Methylation-sensitive Enzyme Research Articles

Difference in DNA methylation pattern and expression of active ingredients between garden ginseng and ginseng under forest

Panax ginseng is widely used for its medicinal value. With the decline in wild ginseng resources, garden ginseng (GG) and ginseng under forest (FG) have become the main sources. Their different growth conditions and ages may account for the differences in epigenetics. The genomic DNA of Panax ginseng was digested using the methylation-insensitive enzyme MseⅠ combined with the methylation-sensitive enzymes AciⅠ, PstⅠ, and EcoT22Ⅰ. Then, a sequencing library of CG, CHG, and CHH methylation contexts was constructed and sequenced through amplicon sequencing. Differences in DNA methylation were observed using the methylation context-sensitive enzyme ddRAD (MCSeEd), revealing eight differentially methylated regions (DMRs). The bisulfite sequencing technology revealed that the methylation level of GG was higher than that of FG. The Plant ISOform Sequencing Database was used to predict the regulatory function of CpG island–coding region so as to estimate the relationship between methylation and effective components. The quantitative real-time polymerase chain reaction (qPCR) showed that the expression of functional genes in FG was higher than that in GG. These studies showed that the increase in GG methylation level was associated with the decrease in gene expression, providing a new perspective on epigenetic differences and gene expression in ginseng.

Amelioration effect of 18β-Glycyrrhetinic acid on methylation inhibitors in hepatocarcinogenesis -induced by diethylnitrosamine.

suppression of methylation inhibitors (epigenetic genes) in hepatocarcinogenesis induced by diethylnitrosamine using glycyrrhetinic acid. In the current work, we investigated the effect of sole GA combined with different agents such as doxorubicin (DOX) or probiotic bacteria (Lactobacillus rhamanosus) against hepatocarcinogenesis induced by diethylnitrosamine to improve efficiency. The genomic DNA was isolated from rats' liver tissues to evaluate either methylation-sensitive or methylation-dependent resection enzymes. The methylation activity of the targeting genes DLC-1, TET-1, NF-kB, and STAT-3 was examined using specific primers and cleaved DNA products. Furthermore, flow cytometry was used to determine the protein expression profiles of DLC-1 and TET-1 in treated rats' liver tissue. Our results demonstrated the activity of GA to reduce the methylation activity in TET-1 and DLC-1 by 33.6% and 78%, respectively. As compared with the positive control. Furthermore, the association of GA with DOX avoided the methylation activity by 88% and 91% for TET-1 and DLC-1, respectively, as compared with the positive control. Similarly, the combined use of GA with probiotics suppressed the methylation activity in the TET-1 and DLC-1 genes by 75% and 81% for TET-1 and DLC-1, respectively. Also, GA and its combination with bacteria attenuated the adverse effect in hepatocarcinogenesis rats by altering potential methylomic genes such as NF-kb and STAT3 genes by 76% and 83%, respectively. GA has an ameliorative effect against methylation inhibitors in hepatocellular carcinoma (HCC) by decreasing the methylation activity genes.

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Cystathionine γ-synthase expression in seeds alters metabolic and DNA methylation profiles in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.

Legionellapneumophila induces methylomic changes in ten-eleven translocation to ensure bacterial reproduction in human lung epithelial cells.

Introduction. Legionella pneumophila is a Gram-negative flagellated bacteria that can infect human lungs and cause a severe form of pneumonia named Legionnaires' disease.Hypothesis. We hypothesize that L. pneumophila infection induces methylomic changes in methylcytosine dioxygenases, ten-eleven translocation (TET) genes, and controls DNA methylation following infection.Aim. In the current research, we sought to further investigate DNA methylation changes in human lung epithelial cells upon L. pneumophila infection and determine how methylation inhibitor agents disturb L. pneumophila reproduction.Methodology. A549 cell line was used in L. pneumophila infection and inhibitors' treatment, including 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG).Results. Interestingly, DNA methylation analysis of infected A549 using sodium bisulfite PCR and the methylation-sensitive HpaII enzyme showed potential methylation activity within the promoter regions of ten-eleven translocation (TET) genes located on CpG/397-8 and CpG/385-6 of TET1 and TET3, respectively. Such methylation changes in TET effectors decreased their expression profile following infection, indicated by quantitative real-time PCR (RT-qPCR), immunoblotting and flow cytometry. Furthermore, pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased the bacterial reproduction characterized by the expression of L. pneumophila 16S ribosomal RNA and the c.f.u. ml-1 of bacterial particles. Moreover, both methylation inhibitors showed potent inhibition of methionine synthase (MS) expression, which was further confirmed by the docking analysis of inhibitor ligands and crystal structure of MS protein.Conclusion. These data provide evidence for the methylomic changes in the promoter region of TET1 and TET3 by L. pneumophila infection in the A549 cell line and suggest the anti-bacterial properties of 5-AZA and EGCG, as methylation inhibitors, are due to targeting the epigenetic effector methionine synthase.

SEEMLIS: a flexible semi-automated method for enrichment of methylated DNA from low-input samples

BackgroundDNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS.ResultsWe show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity.ConclusionsSEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

Demethylase FTO activity analysis based on methyl sensitive enzyme MazF and hybridization chain reaction

FTO mediates oxidation demethylation of different RNA types and regulates fat mass and adipogenesis. More and more evidences show that FTO is deeply involved in the development of human diseases, such as acute myeloid leukemia and lung cancer, by regulating the content of m6A. Hence, it is essential to detect FTO in a smart way. At present study, we have developed a simple, sensitive and reliable methyl-sensitive nucleotide biosensor to analyze the activity of demethylase based on the methyl-sensitive RNA restriction enzyme MazF and the hybridization chain reaction (HCR) as a signal amplification unit. In the presence of target FTO, the biotin-modified 3′end of m6A RNA probe was demethylated and became available for MazF digestion, afterwards the trigger sequence obtained by digestion could continue to open hairpin structure for HCR. Under the optimal conditions, the linear range of this method was 20 nM to 200 nM with the detection limit of 2.273 nM. This strategy provided outgoing sensitivity, specificity and anti-interference ability in MDA-MB-231 cell lysates. In addition, we verified the feasibility of this strategy for FTO inhibitors and drug screening. Furthermore, this strategy could be used for analyzing other demethylases of m6A.

Detection of free DNA septin 9 gene methylation in plasma.

To explore the correlation between cytosine-phosphoric-guanylic (CpG) site of Septin 9 gene and colorectal cancer, and to develop a real-time PCR detection system in plasma in patients with colorectal cancer. The methylation of training samples was detected by high-throughput sequencing technology, and the sites highly consistent with the clinical information of colorectal cancer were identified. Then the detection system of real-time PCR was designed to analyze the consistency of plasma and tissue based on methylationa sensitive enzyme digestion. Finally, 100 clinical trials were conducted to evaluate the performance of the detection system with the methylation sensitive enzyme digestion-real-time PCR. The highly consistent sites, which were selected by high-throughput sequencing from 71 training set samples, was the 38th CpG. Based on the detection region, the screened methylation sensitive enzymes were BstU1, HhaI and HinP1I. The limit for the detection of methylation sensitive enzyme digestion-real-time PCR was 0.5%, and the Ct value was 38.5. The sensitivity was 87.27%, the specificity was 91.49%, the positive predictive value was 92.31%, and the negative predictive value was 86.00%. The 38th CpG site of Septin 9 detected by the detection system of methylation sensitive enzyme digestion-real-time PCR can highly predict the occurrence of colorectal cancer with great clinical application value.

Abstract 1976: Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation

Abstract Purpose Studies have shown that MutL Homolog 1 (MLH1) promotor methylation is closely associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. ctDNA methylation analysis has advantages in performance over mutation analysis, such as higher sensitivity, broader dynamic range and increased stability. However, current methods for methylation analysis involving bisulfite treatment possess many challenges which limit broad clinical applications. Here, we report the development of a fit-for-purpose digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. Study Procedure Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to methylation sensitive restrictive enzyme (MSRE) HpaII digestion. The performance of the MSRE ddPCR assay was evaluated as compared to MSRE qPCR assay. Plasma samples from healthy donors and CRC patients were analyzed with the optimal primer/probe sets using ddPCR. Assay performance was evaluated by determining the limit of detection (LOD), linearity of the primer/probe sets and the correlation coefficient. Receiver operating characteristic (ROC) curves were generated to determine the optimal assay sensitivity and specificity. Data Summary Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Three sets of primer/probes were optimized for robust amplification in ddPCR. Using 0.375ng synthetic DNA input, the LOD was measured as 1 positive droplet for set A, 4 positive droplets for set B, and 3 positive droplets for set C . Based on the results of ddPCR assay using 1ng cfDNA input from healthy donors or CRC samples, ROC curves were generated for each primer/probe set with area under the curve values ranging from 0.85 to 0.95. The optimal assay sensitivity and specificity were displayed when 5 positive droplets was used as acceptance criteria (93% sensitivity and 95% specificity). The optimal cutoff value of MLH-1 promoter methylation in ctDNA associated with patient diagnosis remains to be further defined. Conclusion The liquid biopsy assay for MLH-1 promoter methylation detection using purely quantitative ddPCR is a simple and ultrasensitive procedure that provides reliable methylation detection in ctDNA. The MRSE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development. Citation Format: DANYI WANG, Dennis O'Rourke, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH-1promoter methylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1976.

Discovery of a core-panel of markers for a blood-assay for cancer detection utilizing cfDNA methylation changes.

1522 Background: Cancer screening is limited to several cancers despite improved outcome A screening test should be acceptable, safe, and relatively inexpensive1 Tumors shed cfDNA to the blood where abundant tumor-specific methylation changes can be detected 1https://www.who.int/cancer/detection/variouscancer/en/. Methods: This is a prospective, multicenter, observational study under two protocols NCT04264767, NCT04264754. Plasma was collected from 1,255 subjects: 586 treatment-naïve cancer patients and 639 controls, in 21 sites and biobanks. Training set I (211 cases/99 controls) was used to select the 6 final markers for the core panel, training set II (200 controls) was used to lock the algorithm, and set the threshold to a score yielding specificity of 95%. The validation set (342 cases/310 controls) was performed utilizing the pre-specified algorithm and threshold. Plasma was separated from a single EDTA tube within 4 hours of blood draw. EpiCheck’s reagents and methylation-sensitive enzymes (Nucleix, Israel) were used for DNA extraction, digestion, and amplification in real-time PCR (ABI 7500 Fast Dx, Applied Biosystems). Results: Age was comparable but sex and smoking history were different (more women in cases, more smokers in controls). In the validation cohort twelve cancer types were included, with prominent representation of major cancer types (19% Breast, 14% colorectal and 21% lung) and stages I&II (56%). Specificity and sensitivity were maintained high at 94% and 62%. Highest sensitivity was demonstrated in GI cancers (77% colorectal, 83% esophageal, 100% gastric) and non-solid malignancies (83%). Sensitivity in early stage cancers (stages I, II & IIIA) was 71%, led by Sarcoma (83%) esophageal (76%) and colorectal (61%). Conclusions: This 6-marker blood-based methylation assay is a promising initial component in a future cancer screening test, generating significant signal in early cancers and utilizing simple and inexpensive PCR technology. Clinical trial information: NCT04264767, NCT04264754. [Table: see text]

DNA Methylation Tools and Strategies: Methods in a Review

DNA methylation is known as an important epigenetic change in plants and vertebrates genome. In this process, the methyl group transferred by DNA methyl transferase enzymes to cytosine at carbon residue 5 often in the CpG dinucleotide context. DNA methylation plays an important role in the natural development of the organism, genome stability maintenance and processes such as genomic imprinting and chromosome X inactivation in mammals. In addition, changes in DNA methylation pattern have seen in many diseases, including cancer. Analysis of DNA methylation has been useful for rapid disease diagnosis and progression. In recent decades, a revolution has taken place in the methods of DNA methylation analysis, and it is possible to study the pattern of gene methylation at a widespread, short and high resolution level. These methods can be divided into three general categories: (1) cut-based methods by methylation-sensitive enzymes; (2) sodium bisulfide based methods; (3) antibody based methods. Since the existence of different methods makes it difficult to select the appropriate approach, in this review, a number of common methods for examining the methylation pattern with the advantages and disadvantages will be discussed.

Unique Epigenetic Features of Ribosomal RNA Genes (rDNA) in Early Diverging Plants (Bryophytes).

Introduction: In plants, the multicopy genes encoding ribosomal RNA (rDNA) typically exhibit heterochromatic features and high level of DNA methylation. Here, we explored rDNA methylation in early diverging land plants from Bryophyta (15 species, 14 families) and Marchantiophyta (4 species, 4 families). DNA methylation was investigated by methylation-sensitive Southern blot hybridization in all species. We also carried out whole genomic bisulfite sequencing in Polytrichum formosum (Polytrichaceae) and Dicranum scoparium (Dicranaceae) and used available model plant methyloms (Physcomitrella patents and Marchantia polymorpha) to determine rDNA unit-wide methylation patterns. Chromatin structure was analyzed using fluorescence in situ hybridization (FISH) and immunoprecipitation (CHIP) assays.Results: In contrast to seed plants, bryophyte rDNAs were efficiently digested with methylation-sensitive enzymes indicating no or low levels of CG and CHG methylation in these loci. The rDNA methylom analyses revealed variation between species ranging from negligible (<3%, P. formosum, P. patens) to moderate (7 and 17% in M. polymorpha and D. scoparium, respectively) methylation levels. There were no differences between coding and noncoding parts of rDNA units and between gametophyte and sporophyte tissues. However, major satellite repeat and transposable elements were heavily methylated in P. formosum and D. scoparium. In P. formosum rDNA, the euchromatic H3K4m3 and heterochromatic H3K9m2 histone marks were nearly balanced contrasting the angiosperms data where H3K9m2 typically dominates rDNA chromatin. In moss interphase nuclei, rDNA was localized at the nucleolar periphery and its condensation level was high.Conclusions: Unlike seed plants, the rRNA genes seem to escape global methylation machinery in bryophytes. Distinct epigenetic features may be related to rDNA expression and the physiology of these early diverging plants that exist in haploid state for most of their life cycles.

Effect of cold stress on genomic DNA methylation in zebrafish ZF4 cells

PDF HTML阅读 XML下载 导出引用 引用提醒 低温对斑马鱼ZF4细胞基因组DNA甲基化水平的影响 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306;3. 海洋生物科学国际联合研究中心, 中国科学技术部, 上海海洋大学, 上海 201306 作者简介: 侯艳雯(1993-),女,硕士研究生,研究方向为分子生物学.E-mail:471708976@qq.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31372516，81770165）；上海市自然科学基金项目（13ZR1419500）；上海市教育委员会"东方学者"计划支持项目. Effect of cold stress on genomic DNA methylation in zebrafish ZF4 cells Author: Affiliation: 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education;Shanghai Ocean University, Shanghai 201306, China;2. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;3. International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Tech-nology, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:低温压力会导致鱼类生理功能失调、机体损伤甚至死亡，鱼类会产生各种适应性变化来应对低温压力，其中涉及的表观遗传学机制越来越受到重视。为了探讨鱼类低温应激压力下的表观遗传学调控机制，本研究对斑马鱼（）胚胎成纤维细胞ZF4进行短期低温胁迫（18℃处理3 d、5 d和10℃处理3 d、5 d）和长期低温胁迫（18℃处理30 d），然后用具有不同甲基化敏感性的同裂酶Ⅰ对细胞基因组DNA进行酶切以监测细胞基因组DNA甲基化水平变化。结果显示短期低温胁迫下ZF4细胞生长受到抑制甚至死亡，而经过长期低温胁迫的ZF4细胞对低温压力产生了一定适应性。并且低温胁迫下DNA甲基化水平呈现动态变化，短期低温培养细胞的基因组DNA甲基化水平明显增高，但是长期低温培养细胞的DNA甲基化水平反而下降。此外，研究发现抗氧化剂N-乙酰半胱氨酸（N-acetylcysteine，NAC）或者共济失调-毛细血管扩张突变蛋白（ataxia telangiectasia mutated，ATM）抑制剂KU-55933可以抑制18℃ 5 d低温处理后的ZF4细胞DNA甲基化水平的增高，说明活性氧（reactive oxygen species，ROS）的产生和ATM的激活介导了DNA甲基化水平的增高。本研究结果显示，短期低温刺激下ZF4细胞ROS的产生导致DNA损伤，激活DNA损伤修复机制，进而导致基因组DNA甲基化水平上升，该研究为后期斑马鱼低温胁迫分子机制的研究奠定基础。 Abstract:Cold stress causes physiological dysfunction, tissue damage, and finally death in fishes, and increasing studies have suggested that epigenetic mechanisms play essential roles in the cold stress response in fishes. Our previous study showed that cold stress induced the production of reactive oxygen species (ROS) in zebrafish -derived ZF4 cells in a temperature and time-dependent manner and that the genomic DNA methylation level was increased under short-term (18℃ for 5 days) cold exposure and decreased under long-term cold exposure (18℃ for 30 days). However, the relationship among DNA methylation, ROS production, and cold acclimation in fishes remains poorly understood. In the present study, zebrafish ZF4 cells were exposed to short-term (18℃ or 10℃, for 3 or 5 days) and long-term (18℃ for 30 days) cold stress, and then the global DNA methylation level was detected by digestion with the methylation-sensitive enzyme I. The results showed that short-term cold stress caused remarkable growth arrest and cell death in ZF4 cells, and cold acclimation was observed under long-term cold stress. Additionally, global DNA methylation increased remarkably under short-term cold stress ( < 0.05). Co-treatment of ZF4 cells with -acetylcysteine inhibited global DNA methylation induced by short-term cold stress ( < 0.05), suggesting that ROS affects short-term cold stress-induced global DNA methylation levels. Co-treatment of ZF4 cells with the ataxia telangiectasia mutated (ATM) inhibitor KU-55933 also inhibited the induction of global DNA methylation under short-term cold exposure, indicating the involvement of DNA damage repair pathways in this process. Our data indicate that short-term cold stress resulted in ROS production and ataxia telangiectasia mutated activation, which then up-regulated global DNA methylation in ZF4 cells. The present study improves our understanding of the role of DNA methylation under cold stress in fishes. 参考文献 相似文献 引证文献

Role of the Androgen Receptor Gene CAG Repeat Polymorphism on the Sequence of Pubertal Events and Adiposity in Girls with High Dehydroepiandrosterone Sulfate Level.

The androgen receptor (AR) harbors a variable repeat number of glutamine residues codified by (CAG)n, which seems to inversely affect AR transcriptional activity. We assessed whether (CAG)n affects the sequence of the androgen-sensitive pubertal events and body composition in prepubertal girls. Nested case-control study within the Growth and Obesity Cohort Study of 1196 low-middle income children (approximately 50% girls) from a university clinic in Santiago, Chile. Cases were girls with high dehydroepiandrosterone sulfate (DHEAS; >42μg/dL; HD) at age 7.0 (±0.4) years (n=58). On follow-up, 32 of them had thelarche (TB2) before the age of pubarche (PH2) and 26 had PH2 before the age of TB2. As controls, 107 age-matched girls with normal DHEAS (≤42μg/dL; ND) were selected. Methylation-weighted mean (CAG)n (mw[CAG]n) was calculated through X-chromosome methylation-sensitive enzyme restriction and polymerase chain reaction followed by automated capillary electrophoresis in peripheral blood DNA. Girls with HD and PH2 before the age of TB2 showed a trend to higher frequency (7/26, 26.9%) of mw(CAG)n <20 compared with ND girls (12/107; 11.2%; P=.087). Accordingly, a direct correlation between age of PH2 and mw(CAG)n was observed in HD (r=0.352; P=.007) and in ND girls (r=0.207; P=.033). Moreover, HD girls with mw(CAG)n less than 20 had lower waist circumference and waist/height ratio than HD girls with mw(CAG)n from 20 to less than 25 (P=.027 and P=.012, respectively) at age of DHEAS determination. Our results suggest that a greater transcriptional activity of theAR, given by short number of CAG repeats, might favor the onset of pubarche and reduce central adiposity in prepubertal girls with HD.

Epigenetics in Medical Practice in the Twenty First Century

Epigenetics as a field of study is important because it tends to give answers to pertinent questions like why are identical twins behaving differently despite their possession of the same gene? Epigenetics provides a frame work for solving medical problems like mental retardation, neurodegenerative disorder, schizophrenia and social challenges like suicide and addiction. Abnormal DNA methylation pattern in epigenetics has been linked with a huge number of human cancers. However, recent research has revealed that epigenetic pharmaceuticals like vorinostat, decitabine and azacytidine could be a replacement or aid therapy for presently accepted treatment methods such as radiation and chemotherapy or may argument these current treatments. Understanding epigenetics phenomena entails the use of some research methods which include chromatin immunoprecipitation, fluorescent in-situ hybridization, methylation-sensitive restrictive enzyme and the use of bioinformatics methods. Further research work into epigenetics should be encouraged since this discipline of study has the potential to unravel lots of knowledge that will be very beneficial to managing some medical conditions.

Skewed X chromosome inactivation in girls and female adolescents with autoimmune thyroid disease.

Skewed X chromosome inactivation (XCI) was associated with female predominance in adult autoimmune thyroid disease (ATD). In normal females, skewed XCI is increased with age. Whether early-onset skewed XCI is associated with childhood ATD remains unknown. This study aimed to determine XCI skewing in paediatric ATD. Ninety-one female ATD patients, aged 3-20years and 57 age-matched, female controls were enrolled. XCI was analysed by enzymatic digestion of DNA with methylation-sensitive enzymes followed by PCR of the polymorphic CAG repeat in the androgen receptor gene. Skewed XCI was defined as having 80% or greater of the cells preferentially inactivated on the same X chromosome. XCI pattern of the enrolled patients and parental origin of the skewed XCI were determined. After exclusion of samples with homozygous CAG repeats, skewed XCI was analysed in 83 patients (57 Graves' disease and 26 Hashimoto thyroiditis) and 52 controls. There was an increased frequency of skewed XCI in ATD patients as compared with the controls (23% vs 8%, P=0.022). Patients with Hashimoto thyroiditis had greater frequency of skewed XCI than patients with Graves' disease (38% vs 16%, P=0.023). There were no differences in clinical parameters between patients with skewed and random XCI. Analysis of 7 patients with skewed XCI showed a preferential inactivation of paternal X chromosome in 6 patients (86%). Frequency of skewed XCI was increased in childhood ATD. This observation suggests a possible association of skewed XCI in the development of paediatric ATD.

MsgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package (https://bioconductor.org/packages/release/bioc/html/msgbsR.html).

DNA methylation patterns of genes related to immune response in the different clinical forms of oral lichen planus.

The oral lichen planus is a chronic inflammatory disease. Although its aetiology is not well understood, the role of T lymphocytes in its inflammatory events is recognised. Identifying the epigenetic mechanisms involved in the pathogenesis of this immune-mediated condition is fundamental for understanding the inflammatory reaction that occurs in the disease. The purpose of this work was to evaluate the methylation pattern of 21 immune response-related genes in the different clinical forms of oral lichen planus. A cross-sectional study was performed to analyse the DNA methylation patterns in three distinct groups of oral lichen planus: (i) reticular/plaque lesions; (ii) erosive lesions; (iii) normal oral mucosa (control group). After DNA extraction from biopsies, the samples were submitted to digestions by methylation-sensitive and methylation-dependent enzymes and double digestion. The relative percentage of methylated DNA for each gene was provided using real-time polymerase chain reaction arrays. Hypermethylation of the STAT5A gene was observed only in the control group (59.0%). A higher hypermethylation of the ELANE gene was found in reticular/plaque lesions (72.1%) compared to the erosive lesions (50.0%). Our results show variations in the methylation profile of immune response-related genes, according to the clinical type of oral lichen planus after comparing with the normal oral mucosa. Further studies are necessary to validate these findings using gene expression analysis.

Bisulfite Sequencing for DNA Methylation Analysis of Primary Muscle Stem Cells.

In skeletal muscle, DNA methylation contributes to the suppression of gene expression in several biological processes and diseases. A protocol for the detection of methylated cytosine was thus established based on methylation-sensitive enzymes, immunoprecipitation, and bisulfite conversion. DNA methylation analysis, with bisulfite conversion and sequencing, enables the quantification of methylation at each single base position. Here, we describe a basic method of bisulfite sequencing that can be used to analyze local DNA methylation status to confirm genome-wide DNA methylation analysis or correlation of gene expression regulatory mechanisms.

Effects of methylation-sensitive enzymes on the enrichment of genic SNPs and the degree of genome complexity reduction in a two-enzyme genotyping-by-sequencing (GBS) approach: a case study in oil palm (Elaeis guineensis).

Advances in next generation sequencing have facilitated a large-scale single nucleotide polymorphism (SNP) discovery in many crop species. Genotyping-by-sequencing (GBS) approach couples next generation sequencing with genome complexity reduction techniques to simultaneously identify and genotype SNPs. Choice of enzymes used in GBS library preparation depends on several factors including the number of markers required, the desired level of multiplexing, and whether the enrichment of genic SNP is preferred. We evaluated various combinations of methylation-sensitive (AatII, PstI, MspI) and methylation-insensitive (SphI, MseI) enzymes for their effectiveness in genome complexity reduction and enrichment of genic SNPs. We discovered that the use of two methylation-sensitive enzymes effectively reduced genome complexity and did not require a size selection step. On the contrary, the genome coverage of libraries constructed with methylation-insensitive enzymes was quite high, and the additional size selection step may be required to increase the overall read depth. We also demonstrated the effectiveness of methylation-sensitive enzymes in enriching for SNPs located in genic regions. When two methylation-insensitive enzymes were used, only 16% of SNPs identified were located in genes and 18% in the vicinity (± 5 kb) of the genic regions, while most SNPs resided in the intergenic regions. In contrast, a remarkable degree of enrichment was observed when two methylation-sensitive enzymes were employed. Almost two thirds of the SNPs were located either inside (32–36%) or in the vicinity (28–31%) of the genic regions. These results provide useful information to help researchers choose appropriate GBS enzymes in oil palm and other crop species.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-016-0572-x) contains supplementary material, which is available to authorized users.