A pilot examination of the genome-wide DNA methylation signatures of subjects entering and exiting short-term alcohol dependence treatment programs

Alcoholism has a profound impact on millions of people throughout the world. However, the ability to determine if a patient needs treatment is hindered by reliance on self-reporting and the clinician’s capability to monitor the patient’s response to treatment is challenged by the lack of reliable biomarkers. Using a genome-wide approach, we have previously shown that chronic alcohol use is associated with methylation changes in DNA from human cell lines. In this pilot study, we now examine DNA methylation in peripheral mononuclear cell DNA gathered from subjects as they enter and leave short-term alcohol treatment. When compared with abstinent controls, subjects with heavy alcohol use show widespread changes in DNA methylation that have a tendency to reverse with abstinence. Pathway analysis demonstrates that these changes map to gene networks involved in apoptosis. There is no significant overlap of the alcohol signature with the methylation signature previously derived for smoking. We conclude that DNA methylation may have future clinical utility in assessing acute alcohol use status and monitoring treatment response.     

Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2’-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.     

DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter

IntroductionSclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes’ hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST’s expression in OA chondrocytes.MethodsSOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5’-Aza-2-deoxycytidine (5-AzadC) on SOST’s expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).ResultsWe observed that SOST’s expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.ConclusionsWe report novel findings that DNA methylation regulates SOST’s expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes’ expression observed in OA.     

DNA Methylation Dynamics in Blood after Hematopoietic Cell Transplant

Epigenetic deregulation is considered a common hallmark of cancer. Nevertheless, recent publications have demonstrated its association with a large array of human diseases. Here, we explore the DNA methylation dynamics in blood samples during hematopoietic cell transplant and how they are affected by pathophysiological events during transplant evolution. We analyzed global DNA methylation in a cohort of 47 patients with allogenic transplant up to 12 months post-transplant. Recipients stably maintained the donor’s global methylation levels after transplant. Nonetheless, global methylation is affected by chimerism status. Methylation analysis of promoters revealed that methylation in more than 200 genes is altered 1 month post-transplant when compared with non-pathological methylation levels in the donor. This number decreased by 6 months post-transplant. Finally, we analyzed methylation in IFN-γ, FASL, IL-10, and PRF1 and found association with the severity of the acute graft-versus-host disease. Our results provide strong evidence that methylation changes in blood are linked to underlying physiological events and demonstrate that DNA methylation analysis is a viable strategy for the study of transplantation and for development of biomarkers.     

外周全血中与老化相关的DNA甲基化标记的来源

机体老化与癌症、神经退行性疾病等许多复杂疾病相关。目前,研究者已在外周全血中识别了大量的与老化相关的DNA甲基化标记,这些标记可能反映外周血白细胞在机体老化过程中发生的变化,也可能反映外周血中与年龄相关的细胞构成比例的变化。文章利用3组正常个体外周全血DNA甲基化谱,采用Spearman秩相关分析识别了与老化相关的CpG甲基化位点(age-related DNA methylation CpG sites, arCpGs)并评价了其可重复性;利用去卷积算法估计了各外周血样本中髓性和淋巴性细胞的比例并分析了其与年龄的相关性;比较了在外周全血、CD4+T细胞和CD14+单核细胞中识别的arCpGs的一致性。结果显示,在独立外周全血数据中识别的arCpGs具有显著的可重复性(超几何检验,P=1.65×10^-11)。外周血髓性和淋巴性细胞的比例分别与年龄显著正、负相关(Spearman秩相关检验,P<0.05,r≤0.22),它们间DNA甲基化水平差异较大的CpG位点倾向于在外周全血中被识别为arCpGs。在CD4+T细胞中识别的arCpGs与在外周全血中识别的arCpGs显著交叠(超几何检验,P=6.14×10^-12),且99.1%的交叠位点在CD4+T细胞及外周全血中的DNA甲基化水平与年龄的正、负相关性一致。尽管在CD14+单核细胞中识别的arCpGs与在外周全血中识别的arCpGs并不显著交叠,但是在交叠的51个arCpGs中,有90.1%的位点在CD14+单核细胞、外周全血以及CD4+T细胞中的DNA甲基化水平与年龄的正、负相关性一致,提示它们可能主要反映细胞间共同的改变。在外周全血中识别的arCpGs主要反映某些白细胞共同或特异的DNA甲基化改变,但是也有一部分反映外周血细胞比例构成的变化。     

DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10⁻⁷ and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

A new method for accurate assessment of DNA quality after bisulfite treatment

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called ‘bisulfite treatment’, which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies.

The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.

     

SET8 methyltransferase activity during the DNA double-strand break response is required for recruitment of 53BP1

DNA double-strand breaks (DSBs) activate a signaling pathway known as the DNA damage response (DDR) which via protein–protein interactions and post-translational modifications recruit signaling proteins, such as 53BP1, to chromatin flanking the lesion. Depletion of the SET8 methyltransferase prevents accumulation of 53BP1 at DSBs; however, this phenotype has been attributed to the role of SET8 in generating H4K20 methylation across the genome, which is required for 53BP1 binding to chromatin, prior to DNA damage. Here, we report that SET8 acts directly at DSBs during the DNA damage response (DDR). SET8 accumulates at DSBs and is enzymatically active at DSBs. Depletion of SET8 just prior to the induction of DNA damage abrogates 53BP1’s accumulation at DSBs, suggesting that SET8 acts during DDR. SET8’s occupancy at DSBs is regulated by histone deacetylases (HDACs). Finally, SET8 is functionally required for efficient repair of DSBs specifically via the non-homologous end-joining pathway (NHEJ). Our findings reveal that SET8’s active role during DDR at DSBs is required for 53BP1’s accumulation.     

Exploring effects of DNA methylation and gene expression on pan-cancer drug response by mathematical models

Since genetic alteration only accounts for 20%–30% in the drug effect-related factors, the role of epigenetic regulation mechanisms in drug response is gradually being valued. However, how epigenetic changes and abnormal gene expression affect the chemotherapy response remains unclear. Therefore, we constructed a variety of mathematical models based on the integrated DNA methylation, gene expression, and anticancer drug response data of cancer cell lines from pan-cancer levels to identify genes whose DNA methylation is associated with drug response and then to assess the impact of epigenetic regulation of gene expression on the sensitivity of anticancer drugs. The innovation of the mathematical models lies in: Linear regression model is followed by logistic regression model, which greatly shortens the calculation time and ensures the reliability of results by considering the covariates. Second, reconstruction of prediction models based on multiple dataset partition methods not only evaluates the model stability but also optimizes the drug-gene pairs. For 368,520 drug-gene pairs with P < 0.05 in linear models, 999 candidate pairs with both AUC ≥ 0.8 and P < 0.05 were obtained by logistic regression models between drug response and DNA methylation. Then 931 drug-gene pairs with 45 drugs and 491 genes were optimized by model stability assessment. Integrating both DNA methylation and gene expression markedly increased predictive power for 732 drug-gene pairs where 598 drug-gene pairs including 44 drugs and 359 genes were prioritized. Several drug target genes were enriched in the modules of the drug-gene-weighted interaction network. Besides, for cancer driver genes such as EGFR, MET, and TET2, synergistic effects of DNA methylation and gene expression can predict certain anticancer drugs’ responses. In summary, we identified potential drug sensitivity-related markers from pan-cancer levels and concluded that synergistic regulation of DNA methylation and gene expression affect anticancer drug response.     

Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While ∼70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, ∼70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.     

DNA Methylation Signature of Childhood Chronic Physical Aggression in T Cells of Both Men and Women

Background

High frequency of physical aggression is the central feature of severe conduct disorder and is associated with a wide range of social, mental and physical health problems. We have previously tested the hypothesis that differential DNA methylation signatures in peripheral T cells are associated with a chronic aggression trajectory in males. Despite the fact that sex differences appear to play a pivotal role in determining the development, magnitude and frequency of aggression, most of previous studies focused on males, so little is known about female chronic physical aggression. We therefore tested here whether or not there is a signature of physical aggression in female DNA methylation and, if there is, how it relates to the signature observed in males.

Methodology/Principal Findings

Methylation profiles were created using the method of methylated DNA immunoprecipitation (MeDIP) followed by microarray hybridization and statistical and bioinformatic analyses on T cell DNA obtained from adult women who were found to be on a chronic physical aggression trajectory (CPA) between 6 and 12 years of age compared to women who followed a normal physical aggression trajectory. We confirmed the existence of a well-defined, genome-wide signature of DNA methylation associated with chronic physical aggression in the peripheral T cells of adult females that includes many of the genes similarly associated with physical aggression in the same cell types of adult males.

Conclusions

This study in a small number of women presents preliminary evidence for a genome-wide variation in promoter DNA methylation that associates with CPA in women that warrant larger studies for further verification. A significant proportion of these associations were previously observed in men with CPA supporting the hypothesis that the epigenetic signature of early life aggression in females is composed of a component specific to females and another common to both males and females.     

DNA sequence and methylation prescribe the inside-out conformational dynamics and bending energetics of DNA minicircles

Eukaryotic genome and methylome encode DNA fragments’ propensity to form nucleosome particles. Although the mechanical properties of DNA possibly orchestrate such encoding, the definite link between ‘omics’ and DNA energetics has remained elusive. Here, we bridge the divide by examining the sequence-dependent energetics of highly bent DNA. Molecular dynamics simulations of 42 intact DNA minicircles reveal that each DNA minicircle undergoes inside-out conformational transitions with the most likely configuration uniquely prescribed by the nucleotide sequence and methylation of DNA. The minicircles’ local geometry consists of straight segments connected by sharp bends compressing the DNA’s inward-facing major groove. Such an uneven distribution of the bending stress favors minimum free energy configurations that avoid stiff base pair sequences at inward-facing major grooves. Analysis of the minicircles’ inside-out free energy landscapes yields a discrete worm-like chain model of bent DNA energetics that accurately account for its nucleotide sequence and methylation. Experimentally measuring the dependence of the DNA looping time on the DNA sequence validates the model. When applied to a nucleosome-like DNA configuration, the model quantitatively reproduces yeast and human genomes’ nucleosome occupancy. Further analyses of the genome-wide chromatin structure data suggest that DNA bending energetics is a fundamental determinant of genome architecture.     

Lung Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis Exhibit Genome-Wide Differences in DNA Methylation Compared to Fibroblasts from Nonfibrotic Lung

Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n = 6) with those of nonfibrotic patient controls (n = 3) and commercially available normal lung fibroblast cell lines (n = 3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease.     

Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood

Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log₁₀ arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log₁₀ increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.     

Genome‐wide methylomic analyses identify prognostic epigenetic signature in lower grade glioma

Glioma is the most malignant and aggressive type of brain tumour with high heterogeneity and mortality. Although some clinicopathological factors have been identified as prognostic biomarkers, the individual variants and risk stratification in patients with lower grade glioma (LGG) have not been fully elucidated. The primary aim of this study was to identify an efficient DNA methylation combination biomarker for risk stratification and prognosis in LGG. We conducted a retrospective cohort study by analysing whole genome DNA methylation data of 646 patients with LGG from the TCGA and GEO database. Cox proportional hazard analysis was carried out to screen and construct biomarker model that predicted overall survival (OS). The Kaplan‐Meier survival curves and time‐dependent ROC were constructed to prove the efficiency of the signature. Then, another independent cohort was used to further validate the finding. A two‐CpG site DNA methylation signature was identified by multivariate Cox proportional hazard analysis. Further analysis indicated that the signature was an independent survival predictor from other clinical factors and exhibited higher predictive accuracy compared with known biomarkers. This signature was significantly correlated with immune‐checkpoint blockade, immunotherapy‐related signatures and ferroptosis regulator genes. The expression pattern and functional analysis showed that these two genes corresponding with two methylation sites contained in the model were correlated with immune infiltration level, and involved in MAPK and Rap1 signalling pathway. The signature may contribute to improve the risk stratification of patients and provide a more accurate assessment for precision medicine in the clinic.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP)

Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. Techniques currently available for examining such changes either require selection of a region, then examination of methylation changes, or utilise methylation-sensitive restriction enzymes to identify an alteration. We describe here a novel method that identifies genomic regions as a consequence of altered methylation during tumourigenesis. A methyl-CpG binding domain column isolates methylated GC-rich sequences from both tumours and surrounding normal tissue. Subsequent subtractive hybridisation removes sequences common to both, leaving only methylated sequences unique to the tumour. Libraries of sequences generated using DNA derived from a breast tumour (histological grade; poorly differentiated) as ‘tester’ and from matched normal tissue as ‘driver’ were examined; 26% of clones had the sequence criteria of a CpG island (CGI). Analysis using the bisulfite technique revealed that a number of these sequences were methylated in tumour DNA relative to the normal control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved.