between methylation in renal aging and other kidney diseases remains unclear. This study aimed to elucidate the methylation status of peripheral blood mononuclear cells (PBMCs) in the Asian population. Using human whole blood DNA methylation analysis from the Taiwan Biobank, we included participants with both whole blood genome-wide methylation data and follow-up data on serum creatinine. We investigated hyper- and hypomethylated genes in comparison of participants with higher and lower estimated glomerular filtration (eGFR) decline rate in overall cohort as well as in comparison of old and young participants in subgroup of participants with higher eGFR decline rate. Common genes and signaling pathways in both comparative analyses were identified.
RESULTS: Among 1587 participants in the analysis, 187 participants had higher eGFR decline rate. According to the comparison of methylation in participants with different eGFR declines and at different ages, respectively, we identified common hypermethylated genes, including DNMT3A and GGACT, as well as hypomethylated genes such as ARL6IP5, CYB5D1, BCL6, RPRD2, ZNF451, and MIAT in both participants with higher eGFR decline and those of older age. We observed associations between the methylation status of signaling pathways and aging as well as renal function decline. These pathways notably included autophagy, p38 mitogen-activated protein kinases, and sirtuins, which were associated with autophagy process and cytokine production.
CONCLUSIONS: Through methylation analysis of PBMCs, we identified genes and signaling pathways which could play crucial roles in the interplay of renal aging and renal function decline. These findings contribute to the development of novel biomarkers for identifying at-risk groups and even for therapeutic agent discovery.

BACKGROUND: Innate/adaptive immunity is the key to anti-tumor therapy. However, its causal relationship to Gastrointestinal (GI) cancer remains unclear.
METHODS: Immunity genes were extracted from the MSigDB database. The Genome-wide association studies (GWAS) summary data of GI cancer were integrated with expression quantitative trait loci (eQTL) and DNA methylation quantitative trait loci (mQTL) associated with genes. Summary-data-based Mendelian randomization (SMR) and co-localization analysis were used to reveal causal relationships between genes and GI cancer. Two-sample MR analysis was used for sensitivity analysis. Single cell analysis clarified the enrichment of genes.
RESULTS: Three-step SMR analysis showed that a putative mechanism, cg17294865 CpG site regulating HLA-DRA expression was negatively associated with gastric cancer risk. HLA-DRA was significantly differentially expressed in monocyte/macrophage and myeloid cells in gastric cancer.
CONCLUSIONS: This study provides evidence that upregulating the expression level of HLA-DRA can reduce the risk of gastric cancer.

OBJECTIVE: Stroke is the leading cause of adult-onset disability. Although clinical factors influence stroke outcome, there is a significant variability among individuals that may be attributed to genetics and epigenetics, including DNA methylation (DNAm). We aimed to study the association between DNAm and stroke prognosis.
RESULTS: To that aim, we conducted a two-phase study (discovery-replication and meta-analysis) in Caucasian patients with ischemic stroke from two independent centers (BasicMar [discovery, N = 316] and St. Pau [replication, N = 92]). Functional outcome was assessed using the modified Rankin Scale (mRS) at three months after stroke, being poor outcome defined as mRS > 2. DNAm was determined using the 450K and EPIC BeadChips in whole-blood samples collected within the first 24 h. We searched for differentially methylated positions (DMPs) in 370,344 CpGs, and candidates below p-value < 10-5 were subsequently tested in the replication cohort. We then meta-analyzed DMP results from both cohorts and used them to identify differentially methylated regions (DMRs). After doing the epigenome-wide association study, we found 29 DMPs at p-value < 10-5 and one of them was replicated: cg24391982, annotated to thrombospondin-2 (THBS2) gene (p-valuediscovery = 1.54·10-6; p-valuereplication = 9.17·10-4; p-valuemeta-analysis = 6.39·10-9). Besides, four DMRs were identified in patients with poor outcome annotated to zinc finger protein 57 homolog (ZFP57), Arachidonate 12-Lipoxygenase 12S Type (ALOX12), ABI Family Member 3 (ABI3) and Allantoicase (ALLC) genes (p-value < 1·10-9 in all cases).
CONCLUSIONS: Patients with poor outcome showed a DMP at THBS2 and four DMRs annotated to ZFP57, ALOX12, ABI3 and ALLC genes. This suggests an association between stroke outcome and DNAm, which may help identify new stroke recovery mechanisms.

The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

Our study aimed to investigate the association between the transition of the TXNIP gene methylation level and the risk of incident type 2 diabetes mellitus (T2DM). This study included 263 incident cases of T2DM and 263 matched non-T2DM participants. According to the methylation levels of five loci (CpG1-5; chr1:145441102-145442001) on the TXNIP gene, the participants were classified into four transition groups: maintained low, low to high, high to low, and maintained high methylation levels. Compared with individuals whose methylation level of CpG2-5 at the TXNIP gene was maintained low, individuals with maintained high methylation levels showed a 61-87% reduction in T2DM risk (66% for CpG2 [OR: 0.34, 95% CI: 0.14, 0.80]; 77% for CpG3 [OR: 0.23, 95% CI: 0.07, 0.78]; 87% for CpG4 [OR: 0.13, 95% CI: 0.03, 0.56]; and 61% for CpG5 [OR: 0.39, 95% CI: 0.16, 0.92]). Maintained high methylation levels of four loci of the TXNIP gene are associated with a reduction of T2DM incident risk in the current study. Our study suggests that preserving hypermethylation levels of the TXNIP gene may hold promise as a potential preventive measure against the onset of T2DM.