DNA差异甲基化在结核病检测中的应用进展

结核分枝杆菌感染导致的结核病仍然是全球公共卫生的重大挑战。结核病诊断新技术是提高结核病控制水平的关键。本文对全球基于DNA 甲基化诊断结核病新技术的相关进展进行综述,主要包括可能作为诊断靶标基因的甲基化,以及这些差异甲基化基因涉及的信号通路。     

Altered DNA methylation profile in idiopathic pulmonary fibrosis

Rationale: DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF). Objectives: To determine the DNA methylation changes in IPF and their effects on gene expression. Methods: Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genome-wide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels. Measurements and Main Results: DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase expression without observed alterations in global DNA methylation. Genome-wide differences in DNA methylation status and RNA expression were demonstrated by array hybridization. Among the genes whose DNA methylation status and RNA expression were both significantly altered, 16 genes were hypermethylated in DNA associated with decreased mRNA expression or vice versa. We validated CLDN5, ZNF467, TP53INP1, and DDAH1 genes at the level of DNA methylation status, RNA, and protein-level expression. Conclusions: Changes in DNA methylation correspond to altered mRNA expression of a number of genes, some with known and others with previously uncharacterized roles in IPF, suggesting that DNA methylation is important in the pathogenesis of IPF.     

Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression

Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9-25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., Held, W.A., Yoshida, S. & Nagase, H. (2003) Nucleic Acids Res. 31, 4490-4496]. Analysis of 14 TDMs by methylation-specific PCR and by bisulfite genomic sequencing confirms that the regions identified by RLGS are differentially methylated in a tissue-specific manner. The results indicate that 5% or more of the CpG islands are TDMs, disputing the general notion that all CpG islands are unmethylated. Some of the TDMs are within 5' promoter CpG islands of genes, which exhibit a tissue-specific expression pattern that is consistent with methylation status and a role in tissue differentiation.     

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.     

DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.     

Genome-wide DNA methylation analysis of articular chondrocytes identifies TRAF1, CTGF,and CX3CL1 genes as hypomethylated in osteoarthritis

Clinical Rheumatology - The aim of this study is to identify osteoarthritis (OA)-associated differentially methylated genes in human articular chondrocytes from patients with OA. DNA methylation...     

Aberrant DNA methylation profile of chronic and transformed classic Philadelphia-negative myeloproliferative neoplasms

Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.     

Analysis of the Genome-Wide DNA Methylation Profile of Side Population Cells in Hepatocellular Carcinoma

Background

DNA methylation plays an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the DNA methylation profile of stem cells in hepatocellular carcinoma (HCC) remains unclear.

Aims

To investigate the genome-wide DNA methylation profile of side population (SP) cells of HCC, a special subpopulation of cells enriched with cancer stem cells, by DNA methylation microarray analysis and to analyze the functions and signal pathways of the aberrantly methylated genes in SP cells.

Methods

Side population cells were isolated from HCC cell lines Huh7 and PLC/PRF/5 using flow cytometry, and the tumorigenicity of these SP cells was assessed in NOD/SCID mice. The genome-wide DNA methylation status of SP cells and non-SP (NSP) cells was detected and compared by DNA methylation microarray analysis. Genes with differential methylation between SP and NSP cells were further analyzed for their functions and roles in related signaling pathways.

Results

Subcutaneous inoculation of 1 × 10³ SP cells yielded tumors in 60 % NOD/SCID mice, whereas no tumor was developed after the inoculation of 1 × 10⁶ NSP cells. Genome-wide DNA methylation microarray analysis showed that 72 and 181 genes were hypermethylated and hypomethylated, respectively, in both Huh7 and PLC/PRF/5 SP cells as compared with their corresponding NSP cells. Analyses of signaling pathways revealed that hypermethylated and hypomethylated genes were related to four and eight pathways, respectively.

Conclusions

Hepatocellular carcinoma SP cells possessed a differential DNA methylation status compared with NSP cells, and the differentially methylated genes in SP cells were involved in 12 signaling pathways. Our results provide valuable clues for further investigations in elucidating the importance of epigenetic regulation in sustaining HCC SP cells and tumorigenesis.     

Honeybees and cell lines as models of DNA methylation and aging in response to diet

DNA methylation patterns change as individuals grow older, and DNA methylation appears susceptible to modification by the diet. Thus DNA methylation may be a mechanism through which diet can affect aging and longevity. We propose that effects on DNA methylation also contribute to the extension in lifespan observed in response to dietary restriction. Relationships between diet-induced changes in DNA methylation and parallel effects on aging and/or lifespan could, of course, be purely associative. Proof of these ideas requires experimental model systems in which it is possible to manipulate genome methylation status and to measure effects on aging and/or lifespan. Commonly-used short-lived and genetically-malleable metazoan species, such as Caenorhabditis elegans and Drosophila, are not suitable for such studies; the C. elegans genome is not methylated, and DNA methylation in Drosophila is dissimilar from mammalian DNA methylation, occurring at cytosines at sites other than in CpG sequences. The honeybee provides a potentially unique and tractable model for such studies. Female larval development into the long-lived queen phenotype or short-lived worker is determined purely by diet (royal jelly) through an effect on DNA methylation, and honeybee DNA methylation mirrors that of the mammalian genome. Mammalian cell lines and biochemical approaches offer complementary tools to address specific components of hypotheses relating to effects of diet on aging through DNA methylation in a more targeted manner. Our studies using mammalian cell lines are revealing effects of Sirt1 on DNA methylation, and indicate that Sirt1 and resveratrol affect the expression of different sets of genes.     

A meta-analysis on age-associated changes in blood DNA methylation: results from an original analysis pipeline for Infinium 450k data

Aging is characterized by a profound remodeling of the epigenetic architecture in terms of DNA methylation patterns. To date the most effective tool to study genome wide DNA methylation changes is Infinium HumanMethylation450 BeadChip (Infinium 450k). Despite the wealth of tools for Infinium 450k analysis, the identification of the most biologically relevant DNA methylation changes is still challenging. Here we propose an analytical pipeline to select differentially methylated regions (DMRs), tailored on microarray architecture, which is highly effective in highlighting biologically relevant results. The pipeline groups microarray probes on the basis of their localization respect to CpG islands and genic sequences and, depending on probes density, identifies DMRs through a single-probe or a region-centric approach that considers the concomitant variation of multiple adjacent CpG probes. We successfully applied this analytical pipeline on 3 independent Infinium 450k datasets that investigated age-associated changes in blood DNA methylation. We provide a consensus list of genes that systematically vary in DNA methylation levels from 0 to 100 years and that have a potentially relevant role in the aging process.     

Potential epigenetic dysregulation of genes associated with MODY and type 2 diabetes in humans exposed to a diabetic intrauterine environment: An analysis of genome-wide DNA methylation

ObjectiveThe aim of this study is to investigate the potential role of DNA methylation in mediating the increased risk of developing type 2 diabetes in offspring of mothers who had diabetes during pregnancy.Materials and MethodsPeripheral blood leukocytes were collected from non-diabetic Pima Indians who were either offspring of diabetic mothers (ODM; n = 14) or offspring of nondiabetic mothers (ONDM; n = 14). The two groups were matched for age, sex, age of mother, and fraction of Pima ethnicity. Differentially methylated regions were determined using a MeDIP-chip assay on an Affymetrix Human Tiling 2.0R Array. Data were analyzed using the model based analysis of tiling arrays (MAT) algorithm, and 4883 regions overlapping with putative promoters, were identified as differentially methylated, having met an empirically derived threshold (nominal p < 0.0077). The list of genes with differentially methylated promoters was subjected to KEGG pathway analysis to determine canonical metabolic pathways enriched by these genes.ResultsPathway analysis of genes with differentially methylated promoters identified the top 3 enriched pathways as maturity onset diabetes of the young (MODY), type 2 diabetes, and Notch signaling. Several genes in these pathways are known to affect pancreatic development and insulin secretion.ConclusionsThese findings support the hypothesis that epigenetic changes may increase the risk of type 2 diabetes via an effect on β-cell function in the offspring of mothers with diabetes during pregnancy.     

Gestational diabetes mellitus alters DNA methylation profiles in pancreas of the offspring mice

Gestational diabetes mellitus (GDM), which has an increasing global prevalence, contributes to the susceptibility to metabolic dysregulation and obesity in the offspring via epigenetic modifications. However, the underlying mechanism remains largely obscure. The current study established a GDM mice model to investigate the alternations in the metabolic phenotypes and genomic DNA methylation in the pancreas of the offspring. We found that in the GDM offspring, intrauterine hyperglycemia induced dyslipidemia, insulin resistance, and glucose intolerance. Meanwhile, altered DNA methylation patterns were exhibited in the pancreas and many differentially methylated regions (DMRs)-related genes were involved in glycolipids metabolism and related signaling pathways, including Agap2, Plcbr, Hnf1b, Gnas, Fbp2, Cdh13, Wnt2, Kcnq1, Lhcgr, Irx3, etc. Additionally, the overall hypermethylation of Agap2, verified by bisulfite sequencing PCR (BSP), was negatively correlated with its mRNA expression level. In conclusion, these findings suggest that the DNA methylation changes in the pancreatic genome of the GDM offspring may be associated with the glycolipid metabolism abnormalities, T2DM susceptibility, and obesity in the adult GDM offspring.     

Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes.

The relationship between DNA methylation and differential expression of rabbit beta-like globin genes was studied by using restriction enzymes that cleave the sequence C-C-G-G but are differentially inhibited by the presence of 5-methylcytosine. The methylation frequency of 13 C-C-G-G sites that flank a set of four closely linked rabbit beta-like globin genes was determined. This analysis revealed that certain sites surrounding embryonic and adult globin genes are relatively undermethylated in DNA from embryonic and adult erythroid tissues, respectively. This pattern is most pronounced for three sites that are undermethylated in erythroid cells but are totally methylated in nonerythroid cells. We conclude that the degree of CpG methylation in the rabbit beta-like globin gene cluster is correlated with gene activity, but the effect is confined to relatively small regions of DNA.     

Identification of the genomic region under epigenetic regulation during non‐alcoholic fatty liver disease progression

Aim

The progression of non‐alcoholic fatty liver disease (NAFLD) is affected by epigenetics. We undertook co‐methylation and differentially methylated region (DMR) analyses to identify the genomic region that is under epigenetic regulation during NAFLD progression.

Methods

We collected liver biopsy specimens from 60 Japanese patients with NAFLD and classified these into mild (fibrosis stages 0–2) or advanced (fibrosis stages 3–4) NAFLD. We carried out a genome‐wide DNA methylation analysis and identified the differentially methylated CpGs between mild and advanced NAFLD. Differentially methylated regions with multiple consecutive differentially methylated CpGs between mild and advanced NAFLD were extracted.

Results

Co‐methylation analysis showed that individual differentially methylated CpG sites were clustered into three modules. The CpG sites clustered in one module were hypomethylated in advanced NAFLD and their annotated genes were enriched for “immune system” function. The CpG sites in another module were hypermethylated and their annotated genes were enriched for “mitochondria” or “lipid particle”, and “lipid metabolism” or “oxidoreductase activity”. Hypomethylated DMRs included tumorigenesis‐related genes (FGFR2, PTGFRN, and ZBTB38), the expressions of which are upregulated in advanced NAFLD. Tumor suppressor MGMT had two DMRs and was downregulated. Conversely, FBLIM1 and CYR61, encoding proteins that reduce cell proliferation, showed hypomethylated DMRs and were upregulated. Expression of the antioxidant gene NQO1 was upregulated, with a hypomethylated DMR. The DMR containing cancer‐related MIR21 was hypomethylated in advanced NAFLD.

Conclusions

Co‐methylation and DMR analyses suggest that the NAFLD liver undergoes mitochondrial dysfunction, decreased lipid metabolism, and impaired oxidoreductase activity, and acquires tumorigenic potential at the epigenetic level.     

Identification of a region of the DNMT1 methyltransferase that regulates the maintenance of genomic imprints

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.