Diagnostic efficacy of SEPT9 and PAX5 gene methylation in gastrointestinal cancer and precancerous lesions.

To assess the diagnostic efficacy of SEPT9 along with PAX5 gene methylation detection in gastrointestinal cancer and precancerous lesions, the peripheral blood of 62 patients with gastric cancer (GC) and 60 patients with no evidence of disease (as the control group) were retrospectively collected. The methylation rates of PAX5 and SEPT9 gene promoters in blood samples of GC group were detected by PCR. At the same time, the differences in methylation rates of genes in the two groups were compared, and the predictive value of plasma methylation PAX5 and SEPT9 in GC was evaluated by receiver operating characteristic (ROC) curve. We found that there were 41 cases of methylated PAX5 gene promoter region and 39 cases of methylated SEPT9 gene promoter region in GC group. The control group contained 14 cases of PAX5 gene promoter methylation and 12 cases of RNF¹80 gene promoter methylation. The occurrence of PAX5 promoter methylation was correlated with age of GC patients. There were statistically significant differences in mSEPT9 gene in patients with different TNM stages. Kaplan-Meier survival curve analysis revealed that the three-year overall survival rate of GC patients with PAX5 methylation was lower than that of GC patients without PAX5 methylation. No significant difference was discovered in 3-year overall survival rate between GC patients with SEPT9 methylation and those without SEPT9 methylation. Combined detection could not improve the diagnostic value of GC, but could promote diagnosis sensitivity. In summary, the risk of PAX5 and SEPT9 gene methylation in GC patients presents higher when compared with healthy people. PAX5 gene methylation is closely related to age, while SEPT9 is closely related to tumor TNM stage, and PAX5 gene methylation can decrease the survival rate of GC patients. Detection of PAX5 gene methylation level can assist in evaluating the prognosis of GC patients.

Diagnostic utility of DNA methylation analysis in genetically unsolved pediatric epilepsies and CHD2 episignature refinement.

Sequence-based genetic testing identifies causative variants in ~ 50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases.

Epigenome-wide association study on the plasma metabolome suggests self-regulation of the glycine and serine pathway through DNA methylation.

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.

DNA methylation markers for oral cancer detection in non- and minimally invasive samples: a systematic review.

More than 50% of oral cancer (OC) patients are diagnosed with advanced-stage disease associated with poor prognosis and quality of life, supporting an urgent need to improve early OC detection. The identification of effective molecular markers by minimally invasive approaches has emerged as a promising strategy for OC screening. This systematic review summarizes and evaluates the performance of the DNA methylation markers identified in non- or minimally invasive samples for OC detection. PubMed's MEDLINE, Scopus, Embase, and Cochrane Library databases were systematically searched for studies that evaluated DNA methylation markers in non-invasive and/or minimally invasive samples (oral rinse/saliva, oral brush, and blood) from OC patients. Two investigators independently extracted data on study population characteristics, candidate methylation markers, testing samples, DNA methylation assay, and performance diagnostic outcomes. Methodological study quality was assessed with the Quality Assessment for Studies of Diagnostic Accuracy-2 tool. Thirty-one studies met the inclusion criteria for this systematic review. DNA methylation markers were evaluated in oral rinse/saliva (n = 17), oral brush (n = 9), and blood (n = 7) samples. Methylation-specific PCR (MSP) and quantitative-MSP were the most common DNA methylation assays. Regarding diagnostic performance values for salivary, oral brush, and blood DNA methylation markers, sensitivity and specificity ranged between 3.4-100% and 21-100%, 9-100% and 26.8-100%, 22-70% and 45.45-100%, respectively. Different gene methylation panels showed good diagnostic performance for OC detection. This systematic review discloses the promising value of testing DNA methylation markers in non-invasive (saliva or oral rinse) or minimally invasive (oral brush or blood) samples as a novel strategy for OC detection. However, further validation in large, multicenter, and prospective study cohorts must be carried out to confirm the clinical value of specific DNA methylation markers in this setting.

CpG Island Definition and Methylation Mapping of the T2T-YAO Genome.

Precisely defining and mapping all cytosine (C) positions and their clusters, known as CpG islands (CGIs), as well as their methylation status, are pivotal for genome-wide epigenetic studies, especially when population-centric reference genomes are ready for timely application. Here, we first align the two high-quality reference genomes, T2T-YAO and T2T-CHM13, from different ethnic backgrounds in a base-by-base fashion and compute their genome-wide density-defined and position-defined CGIs. Second, by mapping some representative genome-wide methylation data from selected organs onto the two genomes, we find that there are about 4.7%-5.8% sequence divergency of variable categories depending on quality cutoffs. Genes among the divergent sequences are mostly associated with neurological functions. Moreover, CGIs associated with the divergent sequences are significantly different with respect to CpG density and observed CpG/expected CpG (O/E) ratio between the two genomes. Finally, we find that the T2T-YAO genome not only has a greater CpG coverage than that of the T2T-CHM13 genome when whole-genome bisulfite sequencing (WGBS) data from the European and American populations are mapped to each reference, but also shows more hyper-methylated CpG sites as compared to the T2T-CHM13 genome. Our study suggests that future genome-wide epigenetic studies of the Chinese populations rely on both acquisition of high-quality methylation data and subsequent precision CGI mapping based on the Chinese T2T reference.

Mendelian randomization implicates causal association between epigenetic age acceleration and age-related eye diseases or glaucoma endophenotypes.

BACKGROUND: Age-related eye diseases (AREDs) have become increasingly prevalent with the aging population, serving as the leading causes of visual impairment worldwide. Epigenetic clocks are generated based on DNA methylation (DNAm) levels and are considered one of the most promising predictors of biological age. This study aimed to investigate the bidirectional causal association between epigenetic clocks and common AREDs or glaucoma endophenotypes. METHODS: Instrumental variables for epigenetic clocks, AREDs, and glaucoma endophenotypes were obtained from corresponding genome-wide association study data of European descent. Bidirectional two-sample Mendelian randomization (MR) was employed to explore the causal relationship between epigenetic clocks and AREDs or glaucoma endophenotypes. Multivariable MR (MVMR) was used to determine whether glaucoma endophenotypes mediated the association of epigenetic clocks with glaucoma. Multiple sensitivity analyses were conducted to confirm the robustness of MR estimates. RESULTS: The results showed that an increased intrinsic epigenetic age acceleration (HorvathAge) was significantly associated with an increased risk of primary open-angle glaucoma (OR = 1.04, 95% CI 1.02 to 1.06, P = 6.1E-04). The epigenetic age acceleration (EEA) of HannumAge was related to a decreased risk of primary angle-closure glaucoma (OR = 0.92, 95% CI 0.86 to 0.99, P = 0.035). Reverse MR analysis showed that age-related cataract was linked to decreased HannumAge (ß = -0.190 year, 95% CI -0.374 to -0.008, P = 0.041). The EEA of HannumAge (ß = -0.85 µm, 95% CI -1.57 to -0.14, P = 0.019) and HorvathAge (ß = -0.63 µm, 95% CI -1.18 to -0.08, P = 0.024) were associated with decreased central corneal thickness (CCT). PhenoAge was related to an increased retinal nerve fiber layer thickness (ß = 0.06 µm, 95% CI 0.01 to 0.11, P = 0.027). MVMR analysis found no mediation effect of CCT in the association of HannumAge and HorvathAge with glaucoma. DNAm-based granulocyte proportions were significantly associated with presbyopia, rhegmatogenous retinal detachment, and intraocular pressure (P < 0.05). DNAm-based plasminogen activator inhibitor-1 levels were significantly related to age-related macular degeneration and intraocular pressure (P < 0.05). CONCLUSION: The present study revealed a causal association between epigenetic clocks and AREDs. More research is warranted to clarify the potential mechanisms of the biological aging process in AREDs.

Association of XRCC2 with breast cancer, a multi-omics analysis at genomic, transcriptomic, and epigenomic level.

One of the main causes of cancer-related mortality for women worldwide is breast cancer (BC). The XRCC2 gene, essential for DNA repair, has been implicated in cancer susceptibility. This study aims to evaluate the association between XRCC2 and BC risk. The study was conducted at Zheen International Hospital in Erbil, Iraq, between 2021 and 2024 with a total of 88 samples, including 44 paired normal and cancer tissue samples. Mutation analysis was performed using Next-Generation Sequencing, coupled with in silico tools for variant impact prediction. Expression levels were assessed through RT-PCR, and methylation status was determined using methylation-sensitive restriction enzyme digestion PCR. The study identified seven inherited germline variants in the XRCC2 gene, with five of these mutations being Uncertain Significance, one being Likely Pathogenic, and one being Likely benign. RNA purity was found high with mean A260/280 ratios of 1.986 ± 0.097 in normal (N) and 1.963 ± 0.092 in tumor (T) samples. Tumor samples exhibited a higher RNA concentration (78.56 ± 40.87 ng/µL) than normal samples (71.44 ± 40.79 ng/µL). XRCC2 gene expression was significantly upregulated in tumor tissue, with marked increases in patients aged 40-55 and >56 years and in higher cancer grades (II and III) and invasive ductal carcinoma (p-values ranging from <0.0001 to 0.0392). DNA methylation rates in tumor tissues were low (7%), suggesting limited regulation by methylation. The study suggests that XRCC2 can be classified as an oncogene and that its structural investigation by targeted NGS and expression evaluation can be used as a potential biomarker in BC.

The DNA methylome of pediatric brain tumors appears shaped by structural variation and predicts survival.

Structural variation heavily influences the molecular landscape of cancer, in part by impacting DNA methylation-mediated transcriptional regulation. Here, using multi-omic datasets involving >2400 pediatric brain and central nervous system tumors of diverse histologies from the Children's Brain Tumor Network, we report hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of somatic structural variant (SV) breakpoints is recurrently associated with altered expression or DNA methylation, respectively, including tumor suppressor genes ATRX and CDKN2A. Altered DNA methylation near enhancers associates with nearby somatic SV breakpoints, including MYC and MYCN. A subset of genes with SV-CGI methylation associations also have expression associations with patient survival, including BCOR, TERT, RCOR2, and PDLIM4. DNA methylation changes in recurrent or progressive tumors compared to the initial tumor within the same patient can predict survival in pediatric and adult cancers. Our comprehensive and pan-histology genomic analyses reveal mechanisms of noncoding alterations impacting cancer genes.

Inflammatory breast cancer microenvironment repertoire based on DNA methylation data deconvolution reveals actionable targets to enhance the treatment efficacy.

BACKGROUND: Although the clinical signs of inflammatory breast cancer (IBC) resemble acute inflammation, the role played by infiltrating immune and stromal cells in this aggressive disease is uncharted. The tumor microenvironment (TME) presents molecular alterations, such as epimutations, prior to morphological abnormalities. These changes affect the distribution and the intricate communication between the TME components related to cancer prognosis and therapy response. Herein, we explored the global DNA methylation profile of IBC and surrounding tissues to estimate the microenvironment cellular composition and identify epigenetically dysregulated markers. METHODS: We used the HiTIMED algorithm to deconvolve the bulk DNA methylation data of 24 IBC and six surrounding non-tumoral tissues (SNT) (GSE238092) and determine their cellular composition. The prognostic relevance of cell types infiltrating IBC and their relationship with clinicopathological variables were investigated. CD34 (endothelial cell marker) and CD68 (macrophage marker) immunofluorescence staining was evaluated in an independent set of 17 IBC and 16 non-IBC samples. RESULTS: We found lower infiltration of endothelial, stromal, memory B, dendritic, and natural killer cells in IBC than in SNT samples. Higher endothelial cell (EC) and stromal cell content were related to better overall survival. EC proportions positively correlated with memory B and memory CD8+ T infiltration in IBC. Immune and EC markers exhibited distinct DNA methylation profiles between IBC and SNT samples, revealing hypermethylated regions mapped to six genes (CD40, CD34, EMCN, HLA-G, PDPN, and TEK). We identified significantly higher CD34 and CD68 protein expression in IBC compared to non-IBC. CONCLUSIONS: Our findings underscored cell subsets that distinguished patients with better survival and dysregulated markers potentially actionable through combinations of immunotherapy and epigenetic drugs.

Epigenetic patterns, accelerated biological aging, and enhanced epigenetic drift detected 6 months following COVID-19 infection: insights from a genome-wide DNA methylation study.

BACKGROUND: The epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role. METHODS AND RESULTS: Our study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR < 0.05), primarily within CpG islands and gene promoters. Dysregulated genes highlighted potential links to glutamate/glutamine metabolism, which may be relevant to PASC symptoms. Key genes with potential significance to COVID-19 infection and long-term effects include GLUD1, ATP1A3, and ARRB2. Furthermore, Horvath's epigenetic clock showed a slight but significant age acceleration in post-COVID-19 patients. We also observed a substantial increase in stochastic epigenetic mutations (SEMs) in the post-COVID-19 group, implying potential epigenetic drift. SEM analysis identified 790 affected genes, indicating dysregulation in pathways related to insulin resistance, VEGF signaling, apoptosis, hypoxia response, T-cell activation, and endothelin signaling. CONCLUSIONS: Our study provides valuable insights into the epigenetic consequences of COVID-19. Results suggest possible associations with accelerated aging, epigenetic drift, and the disruption of critical biological pathways linked to insulin resistance, immune response, and vascular health. Understanding these epigenetic changes could be crucial for elucidating the complex mechanisms behind long-COVID and developing targeted therapeutic interventions.

Gestational DNA methylation age as a marker for fetal development and birth outcomes: findings from the Boston Birth Cohort.

BACKGROUND: Gestational DNA methylation age (GAmAge) has been developed and validated in European ancestry samples. Its applicability to other ethnicities and associations with fetal stress and newborn phenotypes such as inflammation markers are still to be determined. This study aims to examine the applicability of GAmAge developed from cord blood samples of European decedents to a racially diverse birth cohort, and associations with newborn phenotypes. METHODS: GAmAge based on 176 CpGs (Haftorn GAmAge) was calculated for 940 children from a US predominantly urban, low-income, multiethnic birth cohort. Cord blood DNA methylation was profiled by Illumina EPIC array. Newborn phenotypes included anthropometric measurements and, for a subset of newborns (N = 194), twenty-seven cord blood inflammatory markers (sandwich immunoassays). RESULTS: GAmAge had a stronger correlation with GEAA in boys (r = 0.89, 95% confidence interval (CI) [0.87,0.91]) compared with girls (r = 0.83, 95% CI [0.80,0.86]), and was stronger among extremely preterm to very preterm babies (r = 0.91, 95% CI [0.81,0.96]), compared with moderate (r = 0.48, 95% CI [0.34,0.60]) and term babies (r = 0.58, 95% CI [0.53,0.63]). Among White newborns (N = 51), the correlation between GAmAge vs. GEAA was slightly stronger (r = 0.89, 95% CI [0.82,0.94]) compared with Black/African American newborns (N = 668; r = 0.87, 95% CI [0.85,0.89]) or Hispanic (N = 221; r = 0.79, 95% CI [0.74,0.84]). Adjusting for GEAA and sex, GAmAge was associated with anthropometric measurements, cord blood brain-derived neurotrophic factor (BDNF), and monocyte chemoattractant protein-1 (MCP-1) (p < 0.05 for all). CONCLUSIONS: GAmAge estimation is robust across different populations and racial/ethnic subgroups. GAmAge may be utilized as a proxy for GEAA and for assessing fetus development, indicated by inflammatory state and birth outcomes.

Exposure-associated DNA methylation among people exposed to multiple industrial pollutants.

BACKGROUND: Current research on the epigenetic repercussions of exposure to a combination of pollutants is limited. This study aims to discern DNA methylation probes associated with exposure to multiple pollutants, serving as early effect markers, and single-nucleotide polymorphisms (SNPs) as surrogate indicators for population susceptibility. The investigation involved the analysis of urine exposure biomarkers for 11 heavy metals (vanadium, arsenic, mercury, cadmium, chromium, nickel, lead, manganese, copper, strontium, thallium), polycyclic aromatic hydrocarbon (PAHs) (1-hydroxypyrene), genome-wide DNA methylation sequencing, and SNPs array on all study participants. The data were integrated with metabolomics information and analyzed both at a community level based on proximity to home addresses relative to the complex and at an individual level based on exposure biomarker concentrations. RESULTS: On a community level, 67 exposure-related CpG probes were identified, while 70 CpG probes were associated with urine arsenic concentration, 2 with mercury, and 46 with vanadium on an individual level. These probes were annotated to genes implicated in cancers and chronic kidney disease. Weighted quantile sum regression analysis revealed that vanadium, mercury, and 1-hydroxypyrene contributed the most to cg08238319 hypomethylation. cg08238319 is annotated to the aryl hydrocarbon receptor repressor (AHRR) gene, and AHRR hypomethylation was correlated with an elevated risk of lung cancer. AHRR was further linked to deregulations in phenylalanine metabolism, alanine, aspartate, and glutamate metabolism, along with heightened oxidative stress. Additionally, three SNPs (rs11085020, rs199442, and rs10947050) corresponding to exposure-related CpG probes exhibited significant interaction effects with multiple heavy metals and PAHs exposure, and have been implicated in cancer progression and respiratory diseases. CONCLUSION: Our findings underscore the pivotal role of AHRR methylation in gene-environment interactions and highlight SNPs that could potentially serve as indicators of population susceptibility in regions exposed to multiple heavy metals and PAHs.

Multi-locus imprinting disturbance (MLID): interim joint statement for clinical and molecular diagnosis.

BACKGROUND: Imprinting disorders are rare diseases resulting from altered expression of imprinted genes, which exhibit parent-of-origin-specific expression patterns regulated through differential DNA methylation. A subgroup of patients with imprinting disorders have DNA methylation changes at multiple imprinted loci, a condition referred to as multi-locus imprinting disturbance (MLID). MLID is recognised in most but not all imprinting disorders and is also found in individuals with atypical clinical features; the presence of MLID often alters the management or prognosis of the affected person. Some cases of MLID are caused by trans-acting genetic variants, frequently not in the patients but their mothers, which have counselling implications. There is currently no consensus on the definition of MLID, clinical indications prompting testing, molecular procedures and methods for epigenetic and genetic diagnosis, recommendations for laboratory reporting, considerations for counselling, and implications for prognosis and management. The purpose of this study is thus to cover this unmet need. METHODS: A comprehensive literature search was conducted resulting in identification of more than 100 articles which formed the basis of discussions by two working groups focusing on clinical diagnosis (n = 12 members) and molecular testing (n = 19 members). Following eight months of preparations and regular online discussions, the experts from 11 countries compiled the preliminary documentation and determined the questions to be addressed during a face-to-face meeting which was held with the attendance of the experts together with four representatives of patient advocacy organisations. RESULTS: In light of available evidence and expert consensus, we formulated 16 propositions and 8 recommendations as interim guidance for the clinical and molecular diagnosis of MLID. CONCLUSIONS: MLID is a molecular designation, and for patients with MLID and atypical phenotypes, we propose the alternative term multi-locus imprinting syndrome. Due to the intrinsic variability of MLID, the guidelines underscore the importance of involving experts from various fields to ensure a confident approach to diagnosis, counselling, and care. The authors advocate for global, collaborative efforts in both basic and translational research to tackle numerous crucial questions that currently lack answers, and suggest reconvening within the next 3-5 years to evaluate the research advancements and update this guidance as needed.

A case of an Angelman-syndrome caused by an intragenic duplication of UBE3A uncovered by adaptive nanopore sequencing.

Adaptive nanopore sequencing as a diagnostic method for imprinting disorders and episignature analysis revealed an intragenic duplication of Exon 6 and 7 in UBE3A (NM_000462.5) in a patient with relatively mild Angelman-like syndrome. In an all-in-one nanopore sequencing analysis DNA hypomethylation of the SNURF:TSS-DMR, known contributing deletions on the maternal allele and point mutations in UBE3A could be ruled out as disease drivers. In contrast, breakpoints and orientation of the tandem duplication could clearly be defined. Segregation analysis in the family showed that the duplication derived de novo in the maternal grandfather. Our study shows the benefits of an all-in-one nanopore sequencing approach for the diagnostics of Angelman syndrome and other imprinting disorders.

Genome-wide methylation and gene-expression analyses in thalassemia.

Thalassemia is the most common autosomal genetic disorder in humans. The pathogenesis of thalassemia is principally due to the deletion or mutation of globin genes that then leads to disorders in globin-chain synthesis, and its predominant clinical manifestations include chronic forms of hemolytic anemia. However, research on the epigenetics and underlying pathogenesis of thalassemia is in its nascency and not yet been systematically realized. In this study, we compared the results of RNA-seq and the whole-genome bisulfite sequencing (WGBS) on 22 peripheral blood samples from 14 thalassemic patients and eight healthy individuals revealed a genome-wide methylation landscape of differentially methylated regions (DMRs). And functional-enrichment analysis revealed the enriched biological pathways with respect to the differentially expressed genes (DEGs) and differentially methylated genes (DMGs) to include hematopoietic lineage, glucose metabolism, and ribosome. To further analyze the interaction between the transcriptome and methylome, we implemented a comprehensive analysis of overlaps between DEGs and DMGs, and observed that biological processes significantly enriched the immune-related genes (i.e., our hypermethylated and down-regulated gene group). Hypermethylated and hypomethylated regions of thalassemia-related genes exhibited different distribution patterns. We thus, further identified and validated thalassemia-associated DMGs and DEGs by multi-omics integrative analyses of DNA methylation and transcriptomics data, and provided a comprehensive genomic map of thalassemia that will facilitate the exploration of the epigenetics mechanisms and pathogenesis underlying thalassemia.

DNA methylation impacts soybean early development by modulating hormones and metabolic pathways.

Genomic DNA methylation patterns play a crucial role in the developmental processes of plants and mammals. In this study, we aimed to investigate the significant effects of epigenetic mechanisms on the development of soybean seedlings and metabolic pathways. Our analyses show that 5-azaC-treatment affects radicle development from two Days After Imbibition (DAI), as well as both shoot and root development. We examined the expression levels of key genes related to DNA methylation and demethylation pathways, such as DRM2, which encodes RNA-directed DNA Methylation (RdDM) pathway, SAM synthase, responsible for methyl group donation, and ROS1, a DNA demethylase. In treated seedling roots, we observed an increase in DRM2 expression and a decrease in ROS1 expression. Additionally, 5-azaC treatment altered protein accumulation, indicating epigenetic control over stress response while inhibiting nitrogen assimilation, urea cycle, and glycolysis-related proteins. Furthermore, it influenced the levels of various phytohormones and metabolites crucial for seedling growth, such as ABA, IAA, ethylene, polyamines (PUT and Cad), and free amino acids, suggesting that epigenetic changes may shape soybean responses to pathogens, abiotic stress, and nutrient absorption. Our results assist in understanding how hypomethylation shapes soybean responses to pathogens, abiotic stress, and nutrient absorption crucial for seedling growth, suggesting that the plant's assimilation of carbon and nitrogen, along with hormone pathways, may be influenced by epigenetic changes.

Triage performance of PAX1m/JAM3m in opportunistic cervical cancer screening of non‒16/18 human papillomavirus-positive women: a multicenter prospective study in China.

OBJECTIVES: In this study, we aimed to validate the performance of the PAX1 and JAM3 methylation (PAX1m/JAM3m) test as a triage tool for detecting cervical intraepithelial neoplasia grade 3 or worse (CIN3 +) in non-16/18 high-risk human papillomavirus-positive patients (non-16/18 hrHPV +). METHODS: The triage performance of liquid-based cytology (LBC) and the PAX1m/JAM3m test for detecting CIN3 + were compared. RESULTS: In total, 1851 participants had cervical histological outcomes and were included in the analysis. The sensitivity/specificity of the LBC test results with atypical squamous cells of undetermined significance or worse (LBC ≥ ASCUS) and the PAX1m/JAM3m test were 90.1%/26.7% and 84.8%/88.5%, respectively. PAX1m/JAM3m( +) had the highest diagnostic AUC (0.866, 95% confidence interval (CI) 0.837-0.896) in the whole cohort. All cancers (n = 20) were detected by PAX1m/JAM3m(+). Compared with LBC ≥ ASCUS, PAX1m/JAM3m(+) reduced the number of patients who needed referral for colposcopy by 57.21% (74.66% vs. 17.45%). The odds ratios for detecting CIN3 + by LBC ≥ ASCUS and PAX1m/JAM3m(+) were 3.3 (95% CI 2.0-5.9) and 42.6 (27.1-69.6), respectively (p < 0.001). The combination of LBC ≥ ASCUS or PAX1m/JAM3m(+) slightly increased the diagnostic sensitivity (98.0%, 95% CI: 95.8-100%) and referral rate (77.09%) but reduced the diagnostic specificity (24.8%, 22.7-26.8%). CONCLUSIONS: In non-16/18 hrHPV(+) women, PAX1m/JAM3m was superior to cytology for detecting CIN3 + . Compared with LBC ≥ ASCUS, PAX1m/JAM3m(+) reduced the number of significant referrals to colposcopy without compromising diagnostic sensitivity.

Maternal smoking DNA methylation risk score associated with health outcomes in offspring of European and South Asian ancestry.

Background: Maternal smoking has been linked to adverse health outcomes in newborns but the extent to which it impacts newborn health has not been quantified through an aggregated cord blood DNA methylation (DNAm) score. Here, we examine the feasibility of using cord blood DNAm scores leveraging large external studies as discovery samples to capture the epigenetic signature of maternal smoking and its influence on newborns in White European and South Asian populations. Methods: We first examined the association between individual CpGs and cigarette smoking during pregnancy, and smoking exposure in two White European birth cohorts (n=744). Leveraging established CpGs for maternal smoking, we constructed a cord blood epigenetic score of maternal smoking that was validated in one of the European-origin cohorts (n=347). This score was then tested for association with smoking status, secondary smoking exposure during pregnancy, and health outcomes in offspring measured after birth in an independent White European (n=397) and a South Asian birth cohort (n=504). Results: Several previously reported genes for maternal smoking were supported, with the strongest and most consistent association signal from the GFI1 gene (6 CpGs with p<5 × 10-5). The epigenetic maternal smoking score was strongly associated with smoking status during pregnancy (OR = 1.09 [1.07, 1.10], p=5.5 × 10-33) and more hours of self-reported smoking exposure per week (1.93 [1.27, 2.58], p=7.8 × 10-9) in White Europeans. However, it was not associated with self-reported exposure (p>0.05) among South Asians, likely due to a lack of smoking in this group. The same score was consistently associated with a smaller birth size (-0.37±0.12 cm, p=0.0023) in the South Asian cohort and a lower birth weight (-0.043±0.013 kg, p=0.0011) in the combined cohorts. Conclusions: This cord blood epigenetic score can help identify babies exposed to maternal smoking and assess its long-term impact on growth. Notably, these results indicate a consistent association between the DNAm signature of maternal smoking and a small body size and low birth weight in newborns, in both White European mothers who exhibited some amount of smoking and in South Asian mothers who themselves were not active smokers. Funding: This study was funded by the Canadian Institutes of Health Research Metabolomics Team Grant: MWG-146332.

COPS: A novel platform for multi-omic disease subtype discovery via robust multi-objective evaluation of clustering algorithms.

Recent research on multi-view clustering algorithms for complex disease subtyping often overlooks aspects like clustering stability and critical assessment of prognostic relevance. Furthermore, current frameworks do not allow for a comparison between data-driven and pathway-driven clustering, highlighting a significant gap in the methodology. We present the COPS R-package, tailored for robust evaluation of single and multi-omics clustering results. COPS features advanced methods, including similarity networks, kernel-based approaches, dimensionality reduction, and pathway knowledge integration. Some of these methods are not accessible through R, and some correspond to new approaches proposed with COPS. Our framework was rigorously applied to multi-omics data across seven cancer types, including breast, prostate, and lung, utilizing mRNA, CNV, miRNA, and DNA methylation data. Unlike previous studies, our approach contrasts data- and knowledge-driven multi-view clustering methods and incorporates cross-fold validation for robustness. Clustering outcomes were assessed using the ARI score, survival analysis via Cox regression models including relevant covariates, and the stability of the results. While survival analysis and gold-standard agreement are standard metrics, they vary considerably across methods and datasets. Therefore, it is essential to assess multi-view clustering methods using multiple criteria, from cluster stability to prognostic relevance, and to provide ways of comparing these metrics simultaneously to select the optimal approach for disease subtype discovery in novel datasets. Emphasizing multi-objective evaluation, we applied the Pareto efficiency concept to gauge the equilibrium of evaluation metrics in each cancer case-study. Affinity Network Fusion, Integrative Non-negative Matrix Factorization, and Multiple Kernel K-Means with linear or Pathway Induced Kernels were the most stable and effective in discerning groups with significantly different survival outcomes in several case studies.

Differential methylation patterns in paternally imprinted gene promoter regions in sperm from hepatitis B virus infected individuals.

BACKGROUND: Hepatitis B virus (HBV) infection poses a substantial threat to human health, impacting not only infected individuals but also potentially exerting adverse effects on the health of their offspring. The underlying mechanisms driving this phenomenon remain elusive. This study aims to shed light on this issue by examining alterations in paternally imprinted genes within sperm. METHODS: A cohort of 35 individuals with normal semen analysis, comprising 17 hepatitis B surface antigen (HBsAg)-positive and 18 negative individuals, was recruited. Based on the previous research and the Online Mendelian Inheritance in Man database (OMIM, https://www.omim.org/ ), targeted promoter methylation sequencing was employed to investigate 28 paternally imprinted genes associated with various diseases. RESULTS: Bioinformatic analyses revealed 42 differentially methylated sites across 29 CpG islands within 19 genes and four differentially methylated CpG islands within four genes. At the gene level, an increase in methylation of DNMT1 and a decrease in methylation of CUL7, PRKAG2, and TP53 were observed. DNA methylation haplotype analysis identified 51 differentially methylated haplotypes within 36 CpG islands across 22 genes. CONCLUSIONS: This is the first study to explore the effects of HBV infection on sperm DNA methylation and the potential underlying mechanisms of intergenerational influence of paternal HBV infection.