Methylation Patterns Of CpG Sites Research Articles

Postmortem age estimation via DNA methylation analysis in buccal swabs from corpses in different stages of decomposition\u2014a \u201cproof of principle\u201d study

Age estimation based on the analysis of DNA methylation patterns has become a focus of forensic research within the past few years. However, there is little data available regarding postmortem DNA methylation analysis yet, and literature mainly encompasses analysis of blood from corpses without any signs of decomposition. It is not entirely clear yet which other types of specimen are suitable for postmortem epigenetic age estimation, and if advanced decomposition may affect methylation patterns of CpG sites. In living persons, buccal swabs are an easily accessible source of DNA for epigenetic age estimation. In this work, the applicability of this approach (buccal swabs as source of DNA) under different postmortem conditions was tested. Methylation levels of PDE4C were investigated in buccal swab samples collected from 73 corpses (0–90 years old; mean: 51.2) in different stages of decomposition. Moreover, buccal swab samples from 142 living individuals (0–89 years old; mean 41.2) were analysed. As expected, methylation levels exhibited a high correlation with age in living individuals (training set: r2 = 0.87, validation set: r2 = 0.85). This was also the case in postmortem samples (r2 = 0.90), independent of the state of decomposition. Only in advanced putrified cases with extremely low DNA amounts, epigenetic age estimation was not possible. In conclusion, buccal swabs are a suitable and easy to collect source for DNA methylation analysis as long as sufficient amounts of DNA are present.

Prediction of the Prognosis Based on Chromosomal Instability-Related DNA Methylation Patterns of ELOVL2 and UBAC2 in PTCs

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of endocrine systems. Chromosomal instability (CIN) is crucial to the clinical prognoses of tumor patients. DNA methylation plays an important role in the regulation of gene expression and CIN. Based on PTC samples from The Cancer Genome Atlas database, we used multiple regression analyses to identify methylation patterns of CpG sites with the strongest correlation with gene expression. A total of 4,997 genes were obtained through combining the CpG sites, which were represented as featured DNA methylation patterns. In order to identify CIN-related epigenetic markers of PTC survival, we developed a method to characterize CIN based on DNA methylation patterns of genes using the Student’s t statistics. We found that 1,239 genes were highly associated with CIN. With the use of the log-rank test, univariate Cox regression analyses, and the Kaplan-Meier method, DNA methylation patterns of UBAC2 and ELOVL2, highly correlated with CIN, provided potential prognostic values for PTC. The higher these two genes, risk scores were correlated with worse PTC patient prognoses. Moreover, the ELOVL2 risk score was significantly different in the four stages of PTC, suggesting that it was related to the progress of PTC. The DNA methylation pattern associated with CIN may therefore be a good predictor of PTC survival.

Novel Epigenomic Biomarkers of Male Infertility Identified by Methylation Patterns of CpG Sites Within Imprinting Control Regions of H19 and SNRPN Genes.

Male infertility is an important global health burden that can benefit from novel biomarkers and diagnostics innovation. Aberrant methylation of the imprinted genes H19 and SNRPN (small nuclear ribonucleoprotein polypeptide N) in sperm DNA has been implicated in abnormal sperm parameters and male infertility. However, whether certain methylation patterns of one or multiple CpG sites within an imprinted gene are pathological for multiple sperm defects remains poorly understood. To examine the diagnostic potential of certain methylation patterns of CpG sites for multiphenotype defects in human sperm, the sperm DNA methylation patterns of individual CpG sites within imprinting control regions (ICRs) of imprinted genes H19 and SNRPN were measured by bisulfite pyrosequencing in a Han Chinese population sample: 39 oligoasthenozoospermia (OA) patients, 36 asthenoteratozoospermia (AT) patients, and 50 normozoospermia (N) controls. A partial least squares discriminant analysis model was built with the CpG sites as independent variables. Among the 16 CpG sites screened, the methylation patterns of eight CpG sites within H19-ICR (CpG sites 1, 6-9, 12 and 15-16), and eight CpG sites within SNRPN-ICR (CpG sites 2, 5-6, 8-10, 13, and 16) correctly classified 74.4% and 72.0% of the samples in terms of male fertile status, respectively. Furthermore, by combination of these 16 selected CpG sites within ICRs of H19 and SNRPN, 88.0% of the samples could be successfully classified. Our study demonstrates that methylation profiles of CpG sites within ICRs of imprinted genes H19 and SNRPN may potentially serve as epigenomic biomarkers for assessment of infertility in men with multiple sperm defects. Further studies in independent population samples are called for diagnostic significance of methylation patterns of CpG sites within imprinted genes.

The impact of methylation quantitative trait loci (mQTLs) on active smoking-related DNA methylation changes

BackgroundMethylation quantitative trait loci (mQTLs) are the genetic variants that may affect the DNA methylation patterns of CpG sites. However, their roles in influencing the disturbances of smoking-related epigenetic changes have not been well established. This study was conducted to address whether mQTLs exist in the vicinity of smoking-related CpG sites (± 50 kb) and to examine their associations with smoking exposure and all-cause mortality in older adults.ResultsWe obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 BeadChip array of two independent subsamples of the ESTHER study (discovery set, n = 581; validation set, n = 368) and their corresponding genotyping data using the Illumina Infinium OncoArray BeadChip. After correction for multiple testing (FDR), we successfully identified that 70 out of 151 previously reported smoking-related CpG sites were significantly associated with 192 SNPs within the 50 kb search window of each locus. The 192 mQTLs significantly influenced the active smoking-related DNA methylation changes, with percentage changes ranging from 0.01 to 18.96%, especially for the weakly/moderately smoking-related CpG sites. However, these identified mQTLs were not directly associated with active smoking exposure or all-cause mortality.ConclusionsOur findings clearly demonstrated that if not dealt with properly, the mQTLs might impair the power of epigenetic-based models of smoking exposure to a certain extent. In addition, such genetic variants could be the key factor to distinguish between the heritable and smoking-induced impact on epigenome disparities. These mQTLs are of special importance when DNA methylation markers measured by Illumina Infinium assay are used for any comparative population studies related to smoking-related cancers and chronic diseases.

Disturbance of DNA methylation patterns in the early phase of hepatocarcinogenesis induced by a choline‐deficient L‐amino acid‐defined diet in rats

The authors investigated the DNA methylation patterns of the E-cadherin, Connexin 26 (Cx26), Rassf1a and c-fos genes in the early phase of rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet. Six-week-old F344 male rats were continuously fed with the CDAA diet, and three animals were then killed at each of 4 and 8 days and 3 weeks. Genomic DNA was extracted from livers for assessment of methylation status in the 5' upstream regions of E-cadherin, Cx26, Rassf1a and c-fos genes by bisulfite sequencing, compared with normal livers. The livers of rats fed the CDAA diet for 4 and 8 days and 3 weeks were methylated in E-cadherin, Cx26 and Rassf1a genes, while normal livers were all unmethylated. In contrast, normal livers were highly methylated in c-fos gene. Although the livers at 4 days were weakly methylated, those at 8 days and 3 weeks were markedly unmethylated. Methylation patterns of CpG sites in E-cadherin, Cx26 and Rassf1a were sparse and the methylation was not associated with gene repression. These results indicate that gene-specific DNA methylation patterns were found in livers of rats after short-term feeding of the CDAA diet, suggesting gene-specific hypermethylation might be involved in the early phase of rat hepatocarcinogenesis induced by the CDAA diet.

Bisulfite modification of immobilized DNAs for methylation detection

We have developed a novel method for detecting DNA methylation status of multiple samples, in which the DNA samples were firstly immobilized on the slide and treated with bisulfite directly on the chip. In this experiment, DNAs of pUC19 plasmid were restricted by the enzymes, and ligated with a linker bearing 5′-terminal acrylamide group at the sticky ends. Using universal acrylamide gel polymerization technique, a large amount of DNAs could be immobilized on the slide. The immobilized DNAs were converted by soaking the chip in bisulfite reaction mixtures for 16 h. The probes for detection of the methylation patterns of CpG sites hybridized with the converted DNAs on the microarray, and non-specifically bound probes were cleaned by electrophoresis. We have optimized the experimental conditions of both bisulfite treatment and electrophoresis to increase sensitivity and specificity. The results were further validated by bisulfite DNA sequencing. The experiments show that the method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This novel method could be used as a convenient tool to detect the methylation status of the multiple genes for a large amount of samples in the future.

In situ bisulfite modification of membrane-immobilized DNA for multiple methylation analysis

Increasing interest in DNA methylation has resulted in the recent development of array-based methods. However, these method require complex sample treatment processes, such as bisulfite modification in the liquid state of every sample. Here, we describe a microarray-based technology for interrogating DNA methylation status of multiple DNA samples at the same time, in which the DNA samples are first dotted on membranes and then treated with bisulfite directly. In this assay, plasmid pUC19 DNA is immobilized on nylon membranes and soaked in bisulfite reaction mixtures for 16 h to convert unmethylated Cs to Us, and methylated Cs remain unchanged. The probes for detecting the methylation patterns of CpG sites are hybridized with the converted DNA dotted on the membranes, and the signals are revealed by chemiluminescence of DIG-labeled probes. The experiments show that this method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This new method could be used as a convenient tool for detecting the methylation status of multiple genes for a large number of samples in the future.