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  • 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic and predictive biomarkers for coronary artery disease
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-21
    Chaoran Dong; Jiemei Chen; Jilin Zheng; Yiming Liang; Tao Yu; Yupeng Liu; Feng Gao; Jie Long; Hangyu Chen; Qianhui Zhu; Zilong He; Songnian Hu; Chuan He; Jian Lin; Yida Tang; Haibo Zhu

    The 5-hydroxymethylcytosine (5hmC) DNA modification is an epigenetic marker involved in a range of biological processes. Its function has been studied extensively in tumors, neurodegenerative diseases, and atherosclerosis. Studies have reported that 5hmC modification is closely related to the phenotype transformation of vascular smooth muscle cells and endothelial dysfunction. However, its role in coronary artery disease (CAD) has not been fully studied. To investigate whether 5hmC modification correlates with CAD pathogenesis and whether 5hmC can be used as a biomarker, we used a low-input whole-genome sequencing technology based on selective chemical capture (hmC-Seal) to firstly generate the 5hmC profiles in the circulating cell-free DNA(cfDNA) of CAD patients, including stable coronary artery disease (sCAD) patients and acute myocardial infarction (AMI) patients. We detected a significant difference of 5hmC enrichment in gene bodies from CAD patients compared with normal coronary artery (NCA) individuals. Our results showed that CAD patients can be well separated from NCA individuals by 5hmC markers. The prediction performance of the model established by differentially regulated 5hmc modified genes were superior to common clinical indicators for the diagnosis of CAD (AUC = 0.93) and sCAD (AUC = 0.93). Specially, we found that 5hmC markers in cfDNA showed prediction potential for AMI (AUC = 0.95), which was superior to that of cardiac troponin I, muscle/brain creatine kinase, and myoglobin. Our results suggest that 5hmC markers derived from cfDNA can serve as effective epigenetic biomarkers for minimally noninvasive diagnosis and prediction of CAD.

    更新日期:2020-01-22
  • The effects of bariatric surgery on clinical profile, DNA methylation, and ageing in severely obese patients
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-20
    Eliza Fraszczyk; Mirjam Luijten; Annemieke M. W. Spijkerman; Harold Snieder; Paul F. K. Wackers; Vincent W. Bloks; Carolina F. Nicoletti; Carla B. Nonino; Ana B. Crujeiras; Wim A. Buurman; Jan Willem Greve; Sander S. Rensen; Bruce H. R. Wolffenbuttel; Jana V. van Vliet-Ostaptchouk

    Severe obesity is a growing, worldwide burden and conventional therapies including radical change of diet and/or increased physical activity have limited results. Bariatric surgery has been proposed as an alternative therapy showing promising results. It leads to substantial weight loss and improvement of comorbidities such as type 2 diabetes. Increased adiposity is associated with changes in epigenetic profile, including DNA methylation. We investigated the effect of bariatric surgery on clinical profile, DNA methylation, and biological age estimated using Horvath’s epigenetic clock. To determine the impact of bariatric surgery and subsequent weight loss on clinical traits, a cohort of 40 severely obese individuals (BMI = 30–73 kg/m2) was examined at the time of surgery and at three follow-up visits, i.e., 3, 6, and 12 months after surgery. The majority of the individuals were women (65%) and the mean age at surgery was 45.1 ± 8.1 years. We observed a significant decrease over time in BMI, fasting glucose, HbA1c, HOMA-IR, insulin, total cholesterol, triglycerides, LDL and free fatty acids levels, and a significant small increase in HDL levels (all p values < 0.05). Epigenome-wide association analysis revealed 4857 differentially methylated CpG sites 12 months after surgery (at Bonferroni-corrected p value < 1.09 × 10−7). Including BMI change in the model decreased the number of significantly differentially methylated CpG sites by 51%. Gene set enrichment analysis identified overrepresentation of multiple processes including regulation of transcription, RNA metabolic, and biosynthetic processes in the cell. Bariatric surgery in severely obese patients resulted in a decrease in both biological age and epigenetic age acceleration (EAA) (mean = − 0.92, p value = 0.039). Our study shows that bariatric surgery leads to substantial BMI decrease and improvement of clinical outcomes observed 12 months after surgery. These changes explained part of the association between bariatric surgery and DNA methylation. We also observed a small, but significant improvement of biological age. These epigenetic changes may be modifiable by environmental lifestyle factors and could be used as potential biomarkers for obesity and in the future for obesity related comorbidities.

    更新日期:2020-01-21
  • A DNA methylation signature to improve survival prediction of gastric cancer
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-20
    Yaojun Peng; Qiyan Wu; Lingxiong Wang; Huan Wang; Fan Yin

    The current Union International Committee on Cancer or the American Joint Committee on Cancer TNM stage system has shown valuable but insufficient estimation for subsets of gastric cancer and prediction for prognosis patients. Thus, there is an urgent need to identify diagnostic, prognostic, and predictive biomarkers to improve patients’ outcomes. Our aim was to perform an integrative analysis on publicly available datasets to identify epigenetic changes that may play key role in the initiation and progression of gastric cancer, based on which we set to develop a DNA methylation signature to improve survival prediction of gastric cancer. A total of 340 methylation-related differentially expression genes (mrDEGs) were screened in gastric cancer patients from The Cancer Genome Atlas (TCGA) project. Pathway enrichment analysis revealed that they were involved in the biological process related to initiation and progression of gastric cancer. Based on the mrDEGs identified, we developed a DNA methylation signature consisting of ten gene members (SCNN1B, NFE2L3, CLDN2, RBPMS2, JPH2, GBP6, COL4A5, SMKR1, PPP1R14A, and ARL4D) according to their methylation β value. This innovative DNA methylation signature was associated with cancer recurrence, while it showed independence of cancer recurrence and TNM stage for survival prediction. Combination of this DNA methylation signature and TNM stage improved overall survival prediction in the receiver operating characteristic analysis. We also verified that two individual genes (PPP1R14A and SCNN1B) of the identified prognostic signature were regulated by promoter region methylation in a panel of gastric cell lines. This study presents a powerful DNA methylation signature by performing analyses integrating multi-source data including transcriptome, methylome, and clinical outcome of gastric cancer patients from TCGA. The identified DNA methylation signature may be used to refine the current prognostic model and facilitate further stratification of patients in the future clinical trials. Further experimental studies are warranted to unveil the regulatory mechanism and functional role of all the individual genes of the DNA methylation signature. Also, clinical investigations in large GC patient cohorts are greatly needed to validate our findings.

    更新日期:2020-01-21
  • Methylome and transcriptome analyses reveal insights into the epigenetic basis for the good survival of hypomethylated ER-positive breast cancer subtype
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-20
    Xiao-Qiong Chen; Fan Zhang; Qi-Chen Su; Chi Zeng; Fu-Hui Xiao; Yu Peng

    Breast cancer (BRCA) is a heterogeneous disease, characterized by different histopathological and clinical features and responses to various therapeutic measures. Despite the research progress of DNA methylation in classification and diagnosis of BRCA and the close relationship between DNA methylation and hormone receptor status, especially estrogen receptor (ER), the epigenetic mechanisms in various BRCA subtypes and the biomarkers associated with diagnostic characteristics of patients under specific hormone receptor status remain elusive. In this study, we collected and analyzed methylation data from 785 invasive BRCA and 98 normal breast tissue samples from The Cancer Genome Atlas (TCGA) database. Consensus classification analysis revealed that ER-positive BRCA samples were constitutive of two distinct methylation subgroups; with the hypomethylated subgroup showing good survival probability. This finding was further supported by another cohort of ER-positive BRCA containing 30 subjects. Additionally, we identified 977 hypomethylated CpG loci showing significant associations with good survival probability in ER-positive BRCA. Genes with these loci were enriched in cancer-related pathways (e.g., Wnt signaling pathway). Among them, the upregulated 47 genes were also in line with good survival probability of ER-positive BRCA, while they showed significantly negative correlations between their expression and methylation level of certain hypomethylated loci. Functional assay in numerous literatures provided further evidences supporting that some of the loci have close links with the modulation of tumor-suppressive mechanisms via regulation gene transcription (e.g., SFRP1 and WIF1). Our study identified a hypomethylated ER-positive BRCA subtype. Notably, this subgroup presented the best survival probability compared with the hypermethylated ER-positive and hypomethylated ER-negative BRCA subtypes. Specifically, we found that certain upregulated genes (e.g., SFRP1 and WIF1) have great potential to suppress the progression of ER-positive BRCA, concurrently exist negative correlations between their expression and methylation of corresponding hypomethylated CpG loci. Therefore, our study indicates that different epigenetic mechanisms likely exist in ER-positive BRCA and provides novel clinical biomarkers specific to ER-positive BRCA diagnosis and therapy.

    更新日期:2020-01-21
  • Transcriptomic profiling disclosed the role of DNA methylation and histone modifications in tumor-infiltrating myeloid-derived suppressor cell subsets in colorectal cancer
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-15
    Varun Sasidharan Nair; Reem Saleh; Salman M. Toor; Rowaida Z. Taha; Ayman A. Ahmed; Mohamed A. Kurer; Khaled Murshed; Nehad M. Alajez; Mohamed Abu Nada; Eyad Elkord

    Increased numbers of myeloid-derived suppressor cells (MDSCs) are positively correlated with poor prognosis and reduced survivals of cancer patients. They play central roles in tumor immune evasion and tumor metastasis. However, limited data are available on phenotypic/transcriptomic characteristics of the different MDSCs subsets in cancer. These cells include immature (I-MDSCs), monocytic (M-MDSCs), and polymorphonuclear/granulocytic (PMN-MDSCs). Phenotypic characterization of myeloid subsets from 27 colorectal cancer (CRC) patients was assessed by flow cytometric analyses. RNA-sequencing of sorted I-MDSCs, PMN-MDSCs, and antigen-presenting cells (APCs) was also performed. We found that the levels of I-MDSCs and PMN-MDSCs were increased in tumor tissues (TT), compared with normal tissues (NT) in colorectal cancer. Our functional annotation analyses showed that genes associated with histone deacetylase (HDAC) activation- and DNA methylation-mediated transcriptional silencing were upregulated, and histone acetyl transferase (HAT)-related genes were downregulated in tumor-infiltrating I-MDSCs. Moreover, pathways implicated in cell trafficking and immune suppression, including Wnt, interleukin-6 (IL-6), and mitogen-activated protein kinase (MAPK) signaling, were upregulated in I-MDSCs. Notably, PMN-MDSCs showed downregulation in genes related to DNA methylation and HDAC binding. Using an ex vivo model, we found that inhibition of HDAC activation or neutralization of IL-6 in CRC tumor tissues downregulates the expression of genes associated with immunosuppression and myeloid cell chemotaxis, confirming the importance of HDAC activation and IL-6 signaling pathway in MDSC function and chemotaxis. This study provides novel insights into the epigenetic regulations and other molecular pathways in different myeloid cell subsets within the CRC tumor microenvironment (TME), giving opportunities to potential targets for therapeutic benefits.

    更新日期:2020-01-15
  • Longitudinal epigenome-wide association studies of three male military cohorts reveal multiple CpG sites associated with post-traumatic stress disorder
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-13
    Clara Snijders; Adam X. Maihofer; Andrew Ratanatharathorn; Dewleen G. Baker; Marco P. Boks; Elbert Geuze; Sonia Jain; Ronald C. Kessler; Ehsan Pishva; Victoria B. Risbrough; Murray B. Stein; Robert J. Ursano; Eric Vermetten; Christiaan H. Vinkers; Alicia K. Smith; Monica Uddin; Bart P. F. Rutten; Caroline M. Nievergelt

    Epigenetic mechanisms have been suggested to play a role in the development of post-traumatic stress disorder (PTSD). Here, blood-derived DNA methylation data (HumanMethylation450 BeadChip) collected prior to and following combat exposure in three cohorts of male military members were analyzed to assess whether DNA methylation profiles are associated with the development of PTSD. A total of 123 PTSD cases and 143 trauma-exposed controls were included in the analyses. The Psychiatric Genomics Consortium (PGC) PTSD EWAS QC pipeline was used on all cohorts, and results were combined using a sample size weighted meta-analysis in a two-stage design. In stage one, we jointly analyzed data of two new cohorts (N = 126 and 78) for gene discovery, and sought to replicate significant findings in a third, previously published cohort (N = 62) to assess the robustness of our results. In stage 2, we aimed at maximizing power for gene discovery by combining all three cohorts in a meta-analysis. Stage 1 analyses identified four CpG sites in which, conditional on pre-deployment DNA methylation, post-deployment DNA methylation was significantly associated with PTSD status after epigenome-wide adjustment for multiple comparisons. The most significant (intergenic) CpG cg05656210 (p = 1.0 × 10−08) was located on 5q31 and significantly replicated in the third cohort. In addition, 19 differentially methylated regions (DMRs) were identified, but failed replication. Stage 2 analyses identified three epigenome-wide significant CpGs, the intergenic CpG cg05656210 and two additional CpGs located in MAD1L1 (cg12169700) and HEXDC (cg20756026). Interestingly, cg12169700 had an underlying single nucleotide polymorphism (SNP) which was located within the same LD block as a recently identified PTSD-associated SNP in MAD1L1. Stage 2 analyses further identified 12 significant differential methylated regions (DMRs), 1 of which was located in MAD1L1 and 4 were situated in the human leukocyte antigen (HLA) region. This study suggests that the development of combat-related PTSD is associated with distinct methylation patterns in several genomic positions and regions. Our most prominent findings suggest the involvement of the immune system through the HLA region and HEXDC, and MAD1L1 which was previously associated with PTSD.

    更新日期:2020-01-13
  • DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing—a possible predictive biomarker for malignant transformation
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-13
    Shajedul Islam; Osamu Uehara; Hirofumi Matsuoka; Yasuhiro Kuramitsu; Bhoj Raj Adhikari; Daichi Hiraki; Seiko Toraya; Asiri Jayawardena; Ichiro Saito; Malsantha Muthumala; Hiroki Nagayasu; Yoshihiro Abiko; Itsuo Chiba

    DNA hypermethylation of tumor suppressor genes is observed in precancerous lesions and oral cancer of individuals with the habits of betel quid (BQ) chewing. SIRT1 has been identified as playing a role in the maintenance of epithelial integrity, and its alteration is often related to carcinogenesis. However, the methylation and transcription status of SIRT1 in patients with BQ chewing-related oral cancer has not been investigated. We examined the methylation status of SIRT1 in paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC) obtained from BQ chewing and non-chewing patients and in tissue samples from healthy control subjects. In addition, we examined whether the hypermethylation of SIRT1 followed by its transcriptional downregulation in the human gingival epithelial cells could be caused by arecoline, a major component of BQ. Furthermore, we investigated the methylation status of SIRT1 in smear samples of macroscopically healthy buccal mucosa from subjects with a habit of BQ chewing. SIRT1 was significantly hypermethylated in tissue samples of OSCC from BQ chewers and non-chewers than in oral mucosa from healthy control subjects. Results also showed that the hypermethylation level of SIRT1 was significantly higher in OSCC of patients with BQ chewing habits than in those of non-chewing habits (p < 0.05). Our in vitro model showed that hypermethylation is followed by downregulation of the transcriptional level of SIRT1 (p < 0.05). The methylation levels of SIRT1 in the smear samples obtained from BQ chewing individuals were significantly higher than those in the samples obtained from individuals that did not chew BQ. The duration of BQ chewing habits was correlated positively to the frequency of SIRT1 hypermethylation (p < 0.05). Our results suggest that DNA hypermethylation of SIRT1 is involved in the occurrence of oral cancer in BQ chewing patients and that hypermethylation in the oral mucosa of BQ chewers could be a predictive marker for the occurrence of malignant transformation. This is the first report that showed DNA hypermethylation in clinically healthy oral epithelium of BQ chewers. Our study shows evidence that DNA hypermethylation may be an early event of oral carcinogenesis prior to observable clinical changes.

    更新日期:2020-01-13
  • KMT2C/D COMPASS complex-associated diseases [KCDCOM-ADs]: an emerging class of congenital regulopathies
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-10
    William J. Lavery; Artem Barski; Susan Wiley; Elizabeth K. Schorry; Andrew W. Lindsley

    The type 2 lysine methyltransferases KMT2C and KMT2D are large, enzymatically active scaffold proteins that form the core of nuclear regulatory structures known as KMT2C/D COMPASS complexes (complex of proteins associating with Set1). These evolutionarily conserved proteins regulate DNA promoter and enhancer elements, modulating the activity of diverse cell types critical for embryonic morphogenesis, central nervous system development, and post-natal survival. KMT2C/D COMPASS complexes and their binding partners enhance active gene expression of specific loci via the targeted modification of histone-3 tail residues, in general promoting active euchromatic conformations. Over the last 20 years, mutations in five key COMPASS complex genes have been linked to three human congenital syndromes: Kabuki syndrome (type 1 [KMT2D] and 2 [KDM6A]), Rubinstein-Taybi syndrome (type 1 [CBP] and 2 [EP300]), and Kleefstra syndrome type 2 (KMT2C). Here, we review the composition and biochemical function of the KMT2 complexes. The specific cellular and embryonic roles of the KMT2C/D COMPASS complex are highlight with a focus on clinically relevant mechanisms sensitive to haploinsufficiency. The phenotypic similarities and differences between the members of this new family of disorders are outlined and emerging therapeutic strategies are detailed.

    更新日期:2020-01-11
  • Epigenome-wide association study of seizures in childhood and adolescence
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-08
    Doretta Caramaschi; Charlie Hatcher; Rosa H. Mulder; Janine F. Felix; Charlotte A. M. Cecil; Caroline L. Relton; Esther Walton

    The occurrence of seizures in childhood is often associated with neurodevelopmental impairments and school underachievement. Common genetic variants associated with epilepsy have been identified and epigenetic mechanisms have also been suggested to play a role. In this study, we analyzed the association of genome-wide blood DNA methylation with the occurrence of seizures in ~ 800 children from the Avon Longitudinal Study of Parents and Children, UK, at birth (cord blood), during childhood, and adolescence (peripheral blood). We also analyzed the association between the lifetime occurrence of any seizures before age 13 with blood DNA methylation levels. We sought replication of the findings in the Generation R Study and explored causality using Mendelian randomization, i.e., using genetic variants as proxies. The results showed five CpG sites which were associated cross-sectionally with seizures either in childhood or adolescence (1–5% absolute methylation difference at pFDR < 0.05), although the evidence of replication in an independent study was weak. One of these sites was located in the BDNF gene, which is highly expressed in the brain, and showed high correspondence with brain methylation levels. The Mendelian randomization analyses suggested that seizures might be causal for changes in methylation rather than vice-versa. In conclusion, we show a suggestive link between seizures and blood DNA methylation while at the same time exploring the limitations of conducting such study.

    更新日期:2020-01-08
  • DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-08
    Loora Laan; Joakim Klar; Maria Sobol; Jan Hoeber; Mansoureh Shahsavani; Malin Kele; Ambrin Fatima; Muhammad Zakaria; Göran Annerén; Anna Falk; Jens Schuster; Niklas Dahl

    Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages. Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling. The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.

    更新日期:2020-01-08
  • Replication and expansion of epigenome-wide association literature in a black South African population
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-07
    H. Toinét Cronjé; Hannah R. Elliott; Cornelie Nienaber-Rousseau; Marlien Pieters

    DNA methylation is associated with non-communicable diseases (NCDs) and related traits. Methylation data on continental African ancestries are currently scarce, even though there are known genetic and epigenetic differences between ancestral groups and a high burden of NCDs in Africans. Furthermore, the degree to which current literature can be extrapolated to the understudied African populations, who have limited resources to conduct independent large-scale analysis, is not yet known. To this end, this study examines the reproducibility of previously published epigenome-wide association studies of DNA methylation conducted in different ethinicities, on factors related to NCDs, by replicating findings in 120 South African Batswana men aged 45 to 88 years. In addition, novel associations between methylation and NCD-related factors are investigated using the Illumina EPIC BeadChip. Up to 86% of previously identified epigenome-wide associations with NCD-related traits (alcohol consumption, smoking, body mass index, waist circumference, C-reactive protein, blood lipids and age) overlapped with those observed here and a further 13% were directionally consistent. Only 1% of the replicated associations presented with effects opposite to findings in other ancestral groups. The majority of these inconcistencies were associated with population-specific genomic variance. In addition, we identified eight new 450K array CpG associations not previously reported in other ancestries, and 11 novel EPIC CpG associations with alcohol consumption. The successful replication of existing EWAS findings in this African population demonstrates that blood-based 450K EWAS findings from commonly investigated ancestries can largely be extrapolated to ethnicities for which epigenetic data are not yet available. Possible population-specific differences in 14% of the tested associations do, however, motivate the need to include a diversity of ethnic groups in future epigenetic research. The novel associations found with the enhanced coverage of the Illumina EPIC array support its usefulness to expand epigenetic literature.

    更新日期:2020-01-07
  • Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-07
    Andrea Ciolfi; Erfan Aref-Eshghi; Simone Pizzi; Lucia Pedace; Evelina Miele; Jennifer Kerkhof; Elisabetta Flex; Simone Martinelli; Francesca Clementina Radio; Claudia A. L. Ruivenkamp; Gijs W. E. Santen; Emilia Bijlsma; Daniela Barge-Schaapveld; Katrin Ounap; Victoria Mok Siu; R. Frank Kooy; Bruno Dallapiccola; Bekim Sadikovic; Marco Tartaglia

    We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This “episignature” was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis.

    更新日期:2020-01-07
  • Elevated HDAC activity and altered histone phospho-acetylation confer acquired radio-resistant phenotype to breast cancer cells
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-03
    Asmita Sharda; Mudasir Rashid; Sanket Girish Shah; Ajit Kumar Sharma; Saurav Raj Singh; Poonam Gera; Murali Krishna Chilkapati; Sanjay Gupta

    Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G0/G1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor–based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor–based radio-sensitization.

    更新日期:2020-01-04
  • Dysfunctional epigenetic aging of the normal colon and colorectal cancer risk
    Clin. Epigenet. (IF 5.496) Pub Date : 2020-01-03
    Ting Wang; Sean K. Maden; Georg E. Luebeck; Christopher I. Li; Polly A. Newcomb; Cornelia M. Ulrich; Ji-Hoon E. Joo; Daniel D. Buchanan; Roger L. Milne; Melissa C. Southey; Kelly T. Carter; Amber R. Willbanks; Yanxin Luo; Ming Yu; William M. Grady

    Chronological age is a prominent risk factor for many types of cancers including colorectal cancer (CRC). Yet, the risk of CRC varies substantially between individuals, even within the same age group, which may reflect heterogeneity in biological tissue aging between people. Epigenetic clocks based on DNA methylation are a useful measure of the biological aging process with the potential to serve as a biomarker of an individual’s susceptibility to age-related diseases such as CRC. We conducted a genome-wide DNA methylation study on samples of normal colon mucosa (N = 334). Subjects were assigned to three cancer risk groups (low, medium, and high) based on their personal adenoma or cancer history. Using previously established epigenetic clocks (Hannum, Horvath, PhenoAge, and EpiTOC), we estimated the biological age of each sample and assessed for epigenetic age acceleration in the samples by regressing the estimated biological age on the individual’s chronological age. We compared the epigenetic age acceleration between different risk groups using a multivariate linear regression model with the adjustment for gender and cell-type fractions for each epigenetic clock. An epigenome-wide association study (EWAS) was performed to identify differential methylation changes associated with CRC risk. Each epigenetic clock was significantly correlated with the chronological age of the subjects, and the Horvath clock exhibited the strongest correlation in all risk groups (r > 0.8, p < 1 × 10−30). The PhenoAge clock (p = 0.0012) revealed epigenetic age deceleration in the high-risk group compared to the low-risk group. Among the four DNA methylation-based measures of biological age, the Horvath clock is the most accurate for estimating the chronological age of individuals. Individuals with a high risk for CRC have epigenetic age deceleration in their normal colons measured by the PhenoAge clock, which may reflect a dysfunctional epigenetic aging process.

    更新日期:2020-01-04
  • Silencing or inhibition of H3K79 methyltransferase DOT1L induces cell cycle arrest by epigenetically modulating c-Myc expression in colorectal cancer
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-30
    Liqun Yang; Qian Lei; Lin Li; Jie Yang; Zhen Dong; Hongjuan Cui

    Epigenetic regulations play pivotal roles in tumorigenesis and cancer development. Disruptor of telomeric silencing-1-like (DOT1L), also known as KMT4, is the only identified histone methyltransferase that catalyzes the mono-, di-, and tri-methylation of lysine 79 histone 3 (H3K79). However, little is known about the effect of H3K79 methylation on the modulation of colorectal cancer (CRC) development. DOT1L expression profiles in different subgroups of CRC tissues and its clinical significances were analyzed from some online datasheets. DOT1L in CRC cell lines was silenced by either lentivirus-mediated knockdown or inhibited by its specific inhibitor, EPZ004777. Then cell proliferation was detected by MTT assay, BrdU assay, and soft agar assay; cell cycle was detected by cytometry; and tumorigenicity was detected by using nude mice xenograft models. Clinical co-expression was analyzed between DOT1L and c-Myc. Chromatin immunoprecipitation (ChIP) assay was used to determine whether the translation of c-Myc was epigenetically regulated by H3K79me2 induced by DOT1L. c-Myc overexpression was used to rescue the cell cycle arrest and tumor growth induced by DOT1L silencing or inhibition in CRC. We found that DOT1L was highly expressed in colorectal cancer and was negatively related to the prognosis of patients with CRC. Silencing or inhibition of DOT1L blocked cell proliferation, BrdU incorporation, self-renewal capability in vitro, and tumorigenicity in vivo. Besides, inhibition or silencing of DOT1L also induced cell cycle arrest at S phase, as well as decreased the expression of CDK2 and Cyclin A2. Furthermore, in the clinical databases of CRC, we found that the expression of DOT1L was positively correlated with that of c-Myc, a major regulator in the upstream of cell cycle–related factors. Besides, c-Myc expression was downregulated after DOT1L knockdown and c-Myc restoration rescued decrease of cell proliferation, BrdU corporation, self-renewal capability, cell cycle progression in vitro and tumorigenicity in vivo induced by DOT1L silencing. Then we found that H3K79 methylation was decreased after DOT1L knockdown. ChIP assay showed that H3K79me2 was enriched on the – 682~+ 284 region of c-Myc promoter, and the enrichment was decreased after DOT1L inhibition. Our results show that DOT1L epigenetically promotes the transcription of c-Myc via H3K79me2. DOT1L silencing or inhibition induces cell cycle arrest at S phase. DOT1L is a potential marker for colorectal cancer and EPZ004777 may be a potential drug for the treatment of colorectal cancer.

    更新日期:2019-12-31
  • Transient receptor potential ankyrin 1 promoter methylation and peripheral pain sensitivity in Crohn’s disease
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-31
    Sara Gombert; Mathias Rhein; Andreas Winterpacht; Tino Münster; Thomas Hillemacher; Andreas Leffler; Helge Frieling

    Crohn’s disease is a chronic inflammatory disorder of the gastrointestinal tract associated with abdominal pain and diarrhea. Pain caused by Crohn’s disease likely involves neurogenic inflammation which seems to involve the ion channel transient receptor potential ankyrin 1 (TRPA1). Since the promoter methylation of TRPA1 was shown to influence pain sensitivity, we asked if the expression of TRPA1 is dysregulated in patients suffering from Crohn’s disease. The methylation rates of CpG dinucleotides in the TRPA1 promoter region were determined from DNA derived from whole blood samples of Crohn patients and healthy participants. Quantitative sensory testing was used to examine pain sensitivities. Pressure pain thresholds were lower in Crohn patients as compared to healthy participants, and they were also lower in females than in males. They correlated inversely with the methylation rate at the CpG − 628 site of the TRPA1 promoter. This effect was more pronounced in female compared to male Crohn patients. Similar results were found for mechanical pain thresholds. Furthermore, age-dependent effects were detected. Whereas the CpG − 628 methylation rate declined with age in healthy participants, the methylation rate in Crohn patients increased. Pressure pain thresholds increased with age in both cohorts. The TRPA1 promoter methylation appears to be dysregulated in patients suffering from Crohn’s disease, and this effect is most obvious when taking gender and age into account. As TRPA1 is regarded to be involved in pain caused by neurogenic inflammation, its aberrant expression may contribute to typical symptoms of Crohn’s disease.

    更新日期:2019-12-31
  • Obesity-induced nucleosome release predicts poor cardio-metabolic health
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-31
    Oriana Lo Re; Andrea Maugeri; Jana Hruskova; Juraj Jakubik; Jan Kucera; Julie Bienertova-Vasku; Jude A. Oben; Lukas Kubala; Adela Dvorakova; Milan Ciz; Manlio Vinciguerra

    While circulating nucleosome levels are high in obese mouse models, it is unknown where these nucleosomes originate from and whether they are a marker of cardio-metabolic health in humans. Here, we aimed to determine whether an association exists between circulating nucleosomes and the risk of developing obesity, metabolic syndrome (MetS) and/or a dysfunctional cardiovascular performance. We randomly selected 120 participants of the Kardiovize Brno 2030 study across three BMI strata: BMI 18–25, 25–30, and > 30. We assessed the association between circulating nucleosome levels and the risk of obesity, MetS, and poor cardiovascular health. We then cultured human neutrophils, adipocytes, and hepatoma cells to study nucleosome origins in a fat-rich environment. Circulating nucleosome levels positively correlated with BMI (R = 0.602, p < 0.05), fatty liver index (R = 0.622, p < 0.05), left ventricular mass (R = 0.457, p < 0.05), and associated with MetS (p < 0.001) and poor cardiovascular health (p < 0.001). Incubating neutrophils with 1–10 μM free fatty acids triggered nucleosome production without concomitant cell death. Nucleosomes were not produced during pre-adipocyte differentiation or upon incubation of hepatic cells with palmitic acid. Neutrophils are a bona fide source of circulating nucleosomes in an obesogenic environment and in overweight/obese patients. High nucleosome levels are associated with MetS and cardiovascular performance, and might represent novel candidate biomarkers for cardio-metabolic health.

    更新日期:2019-12-31
  • Newborn and childhood differential DNA methylation and liver fat in school-age children
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-31
    Madelon L. Geurtsen; Vincent W. V. Jaddoe; Lucas A. Salas; Susana Santos; Janine F. Felix

    Non-alcoholic fatty liver disease is the most common chronic liver disease in children in western countries. Adverse early-life exposures are associated with higher liver fat percentages in children. Differential DNA methylation may underlie these associations. We aimed to identify differential DNA methylation in newborns and children associated with liver fat accumulation in childhood. We also examined whether DNA methylation at 22 cytosine-phosphate-guanine sites (CpGs) associated with adult non-alcoholic fatty liver disease is associated with liver fat in children. Within a population-based prospective cohort study, we analyzed epigenome-wide DNA methylation data of 785 newborns and 344 10-year-old children in relation to liver fat fraction at 10 years. DNA methylation was measured using the Infinium HumanMethylation450 BeadChip (Illumina). We measured liver fat fraction by Magnetic Resonance Imaging. Associations of single CpG DNA methylation at the two-time points with liver fat accumulation were analyzed using robust linear regression models. We also analyzed differentially methylation regions using the dmrff package. We looked-up associations of 22 known adult CpGs at both ages with liver fat at 10 years. The median liver fat fraction was 2.0% (95% range 1.3, 5.1). No single CpGs and no differentially methylated regions were associated with liver fat accumulation. None of the 22 known adult CpGs were associated with liver fat in children. DNA methylation at birth and in childhood was not associated with liver fat accumulation in 10-year-old children in this study. This may be due to modest sample sizes or DNA methylation changes being a consequence rather than a determinant of liver fat.

    更新日期:2019-12-31
  • Psychotropic drug-induced genetic-epigenetic modulation of CRTC1 gene is associated with early weight gain in a prospective study of psychiatric patients
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-26
    Aurélie Delacrétaz; Anaïs Glatard; Céline Dubath; Mehdi Gholam-Rezaee; Jose Vicente Sanchez-Mut; Johannes Gräff; Armin von Gunten; Philippe Conus; Chin B. Eap

    Metabolic side effects induced by psychotropic drugs represent a major health issue in psychiatry. CREB-regulated transcription coactivator 1 (CRTC1) gene plays a major role in the regulation of energy homeostasis and epigenetic mechanisms may explain its association with obesity features previously described in psychiatric patients. This prospective study included 78 patients receiving psychotropic drugs that induce metabolic disturbances, with weight and other metabolic parameters monitored regularly. Methylation levels in 76 CRTC1 probes were assessed before and after 1 month of psychotropic treatment in blood samples. Significant methylation changes were observed in three CRTC1 CpG sites (i.e., cg07015183, cg12034943, and cg 17006757) in patients with early and important weight gain (i.e., equal or higher than 5% after 1 month; FDR p value = 0.02). Multivariable models showed that methylation decrease in cg12034943 was more important in patients with early weight gain (≥ 5%) than in those who did not gain weight (p = 0.01). Further analyses combining genetic and methylation data showed that cg12034943 was significantly associated with early weight gain in patients carrying the G allele of rs4808844A>G (p = 0.03), a SNP associated with this methylation site (p = 0.03). These findings give new insights on psychotropic-induced weight gain and underline the need of future larger prospective epigenetic studies to better understand the complex pathways involved in psychotropic-induced metabolic side effects.

    更新日期:2019-12-27
  • Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-19
    Maria Desemparats Saenz-de-Juano; Elena Ivanova; Katy Billooye; Anamaria-Cristina Herta; Johan Smitz; Gavin Kelsey; Ellen Anckaert

    In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART). In this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development. Our observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

    更新日期:2019-12-19
  • Epigenetic gestational age and trajectories of weight and height during childhood: a prospective cohort study
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-16
    Harold D. Bright; Laura D. Howe; Jasmine N. Khouja; Andrew J. Simpkin; Matthew Suderman; Linda M. O’Keeffe

    Differences between an individual’s estimated epigenetic gestational age (EGA) and their actual gestational age (GA) are defined as gestational age acceleration (GAA). GAA is associated with increased birthweight and birth length. Whether these associations persist through childhood is yet to be investigated. We examined the association between GAA and trajectories of height and weight from birth to 10 years (n = 785) in a British birth cohort study, the Avon Longitudinal Study of Parents and Children (ALSPAC). EGA of participants was estimated using DNA methylation data from cord blood using a recently developed prediction model. GAA of participants, measured in weeks, was calculated as the residuals from a regression model of EGA on actual GA. Analyses were performed using linear spline multilevel models and adjusted for maternal age, maternal pre-pregnancy BMI, maternal smoking during pregnancy, and maternal education. In adjusted analyses, offspring with a one-week greater GAA were born on average 0.14 kg heavier (95% confidence interval (CI) 0.09, 0.19) and 0.55 cm taller (95% CI 0.33, 0.78) at birth. These differences in weight persisted up to approximately age 9 months but thereafter began to attenuate. From age 5 years onwards, the association between GAA and weight reversed such that GAA was associated with lower weight and this association strengthened with age (mean difference at age 10 years − 0.60 kg, 95% CI − 1.19, − 0.01). Differences in height persisted only up to age 9 months (mean difference at 9 months 0.15 cm, 95% CI − 0.09, 0.39). From age 9 months to age 10 years, offspring with a one-week greater GAA were of comparable height with those with no GAA (mean difference at age 10 years − 0.07 cm, 95% CI − 0.64, 0.50). Gestational age acceleration is associated with increased birth weight and length and these differences persist to age 9 months. From age 5 years onwards, the association of GAA and weight reverses such that by age 10 years, greater GAA is associated with lower childhood weight. Further work is required to examine whether the weight effects of GAA strengthen through adolescence and into early adulthood.

    更新日期:2019-12-17
  • Methylomic profiling in trisomy 21 identifies cognition- and Alzheimer’s disease-related dysregulation
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-16
    Larissa Haertle; Tobias Müller; Roy Lardenoije; Anna Maierhofer; Marcus Dittrich; Renzo J. M. Riemens; Samantha Stora; Mathilde Roche; Markus Leber; Steffi Riedel-Heller; Michael Wagner; Martin Scherer; Aimé Ravel; Clotilde Mircher; Cecile Cieuta-Walti; Sophie Durand; Daniel L. A. van de Hove; Per Hoffmann; Alfredo Ramirez; Thomas Haaf; Nady El Hajj; André Mégarbané

    Trisomy 21 (T21) is associated with intellectual disability that ranges from mild to profound with an average intellectual quotient of around 50. Furthermore, T21 patients have a high risk of developing Alzheimer’s disease (AD) early in life, characterized by the presence of senile plaques of amyloid protein and neurofibrillary tangles, leading to neuronal loss and cognitive decline. We postulate that epigenetic factors contribute to the observed variability in intellectual disability, as well as at the level of neurodegeneration seen in T21 individuals. A genome-wide DNA methylation study was performed using Illumina Infinium® MethylationEPIC BeadChips on whole blood DNA of 3 male T21 patients with low IQ, 8 T21 patients with high IQ (4 males and 4 females), and 21 age- and sex-matched control samples (12 males and 9 females) in order to determine whether DNA methylation alterations could help explain variation in cognitive impairment between individuals with T21. In view of the increased risk of developing AD in T21 individuals, we additionally investigated the T21-associated sites in published blood DNA methylation data from the AgeCoDe cohort (German study on Ageing, Cognition, and Dementia). AgeCoDe represents a prospective longitudinal study including non-demented individuals at baseline of which a part develops AD dementia at follow-up. Two thousand seven hundred sixteen differentially methylated sites and regions discriminating T21 and healthy individuals were identified. In the T21 high and low IQ comparison, a single CpG located in the promoter of PELI1 was differentially methylated after multiple testing adjustment. For the same contrast, 69 differentially methylated regions were identified. Performing a targeted association analysis for the significant T21-associated CpG sites in the AgeCoDe cohort, we found that 9 showed significant methylation differences related to AD dementia, including one in the ADAM10 gene. This gene has previously been shown to play a role in the prevention of amyloid plaque formation in the brain. The differentially methylated regions may help understand the interaction between methylation alterations and cognitive function. In addition, ADAM10 might be a valuable blood-based biomarker for at least the early detection of AD.

    更新日期:2019-12-17
  • DNA methylation instability by BRAF-mediated TET silencing and lifestyle-exposure divides colon cancer pathways
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-16
    Faiza Noreen; Taya Küng; Luigi Tornillo; Hannah Parker; Miguel Silva; Stefan Weis; Giancarlo Marra; Roland Rad; Kaspar Truninger; Primo Schär

    Aberrations in DNA methylation are widespread in colon cancer (CC). Understanding origin and progression of DNA methylation aberrations is essential to develop effective preventive and therapeutic strategies. Here, we aimed to dissect CC subtype-specific methylation instability to understand underlying mechanisms and functions. We have assessed genome-wide DNA methylation in the healthy normal colon mucosa (HNM), precursor lesions and CCs in a first comprehensive study to delineate epigenetic change along the process of colon carcinogenesis. Mechanistically, we used stable cell lines, genetically engineered mouse model of mutant BRAFV600E and molecular biology analysis to establish the role of BRAFV600E-mediated-TET inhibition in CpG-island methylator phenotype (CIMP) inititation. We identified two distinct patterns of CpG methylation instability, determined either by age–lifestyle (CC-neutral CpGs) or genetically (CIMP-CpGs). CC-neutral-CpGs showed age-dependent hypermethylation in HNM, all precursors, and CCs, while CIMP-CpGs showed hypermethylation specifically in sessile serrated adenomas/polyps (SSA/Ps) and CIMP-CCs. BRAFV600E-mutated CCs and precursors showed a significant downregulation of TET1 and TET2 DNA demethylases. Stable expression of BRAFV600E in nonCIMP CC cells and in a genetic mouse model was sufficient to repress TET1/TET2 and initiate hypermethylation at CIMP-CpGs, reversible by BRAFV600E inhibition. BRAFV600E-driven CIMP-CpG hypermethylation occurred at genes associated with established CC pathways, effecting functional changes otherwise achieved by genetic mutation in carcinogenesis. Hence, while age–lifestyle-driven hypermethylation occurs generally in colon carcinogenesis, BRAFV600E-driven hypermethylation is specific for the “serrated” pathway. This knowledge will advance the use of epigenetic biomarkers to assess subgroup-specific CC risk and disease progression.

    更新日期:2019-12-17
  • DNA methylation data by sequencing: experimental approaches and recommendations for tools and pipelines for data analysis
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-12
    Ieva Rauluseviciute; Finn Drabløs; Morten Beck Rye

    Sequencing technologies have changed not only our approaches to classical genetics, but also the field of epigenetics. Specific methods allow scientists to identify novel genome-wide epigenetic patterns of DNA methylation down to single-nucleotide resolution. DNA methylation is the most researched epigenetic mark involved in various processes in the human cell, including gene regulation and development of diseases, such as cancer. Increasing numbers of DNA methylation sequencing datasets from human genome are produced using various platforms—from methylated DNA precipitation to the whole genome bisulfite sequencing. Many of those datasets are fully accessible for repeated analyses. Sequencing experiments have become routine in laboratories around the world, while analysis of outcoming data is still a challenge among the majority of scientists, since in many cases it requires advanced computational skills. Even though various tools are being created and published, guidelines for their selection are often not clear, especially to non-bioinformaticians with limited experience in computational analyses. Separate tools are often used for individual steps in the analysis, and these can be challenging to manage and integrate. However, in some instances, tools are combined into pipelines that are capable to complete all the essential steps to achieve the result. In the case of DNA methylation sequencing analysis, the goal of such pipeline is to map sequencing reads, calculate methylation levels, and distinguish differentially methylated positions and/or regions. The objective of this review is to describe basic principles and steps in the analysis of DNA methylation sequencing data that in particular have been used for mammalian genomes, and more importantly to present and discuss the most pronounced computational pipelines that can be used to analyze such data. We aim to provide a good starting point for scientists with limited experience in computational analyses of DNA methylation and hydroxymethylation data, and recommend a few tools that are powerful, but still easy enough to use for their own data analysis.

    更新日期:2019-12-13
  • The phenotypic variations of multi-locus imprinting disturbances associated with maternal-effect variants of NLRP5 range from overt imprinting disorder to apparently healthy phenotype
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-11
    Angela Sparago; Ankit Verma; Maria Grazia Patricelli; Laura Pignata; Silvia Russo; Luciano Calzari; Naomi De Francesco; Rosita Del Prete; Orazio Palumbo; Massimo Carella; Deborah J. G. Mackay; Faisal I. Rezwan; Claudia Angelini; Flavia Cerrato; Maria Vittoria Cubellis; Andrea Riccio

    A subset of individuals affected by imprinting disorders displays multi-locus imprinting disturbances (MLID). MLID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (gDMRs) in early embryogenesis. Pedigrees of individuals with MLID also include siblings with healthy phenotype. However, it is unknown if these healthy individuals have MLID themselves or if their methylation patterns differ from those associated with imprinting disorders, and in general, if MLID affects the clinical phenotype. We have investigated gDMR methylation by locus-specific and whole-genome analyses in a family with multiple pregnancy losses, a child with Beckwith-Wiedemann syndrome (BWS) and a further child with no clinical diagnosis of imprinting disorder or other pathologies. We detected MLID with different methylation profiles in the BWS-affected and healthy siblings. Whole-exome sequencing demonstrated the presence of novel loss-of-function variants of NLRP5 in compound heterozygosity in the mother. The methylation profiles of the two siblings were compared with those of other cases with MLID and control groups by principal component analysis and unsupervised hierarchical clustering, but while their patterns were clearly separated from those of controls, we were unable to cluster those associated with specific clinical phenotypes among the MLID cases. The identification of two novel maternal-effect variants of NLRP5 associated with poly-abortivity and MLID adds further evidence to the role of this gene in the maintenance of genomic imprinting in early embryos. Furthermore, our results demonstrate that within these pedigrees, MLID can also be present in the progeny with healthy phenotype, indicating that some sort of compensation occurs between altered imprinted loci in these individuals. The analysis of larger cohorts of patients with MLID is needed to formulate more accurate epigenotype-phenotype correlations.

    更新日期:2019-12-11
  • Distinct DNA methylation targets by aging and chronic inflammation: a pilot study using gastric mucosa infected with Helicobacter pylori
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-11
    Satoshi Yamashita; Sohachi Nanjo; Emil Rehnberg; Naoko Iida; Hideyuki Takeshima; Takayuki Ando; Takao Maekita; Toshiro Sugiyama; Toshikazu Ushijima

    Aberrant DNA methylation is induced by aging and chronic inflammation in normal tissues. The induction by inflammation is widely recognized as acceleration of age-related methylation. However, few studies addressed target genomic regions and the responsible factors in a genome-wide manner. Here, we analyzed methylation targets by aging and inflammation, taking advantage of the potent methylation induction in human gastric mucosa by Helicobacter pylori infection-triggered inflammation. DNA methylation microarray analysis of 482,421 CpG probes, grouped into 270,249 genomic blocks, revealed that high levels of methylation were induced in 44,461 (16.5%) genomic blocks by inflammation, even after correction of the influence of leukocyte infiltration. A total of 61.8% of the hypermethylation was acceleration of age-related methylation while 21.6% was specific to inflammation. Regions with H3K27me3 were frequently hypermethylated both by aging and inflammation. Basal methylation levels were essential for age-related hypermethylation while even regions with little basal methylation were hypermethylated by inflammation. When limited to promoter CpG islands, being a microRNA gene and high basal methylation levels strongly enhanced hypermethylation while H3K27me3 strongly enhanced inflammation-induced hypermethylation. Inflammation was capable of overriding active transcription. In young gastric mucosae, genes with high expression and frequent mutations in gastric cancers were more frequently methylated than in old ones. Methylation by inflammation was not simple acceleration of age-related methylation. Targets of aberrant DNA methylation were different between young and old gastric mucosae, and driver genes were preferentially methylated in young gastric mucosa.

    更新日期:2019-12-11
  • Drug screening approach combines epigenetic sensitization with immunochemotherapy in cancer
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-11
    Chiara Facciotto; Julia Casado; Laura Turunen; Suvi-Katri Leivonen; Manuela Tumiati; Ville Rantanen; Liisa Kauppi; Rainer Lehtonen; Sirpa Leppä; Krister Wennerberg; Sampsa Hautaniemi

    The epigenome plays a key role in cancer heterogeneity and drug resistance. Hence, a number of epigenetic inhibitors have been developed and tested in cancers. The major focus of most studies so far has been on the cytotoxic effect of these compounds, and only few have investigated the ability to revert the resistant phenotype in cancer cells. Hence, there is a need for a systematic methodology to unravel the mechanisms behind epigenetic sensitization. We have developed a high-throughput protocol to screen non-simultaneous drug combinations, and used it to investigate the reprogramming potential of epigenetic inhibitors. We demonstrated the effectiveness of our protocol by screening 60 epigenetic compounds on diffuse large B-cell lymphoma (DLBCL) cells. We identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with doxorubicin and rituximab. These two classes of epigenetic inhibitors achieved sensitization by disrupting DNA repair, cell cycle, and apoptotic signaling. The data used to perform these analyses are easily browsable through our Results Explorer. Additionally, we showed that these inhibitors achieve sensitization at lower doses than those required to induce cytotoxicity. Our drug screening approach provides a systematic framework to test non-simultaneous drug combinations. This methodology identified HDAC and HMT inhibitors as successful sensitizing compounds in treatment-resistant DLBCL. Further investigation into the mechanisms behind successful epigenetic sensitization highlighted DNA repair, cell cycle, and apoptosis as the most dysregulated pathways. Altogether, our method adds supporting evidence in the use of epigenetic inhibitors as sensitizing agents in clinical settings.

    更新日期:2019-12-11
  • Comprehensive longitudinal study of epigenetic mutations in aging
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-09
    Yunzhang Wang; Robert Karlsson; Juulia Jylhävä; Åsa K. Hedman; Catarina Almqvist; Ida K. Karlsson; Nancy L. Pedersen; Malin Almgren; Sara Hägg

    The role of DNA methylation in aging has been widely studied. However, epigenetic mutations, here defined as aberrant methylation levels compared to the distribution in a population, are less understood. Hence, we investigated longitudinal accumulation of epigenetic mutations, using 994 blood samples collected at up to five time points from 375 individuals in old ages. We verified earlier cross-sectional evidence on the increase of epigenetic mutations with age, and identified important contributing factors including sex, CD19+ B cells, genetic background, cancer diagnosis, and technical artifacts. We further classified epigenetic mutations into High/Low Methylation Outliers (HMO/LMO) according to their changes in methylation, and specifically studied methylation sites (CpGs) that were prone to mutate (frequently mutated CpGs). We validated four epigenetically mutated CpGs using pyrosequencing in 93 samples. Furthermore, by using twins, we concluded that the age-related accumulation of epigenetic mutations was not related to genetic factors, hence driven by stochastic or environmental effects. Here we conducted a comprehensive study of epigenetic mutation and highlighted its important role in aging process and cancer development.

    更新日期:2019-12-11
  • Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma, or leukemia
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-10
    Cornelis M. van Tilburg; Till Milde; Ruth Witt; Jonas Ecker; Thomas Hielscher; Angelika Seitz; Jens-Peter Schenk; Juliane L. Buhl; Dennis Riehl; Michael C. Frühwald; Arnulf Pekrun; Claudia Rossig; Regina Wieland; Christian Flotho; Uwe Kordes; Bernd Gruhn; Thorsten Simon; Christin Linderkamp; Felix Sahm; Lenka Taylor; Angelika Freitag; Jürgen Burhenne; Kathrin I. Foerster; Andreas D. Meid; Stefan M. Pfister; Irini Karapanagiotou-Schenkel; Olaf Witt

    Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3–18 years) with relapsed or therapy-refractory malignancies. A phase I intra-patient dose (de)escalation was performed until individual maximum tolerated dose (MTD). The starting dose was 180 mg/m2/day with weekly dose escalations of 50 mg/m2 until DLT/maximum dose. After MTD determination, patients seamlessly continued in phase II with disease assessments every 3 months. PK and plasma cytokine profiles were determined. Fifty of 52 patients received treatment. n = 27/50 (54%) completed the intra-patient (de)escalation and entered phase II. An SDR of 130 mg/m2/day was determined (maximum, 580 mg/m2/day). n = 46/50 (92%) patients experienced treatment-related AEs which were mostly reversible and included thrombocytopenia, fatigue, nausea, diarrhea, anemia, and vomiting. n = 6/50 (12%) had treatment-related SAEs. No treatment-related deaths occurred. Higher dose levels resulted in higher Cmax. Five patients achieved prolonged disease control (> 12 months) and showed a higher Cmax (> 270 ng/mL) and MTDs. Best overall response (combining PR and SD, no CR observed) rate in phase II was 6/27 (22%) with a median PFS and OS of 5.3 and 22.4 months. Low levels of baseline cytokine expression were significantly correlated with favorable outcome. An SDR of 130 mg/m2/day for individual dose escalation was determined. Higher drug exposure was associated with responses and long-term disease stabilization with manageable toxicity. Patients with low expression of plasma cytokine levels at baseline were able to tolerate higher doses of vorinostat and benefited from treatment. Baseline cytokine profile is a promising potential predictive biomarker. ClinicalTrials.gov, NCT01422499. Registered 24 August 2011,

    更新日期:2019-12-11
  • Overall and sex-specific associations between methylation of the ABCG1 and APOE genes and ischemic stroke or other atherosclerosis-related traits in a sibling study of Chinese population
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-10
    Xueying Qin; Jin Li; Tao Wu; Yiqun Wu; Xun Tang; Pei Gao; Lin Li; Mengying Wang; Yao Wu; Xiaowen Wang; Dafang Chen; Yonghua Hu

    Identifying subjects with a high risk of ischemic stroke is fundamental for prevention of the disease. Both genetic and environmental risk factors contribute to ischemic stroke, but the underlying epigenetic mechanisms which mediate genetic and environmental risk effects are not fully understood. The aim of this study was to explore whether DNA methylation loci located in the ATP-binding cassette G1 (ABCG1) and apolipoprotein E (APOE) genes, both involved in the metabolism of lipids in the body, are related to ischemic stroke, using the Fangshan/Family-based Ischemic Stroke Study in China. We also tested if these CpG sites were associated with early signs of cardiovascular atherosclerosis (carotid intima–media thickness (cIMT), ankle–brachial index (ABI), and brachial–ankle pulse wave velocity (baPWV)). DNA methylation at the cg02494239 locus in ABCG1 was correlated with ischemic stroke after adjusting for gender, previous history of diabetes and hypertension, smoking, drinking, body mass index, and blood lipid levels (above vs below mean, OR = 2.416, 95% CI 1.024–5.700, P = 0.044; 75–100% percentile vs 0–25% percentile, OR = 4.461, 95% CI 1.226–16.225, P = 0.023). No statistically significant associations were observed for the cg06500161 site in ABCG1 and the cg14123992 site in APOE with ischemic stroke. The study detected that hypermethylation of the ABCG1 gene was significantly associated with cIMT, hypermethylation of the APOE gene was significantly related to ABI, and methylation of the APOE gene was statistically negatively correlated with baPWV. The above relationships demonstrated gender differences. These findings suggest that epigenetic modification of ABCG1 and APOE may play a role in the pathway from disturbed blood lipid levels to the development of cardiovascular diseases. Future prospective validation of these findings is warranted.

    更新日期:2019-12-11
  • DNMT1 recruited by EZH2-mediated silencing of miR-484 contributes to the malignancy of cervical cancer cells through MMP14 and HNF1A
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-07
    Yang Hu; Fuxia Wu; Yankun Liu; Qian Zhao; Hua Tang

    Emerging evidence indicates that dysregulation of microRNAs (miRNAs) contributes to cervical cancer (CC) tumorigenesis and development. Previous work showed that miR-484 which regulated the EMT process was obviously downregulated in CC. However, little is known about the precise mechanism. We found that the deficiency of EZH2-recruited DNA methyltransferases DNMT1 reduced the CpG methylation of miR-484 promoter and then increased the miR-484 expression. Furthermore, the cell membrane-bound matrix metalloproteinase (MMP14) and the hepatocyte nuclear factor 1A (HNF1A) were found to be downregulated by miR-484. miR-484 repressed the expression of MMP14 and HNF1A inhibiting CC growth and metastasis in vitro and in vivo. Upregulation of MMP14 and HNF1A promotes the CC cell adhesion and EMT, all of which contribute to cell motility and metastasis. Moreover, miR-484 negatively regulates the WNT/MAPK and TNF signaling pathway by downregulating HNF1A and MMP14 respectively. Thus, miR-484, who is downregulated by DNMT1-mediated hypermethylation in its promoter, functions as a tumor suppressor by inhibiting MMP14 and HNF1A expression in CC. Our finding characterizes miR-484 as a key suppressive regulator in CC metastasis and reveals a DNMT1-mediated epigenetic mechanism for miR-484 silencing, expanding our understanding of the molecular mechanism underlying CC progression and metastasis.

    更新日期:2019-12-07
  • Epigenome-wide base-resolution profiling of DNA methylation in chorionic villi of fetuses with Down syndrome by methyl-capture sequencing
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-04
    Ji Hyae Lim; Yu-Jung Kang; Bom Yi Lee; You Jung Han; Jin Hoon Chung; Moon Young Kim; Min Hyoung Kim; Jin Woo Kim; Youl-Hee Cho; Hyun Mee Ryu

    Epigenetic mechanisms provide an interface between environmental factors and the genome and are influential in various diseases. These mechanisms, including DNA methylation, influence the regulation of development, differentiation, and establishment of cellular identity. Here, we performed high-throughput methylome profiling to determine whether differential patterns of DNA methylation correlate with Down syndrome (DS). We extracted DNA from the chorionic villi cells of five normal and five DS fetuses at the early developmental stage (12–13 weeks of gestation). Methyl-capture sequencing (MC-Seq) was used to investigate the methylation levels of CpG sites distributed across the whole genome to identify differentially methylated CpG sites (DMCs) and regions (DMRs) in DS. New functional annotations of DMR genes using bioinformatics tools were predicted. DNA hypermethylation was observed in DS fetal chorionic villi cells. Significant differences were evident for 4,439 DMCs, including hypermethylation (n = 4,261) and hypomethylation (n = 178). Among them, 140 hypermethylated DMRs and only 1 hypomethylated DMR were located on 121 genes and 1 gene, respectively. One hundred twenty-two genes, including 141 DMRs, were associated with heart morphogenesis and development of the ear, thyroid gland, and nervous systems. The genes were significantly associated with DS and various diseases, including hepatopulmonary syndrome, conductive hearing loss, holoprosencephaly, heart diseases, glaucoma, and musculoskeletal abnormalities. This is the first study to compare the whole-epigenome DNA methylation pattern of the chorionic villi cells from normal and DS fetuses at the early developmental-stage using MC-seq. Overall, our results indicate that the chorionic villi cells of DS fetuses are hypermethylated in all autosomes and suggested that altered DNA methylation may be a recurrent and functionally relevant downstream response to DS in human cells. This study provides basic information for future research focused on the pathophysiology of the DS and its potential effects, as well as the role DNA methylation plays in the early developmental stage of DS fetuses.

    更新日期:2019-12-05
  • Epigenetic silencing of the ANKRD26 gene correlates to the pro-inflammatory profile and increased cardio-metabolic risk factors in human obesity
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-04
    Antonella Desiderio; Michele Longo; Luca Parrillo; Michele Campitelli; Giuseppe Cacace; Sonia de Simone; Rosa Spinelli; Federica Zatterale; Serena Cabaro; Pasquale Dolce; Pietro Formisano; Marco Milone; Claudia Miele; Francesco Beguinot; Gregory A. Raciti

    Obesity is a major worldwide threat to human health. Increasing evidence indicates that epigenetic modifications have a major impact on the natural history of this disorder. Ankyrin Repeat Domain 26 (Ankrd26) is involved in the development of both obesity and diabetes in mice and is modulated by environmentally induced epigenetic modifications. This study aims at investigating whether impaired ANKRD26 gene expression and methylation occur in human obesity and whether they correlate to the phenotype of these subjects. We found that downregulation of ANKRD26 mRNA and hyper-methylation of a specific region of the ANKRD26 promoter, embedding the CpG dinucleotides − 689, − 659, and − 651 bp, occur in peripheral blood leukocytes from obese compared with the lean subjects. ANKRD26 gene expression correlates inversely to the percentage of DNA methylation at these 3 CpG sites. Luciferase assays reveal a cause-effect relationship between DNA methylation at the 3 CpG sites and ANKRD26 gene expression. Finally, both ANKRD26 mRNA levels and CpG methylation correlate to body mass index and to the pro-inflammatory status and the increased cardio-metabolic risk factors of these same subjects. Downregulation of the ANKRD26 gene and hyper-methylation at specific CpGs of its promoter are common abnormalities in obese patients. These changes correlate to the pro-inflammatory profile and the cardio-metabolic risk factors of the obese individuals, indicating that, in humans, they mark adverse health outcomes.

    更新日期:2019-12-05
  • Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-04
    Verena Deutschmeyer; Janina Breuer; Sara K. Walesch; Anna M. Sokol; Johannes Graumann; Marek Bartkuhn; Thomas Boettger; Oliver Rossbach; Antje M. Richter

    Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation. We are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines. ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.

    更新日期:2019-12-05
  • Effect of tobacco smoking on the epigenetic age of human respiratory organs
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-04
    Xiaohui Wu; Qingsheng Huang; Ruheena Javed; Jiayong Zhong; Huan Gao; Huiying Liang

    Smoking leads to the aging of organs. However, no studies have been conducted to quantify the effect of smoking on the aging of respiratory organs and the aging-reversing ability of smoking cessation. We collected genome-wide methylation datasets of buccal cells, airway cells, esophagus tissue, and lung tissue from non-smokers, smokers, and ex-smokers. We used the “epigenetic clock” method to quantify the epigenetic age acceleration in the four organs. The statistical analyses showed the following: (1) Smoking increased the epigenetic age of airway cells by an average of 4.9 years and lung tissue by 4.3 years. (2) After smoking ceased, the epigenetic age acceleration in airway cells (but not in lung tissue) slowed to a level that non-smokers had. (3) The epigenetic age acceleration in airway cells and lung tissue showed no gender difference. Smoking can accelerate the epigenetic age of human respiratory organs, but the effect varies among organs and can be reversed by smoking cessation. Our study provides a powerful incentive to reduce tobacco consumption autonomously.

    更新日期:2019-12-05
  • ZMYND10, an epigenetically regulated tumor suppressor, exerts tumor-suppressive functions via miR145-5p/NEDD9 axis in breast cancer
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-04
    Yan Wang; Liangying Dan; Qianqian Li; Lili Li; Lan Zhong; Bianfei Shao; Fang Yu; Sanxiu He; Shaorong Tian; Jin He; Qian Xiao; Thomas C. Putti; Xiaoqian He; Yixiao Feng; Yong Lin; Tingxiu Xiang

    Recent studies suggested that ZMYND10 is a potential tumor suppressor gene in multiple tumor types. However, the mechanism by which ZMYND10 inhibits breast cancer remains unclear. Here, we investigated the role and mechanism of ZMYND10 in breast cancer inhibition. ZMYND10 was dramatically reduced in multiple breast cancer cell lines and tissues, which was associated with promoter hypermethylation. Ectopic expression of ZMYND10 in silenced breast cancer cells induced cell apoptosis while suppressed cell growth, cell migration and invasion in vitro, and xenograft tumor growth in vivo. Furthermore, molecular mechanism studies indicated that ZMYND10 enhances expression of miR145-5p, which suppresses the expression of NEDD9 protein through directly targeting the 3'-untranslated region of NEDD9 mRNA. Results from this study show that ZMYND10 suppresses breast cancer tumorigenicity by inhibiting the miR145-5p/NEDD9 signaling pathway. This novel discovered signaling pathway may be a valid target for small molecules that might help to develop new therapies to better inhibit the breast cancer metastasis.

    更新日期:2019-12-05
  • The central nervous system tumor methylation classifier changes neuro-oncology practice for challenging brain tumor diagnoses and directly impacts patient care
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-05
    Shirin Karimi; Jeffrey A. Zuccato; Yasin Mamatjan; Sheila Mansouri; Suganth Suppiah; Farshad Nassiri; Phedias Diamandis; David G. Munoz; Kenneth D. Aldape; Gelareh Zadeh

    Molecular signatures are being increasingly incorporated into cancer classification systems. DNA methylation-based central nervous system (CNS) tumor classification is being recognized as having the potential to aid in cases of difficult histopathological diagnoses. Here, we present our institutional clinical experience in integrating a DNA-methylation-based classifier into clinical practice and report its impact on CNS tumor patient diagnosis and treatment. Prospective case review was undertaken at CNS tumor board discussions over a 3-year period and 55 tumors with a diagnosis that was not certain to two senior neuropathologists were recommended for methylation profiling based on diagnostic needs. Tumor classification, calibrated scores, and copy number variant (CNV) plots were obtained for all 55 cases. These results were integrated with histopathological findings to reach a final diagnosis. We retrospectively reviewed each patient's clinical course to determine final neuro-pathology diagnoses and the impact of methylation profiling on their clinical management, with a focus on changes that were made to treatment decisions. Following methylation profiling, 46 of the 55 (84%) challenging cases received a clinically relevant diagnostic alteration, with two-thirds having a change in the histopathological diagnosis and the other one-third obtaining clinically important molecular diagnostic or subtyping alterations. WHO grading changed by 27% with two-thirds receiving a higher grade. Patient care was directly changed in 15% of all cases with major changes in clinical decision-making being made for these patients to avoid unnecessary or insufficient treatment. The integration of methylation-based CNS tumor classification into diagnostics has a substantial clinical benefit for patients with challenging CNS tumors while also avoiding unnecessary health care costs. The clinical impact shown here may prompt the expanded use of DNA methylation profiling for CNS tumor diagnostics within prominent neuro-oncology centers globally.

    更新日期:2019-12-05
  • Cisplatin treatment of testicular cancer patients introduces long-term changes in the epigenome
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-03
    Cecilie Bucher-Johannessen; Christian M. Page; Trine B. Haugen; Marcin W. Wojewodzic; Sophie D. Fosså; Tom Grotmol; Hege S. Haugnes; Trine B. Rounge

    Cisplatin-based chemotherapy (CBCT) is part of standard treatment of several cancers. In testicular cancer (TC) survivors, an increased risk of developing metabolic syndrome (MetS) is observed. In this epigenome-wide association study, we investigated if CBCT relates to epigenetic changes (DNA methylation) and if epigenetic changes render individuals susceptible for developing MetS later in life. We analyzed methylation profiles, using the MethylationEPIC BeadChip, in samples collected ~ 16 years after treatment from 279 Norwegian TC survivors with known MetS status. Among the CBCT treated (n = 176) and non-treated (n = 103), 61 and 34 developed MetS, respectively. We used two linear regression models to identify if (i) CBCT results in epigenetic changes and (ii) epigenetic changes play a role in development of MetS. Then we investigated if these changes in (i) and (ii) links to genes, functional networks, and pathways related to MetS symptoms. We identified 35 sites that were differentially methylated when comparing CBCT treated and untreated TC survivors. The PTK6–RAS–MAPk pathway was significantly enriched with these sites and infers a gene network of 13 genes with CACNA1D (involved in insulin release) as a network hub. We found nominal MetS-associations and a functional gene network with ABCG1 and NCF2 as network hubs. Our results suggest that CBCT has long-term effects on the epigenome. We could not directly link the CBCT effects to the risk of developing MetS. Nevertheless, since we identified differential methylation occurring in genes associated with conditions pertaining to MetS, we hypothesize that epigenomic changes may also play a role in the development of MetS in TC survivors. Further studies are needed to validate this hypothesis.

    更新日期:2019-12-04
  • Body mass index is associated with epigenetic age acceleration in the visceral adipose tissue of subjects with severe obesity
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    Juan de Toro-Martín; Frédéric Guénard; André Tchernof; Frédéric-Simon Hould; Stéfane Lebel; François Julien; Simon Marceau; Marie-Claude Vohl

    There is solid evidence that obesity induces the acceleration of liver epigenetic aging. However, unlike easily accessible blood or subcutaneous adipose tissue, little is known about the impact of obesity on epigenetic aging of metabolically active visceral adipose tissue (VAT). Herein, we aimed to test whether obesity accelerates VAT epigenetic aging in subjects with severe obesity. A significant and positive correlation between chronological age and epigenetic age, estimated with a reduced version of the Horvath’s epigenetic clock, was found in both blood (r = 0.78, p = 9.4 × 10−12) and VAT (r = 0.80, p = 1.1 × 10−12). Epigenetic age acceleration, defined as the residual resulting from regressing epigenetic age on chronological age, was significantly correlated with body mass index (BMI) in VAT (r = 0.29, p = 0.037). Multivariate linear regression analysis showed that, after adjusting for chronological age, sex and metabolic syndrome status, BMI remained significantly associated with epigenetic age acceleration in VAT (beta = 0.15, p = 0.035), equivalent to 2.3 years for each 10 BMI units. Binomial logistic regression showed that BMI-adjusted epigenetic age acceleration in VAT was significantly associated with a higher loss of excess body weight following biliopancreatic diversion with duodenal switch surgery (odds ratio = 1.21; 95% CI = 1.04–1.48; p = 0.03). Epigenetic age acceleration increases with BMI in VAT, but not in blood, as previously reported in liver. These results suggest that obesity is associated with epigenetic age acceleration of metabolically active tissues. Further studies that deepen the physiological relevance of VAT epigenetic aging will help to better understand the onset of metabolic syndrome and weight loss dynamics following bariatric surgery.

    更新日期:2019-12-02
  • Dissecting the role of novel EZH2 inhibitors in primary glioblastoma cell cultures: effects on proliferation, epithelial-mesenchymal transition, migration, and on the pro-inflammatory phenotype
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    Giulia Stazi; Ludovica Taglieri; Alice Nicolai; Annalisa Romanelli; Rossella Fioravanti; Stefania Morrone; Manuela Sabatino; Rino Ragno; Samanta Taurone; Marcella Nebbioso; Raffaella Carletti; Marco Artico; Sergio Valente; Susanna Scarpa; Antonello Mai

    Glioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM. Two new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10. The two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

    更新日期:2019-12-02
  • The timeline of epigenetic drug discovery: from reality to dreams
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    A. Ganesan; Paola B. Arimondo; Marianne G. Rots; Carmen Jeronimo; María Berdasco

    The flexibility of the epigenome has generated an enticing argument to explore its reversion through pharmacological treatments as a strategy to ameliorate disease phenotypes. All three families of epigenetic proteins—readers, writers, and erasers—are druggable targets that can be addressed through small-molecule inhibitors. At present, a few drugs targeting epigenetic enzymes as well as analogues of epigenetic modifications have been introduced into the clinic use (e.g. to treat haematological malignancies), and a wide range of epigenetic-based drugs are undergoing clinical trials. Here, we describe the timeline of epigenetic drug discovery and development beginning with the early design based solely on phenotypic observations to the state-of-the-art rational epigenetic drug discovery using validated targets. Finally, we will highlight some of the major aspects that need further research and discuss the challenges that need to be overcome to implement epigenetic drug discovery into clinical management of human disorders. To turn into reality, researchers from various disciplines (chemists, biologists, clinicians) need to work together to optimise the drug engineering, read-out assays, and clinical trial design.

    更新日期:2019-12-02
  • Early detection of the major male cancer types in blood-based liquid biopsies using a DNA methylation panel
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    Vera Constâncio; Sandra P. Nunes; Catarina Moreira-Barbosa; Rui Freitas; Jorge Oliveira; Inês Pousa; Júlio Oliveira; Marta Soares; Carlos Gonçalves Dias; Teresa Dias; Luís Antunes; Rui Henrique; Carmen Jerónimo

    Lung (LC), prostate (PCa) and colorectal (CRC) cancers are the most incident in males worldwide. Despite recent advances, optimal population-based cancer screening methods remain an unmet need. Due to its early onset, cancer specificity and accessibility in body fluids, aberrant DNA promoter methylation might be a valuable minimally invasive tool for early cancer detection. Herein, we aimed to develop a minimally invasive methylation-based test for simultaneous early detection of LC, PCa and CRC in males, using liquid biopsies. Circulating cell-free DNA was extracted from 102 LC, 121 PCa and 100 CRC patients and 136 asymptomatic donors’ plasma samples. Sodium-bisulfite modification and whole-genome amplification was performed. Promoter methylation levels of APCme, FOXA1me, GSTP1me, HOXD3me, RARβ2me, RASSF1Ame, SEPT9me and SOX17me were assessed by multiplex quantitative methylation-specific PCR. SEPT9me and SOX17me were the only biomarkers shared by all three cancer types, although they detected CRC with limited sensitivity. A “PanCancer” panel (FOXA1me, RARβ2me and RASSF1Ame) detected LC and PCa with 64% sensitivity and 70% specificity, complemented with “CancerType” panel (GSTP1me and SOX17me) which discriminated between LC and PCa with 93% specificity, but with modest sensitivity. Moreover, a HOXD3me and RASSF1Ame panel discriminated small cell lung carcinoma from non-small cell lung carcinoma with 75% sensitivity, 88% specificity, 6.5 LR+ and 0.28 LR–. An APCme and RASSF1Ame panel independently predicted disease-specific mortality in LC patients. We concluded that a DNA methylation-based test in liquid biopsies might enable minimally invasive screening of LC and PCa, improving patient compliance and reducing healthcare costs. Moreover, it might assist in LC subtyping and prognostication.

    更新日期:2019-12-02
  • Changes in DNA methylation from pre- to post-adolescence are associated with pubertal exposures
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    Luhang Han; Hongmei Zhang; Akhilesh Kaushal; Faisal I. Rezwan; Latha Kadalayil; Wilfried Karmaus; A. John Henderson; Caroline L. Relton; Susan Ring; S. Hasan Arshad; Susan L. Ewart; John W. Holloway

    Adolescence is a period characterized by major biological development, which may be associated with changes in DNA methylation (DNA-M). However, it is unknown to what extent DNA-M varies from pre- to post-adolescence, whether the pattern of changes is different between females and males, and how adolescence-related factors are associated with changes in DNA-M. Genome-scale DNA-M at ages 10 and 18 years in whole blood of 325 subjects (n = 140 females) in the Isle of Wight (IOW) birth cohort was analyzed using Illumina Infinium arrays (450K and EPIC). Linear mixed models were used to examine DNA-M changes between pre- and post-adolescence and whether the changes were gender-specific. Adolescence-related factors and environmental exposure factors were assessed on their association with DNA-M changes. Replication of findings was attempted in the comparable Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. In the IOW cohort, after controlling for technical variation and cell compositions at both pre- and post-adolescence, 15,532 cytosine–phosphate–guanine (CpG) sites (of 400,825 CpGs, 3.88%) showed statistically significant DNA-M changes from pre-adolescence to post-adolescence invariant to gender (false discovery rate (FDR) = 0.05). Of these 15,532 CpGs, 10,212 CpGs (66%) were replicated in the ALSPAC cohort. Pathway analysis using Ingenuity Pathway Analysis (IPA) identified significant biological pathways related to growth and development of the reproductive system, emphasizing the importance of this period of transition on epigenetic state of genes. In addition, in IOW, we identified 1179 CpGs with gender-specific DNA-M changes. In the IOW cohort, body mass index (BMI) at age 10 years, age of growth spurt, nonsteroidal drugs use, and current smoking status showed statistically significant associations with DNA-M changes at 15 CpGs on 14 genes such as the AHRR gene. For BMI at age 10 years, the association was gender-specific. Findings on current smoking status were replicated in the ALSPAC cohort. Adolescent transition is associated with changes in DNA-M at more than 15K CpGs. Identified pathways emphasize the importance of this period of transition on epigenetic state of genes relevant to cell growth and immune system development.

    更新日期:2019-12-02
  • DNA methylation profiles in chronic lymphocytic leukemia patients treated with chemoimmunotherapy
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-12-02
    Maria Tsagiopoulou; Nikos Papakonstantinou; Theodoros Moysiadis; Larry Mansouri; Viktor Ljungström; Martí Duran-Ferrer; Andigoni Malousi; Ana C. Queirós; Karla Plevova; Sujata Bhoi; Panagoula Kollia; David Oscier; Achilles Anagnostopoulos; Livio Trentin; Matthias Ritgen; Sarka Pospisilova; Niki Stavroyianni; Paolo Ghia; Jose I. Martin-Subero; Christiane Pott; Richard Rosenquist; Kostas Stamatopoulos

    In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed “epigenetic burden” (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed “relapse changes” (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

    更新日期:2019-12-02
  • Validation of an epigenetic field of susceptibility to detect significant prostate cancer from non-tumor biopsies
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-11-28
    Bing Yang; Tyler Etheridge; Johnathon McCormick; Adam Schultz; Tariq A. Khemees; Nathan Damaschke; Glen Leverson; Kaitlin Woo; Geoffrey A. Sonn; Eric A. Klein; Mike Fumo; Wei Huang; David F. Jarrard

    An epigenetic field of cancer susceptibility exists for prostate cancer (PC) that gives rise to multifocal disease in the peripheral prostate. In previous work, genome-wide DNA methylation profiling identified altered regions in the normal prostate tissue of men with PC. In the current multicenter study, we examined the predictive strength of a panel of loci to detect cancer presence and grade in patients with negative biopsy tissue. Four centers contributed benign prostate biopsy tissues blocks from 129 subjects that were either tumor associated (TA, Grade Group [GG] ≥ 2, n = 77) or non-tumor associated (NTA, n = 52). Biopsies were analyzed using pyrosequencing for DNA methylation encompassing CpG loci near CAV1, EVX1, FGF1, NCR2, PLA2G16, and SPAG4 and methylation differences were detected within all gene regions (p < 0.05). A multiplex regression model for biomarker performance incorporating a gene combination discriminated TA from NTA tissues (area under the curve [AUC] 0.747, p = 0.004). A multiplex model incorporating all the above genes and clinical information (PSA, age) identified patients with GG ≥ 2 PC (AUC 0.815, p < 0.0001). In patients with cancer, increased variation in gene methylation levels occurs between biopsies across the prostate. A widespread epigenetic field defect is utilized to detect GG ≥ 2 PC in patients with histologically negative biopsies. These alterations in non-tumor cells display increased heterogeneity of methylation extent and are spatially distant from tumor foci. These findings have the potential to decrease the need for repeated prostate biopsy.

    更新日期:2019-11-29
  • DNA methylation associated with postpartum depressive symptoms overlaps findings from a genome-wide association meta-analysis of depression
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-11-28
    Dana M. Lapato; Roxann Roberson-Nay; Robert M. Kirkpatrick; Bradley T. Webb; Timothy P. York; Patricia A. Kinser

    Perinatal depressive symptoms have been linked to adverse maternal and infant health outcomes. The etiology associated with perinatal depressive psychopathology is poorly understood, but accumulating evidence suggests that understanding inter-individual differences in DNA methylation (DNAm) patterning may provide insight regarding the genomic regions salient to the risk liability of perinatal depressive psychopathology. Genome-wide DNAm was measured in maternal peripheral blood using the Infinium MethylationEPIC microarray. Ninety-two participants (46% African-American) had DNAm samples that passed all quality control metrics, and all participants were within 7 months of delivery. Linear models were constructed to identify differentially methylated sites and regions, and permutation testing was utilized to assess significance. Differentially methylated regions (DMRs) were defined as genomic regions of consistent DNAm change with at least two probes within 1 kb of each other. Maternal age, current smoking status, estimated cell-type proportions, ancestry-relevant principal components, days since delivery, and chip position served as covariates to adjust for technical and biological factors. Current postpartum depressive symptoms were measured using the Edinburgh Postnatal Depression Scale. Ninety-eight DMRs were significant (false discovery rate < 5%) and overlapped 92 genes. Three of the regions overlap loci from the latest Psychiatric Genomics Consortium meta-analysis of depression. Many of the genes identified in this analysis corroborate previous allelic, transcriptomic, and DNAm association results related to depressive phenotypes. Future work should integrate data from multi-omic platforms to understand the functional relevance of these DMRs and refine DNAm association results by limiting phenotypic heterogeneity and clarifying if DNAm differences relate to the timing of onset, severity, duration of perinatal mental health outcomes of the current pregnancy or to previous history of depressive psychopathology.

    更新日期:2019-11-29
  • Combined genetic mutations and DNA-methylated genes as biomarkers for endometrial cancer detection from cervical scrapings
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-11-28
    Phui-Ly Liew; Rui-Lan Huang; Tzu-I Wu; Chi-Chun Liao; Chien-Wen Chen; Po-Hsuan Su; Hui-Chen Wang; Yu-Chun Weng; Hung-Cheng Lai

    Endometrial cancer is a common gynecologic cancer. Noninvasive molecular biomarkers for triage of high-risk patients for invasive procedures are needed. Based on the success of cytological Pap smear screening, cervical scrapings are a good source of DNA for molecular testing. In addition to genetic lesions, DNA methylation is a promising biomarker. We assessed the usefulness of combining genetic and epigenetic biomarkers from cervical scrapings to detect endometrial carcinomas. We performed a retrospective case–control study of 96 consecutive cervical scrapings from patients with abnormal uterine bleeding who underwent surgery for diagnostic evaluation. Thirty and 16 cases were diagnosed with type I and type II endometrial cancers, respectively. The remaining non-cancer cases included normal endometrium (n = 12), benign uterine lesions (n = 20), and endometrial hyperplasia (n = 18). Quantitative methylation-specific PCR and mass spectrometry were used for DNA methylation and genetic mutation analysis. Logistic regression was used to evaluate the clinical performance of these candidate biomarkers. We tested the effectiveness of the methylation status of four genes (BHLHE22, CDO1, TBX5, and HAND2) in endometrial cancer detection. The area under the receiver operating characteristic curves ranged from 0.703 to 0.878, and panels of hypermethylated BHLHE22/CDO1/HAND2 (87.0% sensitivity and 86.0% specificity) and BHLHE22/CDO1/TBX5 (89.1% sensitivity and 80.0% specificity) showed significant differences and could distinguish benign from malignant endometrial lesions. The sensitivity and specificity in endometrial cancer detection for BHLHE22/CDO1 were 84.8% and 88.0%, respectively. Both type I and II endometrial carcinomas could be detected using a BHLHE22/CDO1-based methylation profile, suggesting that they may have common epigenomes. Moreover, PTEN and TP53 mutations were found in 63.3% of type I and 93.6% of type II endometrial cancers. Unexpectedly, PTEN and TP53 mutations were commonly found in cervical scrapings of the normal endometrium (25% and 33.3%, respectively) and in cases with benign uterine lesions (10% and 50%, respectively). Finally, combinations of any one mutation of PTEN and TP53 mutations had a sensitivity of 91.3%, but a specificity of only 42.0%. Adding PTEN/TP53 mutation testing to BHLHE22/CDO1-based methylation testing did not improve the detection of endometrial cancer.

    更新日期:2019-11-29
  • Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-11-28
    Estela Dámaso; Júlia Canet-Hermida; Gardenia Vargas-Parra; Àngela Velasco; Fátima Marín; Esther Darder; Jesús del Valle; Anna Fernández; Àngel Izquierdo; Gemma Mateu; Glòria Oliveras; Carmen Escribano; Virgínia Piñol; Hugo-Ikuo Uchima; José Luis Soto; Megan Hitchins; Ramon Farrés; Conxi Lázaro; Bernat Queralt; Joan Brunet; Gabriel Capellá; Marta Pineda

    Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer. Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches. MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient’s tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative. Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

    更新日期:2019-11-29
  • DNA methyltransferase isoforms expression in the temporal lobe of epilepsy patients with a history of febrile seizures
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-19
    Laurence de Nijs; Kyonghwan Choe; Hellen Steinbusch; Olaf E. M. G. Schijns; Jim Dings; Daniel L. A. van den Hove; Bart P. F. Rutten; Govert Hoogland

    Temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) is a common pharmaco-resistant epilepsy referred for adult epilepsy surgery. Though associated with prolonged febrile seizures (FS) in childhood, the neurobiological basis for this relationship is not fully understood and currently no preventive or curative therapies are available. DNA methylation, an epigenetic mechanism catalyzed by DNA methyltransferases (DNMTs), potentially plays a pivotal role in epileptogenesis associated with FS. In an attempt to start exploring this notion, the present cross-sectional pilot study investigated whether global DNA methylation levels (5-mC and 5-hmC markers) and DNMT isoforms (DNMT1, DNMT3a1, and DNMT3a2) expression would be different in hippocampal and neocortical tissues between controls and TLE patients with or without a history of FS. We found that global DNA methylation levels and DNMT3a2 isoform expression were lower in the hippocampus for all TLE groups when compared to control patients, with a more significant decrease amongst the TLE groups with a history of FS. Interestingly, we showed that DNMT3a1 expression was severely diminished in the hippocampus of TLE patients with a history of FS in comparison with control and other TLE groups. In the neocortex, we found a higher expression of DNMT1 and DNMT3a1 as well as increased levels of global DNA methylation for all TLE patients compared to controls. Together, the findings of this descriptive cross-sectional pilot study demonstrated brain region-specific changes in DNMT1 and DNMT3a isoform expression as well as global DNA methylation levels in human TLE with or without a history of FS. They highlighted a specific implication of DNMT3a isoforms in TLE after FS. Therefore, longitudinal studies that aim at targeting DNMT3a isoforms to evaluate the potential causal relationship between FS and TLE or treatment of FS-induced epileptogenesis seem warranted.

    更新日期:2019-11-28
  • Novel age-associated DNA methylation changes and epigenetic age acceleration in middle-aged African Americans and whites
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-19
    Salman M. Tajuddin; Dena G. Hernandez; Brian H. Chen; Nicole Noren Hooten; Nicolle A. Mode; Mike A. Nalls; Andrew B. Singleton; Ngozi Ejiogu; Kumaraswamy Naidu Chitrala; Alan B. Zonderman; Michele K. Evans

    African Americans (AAs) experience premature chronic health outcomes and longevity disparities consistent with an accelerated aging phenotype. DNA methylation (DNAm) levels at specific CpG positions are hallmarks of aging evidenced by the presence of age-associated differentially methylated CpG positions (aDMPs) that are the basis for the epigenetic clock for measuring biological age acceleration. Since DNAm has not been widely studied among non-European populations, we examined the association between DNAm and chronological age in AAs and whites, and the association between race, poverty, sex, and epigenetic age acceleration. We measured genome-wide DNA methylation (866,836 CpGs) using the Illumina MethylationEPIC BeadChip in blood DNA extracted from 487 middle-aged AA (N = 244) and white (N = 243), men (N = 248), and women (N = 239). The mean (sd) age was 48.4 (8.8) in AA and 49.0 (8.7) in whites (p = 0.48). We identified 4930 significantly associated aDMPs in AAs and 469 in whites. Of these, 75.6% and 53.1% were novel, largely driven by the increased number of measured CpGs in the EPIC array, in AA and whites, respectively. AAs had more age-associated DNAm changes than whites in genes implicated in age-related diseases and cellular pathways involved in growth and development. We assessed three epigenetic age acceleration measures (universal, intrinsic, and extrinsic). AAs had a significantly slower extrinsic aging compared to whites. Furthermore, compared to AA women, both AA and white men had faster aging in the universal age acceleration measure (+ 2.04 and + 1.24 years, respectively, p < 0.05). AAs have more wide-spread methylation changes than whites. Race and sex interact to underlie biological age acceleration suggesting altered DNA methylation patterns may be important in age-associated health disparities.

    更新日期:2019-11-28
  • Promoter methylation of SEPT9 as a potential biomarker for early detection of cervical cancer and its overexpression predicts radioresistance
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-19
    Xinlin Jiao; Siying Zhang; Jun Jiao; Teng Zhang; Wenjie Qu; Guy Mutangala Muloye; Beihua Kong; Qing Zhang; Baoxia Cui

    Cervical cancer screening by combined cytology and HPV test has reduced the incidence of cervical cancer, but cytological screening lacks a higher sensitivity while HPV testing possesses a lower specificity. Most patients with invasive cervical cancer are treated with radiotherapy. However, insensitivity to radiotherapy leads to poor efficacy. Illumina Methylation EPIC 850k Beadchip was used for genomic screening. We detected methylation of SEPT9 and mRNA expression in different cervical tissues by using methylation-specific PCR and qRT-PCR. Then using CCK8, migration assay, and flow cytometry to detect the biological function and irradiation resistance of SEPT9 in vitro and in vivo. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation (CoIP) were used to find the interacting gene with SEPT9. Immunostaining of CD206 in cervical cancer and polarization of macrophages (M2) were evaluated by immunofluorescence and WB. The Cancer Genome Atlas (TCGA) database was used for screening the potential miRNAs induced by SEPT9. Hyper-methylation of SEPT9 detects cervical cancer and normal tissues, normal+CIN1 and CIN2+CIN3+cancer with high sensitivity and specificity (AUC = 0.854 and 0.797, respectively, P < 0.001). The mRNA and protein expression of SEPT9 was upregulated in cervical cancer tissues when compared to para-carcinoma tissues. SEPT9 promotes proliferation, invasion, migration, and influences the cell cycle of cervical cancer. SEPT9 interacted with HMGB1-RB axis increases irradiation resistance. Furthermore, SEPT9 mediated miR-375 via the tumor-associated macrophages (TAMs) polarization, affecting the resistance to radiotherapy in cervical cancer. These findings give us the evidence that SEPT9 methylation could be a biomarker for cervical cancer diagnoses. It promotes tumorigenesis and radioresistance of cervical cancer by targeting HMGB1-RB axis and causes polarization of macrophages by mediating miR-375. We suggest SEPT9 could be a potential screening and therapeutic biomarker for cervical cancer.

    更新日期:2019-11-28
  • Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-22
    Oxana Bereshchenko; Oriana Lo Re; Fedor Nikulenkov; Sara Flamini; Jana Kotaskova; Tommaso Mazza; Marguerite-Marie Le Pannérer; Marcus Buschbeck; Cesarina Giallongo; Giuseppe Palumbo; Giovanni Li Volti; Valerio Pazienza; Libor Cervinek; Carlo Riccardi; Lumir Krejci; Sarka Pospisilova; A. Francis Stewart; Manlio Vinciguerra

    Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies.

    更新日期:2019-11-28
  • Replicated methylation changes associated with eczema herpeticum and allergic response
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-23
    Meher Preethi Boorgula; Margaret A. Taub; Nicholas Rafaels; Michelle Daya; Monica Campbell; Sameer Chavan; Aniket Shetty; Chris Cheadle; Sangjucta Barkataki; Jinshui Fan; Gloria David; Terri H. Beaty; Ingo Ruczinski; Jon Hanifin; Lynda C. Schneider; Richard L. Gallo; Amy S. Paller; Lisa A. Beck; Donald Y. Leung; Rasika A. Mathias; Kathleen C. Barnes

    Although epigenetic mechanisms are important risk factors for allergic disease, few studies have evaluated DNA methylation differences associated with atopic dermatitis (AD), and none has focused on AD with eczema herpeticum (ADEH+). We will determine how methylation varies in AD individuals with/without EH and associated traits. We modeled differences in genome-wide DNA methylation in whole blood cells from 90 ADEH+, 83 ADEH−, and 84 non-atopic, healthy control subjects, replicating in 36 ADEH+, 53 ADEH−, and 55 non-atopic healthy control subjects. We adjusted for cell-type composition in our models and used genome-wide and candidate-gene approaches. We replicated one CpG which was significantly differentially methylated by severity, with suggestive replication at four others showing differential methylation by phenotype or severity. Not adjusting for eosinophil content, we identified 490 significantly differentially methylated CpGs (ADEH+ vs healthy controls, genome-wide). Many of these associated with severity measures, especially eosinophil count (431/490 sites). We identified a CpG in IL4 associated with serum tIgE levels, supporting a role for Th2 immune mediating mechanisms in AD. Changes in eosinophil level, a measure of disease severity, are associated with methylation changes, providing a potential mechanism for phenotypic changes in immune response-related traits.

    更新日期:2019-11-28
  • Statistical predictions with glmnet
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-23
    Solveig Engebretsen; Jon Bohlin

    Elastic net type regression methods have become very popular for prediction of certain outcomes in epigenome-wide association studies (EWAS). The methods considered accept biased coefficient estimates in return for lower variance thus obtaining improved prediction accuracy. We provide guidelines on how to obtain parsimonious models with low mean squared error and include easy to follow walk-through examples for each step in R.

    更新日期:2019-11-28
  • Systematic evaluation and validation of reference and library selection methods for deconvolution of cord blood DNA methylation data
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-27
    Kristina Gervin; Lucas A. Salas; Kelly M. Bakulski; Menno C. van Zelm; Devin C. Koestler; John K. Wiencke; Liesbeth Duijts; Henriëtte A. Moll; Karl T. Kelsey; Michael S. Kobor; Robert Lyle; Brock C. Christensen; Janine F. Felix; Meaghan J. Jones

    Umbilical cord blood (UCB) is commonly used in epigenome-wide association studies of prenatal exposures. Accounting for cell type composition is critical in such studies as it reduces confounding due to the cell specificity of DNA methylation (DNAm). In the absence of cell sorting information, statistical methods can be applied to deconvolve heterogeneous cell mixtures. Among these methods, reference-based approaches leverage age-appropriate cell-specific DNAm profiles to estimate cellular composition. In UCB, four reference datasets comprising DNAm signatures profiled in purified cell populations have been published using the Illumina 450 K and EPIC arrays. These datasets are biologically and technically different, and currently, there is no consensus on how to best apply them. Here, we systematically evaluate and compare these datasets and provide recommendations for reference-based UCB deconvolution. We first evaluated the four reference datasets to ascertain both the purity of the samples and the potential cell cross-contamination. We filtered samples and combined datasets to obtain a joint UCB reference. We selected deconvolution libraries using two different approaches: automatic selection using the top differentially methylated probes from the function pickCompProbes in minfi and a standardized library selected using the IDOL (Identifying Optimal Libraries) iterative algorithm. We compared the performance of each reference separately and in combination, using the two approaches for reference library selection, and validated the results in an independent cohort (Generation R Study, n = 191) with matched Fluorescence-Activated Cell Sorting measured cell counts. Strict filtering and combination of the references significantly improved the accuracy and efficiency of cell type estimates. Ultimately, the IDOL library outperformed the library from the automatic selection method implemented in pickCompProbes. These results have important implications for epigenetic studies in UCB as implementing this method will optimally reduce confounding due to cellular heterogeneity. This work provides guidelines for future reference-based UCB deconvolution and establishes a framework for combining reference datasets in other tissues.

    更新日期:2019-11-28
  • Childhood traumatization is associated with differences in TRPA1 promoter methylation in female patients with multisomatoform disorder with pain as the leading bodily symptom
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-28
    Johannes Achenbach; Mathias Rhein; Sara Gombert; Fiona Meyer-Bockenkamp; Miro Buhck; Mirjam Eberhardt; Andreas Leffler; Helge Frieling; Matthias Karst

    The construct of multisomatoform disorder (MSD) is a common point of reference for patients in different somatic and psychosomatic specialties and therefore useful in studying large well-characterized cohorts of a prototype of a somatoform disorder and in parallel as a functional somatic syndrome (FSS). This disorder is characterized by distressing and functionally disabling somatic symptoms with chronic pain as the most frequent and clinically relevant complaint. Pain is perceived by nociceptive nerve fibers and transferred through the generation of action potentials by different receptor molecules known to determine pain sensitivity in pathophysiological processes. Previous studies have shown that for the transient receptor potential ankyrin 1 (TRPA1), receptor methylation of a particular CpG dinucleotide in the promoter region is inversely associated with both heat pain and pressure pain thresholds. In this study, we hypothesized that TRPA1 promoter methylation regulates pain sensitivity of patients with multisomatoform disorder (MSD). A cohort of 151 patients with MSD and 149 matched healthy volunteers were evaluated using quantitative sensory testing, clinical and psychometric assessment, and methylation analysis using DNA isolated from whole blood. We found CpG -628 to be correlated with mechanical pain threshold and CpG -411 to be correlated with mechanical pain threshold in female volunteers, i.e., higher methylation levels lead to higher pain thresholds. A novel finding is that methylation levels were significantly different between patients with no and severe levels of childhood trauma. CpG methylation also correlated with psychometric assessment of pain and pain levels rated on a visual analog scale. Our findings support the hypothesis that epigenetic regulation of TRPA1 plays a role in mechanical pain sensitivities in healthy volunteers. They further provide evidence for the possible influence of childhood traumatic experiences on the epigenetic regulation of TRPA1 in patients with MSD.

    更新日期:2019-11-28
  • High-resolution analysis of germ cells from men with sex chromosomal aneuploidies reveals normal transcriptome but impaired imprinting
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-28
    Sandra Laurentino; Laura Heckmann; Sara Di Persio; Xiaolin Li; Gerd Meyer zu Hörste; Joachim Wistuba; Jann-Frederik Cremers; Jörg Gromoll; Sabine Kliesch; Stefan Schlatt; Nina Neuhaus

    The most common sex chromosomal aneuploidy in males is Klinefelter syndrome, which is characterized by at least one supernumerary X chromosome. While these men have long been considered infertile, focal spermatogenesis can be observed in some patients, and sperm can be surgically retrieved and used for artificial reproductive techniques. Although these gametes can be used for fertility treatments, little is known about the molecular biology of the germline in Klinefelter men. Specifically, it is unclear if germ cells in Klinefelter syndrome correctly establish the androgenetic DNA methylation profile and transcriptome. This is due to the low number of germ cells in the Klinefelter testes available for analysis. Here, we overcame these difficulties and successfully investigated the epigenetic and transcriptional profiles of germ cells in Klinefelter patients employing deep bisulfite sequencing and single-cell RNA sequencing. On the transcriptional level, the germ cells from Klinefelter men clustered together with the differentiation stages of normal spermatogenesis. Klinefelter germ cells showed a normal DNA methylation profile of selected germ cell-specific markers compared with spermatogonia and sperm from men with normal spermatogenesis. However, germ cells from Klinefelter patients showed variations in the DNA methylation of imprinted regions. These data indicate that Klinefelter germ cells have a normal transcriptome but might present aberrant imprinting, showing impairment in germ cell development that goes beyond mere germ cell loss.

    更新日期:2019-11-28
  • Duration of breastfeeding is associated with leptin (LEP) DNA methylation profiles and BMI in 10-year-old children
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-29
    William B. Sherwood; Victoria Bion; Gabrielle A. Lockett; Ali H. Ziyab; Nelís Soto-Ramírez; Nandini Mukherjee; Ramesh J. Kurukulaaratchy; Susan Ewart; Hongmei Zhang; S. Hasan Arshad; Wilfried Karmaus; John W. Holloway; Faisal I. Rezwan

    Breastfeeding is protective against many long-term diseases, yet the mechanisms involved are unknown. Leptin gene (LEP) is reported to be associated with body mass index (BMI). On the other hand, breastfeeding duration has been found to be associated with DNA methylation (DNAm) of the LEP gene. Therefore, epigenetic regulation of LEP may represent the mechanism underlying the protective effect of breastfeeding duration against obesity. In the Isle of Wight Birth Cohort, peripheral blood DNAm at 23 cytosine-phosphate-guanine sites (CpGs) in the LEP locus in 10-year-old (n = 297) samples and 16 CpGs in 18-year-old (n = 305) samples, were generated using the Illumina Infinium MethylationEPIC and HumanMethylation450 Beadchips respectively and tested for association with breastfeeding duration (total and exclusive) using linear regression. To explore the association between breastfeeding durations and genome-wide DNAm, epigenome-wide association studies (EWASs) and differential methylation region (DMR) analyses were performed. BMI trajectories spanning the first 18 years of life were used as the outcome to test the association with breastfeeding duration (exposure) using multi-nominal logistic regression. Mediation analysis was performed for significant CpG sites. Both total and exclusive breastfeeding duration were associated with DNAm at four LEP CpG sites at 10 years (P value < 0.05), and not at 18 years. Though no association was observed between breastfeeding duration and genome-wide DNAm, DMR analyses identified five significant differentially methylated regions (Sidak adjusted P value < 0.05). Breastfeeding duration was also associated with the early transient overweight trajectory. Furthermore, DNAm of LEP was associated with this trajectory at one CpG site and early persistent obesity at another, though mediation analysis was not significant. Breastfeeding duration is associated with LEP methylation at age 10 years and BMI trajectory. LEP DNAm is also significantly associated with BMI trajectories throughout childhood, though sample sizes were small. However, mediation analysis did not demonstrate that DNAm of LEP explained the protective effect of breastfeeding against childhood obesity.

    更新日期:2019-11-28
  • Particulate matter exposure shapes DNA methylation through the lifespan
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-08-30
    L. Ferrari; M. Carugno; V. Bollati

    Exposure to airborne particulate matter (PM) has been associated with detrimental health effects. DNA methylation represents the most well-studied epigenetic factor among the possible mechanisms underlying this association. Interestingly, changes of DNA methylation in response to environmental stimuli are being considered for their role in the pathogenic mechanism, but also as mediators of the body adaptation to air pollutants. Several studies have evaluated both global and gene-specific methylation in relation to PM exposure in different clinical conditions and life stages. The purpose of the present literature review is to evaluate the most relevant and recent studies in the field in order to analyze the available evidences on long- and short-term PM exposure and DNA methylation changes, with a particular focus on the different life stages when the alteration occurs. PM exposure modulates DNA methylation affecting several biological mechanisms with marked effects on health, especially during susceptible life stages such as pregnancy, childhood, and the older age. Although many cross-sectional investigations have been conducted so far, only a limited number of prospective studies have explored the potential role of DNA methylation. Future studies are needed in order to evaluate whether these changes might be reverted.

    更新日期:2019-11-28
  • Peripheral blood DNA methylation differences in twin pairs discordant for Alzheimer’s disease
    Clin. Epigenet. (IF 5.496) Pub Date : 2019-09-02
    Mikko Konki; Maia Malonzo; Ida K. Karlsson; Noora Lindgren; Bishwa Ghimire; Johannes Smolander; Noora M. Scheinin; Miina Ollikainen; Asta Laiho; Laura L. Elo; Tapio Lönnberg; Matias Röyttä; Nancy L. Pedersen; Jaakko Kaprio; Harri Lähdesmäki; Juha O. Rinne; Riikka J. Lund

    Alzheimer’s disease results from a neurodegenerative process that starts well before the diagnosis can be made. New prognostic or diagnostic markers enabling early intervention into the disease process would be highly valuable. Environmental and lifestyle factors largely modulate the disease risk and may influence the pathogenesis through epigenetic mechanisms, such as DNA methylation. As environmental and lifestyle factors may affect multiple tissues of the body, we hypothesized that the disease-associated DNA methylation signatures are detectable in the peripheral blood of discordant twin pairs. Comparison of 23 disease discordant Finnish twin pairs with reduced representation bisulfite sequencing revealed peripheral blood DNA methylation differences in 11 genomic regions with at least 15.0% median methylation difference and FDR adjusted p value ≤ 0.05. Several of the affected genes are primarily associated with neuronal functions and pathologies and do not display disease-associated differences in gene expression in blood. The DNA methylation mark in ADARB2 gene was found to be differentially methylated also in the anterior hippocampus, including entorhinal cortex, of non-twin cases and controls. Targeted bisulfite pyrosequencing of the DNA methylation mark in ADARB2 gene in 62 Finnish and Swedish twin pairs revealed that, in addition to the disease status, DNA methylation of this region is influenced by gender, age, zygosity, APOE genotype, and smoking. Further analysis of 120 Swedish twin pairs indicated that this specific DNA methylation mark is not predictive for Alzheimer’s disease and becomes differentially methylated after disease onset. DNA methylation differences can be detected in the peripheral blood of twin pairs discordant for Alzheimer’s disease. These DNA methylation signatures may have value as disease markers and provide insights into the molecular mechanisms of pathogenesis. We found no evidence that the DNA methylation marks would be associated with gene expression in blood. Further studies are needed to elucidate the potential importance of the associated genes in neuronal functions and to validate the prognostic or diagnostic value of the individual marks or marker panels.

    更新日期:2019-11-28
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