Differentially Methylated Positions Research Articles

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Altered DNA methylome profiles of blood leukocytes in Chinese patients with mild cognitive impairment and Alzheimer's disease.

Objective: DNA methylation plays a potential role in the pathogenesis of Alzheimer's disease (AD). However, little is known about the global changes of blood leukocyte DNA methylome profiles from Chinese patients with mild cognitive impairment (MCI) and with AD, or the specific DNA methylation-based signatures associated with MCI and AD. In this study, we sought to dissect the characteristics of blood DNA methylome profiles in MCI- and AD-affected Chinese patients with the aim of identifying novel DNA methylation biomarkers for AD. Methods: In this study, we profiled the DNA methylome of peripheral blood leukocytes from 20 MCI- and 20 AD-affected Chinese patients and 20 cognitively healthy controls (CHCs) with the Infinium Methylation EPIC BeadChip array. Results: We identified significant alterations of the methylome profiles in MCI and AD blood leukocytes. A total of 2,582 and 20,829 CpG sites were significantly and differentially methylated in AD and MCI compared with CHCs (adjusted p < 0.05), respectively. Furthermore, 441 differentially methylated positions (DMPs), aligning to 213 unique genes, were overlapped by the three comparative groups of AD versus CHCs, MCI versus CHCs, and AD versus MCI, of which 6 and 5 DMPs were continuously hypermethylated and hypomethylated in MCI and AD relative to CHCs (adjusted p < 0.05), respectively, such as FLNC cg20186636 and AFAP1 cg06758191. The DMPs with an area under the curve >0.900, such as cg18771300, showed high potency for predicting MCI and AD. In addition, gene ontology and pathway enrichment results showed that these overlapping genes were mainly involved in neurotransmitter transport, GABAergic synaptic transmission, signal release from synapse, neurotransmitter secretion, and the regulation of neurotransmitter levels. Furthermore, tissue expression enrichment analysis revealed a subset of potentially cerebral cortex-enriched genes associated with MCI and AD, including SYT7, SYN3, and KCNT1. Conclusion: This study revealed a number of potential biomarkers for MCI and AD, also highlighted the presence of epigenetically dysregulated gene networks that may engage in the underlying pathological events resulting in the onset of cognitive impairment and AD progression. Collectively, this study provides prospective cues for developing therapeutic strategies to improve cognitive impairment and AD course.

Prenatal maternal stress is associated with site-specific and age acceleration changes in maternal and newborn DNA methylation

ABSTRACT Prenatal maternal stress has a negative impact on child health but the mechanisms through which maternal stress affects child health are unclear. Epigenetic variation, such as DNA methylation, is a likely mechanistic candidate as DNA methylation is sensitive to environmental insults and can regulate long-term changes in gene expression. We recruited 155 mother-newborn dyads in the Democratic Republic of Congo to investigate the effects of maternal stress on DNA methylation in mothers and newborns. We used four measures of maternal stress to capture a range of stressful experiences: general trauma, sexual trauma, war trauma, and chronic stress. We identified differentially methylated positions (DMPs) associated with general trauma, sexual trauma, and war trauma in both mothers and newborns. No DMPs were associated with chronic stress. Sexual trauma was positively associated with epigenetic age acceleration across several epigenetic clocks in mothers. General trauma and war trauma were positively associated with newborn epigenetic age acceleration using the extrinsic epigenetic age clock. We tested the top DMPs for enrichment of DNase I hypersensitive sites (DHS) and found no enrichment in mothers. In newborns, top DMPs associated with war trauma were enriched for DHS in embryonic and foetal cell types. Finally, one of the top DMPs associated with war trauma in newborns also predicted birthweight, completing the cycle from maternal stress to DNA methylation to newborn health outcome. Our results indicate that maternal stress is associated with site-specific changes in DNAm and epigenetic age acceleration in both mothers and newborns.

Epigenome-wide DNA methylation analysis of myasthenia gravis.

Myasthenia gravis (MG) is a common neuromuscular junction (NMJ) disorder and autoimmune disease mediated by several antibodies. Several studies have shown that genetic factors play an important role in MG pathogenesis. To gain insight into the epigenetic factors affecting MG, we report here genome-scale DNA methylation profiles of MG. DNA was extracted from 8 MG patients and 4 healthy controls for genome-wide DNA methylation analysis using the Illumina HumanMethylation 850K BeadChip. Verification of pyrosequencing was conducted based on differential methylation positions (DMPs). Subsequently, C2C12 and HT22 cell lines (derived from mouse) were treated with demethylation drugs. Transcribed mRNA of the screened differential genes were detected using quantitative Real-time PCR. The control and MG group were compared, and two key probe positions were selected. The corresponding genes were CAMK1D and CREB5 (P<0.05). Similarly, the myasthenic crisis (MC) and non-MC group were compared and four key probe positions were selected. The corresponding genes were SAV1, STK3, YAP1 and WWTR1 (P<0.05). Subsequently, pyrosequencing was performed for verification, revealing that hypomethylation of CAMK1D was significantly different between the MG and control group (P<0.001). Moreover, transcription of CREB5, PKD, YAP1 and STK3 genes in the C2C12 cells was downregulated (P<0.05) after drug treatment, but only YAP1 mRNA was downregulated in HT22 cells (P<0.05). This is the first study to investigate genome-scale DNA methylation profiles of MG using 850K BeadChip. The identified molecular markers of methylation may aid in the prevention, diagnosis, treatment and prognosis of MG.

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 &lt;0.05). Six additional CpG sites met the threshold for suggestive significance (p&lt;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.

A PROOF-OF-PRINCIPLE EPIGENOME-WIDE ASSOCIATION STUDY OF PLASMA RENIN AND ALDOSTERONE CONCENTRATION IN A GHANAIAN POPULATION: THE RODAM STUDY

Objective: The epigenetic regulation of the renin-angiotensin-aldosterone system (RAAS) potentially plays a role in the pathophysiology underlying the high burden of hypertension in sub-Saharan Africans (SSA). In this first epigenome-wide association study (EWAS) of plasma renin and aldosterone concentrations, we explored the association between DNA methylation and concentrations of renin and aldosterone. Design and method: Epigenome-wide DNA methylation was measured using the Illumina 450k array on whole blood samples of 68 Ghanaian participants of the Research on Obesity and Diabetes among African Migrants Study (RODAM). Differentially methylated positions (DMPs) were assessed for plasma renin and aldosterone concentration using linear regression models adjusted for age, sex, body mass index, diabetes mellitus, hypertension, and technical covariates. Additionally, we extracted methylation loci previously associated with hypertension, kidney function, or that were annotated to RAAS-related genes, and associated these with renin and aldosterone concentration. Results: We identified one epigenome-wide significant DMP associated with renin, and ten DMPs associated with aldosterone. Top DMPs were annotated to the PTPRN2, SKIL, and KCNT1 genes, which have been reported in relation to cardiometabolic risk factors, atherosclerosis, and sodium-potassium handling. Moreover, EWAS loci previously associated with hypertension, kidney function, or RAAS-related genes, were also associated with renin and aldosterone concentration. Conclusions: In this first EWAS on RAAS hormones, we identified DMPs associated with renin and aldosterone in a SSA population. These findings are a first step in understanding the role of DNA methylation in regulation of the RAAS in general, and in a SSA population specifically. The results of this proof-of-principle study will inform new analyses including a larger sample size and a new methylation array (Illumina Infinium MethylationEPIC), which will we use to replicate these initial findings. This will help to further establish the role of DNA methylation and the RAAS system in the hypertension burden in SSA populations.

Blood DNA methylation biomarkers for cognitive decline in older adults with type 2 diabetes

AbstractBackgroundIndividuals with type 2 diabetes (T2D) are at particularly high risk for cognitive decline and dementia. Finding early, pre‐clinical biomarkers for detecting and tracking cognitive impairment and dementia in this population is important for personalized medicine approach. Our study aims to find blood DNA methylation biomarkers for cognitive decline among elderly T2D patients.MethodTwo groups of 30 T2D subjects each, from the Israel Diabetes and Cognitive Decline (IDCD) cohort, were included. One group (converters) was cognitively normal at baseline, yet at 36‐54 months follow‐up deteriorated from a Clinical Dementia Rating scale CDR = 0 (no dementia) to CDR≥0.5 (questionable dementia or frank dementia). The control group (non‐converters) was cognitively normal at baseline and maintained normal cognition during the follow‐up period (CDR = 0). We performed an epigenome‐wide association study, using the Illumina Infinium MethylationEPIC microarray, and searched for differentially methylated positions (DMPs) with significant methylation alterations (≥ 2 fold and FDR corrected p value &lt;0.05) between the baseline and the follow‐up time points for each of the two groups.ResultParticipants were 65‐83 years of age (mean‐73.6 ± 4.1), 72% were men. The mean hemoglobin A1C value was 6.9 ± 0.8%, and T2D duration was 9.4 ± 4.7 years. Converters and non‐converters did not differ in any of these characteristics. Results show significant changes in multiple DMPs among the converters, but only in a few DMPs in the non‐converters. Gene ontology analyses indicates primarily involvement of central nervous system‐related pathways, mainly neurodevelopmental disorders.ConclusionThese preliminary results, employing a high‐throughput approach for methylation profile in blood, suggest methylomic alterations as a potential biomarker for cognitive decline in T2D elderly. A priori identification of patients at risk for cognitive decline may lead to early intervention in modifiable risk factors in this population, which is highly susceptible to dementia.

CREB5 hypermethylation involved in the ganglioside GM1 therapy of Parkinson's disease.

The treatment with monosialotetrahexosylganglioside (GM1) improves the symptoms of Parkinson's disease (PD). The alteration of DNA methylation in the blood was examined to investigate epigenetic modification by GM1 treatment. After a 28-day continuous intravenous infusion of GM1 (100mg), the motor and non-motor symptoms were evaluated by UPDRS III, Mini-mental state examination (MMSE) scores, FS-14, SCOPA-AUT, and PDQ-8. Moreover, blood samples were collected and PBMC was isolated. Genome-wide DNA methylation was performed by an 850K BeadChip. RNA levels and apoptosis were examined by RT-PCR and flow cytometry in rotenone-based cell models. The CREB5 plasmid was transfected by electroporation into SH-SY5Y cells. We also identified 235 methylation variable positions achieving genome-wide significance in 717558 differentially methylated positions (DMPs) (P = 0.0003) in comparison of pre-treatment with post-treatment measurements (statistical analysis paired-samples t-test). By searching the Gene Expression Omnibus (GEO) dataset and GWAS, 23 methylation variable positions were screened. Moreover, there are 7 hypomethylated methylation variable positions correlated with the scores of motor symptoms (UPDRS III scale). According to KEGG pathways enrichment analysis, the methylated genes CACNA1B (hypomethylated), CREB5 (hypermethylated), GNB4 (hypomethylated), and PPP2R5A (hypomethylated) were enriched in the dopaminergic synapse pathway. Pretreated with GM1 (80 μM) for 1 h, cell apoptosis and impaired neurite outgrowth were inhibited in rotenone-induced PD cell models. The RNA expression of CREB5 was increased in rotenone-treated SH-SY5Y cells. GM1 treatment decreased rotenone-induced CREB5 gene expression. The enhancement of CREB5 gene expression suppressed the protective role of GM1 in rotenone-induced cell apoptosis. The application of GM1 improves the motor and non-motor symptoms of PD associated with the decreased CREB5 expression and the hypermethylation of CREB5. https://www.chictr.org.cn/showproj.html?proj=120582t, identifier ChiCTR2100042537.

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.

Genome-wide DNA methylation analysis in families with multiple individuals diagnosed with schizophrenia and intellectual disability

Objectives Schizophrenia (SZ) and intellectual disability (ID) are both included in the continuum of neurodevelopmental disorders (NDDs). DNA methylation is known to be important in the occurrence of NDDs. The family study is conducive to eliminate the effects of relative epigenetic backgrounds, and to screen for differentially methylated positions (DMPs) and regions (DMRs) that are truly associated with NDDs. Methods Four monozygotic twin families were recruited, and both twin individuals suffered from NDDs (either SZ, ID, or SZ plus ID). Genome-wide methylation analysis was performed in all samples and each family. DMPs and DMRs between NDD patients and unaffected individuals were identified. Functional and pathway enrichment analyses were performed on the annotated genes. Results Two significant DMPs annotated to CYP2E1 were found in all samples. In Family One, 1476 DMPs mapped to 880 genes, and 162 DMRs overlapping with 153 unique genes were recognised. Our results suggested that the altered methylation levels of FYN, STAT3, RAC1, and NR4A2 were associated with the development of SZ and ID. Neurodevelopment and the immune system may participate in the occurrence of SZ and ID. Conclusions Our findings suggested that DNA methylation participated in the development of NDDs by affecting neurodevelopment and the immune system.

Sex differences in methylation profiles are apparent in medulloblastoma, particularly among SHH tumors.

Medulloblastoma, the most common malignant pediatric brain tumor, displays marked sex differences in prevalence of the four main molecular subgroups: SHH, WNT, Group 3 and Group 4. Males are more frequently diagnosed with SHH, Group 3 and 4 tumors, which have worse prognoses than WNT tumors. Little is known about sex differences in methylation profiles within subgroups. Using publicly available methylation data (Illumina HumanMethylation450K array), we compared beta values for males versus females. Differentially methylated positions (DMP) by sex within medulloblastoma subgroups were identified on the autosomes. DMPs were mapped to genes and Reactome pathway analysis was run by subgroup. Kaplan-Meier survival curves (Log-Rank p-values) were assessed for each sex within subgroup. MethylCIBERSORT was used to investigate the tumor microenvironment using deconvolution to estimate the abundances of immune cell types using DNA methylation data. There were statistically significant differences in sex by medulloblastoma subgroups (chi-squared p-value=0.00004): Group 3 (n=144; 65% male), Group 4 (n=326; 67% male), SHH (n=223; 57% male) and WNT (n=70; 41% male). Females had worse survival than males for SHH (p-value=0.02). DMPs by sex were identified within subgroups: SHH (n=131), Group 4 (n=29), Group 3 (n=19), and WNT (n=16) and validated in an independent dataset. Unsupervised hierarchical clustering showed that sex-DMPs in SHH did not correlate with other tumor attributes. Ten genes with sex DMPs (RFTN1, C1orf103, FKBP1B, COL25A1, NPDC1, B3GNT1, FOXN3, RNASEH2C, TLE1, and PHF17) were shared across subgroups. Significant pathways (p<0.05) associated with DMPs were identified for SHH (n=22) and Group 4 (n=4) and included signaling pathways for RET proto-oncogene, advanced glycosylation end product receptor, regulation of KIT, neurotrophic receptors, NOTCH, and TGF-β. In SHH, we identified DMPs in four genes (CDK6, COL25A1, MMP16, PRIM2) that encode proteins which are the target of therapies in clinical trials for other cancers. There were few sex differences in immune cell composition within tumor subgroups. There are sexually dimorphic methylation profiles for SHH medulloblastoma where survival differences were observed. Sex-specific therapies in medulloblastoma may impact outcomes.

Genome-wide DNA methylation analysis of aggressive behaviour: a longitudinal population-based study.

Human aggression is influenced by an interplay between genetic predisposition and experience across the life span. This interaction is thought to occur through epigenetic mechanisms, inducing differential gene expression, thereby moderating neuronal cell and circuit function, and thus shaping aggressive behaviour. Genome-wide DNA methylation (DNAm) levels were measured in peripheral blood obtained from 95 individuals participating in the Estonian Children Personality Behaviours and Health Study (ECPBHS) at 15 and 25 years of age. We examined the association between aggressive behaviour, as measured by Life History of Aggression (LHA) total score and DNAm levels both assessed at age 25. We further examined the pleiotropic effect of genetic variants regulating LHA-associated differentially methylated positions (DMPs) and multiple traits related to aggressive behaviours. Lastly, we tested whether the DNA methylomic loci identified in association with LHA at age 25 were also present at age 15. We found one differentially methylated position (DMP) (cg17815886; p= 1.12 × 10-8 ) and five differentially methylated regions (DMRs) associated with LHA after multiple testing adjustments. The DMP annotated to the PDLIM5 gene, and DMRs resided in the vicinity of four protein-encoding genes (TRIM10, GTF2H4, SLC45A4, B3GALT4) and a long intergenic non-coding RNA (LINC02068). We observed evidence for the colocalization of genetic variants associated with top DMPs and general cognitive function, educational attainment and cholesterol levels. Notably, a subset of the DMPs associated with LHA at age 25 also displayed altered DNAm patterns at age 15 with high accuracy in predicting aggression. Our findings highlight the potential role of DNAm in the development of aggressive behaviours. We observed pleiotropic genetic variants associated with identified DMPs, and various traits previously established to be relevant in shaping aggression in humans. The concordance of DNAm signatures in adolescents and young adults may have predictive value for inappropriate and maladaptive aggression later in life.

Epigenome-wide association study of plasma lipids in West Africans: the RODAM study.

DNA-methylation has been associated with plasma lipid concentration in populations of diverse ethnic backgrounds, but epigenome-wide association studies (EWAS) in West-Africans are lacking. The aim of this study was to identify DNA-methylation loci associated with plasma lipids in Ghanaians. We conducted an EWAS using Illumina 450k DNA-methylation array profiles of extracted DNA from 663 Ghanaian participants. Differentially methylated positions (DMPs) were examined for association with plasma total cholesterol (TC), LDL-cholesterol, HDL-cholesterol, and triglycerides concentrations using linear regression models adjusted for age, sex, body mass index, diabetes mellitus, and technical covariates. Findings were replicated in independent cohorts of different ethnicities. We identified one significantly associated DMP with triglycerides (cg19693031 annotated to TXNIP, regression coefficient beta-0.26, false discovery rate adjusted p-value 0.001), which replicated in-silico in South African Batswana, African American, and European populations. From the top five DMPs with the lowest nominal p-values, two additional DMPs for triglycerides (CPT1A, ABCG1), two DMPs for LDL-cholesterol (EPSTI1, cg13781819), and one for TC (TXNIP) replicated. With the exception of EPSTI1, these loci are involved in lipid transport/metabolism or are known GWAS-associated loci. The top 5 DMPs per lipid trait explained 9.5% in the variance of TC, 8.3% in LDL-cholesterol, 6.1% in HDL-cholesterol, and 11.0% in triglycerides. The top DMPs identified in this study are in loci that play a role in lipid metabolism across populations, including West-Africans. Future studies including larger sample size, longitudinal study design and translational research is needed to increase our understanding on the epigenetic regulation of lipid metabolism among West-African populations. European Commission under the Framework Programme (grant number: 278901).

Effect of mechanical unloading on genome-wide DNA methylation profile of the failing human heart.

Heart failure (HF) is characterized by global alterations in myocardial DNA methylation, yet little is known about the epigenetic regulation of the noncoding genome and potential reversibility of DNA methylation with left ventricular assist device (LVAD) therapy. Genome-wide mapping of myocardial DNA methylation in 36 patients with HF at LVAD implantation, 8 patients at LVAD explantation, and 7 nonfailing (NF) donors using a high-density bead array platform identified 2,079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in nonischemic cardiomyopathy (NICM). LVAD support resulted in normalization of 3.2% of HF-associated DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FBXO16, EFCAB13, and AKAP13) or hypermethylated and downregulated (TBX3) in HF. A potentially novel cardiac-specific super-enhancer long noncoding RNA (lncRNA) (LINC00881) is hypermethylated and downregulated in human HF. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown reduces peak calcium amplitude in the beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These data suggest that HF-associated changes in myocardial DNA methylation within coding and noncoding genomes are minimally reversible with mechanical unloading. Epigenetic reprogramming strategies may be necessary to achieve sustained clinical recovery from heart failure.

DNA methylation differences in monozygotic twins with Van der Woude syndrome.

Van der Woude Syndrome (VWS) is an autosomal dominant disorder responsible for 2% of all syndromic orofacial clefts (OFCs) with IRF6 being the primary causal gene (70%). Cases may present with lip pits and either cleft lip, cleft lip with cleft palate, or cleft palate, with marked phenotypic discordance even among individuals carrying the same mutation. This suggests that genetic or epigenetic modifiers may play additional roles in the syndrome's etiology and variability in expression. We report the first DNA methylation profiling of 2 pairs of monozygotic twins with VWS. Our goal is to explore epigenetic contributions to VWS etiology and variable phenotypic expressivity by comparing DNAm profiles in both twin pairs. While the mutations that cause VWS in these twins are known, the additional mechanism behind their phenotypic risk and variability in expression remains unclear. We generated whole genome DNAm data for both twin pairs. Differentially methylated positions (DMPs) were selected based on: (1) a coefficient of variation in DNAm levels in unaffected individuals < 20%, and (2) intra-twin pair absolute difference in DNAm levels >5% (delta beta > | 0.05|). We then divided the DMPs in two subgroups for each twin pair for further analysis: (1) higher methylation levels in twin A (Twin A > Twin B); and (2) higher methylation levels in twin B (Twin B >Twin A). Gene ontology analysis revealed a list of enriched genes that showed significant differential DNAm, including clef-associated genes. Among the cleft-associated genes, TP63 was the most significant hit (p=7.82E-12). Both twin pairs presented differential DNAm levels in CpG sites in/near TP63 (Twin 1A > Twin 1B and Twin 2A < Twin 2B). The genes TP63 and IRF6 function in a biological regulatory loop to coordinate epithelial proliferation and differentiation in a process that is critical for palatal fusion. The effects of the causal mutations in IRF6 can be further impacted by epigenetic dysregulation of IRF6 itself, or genes in its pathway. Our data shows evidence that changes in DNAm is a plausible mechanism that can lead to markedly distinct phenotypes, even among individuals carrying the same mutation.

Integrative analysis of DNA methylation and gene expression through machine learning identifies stomach cancer diagnostic and prognostic biomarkers.

DNA methylation is an early event in tumorigenesis. Here, by integrative analysis of DNA methylation and gene expression and utilizing machine learning approaches, we introduced potential diagnostic and prognostic methylation signatures for stomach cancer. Differentially-methylated positions (DMPs) and differentially-expressed genes (DEGs) were identified using The Cancer Genome Atlas (TCGA) stomach adenocarcinoma (STAD) data. A total of 256 DMPs consisting of 140 and 116 hyper- and hypomethylated positions were identified between 443 tumour and 27 nontumour STAD samples. Gene expression analysis revealed a total of 2821 DEGs with 1247 upregulated and 1574 downregulated genes. By analysing the impact of cis and trans regulation of methylation on gene expression, a dominant negative correlation between methylation and expression was observed, while for trans regulation, in hypermethylated and hypomethylated genes, there was mainly a negative and positive correlation with gene expression, respectively. To find diagnostic biomarkers, we used 28 hypermethylated probes locating in the promoter of 27 downregulated genes. By implementing a feature selection approach, eight probes were selected and then used to build a support vector machine diagnostic model, which had an area under the curve of 0.99 and 0.97 in the training and validation (GSE30601 with 203 tumour and 94 nontumour samples) cohorts, respectively. Using 412 TCGA-STAD samples with both methylation and clinical data, we also identified four prognostic probes by implementing univariate and multivariate Cox regression analysis. In summary, our study introduced potential diagnostic and prognostic biomarkers for STAD, which demands further validation.

Effect of HIV infection and antiretroviral therapy initiation on genome-wide DNA methylation patterns.

Previous epigenome-wide association studies have shown that HIV infection can disrupt the host DNA methylation landscape. However, it remains unclear how antiretroviral therapy (ART) affects the HIV-induced epigenetic modifications. 184 individuals with HIV from the NEAT001/ANRS143 clinical trial (with pre-ART and post-ART samples [96 weeks of follow-up]) and 44 age-and-sex matched individuals without HIV were included. We compared genome-wide DNA methylation profiles in whole blood between groups adjusting for age, sex, batch effects, and DNA methylation-based estimates of leucocyte composition. We identified 430 differentially methylated positions (DMPs) between HIV+pre-ART individuals and HIV-uninfected controls. In participants with HIV, ART initiation modified the DNA methylation levels at 845 CpG positions and restored 49.3% of the changes found between HIV+pre-ART and HIV-uninfected individuals. We only found 15 DMPs when comparing DNA methylation profiles between HIV+post-ART individuals and participants without HIV. The Gene Ontology enrichment analysis of DMPs associated with untreated HIV infection revealed an enrichment in biological processes regulating the immune system and antiviral responses. In participants with untreated HIV infection, DNA methylation levels at top HIV-related DMPs were associated with CD4/CD8 ratios and viral loads. Changes in DNA methylation levels after ART initiation were weakly correlated with changes in CD4+ cell counts and the CD4/CD8 ratio. Control of HIV viraemia after 96 weeks of ART initiation partly restores the host DNA methylation changes that occurred before antiretroviral treatment of HIV infection. NEAT-ID Foundation and Instituto de Salud Carlos III, co-funded by European Union.

DNA Methylation Profile of CD14+ Monocytes Changes in Primary Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune inflammatory and neurodegenerative disease of the central nervous system, which is characterized by significant clinical heterogeneity. Primary progressive MS (PPMS) develops in 10-15% of patients. Unlike the most common relapsing-remitting form of MS, PPMS involves steady progress of neurodegeneration and, as a consequence, a persistent gradual increase in neurological symptoms. The peculiarities of epigenetic regulation of gene expression may be one of the reasons for the differences in the pathogenesis of the two MS forms. DNA methylation is one of the key epigenetic mechanisms, which remains almost unexplored in different cell populations of PPMS patients. The goal of this work was to identify differential methylation profiles of the CpG sites in the CD14+ monocyte DNA, which characterize PPMS. A genome-wide analysis of DNA methylation in PPMS patients and healthy individuals has identified 169 differentially methylated positions (DMPs), 90.5% of which were hypermethylated in PPMS patients. More than half of all DMPs are located in/near known genes and within CpG islands and their neighboring regions, which indicates their high functional significance. We have found six differentially methylated regions (DMRs) in the OR2L13, CAT, LCLAT1, HOXA5, RNF39, and CRTAC1 genes involved in inflammation and neurodegeneration, which indicates active epigenetic regulation of their expression.

DNA methylation-based patterns for early diagnostic prediction and prognostic evaluation in colorectal cancer patients with high tumor mutation burden.

Immune checkpoint inhibitor (ICI) therapy has proven to be a promising treatment for colorectal cancer (CRC). We aim to investigate the relationship between DNA methylation and tumor mutation burden (TMB) by integrating genomic and epigenetic profiles to precisely identify clinical benefit populations and to evaluate the effect of ICI therapy. A total of 536 CRC tissues from the Cancer Genome Atlas (TCGA) with mutation data were collected and subjected to calculate TMB. 80 CRC patients with high TMB and paired normal tissues were selected as training sets and developed the diagnostic and prognostic methylation models, respectively. In the validation set, the diagnostic model was validated in our in-house 47 CRC tissues and 122 CRC tissues from the Gene Expression Omnibus (GEO) datasets, respectively. And a total of 38 CRC tissues with high TMB from the COLONOMICS dataset verified the prognostic model. A positive correlation between differential methylation positions and TMB level was observed in TCGA CRC cohort (r=0.45). The diagnostic score that consisted of methylation levels of four genes (ADHFE1, DOK6, GPR75, and MAP3K14-AS1) showed high diagnostic performance in the discovery (AUC=1.000) and two independent validation (AUC=0.946, AUC=0.857) datasets. Additionally, these four genes showed significant positive correlations with NK cells. The prognostic score containing three genes (POU3F3, SYN2, and TMEM178A) had significantly poorer survival in the high-risk TMB samples than those in the low-risk TMB samples (P=0.016). CRC patients with low-risk scores combined with TMB levels represent a favorable survival. By integrating analyses of methylation and mutation data, it is suggested that DNA methylation patterns combined with TMB serve as a novel potential biomarker for early screening in more high-TMB populations and for evaluating the prognostic effect of CRC patients with ICI therapy.

Genome-wide DNA methylation profiling in chronic lymphocytic leukaemia.

Background: DNA methylation aberrations are widespread among the malignant B lymphocytes of patients with chronic lymphocytic leukaemia (CLL), suggesting that DNA methylation might contribute to the pathogenesis of CLL. Aim: We aimed to explore the differentially methylated positions (DMPs) associated with CLL and screen the differentially methylated and expressed genes (DMEGs) by combining public databases. We aimed to observe the direction of each DMEG in CLL based on the DMPs in the promoter and the body region respectively to narrow down DMEGs. We also aimed to explore the methylation heterogeneity of CLL subgroups and the effect of B cells maturation on CLL. Methods: In this population-based case control study, we reported a genome-wide DNA methylation association study using the Infinium HumanMethylation450 BeadChip, profiling the DNA methylation of CD19+ B Cells from 48 CLL cases and 28 healthy controls. By integrating methylation data and expression data from public databases, gene sets were jointly screened, and then the relationship between methylation sites in promoter and body region and expression of each gene was explored. In addition, support vector machine (SVM) classification algorithm was used to identify subgroups of CLL cases based on methylation pattern, and the effect of B-cell differentiation related methylation sites on CLL-related sites was observed. Results: We identified 34,797 DMPs related to CLL across the genome, most of which were hypomethylated; the majority were located in gene body regions. By combining these DMPs with published DNA methylation and RNA sequencing data, we detected 26,244 replicated DMPs associated with 1,130 genes whose expression were significantly different in CLL cases. Among these DMEGs, nine low expressed DMEGs were selected with hypermethylated in promoter and hypomethylated in body region, and 83 high expressed DMEGs were selected with both hypomethylated in promoter and body region. The 48 CLL cases were divided into 3 subgroups based on methylation site by SVM algorithm. Over 92% of CpGs associated with B cell subtypes were found in CLL-related DMPs. Conclusion: The DNA methylation pattern was altered across the genome in CLL patients. The methylation of ZAP70, FMOD, and ADAMTS17 was significantly different between CLL cases and controls. Further studies are warranted to confirm our findings and identify the underlying mechanisms through which these methylation markers are associated with CLL.