Epigenome-wide Association Studies Research Articles

Analysis of epigenetic mediators for the effect of Polyunsaturated fatty acids Omega6/3 ratio on cognitive function and progression to dementia

AbstractBackgroundPrevious research reported the association between circulating Omega‐3 polyunsaturated fatty acids (PUFA) in blood and the risk of dementia and Alzheimer’s disease (AD). However, a large intervention study supplementing Omega‐3 reported that this supplementation has only a marginal effect on improvement of cognitive performance after the supplementation. Since PUFA metabolism shared the processing enzymes, we explored whether the relationship between two main families of PUFA, i.e., Omega‐3 and Omega‐6, might associate with cognitive performance. Hence, we observed that the ratio Omega‐6/Omega‐3 in blood is associated with cognitive decline and dementia. To follow up on this finding, we sought to explore whether modulation of immune response and inflammation may underlie these effects.MethodThree independent cohort were used for this study: i) the observational memory‐clinic‐based cohort DELCODE (n = 1036); ii) the primary care‐derived cohort AgeCoDe (n = 1252); iii) the Multidomain Alzheimer Preventive Trial (MAPT study) cohort (n = 1679). Omega‐3 and PUFA levels were quantified specifically in plasma from each cohort. COX regressions and linear mixed models were applied to evaluate PUFA ratio effect on cognitive performance and progression to dementia. Genomic and epigenomic modulators for the observed effects were further explored using the previously published tool (MetaMeth). Results were confirmed using our pipeline for epigenome‐wide association study (EWAS).ResultOmega‐6/Omega‐3 ratio is associated with cognitive performance and risk of progressing to AD in DELCODE and AgeCoDe. In MAPT, the improvement of this ratio is associated with improvement on cognitive performance. Moreover, levels of Omega‐3 in blood modulate methylation of genes coding for key enzymes of PUFA metabolism. Our data showed that the biological effect for some genetic susceptibility signals for AD is mediated by changes in DNA methylation, including immune‐related genes like MS4A and TREM2.ConclusionOur data suggests that balance between PUFA is relevant on cognitive decline and dementia. In addition, the genetic effect of risk variants located at the AD risk loci containing the MS4A gene cluster and TREM2 are mediated by changes in methylation. To provide a connection between PUFA and neuroinflammation in the context of AD, we are currently exploring the effect of PUFA levels on DNA methylation at MS4A and TREM2 loci.

GENOME-WIDE DNA METHYLATION ANALYSIS IDENTIFIES SERUM HOMOCYSTEINE-ASSOCIATED CPG SITES IN HYPERTENSIVES

Objective: Homocysteine (Hcy) is associated with DNA methylation in cardiovascular disease (CVD). Moreover, the relationship between Hyperhomocysteine (HHcy) and CVD risk is stronger in individuals with hypertension. However, no research has focused on the influence of Hcy on DNA methylation status in patients with hypertension. We performed an epigenome-wide association study to identify DNA methylation loci associated with Hcy among 218 hypertensive patients using the Illumina Infinium Methylation EPIC BeadChip (850k chip) and further explored their effects on arteriosclerosis. Design and method: Epigenome-wide methylation profiles were assessed using the Illumina Infinium Methylation EPIC BeadChip (850k chip) in peripheral leukocyte DNA from 218 patients with hypertension. Differentially methylated positions (DMPs) associated with serum homocysteine levels were identified using a mixed linear regression with adjustment for potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted to explore the possible functions of the identified DMPs. The association of these differentially methylated loci with hypertension- and Hcy- related arteriosclerosis, Cf-PWV (Carotid-femoral pulse wave velocity) was further analyzed. Results: Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (FDR-corrected p <0.05). Six additional CpG sites met the threshold for suggestive significance (p<1 × 10-6), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to the THUMPD3 gene. Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Conclusions: Our study provides preliminary evidence for an association between DNA methylation and serum Hcy levels in patients with hypertension. This study may give an insight into the role of DNA methylation in cardiovascular injury underlying HHcy in patients with hypertension.

Genetic and epigenetic background of diabetic kidney disease.

Diabetic kidney disease (DKD) is a severe diabetic complication that affects up to half of the individuals with diabetes. Elevated blood glucose levels are a key underlying cause of DKD, but DKD is a complex multifactorial disease, which takes years to develop. Family studies have shown that inherited factors also contribute to the risk of the disease. During the last decade, genome-wide association studies (GWASs) have emerged as a powerful tool to identify genetic risk factors for DKD. In recent years, the GWASs have acquired larger number of participants, leading to increased statistical power to detect more genetic risk factors. In addition, whole-exome and whole-genome sequencing studies are emerging, aiming to identify rare genetic risk factors for DKD, as well as epigenome-wide association studies, investigating DNA methylation in relation to DKD. This article aims to review the identified genetic and epigenetic risk factors for DKD.

Dietary and supplemental intake of vitamins C and E is associated with altered DNA methylation in an epigenome-wide association study meta-analysis

ABSTRACT Background: Dietary intake of antioxidants such as vitamins C and E protect against oxidative stress, and may also be associated with altered DNA methylation patterns. Methods: We meta-analysed epigenome-wide association study (EWAS) results from 11,866 participants across eight population-based cohorts to evaluate the association between self-reported dietary and supplemental intake of vitamins C and E with DNA methylation. EWAS were adjusted for age, sex, BMI, caloric intake, blood cell type proportion, smoking status, alcohol consumption, and technical covariates. Significant results of the meta-analysis were subsequently evaluated in gene set enrichment analysis (GSEA) and expression quantitative trait methylation (eQTM) analysis. Results: In meta-analysis, methylation at 4,656 CpG sites was significantly associated with vitamin C intake at FDR ≤ 0.05. The most significant CpG sites associated with vitamin C (at FDR ≤ 0.01) were enriched for pathways associated with systems development and cell signalling in GSEA, and were associated with downstream expression of genes enriched in the immune response in eQTM analysis. Furthermore, methylation at 160 CpG sites was significantly associated with vitamin E intake at FDR ≤ 0.05, but GSEA and eQTM analysis of the top most significant CpG sites associated with vitamin E did not identify significant enrichment of any biological pathways investigated. Conclusions: We identified significant associations of many CpG sites with vitamin C and E intake, and our results suggest that vitamin C intake may be associated with systems development and the immune response.

Epigenomic profiling of isolated blood cell types reveals highly specific B cell smoking signatures and links to disease risk

BackgroundTobacco smoking alters the DNA methylation profiles of immune cells which may underpin some of the pathogenesis of smoking-associated diseases. To link smoking-driven epigenetic effects in specific immune cell types with disease risk, we isolated six leukocyte subtypes, CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells, from whole blood of 67 healthy adult smokers and 74 nonsmokers for epigenome-wide association study (EWAS) using Illumina 450k and EPIC methylation arrays.ResultsNumbers of smoking-associated differentially methylated sites (smCpGs) at genome-wide significance (p < 1.2 × 10−7) varied widely across cell types, from 5 smCpGs in CD8+ T cells to 111 smCpGs in CD19+ B cells. We found unique smoking effects in each cell type, some of which were not apparent in whole blood. Methylation-based deconvolution to estimate B cell subtypes revealed that smokers had 7.2% (p = 0.033) less naïve B cells. Adjusting for naïve and memory B cell proportions in EWAS and RNA-seq allowed the identification of genes enriched for B cell activation-related cytokine signaling pathways, Th1/Th2 responses, and hematopoietic cancers. Integrating with large-scale public datasets, 62 smCpGs were among CpGs associated with health-relevant EWASs. Furthermore, 74 smCpGs had reproducible methylation quantitative trait loci single nucleotide polymorphisms (SNPs) that were in complete linkage disequilibrium with genome-wide association study SNPs, associating with lung function, disease risks, and other traits.ConclusionsWe observed blood cell-type-specific smCpGs, a naïve-to-memory shift among B cells, and by integrating genome-wide datasets, we identified their potential links to disease risks and health traits.

Large epigenome-wide association study identifies multiple novel differentially methylated CpG sites associated with suicidal thoughts and behaviors in veterans.

The U.S. suicide mortality rate has steadily increased during the past two decades, particularly among military veterans; however, the epigenetic basis of suicidal thoughts and behaviors (STB) remains largely unknown. To address this issue, we conducted an epigenome-wide association study of DNA methylation (DNAm) of peripheral blood samples obtained from 2,712 U.S. military veterans. Three DNAm probes were significantly associated with suicide attempts, surpassing the multiple testing threshold (FDR q-value <0.05), including cg13301722 on chromosome 7, which lies between the genes SLC4A2 and CDK5; cg04724646 in PDE3A; and cg04999352 in RARRES3. cg13301722 was also found to be differentially methylated in the cerebral cortex of suicide decedents in a publicly-available dataset (p = 0.03). Trait enrichment analysis revealed that the CpG sites most strongly associated with STB in the present sample were also associated with smoking, alcohol consumption, maternal smoking, and maternal alcohol consumption, whereas pathway enrichment analysis revealed significant associations with circadian rhythm, adherens junction, insulin secretion, and RAP-1 signaling, each of which was recently associated with suicide attempts in a large, independent genome-wide association study of suicide attempts of veterans. Taken together, the present findings suggest that SLC4A2, CDK5, PDE3A, and RARRES3 may play a role in STB. CDK5, a member of the cyclin-dependent kinase family that is highly expressed in the brain and essential for learning and memory, appears to be a particularly promising candidate worthy of future study; however, additional work is still needed to replicate these finding in independent samples.

Epigenetic Association Analyses and Risk Prediction of RLS.

As opposed to other neurobehavioral disorders, epigenetic analyses and biomarkers are largely missing in the case of idiopathic restless legs syndrome (RLS). Our aims were to develop a biomarker for RLS based on DNA methylation in blood and to examine DNA methylation in brain tissues for dissecting RLS pathophysiology. Methylation of blood DNA from three independent cohorts (n = 2283) and post-mortem brain DNA from two cohorts (n = 61) was assessed by Infinium EPIC 850 K BeadChip. Epigenome-wide association study (EWAS) results of individual cohorts were combined by random-effect meta-analysis. A three-stage selection procedure (discovery, n = 884; testing, n = 520; validation, n = 879) established an epigenetic risk score including 30 CpG sites. Epigenetic age was assessed by Horvath's multi-tissue clock and Shireby's cortical clock. EWAS meta-analysis revealed 149 CpG sites linked to 136 genes (P < 0.05 after Bonferroni correction) in blood and 23 CpG linked to 18 genes in brain (false discovery rate [FDR] < 5%). Gene-set analyses of blood EWAS results suggested enrichments in brain tissue types and in subunits of the kainate-selective glutamate receptor complex. Individual candidate genes of the brain EWAS could be assigned to neurodevelopmental or metabolic traits. The blood epigenetic risk score achieved an area under the curve (AUC) of 0.70 (0.67-0.73) in the validation set, comparable to analogous scores in other neurobehavioral disorders. A significant difference in biological age in blood or brain of RLS patients was not detectable. DNA methylation supports the notion of altered neurodevelopment in RLS. Epigenetic risk scores are reliably associated with RLS but require even higher accuracy to be useful as biomarkers. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Impact of tobacco, alcohol, and marijuana on genome-wide DNA methylation and its relationship with hypertension

ABSTRACT Tobacco, alcohol, and marijuana consumption is an important public health problem because of their high use worldwide and their association with the risk of mortality and many health conditions, such as hypertension, which is the commonest risk factor for death throughout the world. A likely pathway of action of substance consumption leading to persistent hypertension is DNA methylation. Here, we evaluated the effects of tobacco, alcohol, and marijuana on DNA methylation in the same cohort (N = 3,424). Three epigenome-wide association studies (EWAS) were assessed in whole blood using the InfiniumHumanMethylationEPIC BeadChip. We also evaluated the mediation of the top CpG sites in the association between substance consumption and hypertension. Our analyses showed 2,569 CpG sites differentially methylated by alcohol drinking and 528 by tobacco smoking. We did not find significant associations with marijuana consumption after correcting for multiple comparisons. We found 61 genes overlapping between alcohol and tobacco that were enriched in biological processes involved in the nervous and cardiovascular systems. In the mediation analysis, we found 66 CpG sites that significantly mediated the effect of alcohol consumption on hypertension. The top alcohol-related CpG site (cg06690548, P-value = 5.9·10−83) mapped to SLC7A11 strongly mediated 70.5% of the effect of alcohol consumption on hypertension (P-value = 0.006). Our findings suggest that DNA methylation should be considered for new targets in hypertension prevention and management, particularly concerning alcohol consumption. Our data also encourage further research into the use of methylation in blood to study the neurological and cardiovascular effects of substance consumption.

Profiling the immune epigenome across global cattle breeds

BackgroundUnderstanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages.ResultsWe find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states.ConclusionsOur study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds.

Epigenome-wide association study of diabetic chronic kidney disease progression in the Korean population: the KNOW-CKD study

Since the etiology of diabetic chronic kidney disease (CKD) is multifactorial, studies on DNA methylation for kidney function deterioration have rarely been performed despite the need for an epigenetic approach. Therefore, this study aimed to identify epigenetic markers associated with CKD progression based on the decline in the estimated glomerular filtration rate in diabetic CKD in Korea. An epigenome-wide association study was performed using whole blood samples from 180 CKD recruited from the KNOW-CKD cohort. Pyrosequencing was also performed on 133 CKD participants as an external replication analysis. Functional analyses, including the analysis of disease-gene networks, reactome pathways, and protein–protein interaction networks, were conducted to identify the biological mechanisms of CpG sites. A phenome-wide association study was performed to determine the associations between CpG sites and other phenotypes. Two epigenetic markers, cg10297223 on AGTR1 and cg02990553 on KRT28 indicated a potential association with diabetic CKD progression. Based on the functional analyses, other phenotypes (blood pressure and cardiac arrhythmia for AGTR1) and biological pathways (keratinization and cornified envelope for KRT28) related to CKD were also identified. This study suggests a potential association between the cg10297223 and cg02990553 and the progression of diabetic CKD in Koreans. Nevertheless, further validation is needed through additional studies.

Association of DNA methylation signatures with premature ageing and cardiovascular death in patients with end-stage kidney disease: a pilot epigenome-wide association study

ABSTRACT Patients with end-stage kidney disease (ESKD) display features of premature aging. There is strong evidence that changes in DNA methylation (DNAm) contribute to age-related pathologies; however, little is known about their association with premature aging and cardiovascular mortality in patients with ESKD. We assayed genome-wide DNAm in a pilot case-control study of 60 hemodialysis patients with (n=30, cases) and without (n=30, controls) a fatal cardiovascular event. DNAm was profiled on the Illumina EPIC BeadChip. Four established DNAm clocks (i.e., Horvath-, Hannum-, Pheno-, and GrimAge) were used to estimate epigenetic age (DNAmAge). Epigenetic age acceleration (EAA) was derived as the residuals of regressing DNAmAge on chronological age (chroAge), and its association with cardiovascular death was examined using multivariable conditional logistic regression. An epigenome-wide association study (EWAS) was performed to identify differentially methylated CpGs associated with cardiovascular death. All clocks performed well at predicting chroAge (correlation between DNAmAges and chroAge of r=0.76-0.89), with GrimAge showing the largest deviation from chroAge (a mean of +21.3 years). There was no significant association of EAAs with cardiovascular death. In the EWAS, a CpG (cg22305782) in the FBXL19 gene had the strongest association with cardiovascular death with significantly lower DNAm in cases vs. controls (P FDR=2.0x10−6). FBXL19 is involved in cell apoptosis, inflammation, and adipogenesis. Overall, we observed more accelerated aging in patients with ESKD, although there was no significant association of EAAs with cardiovascular death. EWAS suggests a potential novel DNAm biomarker for premature cardiovascular mortality in ESKD.

Investigating causality in the association between DNA methylation and type 2 diabetes using bidirectional two-sample Mendelian randomisation

Aims/hypothesisSeveral studies have identified associations between type 2 diabetes and DNA methylation (DNAm). However, the causal role of these associations remains unclear. This study aimed to provide evidence for a causal relationship between DNAm and type 2 diabetes.MethodsWe used bidirectional two-sample Mendelian randomisation (2SMR) to evaluate causality at 58 CpG sites previously detected in a meta-analysis of epigenome-wide association studies (meta-EWAS) of prevalent type 2 diabetes in European populations. We retrieved genetic proxies for type 2 diabetes and DNAm from the largest genome-wide association study (GWAS) available. We also used data from the Avon Longitudinal Study of Parents and Children (ALSPAC, UK) when associations of interest were not available in the larger datasets. We identified 62 independent SNPs as proxies for type 2 diabetes, and 39 methylation quantitative trait loci as proxies for 30 of the 58 type 2 diabetes-related CpGs. We applied the Bonferroni correction for multiple testing and inferred causality based on p<0.001 for the type 2 diabetes to DNAm direction and p<0.002 for the opposing DNAm to type 2 diabetes direction in the 2SMR analysis.ResultsWe found strong evidence of a causal effect of DNAm at cg25536676 (DHCR24) on type 2 diabetes. An increase in transformed residuals of DNAm at this site was associated with a 43% (OR 1.43, 95% CI 1.15, 1.78, p=0.001) higher risk of type 2 diabetes. We inferred a likely causal direction for the remaining CpG sites assessed. In silico analyses showed that the CpGs analysed were enriched for expression quantitative trait methylation sites (eQTMs) and for specific traits, dependent on the direction of causality predicted by the 2SMR analysis.Conclusions/interpretationWe identified one CpG mapping to a gene related to the metabolism of lipids (DHCR24) as a novel causal biomarker for risk of type 2 diabetes. CpGs within the same gene region have previously been associated with type 2 diabetes-related traits in observational studies (BMI, waist circumference, HDL-cholesterol, insulin) and in Mendelian randomisation analyses (LDL-cholesterol). Thus, we hypothesise that our candidate CpG in DHCR24 may be a causal mediator of the association between known modifiable risk factors and type 2 diabetes. Formal causal mediation analysis should be implemented to further validate this assumption.Graphical

Environment- and epigenome-wide association study of obesity in 'Children of 1997' birth cohort.

Increasing childhood obesity is a global issue requiring potentially local solutions to ensure it does not continue into adulthood. We systematically identified potentially modifiable targets of obesity at the onset and end of puberty in Hong Kong, the most economically developed major Chinese city. We conducted an environment-wide association study (EWAS) and an epigenome-wide association study of obesity to systematically assess associations with body mass index (BMI) and waist-hip ratio (WHR) in Hong Kong's population-representative 'Children of 1997' birth cohort. Univariable linear regression was used to select exposures related to obesity at ~11.5 years (BMI and obesity risk n ≤ 7119, WHR n = 5691) and ~17.6 years (n = 3618) at Bonferroni-corrected significance, and multivariable regression to adjust for potential confounders followed by replicated multivariable regression (n = 308) and CpG by CpG analysis (n = 286) at ~23 years. Findings were compared with evidence from published randomized controlled trials (RCTs) and Mendelian randomization (MR) studies. At ~11.5 and~17.6 years the EWAS identified 14 and 37 exposures associated with BMI, as well as 7 and 12 associated with WHR, respectively. Most exposures had directionally consistent associations at ~23 years. Maternal second-hand smoking, maternal weight, and birth weight were consistently associated with obesity. Diet (including dairy intake and artificially sweetened beverages), physical activity, snoring, binge eating, and earlier puberty were positively associated with BMI at ~17.6 years, while eating before sleep was inversely associated with BMI at ~17.6 years. Findings for birth weight, dairy intake, and binge eating are consistent with available evidence from RCTs or MR studies. We found 17 CpGs related to BMI and 17 to WHR. These novel insights into potentially modifiable factors associated with obesity at the outset and the end of puberty could, if causal, inform future interventions to improve population health in Hong Kong and similar Chinese settings. This study including the follow-up survey and epigenetics testing was supported by the Health and Medical Research Fund Research Fellowship, Food and Health Bureau, Hong Kong SAR Government (#04180097). The DNA extraction of the samples used for epigenetic testing was supported by CFS-HKU1.

Epigenome wide association study in peripheral blood of pregnant women identifies potential metabolic pathways related to gestational diabetes

ABSTRACT Gestational diabetes mellitus (GDM) increases the risk of developing metabolic disorders in both pregnant women and their offspring. Factors such as nutrition or the intrauterine environment may play an important role, through epigenetic mechanisms, in the development of GDM. The aim of this work is to identify epigenetic marks involved in the mechanisms or pathways related to gestational diabetes. A total of 32 pregnant women were selected, 16 of them with GDM and 16 non-GDM. DNA methylation pattern was obtained from Illumina Methylation Epic BeadChip, from peripheral blood samples at the diagnostic visit (26–28 weeks). Differential methylated positions (DMPs) were extracted using ChAMP and limma package in R 2.9.10, with a threshold of FDR <0.05, deltabeta >|5|% and B >0. A total of 1.141 DMPs were found, and 714 were annotated in genes. A functional analysis was performed, and we found 23 genes significantly related to carbohydrate metabolism. Finally, a total of 27 DMPs were correlated with biochemical variables such as glucose levels at different points of oral glucose tolerance test, fasting glucose, cholesterol, HOMAIR and HbA1c, at different visits during pregnancy and postpartum. Our results show that there is a differentiated methylation pattern between GDM and non-GDM. Furthermore, the genes annotated to the DMPs could be implicated in the development of GDM as well as in alterations in related metabolic variables.

Epigenome-wide methylation study identified 2 novel CpGs associated with T2DM risk and a network of co-methylated CpGs capable of patient's classifications.

Prevention of Type 2 diabetes mellitus (T2DM) pandemic needs markers that can precisely predict the disease risk in an individual. Alterations in DNA methylations due to exposure towards environmental risk factors are widely sought markers for T2DM risk prediction. To identify such individual DNA methylation signatures and their effect on disease risk, we performed an epigenome-wide association study (EWAS) in 844 Indian individuals of Indo-European origin. We identified and validated methylation alterations at 2 novel CpG sites in MIR1287 (cg01178710), and EDN2-SCMH1 (cg04673737) genes associated with T2DM risk at the epigenome-wide-significance-level (p < 1.2x10-7). Further, we also replicated the association of 2 known CpG sites in TXNIP, and CPT1A in the Indian population. With 535 EWAS significant CpGs (p < 1.2x10-7) identified in the discovery phase samples, we created a co-methylation network using weighted correlation network analysis and identified 4 modules among the CpGs. We observed that methylation of one of the module associates with T2DM risk factors (e.g. BMI, insulin, C-peptide) and can be used as markers to segregate T2DM patients with good glycemic control (e.g. low HbA1c) and dyslipidemia (low HDL and high TG) from the other patients. Additionally, an intronic SNP (rs6503650) in the JUP gene, a member of the same module, associated with methylation at all the 14 hub CpG sites of that module as methQTL. Our network-assisted epigenome-wide association study is the first to systematically explore DNA methylation variations conferring risks to T2DM in Indians and use the identified risk CpG sites for patient segregation with different clinical outcomes. These findings can be useful for better stratification of patients to improve the clinical management and treatment effects.

DNA methylation markers for kidney function and progression of diabetic kidney disease

Epigenetic markers are potential biomarkers for diabetes and related complications. Using a prospective cohort from the Hong Kong Diabetes Register, we perform two independent epigenome-wide association studies to identify methylation markers associated with baseline estimated glomerular filtration rate (eGFR) and subsequent decline in kidney function (eGFR slope), respectively, in 1,271 type 2 diabetes subjects. Here we show 40 (30 previously unidentified) and eight (all previously unidentified) CpG sites individually reach epigenome-wide significance for baseline eGFR and eGFR slope, respectively. We also develop a multisite analysis method, which selects 64 and 37 CpG sites for baseline eGFR and eGFR slope, respectively. These models are validated in an independent cohort of Native Americans with type 2 diabetes. Our identified CpG sites are near genes enriched for functional roles in kidney diseases, and some show association with renal damage. This study highlights the potential of methylation markers in risk stratification of kidney disease among type 2 diabetes individuals.

Brain DNA methylomic analysis of frontotemporal lobar degeneration reveals OTUD4 in shared dysregulated signatures across pathological subtypes

Frontotemporal lobar degeneration (FTLD) is an umbrella term describing the neuropathology of a clinically, genetically and pathologically heterogeneous group of diseases, including frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP). Among the major FTLD pathological subgroups, FTLD with TDP-43 positive inclusions (FTLD-TDP) and FTLD with tau-positive inclusions (FTLD-tau) are the most common, representing about 90% of the cases. Although alterations in DNA methylation have been consistently associated with neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, little is known for FTLD and its heterogeneous subgroups and subtypes. The main goal of this study was to investigate DNA methylation variation in FTLD-TDP and FTLD-tau. We used frontal cortex genome-wide DNA methylation profiles from three FTLD cohorts (142 FTLD cases and 92 controls), generated using the Illumina 450K or EPIC microarrays. We performed epigenome-wide association studies (EWAS) for each cohort followed by meta-analysis to identify shared differentially methylated loci across FTLD subgroups/subtypes. In addition, we used weighted gene correlation network analysis to identify co-methylation signatures associated with FTLD and other disease-related traits. Wherever possible, we also incorporated relevant gene/protein expression data. After accounting for a conservative Bonferroni multiple testing correction, the EWAS meta-analysis revealed two differentially methylated loci in FTLD, one annotated to OTUD4 (5’UTR-shore) and the other to NFATC1 (gene body-island). Of these loci, OTUD4 showed consistent upregulation of mRNA and protein expression in FTLD. In addition, in the three independent co-methylation networks, OTUD4-containing modules were enriched for EWAS meta-analysis top loci and were strongly associated with the FTLD status. These co-methylation modules were enriched for genes implicated in the ubiquitin system, RNA/stress granule formation and glutamatergic synaptic signalling. Altogether, our findings identified novel FTLD-associated loci, and support a role for DNA methylation as a mechanism involved in the dysregulation of biological processes relevant to FTLD, highlighting novel potential avenues for therapeutic development.

Entorhinal cortex epigenome-wide association study highlights four novel loci showing differential methylation in Alzheimer’s disease

BackgroundStudies on DNA methylation (DNAm) in Alzheimer’s disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression.MethodsHere, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n = 337).ResultsWe identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case–control status or Braak’s tau-staging. Four of these CpGs, located in proximity to CNFN/LIPE, TENT5A, PALD1/PRF1, and DIRAS1, represent novel findings. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. Lastly, by calculating rates of epigenetic age acceleration using two recently proposed “epigenetic clock” estimators we found a significant association with accelerated epigenetic aging in the brains of AD patients vs. controls.ConclusionIn summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression.

DNA methylation at birth and fine motor ability in childhood: an epigenome-wide association study with replication

ABSTRACT Lower fine motor performance in childhood has been associated with poorer cognitive development and neurodevelopmental conditions such as autism spectrum disorder, yet, biological underpinnings remain unclear. DNA methylation (DNAm), an essential process for healthy neurodevelopment, is a key molecular system of interest. In this study, we conducted the first epigenome-wide association study of neonatal DNAm with childhood fine motor ability and further examined the replicability of epigenetic markers in an independent cohort. The discovery study was embedded in Generation R, a large population-based prospective cohort, including a subsample of 924 ~ 1026 European-ancestry singletons with available data on DNAm in cord blood and fine motor ability at a mean (SD) age of 9.8 (0.4) years. Fine motor ability was measured using a finger-tapping test (3 subtests including left-, right-hand and bimanual), one of the most frequently used neuropsychological instruments of fine motor function. The replication study comprised 326 children with a mean (SD) age of 6.8 (0.4) years from an independent cohort, the INfancia Medio Ambiente (INMA) study. Four CpG sites at birth were prospectively associated with childhood fine motor ability after genome-wide correction. Of these, one CpG (cg07783800 in GNG4) was replicated in INMA, showing that lower levels of methylation at this site were associated with lower fine motor performance in both cohorts. GNG4 is highly expressed in the brain and has been implicated in cognitive decline. Our findings support a prospective, reproducible association between DNAm at birth and fine motor ability in childhood, pointing to GNG4 methylation at birth as a potential biomarker of fine motor ability.

Gene Set Based Integrated Methylome and Transcriptome Analysis Reveals Potential Molecular Mechanisms Linking Cigarette Smoking and Related Diseases.

Advanced integrative analysis of DNA methylation and transcriptomics data may provide deeper insights into smoke-induced epigenetic alterations, their effects on gene expression and related biological processes, linking cigarette smoking and related diseases. We hypothesize that accumulation of DNA methylation changes in CpG sites across genomic locations of different genes might have biological significance. We tested the hypothesis by performing gene set based integrative analysis of blood DNA methylation and transcriptomics data to identify potential transcriptomic consequences of smoking via changes in DNA methylation in the Young Finns Study (YFS) participants (n = 1114, aged 34-49 years, women: 54%, men: 46%). First, we performed epigenome-wide association study (EWAS) of smoking. We then defined sets of genes based on DNA methylation status within their genomic regions, for example, sets of genes containing hyper- or hypomethylated CpG sites in their body or promoter regions. Gene set analysis was performed using transcriptomics data from the same participants. Two sets of genes, one containing 49 genes with hypomethylated CpG sites in their body region and the other containing 33 genes with hypomethylated CpG sites in their promoter region, were differentially expressed among the smokers. Genes in the two gene sets are involved in bone formation, metal ion transport, cell death, peptidyl-serine phosphorylation, and cerebral cortex development process, revealing epigenetic-transcriptomic pathways to smoking-related diseases such as osteoporosis, atherosclerosis, and cognitive impairment. These findings contribute to a deeper understanding of the pathophysiology of smoking-related diseases and may provide potential therapeutic targets.