Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2'-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.     

A microarray to analyze methylation patterns of p16(Ink4a) gene 5'-CpG islands

OBJECTIVES: Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16(Ink4a) gene. DESIGN AND METHODS: We selected a 336 bp segment of the 5' untranslated region and the first exon of the p16(Ink4a) gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites. RESULTS: Methylation patterns of human p16(Ink4a) gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments. CONCLUSIONS: The methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites.     

Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome

BACKGROUND: Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are 2 distinct neurodevelopmental disorders caused primarily by deficiency of specific parental contributions at an imprinted domain within the chromosomal region 15q11.2-13. In most cases, lack of paternal contribution leads to PWS either by paternal deletion (approximately 70%) or maternal uniparental disomy (UPD; approximately 30%). Most cases of AS result from the lack of a maternal contribution from this same region by maternal deletion (approximately 70%) or by paternal UPD (approximately 5%). Analysis of allelic methylation differences at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus can differentiate the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS. METHODS: Sodium bisulfite-treated genomic DNA was PCR-amplified for the SNRPN gene. We used pyrosequencing to individually quantify the resulting artificial C/T sequence variation at CpG sites. Anonymized DNA samples from PWS patients (n = 40), AS patients (n = 31), and controls (n = 81) were analyzed in a blinded fashion with 2 PCR and 3 pyrosequencing reactions. We compared results from the pyrosequencing assays with those obtained with a commonly used methylation-specific PCR (MS-PCR) diagnostic protocol. RESULTS: The pyrosequencing assays had a sensitivity and specificity of 100% and provided quantification of methylation at 12 CpG sites within the SNRPN locus. The resulting diagnoses were 100% concordant with those obtained from the MS-PCR protocol. CONCLUSIONS: Pyrosequencing is a rapid and robust method for quantitative methylation analysis of the SNRPN locus and can be used as a diagnostic test for PWS and AS.     

In-tube DNA methylation profiling by fluorescence melting curve analysis.

BACKGROUND: Most PCR assays for detection of 5-methylcytosine in genomic DNA entail a two-step procedure, comprising initial PCR amplification and subsequent product analysis in separate operations that usually require manual transfer. These methods generally provide information about methylation of only a few CpG dinucleotides within the target sequence. METHODS: An in-tube methylation assay is described that integrates amplification of bisulfite-treated DNA and melting analysis by using a thermal cycler coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR Green I) during a linear temperature transition. RESULTS: Analysis of a region comprising 11 CpG sites at the SNRPN promoter CpG island showed that the melting temperature (T(m)) differed by approximately 3 degrees C between unmethylated and fully methylated alleles. This assay could easily distinguish patients with Prader-Willi syndrome or Angelman syndrome from individuals without these conditions. Melting curve analysis also allowed resolution of methylation "mosaicism" at the p15(Ink4b) promoter in bone marrow samples from patients with acute myeloid leukemia (AML). AML samples representing pools of heterogeneously methylated p15(Ink4b) alleles showed broadened melting peaks with overall T(m)s between those of the unmethylated and fully methylated alleles. CONCLUSIONS: Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.     

恶性血液病中的P15^INK4B基因甲基化

目的 探索特定基因操纵区ＣｐＧ岛高甲基化对人类造血系统恶性肿瘤的作用。方法 利用甲基化特异性聚合酶链反应（ＭＳＰ）的方法,用亚硫酸氢钠修饰后的ＤＮＡ为模板进行甲基化特异性聚合酶链反应（ＰＣＲ）,测定了２５例急性髓系白血病（ＡＭＬ）,１５例慢性粒细胞白血病（ＣＭＬ）,１６例骨髓增生异常综合征（ＭＤＳ）和１２例多发性骨髓瘤（ＭＭ）患Ｐ１５＾ＩＮＫ４Ｂ基因在操纵区５′－ＣｐＧ岛异常甲基化的发生率。结果 Ｐ１５＾ＩＮＫ４Ｂ基因异常甲基化的发生率：ＡＭＬ为８４％,ＣＭＬ为０,ＭＤＳ为５０％,ＭＭ为７５％。高危型的ＭＤＳ较低危型更易发生甲基化,在ＭＭ中,Ｐ１５＾ＩＮＫ４Ｂ甲基化一般发生在早期,与骨髓象的幼稚程度关系密切。结论 调节细胞生长和分化的Ｐ１５＾ＩＮＫ４Ｂ基因高甲基化是使Ｐ１５＾ＩＮＫ４Ｂ基因失活的主要原因之一,     

脐血CD34＋细胞和外周血单个核细胞中HOXB4基因表达及其启动子区CpG岛甲基化状态的研究

本研究旨在通过检测脐血CD34＋细胞和正常人外周血单个核细胞（PBMNC）中HOXB4基因表达水平及其启动子区CpG岛的甲基化水平,初步探讨造血系统中HOXB4基因的表达水平与其启动子区甲基化的相关性。采用半定量RT—PCR检测脐血CD34＋细胞和PBMNC中HOXB4基因的表达,应用亚硫酸氢盐测序法检测这两种细胞中HOXB4基因启动子区CpG岛的甲基化位点。结果发现,CD34＋细胞高表达HOXB4,HOXB4基因启动子区CpG岛未发生甲基化;PBMNc不表达HOXB4,HOXB4基因启动子区在ATG上游-129bp的C碱基发生甲基化。结论：HOXB4基因启动子甲基化程度是其基因表达水平的负性调节机制之一。HOXIM在CD34＋细胞中的高表达与其基因启动子低甲基化有关,而HOXB4在PBMNC中的基因沉默则可能与其启动子区的甲基化密切相关。初步筛选出的HOXB4基因启动子区CpG岛甲基化位点,为后续研究奠定了基础,也为扩增造血祖细胞提供一条新途径。     

5' CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3 genes as potential markers of HCC

OBJECTIVES: The change in DNA methylation patterns can be used to distinguish between normal and cancer cells. The aim of the present study was to examine the 5' CpG island methylation patterns of the cancer-testis antigen (CT antigen) gene family, MAGE-As, in hepatocellular carcinoma (HCC), and to develop the DNA demethylation pattern as a novel tumor biomarker. METHODS: We used bisulfite-sequencing PCR (BSP) to map the methylation status of the CpG site among the promoter of the MAGE-A gene family in several HCC cell lines including Hep G2, BEL7402, BEL7404, and BEL7407, and normal peripheral blood white blood cells (WBCs). According to differences of the methylation pattern between HCC cell lines and the control, methylation-special PCRs (MSP) have been developed. The developed MSPs were used to detect the paraffin-embedded slices that were pathologically diagnosed as HCC, hepatocirrhosis, hepatitis, and healthy. RESULTS: We found that several CpG sites among the MAGE-A1 and MAGE-A3 promoters have different methylation patterns in the HCC cell lines as compared to those in normal WBCs. Two sets of MSP primers were designed to distinguish the HCC genomic DNA and normal control cell genomic DNA as novel tumor biomarkers, and the biomarkers were validated on the archived paraffin sections of liver primary tissue. In the detection of 34 HCCs and 17 tumor-free liver tissues, the clinical sensitivity and specificity were 91.2% and 100%, respectively. CONCLUSION: Detection of aberrant methylation patterns of MAGEs CpG islands using MSP may be useful for diagnosis of HCC.     

重型β地中海贫血γ珠蛋白基因启动子区甲基化状态

本研究旨在通过对广西省重型β地中海贫血患者和正常人外周血中γ珠蛋白基因启动子区CpG岛的甲基化位点的确认和对各个CpG位点甲基化率在重型β地中海贫血患者与正常人之间的差异性分析,初步筛选出可能影响γ珠蛋白表达的主要甲基化位点。采用重亚硫酸盐测序法修饰,递降PCR扩增,最后对PCR产物进行DNA克隆测序,得到目的片段中每个CG位点的甲基化情况,定量检测甲基化的确切位置与程度。结果表明:重型β地中海贫血患者与正常人γ珠蛋白基因启动子区存在4个CpG甲基化位点,在序列中分别位于28、122、231和234bp,均呈高度甲基化,其中122和231 bp位点甲基化率明显低于正常对照组,差异均有统计学意义。28和234 bp位点甲基化率与正常对照组均无显著性差异。结论:证实γ基因启动子区存在甲基化位点、明确位点位置且经定量分析证实无论地中海贫血患者还是正常人各个甲基化位点均呈高甲基化状态;初步筛选出影响γ珠蛋白表达的主要位点可能在122和231 bp,为后续通过调节γ珠蛋白的表达缓解重型β地中海贫血的临床症状、靶向基因治疗研究提供了实验依据。     

恶性血液病细胞株中IEX-1基因启动子区CpG岛甲基化状态的初探

本研究探讨基因IEX-1启动子区CpG岛甲基化状态的改变及其与恶性血液病发生的相关性.应用MSP的方法检测9种恶性血液病细胞系中IEX-1基因启动子区CpG岛甲基化状态,并把经M.sssI酶处理过的正常人外周血单个核细胞中IEX-1 CpG岛甲基化状态作为阳性对照,把正常人外周血单个核细胞中IEX-1 CpG岛甲基化状态作为阴性对照.结果显示:在NB4、Molt4.、Raji细胞系中IEX-1基因启动子区CpG岛呈高甲基化状态;在CA46、CEM、U937、K562、HL-60、Jurkat细胞系中IEX-1基因启动子区CpG岛呈部分甲基化状态;在正常人外周血单个核细胞中IEX-1基因启动子区CpG岛呈非甲基化状态;经M.sssI酶处理过的正常人外周血单个核细胞中IEX-1基因启动子CpG岛呈高甲基化状态.结论:IEX-1基因启动子区CpG岛甲基化状态的改变与恶性血液病有一定的相关性.     

Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools

Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.     

肾透明细胞癌组织中miR-34a和miR-34b/c基因CpG岛甲基化状态的变化及意义

目的 探讨肾透明细胞癌组织中miR-34a和miR-34b/c基因CpG岛甲基化状态的变化及其意义.方法 将30例肾透明细胞癌手术患者的肾癌组织和癌旁正常组织标本按是否有淋巴结转移分为2组:A组15例为无淋巴结和(或)远处转移,B组15例为有淋巴结和(或)远处转移.提取各标本中的DNA,经甲基化处理后,用PCR的方法检测miR-34a和miR-34b/c基因CpG岛甲基化状态在2组肾癌组织和癌旁正常组织中的表达情况.结果 电泳结果显示,miRNA-34a和miR-34b/c基因成单一条带,其所处的位置及其在正常组织和癌组织中的甲基化情况符合前期的预计.miR-34a和miR-34b/c基因CpG岛甲基化状态如下:A组肾癌组织中miR-34a 4例,占26.7％,miR-34b/c 15例,占100.0％,癌旁正常组织均为0;B组肾癌组织中miR-34a 13例,占86.7％,miR-34b/c15例,占100.0％,癌旁正常组织均为0.miR-34a和miR-34b/c基因CpG岛甲基化状态在肾癌组织中明显增高;伴有淋巴结转移的肾癌组织中,miR-34a基因CpG岛甲基化明显高于无淋巴结转移的肾癌组织(86.7％比26.7％,P＜0.05).结论 miR-34a和miR-34b/c基因CpG岛甲基化状态在肾癌组织中明显增高,提示其与肾透明细胞癌的发生与进展有关,尤其是miR-34a基因CpG岛高甲基化与肾透明细胞癌的转移可能存在密切联系,有望成为指导肾透明细胞癌诊断、治疗及判断预后的新靶点.     

Evaluation of markers for CpG island methylator phenotype (CIMP) in colorectal cancer by a large population-based sample

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation is a distinct phenotype in colorectal cancer. However, a choice of markers for CIMP has been controversial. A recent extensive investigation has selected five methylation markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) as surrogate markers for epigenomic aberrations in tumor. The use of these markers as a CIMP-specific panel needs to be validated by an independent, large dataset. Using MethyLight assays on 920 colorectal cancers from two large prospective cohort studies, we quantified DNA methylation in eight CIMP-specific markers [the above five plus CDKN2A (p16), CRABP1, and MLH1]. A CIMP-high cutoff was set at > or = 6/8 or > or = 5/8 methylated promoters, based on tumor distribution and BRAF/KRAS mutation frequencies. All but two very specific markers [MLH1 (98% specific) and SOCS1 (93% specific)] demonstrated > or = 85% sensitivity and > or = 80% specificity, indicating overall good concordance in methylation patterns and good performance of these markers. Based on sensitivity, specificity, and false positives and negatives, the eight markers were ranked in order as: RUNX3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1, and CDKN2A. In conclusion, a panel of markers including at least RUNX3, CACNA1G, IGF2, and MLH1 can serve as a sensitive and specific marker panel for CIMP-high.     

急性髓系白血病E-cadherin基因表达和CpG岛甲基化状态的研究

目的 探讨急性髓系白血病（AML）患者中上皮钙黏附素（E-cadherin）基因表达及其甲基化状态。方法 分别用RT-PCR和流式细胞术方法检测55例AML患者骨髓细胞和7份正常对照骨髓细胞中E—cadherin基凶和蛋白的表达，并用甲基化特异性PCR方法分析E—cadherin基因5’端CpG岛的甲基化状况。结果 55例AML患者骨髓细胞中E-cadherinmRNA和蛋白表达阳性率分别为23．6％和18．2％，较正常对照组明显下调（P值均〈0．01）；E—cadherin基因5’端甲基化的阳性率为69．1％。E-cadherin mRNA和蛋白表达阴性的31例患者中，29例（93．5％）E—cadherin甲基化阳性，其蛋白水平和mRNA表达水平与启动子甲基化呈明显负相关（P〈0．01）。7份正常对照骨髓细胞E—cadherin mRNA和蛋白表达均为阳性，E—eadherin基因的CpG岛无明显甲基化。结论 AML的粒系分化成熟障碍可能与E—cadherin基因表达下调有关。5’端CpG岛的甲基化可能影响E-cadherin基因表达。     

Sensitive sequencing method for KRAS mutation detection by Pyrosequencing

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.     

结直肠癌C-erbB-2基因启动子CpG岛甲基化状态及其与C-erbB-2蛋白表达的关系

目的了解结直肠癌组织C-erbB-2基因启动子区CpG岛甲基化状态、C-erbB-2蛋白表达水平及两者之间的关系。方法取经病理确诊的43例结直肠癌组织和相应癌旁组织,分别用甲基化特异性聚合酶链反应(MSP)和免疫组织化学法(IHC)检测C-erbB-2启动子区CpG岛的甲基化和C-erbB-2蛋白表达水平。结果癌组织和癌旁组织中C-erbB-2启动子区CpG岛甲基化率分别为44.2%和74.4%,差异具有统计学意义(P=0.004)。癌组织和癌旁组织中C-erbB-2蛋白过度表达率分别为67.4%和27.9%,差异具有统计学意义(P=0.000)。C-erbB-2蛋白表达水平与肿瘤分期相关。结直肠癌组织C-erbB-2启动子区CpG岛甲基化状态与C-erbB-2蛋白表达水平呈显著相关(r=0.331,P=0.03)。结论结直肠癌中C-erbB-2基因启动子区CpG岛的低甲基化可能在C-erbB-2蛋白过度表达中起一定作用,有望成为有用的肿瘤分子诊断标记和治疗靶点。     

结直肠癌C-erbB-2基因启动子CpG岛甲基化状态及其与C-erbB-2蛋白表达的关系

目的了解结直肠癌组织C—erbB-2基因启动子区CpG岛甲基化状态、C-erbB-2蛋白表达水平及两者之间的关系。方法取经病理确诊的43例结直肠癌组织和相应癌旁组织，分别用甲基化特异性聚合酶链反应（MSP）和免疫组织化学法（IHC）检测C-erbB-2启动子区CpG岛的甲基化和C-erbB-2蛋白表达水平。结果癌组织和癌旁组织中C—erbB-2启动子区CpG岛甲基化率分别为44．2％和74．4％，差异具有统计学意义（P=0．004）。癌组织和癌旁组织中C—erbB-2蛋白过度表达率分别为67．4％和27．9％，差异具有统计学意义（P=0．000）。C—erbB-2蛋白表达水平与肿瘤分期相关。结直肠癌组织C—erbB-2启动子区CpG岛甲基化状态与C—erbB-2蛋白表达水平呈显著相关（r=0．331，P=0．03）。结论结直肠癌中C—erbB-2基因启动子区CpG岛的低甲基化可能在C—erbB-2蛋白过度表达中起一定作用，有望成为有用的肿瘤分子诊断标记和治疗靶点。     

乳腺癌C-erbB-2蛋白表达与基因CpG岛甲基化状态和mRNA表达的关系

目的 探讨乳腺癌C-erbB-2蛋白表达与基因CpG岛甲基化状态和mRNA表达的关系.方法 取经病理确诊的52例乳腺癌组织,分别用甲基化特异性聚合酶链反应( MSP)、免疫组织化学法(IHC)和逆转录-聚合酶链反应(RT-PCR)检测C-erbB-2启动子CpG岛甲基化状态、C-erbB-2蛋白表达水平和C-er6B-...     

Pyrosequencing: nucleotide sequencing technology with bacterial genotyping applications

Pyrosequencing is a relatively new method for real-time nucleotide sequencing. It has rapidly found applications in DNA sequencing, genotyping, single nucleotide polymorphism analysis, allele quantification and whole-genome sequencing within the areas of microbiology, clinical genetics and pharmacogenetics. It is fast becoming a real alternative to the traditional Sanger sequencing method although, at present, read lengths are normally limited to approximately 70 nucleotides. The pyrosequencing method involves four main stages: first, target DNA is amplified using PCR; second, double-stranded DNA is converted to single-stranded DNA templates; third, oligonucleotide primers are hybridized to a complementary sequence of interest; and, finally, the pyrosequencing reaction itself, in which a reaction mixture of enzymes and substrates catalyses the synthesis of complementary nucleotides. Data are shown as a collection of signal peaks in a pyrogram. Pyrosequencing is increasingly used for bacterial detection, identification and typing, and, recently, a commercial system became available for the identification of bacterial isolates. Pyrosequencing can also be partially or fully automated, thus enabling the high-throughput analysis of samples. Wider use of pyrosequencing may occur in the future if longer nucleotide reads are made possible, which will enable its expansion into larger nucleotide sequencing such as multilocus sequence typing and whole-genome sequencing.     

DNA甲基化调控K562细胞系kir3dl1基因表达

为分析kir3dl1基因启动子区的甲基化模式及其与基因表达的关系，探讨kir3dl1基因的表达调控机制，采用亚硫酸氢盐测序法检测K562细胞中kir3dl1基因启动子区的甲基化状况，应用甲基化抑制剂5．氮胞苷处理K562细胞以诱导CpG岛去甲基化，观察启动子区CpG岛甲基化与kir3dl1基因表达的关系。结果显示：K562细胞中kir3dl1基因核心启动子区CpG二核苷酸甲基化频率在20％-100％之间；K562细胞中kir3dl1基因启动子序列在转录因子E2F可能的结合位点上存在1个碱基的突变，并形成1个新的被甲基化的CpG二核苷酸；应用甲基化抑制剂5．氮胞苷可以诱导不表达kir3dl1基因的K562细胞重新表达该基因。结论：K562细胞中kir3dl1基因表达受启动子甲基化调控，对转录因子E2F的深入研究有助于了解其在kir3dl1基因表达调控中可能的作用。