BACKGROUND: Obesity is a global public health concern linked to chronic diseases such as cardiovascular disease and type 2 diabetes (T2D). Emerging evidence suggests that epigenetic modifications, particularly DNA methylation, may contribute to obesity. However, the molecular mechanism underlying the longitudinal change of BMI has not been well-explored, especially in East Asian populations.
METHODS: This study performed a longitudinal epigenome-wide association analysis of DNA methylation to uncover novel loci associated with BMI change in 533 individuals across two Chinese cohorts with repeated DNA methylation and BMI measurements over four years.
RESULTS: We identified three novel CpG sites (cg14671384, cg25540824, and cg10848724) significantly associated with BMI change. Two of the identified CpG sites were located in regions previously associated with body shape and basal metabolic rate. Annotation of the top 20 BMI change-associated CpGs revealed strong connections to obesity and T2D. Notably, these CpGs exhibited active regulatory roles and located in genes with high expression in the liver and digestive tract, suggesting a potential regulatory pathway from genome to phenotypes of energy metabolism and absorption via DNA methylation. Cross-sectional and longitudinal EWAS comparisons indicated different mechanisms between CpGs related to BMI and BMI change.
CONCLUSIONS: This study enhances our understanding of the epigenetic dynamics underlying BMI change and emphasizes the value of longitudinal analyses in deciphering the complex interplay between epigenetics and obesity.

Background: Patients with late-stage mild cognitive impairment (LMCI) have a higher risk of progression to Alzheimer\'s disease (AD) than those with early-stage mild cognitive impairment (EMCI). However, previous studies have often pooled EMCI and LMCI patients into a single MCI group, with limited independent investigation into the pathogenesis of LMCI. Methods: In this study, we employed whole-genome methylation association analysis to determine the differences in peripheral blood methylation profiles between 663 cognitive aging (CN) and 554 LMCI patients. Results: Our results revealed 2,333 differentially methylated probes (DMPs) and 85 differentially methylated regions (DMRs) specific to LMCI. The top hit methylation sites or regions were associated with genes such as SNED1, histone deacetylases coding gene HDACs, and HOX and ZNF gene family. The DNA methylations upregulated the expression of HDAC4, HDAC8, and HOX family genes HOXC5 and HOXC9, but they downregulated the expression of SNED1, ADCYAP1, and ZNF family genes ZNF415 and ZNF502. Gene Ontology (GO) and KEGG analysis showed that the genes associated with these methylation sites were predominantly related to the processes of addiction disorders, neurotransmission, and neurogenesis. Out of the 554 LMCI patients included in this study, 358 subjects (65%) had progressed to AD. Further association analysis between the LMCI subjects with a stable course (sLMCI) and those who progressed to AD (pLMCI) indicated that the methylation signal intensities of HDAC6, ZNF502, HOXC5, HOXC6, and HOXD8 were associated with increased susceptibility to AD. Protective effects against progression to AD were noticed when the methylation of SNED1 and ZNF727 appeared in LMCI patients. Conclusion: Our findings highlight a substantial number of LMCI-specific methylated biomarkers that differ from those identified in previous MCI case-control studies. These biomarkers have the potential to contribute to a better understanding of the pathogenesis of LMCI.

To control genetic background and early life milieu in genome-wide DNA methylation analysis for blood lipids, we recruited Chinese discordant monozygotic twins to explore the relationships between DNA methylations and total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG). 132 monozygotic (MZ) twins were included with discordant lipid levels and completed data. A linear mixed model was conducted in Epigenome-wide association study (EWAS). Generalized estimating equation model was for gene expression analysis. We conducted Weighted correlation network analysis (WGCNA) to build co-methylated interconnected network. Additional Qingdao citizens were recruited for validation. Inference about Causation through Examination of Familial Confounding (ICE FALCON) was used to infer the possible direction of these relationships. A total of 476 top CpGs reached suggestively significant level (P < 10-4), of which, 192 CpGs were significantly associated with TG (FDR < 0.05). They were used to build interconnected network and highlight crucial genes from WGCNA. Finally, four CpGs in GATA4 were validated as risk factors for TC; six CpGs at ITFG2-AS1 were negatively associated with TG; two CpGs in PLXND1 played protective roles in HDL-C. ICE FALCON indicated abnormal TC was regarded as the consequence of DNA methylation in CpGs at GATA4, rather than vice versa. Four CpGs in ITFG2-AS1 were both causes and consequences of modified TG levels. Our results indicated that DNA methylation levels of 12 CpGs in GATA4, ITFG2-AS1, and PLXND1 were relevant to TC, TG, and HDL-C, respectively, which might provide new epigenetic insights into potential clinical treatment of dyslipidemia.

Epidemiological and epigenetic studies have acknowledged ambient ozone exposure associated with inflammatory and cardiovascular disease. However, the molecular mechanisms still remained unclear, and epigenome-wide analysis in cohort were lacking, especially in Chinese. We included blood-derived DNA methylation for 3365 Chinese participants from the NSPT cohort and estimated individual ozone exposure level of short-, intermediate- and long-term, based on a validated prediction model. We performed epigenome-wide association studies which identified 59 CpGs and 30 DMRs at a strict genome-wide significance (P < 5 ×10-8). We also conducted comparison on the DNA methylation alteration corresponding to different time windows, and observed an enhanced differentiated methylation trend for intermediate- and long-term exposure, while the short-term exposure associated methylation changes did not retain. The targeted genes of methylation alteration were involved in mechanism related to aging, inflammation disease, metabolic syndrome, neurodevelopmental disorders, and oncogenesis. Underlying pathways were enriched in biological activities including telomere maintenance process, DNA damage response and megakaryocyte differentiation. In conclusion, our study is the first EWAS on ozone exposure conducted in large-scale Han Chinese cohort and identified associated DNA methylation change on CpGs and regions, as well as related gene functions and pathways.

DNA methylation has previously been associated with ischemic stroke, but the specific genes and their functional roles in ischemic stroke remain to be determined. Here we aimed to identify differentially methylated genes that play a functional role in ischemic stroke in a Chinese population.
Genome-wide DNA methylation assessed with the Illumina Methylation EPIC Array in a discovery sample including 80 Chinese adults (40 cases vs. 40 controls) found that patients with ischemic stroke were characterized by increased DNA methylation at six CpG loci (individually located at TRIM6, FLRT2, SOX1, SOX17, AGBL4, and FAM84A, respectively) and decreased DNA methylation at one additional locus (located at TLN2). Targeted bisulfite sequencing confirmed six of these differentially methylated probes in an independent Chinese population (853 cases vs. 918 controls), and one probe (located at TRIM6) was further verified in an external European cohort (207 cases vs. 83 controls). Experimental manipulation of DNA methylation in engineered human umbilical vein endothelial cells indicated that the identified differentially methylated probes located at TRIM6, TLN2, and FLRT2 genes may play a role in endothelial cell adhesion and atherosclerosis.
Altered DNA methylation of the TRIM6, TLN2, and FLRT2 genes may play a functional role in ischemic stroke in Chinese populations.

DNA methylation data generated from bulk tissue represents a mixture of many different cell types. Variation in the cell-type composition of tissues is thus a major confounder when inferring differential DNA methylation. Due to the high cost of single-cell methylome sequencing, computational methods that can dissect the cell-type heterogeneity of bulk DNA methylomes offer an efficient and cost-effective solution, especially in the context of large-scale EWAS. In this chapter, we present a step-by-step tutorial of Epigenetic cell-type deconvolution using Single-Cell Omic References (EpiSCORE), a reference-based method that leverages the high-resolution nature of single-cell RNA-Seq datasets to facilitate microdissection of bulk-tissue DNA methylomes.

DNA methylation (DNAm) plays an important role in lipid metabolism, however, no epigenome-wide association study (EWAS) of lipid levels has been conducted among childhood cancer survivors. Here, we performed EWAS analysis with longitudinally collected blood lipid data from survivors in the St. Jude lifetime cohort study.
Among 2052 childhood cancer survivors of European ancestry (EA) and 370 survivors of African ancestry (AA), four types of blood lipids, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglycerides (TG), were measured during follow-up beyond 5-years from childhood cancer diagnosis. For the exposure EWAS (i.e., lipids measured before blood draw for DNAm), the DNAm level was an outcome variable and each of the blood lipid level was an exposure variable; vice versa for the outcome EWAS (i.e., lipids measured after blood draw for DNAm).
Among EA survivors, we identified 43 lipid-associated CpGs in the HDL (n = 7), TC (n = 3), and TG (n = 33) exposure EWAS, and 106 lipid-associated CpGs in the HDL (n = 5), LDL (n = 3), TC (n = 4), and TG (n = 94) outcome EWAS. Among AA survivors, we identified 15 lipid-associated CpGs in TG exposure (n = 6), HDL (n = 1), LDL (n = 1), TG (n = 5) and TC (n = 2) outcome EWAS with epigenome-wide significance (P < 9 × 10-8). There were no overlapping lipids-associated CpGs between exposure and outcome EWAS among EA and AA survivors, suggesting that the DNAm changes of different CpGs could be the cause or consequence of blood lipid levels. In the meta-EWAS, 12 additional CpGs reached epigenome-wide significance. Notably, 32 out of 74 lipid-associated CpGs showed substantial heterogeneity (Phet < 0.1 or I2 > 70%) between EA and AA survivors, highlighting differences in DNAm markers of blood lipids between populations with diverse genetic ancestry. Ten lipid-associated CpGs were cis-expression quantitative trait methylation with their DNAm levels associated with the expression of corresponding genes, out of which seven were negatively associated.
We identified distinct signatures of DNAm for blood lipids as exposures or outcomes and between EA and AA survivors, revealing additional genes involved in lipid metabolism and potential novel targets for controlling blood lipids in childhood cancer survivors.

In this study, we aimed to identified CpG sites at which DNA methylation levels are associated with meat quality traits in 140 Yorkshire pigs, including pH at 45 min (pH45min), pH at 24 h (pH24h), drip loss (DL), meat redness value (a*), yellowness (b*) and lightness (L*). Genome-wide methylation levels were measured in muscular tissue using reduced representation bisulfite sequencing (RRBS). Associations between DNA methylation levels and meat quality traits were examined using linear mixed-effect models that were adjusted for gender, year, month and body weight. A Bonferroni-corrected p-value lower than 7.79 × 10 - 8 was considered statistically significant threshold. Eight CpG sites were associated with DL, including CpG sites annotated to RBM4 gene (cpg301054, cpg301055, cpg301058, cpg301059, cpg301066, cpg301072 and cpg301073) and NCAM1 gene (cpg1802985). Two CpG sites were associated with b*, including RNFT1 and MED13 (cpg2272837) and TRIM37 gene (cpg2270611). Five CpG sites were associated with L*, including GSDMA and LRRC3C gene (cpg2252750) and ENSSSCG00000043539 and IRX1 gene (cpg2820178, cpg2820179, cpg2820181 and cpg2820182). No significant associations were observed with pH45min, pH24h or a*. We reported associations of meat quality traits with DNA methylation and identified some candidate genes associated with these traits, such as NCAM1, MED13 and TRIM37 gene. These results provide new insight into the epigenetic molecular mechanisms of meat quality traits in pigs.

Little information is known about DNA methylation variation in shaping environment-specific drought resistance and resilience for tree adaptation. In this study, we leveraged RNA sequencing and whole-genome bisulfite sequencing data to dissect the distinction of epigenetic regulation under drought stress and rewater condition of Populus tomentosa accessions from three geographical regions. We demonstrated low resistance and high resilience for accessions from South. Non-CG methylation levels in promoter regions of Southern accessions were lower than accessions from higher latitudes and negatively regulated gene expression. CHH context methylation was more sensitive to drought stress, and the geographical-specific differentially methylated regions were scarcely changed by environmental fluctuation. We identified 60 conserved hub genes within the co-expression networks that correlate with photosynthetic and stomatal morphological traits. Epigenome-wide association studies and genome-wide association studies of these 60 hub genes revealed the interdependency between genetic and epigenetic variation in GATA9 and LECRK-VIII.2, which was associated with stomatal morphology and chlorophyll content. The natural epigenetic variation in GATA9 was also faithfully transmitted to progenies in two family-based F1 populations. This study indicates a functional relationship of DNA methylation diversity with drought resistance and resilience which offers new insights into plants\' local adaptation to a stressful environment.

The associations between blood lipids and DNA methylation have been investigated in epigenome-wide association studies mainly among European ancestry populations. Several studies have explored the direction of the association using cross-sectional data, while evidence of longitudinal data is still lacking.
We tested the associations between peripheral blood leukocytes DNA methylation and four lipid measures from Illumina 450 K or EPIC arrays in 1084 participants from the Chinese National Twin Registry and replicated the result in 988 participants from the China Kadoorie Biobank. A total of 23 associations of 19 CpG sites were identified, with 4 CpG sites located in or adjacent to 3 genes (TMEM49, SNX5/SNORD17 and CCDC7) being novel. Among the validated associations, we conducted a cross-lagged analysis to explore the temporal sequence and found temporal associations of methylation levels of 2 CpG sites with triglyceride and 2 CpG sites with high-density lipoprotein-cholesterol (HDL-C) in all twins. In addition, methylation levels of cg11024682 located in SREBF1 at baseline were temporally associated with triglyceride at follow-up in only monozygotic twins. We then performed a mediation analysis with the longitudinal data and the result showed that the association between body mass index and HDL-C was partially mediated by the methylation level of cg06500161 (ABCG1), with a mediation proportion of 10.1%.
Our study indicated that the DNA methylation levels of ABCG1, AKAP1 and SREBF1 may be involved in lipid metabolism and provided evidence for elucidating the regulatory mechanism of lipid homeostasis.