Quantitative DNA Methylation Analysis Research Articles

The Combination of SHOX2 and RASSF1A DNA Methylation Had a Diagnostic Value in Pulmonary Nodules and Early Lung Cancer

Introduction: The study explored the effects of SHOX2 and RASSF1A DNA methylation in lung cancer (LC). Method: Bronchoalveolar lavage fluid (BALF) samples as well as LC and normal adjacent tissues were collected from 72 LC patients and 35 patients with benign pulmonary nodules. Quantitative analysis of SHOX2 and RASSF1A DNA methylation was performed in benign pulmonary nodules and different stages of LC. The diagnostic value of SHOX2 and RASSF1A DNA methylation in LC and benign pulmonary nodules was determined by receiver operating characteristics analysis. Gain/loss-of-function experiments were constructed in LC cells and mouse models of xenograft and pulmonary nodule metastasis. The levels of SHOX2 and transfer-associated genes were tested through quantitative reverse transcription polymerase chain reaction and Western blot. Malignant phenotype of LC cells was assessed by functional experiment. The tumor volume and weight of mice in xenograft models were measured. Pulmonary nodule metastasis was determined through HE staining assay. 5-azacytidine appeared as a positive control drug. Result: SHOX2 DNA methylation or RASSF1A DNA methylation had diagnostic efficiency in pulmonary nodules and early LC, with the two combined having better diagnostic value. SHOX2 expression was upregulated in LC. Similar to 5-azacytidine, SHOX2 knockdown inhibited LC cell viability, migration, and invasion in vitro as well as restrained LC tumorigenesis and pulmonary nodule metastasis in vivo, whereas overexpressed SHOX2 had the opposite effects. Conclusion: The combination of SHOX2 and RASSF1A DNA methylation had a diagnostic value in pulmonary nodules and early LC. SHOX2 positively modulated the tumorigenesis and metastasis of LC by regulating DNA methylation processes.

Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Silver-Russell Syndrome.

Background: Silver-Russell syndrome (SRS; OMIM #180860) is a clinically and genetically heterogeneous imprinting disorder characterized by prenatal and postnatal growth failure. The aim of this study was to identify the epigenotype-phenotype correlations in these patients using quantitative DNA methylation analysis. Methods: One hundred and eighty-three subjects clinically suspected of having SRS were referred for diagnostic testing by the methylation profiling of H19-associated imprinting center (IC) 1 and imprinted PEG1/MEST regions using methylation-specific high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between quantitative DNA methylation status and clinical manifestations of the subjects according to the Netchine-Harbison (N-H) clinical scoring system for SRS were analyzed. Results: Among the 183 subjects, 90 had a clinical diagnosis of SRS [N-H score ≥ 4 (maximum = 6)] and 93 had an SRS score < 4. Molecular lesions were detected in 41% (37/90) of the subjects with a clinical diagnosis of SRS, compared with 3% (3/93) of those with an N-H score < 4. The IC1 methylation level was negatively correlated with the N-H score. The molecular diagnosis rate was positively correlated with the N-H score. Thirty-one subjects had IC1 hypomethylation (IC1 methylation level <35% by the MassARRAY assay), seven had maternal uniparental disomy 7, and two had pathogenic copy number variants. Among the 90 subjects with an N-H score ≥ 4, the IC1 methylation level was significantly different between those with or without some clinical SRS features, including birth length ≤ 10th centile, relative macrocephaly at birth, normal cognitive development, body asymmetry, clinodactyly of the fifth finger, and genital abnormalities. Conclusions: This study confirmed the suitability of the N-H clinical scoring system as clinical diagnostic criteria for SRS. Quantitative DNA methylation analysis using the MassARRAY assay can improve the detection of epigenotype-phenotype correlations, further promoting better genetic counseling and multidisciplinary management for these patients.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

Artificial Intelligence‐Assisted Label‐Free Spectroscopic Quantification of Global DNA Cytosine Methylation in a Miniature Plasmonic Pickering Emulsion

AbstractEpigenetic DNA methylations are early and frequently observed events in a diversity of diseases such as cancer. Despite the considerable clinical values for cancer liquid biopsy, quantitative analysis of DNA methylations remains a major challenge due to the lack of rapid, sensitive detection techniques. Here, an artificial intelligence‐assisted label‐free surface‐enhanced Raman spectroscopy (SERS) (iMeSERS) biosensor is reported for simultaneous quantification of C5‐methylcytosine (5mC) level and methylation ratio in DNA samples. This method utilizes the plasmonic Pickering emulsions as the biosensing platform for label‐free SERS detection, formed upon the addition of a sub‐microliter DNA sample to the hydrophobic Au nanostar‐containing n‐decane. Distinct spectral signatures of monophosphates of canonical deoxyribonucleotides (dNMPs) and the common methylation modification 5‐methyl‐2′‐deoxycytidine (d5mCMP) are identified and distinguished by the iMeSERS biosensor. The deep learning algorithms trained with SERS signatures of dNMPs and d5mCMP are then applied to the quantitative analysis of global DNA methylation. The exceptional capability of the deep learning‐driven approach is demonstrated for simultaneous quantification of the methylation ratio and level using a sub‐microliter volume of DNA samples. This work shows the power of label‐free SERS techniques combined with deep learning algorithms for quantitative analysis of epigenetic DNA modifications with great promises for clinical diagnosis.

Status of TWEAK DNA methylation and mRNA expression in systemic lupus erythematosus.

Draw upon research into the serum concentration, mRNA expression, and DNA methylation of TNF-like weak inducer of apoptosis (TWEAK) in the peripheral blood of systemic lupus erythematosus patients and healthy controls in an attempt to investigate the epigenetics associated with TWEAK in the pathogenesis of systemic lupus erythematosus (SLE). A total of 178 SLE patients (SLE group) and 131 sex-age matched healthy controls (HC group) were recruited. Enzyme-linked immunosorbent assays (ELISA) was used to detect serum protein concentration of TWEAK. TWEAK mRNA expression was analyzed by Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Methylation levels of the promotor of TWEAK were measured using quantitative DNA methylation analysis on the MassARRAY spectrometry. Serum TWEAK concentrations were not statistically significant in SLE patients and HCs. Nevertheless, serum TWEAK concentrations were significantly lower in patients with renal involvement when compared to those without it. Serum TWEAK concentrations were reduced in clinically active patients (SLEDAI ≥ 10) compared with clinically stable patients (SLEDAI < 10). It was also significantly associated with SLEDAI. Compared with the HC group, the TWEAK mRNA expression in the SLE group was significantly lower. The global DNA methylation levels of TWEAK in the SLE group were observed to be significantly higher than the HC group. SLE patients with renal involvement, and the clinically active patients had higher TWEAK global methylation as well as exhibited variation in certain CpG island methylation. Furthermore, TWEAK methylation negatively correlated with TWEAK mRNA expression. This study suggests that TWEAK DNA methylation is a valuable as a focus for epigenetic studies because of it potentially influencing TWEAK gene expression in SLE patients. Aberrant DNA methylation of TWEAK may be involved in the initiation and development of SLE.

Quantitative analysis of DNA methylation using sequence-specific, real-time loop-mediated isothermal amplification

A number of genetic diseases and in the majority of tumor diseases have been reported to be associated with anomalous methylation levels of DNA. Accurate determine of the methylation level is an essential prerequisite for epigenetic diagnostics. Although some studies have used probe with a quencher enable real-time monitor of loop-mediated isothermal amplification (LAMP), most of them lack systematic investigations of design principle and applicable ambient when applied in LAMP based methylation detection. Herein, a sequence-specific, real-time loop-mediated isothermal amplification was developed for the quantitative analysis of DNA methylation. An oligonucleotide dimer was designed to implement three functions at the same time, including primer, sense probe and signal probe (PSS probe). Systematic investigations were performed to optimize the performance of PSS probe. After that, our method can accurately determine as low as 0.1% synthetic methylated DNA in the presence of numerous unmethylated DNA. Moreover, a higher methylated level of kazrin periplakin interacting protein (KAZN) gene was detected in colorectal cancer cells with a stable housekeeping gene β-actin used as an endogenous control. The developed PSS probe for LAMP provide an option to analyze DNA methylation levels in resource-limited environments, and hold great potential for the diagnosis of methylation-related diseases.

Decitabine attenuates ischemic stroke by reducing astrocytes proliferation in rats.

DNA methylation regulates epigenetic gene expression in ischemic stroke. Decitabine attenuates ischemic stroke by inhibiting DNA methylation. However, the underlying mechanism of this effect is not known. A model of ischemic stroke in Sprague-Dawley rats was induced through middle cerebral artery occlusion followed by reperfusion step. The rats were randomly treated with decitabine or vehicle by a one-time intraperitoneal injection. Sham rats received similar treatments. Four days after treatment, the rats were perfused with saline or 4% paraformaldehyde after which the brain was excised. DNA methylation level and brain infarct volume were determined by dot blot and histochemistry, respectively. The cellular co-localization and quantitative analysis of DNA methylation were assessed by immunohistochemistry and expression levels of cdkn1b (p27) mRNA and protein were measured by qRT-PCR and immunohistochemistry, respectively. The proliferation of astrocytes and number of neurons were determined by immunohistochemistry. Rats treated with decitabine showed hypomethylation and reduced infarct volume in the cortex. DNA methylation was decreased in astrocytes. Decitabine upregulated p27 mRNA and protein expression levels and attenuated the proliferation of astrocytes in vivo and vitro. Decitabine promotes p27 gene expression possibly by inhibiting its DNA methylation, thereby decreases the proliferation of astrocytes, neuronal death and infarct volume after ischemic stroke.

Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.).

Simple SummarySesame (Sesamum indicum L.) is an important oilseed crop that is well known for its highly nutritious oil content. Due to its high oil content and nutritional properties, sesame is also known as the ‘queen of oilseeds’. Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. Sesame phyllody is the leading biotic constraint drastically affecting sesame productivity and resulting in yield losses of up to 80% in major sesame-producing countries in the world. In this study, we explored the role of DNA methylation in phytoplasma infection. Whole Genome Bisulfite Sequencing and a Quantitative analysis of DNA methylation using real-time PCR (qAMP) revealed an alteration of methylation pattern upon phytoplasma infection. Few selected development and defense-related loci were either hypo- or hypermethylated. We hereby report the first methylome study in healthy and phytoplasma-infected sesame. Our study provides fundamental insights into the role of DNA methylation in the symptom development of phytoplasma infection in sesame plants.Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

Abstract 6079: Spatial deconvolution from bulk DNA methylation profiles determines intratumoral epigenetic heterogeneity

Abstract Purpose: Intratumoral heterogeneity emerges from accumulating genetic and epigenetic changes during tumorigenesis, which may contribute to therapeutic failure and drug resistance. We aimed to develop a tool that can evaluate the intratumoral epigenetic heterogeneity using the DNA methylation profiles from bulk tumors. Methods: Ggenomic DNA of three laser micro-dissected tumor regions, including digestive tract surface (DTS), central bulk (CB), and invasive margin (IM), was extracted from formalin-fixed paraffin-embedded (FFPE) sections of 98 colorectal cancer patients. The genome-wide methylation profiles were generated with methylation array. The most variable methylated probes were selected to construct a DNA methylation-based heterogeneity (MeHEG) estimation tool that can deconvolve the proportion of each reference tumor region with the support vector machine model-based method. A PCR-based assay for quantitative analysis of DNA methylation (QASM) was developed to specifically determine the methylation status of each CpG in MeHEG assay at single-base resolution to realize fast evaluation of epigenetic heterogeneity. Results: In the discovery set with 79 patients, the differentially methylated CpGs among the three tumor regions were found. The 7 most representative CpGs were identified by random forest analysis and subsequently selected to develop the MeHEG algorithm. We validated its performance of deconvolution of tumor regions in an independent cohort with 19 patients. In addition, we showed the significant association of MeHEG-based epigenetic heterogeneity with the genomic heterogeneity in mutation and copy number variation in our in-house and TCGA cohorts. Finally, we found that the patients with higher MeHEG score had worse disease-free and overall survival outcomes. Conclusion: By constructing a 7-loci panel with a machine learning approach and QASM assay to facilitate its use in a PCR manner, we developed a valuable method to evaluate the intratumoral epigenetic heterogeneity. The MeHEG algorithm provides innovative insights into the intratumoral heterogeneity from the epigenetic perspective that may add valuable sources to current knowledge about tumor heterogeneity. Citation Format: Yumo Xie, Yu Zhang, Guannan Tang, Xiaolin Wang, Meijin Huang, Yanxin Luo, Huichuan Yu. Spatial deconvolution from bulk DNA methylation profiles determines intratumoral epigenetic heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6079.

Liquid biopsy, using a novel DNA methylation signature, distinguishes pancreatic adenocarcinoma from benign pancreatic disease

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cell-free DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

Comprehensive Analysis of Epigenetic Modifications and Immune-Cell Infiltration In Tissues from Patients With Systemic Lupus Erythematosus

Aim: To explore potential abnormal epigenetic modifications and immune-cell infiltration in tissues from systemic lupus erythematosus (SLE) patients. Materials & methods: To utilize bioinformatics analysis and 'wet lab'methods to identify and verify differentially expressed genes in multiple targeted organs in SLE. Results: Seven key genes, IFI44, IFI44L, IFIT1, IFIT3, PLSCR1, RSAD2 and OAS2, which are regulated by epigenetics and may be involved in the pathogenesis of SLE, are identified by combined longnoncoding RNA-miRNA-mRNA network analysis and DNA methylation analysis. The results of quantitative reverse transcription PCR, immunohistochemistry and DNA methylation analysis confirmed the potential of these genes as biomarkers. Conclusion: This study reveals the potential mechanisms in SLE from epigenetic modifications and immune-cell infiltration, providing diagnostic biomarkers and therapeutic targets for SLE.

Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Beckwith-Wiedemann Syndrome.

Background: Beckwith-Wiedemann syndrome (BWS; OMIM 130650) is a rare overgrowth syndrome with tumor predisposition resulting from the abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. The aim of this study was to identify the epigenotype-phenotype correlations of these patients using quantitative DNA methylation analysis. Methods: One hundred and four subjects with clinically suspected BWS were enrolled in this study. All of the subjects had been referred for diagnostic testing which was conducted using methylation profiling of H19-associated imprinting center (IC) 1 and KCNQ1OT1-associated IC2 in high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between the quantitative DNA methylation status and clinical manifestations of the enrolled subjects were analyzed. Results: Among the 104 subjects, 19 had IC2 hypomethylation, 2 had IC1 hypermethylation, and 10 had paternal uniparental disomy (pUPD). The subjects with IC2 hypomethylation were characterized by significantly more macroglossia but less hemihypertrophy compared to the subjects with pUPD (p < 0.05). For 19 subjects with IC2 hypomethylation, the IC2 methylation level was significantly different (p < 0.05) between the subjects with and without features including macroglossia (IC2 methylation level: 11.1% vs. 30.0%) and prenatal or postnatal overgrowth (8.5% vs. 16.9%). The IC2 methylation level was negatively correlated with birth weight z score (p < 0.01, n = 19) and birth height z score (p < 0.05, n = 13). For 36 subjects with clinically diagnosed BWS, the IC2 methylation level was negatively correlated with the BWS score (r = −0.592, p < 0.01). The IC1 methylation level showed the tendency of positive correlation with the BWS score without statistical significance (r = 0.137, p > 0.05). Conclusions: Lower IC2 methylation and higher IC1 methylation levels were associated with greater disease severity in the subjects with clinically diagnosed BWS. Quantitative DNA methylation analysis using the MassARRAY assay could improve the detection of epigenotype-phenotype correlations, which could further promote better genetic counseling and medical care for these patients.

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

Acute sleep loss alters circulating fibroblast growth factor 21 levels in humans: A randomised crossover trial.

The hormone fibroblast growth factor 21 (FGF21) modulates tissue metabolism and circulates at higher levels in metabolic conditions associated with chronic sleep-wake disruption, such as type 2 diabetes and obesity. In the present study, we investigated whether acute sleep loss impacts circulating levels of FGF21 and tissue-specific production, and response pathways linked to FGF21. A total of 15 healthy normal-weight young men participated in a randomised crossover study with two conditions, sleep loss versus an 8.5-hr sleep window. The evening before each intervention, fasting blood was collected. Fasting, post-intervention morning skeletal muscle and adipose tissue samples underwent quantitative polymerase chain reaction and DNA methylation analyses, and serum FGF21 levels were measured before and after an oral glucose tolerance test. Serum levels of FGF21 were higher after sleep loss compared with sleep, both under fasting conditions and following glucose intake (~27%-30%, p=0.023). Fasting circulating levels of fibroblast activation protein, a protein which can degrade circulating FGF21, were not altered by sleep loss, whereas DNA methylation in the FGF21 promoter region increased only in adipose tissue. However, even though specifically the muscle exhibited transcriptional changes indicating adverse alterations to redox and metabolic homeostasis, no tissue-based changes were observed in expression of FGF21, its receptors, or selected signalling targets, in response to sleep loss. In summary, we found that acute sleep loss resulted in increased circulating levels of FGF21 in healthy young men, which may occur independent of a tissue-based stress response in metabolic peripheral tissues. Further studies may decipher whether changes in FGF21 signalling after sleep loss modulate metabolic outcomes associated with sleep or circadian disruption.

DNA methylation-based signature of CD8+ tumor-infiltrating lymphocytes enables evaluation of immune response and prognosis in colorectal cancer

BackgroundTumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, can be used for predicting immunotherapy responsiveness and survival outcome. However, the evaluation of CD8+ TILs currently relies on histopathological methodology with high variability....

Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

Methyl donor micronutrients, CD40-ligand methylation and disease activity in systemic lupus erythematosus: A cross-sectional association study.

ObjectiveHypomethylation of CD40-ligand (CD40L) in T-cells is associated with increased disease activity in systemic lupus erythematosus (SLE). We therefore investigated possible associations of dietary methyl donors and products with CD40L methylation status in SLE.MethodsFood frequency questionnaires were employed to calculate methyl donor micronutrients in 61 female SLE patients (age 45.7 ± 12.0 years, disease duration 16.2 ± 8.4 years) and compared to methylation levels of previously identified key DNA methylation sites (CpG17 and CpG22) within CD40L promotor of T-cells using quantitative DNA methylation analysis on the EpiTYPER mass spectrometry platform. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Linear regression modelling was used. P values were adjusted according to Benjamini & Hochberg.ResultsAmongst the micronutrients assessed (g per day), methionine and cysteine were associated with methylation of CpG17 (β = 5.0 (95%CI: 0.6-9.4), p = 0.04; and β = 2.4 (0.6-4.1), p = 0.02, respectively). Methionine, choline, and cysteine were additionally associated with the mean methylation of the entire CD40L (β = 9.5 (1.0-18.0), p = 0.04; β = 1.6 (0.4-3.0), p = 0.04; and β = 4.3 (0.9-7.7), p = 0.02, respectively). Associations of the SLEDAI with hypomethylation were confirmed for CpG17 (β=-32.6 (-60.6 to -4.6), p = 0.04) and CpG22 (β=-38.3 (-61.2 to -15.4), p = 0.004), but not the mean methylation of CD40L. Dietary products with the highest impact on methylation included meat, ice cream, white bread, and cooked potatoes.ConclusionsDietary methyl donors may influence DNA methylation levels and thereby disease activity in SLE.

Adverse effects of maternal rheumatoid arthritis during pregnancy on children.

Adverse effects of maternal rheumatoid arthritis during pregnancy on children.

Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation.

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

Methylation of TGM-3 Promoter and Its Association with Oral Squamous Cell Carcinoma (OSCC).

Background:Oral Squamous Cell Carcinoma (OSCC) is among the ten most common cancers worldwide. Hypermethylation of CpG sites in the promoter region and subsequent down-regulation of a tumor suppressor gene, TGM-3 has been proposed to be linked to different types of human cancers including OSCC. In this study, methylation status of CpG sites in the promoter region of TGM-3 has been evaluated in a cohort of patients with OSCC compared to normal controls.Methods:Forty fresh tissue samples were obtained from newly diagnosed OSCC patients and normal individuals referred to dentistry clinic for tooth extraction. DNA was extracted, bisulfite conversion was performed and it was subjected to PCR using bisulfite-sequencing PCR (BSP) primers. Prepared samples were sequenced on a DNA analyzer with both forward and reverse primers of the region of interest. The peak height values of cytosine and thymine were calculated and methylation levels for each CpG site within the DNA sequence was quantified.Results:Quantitative DNA methylation analyses in CpG islands revealed that it was significantly higher in OSCC patients compared to controls. DNA methylation at CpG1/CpG3/CpG5 (p=0.004–0.01) and CpG1/CpG3 (p=0.001–0.019) sites was associated with tumor stage and grade, respectively. Male OSCC patients had higher methylation rate at CpG3 (p=0.032), while smoker patients showed higher methylation rate at CpG6 (p=0.045).Conclusion:These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC.