Infinium HumanMethylation450 BeadChip Research Articles

O03 Genetic impacts on the skin methylome

Abstract Introduction and aims DNA methylation at CpG dinucleotides is a dynamic epigenetic mechanism that has been linked to numerous skin phenotypes and diseases. Previous studies in blood show that a substantial proportion of the human DNA methylome exhibits evidence for genetic influence, and that 15–17% of narrow-sense methylation heritability is explained by genetic variants, or methylation quantitative trait loci (meQTLs). However, no genome-wide meQTL studies have previously been performed in skin. The primary purpose of this work was to identify the genetic basis of DNA methylation levels in whole skin. Methods Utilizing a twin-based heritability model, we estimated DNA methylation heritability using 414 bulk skin tissue samples from healthy older female twins from the TwinsUK cohort, profiled using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA). We then integrated genomic data for 394 of these twins to perform genome-wide meQTL analyses. Finally, we integrated these meQTL Results with skin eQTL Results from an overlapping sample (N = 664). Results Over 8.2% of measured CpG sites showed evidence for strong heritability (h2 > 0.4), with a mean additive heritability estimate of 11.07% across all tested sites. We identified 76 442 CpG sites (18.8% of measured CpG sites) associated with a meQTL in cis (< 1 MB variant-CpG distance) and 1438 (0.35%) in trans. Both CpG sites under meQTL effects, and genetic variants underlying these meQTL effects in skin, are enriched in regions previously identified from genome-wide and epigenome-wide association studies for skin-related phenotypes, including psoriasis, melanoma and vitiligo. Additionally, some meQTLs influence gene expression via mediation of expression quantitative trait loci effects in skin, including in genes linked to melanoma and psoriasis. Conclusions Our study is the first genome-wide DNA methylation heritability and meQTL analysis of whole skin, giving insights into the regulatory landscape of epigenomic variation in skin.

Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

Investigation of Genetic Markers for Predicting Oral Cancer Progression and Patient Outcomes

ABSTRACT Background: Genomic methylation being used as a sensitive indicator for identifying oral potentially malignant disorders (OPMDS) and oral cancer could be a viable option. DNA methylation is a potential approach for “cancer therapeutics” and can help researchers truly understand the genomic mechanism that leads to cancer. Aim: Investigation of genetic markers for predicting oral cancer progression and patient outcomes. Methods and Materials: One hundred and twelve patients having oral premalignant lesions and malignant lesions were included in this study. Saliva samples were obtained for analysis of the expression of genetic markers, such as p16, DAP-K, and MGMT. The Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc., USA) would be used to assess genome-wide DNA methylation according to the manufacturer’s instructions. Results: Methylation of all the genes, that is, p16, DAP-K, and MGMT, was observed in 12.7% of oral cancer lesions. 41.94% of oral cancer lesions were found to have methylation of two genes. 13.23% cases were found to be associated with methylation of p16 and DAP-K, 12.12% cases were found to have methylation in the p16 and MGMT, and 15.27% cases of oral cancer lesions showed methylation of DAP-K and MGMT. Conclusion: Genetic markers, such as p53, DAP-K, and MGMT, can be used for predicting oral cancer progression and patient outcomes.

Sex-Specific Associations between Prenatal Exposure to Di(2-ethylhexyl) Phthalate, Epigenetic Age Acceleration, and Susceptibility to Early Childhood Upper Respiratory Infections.

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that can affect immune system development and susceptibility to infection. Aging processes (measured as epigenetic age acceleration (EAA)) may mediate the immune-related effects of prenatal exposure to DEHP. This study's objective was to examine associations between prenatal DEHP exposure, EAA at three months of age, and the number of upper respiratory infections (URIs) from 12 to 18 months of age using a sample of 69 maternal-child pairs from a Canadian pregnancy cohort. Blood DNA methylation data were generated using the Infinium HumanMethylation450 BeadChip; EAA was estimated using Horvath's pan-tissue clock. Robust regressions examined overall and sex-specific associations. Higher prenatal DEHP exposure (B = 6.52, 95% CI = 1.22, 11.81) and increased EAA (B = 2.98, 95% CI = 1.64, 4.32) independently predicted more URIs. In sex-specific analyses, some similar effects were noted for boys, and EAA mediated the association between prenatal DEHP exposure and URIs. In girls, higher prenatal DEHP exposure was associated with decreased EAA, and no mediation was noted. Higher prenatal DEHP exposure may be associated with increased susceptibility to early childhood URIs, particularly in boys, and aging biomarkers such as EAA may be a biological mechanism. Larger cohort studies examining the potential developmental immunotoxicity of phthalates are needed.

Metabolomic biomarkers of habitual B vitamin intakes unveil novel differentially methylated positions in the human epigenome

BackgroundB vitamins such as folate (B9), B6, and B12 are key in one carbon metabolism, which generates methyl donors for DNA methylation. Several studies have linked differential methylation to self-reported intakes of folate and B12, but these estimates can be imprecise, while metabolomic biomarkers can offer an objective assessment of dietary intakes. We explored blood metabolomic biomarkers of folate and vitamins B6 and B12, to carry out epigenome-wide analyses across up to three European cohorts. Associations between self-reported habitual daily B vitamin intakes and 756 metabolites (Metabolon Inc.) were assessed in serum samples from 1064 UK participants from the TwinsUK cohort. The identified B vitamin metabolomic biomarkers were then used in epigenome-wide association tests with fasting blood DNA methylation levels at 430,768 sites from the Infinium HumanMethylation450 BeadChip in blood samples from 2182 European participants from the TwinsUK and KORA cohorts. Candidate signals were explored for metabolite associations with gene expression levels in a subset of the TwinsUK sample (n = 297). Metabolomic biomarker epigenetic associations were also compared with epigenetic associations of self-reported habitual B vitamin intakes in samples from 2294 European participants.ResultsEighteen metabolites were associated with B vitamin intakes after correction for multiple testing (Bonferroni-adj. p < 0.05), of which 7 metabolites were available in both cohorts and tested for epigenome-wide association. Three metabolites — pipecolate (metabolomic biomarker of B6 and folate intakes), pyridoxate (marker of B6 and folate) and docosahexaenoate (DHA, marker of B6) — were associated with 10, 3 and 1 differentially methylated positions (DMPs), respectively. The strongest association was observed between DHA and DMP cg03440556 in the SCD gene (effect = 0.093 ± 0.016, p = 4.07E−09). Pyridoxate, a catabolic product of vitamin B6, was inversely associated with CpG methylation near the SLC1A5 gene promoter region (cg02711608 and cg22304262) and with SLC7A11 (cg06690548), but not with corresponding changes in gene expression levels. The self-reported intake of folate and vitamin B6 had consistent but non-significant associations with the epigenetic signals.ConclusionMetabolomic biomarkers are a valuable approach to investigate the effects of dietary B vitamin intake on the human epigenome.

An epigenome-wide association study of child appetitive traits and DNA methylation

The etiology of childhood appetitive traits is poorly understood. Early-life epigenetic processes may be involved in the developmental programming of appetite regulation in childhood. One such process is DNA methylation (DNAm), whereby a methyl group is added to a specific part of DNA, where a cytosine base is next to a guanine base, a CpG site. We meta-analyzed epigenome-wide association studies (EWASs) of cord blood DNAm and early-childhood appetitive traits. Data were from two independent cohorts: the Generation R Study (n = 1,086, Rotterdam, the Netherlands) and the Healthy Start study (n = 236, Colorado, USA). DNAm at autosomal methylation sites in cord blood was measured using the Illumina Infinium HumanMethylation450 BeadChip. Parents reported on their child's food responsiveness, emotional undereating, satiety responsiveness and food fussiness using the Children's Eating Behaviour Questionnaire at age 4–5 years. Multiple regression models were used to examine the association of DNAm (predictor) at the individual site- and regional-level (using DMRff) with each appetitive trait (outcome), adjusting for covariates. Bonferroni-correction was applied to adjust for multiple testing. There were no associations of DNAm and any appetitive trait when examining individual CpG-sites. However, when examining multiple CpGs jointly in so-called differentially methylated regions, we identified 45 associations of DNAm with food responsiveness, 7 associations of DNAm with emotional undereating, 13 associations of DNAm with satiety responsiveness, and 9 associations of DNAm with food fussiness. This study shows that DNAm in the newborn may partially explain variation in appetitive traits expressed in early childhood and provides preliminary support for early programming of child appetitive traits through DNAm. Investigating differential DNAm associated with appetitive traits could be an important first step in identifying biological pathways underlying the development of these behaviors.

Novel insights into the whole-blood DNA methylome of asthma in ethnically diverse children and youth.

The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.

Associations between epigenome-wide DNA methylation and height-related traits among Sub-Saharan Africans: the RODAM study.

Human height and related traits are highly complex, and extensively research has shown that these traits are determined by both genetic and environmental factors. Such factors may partially affect these traits through epigenetic programing. Epigenetic programing is dynamic and plays an important role in controlling gene expression and cell differentiation during (early) development. DNA methylation (DNAm) is the most commonly studied epigenetic feature. In this study we conducted an epigenome-wide DNAm association analysis on height-related traits in a Sub-Saharan African population, in order to detect DNAm biomarkers across four height-related traits. DNAm profiles were acquired in whole blood samples of 704 Ghanaians, sourced from the Research on Obesity and Diabetes among African Migrants study, using the Illumina Infinium HumanMethylation450 BeadChip. Linear models were fitted to detect differentially methylated positions (DMPs) and regions (DMRs) associated with height, leg-to-height ratio (LHR), leg length, and sitting height. No epigenome-wide significant DMPs were recorded. However we did observe among our top DMPs five informative probes associated with the height-related traits: cg26905768 (leg length), cg13268132 (leg length), cg19776793 (height), cg23072383 (LHR), and cg24625894 (sitting height). All five DMPs are annotated to genes whose functions were linked to bone cell regulation and development. DMR analysis identified overlapping DMRs within the gene body of HLA-DPB1 gene, and the HOXA gene cluster. In this first epigenome-wide association studies of these traits, our findings suggest DNAm associations with height-related heights, and might influence development and maintenance of these traits. Further studies are needed to replicate our findings, and to elucidate the molecular mechanism underlying human height-related traits.

DNA methylation-based depiction of the immune microenvironment and immune-associated long non-coding RNAs in oral cavity squamous cell carcinomas

Oral cavity squamous cell carcinoma (OSCC) is a complex and dynamic disease characterized by clinicopathological and molecular heterogeneity. Spatial and temporal heterogeneity of cell subpopulations has been associated with cancer progression and implicated in the prognosis and therapy response. Emerging evidence indicates that aberrant epigenetic profiles in OSCC may foster an immunosuppressive tumor microenvironment by modulating the expression of immune-related long non-coding RNAs (lncRNAs). DNA methylation analysis was performed in 46 matched OSCC and normal adjacent tissue samples using a genome-wide platform (Infinium HumanMethylation450 BeadChip). Reference-based computational deconvolution (MethylCIBERSORT) was applied to infer the immune cell composition of the bulk samples. The expression levels of genes encoding immune markers and differentially methylated lncRNAs were investigated using The Cancer Genome Atlas dataset. OSCC specimens presented distinct immune cell composition, including the enrichment of monocyte lineage cells, natural killer cells, cytotoxic T-lymphocytes, regulatory T-lymphocytes, and neutrophils. In contrast, B-lymphocytes, effector T-lymphocytes, and fibroblasts were diminished in tumor samples. The hypomethylation of three immune-associated lncRNAs (MEG3, MIR155HG, and WFDC21P) at individual CpG sites was confirmed by bisulfite-pyrosequencing. Also, the upregulation of a set of immune markers (FOXP3, GZMB, IL10, IL2RA, TGFB, IFNG, TDO2, IDO1, and HIF1A) was detected. The immune cell composition, immune markers alteration, and dysregulation of immune-associated lncRNAs reinforce the impact of the immune microenvironment in OSCC. These concurrent factors contribute to tumor heterogeneity, suggesting that epi-immunotherapy could be an efficient alternative to treat OSCC.

Subgrouping of Burkitt lymphoma variants by DNA methylation is driven by an EBV‐associated epigenotype

Introduction: Burkitt lymphoma (BL) is an aggressive mature B-cell lymphoma, which traditionally has been separated into three epidemiologic variants: endemic (eBL), sporadic (sBL) and immunodeficiency-associated BL (iBL). This subgrouping is strongly confounded by EBV-infection. Indeed, there is evidence from molecular studies that subtyping of BL based on EBV status might better reflect the pathogenetic heterogeneity than epidemiology. As EBV is known to influence epigenetic regulation, we here aimed at a comparative DNA methylation (DNAme) profiling of BL variants with respect to their geographic origin and EBV status. Methods: We collected DNAme profiles (Infinium HumanMethylation450 and Infinium MethylationEPIC BeadChip) of 116 BL patients (80 sBL: 7 EBV+; 29 eBL: 27 EBV+; 7 iBL: 4 EBV+), 17 BL cell lines and 6 EBV-transformed LCLs. For group determination unsupervised clustering algorithms (K-means, PGMRA, UMAP) were applied. To investigate potential consequences of EBV-associated DNAme we performed a protein-protein interaction network analysis on genes within the EBV-specific DNAme pattern. Moreover, we mined gene expression values from RNAseq in 21 EBV- sBL and 5 germinal-center B-cell populations. Results: Unsupervised clustering algorithms revealed two distinct DNAme phenotypes, which differentiate EBV+ and EBV- BL based on 1,266 CpGs. This separation is accompanied by a characteristic hypermethylation in EBV+ BLs. Additionally, we identified a subgroup of EBV+ BL with a similar DNAme pattern like the LCLs, possibly indicating latency phase differences within EBV+ BLs. Investigation of the EBV-specific DNAme pattern revealed a network with UBC as a key interactor, indicating the potential importance of the ubiquitin-proteasome system (UPS) in EBV-induced lymphomagenesis. Among genes differentially methylated between EBV+ and EBV- BL, 19 genes have been reported as recurrently mutated in BL. All except 3 of these genes are expressed in sBL and germinal-center B-cells. All these genes were significantly hypermethylated within regulatory regions in EBV+ BLs (σ/σmax = 0.4, q ≤ 0.01), including genes with a lower mutational frequency in EBV+ versus EBV- BLs. Conclusion: The findings of this study show the EBV status to be strongly associated with DNAme subgroups in BL, supporting the subtyping of BL variants based on their EBV status rather than their geographic origin. Moreover, the pattern of DNAme in EBV+ BL might suggest epigenetic silencing and deregulated ubiquitination as means alternative to gene mutation to be involved in the pathogenesis of EBV+ BL. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Genomics, Epigenomics, and Other -Omics, Pathology and Classification of Lymphomas No conflicts of interests pertinent to the abstract.

Time to relapse in chronic lymphocytic leukemia and DNA-methylation-based biological age

Chronic lymphocytic leukemia (CLL) is a mature B cell neoplasm with a predilection for older individuals. While previous studies have identified epigenetic signatures associated with CLL, whether age-related DNA methylation changes modulate CLL relapse remains elusive. In this study, we examined the association between epigenetic age acceleration and time to CLL relapse in a publicly available dataset. DNA methylation profiling of 35 CLL patients prior to initiating chemoimmunotherapy was performed using the Infinium HumanMethylation450 BeadChip. Four epigenetic age acceleration metrics (intrinsic epigenetic age acceleration [IEAA], extrinsic epigenetic age acceleration [EEAA], PhenoAge acceleration [PhenoAA], and GrimAge acceleration [GrimAA]) were estimated from blood DNA methylation levels. Linear, quantile, and logistic regression and receiver operating characteristic curve analyses were conducted to assess the association between each epigenetic age metric and time to CLL relapse. EEAA (p = 0.011) and PhenoAA (p = 0.046) were negatively and GrimAA (p = 0.040) was positively associated with time to CLL relapse. Simultaneous assessment of EEAA and GrimAA in male patients distinguished patients who relapsed early from patients who relapsed later (p = 0.039). No associations were observed with IEAA. These findings suggest epigenetic age acceleration prior to chemoimmunotherapy initiation is associated with time to CLL relapse. Our results provide novel insight into the association between age-related DNA methylation changes and CLL relapse and may serve has biomarkers for treatment relapse, and potentially, treatment selection.

526 Clinical Characteristics and Biomarkers of Aggressive Nonfunctional Pituitary Adenomas and Pituitary Carcinomas

INTRODUCTION: Nonfunctioning pituitary adenomas (NFPAs) are benign tumors of the pituitary gland. While surgery is the treatment of choice, a subset of aggressive NFPAs is associated with multiple recurrences and treatments. Pituitary carcinomas are a rare group of pituitary tumors, comprised of both functional and non-functional, which metastasize to other locations. METHODS: A retrospective review of 601 consecutive patients who underwent endoscopic transsphenoidal surgery between 2010 and 2018 at one institution was conducted. Among 209 NFPA patients, 29 patients had aggressive NFPAs, with ≥2 recurrences and ≥3 treatments. We used Infinium HumanMethylation450 BeadChip arrays along with R (R Core 2014) and Bioconductor for methylation analysis of patients with indolent NFPAs (n = 6), aggressive NFPAs (n = 8), pituitary carcinomas (n = 9), and normal pituitary glands (n=3). RESULTS: In multivariate analysis of clinical features, tumor volume and proliferative index were higher in aggressive NFPAs compared to indolent NFPAs. Although significantly different, these features were not specific to aggressive NFPAs. Methylation analysis showed no significant global methylation differences among all samples. However, we identified significant probe-wise differentiation in methylation of over 100 genes between indolent and aggressive NFPA groups. Heat map representation of the most significant differentially methylated genes demonstrated similarity in methylation patterns between the aggressive NFPA and pituitary carcinoma group, which were distinct from the indolent group. CONCLUSIONS: Tumor volume and proliferative index were significantly increased in the aggressive compared to the indolent group. In DNA methylation analyses, we identified numerous hypermethylated genes in the aggressive and pituitary carcinoma groups, which were distinct from the indolent group. DNA methylation may provide a useful predictive biomarker to identify aggressive NFPA types and potentially reveal common mechanistic links between aggressive NFPAs and pituitary carcinomas.

DNA methylation patterns associated with breast cancer prognosis that are specific to tumor subtype and menopausal status.

Tumor subtype and menopausal status are strong predictors of breast cancer (BC) prognosis. We aimed to find and validate subtype- or menopausal-status-specific changes in tumor DNA methylation (DNAm) associated with all-cause mortality or BC progression. Associations between site-specific tumor DNAm and BC prognosis were estimated among The Cancer Genome Atlas participants (n = 692) with Illumina Infinium HumanMethylation450 BeadChip array data. All-cause mortality and BC progression were modeled using Cox proportional hazards models stratified by tumor subtypes, adjusting for age, race, stage, menopausal status, tumor purity, and cell type proportion. Effect measure modification by subtype and menopausal status were evaluated by incorporating a product term with DNAm. Site-specific inference was used to identify subtype- or menopausal-status-specific differentially methylated regions (DMRs) and functional pathways. The validation of the results was carried out on an independent dataset (GSE72308; n = 180). We identified a total of fifteen unique CpG probes that were significantly associated ( with survival outcomes in subtype- or menopausal-status-specific manner. Seven probes were associated with overall survival (OS) or progression-free interval (PFI) for women with luminal A subtype, and four probes were associated with PFI for women with luminal B subtype. Five probes were associated with PFI for post-menopausal women. A majority of significant probes showed a lower risk of OS or BC progression with higher DNAm. We identified subtype- or menopausal-status-specific DMRs and functional pathways of which top associated pathways differed across subtypes or menopausal status. None of significant probes from site-specific analyses met genome-wide significant level in validation analyses while directions and magnitudes of coefficients showed consistent pattern. We have identified subtype- or menopausal-status-specific DNAm biomarkers, DMRs and functional pathways associated with all-cause mortality or BC progression, albeit with limited validation. Future studies with larger independent cohort of non-post-menopausal women with non-luminal A subtypes are warranted for identifying subtype- and menopausal-status-specific DNAm biomarkers for BC prognosis.

Liver saturated fat content associates with hepatic DNA methylation in obese individuals

BackgroundAccumulation of saturated fatty acids (SFAs) in the liver is known to induce hepatic steatosis and inflammation causing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although SFAs have been shown to affect the epigenome in whole blood, pancreatic islets, and adipose tissue in humans, and genome-wide DNA methylation studies have linked epigenetic changes to NAFLD and NASH, studies focusing on the association of SFAs and DNA methylation in human liver are missing. We, therefore, investigated whether human liver SFA content associates with DNA methylation and tested if SFA-linked alterations in DNA methylation associate with NAFLD-related clinical phenotypes in obese individuals.ResultsWe identified DNA methylation (Infinium HumanMethylation450 BeadChip) of 3169 CpGs to be associated with liver total SFA content (q-value < 0.05) measured using proton NMR spectroscopy in participants of the Kuopio Obesity Surgery Study (n = 51; mean ± SD:49.3 ± 8.5 years old; BMI:43.7 ± 6.2 kg/m2). Of these 3169 sites, 797 overlapped with previously published NASH-associated CpGs (NASH-SFA), while 2372 CpGs were exclusively associated with SFA (Only-SFA). The corresponding annotated genes of these only-SFA CpGs were found to be enriched in pathways linked to satiety and hunger. Among the 54 genes mapping to these enriched pathways, DNA methylation of CpGs mapping to PRKCA and TSPO correlated with their own mRNA expression (HumanHT-12 Expression BeadChip). In addition, DNA methylation of another ten of these CpGs correlated with the mRNA expression of their neighboring genes (p value < 0.05). The proportion of CpGs demonstrating a correlation of DNA methylation with plasma glucose was higher in NASH-SFA and only-SFA groups, while the proportion of significant correlations with plasma insulin was higher in only-NASH and NASH-SFA groups as compared to all CpGs on the Illumina 450 K array (Illumina, San Diego, CA, USA).ConclusionsOur results suggest that one of the mechanisms how SFA could contribute to metabolic dysregulation in NAFLD is at the level of DNA methylation. We further propose that liver SFA-related DNA methylation profile may contribute more to hyperglycemia, while insulin-related methylation profile is more linked to NAFLD or NASH. Further research is needed to elucidate the molecular mechanisms behind these observations.

DNA methylation of GITR, OX40, 4-1BB, CD27 , and CD40 correlates with BAP1 aberrancy and prognosis in uveal melanoma.

Uveal melanoma represents an aggressive tumor that responds mostly poorly to established melanoma treatments. Comprehensive methylation profiling of the next-generation immunotherapeutic target genes, for example, members of the tumor necrosis factor receptor superfamily, might allow for the development of companion predictive biomarkers. We have analyzed CpG sites within the immune checkpoint genes GITR, OX40, 4-1BB, CD 27, and CD40 probed by the Illumina Infinium HumanMethylation450 BeadChip in N = 80 uveal melanomas included in The Cancer Genome Atlas with regard to BAP1 aberrancy, mRNA expression, and overall survival. In all analyzed immune checkpoint genes, BAP1 aberrancy was associated with decreased CpG methylation levels. We identified specific CpG sites that significantly correlated with BAP1 aberrancy, mRNA expression levels, and overall survival. Our results suggest epigenetic regulation of the analyzed immune checkpoint genes via DNA methylation in uveal melanoma and provide rationale for methylation testing in biomarker programs in clinical trials.

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.

A comparison of DNA methylation profiles of blood mononuclear cells in patients with multiple sclerosis in remission and relapse

To study the whole-genome DNA methylation profiles of peripheral blood mononuclear blood cells (PBMCs) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and relapse in order to assess the contribution of this epigenetic mechanism of gene expression regulation to the activity of the pathological process. Eight patients with RRMS in remission and 6 patients in relapse were included in the study. Methylation levels of DNA CpG sites in PBMCs were analyzed using Infinium HumanMethylation450 BeadChip DNA microarrays. Seven differentially methylated positions (DMPs) were identified, of which 3 were hypermethylated (cg02981003, cg18486102, cg19533582) and 4 were hypomethylated (cg16814680, cg1964802, cg18584440, cg08291996) during RRMS relapse. Five DMPs are located in protein-coding genes (GPR123, FAIM2, BTNL2, ZNF8, ASAP2), one in microRNA gene (MIR548N), and one in an intergenic region. For all identified DMPs, we observed a change in DNA methylation levels of more than 20% (range 20.2-57.5%). Hierarchical clustering of DNA samples on the heatmap shows their clear aggregation into separate clusters corresponding to RRMS patients in the stages of relapse and remission. For the first time it was shown that during relapse and remission of RRMS there are differences in the DNA methylation profile that allow discrimination between these clinical stages. These data indicate the involvement of the epigenetic mechanism of DNA methylation in the activation of the pathological process in RRMS.

Pooled analysis of epigenome-wide association studies of food consumption in KORA, TwinsUK and LLS

PurposeExamining epigenetic patterns is a crucial step in identifying molecular changes of disease pathophysiology, with DNA methylation as the most accessible epigenetic measure. Diet is suggested to affect metabolism and health via epigenetic modifications. Thus, our aim was to explore the association between food consumption and DNA methylation.MethodsEpigenome-wide association studies were conducted in three cohorts: KORA FF4, TwinsUK, and Leiden Longevity Study, and 37 dietary exposures were evaluated. Food group definition was harmonized across the three cohorts. DNA methylation was measured using Infinium MethylationEPIC BeadChip in KORA and Infinium HumanMethylation450 BeadChip in the Leiden study and the TwinsUK study. Overall, data from 2293 middle-aged men and women were included. A fixed-effects meta-analysis pooled study-specific estimates. The significance threshold was set at 0.05 for false-discovery rate-adjusted p values per food group.ResultsWe identified significant associations between the methylation level of CpG sites and the consumption of onions and garlic (2), nuts and seeds (18), milk (1), cream (11), plant oils (4), butter (13), and alcoholic beverages (27). The signals targeted genes of metabolic health relevance, for example, GLI1, RPTOR, and DIO1, among others.ConclusionThis EWAS is unique with its focus on food groups that are part of a Western diet. Significant findings were mostly related to food groups with a high-fat content.

Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis

BackgroundIn recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH.ResultsNormal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort.ConclusionsAfter a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

(Peri)Centromeric Rearrangements Are Recurrent Recombination Events in Complex-Karyotype Chronic Lymphocytic Leukemia

Background: A retrospective cytogenetic study encompassing 5290 patients with chronic lymphocytic leukemia (CLL) identified complex karyotype (CK) as adverse prognostic factor. In cases with ≥5 chromosomal aberrations (CAs), this finding was independent of TP53 alterations (Baliakas et al., 2019) Which chromosomal rearrangements dominate CK-CLL is insufficiently defined impeding the identification of genomic events responsible for poor prognosis. To delineate cytogenetic aberrations in CK-CLL, we analysed conventional karyotypes from 125 patients classified as high-risk during the era of chemoimmunotherapy. Methods: Chromosome banding (CBA, CpG oligonucleotide and IL-2 stimulation, N=131), SNP-array analysis (Affymetrix 6.0, N=109), DNA methylation analysis (Illumina Infinium HumanMethylation450 BeadChips, N=45) and telomere length measurement (N=122) was conducted on samples taken at enrolment on the CLL2O trial of the GCLLSG/FCLLSG (NCT01392079). This trial encompassed CLL patients (pts) with TP53 alteration (deletion/mutation, TP53alt; treatment-naïve or purine-analogue relapsed/refractory) and TP53intact purine-analogue refractory disease. CBA and TP53 mutation data from the CLL11 trial of the GCLLSG (NCT01010061, N=152) was integrated for multiple linear regression. Results: An informative karyotype was obtained in 125/131 pts (for clinical/genetic features see Table). The total number of CBA-detected CAs was 618 with an average of 4.9 CAs per case (range 0-21). Pts with TP53alt had a significantly higher karyotype complexity than pts without (5.3 vs 2.7 CAs mean, p=0.02). A normal karyotype was found in 2 pts, a non-CK in 32 pts (≤2 CAs; 20 pts TP53alt), a low- or intermediate-CK in 34 pts (3 or 4 CAs, 30 pts TP53alt), and a high-CK in57 pts (≥5CAs, 54 pts TP53alt). Cytogenetic aberrations comprised 341 derivative chromosomes, 131 interstitial/terminal losses, 65 monosomies, 53 balanced translocations, 21 trisomies, 5 inversions, and 2 interstitial gains. Remarkably, 308/618 CAs had at least one breakpoint in the centromere (p10/q10; N=70) or in p11/q11 (N=238) directing our interest towards (peri-)centromeric breakage. Chr17 was most frequently affected linking (peri-)centromeric breakage to TP53 loss. Whole arm translocations (WAT) constituted the most common cause for TP53 loss (N=47 in 39 pts) with chr18 (N=9), chr17 (N=6), chr8 (N=6) and chr15 (N=6) being the most frequent translocation partners. Other cytogenetic events underlying TP53 loss were complex unbalanced rearrangements (N=40 in 39 pts), simple interstitial/terminal deletion (N=22 in 22 pts) and dicentric chromosomes (DIC, N=8 in 8 pts, 6 with breakpoints in p11/q11). In total, 98/117 aberrations causing TP53 loss had breakpoints in (peri-)centromeric cytobands. Mapping chr17 breakpoints based on SNP-array results revealed that more than half (58%) clustered within a 1 Mb region on both sides of the centromere. In contrast, chr17 breakpoints in CK-AML are random suggesting that chr17 (peri-)centromeric instability is specific for high-risk CLL. (Peri-)centromeric rearrangements in form of WAT and DIC were also found to occur without chr17 involvement (N=24 in 20 pts and N=12 in 8 pts, respectively). Chromosome involvement was random pointing towards general (peri-)centromeric instability. Multiple linear regression revealed only karyotype complexity but not TP53alt or prior treatment as significant risk factor for WAT/DIC substantiating the notion that (peri-)centromeric instability is an intrinsic hallmark of high-risk CLL. DNA methylation patterns in regions adjacent to pericentromeric breakpoint clusters located on chr17p and chr18p showed no abnormalities when compared to cases without chr17/chr18 aberrations. Telomere length was significantly shorter in cases harboring at least one (peri-)centromeric rearrangement (p<0.001), but this association could not be confirmed when restricting the analysis to non-CK cases (N=20 vs 13, p=0.08). Conclusions: Karyotype complexity was confirmed as typical feature of high-risk CLL. (Peri-)centromeric aberrations showed up as new hallmark characteristic of CK-CLL and WAT/DIC as otherwise rare events in hematologic malignancies were frequently observed. The underlying reasons for (peri-)centromeric breakage and its prognostic impact beyond karyotype complexity and TP53 alterations require further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal