Microarray analysis of promoter methylation in lung cancers

Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated + methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated + methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes

DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.     

Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis.

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19-23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the human estrogen receptor alpha (ERalpha) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.     

High-throughput methylation profiling by MCA coupled to CpG island microarray

An abnormal pattern of DNA methylation occurs at specific genes in almost all neoplasms. The lack of high-throughput methods with high specificity and sensitivity to detect changes in DNA methylation has limited its application for clinical profiling. Here we overcome this limitation and present an improved method to identify methylated genes genome-wide by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). We validated this method in three cancer cell lines and 15 primary colorectal tumors, resulting in the discovery of hundreds of new methylated genes in cancer. The sensitivity and specificity of the method to detect hypermethylated loci were 88% and 96%, respectively, according to validation by bisulfite-PCR. Unsupervised hierarchical clustering segregated the tumors into the expected subgroups based on CpG island methylator phenotype classification. In summary, MCAM is a suitable technique to discover methylated genes and to profile methylation changes in clinical samples in a high-throughput fashion.     

APC基因甲基化定量检测芯片的建立

目的 建立抑癌基因APC（adenomatous polyposis coli，APC）启动子1A的甲基化定量芯片检测方法。方法选取一段420bp的APC基因启动子1A CpG密集序列作为靶序列，针对M0、M1、M2、M3、M4 5个CpG靶位点，设计一套检测甲基化与非甲基化的探针。采用脐带血DNA克隆体作为阴性、阳性质控品。结果甲基化阳性、阴性质控的芯片结果与测序吻合。每组探针中荧光强度由强至弱依次为，阳性质控（甲基化）：探针1〉2、3〉4；阴性质控（非甲基化）：探针3〉4、1〉2。5个位点的5条荧光强度标准曲线，尺。范围是0．93～0．99。M0、M1、M2、M3、M4 5个位点甲基化杂合型的检测范围分别为50．0％±3．6％、50．0％±6．9％、50．0％±3．5％、50．0％±8．5％、50．0％±7．3％。结论建立了APC基因启动子5个COG位点的甲基化定量检测芯片。     

Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarray

Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber-type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end-labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non-neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non-neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non-neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation-positive by conventional methylation-specific polymerase chain reaction (MSP). This microarray-based methylation assay is a promising method for the quantitative assessment of gene methylation.     

The CpG island in intron 22 of the factor VIII gene is predominantly methylated on the X chromosome of human males

The inactivation of one of the two X chromosomes in females is a random process associated with methylation principally in CpG islands. The methylation status of a CpG island in intron 22 of the human factor VIII (FVIII) gene was investigated using a novel practical approach. Genomic DNA from men and women was digested with various methylation-sensitive (MS) restriction enzymes, the recognition sequences of which occurred at least once in the FVIII CpG island. Long distance-polymerase chain reaction (LD-PCR) was then used to amplify the island. Successful amplification indicated that the island was methylated and the absence of a PCR product indicated that at least one restriction site was unmethylated. To analyze the relative methylation status of the extragenic and intragenic copies of the island, we used Southern blot with MS restriction enzymes. The MS LD-PCR patterns obtained from male and female DNA samples indicated that at least some copies of the intragenic CG island were fully methylated at all sites investigated. Additionally, the island showed consistent differences among individuals. Southern blot studies using female DNA showed partial resistance to MS digestion for the intragenic and extragenic CpG island homologs. Our observations indicate that this CpG island is predominantly methylated on the X chromosome of males and suggest that its methylation pattern does not correlate with X inactivation of females. This prevents the use of this island coupled with DNA polymorphisms for investigation of X-chromosome inactivation. Received: December 4, 2001 / Accepted: February 12, 2002     

胚胎干细胞分化为表皮样干细胞基因启动子区差异甲化研究

 目的： 用全基因组甲基化芯片探讨可能参与小鼠胚胎干细胞(ESCs)分化为表皮样细胞(ELCs)的基因DNA甲基化调控机制。方法： 利用人羊膜建立体外诱导体系,将小鼠ESCs诱导定向分化为ELCs,分别取未分化的ESCs和诱导分化后的ELCs进行芯片分析,利用甲基化免疫共沉淀技术将每组染色质DNA的甲基化片段共沉淀下来,和本底对照 (input)分别标记Cy3和Cy5荧光,一同上样于Roche NimbleGen高密度(2.1M,芯片覆盖22 425个启动子)甲基化芯片,启动子区采用UCSC数据库进行注释,启动子覆盖转录起始位点上游8 200 bp、下游3 000 bp,通过对这些启动子区甲基化谱的分析,筛选出甲基化调控可能与ESCs向ELCs定向分化相关的基因。结果： 小鼠ESCs和ELCs 两组细胞在基因组水平上有17 500个启动子存在甲基化,其中有3 435个启动子发生甲基化差异变化,高CpG岛的启动子有894个发生差异甲基化,中度CpG岛的启动子有974个发生差异甲基化,低CpG岛的启动子有1 567个发生差异甲基化。结论： 在ESCs向ELCs定向分化的过程中,众多的基因启动子区发生了甲基化程度的变化,说明细胞分化是一个复杂的表观遗传学事件。这些基因启动子的差异甲基化在ESCs向ELCs定向分化过程中所起的作用及其机制尚有待进一步的功能学研究阐明。     

Hypomethylation of the serotonin receptor type‐2A Gene (HTR2A) at T102C polymorphic site in DNA derived from the saliva of patients with schizophrenia and bipolar disorder

Several lines of evidence indicate that dysfunction of serotonin signaling and HTR2A receptor are involved in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BD). DNA methylation of HTR2A at T102C polymorphic site influences HTR2A expression and aberrant DNA methylation of HTR2A promoter was reported in postmortem brain of patients with SCZ and BD. Hypothesizing that the brain's epigenetic alteration of HTR2A may also exist in peripheral tissues that can be used as a diagnostic/therapeutic biomarker, we analyzed HTR2A promoter DNA methylation in DNA extracted from the saliva of patients with SCZ and BD, and their first degree relatives versus normal controls. Bisulfite sequencing was used to screen DNA methylation status of the HTR2A promoter CpGs and qMSP was used to quantify the degree of cytosine methylation at differentially methylated sites. Most of the cytosines of the HTR2A promoter were unmethylated. However, CpGs of the ?1438A/G polymorphism site, ?1420 and ?1223 were >95% methylated. The CpG at T102C polymorphic site and neighboring CpGs were ～70% methylated both in the patients and controls. qMSP analysis revealed that the cytosine of the T102C polymorphic site was significantly hypo‐methylated in SCZ, BD, and their first degree relatives compared to the controls. Cytosine methylation of HTR2A at T102C polymorphic site in DNA derived from the saliva can potentially be used as a diagnostic, prognostic, and/or therapeutic biomarker in SCZ and BD. However, these preliminary observations need to be replicated in other populations with a larger sample size to be considered for clinical applications. © 2011 Wiley‐Liss, Inc.     

Bisulfite genomic sequencing to uncover variability in DNA methylation: Optimized protocol applied to human T cell differentiation genes

DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, is one of several epigenetic mechanisms that cells use to control gene expression. The importance of DNA methylation in a variety of biological processes (i.e., embryonic development, cellular proliferation and differentiation, chromosome stability) has led to a demand for a precise and efficient method to determine the exact DNA methylation status. Bisulfite genomic sequencing is regarded as a gold-standard technology for detection of DNA methylation as it provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine at single base-pair resolution. To optimize the final results of the bisulfite genomic sequencing protocol, numerous modifications have been explored and have significantly improved the sensitivity and accuracy of this procedure. The aim of this methodological report is to give an overview of the bisulfite genomic sequencing protocol, discussing the critical methodological aspects. Since we are interested in studying the methylation status of specific genes involved in T cell development, we applied the bisulfite genomic sequencing to the study of the CD8A T cell co-receptor gene to determine whether the CGIs of this gene were subjected to methylation in different types of tissues. The results show that CD8A gene is differentially methylated depending on the tissue. In conclusion, we described a bisulfite genomic sequencing protocol that can be successfully used for the quantitative analysis of CpG island methylation of specific genes.     

DENV2感染外周血单个核细胞增加TNF-α基因启动子区域CpG位点的甲基化水平

 目的：检测正常人外周血单个核细胞（PBMC）感染2型登革病毒（DENV2）后肿瘤坏死因子α（TNF-α）基因启动子区域CpG位点的甲基化水平。
方法：采用亚硫酸氢盐测序PCR法检测DNA甲基化水平。结果：TNF-α基因启动子区域为-294 bp到+58 bp,覆盖11个散在CpG位点;PCR反应后取PCR产物进行琼脂糖凝胶电泳分析显示,扩增序列大小与理论预测相符合;PBMC感染DENV2 0 h和6 h 在11个甲基化位点中有2个处于甲基化状态,感染12 h有6个甲基化位点甲基化。0 h、6 h和12 h的平均甲基化率分别为103%、121%和255%,且0 h和12 h及6 h和12 h的甲基化率差异有统计学意义。结论：PBMC感染DENV2后会引起TNF-α基因启动子区域的甲基化水平增加。     

Methylation profiles of CpG island loci in major types of human cancers

Several reports have described aberrant methylation in various types of human cancers. However, the interpretation of methylation frequency in various human cancers has some limitations because of the different materials and methods used for methylation analysis. To gain an insight into the role of DNA hypermethylation in human cancers and allow direct comparison of tissue specific methylation, we generated methylation profiles in 328 human cancers, including 24 breast, 48 colon, 61 stomach, 48 liver, 37 larynx, 24 lung, 40 prostate, and 46 uterine cervical cancer samples by analyzing CpG island hypermethylation of 13 genes using methylation-specific PCR. The mean numbers of methylated genes were 6.5, 4.4, 3.6, 3.4, 3.1, 3.1, 3.1, and 2.1 in gastric, liver, prostate, larynx, colon, lung, uterine cervix, and in breast cancer samples, respectively. The number of genes that were methylated at a frequency of more than 40% in each tumor type ranged from nine (stomach) to one (breast). Generally genes frequently methylated in a specific cancer type differed from those methylated in other cancer types. The findings indicate that aberrant CpG island hypermethylation is a frequent finding in human cancers of various tissue types, and each tissue type has its own distinct methylation pattern.     

改进的甲基化特异PCR法在抑癌基因p16检测中的应用

目的 介绍一种ＣｐＧ岛甲基化分析方法,即甲基化特异的ＰＣＲ（ｍｅｔｈｙｌａｔｉｏｎ ｓｐｅｃｉｆｉｃ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ,ＭＳＰ）法以及对该法的改进。方法 用亚硫酸氢盐修饰被测ＤＮＡ后,再进行甲基化与非甲基化特异的ＰＣＲ扩增,并对ＭＳＰ法进行改进。应用该法分析了颌面部鳞癌中ｐ１６基因５′ＣｐＧ岛甲基化状态。结果 发现一些颌面部鳞癌组织中有ｐ１６基因５′ＣｐＧ岛的甲基     

Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease

Lin Z, Hegarty JP, Cappel JA, Yu W, Chen X, Faber P, Wang Y, Kelly AA, Poritz LS, Peterson BZ, Schreiber S, Fan J‐B, Koltun WA. Identification of disease‐associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease. Overwhelming evidence supports the theory that inflammatory bowel disease (IBD) is caused by a complex interplay between genetic predispositions of multiple genes, combined with an abnormal interaction with environmental factors. It is becoming apparent that epigenetic factors can have a significant contribution in the pathogenesis of disease. Changes in the methylation state of IBD‐associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We have explored the role of DNA methylation in IBD pathogenesis. DNA methylation profiles (1505 CpG sites of 807 genes) of matched diseased (n = 26) and non‐diseased (n = 26) intestinal tissues from 26 patients with IBD [Crohn's disease (CD) n = 9, ulcerative colitis (UC) n = 17] were profiled using the GoldenGate? methylation assay. After an initial identification of a panel of 50 differentially methylated CpG sites from a training set (14 non‐diseased and 14 diseased tissues) and subsequent validation with a testing set (12 non‐diseased and 12 diseased tissues), we identified seven CpG sites that are differentially methylated in intestinal tissues of IBD patients. We have also identified changes in DNA methylation associated with the two major IBD subtypes, CD and UC. This study reports IBD‐associated changes in DNA methylation in intestinal tissue, which may be disease subtype‐specific.     

IgA肾病组蛋白H3K4三甲基化和DNA甲基化基因修饰的相关性

目的: 探讨IgA肾病(IgAN)外周血单个核细胞(PBMCs)组蛋白H3赖氨酸4 (H3K4)三甲基化与DNA甲基化之间的关系。方法: 采用染色质免疫共沉淀联合芯片技术(ChIP-chip)对40例IgAN患者和40例健康者的PBMCs H3K4三甲基化进行高通量筛选,染色质免疫共沉淀-实时定量聚合酶链反应 (ChIP-qPCR) 验证芯片结果,定量反转录聚合酶链反应(qRT-PCR)检测阳性基因的mRNA表达水平,采用甲基化DNA免疫共沉淀定量聚合酶链反应(MeDIP-qPCR)检测DNA甲基化水平。结果: IgAN病人PBMCs 基因组H3K4三甲基化水平升高,基因组DNA甲基化水平降低。 4个阳性基因H3K4三甲基化水平和DNA甲基化水平与正常对照组相比,显著差异(P<0.05)。结论: IgAN患者PBMCs基因组H3K4三甲基化和DNA甲基化水平存在显著改变,DNA甲基化和组蛋白H3K4三甲基化基因修饰存在相互作用。     

启动子区域甲基化状态对THP-1细胞肿瘤坏死因子α分泌的影响

目的: 探讨肿瘤坏死因子α( TNF-α )基因启动子区域散在CpG双核苷酸结构甲基化状态对蛋白分泌和基因表达的影响。方法: 检测人单核细胞系THP-1细胞在脂多糖(LPS)刺激状态下,不同时点TNF-α的表达和其基因启动子区域的甲基化状态。采用ELISA法测定细胞培养液上清TNF-α水平;采用重亚硫酸盐转化基因测序法测定DNA甲基化状态。结果: 受到LPS刺激后,THP-1细胞迅速产生 TNF-α ,在4 h达到高峰后快速下降。 TNF-α 基因启动子-344 bp到转录起始位点(TSS)区域内,存在11个散在CpG双核苷酸结构。未受到LPS刺激时均处于甲基化状态;刺激4 h后,有4个处于甲基化状态;刺激8 h后,有9个处于甲基化状态。未刺激与刺激4 h之间,刺激4 h与刺激8 h之间,甲基化率比较均有显著差异(P＜0.01,P<0.05)。结论: 未受到LPS刺激时,THP-1细胞 TNF-α 基因启动子区域甲基化水平较高;LPS刺激后,其该区域甲基化水平降低。该变化与TNF-α表达分泌相关。     

Genomic hypomethylation and CpG island hypermethylation in prostatic intraepithelial neoplasm

Altered DNA methylation in cancer cells is characterized by focal CpG island hypermethylation and diffuse genomic hypomethylation. Both types of aberrant methylation are frequently found in human prostate adenocarcinoma (PCa). Prostatic intraepithelial neoplasm (PIN), a precursor lesion of PCa, has been demonstrated to contain CpG island hypermethylation, but little is known about the role of DNA hypomethylation. We analyzed the methylation status at 12 CpG island loci and at two repetitive DNA elements (LINE-1 and SAT2) from normal prostate (n = 20), PIN (n = 25), and PCa (n = 35) tissues using MethyLight assay or combined bisulfite restriction analysis. The methylation levels in LINE-1 and SAT2 decreased with progression of lesion types from normal prostate to PIN to PCa (P < 0.05), whereas promoter CpG island loci displayed increased methylation. Ten genes were found to be hypermethylated in a cancer-specific manner and were further analyzed in another set of PCa tissues (n = 64). The number of methylated genes was closely associated with TNM stage, Gleason sum, and preoperative serum PSA levels (P = 0.020, 0.073, 0.033, respectively). These results suggest that genomic hypomethylation and CpG island hypermethylation, common among PCas, are early events in prostate carcinogenesis and may be implicated in the development of PIN. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Epigenetic marks by DNA methylation specific to stem,germ and somatic cells in mice

BACKGROUND: DNA methylation is involved in many gene functions such as gene-silencing, X-inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue-dependent and differentially methylated region (T-DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation. RESULTS: We investigated the genome-wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T-DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation. CONCLUSIONS: Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei.