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PubMed2006

Aberrant promoter hypermethylation and silencing of the critical 3p21 tumour suppressor gene, RASSF1A, in Chinese oesophageal squamous cell carcinoma.

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.

Carcinoma, Squamous Cell↗
PubMed1997

Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Chromosome Deletion↗
PubMed1998

Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.

Many aspects of Epstein-Barr virus (EBV) and tumor biology have been studied in Burkitt's lymphoma (BL)-derived cell lines. However, in tissue culture, patterns of gene expression and CpG [corrected] methylation often change and viral strain selection may occur. In this report, 10 cases of snap-frozen endemic BL tumors are characterized in terms of viral gene expression, promoter usage, methylation, and viral strain. EBNA1 and BamHI-A rightward transcripts (BART) were detected in 7 of 7 and LMP2A transcripts in 5 of 7 tumors with well-preserved RNA. Transcripts for the other EBNAs and for LMP1 were not detected in any tumor. These tumors differ from BL cell lines in that they lack a variety of lytic cycle transcripts. This pattern of viral gene expression in endemic BL is similar to that reported in peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive individuals. EBNA1 transcripts originated from the Q promoter (Qp) but not C, W, or F promoters that drive transcription of EBNA1 in other circumstances. Whereas Cp has been previously shown to be entirely CpG methylated in BL, bisulfite genomic sequencing showed virtually no methylation in Qp. Type-A EBV was detected in 6 of 10 and type B in 4 of 10 cases. A previously reported 30bp deletion variant in the carboxyl terminal of LMP1 gene was detected in 5 of 10 cases. The association with both A and B strains contrasts with EBV-associated Hodgkin's disease, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease, which are much more consistently associated with A strain virus.

Burkitt Lymphoma↗
PubMed1998

Ataxia-telangiectasia and T-cell leukemias: no evidence for somatic ATM mutation in sporadic T-ALL or for hypermethylation of the ATM-NPAT/E14 bidirectional promoter in T-PLL.

The ATM gene deficient in ataxia-telangiectasia, a recessive multisystem disease associated with a high risk of lymphomas and leukemias, was found previously to be inactivated in a rare sporadic malignancy, T-cell prolymphocytic leukemia (T-PLL), which is often associated with cytogenetic aberrations of chromosome 14. The ATM gene was shown to sustain frequent loss-of-function mutations in T-PLL tumor cells, consistent with functioning as a tumor suppressor gene in this leukemia. To investigate the possibility of nonmutational or nonrecombinational mechanisms of T-PLL development, we have used bisulfite genomic sequencing to analyze DNA methylation in the putative bidirectional promoter region of the closely linked ATM and NPAT/E14 genes within the CpG island at 11q22-q23. We show that this region is completely demethylated in lymphocytes expressing ATM; however, no extensive hypermethylation was found in 9 T-PLL tumor DNA samples without evidence of ATM/p53 mutations. Because acute T-cell lymphoblastic leukemias (T-ALL) were also observed in ataxia-telangiectasia patients and T-ALL tumor cells contain chromosome 14 abnormalities, 19 presentation samples of T-ALL patients were analyzed for ATM mutations. Although T-ALL patients exhibited rare nucleotide substitutions not previously found in ATM, all were identified in the germ-line, indicating constitutional polymorphisms, potentially confined to ethnic subpopulations. The absence of somatic nucleotide changes in ATM in T-ALL as compared with T-PLL suggests a distinct pattern of genetic events in the development of the two leukemias.

Ataxia Telangiectasia↗
PubMed1998

Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells.

Inactivation of p16INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M0). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16INK4 expression in postselection HMECs. In contrast, p16INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16INK4 CpG island and loss of p16INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

Breast↗
PubMed2006

Oligonucleotide DNA chips are useful adjuncts in epigenetic studies of glioblastomas.

Several studies have suggested that hypermethylation and hypomethylation of CpG islands within the promoters and 5' exons of tumor-related genes are closely associated with carcinogenesis. However, large-scale analysis of candidate genes has been hampered by the lack of a high throughput approach for analyzing methylation patterns. Using methylation-specific oligonucleotide (MSO) chips, we evaluated the methylation patterns of eight samples of fresh frozen glioblastoma tissue. The MSO chip used contained DNA probes with the CpG sites of p16 (p16INK4A, CDKN2A), MGMT (O6-Methylguanine-DNA-methyltransferase), APC (adenomatous polyposis coil), RASSF1A (human RAS effect homolog), which are usually hypermethylated in cancer cells and MAGE (melanoma antigen), which is usually hypomethylated in cancer cells. We selected CpG sites for analysis; 28 CpG sites (263 bp) for p16, 26 CpG sites (249 bp) for MGMT, 16 CpG sites (195 bp) for APC, 22 CpG sites (262 bp) for RASSF1A and 18 CpG sites (235 bp) for MAGE. We then constructed primer sets not including CpG sites. Bisulfite modification of genomic DNA, methylation specific PCR, hybridization and image scan with data analysis and sequencing of the bisulfite modified DNA were carried out. Of the eight glioblastomas, hypermethylation of the 5'-CpG sites of the MGMT were found in two, RASSF1A were found in five, and p16 and APC genes were not found in any cases and hypomethylation of that of the MAGE was found in eight cases. These results obtained from the oligo DNA chip study were correlated well with the sequencing data of bisulfite modified genomic DNA except in regard to the RASSF1A and MAGE genes. The devised MSO DNA chip is a useful tool for studies on methylation.

Base Sequence↗
PubMed2006

[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].

OBJECTIVE: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. METHODS: T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot. RESULTS: The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05). CONCLUSION: The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.

Adult↗
PubMed1994

CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene.

Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.

Animals↗
PubMed1997

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans.

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

Alleles↗
PubMed2002

CpG hypermethylation of promoter region and inactivation of E-cadherin gene in human bladder cancer.

Several studies have shown that E-cadherin expression is lost during malignant transformation. We hypothesized that CpG methylation in the promoter region may inactivate the expression of the E-cadherin gene in human bladder cancer. Normal and bladder cancer samples from 51 patients were compared in terms of E-cadherin gene expression and methylation status by immunohistochemistry, methylation-specific polymerase chain reaction (MSP), and bisulfite genome-sequencing techniques. Ten different CpG sites (nt 863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) in the promoter region were studied. Thirty-five of 51 (69%) bladder cancer samples lacked E-cadherin expression, whereas only six of 51 (12%) normal bladder samples lacked E-cadherin immunoreactivity. MSP analysis of bladder cancer samples suggested that 43 of 51 (84%) showed methylation of the promoter region, whereas only 12 of 51 (24%) normal bladder samples showed hypermethylation. Sodium bisulfite genome-sequencing analysis revealed that of 10 CpG sites, two sites (nt 892 and nt 940) showed 100% methylation in all the cancer samples analyzed. Other CpG sites were partially methylated (47-91%). Normal tissue showed only 12% methylation (range, 1-33%) on various CpG sites. Also supporting these data, E-cadherin-negative bladder cancer cell lines restored expression of the E-cadherin gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The present study showed that CpG hypermethylation was an important mechanism of E-cadherin gene inactivation in bladder cancer and also that specific CpG sites consistently presented higher methylation levels than others. These findings may provide a better strategy for the diagnosis and management of bladder cancer.

Aged↗
PubMed2002

Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives.

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.

Blotting, Southern↗
PubMed2004

Hypermethylation of the retinoic acid receptor-beta(2) gene in head and neck carcinogenesis.

PURPOSE: Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation. EXPERIMENTAL DESIGN: RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. RESULTS: Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. CONCLUSIONS: RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

Adult↗
PubMed2000

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

Amino Acid Sequence↗
PubMed1999

Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Chromosome Mapping↗
PubMed2006

Permissive transcriptional activity at the centromere through pockets of DNA hypomethylation.

DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and "chromatin-boundary activity" are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.

Animals↗
PubMed2004

Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates.

MOTIVATION: Methylation of cytosines in DNA plays an important role in the regulation of gene expression, and the analysis of methylation patterns is fundamental for the understanding of cell differentiation, aging processes, diseases and cancer development. Such analysis has been limited, because technologies for detailed and efficient high-throughput studies have not been available. We have developed a novel quantitative methylation analysis algorithm and workflow based on direct DNA sequencing of PCR products from bisulfite-treated DNA with high-throughput sequencing machines. This technology is a prerequisite for success of the Human Epigenome Project, the first large genome-wide sequencing study for DNA methylation in many different tissues. Methylation in tissue samples which are compositions of different cells is a quantitative information represented by cytosine/thymine proportions after bisulfite conversion of unmethylated cytosines to uracil and PCR. Calculation of quantitative methylation information from base proportions represented by different dye signals in four-dye sequencing trace files needs a specific algorithm handling imbalanced and overscaled signals, incomplete conversion, quality problems and basecaller artifacts. RESULTS: The algorithm we developed has several key properties: it analyzes trace files from PCR products of bisulfite-treated DNA sequenced directly on ABI machines; it yields quantitative methylation measurements for individual cytosine positions after alignment with genomic reference sequences, signal normalization and estimation of effectiveness of bisulfite treatment; it works in a fully automated pipeline including data quality monitoring; it is efficient and avoids the usual cost of multiple sequencing runs on subclones to estimate DNA methylation. The power of our new algorithm is demonstrated with data from two test systems based on mixtures with known base compositions and defined methylation. In addition, the applicability is proven by identifying CpGs that are differentially methylated in real tissue samples.

Algorithms↗
PubMed2006

A single nucleotide polymorphism chip-based method for combined genetic and epigenetic profiling: validation in decitabine therapy and tumor/normal comparisons.

Progress on several unresolved issues in cancer epigenetics will benefit from rapid and standardized methods for profiling DNA methylation genome-wide. In the area of epigenetic therapy, the demethylating drug decitabine (5-aza-2'-deoxycytidine) is increasingly used to treat acute myelogenous leukemia and myelodysplastic syndrome, but the mechanisms of its anticancer activity have remained unclear. Given the clinical efficacy of decitabine and the uncertainties about its mode of action, it will be useful to optimize methods for following DNA methylation as a biochemical response in individual patients. Here, we describe a single nucleotide polymorphism (SNP) chip-based method (MSNP) for profiling DNA methylation. Using this procedure, the extent of demethylation in bone marrow aspirates from patients with leukemia receiving decitabine can be assessed genome-wide using commercially available (Affymetrix) SNP chips. We validated the accuracy of MSNP by comparing the results with combined bisulfite restriction analysis and by sequencing cloned PCR products from bisulfite-converted DNA. We further validated MSNP in a Wilms' tumor/normal kidney comparison, comparing the results with methylation-sensitive Southern blotting. MSNP simultaneously detects aberrations in DNA copy number and loss of heterozygosity, making it a generally useful approach for combined genetic and epigenetic profiling in tissue samples from cancer patients.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

Analysis of methylation in the 5' region of the human alpha-galactosidase A gene containing a binding site for methylated DNA-binding protein/RFX1-4.

We examined methylation in the 5' untranslated region and surrounding sequences of the X-linked human alpha-galactosidase A gene. This region includes a methylation-dependent binding site for the MDBP/RFX1-4 transcription factor family. By bisulfite-based genomic sequencing, we determined the distribution of 5-methylcytosine residues from position +28 to +143 relative to the major transcription start site of the gene in almost 500 molecular clones from different tissues and individuals. The CpG methylation patterns in DNA from females differed from molecule to molecule with no indication of tissue-specificity or age-dependence. In contrast, the CG dinucleotides within this region were completely unmethylated in almost all of the DNA molecules cloned from male tissues. In female eutherian mammals, 50% of the molecules are expected to be methylated in an X-linked gene region in which methylation is coupled to gene silencing. Of the cloned DNA molecules from female tissues, 40% had 4 or more of the 9 CpG's methylated and the MDBP site was methylated in about one-fifth of the female tissue-derived DNA molecules.

Binding Sites↗