The methylome of buccal epithelial cells is influenced by age, sex, and physiological properties.

Epigenetic modifications, particularly DNA methylation, have emerged as regulators of gene expression and are implicated in various biological processes and disease states. Understanding the factors influencing the epigenome is essential for unraveling its complexity. In this study, we aimed to identify how the methylome of buccal epithelial cells, a noninvasive and easily accessible tissue, is associated with demographic and health-related variables commonly used in clinical settings, such as age, sex, blood immune composition, hemoglobin levels, and others. We developed a model to assess the association of multiple factors with the human methylome and identify the genomic loci significantly impacted by each trait. We demonstrated that DNA methylation variation is accurately modeled by several factors. We confirmed the well-known impact of age and sex and unveiled novel clinical factors associated with DNA methylation, such as blood neutrophils, hemoglobin, red blood cell distribution width, high-density lipoprotein cholesterol, and urea. Genomic regions significantly associated with these traits were enriched in relevant transcription factors, drugs, and diseases. Among our findings, we showed that neutrophil-impacted loci were involved in neutrophil functionality and maturation. Similarly, hemoglobin-influenced sites were associated with several diseases, including aplastic anemia, and the genomic loci affected by urea were related to congenital anomalies of the kidney and urinary tract. Our findings contribute to a better understanding of the human methylome plasticity and provide insights into novel factors shaping DNA methylation patterns, highlighting their potential clinical implications as biomarkers and the importance of considering these physiological traits in future medical epigenomic investigations.NEW & NOTEWORTHY We have developed a quantitative model to assess how the human methylome is associated with several factors and to identify the genomic loci significantly impacted by each trait. We reported novel health-related factors driving DNA methylation patterns and new site-specific regulations that further elucidate methylome dynamics. Our study contributes to a better understanding of the plasticity of the human methylome and unveils novel physiological traits with a potential role in future medical epigenomic investigations.

Fine-mapping and replication of EWAS loci harboring putative epigenetic alterations associated with AD neuropathology in a large collection of human brain tissue samples.

INTRODUCTION: Our previous epigenome-wide association study (EWAS) of Alzheimer's disease (AD) in human brain identified 71 CpGs associated with AD pathology. However, due to low coverage of the Illumina platform, many important CpGs might have been missed. METHODS: In a large collection of human brain tissue samples (N = 864), we fine-mapped previous EWAS loci by targeted bisulfite sequencing and examined their associations with AD neuropathology. DNA methylation was also linked to gene expression of the same brain cortex. RESULTS: Our targeted sequencing captured 130 CpGs (∼1.2 kb), 93 of which are novel. Of the 130 CpGs, 57 sites (only 17 included in previous EWAS) and 12 gene regions (e.g., ANK1, BIN1, RHBDF2, SPG7, PODXL) were significantly associated with amyloid load. DNA methylation in some regions was associated with expression of nearby genes. DISCUSSION: Targeted methylation sequencing can validate previous EWAS loci and discover novel CpGs associated with AD pathology.

Evaluation of clinical formalin-fixed paraffin-embedded tissue quality for targeted-bisulfite sequencing.

Formalin-fixed paraffin-embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for multiple sequencing methods. However, fundamental research addressing the application of FFPE-derived DNA for targeted-bisulfite sequencing (TB-seq) is lacking. Here, we evaluated the suitability of FFPE-derived DNA for TB-seq. We conducted TB-seq using FFPE-derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB-seq statistics between the two preservation methods. The approximately 600-bp average fragment size of the FFPE-derived DNA was significantly shorter than that of the FF-derived DNA. The sequencing libraries constructed using FFPE-derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF-derived DNA. In the mapped data of FFPE-derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on-target coverage. Therefore, the standard TB-seq protocol is inadequate for obtaining high-quality data for epigenetic analysis from FFPE-derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.

Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine.

BACKGROUND: Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing. RESULTS: Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods. CONCLUSIONS: This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.

Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia.

STUDY QUESTION: Is there an association between sperm DNA methylation profiles and asthenozoospermia? SUMMARY ANSWER: DNA methylation, at specific CpGs but not at the global level, was significantly different between low motile sperm cells of asthenozoospermic individuals and high motile sperm cells of normozoospermic controls. WHAT IS KNOWN ALREADY: Aberrant DNA methylation, both globally and restricted to a specific gene locus, has been associated with male infertility and abnormal semen parameters. STUDY DESIGN, SIZE, DURATION: This was a case-control study investigating the differences in DNA methylation at CpGs in promoter regions between high and low motile sperm cells from eight normozoospermic controls and seven asthenozoospermic patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: The liquid hybridization capture-based bisulfite sequencing method was used to determine DNA methylation at CpGs in promoter regions. The global inter-individual and intra-individual methylation variability were estimated by evaluating the methylation variance between and within different motile sperm fractions from the same or different individuals. Asthenozoospermia-associated differentially methylated or variable CpGs and differentially methylated regions were identified by comparing the DNA methylation of high motile sperm cells from normozoospermic controls with that of low motile sperm cells from asthenozoospermic patients. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, we determined the global DNA methylation level (24.7%), inter-individual variance (14.4%) and intra-individual differences between high and low motile sperm fractions (3.9%). We demonstrated that there were no statistically significant differences in either the global DNA methylation level or global methylation variability between sperm from men with normozoospermia or asthenozoospermia. Between high motile sperm from men with normozoospermia and low motile sperm from men with asthenozoospermia, we identified 134 differentially methylated CpGs, 41 differentially methylated regions and 134 differentially variable CpGs. The genomic distribution patterns of the differential methylation spectrum suggested that gene expression may be affected in low motile sperm cells of asthenozoospermic patients. Finally, through a functional analysis, we detected 16 differentially methylated or variable genes that are required for spermatogenesis and sperm motility or dominantly expressed in testis. LIMITATIONS, REASONS FOR CAUTION: The sample size in this study was limited, although the participants in the two groups were carefully selected and well matched. Our results must be verified in larger cohorts with the use of different techniques. Furthermore, our results were descriptive, and follow-up studies will be needed to elucidate the effect of differential methylation profiles on asthenozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: Our study identified asthenozoospermia-associated DNA methylation profiles and proposed a list of genes, which were suggested to be involved in the regulation of sperm motility through an alteration of DNA methylation. These results will provide promising clues for understanding the effect of DNA methylation on sperm motility and asthenozoospermia. STUDY FUNDING/COMPETING INTERESTS: This study was funded primarily by the National Natural Science Foundation of China, Shenzhen Project of Science and Technology and the National Basic Research Program of China. The authors have no competing interests.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

Background: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Results: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Conclusions: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000.

BACKGROUND: An accurate and reproducible next-generation sequencing platform is essential to identify malignancy-related abnormal DNA methylation changes and translate them into clinical applications including cancer detection, prognosis, and surveillance. However, high-quality DNA methylation sequencing has been challenging because poor sequence diversity of the bisulfite-converted libraries severely impairs sequencing quality and yield. In this study, we tested MGISEQ-2000 Sequencer's capability of DNA methylation sequencing with a published non-invasive pancreatic cancer detection assay, using NovaSeq6000 as the benchmark. RESULTS: We sequenced a series of synthetic cell-free DNA (cfDNA) samples with different tumor fractions and found MGISEQ-2000 yielded data with similar quality as NovaSeq6000. The methylation levels measured by MGISEQ-2000 demonstrated high consistency with NovaSeq6000. Moreover, MGISEQ-2000 showed a comparable analytic sensitivity with NovaSeq6000, suggesting its potential for clinical detection. As to evaluate the clinical performance of MGISEQ-2000, we sequenced 24 clinical samples and predicted the pathology of the samples with a clinical diagnosis model, PDACatch classifier. The clinical model performance of MGISEQ-2000's data was highly consistent with that of NovaSeq6000's data, with the area under the curve of 1. We also tested the model's robustness with MGISEQ-2000's data when reducing the sequencing depth. The results showed that MGISEQ-2000's data showed matching robustness of the PDACatch classifier with NovaSeq6000's data. CONCLUSIONS: Taken together, MGISEQ-2000 demonstrated similar data quality, consistency of the methylation levels, comparable analytic sensitivity, and matching clinical performance, supporting its application in future non-invasive early cancer detection investigations by detecting distinct methylation patterns of cfDNAs.

Age-associated DNA methylation changes in Xenopus frogs.

Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

DNA methylation profiles in pneumonia patients reflect changes in cell types and pneumonia severity.

Immune cell-type composition changes with age, potentially weakening the response to infectious diseases. Profiling epigenetics marks of immune cells can help us understand the relationship with disease severity. We therefore leveraged a targeted DNA methylation method to study the differences in a cohort of pneumonia patients (both COVID-19 positive and negative) and unaffected individuals from peripheral blood.This approach allowed us to predict the pneumonia diagnosis with high accuracy (AUC = 0.92), and the PCR positivity to the SARS-CoV-2 viral genome with moderate, albeit lower, accuracy (AUC = 0.77). We were also able to predict the severity of pneumonia (PORT score) with an R2 = 0.69. By estimating immune cellular frequency from DNA methylation data, patients under the age of 65 positive to the SARS-CoV-2 genome (as revealed by PCR) showed an increase in T cells, and specifically in CD8+ cells, compared to the negative control group. Conversely, we observed a decreased frequency of neutrophils in the positive compared to the negative group. No significant difference was found in patients over the age of 65. The results suggest that this DNA methylation-based approach can be used as a cost-effective and clinically useful biomarker platform for predicting pneumonias and their severity.

Preliminary Study on the Sequencing of Whole Genomic Methylation and Transcriptome-Related Genes in Thyroid Carcinoma.

Thyroid carcinoma is the most prevalent endocrine cancer globally and the primary cause of cancer-related mortality. Epigenetic modifications are progressively being linked to metastasis. This study aimed to examine whole-genome DNA methylation patterns and the gene expression profiles in thyroid cancer tissue samples using a MethylationEPIC BeadChip (850K), RNA sequencing, and a targeted bisulfite sequencing assay. The results of the Illumina Infinium human methylation kit (850K) analyses identified differentially methylated CpG locations (DMPs) and differentially methylated CpG regions (DMRs) encompassing nearly the entire genome with high resolution and depth. Gene ontology and KEGG pathway analyses revealed that the genes associated with DMRs belonged to various domain-specific ontologies, including cell adhesion, molecule binding, and proliferation. The RNA-Seq study found 1627 differentially expressed genes, 1174 of which that were up-regulated and 453 of which that were down-regulated. The targeted bisulfite sequencing assay revealed that CHST2, DPP4, DUSP6, ITGA2, SLC1A5, TIAM1, TNIK, and ABTB2 methylation levels were dramatically lowered in thyroid cancer patients when compared to the controls, but GALNTL6, HTR7, SPOCD1, and GRM5 methylation levels were significantly raised. Our study revealed that the whole-genome DNA methylation patterns and gene expression profiles in thyroid cancer shed new light on the tumorigenesis of thyroid cancer.

FKBP5 intron 7 methylation is associated with higher anxiety proneness and smaller right thalamus volume in adolescents.

Dysregulation of stress response systems may mediate the detrimental effects of childhood trauma (CT) on mental health. FKBP5 regulates glucocorticoid receptor sensitivity and exerts pleiotropic effects on intracellular signaling, neurobiology and behavior. We investigated whether CT, alone and in combination with rs1360780 genotype, is associated with altered FKBP5 methylation and whether CT-associated methylation profiles are associated with anxiety proneness (AP) and structural brain volumes. Ninety-four adolescents completed the Childhood Trauma Questionnaire, and a composite AP score was generated from the Childhood Anxiety Sensitivity Index and the State-Trait Anxiety Inventory-Trait measure. Mean methylation values for 12 regulatory regions and 25 individual CpG sites were determined using high-accuracy measurement via targeted bisulfite sequencing. FKBP5 rs1360780 genotype and structural MRI data were available for a subset of participants (n = 71 and n = 75, respectively). Regression models revealed an inverse association between methylation of three intron 7 CpG sites (35558438, 35558566 and 35558710) and right thalamus volume. CpG35558438 methylation was positively associated with AP scores. Our data indicate that an intron 7 methylation profile, consistent with lower FKBP5 expression and elevated high sensitivity glucocorticoid receptor levels, is associated with higher AP and smaller right thalamus volume. Research into the mechanisms underlying the intron 7 methylation-thalamus volume relationship, and whether it confers increased risk for long-term psychopathology by altering the regulatory threshold of stress responding, is required.

Betamethasone administration during pregnancy is associated with placental epigenetic changes with implications for inflammation.

BACKGROUND: Glucocorticoids (GCs) play a pivotal role in fetal programming. Antenatal treatment with synthetic GCs (sGCs) in individuals in danger of preterm labor is common practice. Adverse short- and long-term effects of antenatal sGCs have been reported, but their effects on placental epigenetic characteristics have never been systematically studied in humans. RESULTS: We tested the association between exposure to the sGC betamethasone (BET) and placental DNA methylation (DNAm) in 52 exposed cases and 84 gestational-age-matched controls. We fine-mapped associated loci using targeted bisulfite sequencing. The association of placental DNAm with gene expression and co-expression analysis on implicated genes was performed in an independent cohort including 494 placentas. Exposure to BET was significantly associated with lower placenta DNAm at an enhancer of FKBP5. FKBP5 (FK506-binding protein 51) is a co-chaperone that modulates glucocorticoid receptor activity. Lower DNAm at this enhancer site was associated with higher expression of FKBP5 and a co-expressed gene module. This module is enriched for genes associated with preeclampsia and involved in inflammation and immune response. CONCLUSIONS: Our findings suggest that BET exposure during pregnancy associates with few but lasting changes in placental DNAm and may promote a gene expression profile associated with placental dysfunction and increased inflammation. This may represent a pathway mediating GC-associated negative long-term consequences and health outcomes in offspring.

Evaluating the Consistency of Gene Methylation in Liver Cancer Using Bisulfite Sequencing Data.

Bisulfite sequencing is considered as the gold standard approach for measuring DNA methylation, which acts as a pivotal part in regulating a variety of biological processes without changes in DNA sequences. In this study, we introduced the most prevalent methods for processing bisulfite sequencing data and evaluated the consistency of the data acquired from different measurements in liver cancer. Firstly, we introduced three commonly used bisulfite sequencing assays, i.e., reduced-representation bisulfite sequencing (RRBS), whole-genome bisulfite sequencing (WGBS), and targeted bisulfite sequencing (targeted BS). Next, we discussed the principles and compared different methods for alignment, quality assessment, methylation level scoring, and differentially methylated region identification. After that, we screened differential methylated genes in liver cancer through the three bisulfite sequencing assays and evaluated the consistency of their results. Ultimately, we compared bisulfite sequencing to 450 k beadchip and assessed the statistical similarity and functional association of differentially methylated genes (DMGs) among the four assays. Our results demonstrated that the DMGs measured by WGBS, RRBS, targeted BS and 450 k beadchip are consistently hypo-methylated in liver cancer with high functional similarity.

Thousands of CpGs Show DNA Methylation Differences in ACPA-Positive Individuals.

High levels of anti-citrullinated protein antibodies (ACPA) are often observed prior to a diagnosis of rheumatoid arthritis (RA). We undertook a replication study to confirm CpG sites showing evidence of differential methylation in subjects positive vs. negative for ACPA, in a new subset of 112 individuals sampled from the population cohort and biobank CARTaGENE in Quebec, Canada. Targeted custom capture bisulfite sequencing was conducted at approximately 5.3 million CpGs located in regulatory or hypomethylated regions from whole blood; library and protocol improvements had been instituted between the original and this replication study, enabling better coverage and additional identification of differentially methylated regions (DMRs). Using binomial regression models, we identified 19,472 ACPA-associated differentially methylated cytosines (DMCs), of which 430 overlapped with the 1909 DMCs reported by the original study; 814 DMRs of relevance were clustered by grouping adjacent DMCs into regions. Furthermore, we performed an additional integrative analysis by looking at the DMRs that overlap with RA related loci published in the GWAS Catalog, and protein-coding genes associated with these DMRs were enriched in the biological process of cell adhesion and involved in immune-related pathways.

Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage.

DNA methylation analysis is increasingly used in stress research. Available methods are expensive, laborious and often limited by either the analysis of short CpG stretches or low assay sensitivity. Here, we present a cost-efficient next generation sequencing-based strategy for the simultaneous investigation of multiple candidate genes in large cohorts. To illustrate the method, we present analysis of four candidate genes commonly assessed in psychoneuroendocrine research: Glucocorticoid receptor (NR3C1), Serotonin transporter (SLC6A4), FKBP Prolyl isomerase 5 (FKBP5), and the Oxytocin receptor (OXTR). DNA methylation standards (100 %; 75 %; 50 %; 25 % and 0 %) and DNA of a female and male donor were bisulfite treated in three independent trials and were used to generate sequencing libraries for 42 CpGs from the NR3C1 1 F promoter region, 84 CpGs of the SLC6A4 5' regulatory region, 5 CpGs located in FKBP5 intron 7, and additional 12 CpGs located in a potential enhancer element in intron 3 of the OXTR. In addition, DNA of 45 patients with borderline personality disorder (BPD) and 45 healthy controls was assayed. Multiplex libraries of all samples were sequenced on a MiSeq system and analyzed for mean methylation values of all CpG sites using amplikyzer2 software. Results indicated excellent accuracy of the assays when investigating replicates generated from the same bisulfite converted DNA, and very high linearity (R2 > 0.9) of the assays shown by the analysis of differentially methylated DNA standards. Comparing DNA methylation between BPD and healthy controls revealed no biologically relevant differences. The technical approach as described here facilitates targeted DNA methylation analysis and represents a highly sensitive, cost-efficient and high throughput tool to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes.

Development and optimization of the VISAGE basic prototype tool for forensic age estimation.

The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay's reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20 ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicates.

Novel DNA methylation biomarkers for hexavalent chromium exposure: an epigenome-wide analysis.

Aim: We aimed to identify differential methylation of genes that could illuminate the biological mechanisms of chromium (VI) toxicity in this exposure-control study. Materials & methods: DNA methylation was measured in blood samples collected from electroplating workers and controls using a combination of Infinium Methylation450K Chip and targeted-bisulfite sequencing. QuantiGene assay was used to detect the mRNA expression of differentially methylated genes. Inductively coupled plasma-mass spectrometry was used to quantify metals in blood and urine samples. The cytosine-phosphate-guanine sites methylation and gene expression were confirmed in a human lymphoblastoid cell line. Results & conclusion: A total of 131 differentially methylated cytosine-phosphate-guanine sites were found between exposures and controls. DNA methylation of SEMA4B may serve as a potential biomarker for chromium (VI) exposure.

Whole Blood Targeted Bisulfite Sequencing and Differential Methylation in the C6ORF10 Gene of Patients with Rheumatoid Arthritis.

OBJECTIVE: Polymorphisms in human major histocompatibility complex (MHC) are the strongest genetic associations with rheumatoid arthritis (RA). Epigenome-wide methylation studies suggest DNA methylation changes within MHC may contribute to disease susceptibility. We profiled MHC-specific methylated CpG (5'-C-phosphate-G-3') in autoantibody-positive patients with RA and matched unaffected anticitrullinated protein antibodies-negative first-degree relatives (ACPA-/FDR) from an indigenous North American (INA) population that is known to have prevalent RA. METHODS: DNA was isolated from whole blood and targeted bisulfite sequencing was used to profile methylated CpG in patients with RA and ACPA-/FDR. Differentially methylated CpG loci (DML) were mapped and gene annotated. Ingenuity pathway analysis (IPA) was used for curating biomolecular networks of mapped genes. Transcript abundance was determined by quantitative (q)PCR. RESULTS: We identified 74 uniquely methylated CpG sites within the MHC region that were differentially methylated in patients with RA (p < 0.05), compared to ACPA-/FDR. Of these, 32 DML were located on 22 genes. IPA showed these genes are involved in regulating the nuclear factor-κB complex and processes involved in antigen presentation, and immune cell crosstalk in autoimmunity. Pearson correlation analysis demonstrated a negative association between differentially methylated CpG in the C6ORF10 gene and risk factors associated with RA. Analysis by qPCR confirmed differential abundance of C6ORF10, TNXB, and HCG18 mRNA in patients with RA compared to ACPA-/FDR. CONCLUSION: Our results confirm the presence of differential methylation at specific gene loci within the MHC region of INA patients with RA. These epigenetic signatures may precede disease onset, or alternatively, may be a result of developing RA.

Identification of dynamic glucocorticoid-induced methylation changes at the FKBP5 locus.

BACKGROUND: Epigenetic mechanisms may play a major role in the biological embedding of early-life stress (ELS). One proposed mechanism is that glucocorticoid (GC) release following ELS exposure induces long-lasting alterations in DNA methylation (DNAm) of important regulatory genes of the stress response. Here, we investigate the dynamics of GC-dependent methylation changes in key regulatory regions of the FKBP5 locus in which ELS-associated DNAm changes have been reported. RESULTS: We repeatedly measured DNAm in human peripheral blood samples from 2 independent cohorts exposed to the GC agonist dexamethasone (DEX) using a targeted bisulfite sequencing approach, complemented by data from Illumina 450K arrays. We detected differentially methylated CpGs in enhancers co-localizing with GC receptor binding sites after acute DEX treatment (1 h, 3 h, 6 h), which returned to baseline levels within 23 h. These changes withstood correction for immune cell count differences. While we observed main effects of sex, age, body mass index, smoking, and depression symptoms on FKBP5 methylation levels, only the functional FKBP5 SNP (rs1360780) moderated the dynamic changes following DEX. This genotype effect was observed in both cohorts and included sites previously shown to be associated with ELS. CONCLUSION: Our study highlights that DNAm levels within regulatory regions of the FKBP5 locus show dynamic changes following a GC challenge and suggest that factors influencing the dynamics of this regulation may contribute to the previously reported alterations in DNAm associated with current and past ELS exposure.